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## **Meet the editors**

Dr. Zhiwei Wang is a professor in Cyrus Tang Hematology Center, Soochow University, P.R. China. He received his MD from Anhui Bengbu Medical College, P.R. China, and his Ph.D. from the Wayne State University, Michigan, USA. He has completed postdoctoral fellowships at Karmanos Cancer Institute, Wayne State University, Michigan, USA. Then, he was an instructor in Beth

Israel Deaconess Medical Center, Harvard medical School, USA. He has published more than 140 papers in peer-reviewed journals with Hirsh Factor 31, and citations more than 3000. He has served as an ad hoc reviewer of grant and as an ad hoc reviewer of papers for more than 40 Journals. Dr. Wang's research interests are focused on the roles of cell signaling pathways such as Notch, FoxM1 and E3 ubiquitin ligases in senescence and tumorigenesis. Moreover, He is also interested in roles of microRNA and stem cells in drug resistance.

Dr. Hiroyuki Inuzuka is an instructor at Beth Israel Deaconess Medical Center and Harvard Medical School. He earned his Ph.D. in molecular biology from the University of Tokyo. His current research focuses on the molecular mechanisms of human malignancy including age-related diseases. Specifically, his studies involve uncovering the molecular processes involved in cell

cycle regulation and cellular senescence. Ultimately, he seeks to apply his findings to help find a way to extend human healthy life expectancy.

Contents

**Preface VII**

Wenyi Wei

**Section 2 Cellular Senescence 23**

**Section 3 Plant Senescence 51**

**Signaling 53**

**Population 87**

**Section 5 Mastication and Cognition 113**

**Section 1 Aging and Vascular Diseases 1**

Chapter 1 **Endothelium Aging and Vascular Diseases 3**

Chapter 2 **Molecular Mechanisms of Cellular Senescence 25** Therese Becker and Sebastian Haferkamp

Chapter 3 **Plant Senescence and Nitrogen Mobilization and**

Chapter 4 **Immunosenescence and Senescence Immunosurveillance: One**

Chapter 5 **The Relationship Between Mastication and Cognition 115** Kin-ya Kubo, Huayue Chen and Minoru Onozuka

**of the Possible Links Explaining the Cancer Incidence in Ageing**

Stefan Bieker and Ulrike Zentgraf

Arnaud Augert and David Bernard

**Section 4 Immunosenescence and Cancer 85**

Shavali Shaik, Zhiwei Wang, Hiroyuki Inuzuka, Pengda Liu and

## Contents



#### **Section 6 Neurodegenerative Disease 133**

#### Chapter 6 **On the Way to Longevity: How to Combat Neuro-Degenerative Disease 135** Patrizia d'Alessio, Rita Ostan, Miriam Capri and Claudio Franceschi

Preface

nism of senescence.

complex regulatory network.

This book discusses in detail regarding senescence and its related diseases. Each chapter is written by distinguished researchers and practicing clinicians, which provides unique, in‐ dividual knowledge based on the expertise of the authors. This book should build fur‐ ther the endeavors of the readers in senescence field. Therefore, I wish that this book would serve as a basis for further discussions and developments in exploring molecular mecha‐

The first chapter, written by Dr. Shavali Shaik and his colleagues from Beth Israel Deaconess Medical Center Harvard Medical School, describes the molecular changes that occur in the vascular system due to aging, and defines how age-induced changes in the endothelium ul‐ timately lead to the development of various vascular diseases. Dr. Shaik et al. highlighted particularly how the age-induced oxidative stress plays a major role in causing loss of endo‐ thelial function, and described the underlying mechanisms responsible for the development of various vascular diseases including cardiovascular, peripheral vascular and diabetic ret‐

The second chapter in this book, Dr. Becker Therese and colleagues provide detailed insight into the molecular mechanisms of how the two tumor suppressor pathways, p53-p21 and the p16INK4a-pRb, regulate the onset and maintenance of cellular senescence. They furthermore explain the molecular network regulating chromatin remodeling and the formation of senes‐ cence associated heterochromatin foci, with emphasis on the above mentioned pathways.

In the next chapter, Dr. Stefan Bieker et al. give an overview on the current knowledge on regulatory mechanisms of senescence in general and their impact on nitrogen metabolism, including uptake, assimilation, and distribution within the plant. Special attention was also given to reactive nitrogen and reactive oxygen molecules as signalling components in this

In the following chapter, Dr. David Bernard et al. introduce features, markers, triggers and molecular regulators of cellular senescence and discuss their role in tumorigenesis. More‐ over, Dr. Bernard describes the role of immunosenescence in the development of cancer,

The next chapter, by Dr. Kubo Kin-ya et al., tries to provide evidence supporting the interac‐ tion between mastication and learning and memory. Dr. Kubo et al. briefly describe recent progress in understanding how mastication affects learning and memory. Moreover, they highlight the impaired function and pathology of the hippocampus in an animal model of reduced mastication. More importantly, they discuss how occlusal disharmony is a chronic

suggesting that senescence immunosurveillance is pivotal for tumor eradication.

stressor that suppresses hippocampal-mediated learning and memory.

inopathy which are highly observed in aged population.

## Preface

**Section 6 Neurodegenerative Disease 133**

**VI** Contents

Chapter 6 **On the Way to Longevity: How to Combat Neuro-Degenerative Disease 135**

Patrizia d'Alessio, Rita Ostan, Miriam Capri and Claudio Franceschi

This book discusses in detail regarding senescence and its related diseases. Each chapter is written by distinguished researchers and practicing clinicians, which provides unique, in‐ dividual knowledge based on the expertise of the authors. This book should build fur‐ ther the endeavors of the readers in senescence field. Therefore, I wish that this book would serve as a basis for further discussions and developments in exploring molecular mecha‐ nism of senescence.

The first chapter, written by Dr. Shavali Shaik and his colleagues from Beth Israel Deaconess Medical Center Harvard Medical School, describes the molecular changes that occur in the vascular system due to aging, and defines how age-induced changes in the endothelium ul‐ timately lead to the development of various vascular diseases. Dr. Shaik et al. highlighted particularly how the age-induced oxidative stress plays a major role in causing loss of endo‐ thelial function, and described the underlying mechanisms responsible for the development of various vascular diseases including cardiovascular, peripheral vascular and diabetic ret‐ inopathy which are highly observed in aged population.

The second chapter in this book, Dr. Becker Therese and colleagues provide detailed insight into the molecular mechanisms of how the two tumor suppressor pathways, p53-p21 and the p16INK4a-pRb, regulate the onset and maintenance of cellular senescence. They furthermore explain the molecular network regulating chromatin remodeling and the formation of senes‐ cence associated heterochromatin foci, with emphasis on the above mentioned pathways.

In the next chapter, Dr. Stefan Bieker et al. give an overview on the current knowledge on regulatory mechanisms of senescence in general and their impact on nitrogen metabolism, including uptake, assimilation, and distribution within the plant. Special attention was also given to reactive nitrogen and reactive oxygen molecules as signalling components in this complex regulatory network.

In the following chapter, Dr. David Bernard et al. introduce features, markers, triggers and molecular regulators of cellular senescence and discuss their role in tumorigenesis. More‐ over, Dr. Bernard describes the role of immunosenescence in the development of cancer, suggesting that senescence immunosurveillance is pivotal for tumor eradication.

The next chapter, by Dr. Kubo Kin-ya et al., tries to provide evidence supporting the interac‐ tion between mastication and learning and memory. Dr. Kubo et al. briefly describe recent progress in understanding how mastication affects learning and memory. Moreover, they highlight the impaired function and pathology of the hippocampus in an animal model of reduced mastication. More importantly, they discuss how occlusal disharmony is a chronic stressor that suppresses hippocampal-mediated learning and memory.

The last chapter is by Dr. Claudio Franceschi and colleagues. They describe how to combat neurodegenerative disease using results obtained with ad hoc models such as centenarians and their offspring compared with subjects affected by Down syndrome. They also discuss future perspectives on the reversibility of early stages of degenerative diseases by non antiinflammatory approaches including physical exercise, motivational implementation, nutrition and nutraceutic approaches. Importantly, they conclude the development of novel tools to be integrated in daily life of elderly people which is critical for reducing degenerative diseases.

Lastly, as the editors, we are grateful to the contributors for their promptness in preparing their chapters. We are also impressed by their dedication and diligent work. We are thank‐ ful to Dr. Wenyi Wei for strong support during publishing this book. We also appreciate receiving help from Ms. Danijela Duric.

> **Zhiwei Wang, Ph.D M.D** Harvard Medical School Department of Pathology Beth Israel Deaconess Medical Center Boston, USA

**Section 1**

**Aging and Vascular Diseases**

**Hiroyuki Inuzuka, PhD** Harvard Medical School Department of Pathology Beth Israel Deaconess Medical Center Boston, USA **Aging and Vascular Diseases**

The last chapter is by Dr. Claudio Franceschi and colleagues. They describe how to combat neurodegenerative disease using results obtained with ad hoc models such as centenarians and their offspring compared with subjects affected by Down syndrome. They also discuss future perspectives on the reversibility of early stages of degenerative diseases by non antiinflammatory approaches including physical exercise, motivational implementation, nutrition and nutraceutic approaches. Importantly, they conclude the development of novel tools to be integrated in daily life of elderly people which is critical for reducing degenerative diseases. Lastly, as the editors, we are grateful to the contributors for their promptness in preparing their chapters. We are also impressed by their dedication and diligent work. We are thank‐ ful to Dr. Wenyi Wei for strong support during publishing this book. We also appreciate

> **Zhiwei Wang, Ph.D M.D** Harvard Medical School Department of Pathology

> **Hiroyuki Inuzuka, PhD** Harvard Medical School Department of Pathology

Boston, USA

Boston, USA

Beth Israel Deaconess Medical Center

Beth Israel Deaconess Medical Center

receiving help from Ms. Danijela Duric.

VIII Preface

**Chapter 1**

**Endothelium Aging and Vascular Diseases**

Aging is a biological process that causes a progressive deterioration of structure and func‐ tion of all organs over the time [1]. According to the United Nation's report, the number of people aged 60 and over in the world has increased from 8% (200 million) in 1950 to 11% (760 million) in 2005, and it is estimated that this number will further increase to 22% (2 bil‐ lion) in 2050. It is expected that in the US alone, the aged population of 65 and over will grow rapidly and reach 81 million by 2050 [2,3]. This rapidly increasing aging population will not only cause a decline of productive workforce but also negatively affect the country's economy. Furthermore, aging is one of the major risk factors for the development of many diseases including cardiovascular diseases [4], stroke [5] and cancer [6]. Moreover, the epi‐ demiological data strongly suggests that more often these diseases are diagnosed in older people compared to younger individuals. In addition to the huge economical impact, these diseases also cause loss of productivity and disability in the elderly population. Therefore, it is extremely important to give high priorities to aging and age-associated disease research in order to develop novel therapies to slow the aging process as well as to prevent and /or treat the age-associated diseases more effectively. It has been found that many factors including genetics [7,8], metabolism [9], diet [10] and stress [11] can in part contribute to the aging process. Similar to other organs, the vascular system, which provides oxygen and nutrients to all the organs in the body, is also affected by the aging process and becomes more vulner‐ able to disease development in the elders [12,13]. For example, vascular diseases such as cor‐ onary artery disease, peripheral arterial disease, stroke and microvascular disease are more often found in the aged population. This is in part due to the structural and functional changes that occur in the vascular system of aged people. In this review, we highlighted (i) the changes that occur in the vascular system, particularly in the endothelium due to aging; (ii) the mechanisms by which the age-associated changes lead to decreased angiogenesis;

> © 2013 Shaik et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Shavali Shaik, Zhiwei Wang, Hiroyuki Inuzuka,

Additional information is available at the end of the chapter

Pengda Liu and Wenyi Wei

http://dx.doi.org/10.5772/53065

**1. Introduction**

## **Endothelium Aging and Vascular Diseases**

Shavali Shaik, Zhiwei Wang, Hiroyuki Inuzuka, Pengda Liu and Wenyi Wei

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53065

#### **1. Introduction**

Aging is a biological process that causes a progressive deterioration of structure and func‐ tion of all organs over the time [1]. According to the United Nation's report, the number of people aged 60 and over in the world has increased from 8% (200 million) in 1950 to 11% (760 million) in 2005, and it is estimated that this number will further increase to 22% (2 bil‐ lion) in 2050. It is expected that in the US alone, the aged population of 65 and over will grow rapidly and reach 81 million by 2050 [2,3]. This rapidly increasing aging population will not only cause a decline of productive workforce but also negatively affect the country's economy. Furthermore, aging is one of the major risk factors for the development of many diseases including cardiovascular diseases [4], stroke [5] and cancer [6]. Moreover, the epi‐ demiological data strongly suggests that more often these diseases are diagnosed in older people compared to younger individuals. In addition to the huge economical impact, these diseases also cause loss of productivity and disability in the elderly population. Therefore, it is extremely important to give high priorities to aging and age-associated disease research in order to develop novel therapies to slow the aging process as well as to prevent and /or treat the age-associated diseases more effectively. It has been found that many factors including genetics [7,8], metabolism [9], diet [10] and stress [11] can in part contribute to the aging process. Similar to other organs, the vascular system, which provides oxygen and nutrients to all the organs in the body, is also affected by the aging process and becomes more vulner‐ able to disease development in the elders [12,13]. For example, vascular diseases such as cor‐ onary artery disease, peripheral arterial disease, stroke and microvascular disease are more often found in the aged population. This is in part due to the structural and functional changes that occur in the vascular system of aged people. In this review, we highlighted (i) the changes that occur in the vascular system, particularly in the endothelium due to aging; (ii) the mechanisms by which the age-associated changes lead to decreased angiogenesis;

© 2013 Shaik et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(iii) how the ubiquitin proteasome system plays important roles in regulating vascular endo‐ thelium function; (iv) the mechanisms by which the age-associated increase in oxidative stress might cause endothelial dysfunction; and finally, (iv) how the age-associated changes in the vascular system lead to the development of various vascular diseases such as coro‐ nary artery disease, peripheral artery disease and diabetic retinopathy.

expressed in the lymphatic endothelial cells [16]. The VEGF/VEGFR2 signaling is critical for vasculogenesis as well as angiogenesis [16]. Disruption or loss of VEGF and VEGFR2 genes is associated with severe vascular abnormalities or embryonic lethality [17]. Furthermore, the endothelial cells produce other growth factors known as angiopoitins (Ang), which are required to remodel and stabilize the immature blood vessels induced by VEGF/VEGFR2. Moreover, molecules such as neuropilines are involved in modulating the binding as well as responses to VEGF receptors [16]. Furthermore, endothelial cells express endothelial nitric oxide synthase (eNOS), which produces nitric oxide (NO). NO has many important physio‐ logical functions. For example, NO promotes vasodilation [18], as well as inhibits leukocyte adhesion [19], thrombocyte aggregation [20] and smooth muscle cell proliferation [21]. Un‐ der basal conditions eNOS is found inactive, however its activity is increased by many fac‐ tors including acetyl choline, bradykinin, thrombin and histamine that lead to increased

Endothelium Aging and Vascular Diseases http://dx.doi.org/10.5772/53065 5

**Figure 1. Age-associated changes that occur in the heart and the vascular system.** Normal young heart has highly functional cardiomyocytes, and normal atrium and ventricles (A). Young artery has normal lumen, normal arterial thickness and efficient contractile and relaxation properties (B). However, aged heart has increased thickness in the heart muscle due to hypertrophy. Specifically, cardiomyocytes from aged heart show hyperplasia along with some car‐ diomyocytes undergoing senescence (C). Aged artery also has increased thickness, reduced lumen and less efficient contractile and relaxation properties (D). These age-associated changes ultimately lead to reduced cardiac as well as

production of NO.

vascular functions in the elders.

#### **2. Age-associated changes in the vascular system**

Many changes are known to occur due to aging in the entire vascular system that includes heart, coronary arteries, peripheral arteries and small blood vessels known as capillaries (Figure 1). There will be an increase in the overall size of the heart, due to an increase in the heart wall thickness in the aging heart. The heart valves, which control the unidirectional of blood flow, will also become stiffer. There is also deposition of the pigments known as lipo‐ fuscin in the aged heart along with possible loss of cardiomyocytes as well as cells present in the sinoatrial node (SA node). Furthermore, there is an increase in the size of cardiomyo‐ cytes to compensate for the loss of the heart cells. These changes altogether cause a progres‐ sive decline in the physiological functions of the heart in the elderly population. In addition to these changes in the heart, the blood vessels also undergo significant changes. For exam‐ ple, the aorta, the large artery that originates from the heart becomes thicker, stiffer and less flexible. Smaller blood vessels also become thicker and stiffer. These changes are due to al‐ terations that occur in the cells present in the blood vessels and also in the connective tissue of the blood vessel wall. All these changes ultimately lead to hypertrophy of the heart and causes an increase in the blood pressure [14]. There seems to be an interconnection between changes in the blood vessels and changes in the heart. Changes such as thickening of the blood vessels lead to increase in the blood pressure, which further affects the heart function. In that condition, the heart tries to function more efficiently by becoming larger in size (hy‐ pertrophy) and by enhancing its pumping capacity.

#### **3. Changes that occur in the vascular endothelium**

The vascular endothelium is comprised of a layer of endothelial cells that are positioned in the inner surface of blood vessels. The endothelium forms an interface between circulating blood and vessel wall, hence has a direct contact with circulating blood. In addition to serv‐ ing as a barrier, endothelial cells participate in many physiological functions. They control vascular homeostasis, regulate blood pressure by vasoconstriction and vasodilatory mecha‐ nisms and promote angiogenesis when body requires. They also secrete anti-coagulatory factors to prevent clotting [15]. Importantly, vascular endothelial cells express many impor‐ tant molecules such as vascular endothelial growth factor (VEGF) and its receptors vascular endothelial growth factor receptor-1 (VEGFR1), vascular endothelial growth factor recep‐ tor-2 (VEGFR2) and vascular endothelial growth factor receptor-3 (VEGFR3). VEGFR1 and VEGFR2 are expressed exclusively in vascular endothelial cells, whereas VEGFR3 is mainly expressed in the lymphatic endothelial cells [16]. The VEGF/VEGFR2 signaling is critical for vasculogenesis as well as angiogenesis [16]. Disruption or loss of VEGF and VEGFR2 genes is associated with severe vascular abnormalities or embryonic lethality [17]. Furthermore, the endothelial cells produce other growth factors known as angiopoitins (Ang), which are required to remodel and stabilize the immature blood vessels induced by VEGF/VEGFR2. Moreover, molecules such as neuropilines are involved in modulating the binding as well as responses to VEGF receptors [16]. Furthermore, endothelial cells express endothelial nitric oxide synthase (eNOS), which produces nitric oxide (NO). NO has many important physio‐ logical functions. For example, NO promotes vasodilation [18], as well as inhibits leukocyte adhesion [19], thrombocyte aggregation [20] and smooth muscle cell proliferation [21]. Un‐ der basal conditions eNOS is found inactive, however its activity is increased by many fac‐ tors including acetyl choline, bradykinin, thrombin and histamine that lead to increased production of NO.

(iii) how the ubiquitin proteasome system plays important roles in regulating vascular endo‐ thelium function; (iv) the mechanisms by which the age-associated increase in oxidative stress might cause endothelial dysfunction; and finally, (iv) how the age-associated changes in the vascular system lead to the development of various vascular diseases such as coro‐

Many changes are known to occur due to aging in the entire vascular system that includes heart, coronary arteries, peripheral arteries and small blood vessels known as capillaries (Figure 1). There will be an increase in the overall size of the heart, due to an increase in the heart wall thickness in the aging heart. The heart valves, which control the unidirectional of blood flow, will also become stiffer. There is also deposition of the pigments known as lipo‐ fuscin in the aged heart along with possible loss of cardiomyocytes as well as cells present in the sinoatrial node (SA node). Furthermore, there is an increase in the size of cardiomyo‐ cytes to compensate for the loss of the heart cells. These changes altogether cause a progres‐ sive decline in the physiological functions of the heart in the elderly population. In addition to these changes in the heart, the blood vessels also undergo significant changes. For exam‐ ple, the aorta, the large artery that originates from the heart becomes thicker, stiffer and less flexible. Smaller blood vessels also become thicker and stiffer. These changes are due to al‐ terations that occur in the cells present in the blood vessels and also in the connective tissue of the blood vessel wall. All these changes ultimately lead to hypertrophy of the heart and causes an increase in the blood pressure [14]. There seems to be an interconnection between changes in the blood vessels and changes in the heart. Changes such as thickening of the blood vessels lead to increase in the blood pressure, which further affects the heart function. In that condition, the heart tries to function more efficiently by becoming larger in size (hy‐

The vascular endothelium is comprised of a layer of endothelial cells that are positioned in the inner surface of blood vessels. The endothelium forms an interface between circulating blood and vessel wall, hence has a direct contact with circulating blood. In addition to serv‐ ing as a barrier, endothelial cells participate in many physiological functions. They control vascular homeostasis, regulate blood pressure by vasoconstriction and vasodilatory mecha‐ nisms and promote angiogenesis when body requires. They also secrete anti-coagulatory factors to prevent clotting [15]. Importantly, vascular endothelial cells express many impor‐ tant molecules such as vascular endothelial growth factor (VEGF) and its receptors vascular endothelial growth factor receptor-1 (VEGFR1), vascular endothelial growth factor recep‐ tor-2 (VEGFR2) and vascular endothelial growth factor receptor-3 (VEGFR3). VEGFR1 and VEGFR2 are expressed exclusively in vascular endothelial cells, whereas VEGFR3 is mainly

nary artery disease, peripheral artery disease and diabetic retinopathy.

**2. Age-associated changes in the vascular system**

4 Senescence and Senescence-Related Disorders

pertrophy) and by enhancing its pumping capacity.

**3. Changes that occur in the vascular endothelium**

**Figure 1. Age-associated changes that occur in the heart and the vascular system.** Normal young heart has highly functional cardiomyocytes, and normal atrium and ventricles (A). Young artery has normal lumen, normal arterial thickness and efficient contractile and relaxation properties (B). However, aged heart has increased thickness in the heart muscle due to hypertrophy. Specifically, cardiomyocytes from aged heart show hyperplasia along with some car‐ diomyocytes undergoing senescence (C). Aged artery also has increased thickness, reduced lumen and less efficient contractile and relaxation properties (D). These age-associated changes ultimately lead to reduced cardiac as well as vascular functions in the elders.

Aging also influences endothelial cells and causes a progressive deterioration of their func‐ tion. Previous studies have shown that endothelium-mediated vasodilatory function pro‐ gressively declines with age [22]. This is associated with decreases in eNOS expression and NO production by aging endothelial cells [23,24]. Recently, Yoon et al. have shown that de‐ creased expression of eNOS in aged human umblical vein endothelial cells [24]. However, the precise mechanisms for the age-associated decreases of these molecules remain un‐ known. Interestingly, it has been observed that the aging endothelial cells produce increased amount of O2-anions [25], which scavenge NO to form peroxinitrite, a potent form of free radical. Peroxinitrite further inactivates eNOS and decreases its activity [26]. These descri‐ bed mechansims in part explain oxidative stress-mediated decrease of eNOS and NO in ag‐ ing endothelial cells. On the other hand, it has been suggested that the age-associated changes that occur in eNOS regulatory proteins such as caveolin-1, pAkt, and heat shock protein 90 (Hsp90) contribute to the decreased activity of eNOS in aged endothelial cells [24]. In addition to these regulatory mechanisms, several other factors also regulate eNOS activity. For example, shear stress [27], estrogens [28], and growth factors [29] could also positively regulate eNOS expression. However, as their expression levels decrease with ad‐ vancing in age, these changes might cause a subsequent decrease in eNOS expression. Taken together, these alterations finally lead to both a decreased expression of eNOS and de‐ creased levels of NO in aged endothelial cells. In addition to these changes in endothelial cells, aging also causes several other changes in vascular smooth muscle cells (VSMCs). Dur‐ ing the aging process, VSMCs migrate from tunica media to tunica intima and start accumu‐ lating there. These cells become less functional and less responsive to growth factors such as transforming growth factor-beta1 [30]. As VSMCs are important regulatory cells that control the vascular wall by vasoconstriction and vasodilatory mechanisms, progressive loss of their physiological functions might lead to changes in vascular endothelium and impaired vascu‐ lar function in the aged blood vessels.

individuals [34]. Several studies were attempted to find the age-associated changes that might cause impaired angiogenesis. To this end, it has been observed that aging endothelial cells are functionally less angiogenic and less responsive to growth factors [32]. Rivard et al. [32] have found that VEGF levels were markedly reduced in aging mice. During hind limb ischemia, the old mice are unable to produce sufficient VEGF levels compared to younger mice, which are critically necessary for neovascularization and proper wound healing. Fur‐ thermore, the T lymphocyte-derived VEGF also markedly reduced in old mice, which com‐ promised the angiogenesis-mediated wound healing process during the hind limb ischemia. This study, therefore, identified loss of VEGF as one of the key factors for the impaired an‐ giogenesis observed in aged mice [32]. Furthermore, Qian et al. found that in addition to VEGF decrease, its key receptor VEGFR2 levels were also significantly decreased in eNOS knockout old mice [35]. Since the VEGF/VEGFR2 signaling is crucial for the survival, prolif‐ eration and migration of endothelial cells, a decrease of this pivotal signaling pathway may lead to impaired angiogenesis and delayed wound healing in aged subjects. Even in the eNOS knockout mice, which produce significantly less NO, the angiogenic response was markedly less in older mice due to decreased expression of VEGFR2. This partially explains that VEGFR2 plays an important role in neovascularization even in the absence of eNOS and

Endothelium Aging and Vascular Diseases http://dx.doi.org/10.5772/53065 7

Importantly, in addition to the loss of pro-angiogenic molecules, the anti-angiogenic mole‐ cules such as thrombospondin-2 (TSP2) levels were also affected by aging. To demonstrate the significance of TSP2 in aging and wound healing process, Agah et al. created full thick‐ ness excisional wounds in TSP2 null young and TSP2 null old mice and observed the wound healing process [36]. Consistent with other groups [33], they found that regardless of TSP genetic status, the would healing is delayed in old mice in comparison with young mice. However, interestingly, they found that the wound healing was faster in TSP2 null, old mice compared to wild-type, old mice suggesting that increased TSP2 in older mice might delay the angiogenesis and wound healing process. Correspondingly, there was also impaired ex‐ pression of matrix metalloproteinase-2 (MMP2) found in TSP2 null old mice. These age-as‐ sociated increase in expression of TSP2 and impaired MMP2 expression in older mice together might cause impaired angiogenesis and delay the wound healing process [36]. In addition to these changes observed in older mice, there are also changes observed in cell cy‐ cle-related molecules, which may affect the proliferation of aged endothelial cells. For exam‐ ple, aged endothelial cells undergo senescence and cease proliferation, which may limit neovascularization. Indeed, after certain passages, human umbilical vein endothelial cells (HUVECs) known to undergo senescence and loose their proliferative capacity [37]. As NO is known to prevent endothelial cell senescence, age associated decreases in eNOS and NO may be in part responsible for the senescence observed in HUVECs. Interestingly, the telo‐ merase reverse transcriptase (TERT), which prevents senescence by counteracting telomere shortening process is active in human endothelial cells. However, after several passages, en‐ dothelial cells display a decrease of NO and loss of TERT activity that further lead to endo‐ thelial senescence. Indeed, ectopic overexpression of TERT protects from endothelial cells from undergoing senescence and preserve the angiogenic function of endothelial cells [38]. Furthermore, TERT overexpression increased eNOS function and enhanced precursor endo‐

corresponding NO [35].

#### **4. Aging causes impaired angiogenesis**

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a physiologi‐ cally an important process during growth, menstrual cycle and wound healing. Several fac‐ tors are known to influence angiogenesis. The most important one is hypoxia, which activates the transcription factors such as hypoxia-inducible factor-1 alpha (HIF-1 alpha) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) [31]. These transcription factors increase the production of VEGF and other growth factors that promote proliferation and migration of vascular endothelial cells. During angiogenesis, matrix metalloproteinases, the enzymes that degrade the capillary basement membrane and extra-cellular matrix, will be increased in order to facilitate endothelial cell migration. There‐ fore, angiogenesis is a complex process, and its timely induction is tightly controlled by co‐ ordination from multiple factors. Unfortunately, angiogenesis is markedly reduced by aging [32]. In keeping with this notion, wound healing, which is associated with angiogenesis, is also markedly impaired in aged mice [33] and significantly delayed and impaired in aged individuals [34]. Several studies were attempted to find the age-associated changes that might cause impaired angiogenesis. To this end, it has been observed that aging endothelial cells are functionally less angiogenic and less responsive to growth factors [32]. Rivard et al. [32] have found that VEGF levels were markedly reduced in aging mice. During hind limb ischemia, the old mice are unable to produce sufficient VEGF levels compared to younger mice, which are critically necessary for neovascularization and proper wound healing. Fur‐ thermore, the T lymphocyte-derived VEGF also markedly reduced in old mice, which com‐ promised the angiogenesis-mediated wound healing process during the hind limb ischemia. This study, therefore, identified loss of VEGF as one of the key factors for the impaired an‐ giogenesis observed in aged mice [32]. Furthermore, Qian et al. found that in addition to VEGF decrease, its key receptor VEGFR2 levels were also significantly decreased in eNOS knockout old mice [35]. Since the VEGF/VEGFR2 signaling is crucial for the survival, prolif‐ eration and migration of endothelial cells, a decrease of this pivotal signaling pathway may lead to impaired angiogenesis and delayed wound healing in aged subjects. Even in the eNOS knockout mice, which produce significantly less NO, the angiogenic response was markedly less in older mice due to decreased expression of VEGFR2. This partially explains that VEGFR2 plays an important role in neovascularization even in the absence of eNOS and corresponding NO [35].

Aging also influences endothelial cells and causes a progressive deterioration of their func‐ tion. Previous studies have shown that endothelium-mediated vasodilatory function pro‐ gressively declines with age [22]. This is associated with decreases in eNOS expression and NO production by aging endothelial cells [23,24]. Recently, Yoon et al. have shown that de‐ creased expression of eNOS in aged human umblical vein endothelial cells [24]. However, the precise mechanisms for the age-associated decreases of these molecules remain un‐ known. Interestingly, it has been observed that the aging endothelial cells produce increased amount of O2-anions [25], which scavenge NO to form peroxinitrite, a potent form of free radical. Peroxinitrite further inactivates eNOS and decreases its activity [26]. These descri‐ bed mechansims in part explain oxidative stress-mediated decrease of eNOS and NO in ag‐ ing endothelial cells. On the other hand, it has been suggested that the age-associated changes that occur in eNOS regulatory proteins such as caveolin-1, pAkt, and heat shock protein 90 (Hsp90) contribute to the decreased activity of eNOS in aged endothelial cells [24]. In addition to these regulatory mechanisms, several other factors also regulate eNOS activity. For example, shear stress [27], estrogens [28], and growth factors [29] could also positively regulate eNOS expression. However, as their expression levels decrease with ad‐ vancing in age, these changes might cause a subsequent decrease in eNOS expression. Taken together, these alterations finally lead to both a decreased expression of eNOS and de‐ creased levels of NO in aged endothelial cells. In addition to these changes in endothelial cells, aging also causes several other changes in vascular smooth muscle cells (VSMCs). Dur‐ ing the aging process, VSMCs migrate from tunica media to tunica intima and start accumu‐ lating there. These cells become less functional and less responsive to growth factors such as transforming growth factor-beta1 [30]. As VSMCs are important regulatory cells that control the vascular wall by vasoconstriction and vasodilatory mechanisms, progressive loss of their physiological functions might lead to changes in vascular endothelium and impaired vascu‐

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a physiologi‐ cally an important process during growth, menstrual cycle and wound healing. Several fac‐ tors are known to influence angiogenesis. The most important one is hypoxia, which activates the transcription factors such as hypoxia-inducible factor-1 alpha (HIF-1 alpha) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) [31]. These transcription factors increase the production of VEGF and other growth factors that promote proliferation and migration of vascular endothelial cells. During angiogenesis, matrix metalloproteinases, the enzymes that degrade the capillary basement membrane and extra-cellular matrix, will be increased in order to facilitate endothelial cell migration. There‐ fore, angiogenesis is a complex process, and its timely induction is tightly controlled by co‐ ordination from multiple factors. Unfortunately, angiogenesis is markedly reduced by aging [32]. In keeping with this notion, wound healing, which is associated with angiogenesis, is also markedly impaired in aged mice [33] and significantly delayed and impaired in aged

lar function in the aged blood vessels.

6 Senescence and Senescence-Related Disorders

**4. Aging causes impaired angiogenesis**

Importantly, in addition to the loss of pro-angiogenic molecules, the anti-angiogenic mole‐ cules such as thrombospondin-2 (TSP2) levels were also affected by aging. To demonstrate the significance of TSP2 in aging and wound healing process, Agah et al. created full thick‐ ness excisional wounds in TSP2 null young and TSP2 null old mice and observed the wound healing process [36]. Consistent with other groups [33], they found that regardless of TSP genetic status, the would healing is delayed in old mice in comparison with young mice. However, interestingly, they found that the wound healing was faster in TSP2 null, old mice compared to wild-type, old mice suggesting that increased TSP2 in older mice might delay the angiogenesis and wound healing process. Correspondingly, there was also impaired ex‐ pression of matrix metalloproteinase-2 (MMP2) found in TSP2 null old mice. These age-as‐ sociated increase in expression of TSP2 and impaired MMP2 expression in older mice together might cause impaired angiogenesis and delay the wound healing process [36]. In addition to these changes observed in older mice, there are also changes observed in cell cy‐ cle-related molecules, which may affect the proliferation of aged endothelial cells. For exam‐ ple, aged endothelial cells undergo senescence and cease proliferation, which may limit neovascularization. Indeed, after certain passages, human umbilical vein endothelial cells (HUVECs) known to undergo senescence and loose their proliferative capacity [37]. As NO is known to prevent endothelial cell senescence, age associated decreases in eNOS and NO may be in part responsible for the senescence observed in HUVECs. Interestingly, the telo‐ merase reverse transcriptase (TERT), which prevents senescence by counteracting telomere shortening process is active in human endothelial cells. However, after several passages, en‐ dothelial cells display a decrease of NO and loss of TERT activity that further lead to endo‐ thelial senescence. Indeed, ectopic overexpression of TERT protects from endothelial cells from undergoing senescence and preserve the angiogenic function of endothelial cells [38]. Furthermore, TERT overexpression increased eNOS function and enhanced precursor endo‐ thelial cell proliferation and migration that effectively promoted angiogenesis [39,40]. In fact, TERT expression decreased p16 and p21 activities that are significantly increased in senescent endothelial cells. These findings indicate that loss of telomerase-induced senes‐ cence also plays a role in affecting angiogenesis in aged endothelial cells. Interestingly, in a separate set of experiments, it has been demonstrated that VEGF-A, a potent pro-angiogenic factor, suppresses both p16 and p21 activities in endothelial cells, suggesting that VEGF-A could serve as an anti-senescence agent [41]. However, it remains unclear whether VEGF-A activates the VEGFR2 kinase to influence hTERT activity to exert this anti-senescence capaci‐ ty. Taken together, these findings indicate that even though there is a shift between pro-an‐ giogenic and anti-angiogenic molecules in aged endothelial cells, it remains to be determined whether increasing pro-angiogenic factors or inhibiting anti-angiogenic mole‐ cules restores angiogenesis and accelerate wound healing process especially by aged endo‐ thelial cells. Future research are therefore warranted to thoroughly address these important questions.

#### **5. Aging-induced oxidative stress and vascular endothelial dysfunction**

**Figure 2. Oxidative stress in aged endothelial cells.** Compared to younger endothelial cells, aged endothelial cells produce increased levels of free radicals. In the presence of nitric oxide (NO), which is originated from iNOS in aged

increased oxidative stress that damages macromolecules and ultimately lead to loss of endothelial cell function in

The potential role of oxidative stress in vascular endothelium aging is also evident from the experiments carried out with antioxidants. For example, Vitamin C has been shown to de‐ crease telomere shortening and increase the longevity of endothelial cells in culture [45]. N-Acetylcysteine, a potent antioxidant known to decrease endothelial cell senescence by preserving TERT activity and preventing its nuclear export [46]. Interestingly, it has been demonstrated that p66shc deletion protects endothelial cells from aging-associated vascular dysfunction [43] and sirtuins decrease the p66shc expression [47]. Although human clinical trials with antioxidants such as Vitamin C and E have not yielded beneficial effects on im‐ proving cardiovascular function [48,49], future studies with other antioxidants such as Nacetylcysteine may yield positive results in improving endothelial dysfunction associated

**6. Ubiquitin-proteasome system regulates endothelial cell function**

The ubiquitin-proteasome system (UPS) plays important roles in a variety of key cellular functions including cellular protein homeostasis, signal transduction, cell cycle control, im‐ mune function, cellular senescence and apoptosis. This system targets specific proteins in the cell for degradation via ubiquitination-mediated destruction mechanism by specific ubiquitin E3 ligases [50,51]. Two major complexes, Skp1-Cul-1-F-box protein complex (SCF) and Anaphase Promoting Complex/Cyclosome (APC/C) are involved in the regulation of

). These changes lead to

Endothelium Aging and Vascular Diseases http://dx.doi.org/10.5772/53065 9


endothelial cells, O2

with aging and oxidative stress.

aged cells.

Oxidative stress is implicated in causing aging of endothelium and endothelial dysfunc‐ tions. In turn, aged endothelium produces increased free radicals, which might further ac‐ celerates aging. Based upon biomarkers of oxidant damage, increased levels of nitrotyrosine were observed in human aged vascular endothelial cells [42], Moreover, oxidative stress markers were also observed in the arteries of aged animals [26,43], suggesting that aging is indeed associated with increased formation of reactive oxygen species (ROS). Many differ‐ ent mechanisms are responsible for causing oxidative stress in endothelial cells that includes mitochondria-mediated production of ROS, decreases in free radical scavengers and in‐ creased susceptibility of macromolecules to free radical damage. Similar to other cells, oxi‐ dative stress damages proteins, lipids and DNA in vascular endothelial cells, thus causing loss of endothelial cell function. One of the major free radicals is super oxide anion (O2 - ), which is produced by aging mitochondria due to increased mitochondrial DNA damage. It has been demonstrated that NADPH contributes to O2 generation in vascular endothelial cells. Usually, the O2 anions are detoxified to H2O2 by manganese super oxide dismutase (MnSOD), which is present in the mitochondria. However, in the presence of NO, O2 - leads to formation of a potent free radical known as peroxinitrite (ONOO- ) that further damages macromolecules in the endothelial cells. It has been demonstrated that ONOO can inacti‐ vate both MnSOD and eNOS in the endothelial cells [44]. The switch of eNOS from an NO generating enzyme to an O2 generating enzyme (NO synthase uncoupling) leads to in‐ creased production of O2 and enhanced oxidative stress in aged endothelial cells (Figure 2). Taken together, NADPH and eNOS are important contributors for O2 generation in aged endothelial cells, since inhibition of NADPH and eNOS attenuates O2 - production in the aor‐ ta of aged Wistar-Kyoto rats [25].

thelial cell proliferation and migration that effectively promoted angiogenesis [39,40]. In fact, TERT expression decreased p16 and p21 activities that are significantly increased in senescent endothelial cells. These findings indicate that loss of telomerase-induced senes‐ cence also plays a role in affecting angiogenesis in aged endothelial cells. Interestingly, in a separate set of experiments, it has been demonstrated that VEGF-A, a potent pro-angiogenic factor, suppresses both p16 and p21 activities in endothelial cells, suggesting that VEGF-A could serve as an anti-senescence agent [41]. However, it remains unclear whether VEGF-A activates the VEGFR2 kinase to influence hTERT activity to exert this anti-senescence capaci‐ ty. Taken together, these findings indicate that even though there is a shift between pro-an‐ giogenic and anti-angiogenic molecules in aged endothelial cells, it remains to be determined whether increasing pro-angiogenic factors or inhibiting anti-angiogenic mole‐ cules restores angiogenesis and accelerate wound healing process especially by aged endo‐ thelial cells. Future research are therefore warranted to thoroughly address these important

**5. Aging-induced oxidative stress and vascular endothelial dysfunction**

Oxidative stress is implicated in causing aging of endothelium and endothelial dysfunc‐ tions. In turn, aged endothelium produces increased free radicals, which might further ac‐ celerates aging. Based upon biomarkers of oxidant damage, increased levels of nitrotyrosine were observed in human aged vascular endothelial cells [42], Moreover, oxidative stress markers were also observed in the arteries of aged animals [26,43], suggesting that aging is indeed associated with increased formation of reactive oxygen species (ROS). Many differ‐ ent mechanisms are responsible for causing oxidative stress in endothelial cells that includes mitochondria-mediated production of ROS, decreases in free radical scavengers and in‐ creased susceptibility of macromolecules to free radical damage. Similar to other cells, oxi‐ dative stress damages proteins, lipids and DNA in vascular endothelial cells, thus causing loss of endothelial cell function. One of the major free radicals is super oxide anion (O2

which is produced by aging mitochondria due to increased mitochondrial DNA damage. It

vate both MnSOD and eNOS in the endothelial cells [44]. The switch of eNOS from an NO

(MnSOD), which is present in the mitochondria. However, in the presence of NO, O2

macromolecules in the endothelial cells. It has been demonstrated that ONOO-


anions are detoxified to H2O2 by manganese super oxide dismutase

generating enzyme (NO synthase uncoupling) leads to in‐

and enhanced oxidative stress in aged endothelial cells (Figure 2).

has been demonstrated that NADPH contributes to O2



to formation of a potent free radical known as peroxinitrite (ONOO-

Taken together, NADPH and eNOS are important contributors for O2

endothelial cells, since inhibition of NADPH and eNOS attenuates O2




can inacti‐

generation in vascular endothelial


) that further damages

generation in aged


questions.

8 Senescence and Senescence-Related Disorders

cells. Usually, the O2

generating enzyme to an O2

ta of aged Wistar-Kyoto rats [25].

creased production of O2

**Figure 2. Oxidative stress in aged endothelial cells.** Compared to younger endothelial cells, aged endothelial cells produce increased levels of free radicals. In the presence of nitric oxide (NO), which is originated from iNOS in aged endothelial cells, O2 - lead to formation of a potent free radical known as peroxinitrite (ONOO- ). These changes lead to increased oxidative stress that damages macromolecules and ultimately lead to loss of endothelial cell function in aged cells.

The potential role of oxidative stress in vascular endothelium aging is also evident from the experiments carried out with antioxidants. For example, Vitamin C has been shown to de‐ crease telomere shortening and increase the longevity of endothelial cells in culture [45]. N-Acetylcysteine, a potent antioxidant known to decrease endothelial cell senescence by preserving TERT activity and preventing its nuclear export [46]. Interestingly, it has been demonstrated that p66shc deletion protects endothelial cells from aging-associated vascular dysfunction [43] and sirtuins decrease the p66shc expression [47]. Although human clinical trials with antioxidants such as Vitamin C and E have not yielded beneficial effects on im‐ proving cardiovascular function [48,49], future studies with other antioxidants such as Nacetylcysteine may yield positive results in improving endothelial dysfunction associated with aging and oxidative stress.

#### **6. Ubiquitin-proteasome system regulates endothelial cell function**

The ubiquitin-proteasome system (UPS) plays important roles in a variety of key cellular functions including cellular protein homeostasis, signal transduction, cell cycle control, im‐ mune function, cellular senescence and apoptosis. This system targets specific proteins in the cell for degradation via ubiquitination-mediated destruction mechanism by specific ubiquitin E3 ligases [50,51]. Two major complexes, Skp1-Cul-1-F-box protein complex (SCF) and Anaphase Promoting Complex/Cyclosome (APC/C) are involved in the regulation of cell cycle as well as other key regulatory processes in the cell. Dysfunction of UPS leads to development of many diseases including cancer and cardiovascular disease. Therefore, how UPS regulates endothelial cell function and endothelial cell cycle is crucial in order to under‐ stand the underlying mechanisms involved in vascular disease development, and will also provide important insights into developing novel therapies for many vascular diseases asso‐ ciated with aging. Increasing evidence suggests that UPS regulates endothelial function by specifically regulating the key proteins present in endothelial cells. For example, the halflives of both eNOS and inducible nitric oxide synthase (iNOS) are regulated by proteasomedependent degradation [52,53]. Furthermore, the von Hippel-Lindau protein (pVHL) regulates HIF-1 alpha, which is a critical factor involved in regulating angiogenesis [54] (Fig‐ ure 3). Consistent with the key role of UPS in endothelial function, treatment with low doses of proteasome inhibitor increases endothelial cell function [55]. These findings further sug‐ gest that UPS could be a potential target to improve the physiological functions of vascula‐ ture, hence may be utilized as a valuable drug target to develop novel treatments for agingassociated vascular diseases. However, the specific E3 ligase complexes and the molecular mechanisms that are involved in the regulation of endothelial cell cycle and endothelial cell function remain unknown.

Recent studies indicate that F-box proteins such as SCFFbw7 and SCFβ-TRCP are potentially in‐ volved in regulating endothelial cell function. For example, mice lacking Fbw7 die early (embryonic day 10.5) with developmental defects in vascular and haematopoietic system as well as heart chamber maturation [56,57]. As Fbw7 regulates the key cell cycle regulators in‐ cluding Notch, cyclin E, c-Myc and c-Jun, deletion of Fbw7 leads to accumulation of these substrates in the endothelial and /or hematopoitic cells. Indeed, elevated Notch protein lev‐ els were observed in Fbw7-deficient embryos that lead to the deregulation of the transcrip‐ tional repressor, Hey1, which is an important factor for cardiovascular development [56]. Therefore, these findings suggest that Fbw7 is an important E3 ligase governing the timely destruction of the key substrates involved in cardiovascular development. Furthermore, our laboratory has recently identified SCFβ-TRCP as an E3 ubiquitin ligase that is potentially in‐ volved in regulating VEGFR2 protein levels in microvascular endothelial cells [58]. As stated in above sections, VEGFR2 is the major regulator of angiogenesis. Increased angiogenesis is associated with certain cancers, whereas angiogenesis is markedly decreased in aging indi‐ viduals. Our study, for the first time, revealed that deregulation of β-TRCP leads to stabili‐ zation of VEGFR2 and subsequent increases in angiogenesis, whereas increased β-TRCP activity leads to decreased VEGFR2 levels and reduced angiogenesis. Mechanistically, casein kinase-I (CKI)-induced phosphorylation of VEGFR2 at critical phospho-degrons leads to its ubiquitination by β-TRCP, and subsequent degradation of VEGFR2 through the 26S protea‐ some [58]. However, we are just beginning to understand the critical role of UPS in endothe‐ lial function, future studies are therefore warranted to unravel the important role of various E3 ubiquitin ligases in the regulation of vascular system, which may ultimately, help to pre‐

Endothelium Aging and Vascular Diseases http://dx.doi.org/10.5772/53065 11

Aging vascular endothelium is susceptible to the development of various vascular diseases including cardiovascular disease (CVD) (coronary artery disease; atherosclerosis and hyper‐ tension), peripheral vascular disease (PVD), diabetic retinopathy, renal vascular disease and micro-vascular disease. Importantly, aging-associated changes that occur in the blood ves‐ sels are the major cause for the development of these diseases. Therefore, identifying the molecular changes that occur in the aging-endothelium and elucidating the underlying mo‐ lecular mechanisms responsible for vascular disease development lead to the development

Cardiovascular disease (CVD) is the number one cause of human death in the US as well as in the world. CVD mostly occur in the aged population [59], and according to the World Health Organization, an estimated 17.3 million deaths occurred due to CVD in 2008. Coro‐ nary artery disease (CAD) is the major form of CVD, which occurs when coronary arteries are blocked due to atherosclerosis. Aging endothelium is very susceptible for plaque forma‐ tion that leads to progressive blockage of the coronary arteries. This causes reduced blood

vent vascular diseases in the elderly population.

of novel therapies to treat various vascular diseases.

**7.1. Cardiovascular and peripheral vascular diseases**

**7. Aging and vascular diseases**

**Figure 3. The ubiquitin proteasome system (UPS) regulates the stability of various key proteins in endothelial cells.** The E3 ubiquitin ligases such as SCFβ-TRCP, C-terminus of Hsp70-interacting protein (CHIP), SOCS box-containing protein [ECS(SPSB)] and pVHL, target VEGFR2, eNOS, iNOS and HIF-1 alpha, respectively, for proteasome-dependent degradation. These E3 ligases recognize their respective substrates once the substrates are properly phosphorylated at the critical phosphodegrons by one or more kinases. This is an important regulatory mechanism by which UPS controls the half-lives of various key proteins in endothelial cells to influence the angiogenesis process.

Recent studies indicate that F-box proteins such as SCFFbw7 and SCFβ-TRCP are potentially in‐ volved in regulating endothelial cell function. For example, mice lacking Fbw7 die early (embryonic day 10.5) with developmental defects in vascular and haematopoietic system as well as heart chamber maturation [56,57]. As Fbw7 regulates the key cell cycle regulators in‐ cluding Notch, cyclin E, c-Myc and c-Jun, deletion of Fbw7 leads to accumulation of these substrates in the endothelial and /or hematopoitic cells. Indeed, elevated Notch protein lev‐ els were observed in Fbw7-deficient embryos that lead to the deregulation of the transcrip‐ tional repressor, Hey1, which is an important factor for cardiovascular development [56]. Therefore, these findings suggest that Fbw7 is an important E3 ligase governing the timely destruction of the key substrates involved in cardiovascular development. Furthermore, our laboratory has recently identified SCFβ-TRCP as an E3 ubiquitin ligase that is potentially in‐ volved in regulating VEGFR2 protein levels in microvascular endothelial cells [58]. As stated in above sections, VEGFR2 is the major regulator of angiogenesis. Increased angiogenesis is associated with certain cancers, whereas angiogenesis is markedly decreased in aging indi‐ viduals. Our study, for the first time, revealed that deregulation of β-TRCP leads to stabili‐ zation of VEGFR2 and subsequent increases in angiogenesis, whereas increased β-TRCP activity leads to decreased VEGFR2 levels and reduced angiogenesis. Mechanistically, casein kinase-I (CKI)-induced phosphorylation of VEGFR2 at critical phospho-degrons leads to its ubiquitination by β-TRCP, and subsequent degradation of VEGFR2 through the 26S protea‐ some [58]. However, we are just beginning to understand the critical role of UPS in endothe‐ lial function, future studies are therefore warranted to unravel the important role of various E3 ubiquitin ligases in the regulation of vascular system, which may ultimately, help to pre‐ vent vascular diseases in the elderly population.

#### **7. Aging and vascular diseases**

cell cycle as well as other key regulatory processes in the cell. Dysfunction of UPS leads to development of many diseases including cancer and cardiovascular disease. Therefore, how UPS regulates endothelial cell function and endothelial cell cycle is crucial in order to under‐ stand the underlying mechanisms involved in vascular disease development, and will also provide important insights into developing novel therapies for many vascular diseases asso‐ ciated with aging. Increasing evidence suggests that UPS regulates endothelial function by specifically regulating the key proteins present in endothelial cells. For example, the halflives of both eNOS and inducible nitric oxide synthase (iNOS) are regulated by proteasomedependent degradation [52,53]. Furthermore, the von Hippel-Lindau protein (pVHL) regulates HIF-1 alpha, which is a critical factor involved in regulating angiogenesis [54] (Fig‐ ure 3). Consistent with the key role of UPS in endothelial function, treatment with low doses of proteasome inhibitor increases endothelial cell function [55]. These findings further sug‐ gest that UPS could be a potential target to improve the physiological functions of vascula‐ ture, hence may be utilized as a valuable drug target to develop novel treatments for agingassociated vascular diseases. However, the specific E3 ligase complexes and the molecular mechanisms that are involved in the regulation of endothelial cell cycle and endothelial cell

**Figure 3. The ubiquitin proteasome system (UPS) regulates the stability of various key proteins in endothelial cells.** The E3 ubiquitin ligases such as SCFβ-TRCP, C-terminus of Hsp70-interacting protein (CHIP), SOCS box-containing protein [ECS(SPSB)] and pVHL, target VEGFR2, eNOS, iNOS and HIF-1 alpha, respectively, for proteasome-dependent degradation. These E3 ligases recognize their respective substrates once the substrates are properly phosphorylated at the critical phosphodegrons by one or more kinases. This is an important regulatory mechanism by which UPS controls

the half-lives of various key proteins in endothelial cells to influence the angiogenesis process.

function remain unknown.

10 Senescence and Senescence-Related Disorders

Aging vascular endothelium is susceptible to the development of various vascular diseases including cardiovascular disease (CVD) (coronary artery disease; atherosclerosis and hyper‐ tension), peripheral vascular disease (PVD), diabetic retinopathy, renal vascular disease and micro-vascular disease. Importantly, aging-associated changes that occur in the blood ves‐ sels are the major cause for the development of these diseases. Therefore, identifying the molecular changes that occur in the aging-endothelium and elucidating the underlying mo‐ lecular mechanisms responsible for vascular disease development lead to the development of novel therapies to treat various vascular diseases.

#### **7.1. Cardiovascular and peripheral vascular diseases**

Cardiovascular disease (CVD) is the number one cause of human death in the US as well as in the world. CVD mostly occur in the aged population [59], and according to the World Health Organization, an estimated 17.3 million deaths occurred due to CVD in 2008. Coro‐ nary artery disease (CAD) is the major form of CVD, which occurs when coronary arteries are blocked due to atherosclerosis. Aging endothelium is very susceptible for plaque forma‐ tion that leads to progressive blockage of the coronary arteries. This causes reduced blood supply (decreased supply of oxygen and nutrients) to the affected area of the heart. Al‐ though partial blockages may cause symptoms such as angina, complete loss of blood sup‐ ply leads to heart attack, and if not treated immediately, may lead to sudden death. It has been observed that several age-associated changes in the endothelium-derived factors are responsible for plaque formation in the arteries. Importantly, endothelin (ET), a vascular en‐ dothelium-derived growth factor was found to be significantly increased in the aged endo‐ thelium [60,61,62]. ET mainly acts through its receptors ET-A and ET-B present on endothelial as well as vascular smooth muscle cells (VSMCs). ET-A activation leads to the constriction and proliferation of VSMCs, whereas ET-B activation leads to increased produc‐ tion of NO, which leads to vasodilation and inhibition of platelet aggregation. Studies indi‐ cate that ET-A receptor is mainly involved in the development of atherosclerosis, as inhibition of ET-A receptor prevents atherosclerosis in apolipoprotein-E deficient mice [63]. More importantly, endothelin-1 also decreases eNOS in vascular endothelial cells through ET-A receptor activation [64], suggesting that aging-induced increases in ET-1 as well as in‐ creased activation of ET-A receptor are potentially involved in causing atherosclerosis. Fur‐ thermore, the aging-induced increased expression of various adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) also contribute to the ongoing process of atherosclerosis [65].

occurs in the vascular system will protect the aged population from developing various vas‐

Endothelium Aging and Vascular Diseases http://dx.doi.org/10.5772/53065 13

**Figure 4. Atherosclerosis in the aged artery.** Aged endothelial cells express various adhesion molecules (AM), which facilitate the binding as well as transportation of various inflammatory cells, including monocytes (M) and lympho‐ cytes (L) into the intima. Oxidized low density lipoproteins (OxLDL) play a major role in the formation of foam cells (F). The foam cells secrete several growth factors (GF) and cytokines (C) that lead to increased proliferation of vascular smooth muscle cells (VSMCs). Increased expression of endothelin-1 facilitates atherosclerosis through ET-A receptor activation. The lymphocytes also play a critical role in causing inflammation in the endothelium. Altogether, these

Diabetes affects approximately 200 million people around the world and almost 20 million in the United States. Diabetic retinopathy (DR) is a microvascular disease of the eye and most commonly seen in elderly population [72]. Type I as well as Type II diabetes lead to the development of DR. Importantly, microvessels of the eye are mostly affected by hyper‐ glycemia. Several changes in the blood vessels have been observed including loss of peri‐ cytes, thickening of the basement membrane and increased permeability of blood vessels in DR. Furthermore, as DR progresses from non-proliferative DR to proliferative DR, the new blood vessels start to grow (neovascularization) to compensate for the affected blood ves‐ sels. Although the molecular mechanisms by which diabetes affects blood vessels of the eye

changes facilitate the plaque formation in the blood vessels of aged populations.

**7.2. Diabetic retinopathy, a vascular disease of the eye**

cular diseases.

Inflammation, another major factor that is also known to increase with aging potentially contribute to the process of atherosclerosis [66]. Consistently, the incidence of atherosclero‐ sis is found much higher in patients with autoimmune diseases such as rheumatoid arthritis [67,68] and systemic lupus erythematosus [69]. Several different immune cells and increased expression of adhesion molecules also play a major role in developing atherosclerotic pla‐ que. For instance, adhesion molecules ICAM-1 and VCAM-1 not only facilitate the binding of immune cells such as monocytes and T-cells, but also help to transport these cells into the arterial wall. Once inside, the monocytes differentiate into macrophages, and ultimately be‐ come foam cells by taking up the oxidized LDL. The proteoglycans present in the extra cel‐ lular space of the intima bind with the oxidized LDL molecules. Moreover, the activated Tcells secrete several different cytokines that promote inflammation and activate VSMCs to proliferate. Altogether, this ongoing inflammatory process accelerates the process of athero‐ sclerosis and damages the coronary arterial wall [70] (Figure 4).

Atherosclerosis is also occurs in other arteries other than coronary arteries. If atherosclerosis occurs in the peripheral arteries then it is called peripheral vascular disease or peripheral ar‐ terial disease (PAD). PAD is also influenced by aging and mostly occurs in elderly popula‐ tion. The prevalence increases with age from 3% under 60 years of age to 20% in aged 70 years and over [71]. Several factors influence the development of PAD that includes smok‐ ing, dyslipidemia, hypertension, diabetes and platelet aggregation. Advanced atherosclero‐ sis in coronary arteries leads to angina and heart attack, whereas in cerebral arteries leads to stroke or transient ischemic attacks. If atherosclerosis occurs in peripheral arteries, that will lead to pain during walking or exercising (claudication), and this condition causes defects in the wound healing or ulcers. Preventing or slowing down the age-associated changes that occurs in the vascular system will protect the aged population from developing various vas‐ cular diseases.

supply (decreased supply of oxygen and nutrients) to the affected area of the heart. Al‐ though partial blockages may cause symptoms such as angina, complete loss of blood sup‐ ply leads to heart attack, and if not treated immediately, may lead to sudden death. It has been observed that several age-associated changes in the endothelium-derived factors are responsible for plaque formation in the arteries. Importantly, endothelin (ET), a vascular en‐ dothelium-derived growth factor was found to be significantly increased in the aged endo‐ thelium [60,61,62]. ET mainly acts through its receptors ET-A and ET-B present on endothelial as well as vascular smooth muscle cells (VSMCs). ET-A activation leads to the constriction and proliferation of VSMCs, whereas ET-B activation leads to increased produc‐ tion of NO, which leads to vasodilation and inhibition of platelet aggregation. Studies indi‐ cate that ET-A receptor is mainly involved in the development of atherosclerosis, as inhibition of ET-A receptor prevents atherosclerosis in apolipoprotein-E deficient mice [63]. More importantly, endothelin-1 also decreases eNOS in vascular endothelial cells through ET-A receptor activation [64], suggesting that aging-induced increases in ET-1 as well as in‐ creased activation of ET-A receptor are potentially involved in causing atherosclerosis. Fur‐ thermore, the aging-induced increased expression of various adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1

12 Senescence and Senescence-Related Disorders

(VCAM-1) also contribute to the ongoing process of atherosclerosis [65].

sclerosis and damages the coronary arterial wall [70] (Figure 4).

Inflammation, another major factor that is also known to increase with aging potentially contribute to the process of atherosclerosis [66]. Consistently, the incidence of atherosclero‐ sis is found much higher in patients with autoimmune diseases such as rheumatoid arthritis [67,68] and systemic lupus erythematosus [69]. Several different immune cells and increased expression of adhesion molecules also play a major role in developing atherosclerotic pla‐ que. For instance, adhesion molecules ICAM-1 and VCAM-1 not only facilitate the binding of immune cells such as monocytes and T-cells, but also help to transport these cells into the arterial wall. Once inside, the monocytes differentiate into macrophages, and ultimately be‐ come foam cells by taking up the oxidized LDL. The proteoglycans present in the extra cel‐ lular space of the intima bind with the oxidized LDL molecules. Moreover, the activated Tcells secrete several different cytokines that promote inflammation and activate VSMCs to proliferate. Altogether, this ongoing inflammatory process accelerates the process of athero‐

Atherosclerosis is also occurs in other arteries other than coronary arteries. If atherosclerosis occurs in the peripheral arteries then it is called peripheral vascular disease or peripheral ar‐ terial disease (PAD). PAD is also influenced by aging and mostly occurs in elderly popula‐ tion. The prevalence increases with age from 3% under 60 years of age to 20% in aged 70 years and over [71]. Several factors influence the development of PAD that includes smok‐ ing, dyslipidemia, hypertension, diabetes and platelet aggregation. Advanced atherosclero‐ sis in coronary arteries leads to angina and heart attack, whereas in cerebral arteries leads to stroke or transient ischemic attacks. If atherosclerosis occurs in peripheral arteries, that will lead to pain during walking or exercising (claudication), and this condition causes defects in the wound healing or ulcers. Preventing or slowing down the age-associated changes that

**Figure 4. Atherosclerosis in the aged artery.** Aged endothelial cells express various adhesion molecules (AM), which facilitate the binding as well as transportation of various inflammatory cells, including monocytes (M) and lympho‐ cytes (L) into the intima. Oxidized low density lipoproteins (OxLDL) play a major role in the formation of foam cells (F). The foam cells secrete several growth factors (GF) and cytokines (C) that lead to increased proliferation of vascular smooth muscle cells (VSMCs). Increased expression of endothelin-1 facilitates atherosclerosis through ET-A receptor activation. The lymphocytes also play a critical role in causing inflammation in the endothelium. Altogether, these changes facilitate the plaque formation in the blood vessels of aged populations.

#### **7.2. Diabetic retinopathy, a vascular disease of the eye**

Diabetes affects approximately 200 million people around the world and almost 20 million in the United States. Diabetic retinopathy (DR) is a microvascular disease of the eye and most commonly seen in elderly population [72]. Type I as well as Type II diabetes lead to the development of DR. Importantly, microvessels of the eye are mostly affected by hyper‐ glycemia. Several changes in the blood vessels have been observed including loss of peri‐ cytes, thickening of the basement membrane and increased permeability of blood vessels in DR. Furthermore, as DR progresses from non-proliferative DR to proliferative DR, the new blood vessels start to grow (neovascularization) to compensate for the affected blood ves‐ sels. Although the molecular mechanisms by which diabetes affects blood vessels of the eye remain not completely understood, it is evident from several studies that hyperglycemia di‐ rectly plays a major role in causing DR. The highly elevated blood glucose activates aldose reductase pathway in certain tissues, which converts the sugars into alcohols, mainly sorbi‐ tol. The increased formation of sorbitol further affects the intramural pericytes present in the blood vessels of the retina to cause loss of function of pericytes [73]. As pericytes inhibit the endothelial cell function in occular blood vessels, loss of pericytes function leads to the for‐ mation of microaneurysms and ultimately lead to neovascularization. This pathological con‐ dition is mostly observed at the borders of retina and occurs along the vascular arcades as well as at the optic nerve head. The newly formed blood vessels do not directly affect the retina, however, the blood vessels are susceptible to vitreous traction and lead to hemor‐ rhage into the vitreous cavity or preretinal space. If not treated, this condition may ultimate‐ ly lead to vision loss. Many studies were attempted to understand the underlying molecular mechanisms by which neovascularization occurs in DR. Like in other pathological condi‐ tions described above, it is in part due to aging-associated defects in angiogenesis. Specifi‐ cally, increased shear stress causes enhanced permeability of the blood vessels. On one hand, the blood vessels constantly remodel to adapt such changes induced by shear stress. On the other hand, the increased shear stress also causes activation, proliferation and migra‐ tion of endothelial cells that ultimately cause neovascularization [74]. Furthermore, shear stress also known to cause vasodilatory effects by inhibiting endothelin1, a potent vasocon‐ strictor and increasing the levels of eNOS and prostaglandins which are potent vasodilators. Increased shear stress also increases matrix production by the endothelial cells, which caus‐ es basement thickening. Increased secretion of tissue-type plasminogen activator causes thrombosis and affects microcirculation [75]. Once blood vessels are obscured, the hypoxia generated inside will cause increased dilation of nearby vessels and leads to increased pro‐ duction of growth factors that further promote increased neovascularization.

effects of hyperglycemia on diacylglycerol, which is known to be elevated in DR. Diacylgly‐ cerol is a potent activator of protein kinase C, and in turn protein kinase C increases VEGF-A secretion. The protein kinase C inhibitors are known to have some beneficial effects on DR. Furthermore, as VEGF-A levels are increased in DR, anti-VEGF-A compounds were also

Endothelium Aging and Vascular Diseases http://dx.doi.org/10.5772/53065 15

Aging is one of the major risk factors for the development of various vascular diseases such as cardiovascular disease, peripheral vascular disease and vascular diseases of the eye. Al‐ though exact molecular mechanisms are not clearly known, several molecules are known to be altered in aged endothelial cells. Importantly, reduced expression of eNOS and decreased production of NO, a potent vasodilator, have been observed. Furthermore, decreased ex‐ pression of VEGF and VEGF receptors, and conversely, increased expression of TSP2, a po‐ tent angiogenesis inhibitor, have been observed in aged endothelial cells as well. The imbalance between the pro-angiogenic and the anti-angiogenic molecules seems to be re‐ sponsible for the decreased angiogenesis observed in aged endothelial cells. Importantly, it has been also demonstrated that aging-induced oxidative stress is one of the major contribu‐ ting factors for the loss of endothelial cell function in advanced age. In this regard, novel an‐ tioxidants may prevent aging-induced oxidative stress and thereby improve endothelial cell function in aged cells. As most of the pro-angiogenic and the anti-angiogenic molecules are unstable, recent studies have also established a potential role of UPS in regulating endothe‐ lial cell function. However, further thorough investigations are required to pinpoint the pre‐ cise role of UPS in regulating the aging-associated decline of angiogenesis in the endothelial cells. To this end, it is critical to identify the age-associated molecular signature changes in different cells present in the endothelium such as endothelial cells, smooth muscle cells and pericytes in order to understand how these changes ultimately lead to the loss of endothelial function. This critical information will not only help to identify the crucial signaling path‐ ways through which aging process affects the angiogenesis, but also will aid to develop nov‐

developed to specifically inhibit neovascularization associated with DR [78].

el therapies to combat various vascular diseases associated with aging.

K01 award from National Institute on Aging, NIH (AG041218).

This work is supported by the grants from National Institutes of Health to Wenyi Wei (GM089763; GM094777). Shavali Shaik and Zhiwei Wang are recipients of Ruth L. Kirsch‐ stein National Research Service Award (NRSA) fellowship. Hiroyuki Inuzuka is recipient of

**8. Conclusion**

**Acknowledgements**

Among the various growth factors, VEGF-A seems to be potentially involved in promoting angiogenesis in DR. In fact, Miller et al. demonstrated that increased VEGF-A levels corre‐ late with enhanced angiogenesis in ocular tissue [76]. Moreover, high affinity receptors for VEGF-A have also been identified in endothelial cells as well as the pericytes of blood ves‐ sels located in the eye. This clearly suggests that VEGF-A-induced signaling pathway might play a potential role in promoting angiogenesis in DR. Furthermore, as angiogenesis is pre‐ cisely regulated both by pro-angiogenic and anti-angiogenic factors, Funatsu et al. conduct‐ ed studies to evaluate whether the balance between these two types of molecules is critical in causing angiogenesis in DR [77]. They simultaneously measured pro-angiogenic (VEGF-A) as well as anti-angiogenic molecules (endostatin and PF4) in the vitreous and in the plas‐ ma samples to correlate with DR. Interestingly, these studies revealed that vitreous VEGF-A and endostatin levels clearly correlate with the severity of DR, however, no correlation was found between DR and plasma levels of VEGF-A and endostatin [77]. Therefore, this study suggested that loss of balance between pro- and anti-angiogenic molecules might be respon‐ sible for the neovascularization observed in DR.

Several drugs were investigated to inhibit neovascularization associated with DR. For exam‐ ple, Ruboxistaurin, a protein kinase C inhibitor tested for efficacy. This is based upon the effects of hyperglycemia on diacylglycerol, which is known to be elevated in DR. Diacylgly‐ cerol is a potent activator of protein kinase C, and in turn protein kinase C increases VEGF-A secretion. The protein kinase C inhibitors are known to have some beneficial effects on DR. Furthermore, as VEGF-A levels are increased in DR, anti-VEGF-A compounds were also developed to specifically inhibit neovascularization associated with DR [78].

#### **8. Conclusion**

remain not completely understood, it is evident from several studies that hyperglycemia di‐ rectly plays a major role in causing DR. The highly elevated blood glucose activates aldose reductase pathway in certain tissues, which converts the sugars into alcohols, mainly sorbi‐ tol. The increased formation of sorbitol further affects the intramural pericytes present in the blood vessels of the retina to cause loss of function of pericytes [73]. As pericytes inhibit the endothelial cell function in occular blood vessels, loss of pericytes function leads to the for‐ mation of microaneurysms and ultimately lead to neovascularization. This pathological con‐ dition is mostly observed at the borders of retina and occurs along the vascular arcades as well as at the optic nerve head. The newly formed blood vessels do not directly affect the retina, however, the blood vessels are susceptible to vitreous traction and lead to hemor‐ rhage into the vitreous cavity or preretinal space. If not treated, this condition may ultimate‐ ly lead to vision loss. Many studies were attempted to understand the underlying molecular mechanisms by which neovascularization occurs in DR. Like in other pathological condi‐ tions described above, it is in part due to aging-associated defects in angiogenesis. Specifi‐ cally, increased shear stress causes enhanced permeability of the blood vessels. On one hand, the blood vessels constantly remodel to adapt such changes induced by shear stress. On the other hand, the increased shear stress also causes activation, proliferation and migra‐ tion of endothelial cells that ultimately cause neovascularization [74]. Furthermore, shear stress also known to cause vasodilatory effects by inhibiting endothelin1, a potent vasocon‐ strictor and increasing the levels of eNOS and prostaglandins which are potent vasodilators. Increased shear stress also increases matrix production by the endothelial cells, which caus‐ es basement thickening. Increased secretion of tissue-type plasminogen activator causes thrombosis and affects microcirculation [75]. Once blood vessels are obscured, the hypoxia generated inside will cause increased dilation of nearby vessels and leads to increased pro‐

14 Senescence and Senescence-Related Disorders

duction of growth factors that further promote increased neovascularization.

sible for the neovascularization observed in DR.

Among the various growth factors, VEGF-A seems to be potentially involved in promoting angiogenesis in DR. In fact, Miller et al. demonstrated that increased VEGF-A levels corre‐ late with enhanced angiogenesis in ocular tissue [76]. Moreover, high affinity receptors for VEGF-A have also been identified in endothelial cells as well as the pericytes of blood ves‐ sels located in the eye. This clearly suggests that VEGF-A-induced signaling pathway might play a potential role in promoting angiogenesis in DR. Furthermore, as angiogenesis is pre‐ cisely regulated both by pro-angiogenic and anti-angiogenic factors, Funatsu et al. conduct‐ ed studies to evaluate whether the balance between these two types of molecules is critical in causing angiogenesis in DR [77]. They simultaneously measured pro-angiogenic (VEGF-A) as well as anti-angiogenic molecules (endostatin and PF4) in the vitreous and in the plas‐ ma samples to correlate with DR. Interestingly, these studies revealed that vitreous VEGF-A and endostatin levels clearly correlate with the severity of DR, however, no correlation was found between DR and plasma levels of VEGF-A and endostatin [77]. Therefore, this study suggested that loss of balance between pro- and anti-angiogenic molecules might be respon‐

Several drugs were investigated to inhibit neovascularization associated with DR. For exam‐ ple, Ruboxistaurin, a protein kinase C inhibitor tested for efficacy. This is based upon the Aging is one of the major risk factors for the development of various vascular diseases such as cardiovascular disease, peripheral vascular disease and vascular diseases of the eye. Al‐ though exact molecular mechanisms are not clearly known, several molecules are known to be altered in aged endothelial cells. Importantly, reduced expression of eNOS and decreased production of NO, a potent vasodilator, have been observed. Furthermore, decreased ex‐ pression of VEGF and VEGF receptors, and conversely, increased expression of TSP2, a po‐ tent angiogenesis inhibitor, have been observed in aged endothelial cells as well. The imbalance between the pro-angiogenic and the anti-angiogenic molecules seems to be re‐ sponsible for the decreased angiogenesis observed in aged endothelial cells. Importantly, it has been also demonstrated that aging-induced oxidative stress is one of the major contribu‐ ting factors for the loss of endothelial cell function in advanced age. In this regard, novel an‐ tioxidants may prevent aging-induced oxidative stress and thereby improve endothelial cell function in aged cells. As most of the pro-angiogenic and the anti-angiogenic molecules are unstable, recent studies have also established a potential role of UPS in regulating endothe‐ lial cell function. However, further thorough investigations are required to pinpoint the pre‐ cise role of UPS in regulating the aging-associated decline of angiogenesis in the endothelial cells. To this end, it is critical to identify the age-associated molecular signature changes in different cells present in the endothelium such as endothelial cells, smooth muscle cells and pericytes in order to understand how these changes ultimately lead to the loss of endothelial function. This critical information will not only help to identify the crucial signaling path‐ ways through which aging process affects the angiogenesis, but also will aid to develop nov‐ el therapies to combat various vascular diseases associated with aging.

#### **Acknowledgements**

This work is supported by the grants from National Institutes of Health to Wenyi Wei (GM089763; GM094777). Shavali Shaik and Zhiwei Wang are recipients of Ruth L. Kirsch‐ stein National Research Service Award (NRSA) fellowship. Hiroyuki Inuzuka is recipient of K01 award from National Institute on Aging, NIH (AG041218).

#### **Author details**

Shavali Shaik, Zhiwei Wang, Hiroyuki Inuzuka, Pengda Liu and Wenyi Wei\*

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

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Endothelium Aging and Vascular Diseases http://dx.doi.org/10.5772/53065 17

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20 Senescence and Senescence-Related Disorders

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**Section 2**

**Cellular Senescence**

**Cellular Senescence**

**Chapter 2**

**Molecular Mechanisms of Cellular Senescence**

Normal mammalian cells in culture have a limited life span and will eventually maintain a growth arrested state, referred to as replicative senescence. Usually induced by telomere shortening this form of arrest is irreversible in the sense that cells cannot be triggered to reenter proliferation by physiological mitotic stimuli like growth factors. Senescence may also occur prematurely in response to various stress stimuli such as oxidative stress, DNA dam‐ age or active oncogenes. Thereby premature senescence acts as an important tumor suppres‐ sive mechanism and not surprisingly there is emerging evidence that senescence is indeed not only a result of tissue culture but markers of senescence have been identified in vivo in

The function of the retinoblastoma protein (pRb) is central to the onset of senescence. pRb, in its active hypophosphorylated form, is a potent repressor of genes that function during DNA rep‐ lication and thereby pRb causes cell cycle arrest. The cell cycle inhibitors p16INK4a and p21Waf1 and their homologues work in concert with pRb by inhibiting cyclin dependent kinases (CDKs)

Additionally to the transient role in growth inhibition active, hypophosphorylated pRb co‐ ordinates major changes in direct and epigenetic gene regulation leading to changes in chro‐

Senescent cells display several characteristic morphological and biochemical features. The detection of these markers has been used to identify senescent cells in vitro an in vivo. The

and reproduction in any medium, provided the original work is properly cited.

© 2013 Becker and Haferkamp; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

from phosphorylating pRb and thus maintaining in its active growth inhibitory state.

This chapter provides an insight in these molecular mechanisms of cellular senescence.

matin structure, which are crucial to the onset and maintenance of senescence.

**2. Senescence features and biomarkers**

Therese Becker and Sebastian Haferkamp

http://dx.doi.org/10.5772/54120

**1. Introduction**

human and animal tissue.

Additional information is available at the end of the chapter

## **Molecular Mechanisms of Cellular Senescence**

Therese Becker and Sebastian Haferkamp

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54120

### **1. Introduction**

Normal mammalian cells in culture have a limited life span and will eventually maintain a growth arrested state, referred to as replicative senescence. Usually induced by telomere shortening this form of arrest is irreversible in the sense that cells cannot be triggered to reenter proliferation by physiological mitotic stimuli like growth factors. Senescence may also occur prematurely in response to various stress stimuli such as oxidative stress, DNA dam‐ age or active oncogenes. Thereby premature senescence acts as an important tumor suppres‐ sive mechanism and not surprisingly there is emerging evidence that senescence is indeed not only a result of tissue culture but markers of senescence have been identified in vivo in human and animal tissue.

The function of the retinoblastoma protein (pRb) is central to the onset of senescence. pRb, in its active hypophosphorylated form, is a potent repressor of genes that function during DNA rep‐ lication and thereby pRb causes cell cycle arrest. The cell cycle inhibitors p16INK4a and p21Waf1 and their homologues work in concert with pRb by inhibiting cyclin dependent kinases (CDKs) from phosphorylating pRb and thus maintaining in its active growth inhibitory state.

Additionally to the transient role in growth inhibition active, hypophosphorylated pRb co‐ ordinates major changes in direct and epigenetic gene regulation leading to changes in chro‐ matin structure, which are crucial to the onset and maintenance of senescence.

This chapter provides an insight in these molecular mechanisms of cellular senescence.

### **2. Senescence features and biomarkers**

Senescent cells display several characteristic morphological and biochemical features. The detection of these markers has been used to identify senescent cells in vitro an in vivo. The

typical senescence phenotype consist of enlarged cell with multiple or enlarged nuclei, prominent Golgi apparatus and sometimes a vacuolated cytoplasm (Figure1). Recently a novel method to measure protein levels with fluorescence microscopy confirmed that in‐ deed senescent cells accumulate increased levels of protein in the cytoplasm and nucleus [1]. In addition to the detection of characteristic morphological changes the most common meth‐ od used to identify senescent cells is measurement of the lysosomal beta-galactosidase activ‐ ity with a simple biochemical assay [2]. Due to an expansion of the lysosomes senescent cells show an increased activity of this enzyme, which is therefore often referred to as senescenceassociated beta-galactosidase (SA-beta-gal), [3, 4]. However, it should be noted, that an in‐ creased beta-gal activity is an unreliable marker of senescence since it is also detectable in vitro after prolonged cell culture, serum withdrawal, TGF-beta, heparin or TPA treatment [3, 5-8]. The tumour suppressors p16INK4a and p21Waf1 are mediators of cell cycle arrest and senescence and therefore often used as biomarkers. Since neither p16INK4a nor p21Waf1 is strict‐ ly required for the induction or maintenance of the senescence program their predictive val‐ ue is limited if used individually. A specific feature of senescent cells are condensed heterochromatic regions, known as senescence-associated heterochromatic foci (SAHF). These heterochromatin spots are enriched with i) histone H3-methylated at lysine 9 (H3K9meth), its binding partner ii) heterochromatin protein-1γ (HP- 1γ) and iii) the non-his‐ tone chromatin protein, HMGA2, which all have been used as markers of SAHF [9, 10].

man diploid fibroblasts (HDF) were transduced with lentiviruses expressing N-RASQ61K or copGFP control. The efficiency of transduction was controlled with the co-expression of copGFP and was consistently above 90%. p16Ink4a expression, chromatin condensation (DA‐ PI), and the appearance of increased SA-ß-Gal activity were analyzed and quantified 15 days after infection. Cells enlarged to show DAPI-stained chromatin foci are indicated with arrows. B, C) HDF induced to senesce with oncogenic N-RASQ61K were stained with DAPI and an antibody to H3K9meth or γH2AX to highlight senescence-associated heterochroma‐ tin foci or DNA damage foci respectively. H2AX is a member of the histone H2A family that

Molecular Mechanisms of Cellular Senescence

http://dx.doi.org/10.5772/54120

27

gets instantly phosphorylated after DNA damage and forms foci at DNA break sites.

The retinoblastoma protein (pRb) is often referred to as the "master brake" of the cell cy‐ cle because its main function is to inhibit E2F transcription factors from inducing a range of genes essential for DNA replication and thus proliferation [11]. Consequently active pRb causes cell cycle arrest. In contrast during proliferation when cells are promoted to‐ wards cell division, pRb is sequentially phosphorylated by a series of cyclin dependent kinases (CDKs) and this results in pRb inactivation and consequently derepression of

Initiation of cell proliferation is normally triggered by growth factors. These external mole‐ cules function as ligands to a number of growth factor receptors expressed on the cell sur‐ face and thus activate signalling cascades, most prominently the mitogen activated protein kinase (MAPK) pathway, and ultimately lead to the expression of a number of genes includ‐ ing cyclin D [12]. CDK4 and 6 initiate phosphorylation of pRb in the presence of cyclin D and this leads to de-repression of early cell cycle genes including cyclin E and thus the entry into the cell cycle. Subsequently, CDK2 and CDK1, in co-operation with cyclins E, A and B, continue to stepwise further phosphorylate and inactivate pRb, which leads to cell cycle pro‐ gression and finally cell division. As the "master brake" of the cell cycle pRb is an important tumor suppressor and alterations of its pathway have been associated with the childhood cancer retinoblastoma and are known to occur in over 90% of cancers [13]. There are two important types of cell cycle inhibitors represented most prominently by p16INK4a and p21Waf1. p16INK4a is at the forefront of cell cycle inhibition as it binds specifically to the cyclin D dependent kinases CDK4 and CDK6 and displaces cyclin D and thereby it prevents the entry into the cell cycle and arrests cells in G1 phase (Figure 2). p21Waf1 is more promiscuous and is able to inhibit all CDK molecules at any stage during the cell cycle. p21Waf1 molecules do not necessarily displace cyclin partners from their CDK target and importantly it may re‐ quire several p21Waf1 molecules to effectively inhibit CDKs [14]. In normal cells p16INK4a and p21Waf1 are able to work hand in hand, the accumulation of p16INK4a and binding to CDK4 and 6 frees p21Waf1 molecules from these kinases to bind and inhibit CDK2 and 1 more effi‐ ciently [15]. These basic cell cycle regulatory functions of pRb, p16INK4a and p21Waf1 are essen‐ tial to initiate and maintain senescence and it is not surprising that all three molecules are

**3. pRb in cell cycle regulation**

considered important tumor suppressors.

proliferation genes.

**Figure 1.** Senescence characteristics

A) The typical senescence phenotype consist of enlarged cell with multiple or enlarged nu‐ clei, and an increased SA-beta-gal activity is visible after N-RASQ61K induced senescence. Hu‐ man diploid fibroblasts (HDF) were transduced with lentiviruses expressing N-RASQ61K or copGFP control. The efficiency of transduction was controlled with the co-expression of copGFP and was consistently above 90%. p16Ink4a expression, chromatin condensation (DA‐ PI), and the appearance of increased SA-ß-Gal activity were analyzed and quantified 15 days after infection. Cells enlarged to show DAPI-stained chromatin foci are indicated with arrows. B, C) HDF induced to senesce with oncogenic N-RASQ61K were stained with DAPI and an antibody to H3K9meth or γH2AX to highlight senescence-associated heterochroma‐ tin foci or DNA damage foci respectively. H2AX is a member of the histone H2A family that gets instantly phosphorylated after DNA damage and forms foci at DNA break sites.

#### **3. pRb in cell cycle regulation**

typical senescence phenotype consist of enlarged cell with multiple or enlarged nuclei, prominent Golgi apparatus and sometimes a vacuolated cytoplasm (Figure1). Recently a novel method to measure protein levels with fluorescence microscopy confirmed that in‐ deed senescent cells accumulate increased levels of protein in the cytoplasm and nucleus [1]. In addition to the detection of characteristic morphological changes the most common meth‐ od used to identify senescent cells is measurement of the lysosomal beta-galactosidase activ‐ ity with a simple biochemical assay [2]. Due to an expansion of the lysosomes senescent cells show an increased activity of this enzyme, which is therefore often referred to as senescenceassociated beta-galactosidase (SA-beta-gal), [3, 4]. However, it should be noted, that an in‐ creased beta-gal activity is an unreliable marker of senescence since it is also detectable in vitro after prolonged cell culture, serum withdrawal, TGF-beta, heparin or TPA treatment [3, 5-8]. The tumour suppressors p16INK4a and p21Waf1 are mediators of cell cycle arrest and senescence and therefore often used as biomarkers. Since neither p16INK4a nor p21Waf1 is strict‐ ly required for the induction or maintenance of the senescence program their predictive val‐ ue is limited if used individually. A specific feature of senescent cells are condensed heterochromatic regions, known as senescence-associated heterochromatic foci (SAHF). These heterochromatin spots are enriched with i) histone H3-methylated at lysine 9 (H3K9meth), its binding partner ii) heterochromatin protein-1γ (HP- 1γ) and iii) the non-his‐ tone chromatin protein, HMGA2, which all have been used as markers of SAHF [9, 10].

A) The typical senescence phenotype consist of enlarged cell with multiple or enlarged nu‐ clei, and an increased SA-beta-gal activity is visible after N-RASQ61K induced senescence. Hu‐

**Figure 1.** Senescence characteristics

26 Senescence and Senescence-Related Disorders

The retinoblastoma protein (pRb) is often referred to as the "master brake" of the cell cy‐ cle because its main function is to inhibit E2F transcription factors from inducing a range of genes essential for DNA replication and thus proliferation [11]. Consequently active pRb causes cell cycle arrest. In contrast during proliferation when cells are promoted to‐ wards cell division, pRb is sequentially phosphorylated by a series of cyclin dependent kinases (CDKs) and this results in pRb inactivation and consequently derepression of proliferation genes.

Initiation of cell proliferation is normally triggered by growth factors. These external mole‐ cules function as ligands to a number of growth factor receptors expressed on the cell sur‐ face and thus activate signalling cascades, most prominently the mitogen activated protein kinase (MAPK) pathway, and ultimately lead to the expression of a number of genes includ‐ ing cyclin D [12]. CDK4 and 6 initiate phosphorylation of pRb in the presence of cyclin D and this leads to de-repression of early cell cycle genes including cyclin E and thus the entry into the cell cycle. Subsequently, CDK2 and CDK1, in co-operation with cyclins E, A and B, continue to stepwise further phosphorylate and inactivate pRb, which leads to cell cycle pro‐ gression and finally cell division. As the "master brake" of the cell cycle pRb is an important tumor suppressor and alterations of its pathway have been associated with the childhood cancer retinoblastoma and are known to occur in over 90% of cancers [13]. There are two important types of cell cycle inhibitors represented most prominently by p16INK4a and p21Waf1. p16INK4a is at the forefront of cell cycle inhibition as it binds specifically to the cyclin D dependent kinases CDK4 and CDK6 and displaces cyclin D and thereby it prevents the entry into the cell cycle and arrests cells in G1 phase (Figure 2). p21Waf1 is more promiscuous and is able to inhibit all CDK molecules at any stage during the cell cycle. p21Waf1 molecules do not necessarily displace cyclin partners from their CDK target and importantly it may re‐ quire several p21Waf1 molecules to effectively inhibit CDKs [14]. In normal cells p16INK4a and p21Waf1 are able to work hand in hand, the accumulation of p16INK4a and binding to CDK4 and 6 frees p21Waf1 molecules from these kinases to bind and inhibit CDK2 and 1 more effi‐ ciently [15]. These basic cell cycle regulatory functions of pRb, p16INK4a and p21Waf1 are essen‐ tial to initiate and maintain senescence and it is not surprising that all three molecules are considered important tumor suppressors.

HRAS or NRAS, silencing of p16INK4a did not abolish most senescent features. Interestingly however, the formation of SAHF did only occur in the presence of p16INK4a [29, 36, 37]. These findings show two important points: first there are other redundant mechanisms able to compensate for p16INK4a loss and rescue senescence and second the p16INK4a-pRb pathway has a specific role in SAHF formation and, importantly, these heterochromatin foci have been suggested to abolish expression of proliferation associated genes and secure senescent features so senescence becomes irreversible [9] (see section 7 for more detail). This idea is supported by a report that senescence was only reversible, via p53 inactivation, in fibro‐ blasts and mammary epithelial cells with low but not with high p16INK4a expression [38]. It is noteworthy that the importance of SAHF in securing senescence has been challenged recent‐ ly and SAHF are thought dispensable for senescence by some investigators and/or only as‐ sociated with oncogene-induced senescence [39, 40]. The fact that SAHF formation only occurs in the presence of increased p16INK4a levels remains undebated and it is therefore

Molecular Mechanisms of Cellular Senescence

http://dx.doi.org/10.5772/54120

29

tempting to speculate a direct p16INK4a role in the formation of these structures.

tribute to the melanoma-risk of p16INK4a linked melanoma-prone families [45].

**5. Timing of Senescence by repression and activation of p16INK4a**

In fact, the ability to induce senescence in response to accumulated or sudden genomic stress is probably the most important tumor suppressive function of p16INK4a. In line with this consideration it is not surprising that p16INK4a expression is tightly repressed at the chro‐ matin and transcriptional level in "young" proliferating cells and in cells with extensive re‐ newal capacities, such as stem cells. The polycomb protein Bmi1, which is also known as "stem cell factor" is facilitating repression of the INK4a locus at the chromatin level [48, 49]. Intriguingly, in a functional feedback loop, in human fibroblasts the Bmi1-mediated repres‐ sion of p16INK4a requires active pRb and also H3K27 (histone 3/lysine 27] trimethylation fa‐ cilitated by the histone methyltransferase EZH2 in concert with a second polycomb protein,

**5.1. p16INK4a repression**

Even though cells may be able to compensate for p16INK4a loss and still undergo a growth arrest characterised by most if not all senescent features, the importance of the tumor sup‐ pressor p16INK4a in senescence is clear as p16-null tumor cells can be driven into senescence by the sole re-expression of p16INK4a: Induced p16INK4a expression in glioma cells caused a typical senescent phenotype [41], reversing promoter hypermethylation allowed for the reexpression of endogenous p16INK4a in oral squamous cell carcinoma cells leading to senes‐ cence [42], inducible p16INK4a expression in osteosarcoma cells induced senescence after 3-6 days, potentially irreversible after 6 days [43] and inducible p16INK4a in human melanoma cells caused a senescent phenotype after 3-5 days in the absence of p53 [44, 45]. Moreover, even in normal early passage human fibroblasts the ectopic introduction of p16INK4a or func‐ tional peptides thereof initiated cell cycle arrest and senescent features [46, 47]. In line with this, melanoma associated germline mutations of p16INK4a are impaired in inducing a cellular senescence program in melanoma cells and this disability to promote senescence may con‐

**Figure 2.** The Cell Cycle

This simplified model, focusing on early cell cycle entry, illustrates that hypophosphorylat‐ ed, active pRb represses E2F-mediated transcription. The action of CDKs, exemplified by the cyclin D dependent CDK4 and 6, phosphorylate pRb and thus release E2F to activate tran‐ scription of early DNA replication genes. p16INK4a and p21Waf1, the latter usually activated by p53, inhibit CDKs and retain pRb in its active cell cycle inhibitory state.

#### **4. The role of the tumor suppressor p16INK4a in senescence**

With regard to senescence, it is long known that p16INK4a levels accumulate and cause growth arrest and senescence when cells approach their replicative life span [16-23]. More‐ over, in long term tissue culture studies cells that were able to overcome senescence com‐ monly had lost p16INK4a and p53 expression [24]. Increased p16INK4a expression is also linked to oncogene induced and other forms of premature senescence [25-33].

Interestingly, despite this clear correlation of p16INK4a up-regulation with senescence there is some evidence from p16INK4a is not strictly required for senescence to occur. Evidence form mouse models show that mouse embryonic fibroblasts (MEFs) of p16-null mice undergo a comparable number of cell divisions as wild type MEFs before entering senescence [34] [35]*,* while in primary melanocytes, which were lentivirally transduced to express oncogenic HRAS or NRAS, silencing of p16INK4a did not abolish most senescent features. Interestingly however, the formation of SAHF did only occur in the presence of p16INK4a [29, 36, 37]. These findings show two important points: first there are other redundant mechanisms able to compensate for p16INK4a loss and rescue senescence and second the p16INK4a-pRb pathway has a specific role in SAHF formation and, importantly, these heterochromatin foci have been suggested to abolish expression of proliferation associated genes and secure senescent features so senescence becomes irreversible [9] (see section 7 for more detail). This idea is supported by a report that senescence was only reversible, via p53 inactivation, in fibro‐ blasts and mammary epithelial cells with low but not with high p16INK4a expression [38]. It is noteworthy that the importance of SAHF in securing senescence has been challenged recent‐ ly and SAHF are thought dispensable for senescence by some investigators and/or only as‐ sociated with oncogene-induced senescence [39, 40]. The fact that SAHF formation only occurs in the presence of increased p16INK4a levels remains undebated and it is therefore tempting to speculate a direct p16INK4a role in the formation of these structures.

Even though cells may be able to compensate for p16INK4a loss and still undergo a growth arrest characterised by most if not all senescent features, the importance of the tumor sup‐ pressor p16INK4a in senescence is clear as p16-null tumor cells can be driven into senescence by the sole re-expression of p16INK4a: Induced p16INK4a expression in glioma cells caused a typical senescent phenotype [41], reversing promoter hypermethylation allowed for the reexpression of endogenous p16INK4a in oral squamous cell carcinoma cells leading to senes‐ cence [42], inducible p16INK4a expression in osteosarcoma cells induced senescence after 3-6 days, potentially irreversible after 6 days [43] and inducible p16INK4a in human melanoma cells caused a senescent phenotype after 3-5 days in the absence of p53 [44, 45]. Moreover, even in normal early passage human fibroblasts the ectopic introduction of p16INK4a or func‐ tional peptides thereof initiated cell cycle arrest and senescent features [46, 47]. In line with this, melanoma associated germline mutations of p16INK4a are impaired in inducing a cellular senescence program in melanoma cells and this disability to promote senescence may con‐ tribute to the melanoma-risk of p16INK4a linked melanoma-prone families [45].

#### **5. Timing of Senescence by repression and activation of p16INK4a**

#### **5.1. p16INK4a repression**

**Figure 2.** The Cell Cycle

28 Senescence and Senescence-Related Disorders

This simplified model, focusing on early cell cycle entry, illustrates that hypophosphorylat‐ ed, active pRb represses E2F-mediated transcription. The action of CDKs, exemplified by the cyclin D dependent CDK4 and 6, phosphorylate pRb and thus release E2F to activate tran‐ scription of early DNA replication genes. p16INK4a and p21Waf1, the latter usually activated by

With regard to senescence, it is long known that p16INK4a levels accumulate and cause growth arrest and senescence when cells approach their replicative life span [16-23]. More‐ over, in long term tissue culture studies cells that were able to overcome senescence com‐ monly had lost p16INK4a and p53 expression [24]. Increased p16INK4a expression is also linked

Interestingly, despite this clear correlation of p16INK4a up-regulation with senescence there is some evidence from p16INK4a is not strictly required for senescence to occur. Evidence form mouse models show that mouse embryonic fibroblasts (MEFs) of p16-null mice undergo a comparable number of cell divisions as wild type MEFs before entering senescence [34] [35]*,* while in primary melanocytes, which were lentivirally transduced to express oncogenic

p53, inhibit CDKs and retain pRb in its active cell cycle inhibitory state.

to oncogene induced and other forms of premature senescence [25-33].

**4. The role of the tumor suppressor p16INK4a in senescence**

In fact, the ability to induce senescence in response to accumulated or sudden genomic stress is probably the most important tumor suppressive function of p16INK4a. In line with this consideration it is not surprising that p16INK4a expression is tightly repressed at the chro‐ matin and transcriptional level in "young" proliferating cells and in cells with extensive re‐ newal capacities, such as stem cells. The polycomb protein Bmi1, which is also known as "stem cell factor" is facilitating repression of the INK4a locus at the chromatin level [48, 49]. Intriguingly, in a functional feedback loop, in human fibroblasts the Bmi1-mediated repres‐ sion of p16INK4a requires active pRb and also H3K27 (histone 3/lysine 27] trimethylation fa‐ cilitated by the histone methyltransferase EZH2 in concert with a second polycomb protein, SUZ12 [50]. Crucially, Bmi1 chromatin binding can be inhibited by its phosphorylation through the MAPK and p38 signalling pathways [51]. Hence these pathways are able to di‐ rectly oppose p16INK4a repression and lead to its transcriptional activation via Ets and Sp-1 during oncogene-induced senescence.

ent on the SWI/SNF subunits BRG1 and hSnf5, and the relaxed chromatin structure allows tran‐ scription factor access [71] (Figure 3a). Alterations of hSnf5 are associated with early childhood rhabdoid cancer and re-expression of hSnf5 in rhabdoid cancer cells leads to p16INK4a accumula‐

Molecular Mechanisms of Cellular Senescence

http://dx.doi.org/10.5772/54120

31

The best understood transcription factors driving p16INK4a expression and thereby growth ar‐ rest and senescence are Ets1 and Ets2. They are effectors of MAPK signalling, often in re‐ sponse to oncogenic stress, such as activating N-RAS or B-RAF mutations, which induce an increase in p16INK4a levels [25, 29, 37, 52]. In line with this, human fibroblasts with biallelic mutations in p16INK4a did increase mutant p16INK4a expression in response to RAS signaling or expression of ectopic Ets, but failed to arrest or undergo senescence [74]. Interestingly in‐ creased p16INK4a expression has also been linked to loss of p53 and this appears to be corre‐ lated with increased Ets protein half-life [75]. Another member of the Ets transcription factor family, ESE-3, was independently identified as a down stream target of p38 signalling and caused senescence via p16INK4a up-regulation [76]. p38 signalling has been linked to en‐ hanced p16INK4a expression before and this involved the downstream transcription factor Sp-1, which was proposed to be required for p16INK4a up-regulation during senescence in hu‐ man fibroblasts [77]. Sp-1 was reported to engage the p300 enhancer leading to further

(A) During the proliferative cellular life-time, EZH2 in cooperation with SUZ12 trimethy‐ lates H3K27 at the genomic INK4a locus and these histone modifications attract the poly‐ comb repressor BMI1, which maintains the p16INK4a promoter region inaccessible for

tion, growth arrest and senescence [71, 72] and this requires functional p16INK4a [73].

p16INK4a upregulation [78] (Figure 3b).

**Figure 3.** Schematic presentation of p16INK4a regulation

In concert with Bmi-linked chromatin-remodelling events a number of transcription factors fa‐ cilitate p16INK4a repression during the proliferative life-time of cells. The perhaps most impor‐ tant transcriptional repressors of p16INK4a are Id proteins, with the main representative Id1. Id proteins function by binding to E-box DNA sequences to repress the INK4a promoter and im‐ portantly by interfering with Ets transcriptional complex formation and thereby inhibiting the main INK4a transcriptional activator, Ets, in two ways [52, 53]. In line with this, high Id1 levels were associated with early stage melanoma whereas premalignant and interestingly also more advanced melanoma showed limited Id1 expression [54]. This suggests a role of the Id1/ p16INK4a regulative connection during melanomagenesis and Id1 may be dispensable in later melanoma stages once p16INK4a is either more tightly repressed by engaging repressive histone modifications or inactivated by other mechanisms. Id1 down-regulation on the other hand is usually associated with cell differentiation and senescence [55]. Consequently, ectopic expres‐ sion of Id1 delayed senescence in melanocytes [56] and keratinocytes [57], while MEFs lacking Id1 prematurely senesced due to increased p16INK4a levels [58, 59]. Another way to oppose Ets driven p16INK4a transcription was identified, when Cdh1, an adaptor protein of the anaphase promoting complex, was shown to bind to and promote degradation of Ets2 and thereby in‐ creased the replicative life span of MEFs [60], while the Epstein-Barr virus protein LMP1 re‐ presses p16INK4a by promoting the nuclear export of Ets2 [61, 62]

Interestingly, p16INK4a may also be repressed by the oncogene β-catenin, which has been linked to melanoma. β-catenin binds the INK4a promoter at a conservative β-catenin/Lef/Tcf binding site and thereby directly represses its transcription. Consequently β-catenin silenc‐ ing increased p16INK4a levels in A375P human melanoma cells, while stabilization of β-cate‐ nin together with oncogenic N-RAS led to prevention of senescence and thus, immortalization [63]. Importantly, a role of β-catenin in melanocyte senescence is controver‐ sial as nuclear β-catenin was commonly found in benign melanocytic nevi [64-66] and these lesions were proposed to be senescent by some investigators [29, 67], this again is controver‐ sial, as benign nevi are not be distinguished from normal melanocytes or primary melano‐ mas using a range of common senescence markers [68]. It would clearly be interesting to test whether nuclear β-catenin does overlap with the expression of p16INK4a in benign nevi as the latter is mosaic and not found in all cells [29] and co-localisation or lack of it could help clar‐ ify this debate. Another repressor of p16INK4a is the "T-box transcription factor" Tbx2, this transcription factor binds to corresponding T-box DNA sequence elements [69]. Tbx2 overexpression was identified in melanomas and associated with melanoma progression [70].

#### **5.2. p16INK4a expression**

When cells reach their finite life span the pendulum at the INK4a promoter swings from repres‐ sion to activation and the SWI/SNF chromatin remodeling complex, replaces Bmi1 repressors and relaxes chromatin structures around the INK4 promoter region, which is strictly depend‐ ent on the SWI/SNF subunits BRG1 and hSnf5, and the relaxed chromatin structure allows tran‐ scription factor access [71] (Figure 3a). Alterations of hSnf5 are associated with early childhood rhabdoid cancer and re-expression of hSnf5 in rhabdoid cancer cells leads to p16INK4a accumula‐ tion, growth arrest and senescence [71, 72] and this requires functional p16INK4a [73].

The best understood transcription factors driving p16INK4a expression and thereby growth ar‐ rest and senescence are Ets1 and Ets2. They are effectors of MAPK signalling, often in re‐ sponse to oncogenic stress, such as activating N-RAS or B-RAF mutations, which induce an increase in p16INK4a levels [25, 29, 37, 52]. In line with this, human fibroblasts with biallelic mutations in p16INK4a did increase mutant p16INK4a expression in response to RAS signaling or expression of ectopic Ets, but failed to arrest or undergo senescence [74]. Interestingly in‐ creased p16INK4a expression has also been linked to loss of p53 and this appears to be corre‐ lated with increased Ets protein half-life [75]. Another member of the Ets transcription factor family, ESE-3, was independently identified as a down stream target of p38 signalling and caused senescence via p16INK4a up-regulation [76]. p38 signalling has been linked to en‐ hanced p16INK4a expression before and this involved the downstream transcription factor Sp-1, which was proposed to be required for p16INK4a up-regulation during senescence in hu‐ man fibroblasts [77]. Sp-1 was reported to engage the p300 enhancer leading to further p16INK4a upregulation [78] (Figure 3b).

**Figure 3.** Schematic presentation of p16INK4a regulation

SUZ12 [50]. Crucially, Bmi1 chromatin binding can be inhibited by its phosphorylation through the MAPK and p38 signalling pathways [51]. Hence these pathways are able to di‐ rectly oppose p16INK4a repression and lead to its transcriptional activation via Ets and Sp-1

In concert with Bmi-linked chromatin-remodelling events a number of transcription factors fa‐ cilitate p16INK4a repression during the proliferative life-time of cells. The perhaps most impor‐ tant transcriptional repressors of p16INK4a are Id proteins, with the main representative Id1. Id proteins function by binding to E-box DNA sequences to repress the INK4a promoter and im‐ portantly by interfering with Ets transcriptional complex formation and thereby inhibiting the main INK4a transcriptional activator, Ets, in two ways [52, 53]. In line with this, high Id1 levels were associated with early stage melanoma whereas premalignant and interestingly also more advanced melanoma showed limited Id1 expression [54]. This suggests a role of the Id1/ p16INK4a regulative connection during melanomagenesis and Id1 may be dispensable in later melanoma stages once p16INK4a is either more tightly repressed by engaging repressive histone modifications or inactivated by other mechanisms. Id1 down-regulation on the other hand is usually associated with cell differentiation and senescence [55]. Consequently, ectopic expres‐ sion of Id1 delayed senescence in melanocytes [56] and keratinocytes [57], while MEFs lacking Id1 prematurely senesced due to increased p16INK4a levels [58, 59]. Another way to oppose Ets driven p16INK4a transcription was identified, when Cdh1, an adaptor protein of the anaphase promoting complex, was shown to bind to and promote degradation of Ets2 and thereby in‐ creased the replicative life span of MEFs [60], while the Epstein-Barr virus protein LMP1 re‐

Interestingly, p16INK4a may also be repressed by the oncogene β-catenin, which has been linked to melanoma. β-catenin binds the INK4a promoter at a conservative β-catenin/Lef/Tcf binding site and thereby directly represses its transcription. Consequently β-catenin silenc‐ ing increased p16INK4a levels in A375P human melanoma cells, while stabilization of β-cate‐ nin together with oncogenic N-RAS led to prevention of senescence and thus, immortalization [63]. Importantly, a role of β-catenin in melanocyte senescence is controver‐ sial as nuclear β-catenin was commonly found in benign melanocytic nevi [64-66] and these lesions were proposed to be senescent by some investigators [29, 67], this again is controver‐ sial, as benign nevi are not be distinguished from normal melanocytes or primary melano‐ mas using a range of common senescence markers [68]. It would clearly be interesting to test whether nuclear β-catenin does overlap with the expression of p16INK4a in benign nevi as the latter is mosaic and not found in all cells [29] and co-localisation or lack of it could help clar‐ ify this debate. Another repressor of p16INK4a is the "T-box transcription factor" Tbx2, this transcription factor binds to corresponding T-box DNA sequence elements [69]. Tbx2 overexpression was identified in melanomas and associated with melanoma progression [70].

When cells reach their finite life span the pendulum at the INK4a promoter swings from repres‐ sion to activation and the SWI/SNF chromatin remodeling complex, replaces Bmi1 repressors and relaxes chromatin structures around the INK4 promoter region, which is strictly depend‐

during oncogene-induced senescence.

30 Senescence and Senescence-Related Disorders

**5.2. p16INK4a expression**

presses p16INK4a by promoting the nuclear export of Ets2 [61, 62]

(A) During the proliferative cellular life-time, EZH2 in cooperation with SUZ12 trimethy‐ lates H3K27 at the genomic INK4a locus and these histone modifications attract the poly‐ comb repressor BMI1, which maintains the p16INK4a promoter region inaccessible for transcriptional activation. Once cells reach their finite life span or during premature senes‐ cence the histone modifications at the INK4a locus change from repressive methylations to activating acetylations, which attract the SWI/SNF complex. The SWI/SNF subunits hSnf5 and BRG1 are instrumental in opening the chromatin structure and allowing access of the transcriptional machinery to the p16INK4a promoter. (B) Ets transcription factors are down stream targets of MAPK signalling, exemplified here by RAS, and bind to E-box p16INK4 pro‐ moter motifs to activate gene expression. Id transcription factors can compete with Ets for DNA binding and oppose transcriptional expression. The levels of Id proteins decline with onset of senescence and Ets are able to promote p16INK4a expression.

**6.1. DNA damage and senescence**

**6.2. Drug induced senescence**

level of the stress signal.

**6.3. Oncogene-induced senescence and DNA damage**

The human genome is constantly exposed to genotoxic stress such as ultraviolet light, reactive oxygen species (ROS) as well as chemical and biological mutagens. To ensure the integrity of the genome cells have evolved a sophisticated safety system that can implement a cell cycle ar‐ rest to allow the repair of the incurred damage. The initial step in the complicated DNA dam‐ age repair process is the detection of DNA damage. The Mre11/Rad50/NBS1 (*MRN*) complex function as a DNA damage sensor is localized in nuclear foci at the sites of double strand breaks (DSBs) [102-104]. Here, the complex tethers DNA ends, processes and repairs free strands via endonuclease and exonuclease activities [105-107]. The MRN complex is also involved in the recruitment and activation of ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3 related) protein kinases, which in turn activate checkpoint-1/2 (CHK1/2) kinases leading to phosphorylation and thereby stabilization of a variety of target genes including p53 [108]. In the case of irreparable damage, the cell will be permanently retracted from the pool of dividing

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Several sources of genotoxic stress including cisplatin, cyclophosphamide, doxorubicine, taxol, vincristine, cytarabine, etoposide, hydroxyurea, bromodeoxyuridine, adriamycin, bleomycin mitomycin D, interferon beta, radiation, ROS, H2O2 have been used to trigger senescence-like growth arrest in vitro and in vivo allowing a detailed analysis of the under‐ lying signaling events [28, 111-122]. The crosslining agent cisplatin, for example, induced senescence in primary human fibroblasts and HCT116 colon carcinoma cells in a p53 and dose dependent manner [123, 124]). Interestingly, treatment of HCT116 cells with low cispla‐ tin concentrations lead to DNA damage and senescence whereas high drug concentrations induced apoptosis via superoxide production. Furthermore, in human lung cancer cells in‐ duced p53 expression enhanced the cytotoxic effect of cisplatin whereas p21waf1 overexpres‐ sion surprisingly lead to increased drug resistance [82]. Similarly, cisplatin-resistant human non-small cell lung cancer (NSCLC) cells could be sensitized to drug-induced senescence by re-expressing p16Ink4a [118]. Duale and co-workers compared changes in the gene expression profiles of a cisplatin treated human colon carcinoma cells (HCT116) and cell lines derived from testicular germ cell tumors (TGCTs). The systematic approach combining the acquired gene expression data and other publicly available microarray data identified 1794 genes that were differentially expressed including 29 senescence-related genes such as IGFBP 7, inter‐ leukin 1 and MAPK8 [125]. These findings highlight the notion that cellular response to che‐ motherapeutic agents is generally depending on the cellular context as well as the type and

In contrast to chemotherapeutic agents that directly damage the DNA, activated oncogenes do the harm by forcing the cell in uncontrolled, constitute replication cycles which leads to DNA replication stress and subsequent DNA damage [126, 127]. During this process DNA replication forks stall making the DNA more susceptible to single or double strand breaks.

cells by the induction of apoptosis or senescence (Figure 4] [109, 110].

#### **6. The role of the p53/p21 pathway in senescence**

The transcription factor and tumor suppressor p53 is often referred to as "guardian of the genome" and inactivating mutations in p53 are observed in about half of all human cancer cases. The p53 protein is a critical regulator of cell survival in response to cellular stress sig‐ nals including DNA damage, oncogene activation, hypoxia and viral infection (reviewed in [79]. In the absence of stress stimuli p53 gets rapidly ubiquitinated by one of several E3 li‐ gases including MDM2, MDM4, TOPORS, COP1, and ARF-BP1 and subsequently degraded in the proteasome [80, 81]. Stress signals, on the other hand, induce covalent modification usually by disrupting the interaction between p53 and the E3 ubiquitin ligases which pre‐ vents its degradation. Oncogenic stress, for example, activates the alternative reading frame product of the INK4a locus (p14ARF) that stabilizes p53 by binding and thereby inhibiting its negative regulator MDM2.

Several lines of evidence show convincingly that the p53 and its downstream effector p21Waf1 play a crucial role in the regulation of cell cycle arrest and senescence*.* Overexpression of p53 [82] and p21Waf1 [83-86] autonomously induced senescence in human cells and activation of p53 by either overexpression of p14ARF [87] or nutlin-3 treatment [88] induced senes‐ cence in a p21Waf1 dependent manner in human diploid fibroblasts (HDF) and human glio‐ blastoma cells respectively. Furthermore, inactivation of p53 or p19ARF (mouse homologue of human p14ARF) prevents senescence in mouse embryonic fibroblasts (MEF) [89-91] and human fibroblast lacking p21Waf1 can bypass the senescence growth arrest [83]. Further supporting evidence comes from studies that show that, inactivation of p53 using viral on‐ coproteins, anti-p53 antibodies or anti-sense oligonucleotides can extend the replicative life‐ span or even reverse the senescence growth arrest in human cells [38, 92-94]. However, it should be noted that although inactivation of the p53 pathway can weaken or even reverse the senescence arrest in some cells, there is emerging evidence that it fails to do so in cells with an activated p16INK4a/pRb pathway [38, 95-97]. Despite the clear evidence of p53's role in promoting senescence a study conducted by Demidenko and co-workers suggested that p53 can also act as an inhibitor of senescence [98]. Surprisingly, p53 was able to reverse a p16INK4a and p21Waf1 driven senescence response in human fibroblasts. Although the underly‐ ing mechanisms are not fully understood, inhibition of the mTor (mammalian target of rapa‐ mycin) pathway seems to be involved in the repression of cellular senescence [98-101].

#### **6.1. DNA damage and senescence**

transcriptional activation. Once cells reach their finite life span or during premature senes‐ cence the histone modifications at the INK4a locus change from repressive methylations to activating acetylations, which attract the SWI/SNF complex. The SWI/SNF subunits hSnf5 and BRG1 are instrumental in opening the chromatin structure and allowing access of the transcriptional machinery to the p16INK4a promoter. (B) Ets transcription factors are down stream targets of MAPK signalling, exemplified here by RAS, and bind to E-box p16INK4 pro‐ moter motifs to activate gene expression. Id transcription factors can compete with Ets for DNA binding and oppose transcriptional expression. The levels of Id proteins decline with

The transcription factor and tumor suppressor p53 is often referred to as "guardian of the genome" and inactivating mutations in p53 are observed in about half of all human cancer cases. The p53 protein is a critical regulator of cell survival in response to cellular stress sig‐ nals including DNA damage, oncogene activation, hypoxia and viral infection (reviewed in [79]. In the absence of stress stimuli p53 gets rapidly ubiquitinated by one of several E3 li‐ gases including MDM2, MDM4, TOPORS, COP1, and ARF-BP1 and subsequently degraded in the proteasome [80, 81]. Stress signals, on the other hand, induce covalent modification usually by disrupting the interaction between p53 and the E3 ubiquitin ligases which pre‐ vents its degradation. Oncogenic stress, for example, activates the alternative reading frame product of the INK4a locus (p14ARF) that stabilizes p53 by binding and thereby inhibiting

Several lines of evidence show convincingly that the p53 and its downstream effector p21Waf1 play a crucial role in the regulation of cell cycle arrest and senescence*.* Overexpression of p53 [82] and p21Waf1 [83-86] autonomously induced senescence in human cells and activation of p53 by either overexpression of p14ARF [87] or nutlin-3 treatment [88] induced senes‐ cence in a p21Waf1 dependent manner in human diploid fibroblasts (HDF) and human glio‐ blastoma cells respectively. Furthermore, inactivation of p53 or p19ARF (mouse homologue of human p14ARF) prevents senescence in mouse embryonic fibroblasts (MEF) [89-91] and human fibroblast lacking p21Waf1 can bypass the senescence growth arrest [83]. Further supporting evidence comes from studies that show that, inactivation of p53 using viral on‐ coproteins, anti-p53 antibodies or anti-sense oligonucleotides can extend the replicative life‐ span or even reverse the senescence growth arrest in human cells [38, 92-94]. However, it should be noted that although inactivation of the p53 pathway can weaken or even reverse the senescence arrest in some cells, there is emerging evidence that it fails to do so in cells with an activated p16INK4a/pRb pathway [38, 95-97]. Despite the clear evidence of p53's role in promoting senescence a study conducted by Demidenko and co-workers suggested that p53 can also act as an inhibitor of senescence [98]. Surprisingly, p53 was able to reverse a p16INK4a and p21Waf1 driven senescence response in human fibroblasts. Although the underly‐ ing mechanisms are not fully understood, inhibition of the mTor (mammalian target of rapa‐ mycin) pathway seems to be involved in the repression of cellular senescence [98-101].

onset of senescence and Ets are able to promote p16INK4a expression.

**6. The role of the p53/p21 pathway in senescence**

its negative regulator MDM2.

32 Senescence and Senescence-Related Disorders

The human genome is constantly exposed to genotoxic stress such as ultraviolet light, reactive oxygen species (ROS) as well as chemical and biological mutagens. To ensure the integrity of the genome cells have evolved a sophisticated safety system that can implement a cell cycle ar‐ rest to allow the repair of the incurred damage. The initial step in the complicated DNA dam‐ age repair process is the detection of DNA damage. The Mre11/Rad50/NBS1 (*MRN*) complex function as a DNA damage sensor is localized in nuclear foci at the sites of double strand breaks (DSBs) [102-104]. Here, the complex tethers DNA ends, processes and repairs free strands via endonuclease and exonuclease activities [105-107]. The MRN complex is also involved in the recruitment and activation of ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3 related) protein kinases, which in turn activate checkpoint-1/2 (CHK1/2) kinases leading to phosphorylation and thereby stabilization of a variety of target genes including p53 [108]. In the case of irreparable damage, the cell will be permanently retracted from the pool of dividing cells by the induction of apoptosis or senescence (Figure 4] [109, 110].

#### **6.2. Drug induced senescence**

Several sources of genotoxic stress including cisplatin, cyclophosphamide, doxorubicine, taxol, vincristine, cytarabine, etoposide, hydroxyurea, bromodeoxyuridine, adriamycin, bleomycin mitomycin D, interferon beta, radiation, ROS, H2O2 have been used to trigger senescence-like growth arrest in vitro and in vivo allowing a detailed analysis of the under‐ lying signaling events [28, 111-122]. The crosslining agent cisplatin, for example, induced senescence in primary human fibroblasts and HCT116 colon carcinoma cells in a p53 and dose dependent manner [123, 124]). Interestingly, treatment of HCT116 cells with low cispla‐ tin concentrations lead to DNA damage and senescence whereas high drug concentrations induced apoptosis via superoxide production. Furthermore, in human lung cancer cells in‐ duced p53 expression enhanced the cytotoxic effect of cisplatin whereas p21waf1 overexpres‐ sion surprisingly lead to increased drug resistance [82]. Similarly, cisplatin-resistant human non-small cell lung cancer (NSCLC) cells could be sensitized to drug-induced senescence by re-expressing p16Ink4a [118]. Duale and co-workers compared changes in the gene expression profiles of a cisplatin treated human colon carcinoma cells (HCT116) and cell lines derived from testicular germ cell tumors (TGCTs). The systematic approach combining the acquired gene expression data and other publicly available microarray data identified 1794 genes that were differentially expressed including 29 senescence-related genes such as IGFBP 7, inter‐ leukin 1 and MAPK8 [125]. These findings highlight the notion that cellular response to che‐ motherapeutic agents is generally depending on the cellular context as well as the type and level of the stress signal.

#### **6.3. Oncogene-induced senescence and DNA damage**

In contrast to chemotherapeutic agents that directly damage the DNA, activated oncogenes do the harm by forcing the cell in uncontrolled, constitute replication cycles which leads to DNA replication stress and subsequent DNA damage [126, 127]. During this process DNA replication forks stall making the DNA more susceptible to single or double strand breaks. The subsequent activation of the DNA damage response (DDR) machinery delays cell-cycle progression by initiation of the ATM/ATR-CHK1/2 pathway and stable knockdown of any one of these DDR genes was sufficient to bypass RAS induced senescence in human fibro‐ blasts. Specifically, in the absence of ATM, ATR, Chk1 or Chk2, human fibroblasts continued to proliferate despite Ras expression, and BrdU incorporation increased from approximately 5% in the control sample to 15% in cells deficient for a DNA damage protein. Interestingly, the inhibition of both the p16INK4a and DNA damage pathway enhanced the effect and en‐ abled over 60% of the cells to replicate DNA [126]. In a similar study, Bartkova and co-work‐ ers showed that the suppression of ATM or p53 allowed the MRC5 and BJ human fibroblasts to bypass oncogene-induced senescence using overexpressed Mos (an activator of the MAPK pathway) or Cdc6 (a DNA replication licensing factor). However, in this report p16INK4a depletion alone did not weaken the senescence response [127].

To protect the integrity of their DNA, cells need to be able to sense DNA damage and acti‐ vate response pathways that coordinate cell cycle progression and DNA repair. Ataxia telan‐ giectasia mutated (ATM) and ATM-Rad3-related (ATR) kinases are important DNA damage checkpoints proteins that transduce signals from the DNA damage sensors to the effector proteins that control cell cycle progression, chromatin restructuring, and DNA repair. ATM and ATR activate other kinases such as Chkl (activated by ATR) and Chk2 (activated by ATM) that phosphorylate and activate the tumour suppressor protein p53. ATM and ATR can additionally enhance p53 activity by directing p53 phosphorylation on Ser15. Activated p53 can halt progression of the cell cycle in the G1 phase, allowing DNA repair to occur and

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preventing the transmission of damaged DNA to the daughter cells.

**7. pRb in senescence: maintaining a secure proliferative arrest**

beyond the antiproliferative functions described in the cell cycle section 3.

development of senescence [9].

**7.1. pRb and SAHFs**

In the previous sections we have reviewed the pivotal role of the cell cycle inhibitors p16INK4a and p21Waf1 in keeping pRb in a hypophosphorylated, active state and explored the mecha‐ nisms of timing the expression of these tumor suppressors with the onset and maintenance of senescence. Here we explore the roles of active pRb in the senescence program, which go

There is longstanding evidence that pRb is required for an intact senescence program, for instance re-introduction of pRb into SAOS osteosarcoma cells that have lost its expression can cause senescence [128]. On the other hand inactivation of pRb with the viral oncoprotein E1A prevents senescence, while a mutant form of E1A that is impaired in pRb binding is un‐ able to prevent senescence [9]. Interestingly, another mutant E1A, that is able to bind pRb but is unable to interact with the histone deacetylases p300 and p400 leads to less efficient

These data highlight that chromatin remodeling plays an important role in the cellular sen‐ escence program. Indeed, during senescence E2F responsive genomic promoter regions are stably repressed from transcription by the establishment of heterochromatin regions. As mentioned above (section 3) these regions are visible as microscopical "senescence associat‐ ed heterochromatin foci" (SAHF) when cells are stained with certain DNA intercalating dyes such as DAPI. The term heterochromatin refers to a highly condensed protein-DNA structure, which is facilitated by modifications of the histone core molecules of chromatin and suppresses DNA access by the transcriptional machinery and thus gene expression of these regions is tightly suppressed (reviewed in [129]). The investigation of SAHF remains a progressing research field and is developing forward with improved microscopic imaging technology and the ability to isolate these complex structures and analyse them more pre‐ cisely and we can expect to understand even more about their role and configuration in the future. To date we know that SAHF formation coincides with the recruitment of heterochro‐ matin proteins by pRb when interacting with the histone deacetylase 1 (HDAC1] at E2F-re‐

**Figure 4.** DNA damage pathway in senescence

To protect the integrity of their DNA, cells need to be able to sense DNA damage and acti‐ vate response pathways that coordinate cell cycle progression and DNA repair. Ataxia telan‐ giectasia mutated (ATM) and ATM-Rad3-related (ATR) kinases are important DNA damage checkpoints proteins that transduce signals from the DNA damage sensors to the effector proteins that control cell cycle progression, chromatin restructuring, and DNA repair. ATM and ATR activate other kinases such as Chkl (activated by ATR) and Chk2 (activated by ATM) that phosphorylate and activate the tumour suppressor protein p53. ATM and ATR can additionally enhance p53 activity by directing p53 phosphorylation on Ser15. Activated p53 can halt progression of the cell cycle in the G1 phase, allowing DNA repair to occur and preventing the transmission of damaged DNA to the daughter cells.

#### **7. pRb in senescence: maintaining a secure proliferative arrest**

In the previous sections we have reviewed the pivotal role of the cell cycle inhibitors p16INK4a and p21Waf1 in keeping pRb in a hypophosphorylated, active state and explored the mecha‐ nisms of timing the expression of these tumor suppressors with the onset and maintenance of senescence. Here we explore the roles of active pRb in the senescence program, which go beyond the antiproliferative functions described in the cell cycle section 3.

There is longstanding evidence that pRb is required for an intact senescence program, for instance re-introduction of pRb into SAOS osteosarcoma cells that have lost its expression can cause senescence [128]. On the other hand inactivation of pRb with the viral oncoprotein E1A prevents senescence, while a mutant form of E1A that is impaired in pRb binding is un‐ able to prevent senescence [9]. Interestingly, another mutant E1A, that is able to bind pRb but is unable to interact with the histone deacetylases p300 and p400 leads to less efficient development of senescence [9].

#### **7.1. pRb and SAHFs**

The subsequent activation of the DNA damage response (DDR) machinery delays cell-cycle progression by initiation of the ATM/ATR-CHK1/2 pathway and stable knockdown of any one of these DDR genes was sufficient to bypass RAS induced senescence in human fibro‐ blasts. Specifically, in the absence of ATM, ATR, Chk1 or Chk2, human fibroblasts continued to proliferate despite Ras expression, and BrdU incorporation increased from approximately 5% in the control sample to 15% in cells deficient for a DNA damage protein. Interestingly, the inhibition of both the p16INK4a and DNA damage pathway enhanced the effect and en‐ abled over 60% of the cells to replicate DNA [126]. In a similar study, Bartkova and co-work‐ ers showed that the suppression of ATM or p53 allowed the MRC5 and BJ human fibroblasts to bypass oncogene-induced senescence using overexpressed Mos (an activator of the MAPK pathway) or Cdc6 (a DNA replication licensing factor). However, in this report

p16INK4a depletion alone did not weaken the senescence response [127].

34 Senescence and Senescence-Related Disorders

**Figure 4.** DNA damage pathway in senescence

These data highlight that chromatin remodeling plays an important role in the cellular sen‐ escence program. Indeed, during senescence E2F responsive genomic promoter regions are stably repressed from transcription by the establishment of heterochromatin regions. As mentioned above (section 3) these regions are visible as microscopical "senescence associat‐ ed heterochromatin foci" (SAHF) when cells are stained with certain DNA intercalating dyes such as DAPI. The term heterochromatin refers to a highly condensed protein-DNA structure, which is facilitated by modifications of the histone core molecules of chromatin and suppresses DNA access by the transcriptional machinery and thus gene expression of these regions is tightly suppressed (reviewed in [129]). The investigation of SAHF remains a progressing research field and is developing forward with improved microscopic imaging technology and the ability to isolate these complex structures and analyse them more pre‐ cisely and we can expect to understand even more about their role and configuration in the future. To date we know that SAHF formation coincides with the recruitment of heterochro‐ matin proteins by pRb when interacting with the histone deacetylase 1 (HDAC1] at E2F-re‐ sponsive promoters [130]. A number of heterochromatin-associated histone modifications were characteristically found associated with SAHF. These include the reversion of histone 3/lysine 9 acetylation (H3K9Ac) and histone 3/lysine 4 trimethylation (H3K4me3) and signif‐ icantly, since it is often used as a marker of senescence, the promotion of histone 3/lysine 9 trimethylation (H3K9me3) and H3K27me3. H3K9 trimethylation is a fundamental step dur‐ ing heterochromatin formation as it attracts HP1 proteins, which are pivotal for heterochro‐ matin assembly [9]. It is important to highlight that senescence does not always lead to a net increase in overall H3K9me3, suggesting that SAHF formation is local and indeed targeted via pRb to E2F promoters [131]. H3K9me3 is thought to be very stable and is able to prevent histone acetylases (HATs) to catalyze histone acetylations. Histone acetylations are com‐ monly associated with "euchromatin", which describes actively transcribed chromatin re‐ gions. Accordingly, SAHF formation is thought to "lock" chromatin regions that encode proliferation genes and thus contributes to the very secure silencing of E2F responsive pro‐ liferation genes, which is not reversible by physiological stimuli [9]. H3K9 trimethylation as‐ sociated with SAHF formation is thought to be catalyzed by either the SUV39H1 methyltransferase, which together with HP1 interacts with pRb during E2F promoter silenc‐ ing in senescence [132] or the RIZ1/PRDM2 H3K9 methyltransferase, which was also shown to co-operate in pRb gene repression and is inactivated in colon, breast and gastric carcino‐ ma [133]. Furthermore, a search for H3K9me3 interacting proteins to identify proteins in‐ volved in the senescence program identified JMJD2C, which is a H3K9 specific demethylase. Thereby it is the direct antagonist of the SUV39H1 H3K9 methyltransferase and its over-ex‐ pression is not surprisingly associated with several forms of cancer [134].

overexpressing the H3K9me3 favoring histone demethlyase JMJD2D or by silencing SUZ12, a polycomb protein involved in H3K9 and K27 methylation. Importantly, even without these methylated histone markers RAS induction still led to senescence with the occurrence of DAPI dense SAHF structures. The investigators concluded that SAHF formation is medi‐ ated through spatial rearrangement of pre-existing H3K9me3 and H3K27me3 regions but that these histone marks are not a prerequisite for the SAHF formation process [135]. Clear‐ ly, further studies are needed to fully define the combined roles of histone modifications,

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Consistent with gene silencing and decreased acetylation during senescence is the downregulation of the histone acetylases p300/CBP, which was observed in melanocytes reaching

With regard to the role of chromatin remodelling in senescence, there is also evidence that ATP dependent chromatin remodeling complexes, such as SWI/SNF are required for senes‐ cence onset in roles other than the timely expression of p16INK4a as described in section 5.1.2: The introduction of the ATPase active component of the SWI/SNF complex, BRG1, into various cell lines induced senescent features. This is attributed to the BRG1 ability to interact with pRb and participate in E2F target promopter repression [137]. However development of senescence in response to BRG1 introduction was only convincing in cells that also lack the BRG1 homologue ATPase BRM [138]. Interestingly we found that BRG1 is able to inter‐ act with p16INK4a, and since p16INK4a is required for SAHF formation it might directly be in‐ volved in this process in co-operation with BRG1. However, the absence of BRG1, by specific silencing, does neither prevent p16INK4a induced growth arrest nor senescence associ‐ ated SAHF formation. It should be noted however, that the WMM1175 melanoma cells used in these experiments also express the BRG1 homologue BRM and therefore they still have functional SWI/SNF complexes even in the absence of BRG1. [139]. It still needs to be tested whether BRM also interacts with p16INK4a and whether the BRM-p16INK4a complex is involved in SAHF formation. This idea is in line with a suggested role for BRM in melanocyte senes‐ cence as it was demonstrated that BRM was recruited and required, albeit transiently, by the

Importantly, although the p53-p21 pathway was demonstrated to be capable of inducing a number of senescent features and does so in response to DNA damage, telomere dysfunc‐ tion and oncogene induced senescence [38, 97], E2F promoter silencing via SAHF formation requires an intact p16-pRb pathway [9, 141]. As expected, SAHF formation is prevented by cancer associated p16INK4a mutations [45]. Since p21Waf1 is capable of inhibiting all CDKs it remains unclear why it appears less effective in promoting a secure senescence program in

The current view is that pRb binds HDAC1 and attracts H3K9 and H3K27 methylating com‐ plexes (exemplified by SUV39H1 and EZH2/SUZ12) to promoters of E2F responsive prolif‐ eration genes leading to histone trimethylation and heterochromatin and thereby DAPI dense microscopic structures. Recent work indicates that H3K9 and H3K27, although usual‐ ly associated with SAHF, may not be a strict requirement. Instead the formation of DAPI dense structures is linked to the timing of presenescent replication events [135]. This view

heterochromatin and SAHF in senescence.

pRb-HDAC1 complex during the initiation of SAHF [140].

involving heterochromatin formation (Figure5).

in their finitive lifespan [136].

Lately there is a different view about the importance of SAHF emerging and SAHF forma‐ tion, may according to a recent study only be of major relevance in the oncogene induced senescence process that crucially involves DDR and the corresponding ATR signalling, as knock-down of ATR prevents SAHF formation during RAS induced senescence. The same study also found that SAHF resembling structures containing H3K9me3 do independently occur in proliferating oncogene expressing cells, however the investigators data show that these structures are not associated with E2F target promoters, which remain active. The au‐ thors concluded that SAHF structures might not be an essential feature of senescence [40].

More recently a groundbreaking study by the Narita group has combined microscopic, elec‐ tron microscopic and genomic data analysis to shed more light on SAHF formation and structure. Their significant findings are that H3K9me3 and H3K27me3 may in fact not di‐ rectly be involved in SAHF. Although these markers are usually associated with SAHF, the overall methylation status of histone tail lysines does not change within the genomic DNA during senescence. Moreover, the investigators found that SAHF formation was linked to pre-senescent replication timing and specific histone modifications were characteristic for early-, mid- and late replications genes. Significantly though, when they prevented SAHF formation by silencing of either pRb or the high mobility group AT-hook 1 (HMGA-1), which they and others had previously linked to SAHF formation, there was no change to H3K9me3 in proliferating cells [135] and this may explain the above-mentioned data by Mic‐ co et al [40]. Moreover, Chandra et al. reversed H3K9 and H3K27 trimethylation by either overexpressing the H3K9me3 favoring histone demethlyase JMJD2D or by silencing SUZ12, a polycomb protein involved in H3K9 and K27 methylation. Importantly, even without these methylated histone markers RAS induction still led to senescence with the occurrence of DAPI dense SAHF structures. The investigators concluded that SAHF formation is medi‐ ated through spatial rearrangement of pre-existing H3K9me3 and H3K27me3 regions but that these histone marks are not a prerequisite for the SAHF formation process [135]. Clear‐ ly, further studies are needed to fully define the combined roles of histone modifications, heterochromatin and SAHF in senescence.

sponsive promoters [130]. A number of heterochromatin-associated histone modifications were characteristically found associated with SAHF. These include the reversion of histone 3/lysine 9 acetylation (H3K9Ac) and histone 3/lysine 4 trimethylation (H3K4me3) and signif‐ icantly, since it is often used as a marker of senescence, the promotion of histone 3/lysine 9 trimethylation (H3K9me3) and H3K27me3. H3K9 trimethylation is a fundamental step dur‐ ing heterochromatin formation as it attracts HP1 proteins, which are pivotal for heterochro‐ matin assembly [9]. It is important to highlight that senescence does not always lead to a net increase in overall H3K9me3, suggesting that SAHF formation is local and indeed targeted via pRb to E2F promoters [131]. H3K9me3 is thought to be very stable and is able to prevent histone acetylases (HATs) to catalyze histone acetylations. Histone acetylations are com‐ monly associated with "euchromatin", which describes actively transcribed chromatin re‐ gions. Accordingly, SAHF formation is thought to "lock" chromatin regions that encode proliferation genes and thus contributes to the very secure silencing of E2F responsive pro‐ liferation genes, which is not reversible by physiological stimuli [9]. H3K9 trimethylation as‐ sociated with SAHF formation is thought to be catalyzed by either the SUV39H1 methyltransferase, which together with HP1 interacts with pRb during E2F promoter silenc‐ ing in senescence [132] or the RIZ1/PRDM2 H3K9 methyltransferase, which was also shown to co-operate in pRb gene repression and is inactivated in colon, breast and gastric carcino‐ ma [133]. Furthermore, a search for H3K9me3 interacting proteins to identify proteins in‐ volved in the senescence program identified JMJD2C, which is a H3K9 specific demethylase. Thereby it is the direct antagonist of the SUV39H1 H3K9 methyltransferase and its over-ex‐

36 Senescence and Senescence-Related Disorders

pression is not surprisingly associated with several forms of cancer [134].

Lately there is a different view about the importance of SAHF emerging and SAHF forma‐ tion, may according to a recent study only be of major relevance in the oncogene induced senescence process that crucially involves DDR and the corresponding ATR signalling, as knock-down of ATR prevents SAHF formation during RAS induced senescence. The same study also found that SAHF resembling structures containing H3K9me3 do independently occur in proliferating oncogene expressing cells, however the investigators data show that these structures are not associated with E2F target promoters, which remain active. The au‐ thors concluded that SAHF structures might not be an essential feature of senescence [40].

More recently a groundbreaking study by the Narita group has combined microscopic, elec‐ tron microscopic and genomic data analysis to shed more light on SAHF formation and structure. Their significant findings are that H3K9me3 and H3K27me3 may in fact not di‐ rectly be involved in SAHF. Although these markers are usually associated with SAHF, the overall methylation status of histone tail lysines does not change within the genomic DNA during senescence. Moreover, the investigators found that SAHF formation was linked to pre-senescent replication timing and specific histone modifications were characteristic for early-, mid- and late replications genes. Significantly though, when they prevented SAHF formation by silencing of either pRb or the high mobility group AT-hook 1 (HMGA-1), which they and others had previously linked to SAHF formation, there was no change to H3K9me3 in proliferating cells [135] and this may explain the above-mentioned data by Mic‐ co et al [40]. Moreover, Chandra et al. reversed H3K9 and H3K27 trimethylation by either

Consistent with gene silencing and decreased acetylation during senescence is the downregulation of the histone acetylases p300/CBP, which was observed in melanocytes reaching in their finitive lifespan [136].

With regard to the role of chromatin remodelling in senescence, there is also evidence that ATP dependent chromatin remodeling complexes, such as SWI/SNF are required for senes‐ cence onset in roles other than the timely expression of p16INK4a as described in section 5.1.2: The introduction of the ATPase active component of the SWI/SNF complex, BRG1, into various cell lines induced senescent features. This is attributed to the BRG1 ability to interact with pRb and participate in E2F target promopter repression [137]. However development of senescence in response to BRG1 introduction was only convincing in cells that also lack the BRG1 homologue ATPase BRM [138]. Interestingly we found that BRG1 is able to inter‐ act with p16INK4a, and since p16INK4a is required for SAHF formation it might directly be in‐ volved in this process in co-operation with BRG1. However, the absence of BRG1, by specific silencing, does neither prevent p16INK4a induced growth arrest nor senescence associ‐ ated SAHF formation. It should be noted however, that the WMM1175 melanoma cells used in these experiments also express the BRG1 homologue BRM and therefore they still have functional SWI/SNF complexes even in the absence of BRG1. [139]. It still needs to be tested whether BRM also interacts with p16INK4a and whether the BRM-p16INK4a complex is involved in SAHF formation. This idea is in line with a suggested role for BRM in melanocyte senes‐ cence as it was demonstrated that BRM was recruited and required, albeit transiently, by the pRb-HDAC1 complex during the initiation of SAHF [140].

Importantly, although the p53-p21 pathway was demonstrated to be capable of inducing a number of senescent features and does so in response to DNA damage, telomere dysfunc‐ tion and oncogene induced senescence [38, 97], E2F promoter silencing via SAHF formation requires an intact p16-pRb pathway [9, 141]. As expected, SAHF formation is prevented by cancer associated p16INK4a mutations [45]. Since p21Waf1 is capable of inhibiting all CDKs it remains unclear why it appears less effective in promoting a secure senescence program in involving heterochromatin formation (Figure5).

The current view is that pRb binds HDAC1 and attracts H3K9 and H3K27 methylating com‐ plexes (exemplified by SUV39H1 and EZH2/SUZ12) to promoters of E2F responsive prolif‐ eration genes leading to histone trimethylation and heterochromatin and thereby DAPI dense microscopic structures. Recent work indicates that H3K9 and H3K27, although usual‐ ly associated with SAHF, may not be a strict requirement. Instead the formation of DAPI dense structures is linked to the timing of presenescent replication events [135]. This view would explain the fact that SWI/SNF including BRM has been associated in a transient man‐ ner with the build up of SAHF structures; BRM can also interact with pRb but strictly is as‐ sociated with acetylated histones and replicating chromatin is acetylated [140]. The complete understanding of SAHF formation is still an evolving field. Regardless, it is widely accepted that functional p16INK4a and pRb as well as the HMGA, which accumulate at E2F target pro‐ moters during senescence, are critically required for SAHF arrangement. In regards to p16INK4a it is tempting to speculate that its specific role in SAHF formation goes beyond CDK inhibition and thus maintaining pRb in an active state.

**Author details**

Therese Becker1

burg, Germany

**References**

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and Sebastian Haferkamp2

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1 Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium

Molecular Mechanisms of Cellular Senescence

http://dx.doi.org/10.5772/54120

39

2 Department of Dermatology, Venereology und Allergology, University Hospital Würz‐

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**Figure 5.** Chromatin remodeling and *SAHF formation*

#### **8. Conclusion**

Senescence is a potent means of tumor suppression. The mechanisms of senescence in‐ volve cell cycle regulatory protein functions in concert with the chromatin remodeling machinery to maintain a complex and secure withdrawal from proliferation. The under‐ standing of these mechanisms is still evolving and is predicted to identify novel targets for cancer therapy.

#### **Acknowledgements**

We thank Dr Kavitha Gowrishankar for her assistance with the illustrations and Dr Roland Houben for the critical review of the manuscript.

#### **Author details**

would explain the fact that SWI/SNF including BRM has been associated in a transient man‐ ner with the build up of SAHF structures; BRM can also interact with pRb but strictly is as‐ sociated with acetylated histones and replicating chromatin is acetylated [140]. The complete understanding of SAHF formation is still an evolving field. Regardless, it is widely accepted that functional p16INK4a and pRb as well as the HMGA, which accumulate at E2F target pro‐ moters during senescence, are critically required for SAHF arrangement. In regards to p16INK4a it is tempting to speculate that its specific role in SAHF formation goes beyond CDK

Senescence is a potent means of tumor suppression. The mechanisms of senescence in‐ volve cell cycle regulatory protein functions in concert with the chromatin remodeling machinery to maintain a complex and secure withdrawal from proliferation. The under‐ standing of these mechanisms is still evolving and is predicted to identify novel targets

We thank Dr Kavitha Gowrishankar for her assistance with the illustrations and Dr Roland

inhibition and thus maintaining pRb in an active state.

38 Senescence and Senescence-Related Disorders

**Figure 5.** Chromatin remodeling and *SAHF formation*

**8. Conclusion**

for cancer therapy.

**Acknowledgements**

Houben for the critical review of the manuscript.

Therese Becker1 and Sebastian Haferkamp2

1 Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Westmead Hospital, Westmead, New South Wales, Australia

2 Department of Dermatology, Venereology und Allergology, University Hospital Würz‐ burg, Germany

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**Section 3**

**Plant Senescence**

**Section 3**

**Plant Senescence**

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50 Senescence and Senescence-Related Disorders

duced senescence does not require the p16(INK4a) or p14ARF melanoma tumor sup‐

**Chapter 3**

**Plant Senescence and Nitrogen**

Additional information is available at the end of the chapter

Very early during their reproductive phase, annual plants initiate the process of senescence. Monocarpic senescence describes the last steps in these plants' development; senescence on organ level starts shortly after entering reproductive phase while after anthesis the whole

In the following we will focus on leaf senescence. Two different processes can be distin‐ guished in annual plants relying on different genetic programs. Before anthesis, sequential leaf senescence recycles nutrients from old to developing leaves which is mainly under the control of the growing apex and is arrested when no more new leaves develop and when the plant starts to flower and sets fruit. Monocarpic leaf senescence recovers valuable nutrients from the leaves during flower induction and anthesis to provide these to the developing re‐ productive organs [1, 2]. The latter is crucial for fruit and seed development and has a major impact on yield quantity and quality. In wheat salvaged nitrogen (N) from the leaves ac‐ counts for up to 90% of the total grain N content [3]. A complex regulation of many different metabolic pathways and expression of numerous genes underlies this process. How coordi‐ nation and interplay of many controlling factors, like hormones, genetic reprogramming, bi‐ otic and abiotic stresses are achieved is far from being understood, but it is already clear that

Thousands of genes are differentially regulated during senescence induction and progres‐ sion. To date forward and reverse genetic approaches as well as large-scale transcript profil‐ ing have identified almost 6.500 genes being differentially expressed during the course of leaf senescence including up-regulated as well as down-regulated genes [4]. The high num‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Bieker and Zentgraf; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Mobilization and Signaling**

Stefan Bieker and Ulrike Zentgraf

http://dx.doi.org/10.5772/54392

plant undergoes senescence and dies.

this regulatory network is highly complex and dynamic.

**1. Introduction**

**1.1. Senescence**

**Chapter 3**

## **Plant Senescence and Nitrogen Mobilization and Signaling**

Stefan Bieker and Ulrike Zentgraf

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54392

### **1. Introduction**

#### **1.1. Senescence**

Very early during their reproductive phase, annual plants initiate the process of senescence. Monocarpic senescence describes the last steps in these plants' development; senescence on organ level starts shortly after entering reproductive phase while after anthesis the whole plant undergoes senescence and dies.

In the following we will focus on leaf senescence. Two different processes can be distin‐ guished in annual plants relying on different genetic programs. Before anthesis, sequential leaf senescence recycles nutrients from old to developing leaves which is mainly under the control of the growing apex and is arrested when no more new leaves develop and when the plant starts to flower and sets fruit. Monocarpic leaf senescence recovers valuable nutrients from the leaves during flower induction and anthesis to provide these to the developing re‐ productive organs [1, 2]. The latter is crucial for fruit and seed development and has a major impact on yield quantity and quality. In wheat salvaged nitrogen (N) from the leaves ac‐ counts for up to 90% of the total grain N content [3]. A complex regulation of many different metabolic pathways and expression of numerous genes underlies this process. How coordi‐ nation and interplay of many controlling factors, like hormones, genetic reprogramming, bi‐ otic and abiotic stresses are achieved is far from being understood, but it is already clear that this regulatory network is highly complex and dynamic.

Thousands of genes are differentially regulated during senescence induction and progres‐ sion. To date forward and reverse genetic approaches as well as large-scale transcript profil‐ ing have identified almost 6.500 genes being differentially expressed during the course of leaf senescence including up-regulated as well as down-regulated genes [4]. The high num‐

© 2013 Bieker and Zentgraf; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ber of differentially regulated senescence-associated transcription factors (TF) demonstrates the dimensions of genetic reprogramming taking place. These TFs include 20 distinct fami‐ lies of which NAC-, WRKY-, C2H2-type zinc finger, EREBP- and MYB-families are most abundant [5]. Recently, Breeze et al. (2011) [4] published extremely important results of a high-resolution temporal transcript profiling of senescing Arabidopsis leaves giving insight into the temporal order of genetic events. One of the first steps at the onset of senescence is a shift from anabolic to catabolic processes. Amino acid metabolism and protein synthesis are down-regulated while expression of autophagy- and reactive oxygen species-related, and water-response genes is enhanced. In contrast to the following elevation of abscisic acid (ABA) and jasmonic acid (JA) signaling-related gene expression, cytokinin-mediated signal‐ ing is lowered just as chlorophyll and carotenoid biosynthesis. The next phases include down-regulation of carbon utilization and enhanced expression of cystein-aspartat proteas‐ es, carotene metabolism-associated genes and pectinesterases which is then followed by the reduction of photosynthetic activity and degradation of the photosynthetic apparatus coin‐ ciding with the increased activity of lipid catabolism, ethylene signaling and higher abun‐ dance of cytoskeletal elements [4].

are applied annually worldwide [13-15]. However, 50-70% of these nitrogenous fertilizer are lost to the environment [16], mostly due to volatilization of N2O, NO, N2 and NH3 and leach‐

trients to provide, but it also has a strong detrimental impact on the environment. Since surrounding ecosystems and potable water supply are endangered by oversaturation with nitrogenous compounds, it is necessary to improve application techniques and plant's nitro‐

Several different definitions of nitrogen and nutrient use efficiencies are on hand. The most common is the *nitrogen use efficiency* (NUE), which is defined as shoot dry weight divided by total nitrogen content of the shoots. The *usage index* (UI) takes absolute biomass into account as it is denoted by the shoot fresh weight times the NUE. Likewise, the *N uptake efficiency* (NUpE) takes into account the whole N content of the plant and the N supplied by fertiliza‐ tion per plant. The fraction of the N taken up, which is then distributed to the grain, can be obtained by calculating the *N utilization efficiency* (NUtE) (Grain weight per total N content). Other efficiencies, which seem to be more suitable for the use in applied sciences, are the *agronomic efficiency* (AE), *apparent nitrogen recovery* (AR) and the *physiological efficiency* (PE). AE, AR and PE do require an unfertilized control to be calculated. While AE measures the efficiency to redirect applied nitrogen to the grains, AR defines the efficiency to capture N from the soil. PE puts the N uptake into relationship with the outcome of grain (reviewed in [15]). *Nitrogen remobilization efficiency* (NRE) describes the plant's capacity to translocate al‐ ready assimilated nitrogen to developing organs. Finally, the *harvest index* (HI) and the *nitro‐ gen harvest index* (NHI) are often used terms. HI is the total yield weight per plant mass,

Emission of nitrogen to the environment could be strongly reduced by application of 'best management techniques' in agricultural practice like e.g. rectifying the rate of appliance by accounting for all other possible sources of nitrogen influx (carryover from previous crops, atmospheric deposits etc.), ameliorating the timing and also changing the method of appli‐ ance to reduce atmospheric losses [13]. Food production has doubled in the last 40 years. Most of this increase could be achieved by selection of new strains, breeding and application of greater amounts of fertilizer and pesticides and other techniques [12]. Amending of nu‐ trient use efficiencies of the crop plants was mostly accomplished via breeding programs by now. QTL selection for higher yields, increased oil or protein content has been pursued for decades. In wheat for example, increasing yield and grain protein content has been exten‐ sively studied, but improving both is restrained by the negative genetic relationship be‐

Nitrogen sources vary extremely encompassing organic and inorganic forms, small peptides and single amino acids, thus uptake systems need to be adjusted and well regulated in spa‐ tial and temporal activity. Although the predominant form in which N is taken up mainly

while NHI states the grain N content per whole plant N content.

**2. Nitrogen uptake, assimilation and distribution**

into the water. Thus nitrogen is not only one of the most expensive nu‐

Plant Senescence and Nitrogen Mobilization and Signaling

http://dx.doi.org/10.5772/54392

55

ing of soluble NO3

gen use efficiencies.

tween these traits [17, 18].


Hormonal control of senescence is conveyed especially by ethylene, jasmonic and salicylic acid, cytokinin and auxin. Many mutants with a delayed senescence phenotype could be traced back to impaired or up-regulated ethylene or cytokinin signaling, respectively [6, 7]. In adition, ABA acts as a positive regulator of leaf senescence. Recently a membrane-bound, leucine rich repeat containing receptor kinase (RPK1) has been identified to play an impor‐ tant role in ABA-mediated senescence induction in an age-dependent manner. Strikingly, *rpk1* mutant lines did not show significant alterations in developmental processes, which have been reported for numerous other ABA signaling defective mutant lines [5], except slightly shorter growth [8]. This kinase has been indentified to integrate ABA signals during seed germination, plant growth, stomatal closure and stress responses. Overexpression lines showed enhanced expression of several stress and H2O2-responsive genes [9, 10]. Mutant lines showed a delayed senescence phenotype with slower progression of chlorophyll deg‐ radation and cell death.

Induction and progression of leaf senescence demands a tight regulation of numerous proc‐ esses. Integration of nutritional cues, biotic and abiotic influences, plant development and age has to take place for the correct timing of onset and temporal advance of this complex developmental process. Despite the enormous efforts and achievements in this field, many of the regulatory mechanisms remain elusive.

#### **1.2. Nitrogen and agriculture**

The nowadays growth of population and thus increasing demand for food and oil crops forces agricultural industry to increase quantitative as well as qualitative yields. Until 2050 world's population is predicted to be as high as 9-10 billion people [11] and grain require‐ ment is projected to be doubled, mostly resulting from a higher demand for wheat fed meat [12]. As most of the cropping systems are naturally deficient in nitrogen, there is a funda‐ mental dependency on inorganic nitrogen fertilizers. 85-90 million tons of these fertilizers are applied annually worldwide [13-15]. However, 50-70% of these nitrogenous fertilizer are lost to the environment [16], mostly due to volatilization of N2O, NO, N2 and NH3 and leach‐ ing of soluble NO3 into the water. Thus nitrogen is not only one of the most expensive nu‐ trients to provide, but it also has a strong detrimental impact on the environment. Since surrounding ecosystems and potable water supply are endangered by oversaturation with nitrogenous compounds, it is necessary to improve application techniques and plant's nitro‐ gen use efficiencies.

ber of differentially regulated senescence-associated transcription factors (TF) demonstrates the dimensions of genetic reprogramming taking place. These TFs include 20 distinct fami‐ lies of which NAC-, WRKY-, C2H2-type zinc finger, EREBP- and MYB-families are most abundant [5]. Recently, Breeze et al. (2011) [4] published extremely important results of a high-resolution temporal transcript profiling of senescing Arabidopsis leaves giving insight into the temporal order of genetic events. One of the first steps at the onset of senescence is a shift from anabolic to catabolic processes. Amino acid metabolism and protein synthesis are down-regulated while expression of autophagy- and reactive oxygen species-related, and water-response genes is enhanced. In contrast to the following elevation of abscisic acid (ABA) and jasmonic acid (JA) signaling-related gene expression, cytokinin-mediated signal‐ ing is lowered just as chlorophyll and carotenoid biosynthesis. The next phases include down-regulation of carbon utilization and enhanced expression of cystein-aspartat proteas‐ es, carotene metabolism-associated genes and pectinesterases which is then followed by the reduction of photosynthetic activity and degradation of the photosynthetic apparatus coin‐ ciding with the increased activity of lipid catabolism, ethylene signaling and higher abun‐

Hormonal control of senescence is conveyed especially by ethylene, jasmonic and salicylic acid, cytokinin and auxin. Many mutants with a delayed senescence phenotype could be traced back to impaired or up-regulated ethylene or cytokinin signaling, respectively [6, 7]. In adition, ABA acts as a positive regulator of leaf senescence. Recently a membrane-bound, leucine rich repeat containing receptor kinase (RPK1) has been identified to play an impor‐ tant role in ABA-mediated senescence induction in an age-dependent manner. Strikingly, *rpk1* mutant lines did not show significant alterations in developmental processes, which have been reported for numerous other ABA signaling defective mutant lines [5], except slightly shorter growth [8]. This kinase has been indentified to integrate ABA signals during seed germination, plant growth, stomatal closure and stress responses. Overexpression lines showed enhanced expression of several stress and H2O2-responsive genes [9, 10]. Mutant lines showed a delayed senescence phenotype with slower progression of chlorophyll deg‐

Induction and progression of leaf senescence demands a tight regulation of numerous proc‐ esses. Integration of nutritional cues, biotic and abiotic influences, plant development and age has to take place for the correct timing of onset and temporal advance of this complex developmental process. Despite the enormous efforts and achievements in this field, many

The nowadays growth of population and thus increasing demand for food and oil crops forces agricultural industry to increase quantitative as well as qualitative yields. Until 2050 world's population is predicted to be as high as 9-10 billion people [11] and grain require‐ ment is projected to be doubled, mostly resulting from a higher demand for wheat fed meat [12]. As most of the cropping systems are naturally deficient in nitrogen, there is a funda‐ mental dependency on inorganic nitrogen fertilizers. 85-90 million tons of these fertilizers

dance of cytoskeletal elements [4].

54 Senescence and Senescence-Related Disorders

radation and cell death.

**1.2. Nitrogen and agriculture**

of the regulatory mechanisms remain elusive.

Several different definitions of nitrogen and nutrient use efficiencies are on hand. The most common is the *nitrogen use efficiency* (NUE), which is defined as shoot dry weight divided by total nitrogen content of the shoots. The *usage index* (UI) takes absolute biomass into account as it is denoted by the shoot fresh weight times the NUE. Likewise, the *N uptake efficiency* (NUpE) takes into account the whole N content of the plant and the N supplied by fertiliza‐ tion per plant. The fraction of the N taken up, which is then distributed to the grain, can be obtained by calculating the *N utilization efficiency* (NUtE) (Grain weight per total N content). Other efficiencies, which seem to be more suitable for the use in applied sciences, are the *agronomic efficiency* (AE), *apparent nitrogen recovery* (AR) and the *physiological efficiency* (PE). AE, AR and PE do require an unfertilized control to be calculated. While AE measures the efficiency to redirect applied nitrogen to the grains, AR defines the efficiency to capture N from the soil. PE puts the N uptake into relationship with the outcome of grain (reviewed in [15]). *Nitrogen remobilization efficiency* (NRE) describes the plant's capacity to translocate al‐ ready assimilated nitrogen to developing organs. Finally, the *harvest index* (HI) and the *nitro‐ gen harvest index* (NHI) are often used terms. HI is the total yield weight per plant mass, while NHI states the grain N content per whole plant N content.

Emission of nitrogen to the environment could be strongly reduced by application of 'best management techniques' in agricultural practice like e.g. rectifying the rate of appliance by accounting for all other possible sources of nitrogen influx (carryover from previous crops, atmospheric deposits etc.), ameliorating the timing and also changing the method of appli‐ ance to reduce atmospheric losses [13]. Food production has doubled in the last 40 years. Most of this increase could be achieved by selection of new strains, breeding and application of greater amounts of fertilizer and pesticides and other techniques [12]. Amending of nu‐ trient use efficiencies of the crop plants was mostly accomplished via breeding programs by now. QTL selection for higher yields, increased oil or protein content has been pursued for decades. In wheat for example, increasing yield and grain protein content has been exten‐ sively studied, but improving both is restrained by the negative genetic relationship be‐ tween these traits [17, 18].

#### **2. Nitrogen uptake, assimilation and distribution**

Nitrogen sources vary extremely encompassing organic and inorganic forms, small peptides and single amino acids, thus uptake systems need to be adjusted and well regulated in spa‐ tial and temporal activity. Although the predominant form in which N is taken up mainly depends on the plants adaption to the given environment and influences like fertilization, soil pH, temperature, precipitation and others [14, 19], most plants cover their N demand primarily through soil nitrate being provided by fertilization, bacterial nitrification and oth‐ er processes [15]. However, a wide range of different uptake system has evolved in plants. For example, oligopeptides can be taken up via OPT-proteins (oligopeptide transporters), ammonium via ammonium transporters (AMTs) and amino acids via amino acid transport‐ ers and amino acid permeases. Besides the *AtCLC* (ChLoride Channel) gene family, compris‐ ing 7 members of which two (*AtCLCa* and *AtCLCb*) have been shown to encode tonoplast located NO3 - /H+ antiporters [20, 21], two families of nitrate transporters have been identified in higher plants (NRT1 and NRT2), representing low- and high affinity transporter systems, respectively. Moreover the *NRT1*-family has been shown to comprise also di-/tripeptide transporters (PTR) [22].

low. Additionally, *AtNRT1-4* is predominantly expressed in the leaf petiole and the *atnrt1.4* mutant shows a nitrate content decreased by half in the petiole [22, 31]. *AtNRT1-6* is ex‐ pressed in the silique's and funiculus' vascular tissue and thought to play a nitrate provid‐ ing role in early embryonic development [32]. AtNRT1-8 functions in nitrate unloading from the xylem sap and is mainly located in xylem parenchyma cells within the vasculature [33], whereas AtNRT1-9 facilitates nitrate loading into the root phloem from root phloem com‐

High affinity nitrate uptake is conducted by members of the *NRT2*-family, comprising 7 genes in Arabidopsis. AtNRT2-1 has been shown to be one of the main components of the HATs. Mutant *atnrt2-1* plants displayed a loss of nitrate uptake capacity up to 75% at HAT-

nitrate combined with high sucrose supply, where NRT2-1 acts either as sensor or transduc‐ er [36]. Experiments with *Xenopus* oocytes revealed the requirement of a AtNAR2 protein for AtNRT2-1 function [37]. Mutants of either of these two components showed impaired ni‐ trate uptake at HAT-specific concentrations and hampered growth with display of N-starva‐ tion symptoms, in which, remarkably, the *atnar2* mutant phenotype appeared to be more pronounced [38]. The phenotype of the *atnrt2-7* mutant is similar to the phenotype of the

cumulation of nitrate in the vacuole. Mutation of either of these resulted in lower nitrate content. Ectopic overexpression of *AtNRT2-7* led to higher nitrate contents in dried seeds, where the gene is highly expressed under wild type conditions, and an increase in the ni‐ trate HATs uptake capacity by 2-fold. However, normal development was not impaired in the mutants as well as overexpressor plants [14, 20, 39]. Despite its high homology to *AtNRT2-1, AtNRT2-4* is not dependent on the function of *AtNAR2*. *AtNRT2-4* expression is highly induced upon nitrogen starvation in the outermost layer of young lateral roots [40].

Members of the *AMT1*- and *AMT2*-subfamilies are thought to be the main high affinity am‐ monium transporters in plants. Due to ammonium's toxic nature and convertibility from

(AA). The most abundant transport forms are glutamine, glutamate, asparagine and as‐

lesser extend [46]. Nitrate assimilation thus requires reduction to ammonium. Nitrate re‐

the cytoplasm, while the reduction of nitrite to ammonium is carried out in the plastids.

 to NH4


and NH4

 to NO2 -

activity is regulated posttranscriptionally via the availability of nitrate [42].


ductase (NR) realizes the first step by reducing NO3

partate [45] although direct transport of NO3

Here, nitrite reductase (NiR) converts NO2

 to NH3 and the thus varying membrane permeability, its uptake and transport needs to be tightly regulated [41, 42]. AMTs are regulated transcriptionally by N-supply, sugar and daytime and provide an additive contribution to ammonium transport [41]. AtAMT1-1 contributes 30-35 % as does AtAMT1-3, while AtAMT1-2 provides only 18-25% [43, 44]. AtAMT1-1 transports ammonium as well as its analog methyl-ammonium. Additionally, its

*atclca* mutant. The *AtCLCa* gene has been shown to encode a NO3

concentrations [35]. Furthermore, lateral root growth is repressed under low


Plant Senescence and Nitrogen Mobilization and Signaling

http://dx.doi.org/10.5772/54392

57

+ almost always includes incorporation into amino acids

+ also takes place but to a much

<sup>+</sup> making it readily available for the in‐

. This reaction takes place in

/H+ antiporter enabling ac‐

panion cells [34].

specific NO3

NH4 +

**2.2. Nitrogen assimilation**

Assimilation of NO3


#### **2.1. Nitrogen transporter systems**

Four constituents of nitrate uptake are known, constitutive (c) and inducible (i) high- (HAT) and low-affinity (LAT) transporters, respectively. The high-affinity system's KM ranges from ~ 5-100 µM, varying with plant species, and a maximal influx via this system of 1 µMol \* g-1 \* h-1 has been determined [23, 24]. At nitrate concentrations of 10 mM the influx rate via the LATs can reach up to ~24 µMol \* g-1 \* h-1 [24].

The *NRT1*-family comprises 53 genes in Arabidopsis which are classified as LATs. *AtNRT1-1* (*CHL1*) was the first member to be identified in 1993 and has been shown to en‐ code a proton-coupled nitrate transporter [25]. Studies with *Xenopus* oocytes have shown that this transporter protein possesses two different states, one serving low-affinity and the other one high-affinity nitrate uptake [26, 27], thus the distinction between high and lowaffinity nitrate transporters is overridden in this case. Switching between these two modes of action is conferred by phosphorylation of threonin at position 101 [28]. *AtNRT1-1* is ex‐ pressed in the cortex and endodermis of mature roots and in the epidermis of root tips. Ad‐ ditionally, a nitrate sensing function regulating the plants primary nitrate response has been strongly indicated for the AtNRT1-1 protein by several lines of evidence. The *chl1-5 (atnrt1-1-5)* mutant, a deletion mutant with no detectable *CHL1* transcript, is deficient in ni‐ trate uptake and initiation of the primary nitrate response. The *chl1-9* mutant is defective in nitrate uptake but not in the primary nitrate response. The *chl1-*9 mutant has a point muta‐ tion between two transmembrane domains. When threonine 101 was mutated to mimic or repress phosphorylation and transformed into the *chl1-5* background, primary nitrate re‐ sponse could be repressed or enhanced [29]. Constitutive expression identifies AtNRT1-2 as part of the Arabidopsis cLATs. Its transcript is only found in root epidermal cells [22]. Ex‐ pression of *AtNRT1-5* is nitrate inducible; however, the response to nitrate is much slower than for *AtNRT1-1*. AtNRT1-5 has been shown to be a pH-dependent, bidirectional nitrate transporter, with subcellular localization in the plasma membrane of root pericycle cells near the xylem implicating an involvement in long-distance nitrate transport [30]. Experi‐ mental evidence suggests nitrate storage in leaf petioles to be associated with the function of AtNRT1-4. Here, nitrate content is relatively high, while nitrate reductase (NR) activity is low. Additionally, *AtNRT1-4* is predominantly expressed in the leaf petiole and the *atnrt1.4* mutant shows a nitrate content decreased by half in the petiole [22, 31]. *AtNRT1-6* is ex‐ pressed in the silique's and funiculus' vascular tissue and thought to play a nitrate provid‐ ing role in early embryonic development [32]. AtNRT1-8 functions in nitrate unloading from the xylem sap and is mainly located in xylem parenchyma cells within the vasculature [33], whereas AtNRT1-9 facilitates nitrate loading into the root phloem from root phloem com‐ panion cells [34].

High affinity nitrate uptake is conducted by members of the *NRT2*-family, comprising 7 genes in Arabidopsis. AtNRT2-1 has been shown to be one of the main components of the HATs. Mutant *atnrt2-1* plants displayed a loss of nitrate uptake capacity up to 75% at HATspecific NO3 concentrations [35]. Furthermore, lateral root growth is repressed under low nitrate combined with high sucrose supply, where NRT2-1 acts either as sensor or transduc‐ er [36]. Experiments with *Xenopus* oocytes revealed the requirement of a AtNAR2 protein for AtNRT2-1 function [37]. Mutants of either of these two components showed impaired ni‐ trate uptake at HAT-specific concentrations and hampered growth with display of N-starva‐ tion symptoms, in which, remarkably, the *atnar2* mutant phenotype appeared to be more pronounced [38]. The phenotype of the *atnrt2-7* mutant is similar to the phenotype of the *atclca* mutant. The *AtCLCa* gene has been shown to encode a NO3 - /H+ antiporter enabling ac‐ cumulation of nitrate in the vacuole. Mutation of either of these resulted in lower nitrate content. Ectopic overexpression of *AtNRT2-7* led to higher nitrate contents in dried seeds, where the gene is highly expressed under wild type conditions, and an increase in the ni‐ trate HATs uptake capacity by 2-fold. However, normal development was not impaired in the mutants as well as overexpressor plants [14, 20, 39]. Despite its high homology to *AtNRT2-1, AtNRT2-4* is not dependent on the function of *AtNAR2*. *AtNRT2-4* expression is highly induced upon nitrogen starvation in the outermost layer of young lateral roots [40].

Members of the *AMT1*- and *AMT2*-subfamilies are thought to be the main high affinity am‐ monium transporters in plants. Due to ammonium's toxic nature and convertibility from NH4 + to NH3 and the thus varying membrane permeability, its uptake and transport needs to be tightly regulated [41, 42]. AMTs are regulated transcriptionally by N-supply, sugar and daytime and provide an additive contribution to ammonium transport [41]. AtAMT1-1 contributes 30-35 % as does AtAMT1-3, while AtAMT1-2 provides only 18-25% [43, 44]. AtAMT1-1 transports ammonium as well as its analog methyl-ammonium. Additionally, its activity is regulated posttranscriptionally via the availability of nitrate [42].

#### **2.2. Nitrogen assimilation**

depends on the plants adaption to the given environment and influences like fertilization, soil pH, temperature, precipitation and others [14, 19], most plants cover their N demand primarily through soil nitrate being provided by fertilization, bacterial nitrification and oth‐ er processes [15]. However, a wide range of different uptake system has evolved in plants. For example, oligopeptides can be taken up via OPT-proteins (oligopeptide transporters), ammonium via ammonium transporters (AMTs) and amino acids via amino acid transport‐ ers and amino acid permeases. Besides the *AtCLC* (ChLoride Channel) gene family, compris‐ ing 7 members of which two (*AtCLCa* and *AtCLCb*) have been shown to encode tonoplast

in higher plants (NRT1 and NRT2), representing low- and high affinity transporter systems, respectively. Moreover the *NRT1*-family has been shown to comprise also di-/tripeptide

Four constituents of nitrate uptake are known, constitutive (c) and inducible (i) high- (HAT) and low-affinity (LAT) transporters, respectively. The high-affinity system's KM ranges from ~ 5-100 µM, varying with plant species, and a maximal influx via this system of 1 µMol \* g-1 \* h-1 has been determined [23, 24]. At nitrate concentrations of 10 mM the influx rate via the

The *NRT1*-family comprises 53 genes in Arabidopsis which are classified as LATs. *AtNRT1-1* (*CHL1*) was the first member to be identified in 1993 and has been shown to en‐ code a proton-coupled nitrate transporter [25]. Studies with *Xenopus* oocytes have shown that this transporter protein possesses two different states, one serving low-affinity and the other one high-affinity nitrate uptake [26, 27], thus the distinction between high and lowaffinity nitrate transporters is overridden in this case. Switching between these two modes of action is conferred by phosphorylation of threonin at position 101 [28]. *AtNRT1-1* is ex‐ pressed in the cortex and endodermis of mature roots and in the epidermis of root tips. Ad‐ ditionally, a nitrate sensing function regulating the plants primary nitrate response has been strongly indicated for the AtNRT1-1 protein by several lines of evidence. The *chl1-5 (atnrt1-1-5)* mutant, a deletion mutant with no detectable *CHL1* transcript, is deficient in ni‐ trate uptake and initiation of the primary nitrate response. The *chl1-9* mutant is defective in nitrate uptake but not in the primary nitrate response. The *chl1-*9 mutant has a point muta‐ tion between two transmembrane domains. When threonine 101 was mutated to mimic or repress phosphorylation and transformed into the *chl1-5* background, primary nitrate re‐ sponse could be repressed or enhanced [29]. Constitutive expression identifies AtNRT1-2 as part of the Arabidopsis cLATs. Its transcript is only found in root epidermal cells [22]. Ex‐ pression of *AtNRT1-5* is nitrate inducible; however, the response to nitrate is much slower than for *AtNRT1-1*. AtNRT1-5 has been shown to be a pH-dependent, bidirectional nitrate transporter, with subcellular localization in the plasma membrane of root pericycle cells near the xylem implicating an involvement in long-distance nitrate transport [30]. Experi‐ mental evidence suggests nitrate storage in leaf petioles to be associated with the function of AtNRT1-4. Here, nitrate content is relatively high, while nitrate reductase (NR) activity is

antiporters [20, 21], two families of nitrate transporters have been identified

located NO3


56 Senescence and Senescence-Related Disorders

transporters (PTR) [22].

**2.1. Nitrogen transporter systems**

LATs can reach up to ~24 µMol \* g-1 \* h-1 [24].

Assimilation of NO3 - and NH4 + almost always includes incorporation into amino acids (AA). The most abundant transport forms are glutamine, glutamate, asparagine and as‐ partate [45] although direct transport of NO3 and NH4 + also takes place but to a much lesser extend [46]. Nitrate assimilation thus requires reduction to ammonium. Nitrate re‐ ductase (NR) realizes the first step by reducing NO3 to NO2 - . This reaction takes place in the cytoplasm, while the reduction of nitrite to ammonium is carried out in the plastids. Here, nitrite reductase (NiR) converts NO2 to NH4 <sup>+</sup> making it readily available for the in‐ corporation into AAs in a NADH-dependent manner. Assimilation of ammonium into AAs involves chloroplastic glutamine synthetase 2 (GS2) and glutamate synthase (Fd-GO‐ GAT), which generates glutamine and glutamate (for detailed review see [14]). Glutamine as well as glutamate serve as ammonium donor for the synthesis of all other amino acids including aspartate and asparagine, which in turn function as active NH4 + donor or as long-range nitrogen transport and storage form, respectively [47]. Alternatively carba‐ moylphosphate synthase can be involved in ammonium assimilation by producing carba‐ moylphosphate and successively citrulline and arginine. Assimilation in non-green tissues is achieved in plastids in a similar manner, although here GOGAT depends on NADH in‐ stead of ferredoxin. Carbon skeletons are essential for the acquisition of inorganic nitro‐ gen in AAs. Especially the demand for keto-acids has to be met (see [14] and references within). These are predominantly obtained from the TCA-Cycle in the form of 2-oxogluta‐ rate (2OG) [47, 48]. 2OG is used for incorporation of photorespiratory ammonium, result‐ ing in the production of glutamate, which in turn can be utilized by GS1 and GS2 to produce glutamine. This displays the intricate interconnection between carbon and nitro‐ gen metabolism, in which N uptake and assimilation is also influenced via photosynthet‐ ic rates [47]. Besides direct assimilation into AAs, nitrate can also be stored in the vacuole and in the chloroplast. Vacuolar nitrate concentrations can vary enormously, as vacuolar nitrate also contributes to turgor maintenance and might have a nitrate storage function to maintain the cytosolic nitrate concentrations which are more constant [49].

ply showed a stronger decline of photosynthetic activity and more pronounced senescence symptoms than plants sufficiently supplied with nitrogen [51]. Furthermore these plants showed a more pronounced and earlier drop in (Glu+Asp)/(Gln+Asn) ratio during the pro‐ gression of senescence indicating an additional adaption to low nitrogen conditions through enhanced nitrogen remobilization. In this experiment, also a significant increase in the ratio of hexose to sucrose was observed at the beginning of senescence which was higher in Nstarved plants. This indicates that sugar-related senescence induction is dependent on the availability of nitrogen [51]. However, high sugar contents repress photosynthesis and can induce early SAG expression while late SAG expression is repressed. Diaz et al. (2005) [52] showed sugar accumulation to be lower in some recombinant inbred lines which display early leaf yellowing, thus pointing out a mayor function for sugar accumulation alone but the regulating function of the C/N balance during induction of monocarpic senescence is widely discussed. Recently, trehalose-6-phosphate (T6P) was identified as a main signaling component in this pathway. T6P inhibits the activity of Snf1-related protein kinase (SnRK1). Zhang et al. (2009) [53] showed the T6P/SnRK1 interaction in Arabidopsis seedling extracts and other young tissues treated with T6P. Additionally, Delatte et al. (2011) [54] confirmed the inhibition of SnRK1 by T6P with plants overexpressing the SnRK1 catalytic subunit gene *KIN10.* These plants were insensitive to trehalose treatments. Further verification of T6P as signaling molecule was provided by several studies. In wheat the interaction of T6P and SnRK1 has been suggested to play a role during grain filling [55]. Wingler et al. (2012) [56] conducted a study with *otsA* and *otsB* expressing Arabidopsis plants *otsA* encodes the bacte‐ rial T6P synthase gene, *otsB* the T6P phosphatase gene; therefore, overexpression leads to in‐ creasing or decreasing T6P contents, respectively. A significantly higher accumulation of glucose, fructose and sucrose was observed in *otsB* expressing plants and these plants dis‐ played a delayed senescence phenotype. But most interestingly, these plants were rendered less susceptible to the induction of senescence-associated genes by sugar feeding in combi‐ nation with low nitrogen supply, whereas *otsA* plants induced senescence and anthocyanin

Plant Senescence and Nitrogen Mobilization and Signaling

http://dx.doi.org/10.5772/54392

59

Another signaling component involved in senescence induction is light quantity and quali‐ ty. Senescence can be induced by the darkening of individual leaves. However, darkening of the whole plant resulted in delay rather than in induction of leaf senescence in Arabidopsis and sunflower plants [57, 58]. Brouwer et al. (2012) [59] recently revealed the involvement of photoreceptors in dark and shading induced leaf responses. They applied different shading conditions to single leaves of Arabidopsis plants. Depending on the amount of light per‐ ceived, different biological programs were induced, leading to either acclimation to the new light conditions or leaf senescence. Furthermore, *phytochrome A* mutant lines displayed ac‐ celerated chlorophyll degradation under all shading conditions except darkness, displaying

its involvement in the perception of and adaption to changing light conditions [59].

A tight linkage between stress response and leaf senescence is demonstrated by the function of several members of the NAC- and WRKY-family [60]. For example, *At NTL9 (NAC TRANSCRIPTION FACTOR LIKE 9)* mediates osmotic stress signaling during leaf senes‐

synthesis upon external supply of 2% glucose.

#### **3. Senescence induction and nitrogen mobilization**

As mentioned above, induction of senescence is a highly complex regulated and dynamic process. Besides developmental cues, there are numerous other possible impacts. Nutrition‐ al starvation, photosynthetic activity, pathogen infections, carbon accumulation, carbon to nitrogen ratio, photoperiod and various other cues can lead to senescence induction on ei‐ ther organ or whole plant level. Both natural and stress induced senescence are accompa‐ nied with the remobilization of valuable nutrients from various organs of the plant. In the following we will again focus on the situation in leaves.

#### **3.1. Senescence induction**

Correct timing of leaf senescence is crucial for proper plant development. Too early senes‐ cence induction would decrease the ability to assimilate CO2, while too late induction would reduce the plant's capacity to remobilize nutrients from old leaves to developing organs [50]. Nevertheless, timing of senescence can also be regarded as an active adaption to the given nutritional and environmental conditions. For example under limited nutrition, con‐ tinued growth of vegetative tissues would result in a reduced ability to develop reproduc‐ tive organs.

Nutritional limitation, especially in concerns of nitrogen, has been shown to be able to en‐ hance leaf senescence. Sunflower (*Helianthus annuus*) plants grown under low nitrogen sup‐ ply showed a stronger decline of photosynthetic activity and more pronounced senescence symptoms than plants sufficiently supplied with nitrogen [51]. Furthermore these plants showed a more pronounced and earlier drop in (Glu+Asp)/(Gln+Asn) ratio during the pro‐ gression of senescence indicating an additional adaption to low nitrogen conditions through enhanced nitrogen remobilization. In this experiment, also a significant increase in the ratio of hexose to sucrose was observed at the beginning of senescence which was higher in Nstarved plants. This indicates that sugar-related senescence induction is dependent on the availability of nitrogen [51]. However, high sugar contents repress photosynthesis and can induce early SAG expression while late SAG expression is repressed. Diaz et al. (2005) [52] showed sugar accumulation to be lower in some recombinant inbred lines which display early leaf yellowing, thus pointing out a mayor function for sugar accumulation alone but the regulating function of the C/N balance during induction of monocarpic senescence is widely discussed. Recently, trehalose-6-phosphate (T6P) was identified as a main signaling component in this pathway. T6P inhibits the activity of Snf1-related protein kinase (SnRK1). Zhang et al. (2009) [53] showed the T6P/SnRK1 interaction in Arabidopsis seedling extracts and other young tissues treated with T6P. Additionally, Delatte et al. (2011) [54] confirmed the inhibition of SnRK1 by T6P with plants overexpressing the SnRK1 catalytic subunit gene *KIN10.* These plants were insensitive to trehalose treatments. Further verification of T6P as signaling molecule was provided by several studies. In wheat the interaction of T6P and SnRK1 has been suggested to play a role during grain filling [55]. Wingler et al. (2012) [56] conducted a study with *otsA* and *otsB* expressing Arabidopsis plants *otsA* encodes the bacte‐ rial T6P synthase gene, *otsB* the T6P phosphatase gene; therefore, overexpression leads to in‐ creasing or decreasing T6P contents, respectively. A significantly higher accumulation of glucose, fructose and sucrose was observed in *otsB* expressing plants and these plants dis‐ played a delayed senescence phenotype. But most interestingly, these plants were rendered less susceptible to the induction of senescence-associated genes by sugar feeding in combi‐ nation with low nitrogen supply, whereas *otsA* plants induced senescence and anthocyanin synthesis upon external supply of 2% glucose.

corporation into AAs in a NADH-dependent manner. Assimilation of ammonium into AAs involves chloroplastic glutamine synthetase 2 (GS2) and glutamate synthase (Fd-GO‐ GAT), which generates glutamine and glutamate (for detailed review see [14]). Glutamine as well as glutamate serve as ammonium donor for the synthesis of all other amino acids

long-range nitrogen transport and storage form, respectively [47]. Alternatively carba‐ moylphosphate synthase can be involved in ammonium assimilation by producing carba‐ moylphosphate and successively citrulline and arginine. Assimilation in non-green tissues is achieved in plastids in a similar manner, although here GOGAT depends on NADH in‐ stead of ferredoxin. Carbon skeletons are essential for the acquisition of inorganic nitro‐ gen in AAs. Especially the demand for keto-acids has to be met (see [14] and references within). These are predominantly obtained from the TCA-Cycle in the form of 2-oxogluta‐ rate (2OG) [47, 48]. 2OG is used for incorporation of photorespiratory ammonium, result‐ ing in the production of glutamate, which in turn can be utilized by GS1 and GS2 to produce glutamine. This displays the intricate interconnection between carbon and nitro‐ gen metabolism, in which N uptake and assimilation is also influenced via photosynthet‐ ic rates [47]. Besides direct assimilation into AAs, nitrate can also be stored in the vacuole and in the chloroplast. Vacuolar nitrate concentrations can vary enormously, as vacuolar nitrate also contributes to turgor maintenance and might have a nitrate storage function

+ donor or as

including aspartate and asparagine, which in turn function as active NH4

to maintain the cytosolic nitrate concentrations which are more constant [49].

As mentioned above, induction of senescence is a highly complex regulated and dynamic process. Besides developmental cues, there are numerous other possible impacts. Nutrition‐ al starvation, photosynthetic activity, pathogen infections, carbon accumulation, carbon to nitrogen ratio, photoperiod and various other cues can lead to senescence induction on ei‐ ther organ or whole plant level. Both natural and stress induced senescence are accompa‐ nied with the remobilization of valuable nutrients from various organs of the plant. In the

Correct timing of leaf senescence is crucial for proper plant development. Too early senes‐ cence induction would decrease the ability to assimilate CO2, while too late induction would reduce the plant's capacity to remobilize nutrients from old leaves to developing organs [50]. Nevertheless, timing of senescence can also be regarded as an active adaption to the given nutritional and environmental conditions. For example under limited nutrition, con‐ tinued growth of vegetative tissues would result in a reduced ability to develop reproduc‐

Nutritional limitation, especially in concerns of nitrogen, has been shown to be able to en‐ hance leaf senescence. Sunflower (*Helianthus annuus*) plants grown under low nitrogen sup‐

**3. Senescence induction and nitrogen mobilization**

following we will again focus on the situation in leaves.

**3.1. Senescence induction**

58 Senescence and Senescence-Related Disorders

tive organs.

Another signaling component involved in senescence induction is light quantity and quali‐ ty. Senescence can be induced by the darkening of individual leaves. However, darkening of the whole plant resulted in delay rather than in induction of leaf senescence in Arabidopsis and sunflower plants [57, 58]. Brouwer et al. (2012) [59] recently revealed the involvement of photoreceptors in dark and shading induced leaf responses. They applied different shading conditions to single leaves of Arabidopsis plants. Depending on the amount of light per‐ ceived, different biological programs were induced, leading to either acclimation to the new light conditions or leaf senescence. Furthermore, *phytochrome A* mutant lines displayed ac‐ celerated chlorophyll degradation under all shading conditions except darkness, displaying its involvement in the perception of and adaption to changing light conditions [59].

A tight linkage between stress response and leaf senescence is demonstrated by the function of several members of the NAC- and WRKY-family [60]. For example, *At NTL9 (NAC TRANSCRIPTION FACTOR LIKE 9)* mediates osmotic stress signaling during leaf senes‐ cence [61] and *At VNI2 (ANAC083)* has been shown to integrate abscisic acid (ABA)-mediat‐ ed abiotic stress signals into leaf senescence [62].

ing and flowering [14, 72-74]. Grown under non-limiting nitrogen conditions, Arabidopsis displays a lowered nitrate influx during the reproductive stage in comparison to the influx during the vegetative stage [14]. Although many plants seem to continue N uptake during seed filling, this nitrogen is not always allocated to the seeds, thus rendering nitrogen remo‐ bilization from senescing organs a central component for the proper development of repro‐

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61

Protein degradation is most likely the most important degradation process that occurs dur‐ ing senescence [75]. With a combined 15N tracing/proteomics approach, Desclos et al. (2009) [76] have shown that HSP70, chaperonin10 and disulfide isomerase are synthesized during the whole progression of senescence in *B. napus* illustrating the necessity to prevent the ag‐ gregation of denatured proteins. In addition*,* almost all protease families have been associat‐

The aminopeptidase LAP2 has been characterized as exopeptidase liberating N-terminal leucine, methionine and phenylalanine. Arabidopsis *lap2* mutants displayed a significant change in amino acid contents. In particular, nitrogen rich AAs like glutamate and gluta‐ mine were dramatically reduced while leucine levels were the same as in wild type plants. Furthermore, a premature leaf senescence phenotype was observed in these plants. Different recombinant inbred lines, which are also modified in Glu, Gln, Asp and Asn contents, also show a senescence phenotype tempting the authors to speculate that the senescence pheno‐ type of *lab2* might be related to a decreased turnover of defective proteins and the marked

Chloroplast targeted proteases comprise proteases of the Lon, PreP, Clp, FtsH and DegP type [78-80]. Their substrates include, besides others, chlorophyll apoproteins like LHCII, the D1 protein of the photosystem II reaction center and Rubisco. The Clp protease complex is the most abundant stromal protease, where PreP is also located [78, 79]. Several catalytic subunits of the Clp proteases display up-regulated expression during dark-induced senes‐ cence in Arabidopsis, like e.g. ClpD/ERD1 and ClpC1. They possess sequence similarity to the chaperon HSP100 indicating that they might function as recognition subunit in the Clp protease complex to recruit denatured proteins [80]. FtsH proteases are thylakoid bound fac‐ ing the stroma while Deg proteases are also thylakoid bound but facing stroma as well as thylakoid lumen [81, 82]. DegP2 is responsible for an initial cleavage of the D1 protein, where after FtsH proteases complete the full degradation [80, 83]. These two proteases be‐ long to the family of serine proteases. In wheat, serine proteases are the most important fam‐ ily of proteases participating in N remobilization [84]. Subtilases have been reported to be highly expressed in barley during natural and senescence induced via artificial carbohydrate accumulation. Additionally induced proteases were SAG12, CND41-like, papain-like, serine carboxypeptidase III precursor, aspartic endopeptidases and others [85]. Roberts et al. (2012) [75] suggest a classification of senescence-associated proteases according to their expression

ductive organs.

**3.2. Nitrogen mobilization**

*3.2.1. Senescence associated proteases*

decrease of nitrogen rich AAs [77].

ed with some aspects of plant senescence [75].

Besides various other cues like the stage of plant development, pathogens, extreme tempera‐ tures, source-sink transitions and drought, the action of reactive oxygen species (ROS) has been shown to have a severe impact on the induction of leaf senescence. Cellular H2O2 levels increase at the onset of senescence due to a complex regulation of hydrogen peroxide scav‐ enging enzymes [63]. The increase in intracellular H2O2 levels is initiated via the down-regu‐ lation of the expression of the hydrogen peroxide scavenging enzyme CATALASE2 by the transcription factor GBF1 (G-Box binding factor 1). In *gbf1* knock-out plants, the senescence specific elevation in H2O2 levels is absent leading to a significant delay of leaf senescence [64]. We have demonstrated recently using a specific *in vivo* hydrogen peroxide monitoring and scavenging system that the pivotal role of H2O2 during the induction of developmental leaf senescence in Arabidopsis is depending on the subcellular localization and concentra‐ tion. Furthermore, a similar senescence-specific up-regulation of H2O2 levels and down-reg‐ ulation of the respective scavenging enzymes was also observed in *Brassica napus* [65]. Knock-out and overexpression plants of *AtOSR1* (*ANAC059* or *ATNAC3*) or *AtJUB1* (*ANAC042*), which are both highly inducible by H2O2, were delayed or accelerated, respec‐ tively, concerning the onset of leaf senescence in which JUB1 also modulates cellular H2O2 levels [66, 67]. Besides their important role in disease resistance [68], several WRKY tran‐ scription factors have been suggested to have a striking role in the regulation of leaf senes‐ cence. For example AtWRKY53, a H2O2-responsive transcription factor, has been indicated to have a function as important control element during the onset of leaf senescence [69].

Conclusively, leaf senescence is governed not only by developmental age but a wide range of various different external and internal factors, biotic and abiotic influences, mol‐ ecules and cues, which have to be integrated. Despite its enormous agricultural impor‐ tance, our knowledge of these integrative mechanisms is still limited and needs much more efforts to get complete insight into the regulatory network controlling the onset and progression of leaf senescence.

#### *3.1.1. N-uptake during senescence*

Nitrogen uptake and partitioning after beginning anthesis varies greatly between different species and even between ecotypes. An analysis of different Arabidopsis accessions revealed that the fate of nitrogen absorbed during flowering can be different, depending on general N availability and accession. At low nitrogen concentrations most of the N assimilated postflowering was allocated to the seeds, while under high N regimes the main part of it was distributed to the rosette leaves and successively lost in the dry remains, except for four test‐ ed accessions. N13, Sakata, Bl-1 and Oy-0 allocated the nitrogen taken up post-flowering al‐ so to the seeds under high N supply [70]. In wheat, a minor portion of grain N is derived from N uptake post-flowering, whereas up to 90-95% is remobilized from other plant tissues [3, 71]. In oilseed rape (*Brassica napus*) the induction of the reproductive phase is accompa‐ nied with a drastic down-regulation of nitrogen uptake systems. HATs and HATs + LATs activities are decreased thus almost resulting in an arrest of nitrogen uptake during seed fill‐ ing and flowering [14, 72-74]. Grown under non-limiting nitrogen conditions, Arabidopsis displays a lowered nitrate influx during the reproductive stage in comparison to the influx during the vegetative stage [14]. Although many plants seem to continue N uptake during seed filling, this nitrogen is not always allocated to the seeds, thus rendering nitrogen remo‐ bilization from senescing organs a central component for the proper development of repro‐ ductive organs.

#### **3.2. Nitrogen mobilization**

cence [61] and *At VNI2 (ANAC083)* has been shown to integrate abscisic acid (ABA)-mediat‐

Besides various other cues like the stage of plant development, pathogens, extreme tempera‐ tures, source-sink transitions and drought, the action of reactive oxygen species (ROS) has been shown to have a severe impact on the induction of leaf senescence. Cellular H2O2 levels increase at the onset of senescence due to a complex regulation of hydrogen peroxide scav‐ enging enzymes [63]. The increase in intracellular H2O2 levels is initiated via the down-regu‐ lation of the expression of the hydrogen peroxide scavenging enzyme CATALASE2 by the transcription factor GBF1 (G-Box binding factor 1). In *gbf1* knock-out plants, the senescence specific elevation in H2O2 levels is absent leading to a significant delay of leaf senescence [64]. We have demonstrated recently using a specific *in vivo* hydrogen peroxide monitoring and scavenging system that the pivotal role of H2O2 during the induction of developmental leaf senescence in Arabidopsis is depending on the subcellular localization and concentra‐ tion. Furthermore, a similar senescence-specific up-regulation of H2O2 levels and down-reg‐ ulation of the respective scavenging enzymes was also observed in *Brassica napus* [65]. Knock-out and overexpression plants of *AtOSR1* (*ANAC059* or *ATNAC3*) or *AtJUB1* (*ANAC042*), which are both highly inducible by H2O2, were delayed or accelerated, respec‐ tively, concerning the onset of leaf senescence in which JUB1 also modulates cellular H2O2 levels [66, 67]. Besides their important role in disease resistance [68], several WRKY tran‐ scription factors have been suggested to have a striking role in the regulation of leaf senes‐ cence. For example AtWRKY53, a H2O2-responsive transcription factor, has been indicated to have a function as important control element during the onset of leaf senescence [69].

Conclusively, leaf senescence is governed not only by developmental age but a wide range of various different external and internal factors, biotic and abiotic influences, mol‐ ecules and cues, which have to be integrated. Despite its enormous agricultural impor‐ tance, our knowledge of these integrative mechanisms is still limited and needs much more efforts to get complete insight into the regulatory network controlling the onset

Nitrogen uptake and partitioning after beginning anthesis varies greatly between different species and even between ecotypes. An analysis of different Arabidopsis accessions revealed that the fate of nitrogen absorbed during flowering can be different, depending on general N availability and accession. At low nitrogen concentrations most of the N assimilated postflowering was allocated to the seeds, while under high N regimes the main part of it was distributed to the rosette leaves and successively lost in the dry remains, except for four test‐ ed accessions. N13, Sakata, Bl-1 and Oy-0 allocated the nitrogen taken up post-flowering al‐ so to the seeds under high N supply [70]. In wheat, a minor portion of grain N is derived from N uptake post-flowering, whereas up to 90-95% is remobilized from other plant tissues [3, 71]. In oilseed rape (*Brassica napus*) the induction of the reproductive phase is accompa‐ nied with a drastic down-regulation of nitrogen uptake systems. HATs and HATs + LATs activities are decreased thus almost resulting in an arrest of nitrogen uptake during seed fill‐

ed abiotic stress signals into leaf senescence [62].

60 Senescence and Senescence-Related Disorders

and progression of leaf senescence.

*3.1.1. N-uptake during senescence*

#### *3.2.1. Senescence associated proteases*

Protein degradation is most likely the most important degradation process that occurs dur‐ ing senescence [75]. With a combined 15N tracing/proteomics approach, Desclos et al. (2009) [76] have shown that HSP70, chaperonin10 and disulfide isomerase are synthesized during the whole progression of senescence in *B. napus* illustrating the necessity to prevent the ag‐ gregation of denatured proteins. In addition*,* almost all protease families have been associat‐ ed with some aspects of plant senescence [75].

The aminopeptidase LAP2 has been characterized as exopeptidase liberating N-terminal leucine, methionine and phenylalanine. Arabidopsis *lap2* mutants displayed a significant change in amino acid contents. In particular, nitrogen rich AAs like glutamate and gluta‐ mine were dramatically reduced while leucine levels were the same as in wild type plants. Furthermore, a premature leaf senescence phenotype was observed in these plants. Different recombinant inbred lines, which are also modified in Glu, Gln, Asp and Asn contents, also show a senescence phenotype tempting the authors to speculate that the senescence pheno‐ type of *lab2* might be related to a decreased turnover of defective proteins and the marked decrease of nitrogen rich AAs [77].

Chloroplast targeted proteases comprise proteases of the Lon, PreP, Clp, FtsH and DegP type [78-80]. Their substrates include, besides others, chlorophyll apoproteins like LHCII, the D1 protein of the photosystem II reaction center and Rubisco. The Clp protease complex is the most abundant stromal protease, where PreP is also located [78, 79]. Several catalytic subunits of the Clp proteases display up-regulated expression during dark-induced senes‐ cence in Arabidopsis, like e.g. ClpD/ERD1 and ClpC1. They possess sequence similarity to the chaperon HSP100 indicating that they might function as recognition subunit in the Clp protease complex to recruit denatured proteins [80]. FtsH proteases are thylakoid bound fac‐ ing the stroma while Deg proteases are also thylakoid bound but facing stroma as well as thylakoid lumen [81, 82]. DegP2 is responsible for an initial cleavage of the D1 protein, where after FtsH proteases complete the full degradation [80, 83]. These two proteases be‐ long to the family of serine proteases. In wheat, serine proteases are the most important fam‐ ily of proteases participating in N remobilization [84]. Subtilases have been reported to be highly expressed in barley during natural and senescence induced via artificial carbohydrate accumulation. Additionally induced proteases were SAG12, CND41-like, papain-like, serine carboxypeptidase III precursor, aspartic endopeptidases and others [85]. Roberts et al. (2012) [75] suggest a classification of senescence-associated proteases according to their expression

profile and probable function during natural senescence. Class I includes all proteases being expressed in non-senescent and in senescent tissue. Although no senescence specific expres‐ sion change can be observed, their continued expression in a catabolic environment displays their significance for a normal progression of senescence. Class II contains proteases being expressed at a low level in non-senescent tissue and induced upon senescence onset. Class III comprises proteases which are induced exclusively during senescence. This suggests a role in the late stages of senescence and a probable function in cell death execution. Class IV proteases constitute proteases transiently expressed during onset of senescence which could be involved in early breakdown processes like e.g. chloroplast dismantling. Finally, class V proteases are down-regulated during senescence. These enzymes are likely to fulfill house‐ keeping protein turnover and other proteolytic functions, which are no longer needed dur‐ ing the progression of leaf senescence [75].

However, Rubisco, nitrogen, soluble protein and chlorophyll contents decreased at almost the same rate in wild type plants as in *atg4a4b-1* mutant plants. This suggests alternative, au‐ tophagy-independent protein degradation pathways [96]. Lastly, despite the earlier men‐ tioned 44 kDa Rubisco fragment observed in isolated chloroplasts, oxidative stress conditions might also initiate degradation of Rubisco's large subunit, as under these condi‐

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63

Besides the RCBs, senescence-associated vacuoles (SAVs) have been described. These va‐ cuoles, enclosed by a single membrane layer, are enriched in Rubisco and display a high proteolytic activity at a pH more acidic than the central vacuole´s. SAVs are structurally not related to RCBs which posses a double layer membrane [95, 98]. The double layer mem‐ brane enclosing the RCBs appears to be derived from the chloroplast envelope [95]. Further‐ more, SAV development seems to be autophagy-independent, as Arabidopsis *atapg7-1* mutant lines show normal SAV formation [98]. SAVs are only formed in leaf mesophyll cells. They are approximately 700 nm in diameter and can be labeled with antibodies against the (H+)-pyrophosphatase, an Arabidopsis vacuolar marker indicating these organelles in‐ deed to be vacuoles [98]. Accumulation of stromal proteins in the SAVs was proven via plas‐ tid localized GFP which localized in SAVs in senescing tobacco leaves. In addition, high levels of chloroplastic glutamine synthase could be detected within these vacuoles [89]. Al‐ though the chlorophyll degradation pathway has been elucidated to a large extend and the first steps are regarded to occur within the plastid [88, 99], chlorophyll *a* has been found in SAVs under certain conditions, thus an alternative degradation pathway can be proposed [89]. Despite SAG12 has been shown to localize in SAVs, *sag12* mutant lines did neither show impairment in SAV formation nor in the proteolytic activity within the SAVs [98].

Even though chlorophyll represents about 2% of the total cellular nitrogen content [86], N fixed in chlorophyll is not exported from the leaf but rather remains in the vacuole [100]. However, around 20% N are fixed in proteins associated with or directly binding chloro‐ phyll [88] and removal of chlorophyll seems to be a prerequisite for degradation of the cor‐ responding apoproteins [88]. Pheophorbide a oxygenase (PAO) is an iron-dependent monooxygenase localized to the inner envelope of maturing gerontoplasts and catalyzes the conversion of pheophorbide a to red chlorophyll catabolites, one of the first steps during chlorophyll degradation. It represents a key control point in regulation of chlorophyll degra‐ dation [88, 101, 102]. In *pao* mutants and other stay green mutants affected in PAO activity and thus impaired chlorophyll degradation, this retention is accompanied with the accumu‐

Autophagy plays a crucial role for nitrogen remobilization. The most striking phenotype of all *atg* mutants is hypersensitivity to nitrogen starvation ([103] and references within). Fur‐ thermore, an age dependent early senescence phenotype can be observed. As autophagy is involved in molecule degradation one would expect delayed senescence if this pathway is blocked. One hypothesis explaining this contradiction is that the autophagy pathway is nor‐ mally activated at an early stage of senescence starting to degrade plastid proteins while

lation of chlorophyll apoproteins like LHCII (see [88] and references within).

*3.2.3. Autophagy*

tions the large subunit is split into a 37 and a 16 kDa fragment [97].

#### *3.2.2. Chloroplast dismantling*

Chloroplasts are the first organelles to show visible symptoms of degradation processes dur‐ ing senescence. Containing up to 75% of total leaf nitrogen, chloroplasts are the main source for its remobilization [86]. Four different pathways have been proposed for chloroplast and chloroplastic protein degradation: I) endogenous proteases degrade proteins intra-plastidial, II) degradation of stroma fragments in an extraplastidic, non-autophagic pathway, as well as III) extraplastidic degradation by autophagy-associated pathways, and IV) autophagic deg‐ radation of entire plastids [87]. Chloroplast breakdown is not a chaotic decay, but rather an organized and selective process. As chloroplasts are one of the plants main ROS-producing organelles, and due to the potential phototoxicity of many chloroplastic components and their degradation intermediates, a coordinated dismantling process is necessary to prevent severe cell damage [88, 89].

Within these organelles Rubisco represents the most abundant protein. Its abundance ex‐ ceeds the requirements for photosynthesis by far, thus a nitrogen storage function has been suggested for it [90]. In chloroplasts isolated from senescing leaves a 44 kDa fragment of Ru‐ bisco's large subunit accumulates, but seems not to be degraded further [91]. The chloro‐ plastic aspartate protease CND41 has been shown to degrade denatured Rubisco, but not active Rubisco *in vitro* [92]. This protease might be involved directly in Rubisco degradation, as accumulation of CND41 correlates with loss of Rubisco [93]. However, tobacco CND41 antisense lines also show a dwarf phenotype, reduced gibberellin levels and reduced leaf ex‐ pansion, thus this correlation could be an indirect effect through gibberellin homeostasis [94]. Rubisco containing bodies (RCB) have been found to be shuttling from the chloroplast to the central vacuole via an autophagy-dependent pathway [95]. The autophagy-dependen‐ cy of these bodies was shown using *atg4a4b-1*mutants which are impaired in autophagy. Chloroplast fate was investigated in individually darkened leaves (IDLs) of wild type plants and *atg4a4b-1* mutants since individual darkening of leaves has been shown to rapidly in‐ duce senescence [57]. Wild type plants showed a decrease in chloroplast number and size as well as formation of RCBs. A*tg4a4b-1* mutant lines also displayed a decrease in chloroplast size but RCB formation was abolished and also the count of chloroplasts stayed constant. However, Rubisco, nitrogen, soluble protein and chlorophyll contents decreased at almost the same rate in wild type plants as in *atg4a4b-1* mutant plants. This suggests alternative, au‐ tophagy-independent protein degradation pathways [96]. Lastly, despite the earlier men‐ tioned 44 kDa Rubisco fragment observed in isolated chloroplasts, oxidative stress conditions might also initiate degradation of Rubisco's large subunit, as under these condi‐ tions the large subunit is split into a 37 and a 16 kDa fragment [97].

Besides the RCBs, senescence-associated vacuoles (SAVs) have been described. These va‐ cuoles, enclosed by a single membrane layer, are enriched in Rubisco and display a high proteolytic activity at a pH more acidic than the central vacuole´s. SAVs are structurally not related to RCBs which posses a double layer membrane [95, 98]. The double layer mem‐ brane enclosing the RCBs appears to be derived from the chloroplast envelope [95]. Further‐ more, SAV development seems to be autophagy-independent, as Arabidopsis *atapg7-1* mutant lines show normal SAV formation [98]. SAVs are only formed in leaf mesophyll cells. They are approximately 700 nm in diameter and can be labeled with antibodies against the (H+)-pyrophosphatase, an Arabidopsis vacuolar marker indicating these organelles in‐ deed to be vacuoles [98]. Accumulation of stromal proteins in the SAVs was proven via plas‐ tid localized GFP which localized in SAVs in senescing tobacco leaves. In addition, high levels of chloroplastic glutamine synthase could be detected within these vacuoles [89]. Al‐ though the chlorophyll degradation pathway has been elucidated to a large extend and the first steps are regarded to occur within the plastid [88, 99], chlorophyll *a* has been found in SAVs under certain conditions, thus an alternative degradation pathway can be proposed [89]. Despite SAG12 has been shown to localize in SAVs, *sag12* mutant lines did neither show impairment in SAV formation nor in the proteolytic activity within the SAVs [98].

Even though chlorophyll represents about 2% of the total cellular nitrogen content [86], N fixed in chlorophyll is not exported from the leaf but rather remains in the vacuole [100]. However, around 20% N are fixed in proteins associated with or directly binding chloro‐ phyll [88] and removal of chlorophyll seems to be a prerequisite for degradation of the cor‐ responding apoproteins [88]. Pheophorbide a oxygenase (PAO) is an iron-dependent monooxygenase localized to the inner envelope of maturing gerontoplasts and catalyzes the conversion of pheophorbide a to red chlorophyll catabolites, one of the first steps during chlorophyll degradation. It represents a key control point in regulation of chlorophyll degra‐ dation [88, 101, 102]. In *pao* mutants and other stay green mutants affected in PAO activity and thus impaired chlorophyll degradation, this retention is accompanied with the accumu‐ lation of chlorophyll apoproteins like LHCII (see [88] and references within).

#### *3.2.3. Autophagy*

profile and probable function during natural senescence. Class I includes all proteases being expressed in non-senescent and in senescent tissue. Although no senescence specific expres‐ sion change can be observed, their continued expression in a catabolic environment displays their significance for a normal progression of senescence. Class II contains proteases being expressed at a low level in non-senescent tissue and induced upon senescence onset. Class III comprises proteases which are induced exclusively during senescence. This suggests a role in the late stages of senescence and a probable function in cell death execution. Class IV proteases constitute proteases transiently expressed during onset of senescence which could be involved in early breakdown processes like e.g. chloroplast dismantling. Finally, class V proteases are down-regulated during senescence. These enzymes are likely to fulfill house‐ keeping protein turnover and other proteolytic functions, which are no longer needed dur‐

Chloroplasts are the first organelles to show visible symptoms of degradation processes dur‐ ing senescence. Containing up to 75% of total leaf nitrogen, chloroplasts are the main source for its remobilization [86]. Four different pathways have been proposed for chloroplast and chloroplastic protein degradation: I) endogenous proteases degrade proteins intra-plastidial, II) degradation of stroma fragments in an extraplastidic, non-autophagic pathway, as well as III) extraplastidic degradation by autophagy-associated pathways, and IV) autophagic deg‐ radation of entire plastids [87]. Chloroplast breakdown is not a chaotic decay, but rather an organized and selective process. As chloroplasts are one of the plants main ROS-producing organelles, and due to the potential phototoxicity of many chloroplastic components and their degradation intermediates, a coordinated dismantling process is necessary to prevent

Within these organelles Rubisco represents the most abundant protein. Its abundance ex‐ ceeds the requirements for photosynthesis by far, thus a nitrogen storage function has been suggested for it [90]. In chloroplasts isolated from senescing leaves a 44 kDa fragment of Ru‐ bisco's large subunit accumulates, but seems not to be degraded further [91]. The chloro‐ plastic aspartate protease CND41 has been shown to degrade denatured Rubisco, but not active Rubisco *in vitro* [92]. This protease might be involved directly in Rubisco degradation, as accumulation of CND41 correlates with loss of Rubisco [93]. However, tobacco CND41 antisense lines also show a dwarf phenotype, reduced gibberellin levels and reduced leaf ex‐ pansion, thus this correlation could be an indirect effect through gibberellin homeostasis [94]. Rubisco containing bodies (RCB) have been found to be shuttling from the chloroplast to the central vacuole via an autophagy-dependent pathway [95]. The autophagy-dependen‐ cy of these bodies was shown using *atg4a4b-1*mutants which are impaired in autophagy. Chloroplast fate was investigated in individually darkened leaves (IDLs) of wild type plants and *atg4a4b-1* mutants since individual darkening of leaves has been shown to rapidly in‐ duce senescence [57]. Wild type plants showed a decrease in chloroplast number and size as well as formation of RCBs. A*tg4a4b-1* mutant lines also displayed a decrease in chloroplast size but RCB formation was abolished and also the count of chloroplasts stayed constant.

ing the progression of leaf senescence [75].

*3.2.2. Chloroplast dismantling*

62 Senescence and Senescence-Related Disorders

severe cell damage [88, 89].

Autophagy plays a crucial role for nitrogen remobilization. The most striking phenotype of all *atg* mutants is hypersensitivity to nitrogen starvation ([103] and references within). Fur‐ thermore, an age dependent early senescence phenotype can be observed. As autophagy is involved in molecule degradation one would expect delayed senescence if this pathway is blocked. One hypothesis explaining this contradiction is that the autophagy pathway is nor‐ mally activated at an early stage of senescence starting to degrade plastid proteins while leaving the photosynthetic apparatus intact. However, when autophagy is blocked, it is speculated that autophagy-independent pathways for chloroplast protein degradation might be activated untimely leading to premature chloroplast and chlorophyll degradation and thus to an early senescence phenotype [104]. Recently, Guiboileau et al. (2012) [103] con‐ ducted a study on the impact of *atg* mutants *(atg5, atg9* and RNAi18) on nitrogen remobiliza‐ tion. These plants were grown under ample and low nitrate conditions. In comparison to wild type plants the dry weight as well as the seed weight was lowered. However, when calculating the harvest index, *atg* mutants did not display a significant difference, except for the *atg5* mutant line at low N conditions. When the nitrogen use and remobilization efficien‐ cy was investigated via 15N tracing experiments, all *atg* lines showed a decrease in this fea‐ ture. It was demonstrated that remobilization was significantly impaired, as N contents in the plants dry remains were enriched and 15N previously partitioned to the leaves was not mobilized to the seeds. To verify that this impairment rests on an autophagy defect and not on premature senescence and cell death symptoms, *atg5* mutants were combined with two SA signaling mutants, *sid2* and *nahG,* overriding the early senescence phenotype. These mu‐ tants reached nearly wild type biomass levels, but did not compensate the decrease in NRE. These results and the finding that autophagy regulates SA levels in a negative feedback loop [105] suggest, that the premature senescence phenotype in *atg* mutants is at least in part mediated by increased SA levels [104]. Conclusively, blocked autophagy pathways might re‐ sult in an early senescence phenotype because of the accumulation of damaged and thus po‐ tentially toxic molecules in combination with a missing negative feedback on SA levels leading to cell death and activation of alternative pathways for bulk protein degradation.

profiling revealed that, besides others, the cytosolic GS1, glutamate dehydrogenase (GDH) and asparagine synthetase (AS) are specifically induced during senescence [14]. A series of transamination reactions would result in an accumulation of glutamate, which could serve as substrate for GDH. Deamination of glutamate via GDH provides then 2OG and ammo‐ nia. NH3 could then in turn be used as substrate for cytosolic GS1, giving rise to glutamine, which is one of the major nitrogen transport forms during nutrient remobilization. In fact many studies strengthen a positive correlation between GS activity and yield as well as grain and stem N content. Martin et al. (2006) [107] identified two cytosolic glutamine syn‐ thetase isoforms in maize which have a major impact on kernel size and yield. In wheat, GS activity was also positively linked with grain and stem N content [71]. Recently, two rice va‐ rieties with different levels of GS2 activity were analyzed and plants with higher activity displayed less NH3 emission due to photorespiration and a better ability to recycle and reassimilate ammonia within the plant [108]. In barley amino acid permeases (AAP) seem to play a significant role during N retranslocation and grain filling. Recent RNA-Seq data re‐ vealed an overrepresentation of this gene family in both source and sink tissues. Further‐ more, the grain-specific HvAAP3, which was also identified in this study, has high sequence similarity to Arabidopsis AAP1 and AAP8, which have been already shown to be involved

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65

Asparagine amounts also increase significantly in whole rosettes darkened for several days as well as in senescent leaves (see e.g. [1, 80]). Besides the senescence specific up-regulation of AS, pyruvate orthophosphate dikinase (PPDK) expression is also significantly increased during dark-induced senescence [80]. PPDK might have a role in carbon salvage after lipid degradation, thus Lin and Wu (2004) [80] also investigated other pathways possibly in‐ volved in this process. Remarkably, they found only a few components of these pathways to be up-regulated and many others even down-regulated. Based on their expressional profil‐ ing data, they postulated a alternative pathway for asparagine synthesis, where PPDK deliv‐ ers metabolic precursors [80]. Additionally, seed protein contents were elevated and viability of seedlings was increased on nitrogen-limiting media in Arabidopsis *ASN1* over‐ expressor lines (*35S*::*ASN1*). Furthermore, they observed more Asn to be allocated to flowers

and developing siliques and also higher Asn contents in phloem exudates [80, 110].

Nitrogen is not only remobilized from older leaves via amino acids. Nitrate and ammonia are also translocated to developing sink tissues. Fan et al. (2009) [111] identified a nitrate transporter (NRT1-7) which is involved in remobilization processes. Arabidopsis *nrt1-7* mu‐ tants displayed retarded growth under nitrogen starvation conditions. Also the spatial ex‐ pression of this transporter in phloem tissue of older leaves and the expressional induction upon nitrogen starvation points out its function in nitrogen remobilization. Finally, the in‐ ability of *nrt1-7* mutants to remobilize 15N from old to young leaves and the high accumula‐ tion of nitrate in old leaves in this mutants further underlines this assumption [111]. Another nitrate transporter involved in remobilization is NRT2-4. This transporter acts in the high-affinity range and its expression is also induced upon nitrogen starvation. Addi‐ tionally, *nrt2-4* mutant lines had lower phloem sap nitrate contents. However, *nrt2-4* mu‐ tants were not altered in growth or development, indicating that the decreased NO3-levels

in seed N supply (see [109] and references within).

#### *3.2.4. Re-assimilation and translocation of salvaged nitrogen*

As mentioned above, chloroplastic glutamine synthetase (GS2), GOGAT, NiR and Rubisco are targeted for rapid degradation already during early phases of senescence, disrupting primary nitrogen assimilation. Proteolysis in the vacuole feeds into the cellular pool of free AAs during the progression of senescence. The steady-state concentrations of free AAs de‐ pend on the rate of their release due to proteolysis and their efflux into growing structures [106]. Soudry et al. (2005) [106] have utilized a bioluminescence assay combined with auxo‐ trophic bacteria for the detection of free tryptophan levels. They assumed that tryptophan reflects the overall pool of free AAs, as it is not modified before its export into sink organs. An accumulation of free AAs was observed in detached oat and Arabidopsis leaves. While attached oat leaves showed a gradual decrease in tryptophan levels during further progres‐ sion of senescence, the attached Arabidopsis leaves did not or only due to membrane leak‐ age resulting from the experimental procedure. The authors concluded that not only source strength but also sink strength is important for successful nutrient remobilization and sug‐ gested that the small reproductive organs of Arabidopsis exerted too weak sink strength. However, these findings might be related to the experimental design, as Arabidopsis does indeed remobilize N for seed filling [70] and Diaz et al. (2005) [52] reported decreasing lev‐ els for several AAs during the progression of leaf senescence in Arabidopsis. Protein break‐ down increases free AAs in the cell. While some seem to be exported without prior modification, many are probably modified, hydrolyzed or interconverted. Expressional profiling revealed that, besides others, the cytosolic GS1, glutamate dehydrogenase (GDH) and asparagine synthetase (AS) are specifically induced during senescence [14]. A series of transamination reactions would result in an accumulation of glutamate, which could serve as substrate for GDH. Deamination of glutamate via GDH provides then 2OG and ammo‐ nia. NH3 could then in turn be used as substrate for cytosolic GS1, giving rise to glutamine, which is one of the major nitrogen transport forms during nutrient remobilization. In fact many studies strengthen a positive correlation between GS activity and yield as well as grain and stem N content. Martin et al. (2006) [107] identified two cytosolic glutamine syn‐ thetase isoforms in maize which have a major impact on kernel size and yield. In wheat, GS activity was also positively linked with grain and stem N content [71]. Recently, two rice va‐ rieties with different levels of GS2 activity were analyzed and plants with higher activity displayed less NH3 emission due to photorespiration and a better ability to recycle and reassimilate ammonia within the plant [108]. In barley amino acid permeases (AAP) seem to play a significant role during N retranslocation and grain filling. Recent RNA-Seq data re‐ vealed an overrepresentation of this gene family in both source and sink tissues. Further‐ more, the grain-specific HvAAP3, which was also identified in this study, has high sequence similarity to Arabidopsis AAP1 and AAP8, which have been already shown to be involved in seed N supply (see [109] and references within).

leaving the photosynthetic apparatus intact. However, when autophagy is blocked, it is speculated that autophagy-independent pathways for chloroplast protein degradation might be activated untimely leading to premature chloroplast and chlorophyll degradation and thus to an early senescence phenotype [104]. Recently, Guiboileau et al. (2012) [103] con‐ ducted a study on the impact of *atg* mutants *(atg5, atg9* and RNAi18) on nitrogen remobiliza‐ tion. These plants were grown under ample and low nitrate conditions. In comparison to wild type plants the dry weight as well as the seed weight was lowered. However, when calculating the harvest index, *atg* mutants did not display a significant difference, except for the *atg5* mutant line at low N conditions. When the nitrogen use and remobilization efficien‐ cy was investigated via 15N tracing experiments, all *atg* lines showed a decrease in this fea‐ ture. It was demonstrated that remobilization was significantly impaired, as N contents in the plants dry remains were enriched and 15N previously partitioned to the leaves was not mobilized to the seeds. To verify that this impairment rests on an autophagy defect and not on premature senescence and cell death symptoms, *atg5* mutants were combined with two SA signaling mutants, *sid2* and *nahG,* overriding the early senescence phenotype. These mu‐ tants reached nearly wild type biomass levels, but did not compensate the decrease in NRE. These results and the finding that autophagy regulates SA levels in a negative feedback loop [105] suggest, that the premature senescence phenotype in *atg* mutants is at least in part mediated by increased SA levels [104]. Conclusively, blocked autophagy pathways might re‐ sult in an early senescence phenotype because of the accumulation of damaged and thus po‐ tentially toxic molecules in combination with a missing negative feedback on SA levels leading to cell death and activation of alternative pathways for bulk protein degradation.

As mentioned above, chloroplastic glutamine synthetase (GS2), GOGAT, NiR and Rubisco are targeted for rapid degradation already during early phases of senescence, disrupting primary nitrogen assimilation. Proteolysis in the vacuole feeds into the cellular pool of free AAs during the progression of senescence. The steady-state concentrations of free AAs de‐ pend on the rate of their release due to proteolysis and their efflux into growing structures [106]. Soudry et al. (2005) [106] have utilized a bioluminescence assay combined with auxo‐ trophic bacteria for the detection of free tryptophan levels. They assumed that tryptophan reflects the overall pool of free AAs, as it is not modified before its export into sink organs. An accumulation of free AAs was observed in detached oat and Arabidopsis leaves. While attached oat leaves showed a gradual decrease in tryptophan levels during further progres‐ sion of senescence, the attached Arabidopsis leaves did not or only due to membrane leak‐ age resulting from the experimental procedure. The authors concluded that not only source strength but also sink strength is important for successful nutrient remobilization and sug‐ gested that the small reproductive organs of Arabidopsis exerted too weak sink strength. However, these findings might be related to the experimental design, as Arabidopsis does indeed remobilize N for seed filling [70] and Diaz et al. (2005) [52] reported decreasing lev‐ els for several AAs during the progression of leaf senescence in Arabidopsis. Protein break‐ down increases free AAs in the cell. While some seem to be exported without prior modification, many are probably modified, hydrolyzed or interconverted. Expressional

*3.2.4. Re-assimilation and translocation of salvaged nitrogen*

64 Senescence and Senescence-Related Disorders

Asparagine amounts also increase significantly in whole rosettes darkened for several days as well as in senescent leaves (see e.g. [1, 80]). Besides the senescence specific up-regulation of AS, pyruvate orthophosphate dikinase (PPDK) expression is also significantly increased during dark-induced senescence [80]. PPDK might have a role in carbon salvage after lipid degradation, thus Lin and Wu (2004) [80] also investigated other pathways possibly in‐ volved in this process. Remarkably, they found only a few components of these pathways to be up-regulated and many others even down-regulated. Based on their expressional profil‐ ing data, they postulated a alternative pathway for asparagine synthesis, where PPDK deliv‐ ers metabolic precursors [80]. Additionally, seed protein contents were elevated and viability of seedlings was increased on nitrogen-limiting media in Arabidopsis *ASN1* over‐ expressor lines (*35S*::*ASN1*). Furthermore, they observed more Asn to be allocated to flowers and developing siliques and also higher Asn contents in phloem exudates [80, 110].

Nitrogen is not only remobilized from older leaves via amino acids. Nitrate and ammonia are also translocated to developing sink tissues. Fan et al. (2009) [111] identified a nitrate transporter (NRT1-7) which is involved in remobilization processes. Arabidopsis *nrt1-7* mu‐ tants displayed retarded growth under nitrogen starvation conditions. Also the spatial ex‐ pression of this transporter in phloem tissue of older leaves and the expressional induction upon nitrogen starvation points out its function in nitrogen remobilization. Finally, the in‐ ability of *nrt1-7* mutants to remobilize 15N from old to young leaves and the high accumula‐ tion of nitrate in old leaves in this mutants further underlines this assumption [111]. Another nitrate transporter involved in remobilization is NRT2-4. This transporter acts in the high-affinity range and its expression is also induced upon nitrogen starvation. Addi‐ tionally, *nrt2-4* mutant lines had lower phloem sap nitrate contents. However, *nrt2-4* mu‐ tants were not altered in growth or development, indicating that the decreased NO3-levels are not limiting for the adaption to N starvation and most likely functionally redundant transporter systems exist [40].

ROS production is relatively low; however, during stress, the production of ROS is rapidly

Active production of ROS or the so called "oxidative burst" is initiated upon several stresses and developmental stimuli. The main enzymes generating these ROS are the respiratory

apoplast. This is then converted by SODs to H2O2. The function of the 10 different RBOH proteins identified in Arabidopsis [122] is important in various developmental and regulato‐ ry processes. Root elongation is reduced in *atrbohD/F* mutants [122]. ROS produced upon pathogen attack are generated by RBOHs (see for example [123]). Also the response to heavy metals seems to be at least in part mediated by RBOH proteins. Cadmium treated sunflower leaf discs showed an altered expression and activity of the NADPH oxidase [124]. The func‐ tion of these proteins is often associated with the action of Ca2+. Arabidopsis *rbohC/rhd2* mu‐ tants displayed lowered ROS contents in growing root hairs and a distortion in Ca2+ uptake due to a missing activation of Ca2+ channels [125], although for the rice RBOHB homolog cal‐

Reactive nitrogen species (RNS) comprise NO and NO-derived molecules as di-nitrogen tri‐ oxide, nitrogen dioxide, peroxynitrite, S-nitrosothiols and others [127]. NO production in plant cells is under continuous debate. Especially the existence of a plant nitric oxide syn‐ thase (NOS) is a controversial topic. Until today, there is no clear proof for the existence of NOS in plants although there is indirect evidence through the application of NOS inhibitors, which have been established for mammalian cells (e.g. L-NAME a L-arginine analogue) in combination with NR inhibitors, or the measurement of NOS-like activity, like the conver‐ sion of L-arginine to citrulline, where NO is assumed to be produced at the same time [128-130]. AtNOS1 was identified in 2003 by Guo et al. (2003) [131], but is under controver‐ sial discussion ever since. Indeed, *atnos1* mutant plants do exhibit significantly lower NO contents, a chlorotic phenotype in seedlings which can be rescued by NO application and an early-senescence phenotype, but expression of the corresponding genes from Arabidopsis*,* maize and rice revealed no NOS activity *in-vitro*, and even the mammalian orthologous dis‐ played no NOS function [132]. Thus *AtNOS1* was renamed to *AtNOA1* (NO associated 1). Nevertheless, there are other enzymatic ways known to produce NO. NR was found to be able to generate NO. It was shown to be involved in NO generation during the transition to flowering Arabidopsis *nr1* and *nr2* mutants display a low endogenous NO content [133, 134]. Additionally, a NR- and NiR-independent pathway of NO production has been pro‐ posed via electron carriers of the mitochondrial respiratory chain [135] and an oxidation-as‐ sociated pathway for NO synthesis has been suggested as hydroxylamines can be oxidized

Despite all the controversy on the topic of NO generation, it seems clear, that there are many ways to generate NO in plant cells and the pivotal role in many regulatory pathways cannot be denied. Involvement in fruit ripening, leaf senescence, flowering and stomatal closure

and many other processes has been shown ([129] and references within).

 in the 67

Plant Senescence and Nitrogen Mobilization and Signaling

http://dx.doi.org/10.5772/54392

burst oxidase homologs (RBOH) [121]. In a NADPH-dependent reaction they form O2

cium was needed to activate the oxidase itself [126].

by superoxide and H2O2 generating NO [136].

enhanced [120].

#### **4. Reactive oxygen and nitrogen species in signaling**

Reactive oxygen and nitrogen species (ROS, RNS) play a central role in many aspects of plant development and response to environmental influences. These include among others responses to wounding, pathogen infection, drought and water stress, high salinity, cold and heat. In the case of ROS, research has focused especially on H2O2. As this reactive oxy‐ gen molecule is relatively long lived (~1 ms half life), small and uncharged, and thus is able to pass membranes, a central position in various signaling pathways has been attributed to it. Nitric oxide (NO) has been shown to be involved in many of the H2O2-mediated path‐ ways in either a synergistic or antagonistic mode of action. In the following we will briefly introduce the production and scavenging mechanisms for this two reactive oxygen and ni‐ trogen compounds and their interplay in regulation of developmental processes, stress re‐ sponses and senescence will be outlined.

#### **4.1. ROS and RNS: Molecule types, production and scavenging**

Many of the reactive oxygen species in the cell are formed as toxic byproducts of metabolic processes. Photorespiration and β-oxidation of fatty acids produce H2O2 in peroxisomes and glyoxisomes, which is normally scavenged by an extensive protection system mainly con‐ sisting of catalases (CAT) and ascorbate peroxidases (APX). Xanthine oxidase generates su‐ peroxide anions in the peroxisomes, which is converted by superoxide dismutases (SOD) into O2 and H2O2. Chloroplasts are the main site for ROS production in plants. Due to the photooxidative nature of many of their components they can give rise to superoxide radi‐ cals, hydrogen peroxide, hydroxyl radicals and singlet oxygen. ROS produced in the chloro‐ plasts are mainly scavenged by the ascorbate-glutathione cycle [112]. SODs scavenge superoxide anions and dismutate them to O2 and H2O2, which is then in turn reduced to wa‐ ter by the action of ascorbate peroxidase and ascorbate. The resulting monodehydro-ascor‐ bate (MDHA) is regenerated either via the MDHA reductase (MDHAR) under the use of NADPH or it spontaneously converts into dehydroascorbate (DHA) which is then reduced to ascorbate again via the DHA reductase (DHAR). DHAR uses glutathione (GSH) as second substrate. The reduced state of GSH is reconstituted by glutathione reductase (GR). Excess oxidized GSSG seems to be exported from the cytosol to the central vacuole and the chloro‐ plasts to maintain a reduced environment and redox homeostasis in the cytosol and possibly the nucleus [113]. Finally, superoxide radicals can be produced as a byproduct during respi‐ ration in mitochondria. Here, also SOD and the ascorbate-glutathione cycle removes the ROS. Further ROS scavenging in this organelle is mediated by peroxiredoxins and thiore‐ doxins, as it is also observed in chloroplasts. Additionally, non-enzymatic components like tocopherols, flavonoids, ascorbic acid and others are employed in the extensive and elabo‐ rate ROS detoxification system (reviewed in [114-119]). Under optimal growth conditions, ROS production is relatively low; however, during stress, the production of ROS is rapidly enhanced [120].

are not limiting for the adaption to N starvation and most likely functionally redundant

Reactive oxygen and nitrogen species (ROS, RNS) play a central role in many aspects of plant development and response to environmental influences. These include among others responses to wounding, pathogen infection, drought and water stress, high salinity, cold and heat. In the case of ROS, research has focused especially on H2O2. As this reactive oxy‐ gen molecule is relatively long lived (~1 ms half life), small and uncharged, and thus is able to pass membranes, a central position in various signaling pathways has been attributed to it. Nitric oxide (NO) has been shown to be involved in many of the H2O2-mediated path‐ ways in either a synergistic or antagonistic mode of action. In the following we will briefly introduce the production and scavenging mechanisms for this two reactive oxygen and ni‐ trogen compounds and their interplay in regulation of developmental processes, stress re‐

Many of the reactive oxygen species in the cell are formed as toxic byproducts of metabolic processes. Photorespiration and β-oxidation of fatty acids produce H2O2 in peroxisomes and glyoxisomes, which is normally scavenged by an extensive protection system mainly con‐ sisting of catalases (CAT) and ascorbate peroxidases (APX). Xanthine oxidase generates su‐ peroxide anions in the peroxisomes, which is converted by superoxide dismutases (SOD) into O2 and H2O2. Chloroplasts are the main site for ROS production in plants. Due to the photooxidative nature of many of their components they can give rise to superoxide radi‐ cals, hydrogen peroxide, hydroxyl radicals and singlet oxygen. ROS produced in the chloro‐ plasts are mainly scavenged by the ascorbate-glutathione cycle [112]. SODs scavenge superoxide anions and dismutate them to O2 and H2O2, which is then in turn reduced to wa‐ ter by the action of ascorbate peroxidase and ascorbate. The resulting monodehydro-ascor‐ bate (MDHA) is regenerated either via the MDHA reductase (MDHAR) under the use of NADPH or it spontaneously converts into dehydroascorbate (DHA) which is then reduced to ascorbate again via the DHA reductase (DHAR). DHAR uses glutathione (GSH) as second substrate. The reduced state of GSH is reconstituted by glutathione reductase (GR). Excess oxidized GSSG seems to be exported from the cytosol to the central vacuole and the chloro‐ plasts to maintain a reduced environment and redox homeostasis in the cytosol and possibly the nucleus [113]. Finally, superoxide radicals can be produced as a byproduct during respi‐ ration in mitochondria. Here, also SOD and the ascorbate-glutathione cycle removes the ROS. Further ROS scavenging in this organelle is mediated by peroxiredoxins and thiore‐ doxins, as it is also observed in chloroplasts. Additionally, non-enzymatic components like tocopherols, flavonoids, ascorbic acid and others are employed in the extensive and elabo‐ rate ROS detoxification system (reviewed in [114-119]). Under optimal growth conditions,

**4. Reactive oxygen and nitrogen species in signaling**

**4.1. ROS and RNS: Molecule types, production and scavenging**

transporter systems exist [40].

66 Senescence and Senescence-Related Disorders

sponses and senescence will be outlined.

Active production of ROS or the so called "oxidative burst" is initiated upon several stresses and developmental stimuli. The main enzymes generating these ROS are the respiratory burst oxidase homologs (RBOH) [121]. In a NADPH-dependent reaction they form O2 in the apoplast. This is then converted by SODs to H2O2. The function of the 10 different RBOH proteins identified in Arabidopsis [122] is important in various developmental and regulato‐ ry processes. Root elongation is reduced in *atrbohD/F* mutants [122]. ROS produced upon pathogen attack are generated by RBOHs (see for example [123]). Also the response to heavy metals seems to be at least in part mediated by RBOH proteins. Cadmium treated sunflower leaf discs showed an altered expression and activity of the NADPH oxidase [124]. The func‐ tion of these proteins is often associated with the action of Ca2+. Arabidopsis *rbohC/rhd2* mu‐ tants displayed lowered ROS contents in growing root hairs and a distortion in Ca2+ uptake due to a missing activation of Ca2+ channels [125], although for the rice RBOHB homolog cal‐ cium was needed to activate the oxidase itself [126].

Reactive nitrogen species (RNS) comprise NO and NO-derived molecules as di-nitrogen tri‐ oxide, nitrogen dioxide, peroxynitrite, S-nitrosothiols and others [127]. NO production in plant cells is under continuous debate. Especially the existence of a plant nitric oxide syn‐ thase (NOS) is a controversial topic. Until today, there is no clear proof for the existence of NOS in plants although there is indirect evidence through the application of NOS inhibitors, which have been established for mammalian cells (e.g. L-NAME a L-arginine analogue) in combination with NR inhibitors, or the measurement of NOS-like activity, like the conver‐ sion of L-arginine to citrulline, where NO is assumed to be produced at the same time [128-130]. AtNOS1 was identified in 2003 by Guo et al. (2003) [131], but is under controver‐ sial discussion ever since. Indeed, *atnos1* mutant plants do exhibit significantly lower NO contents, a chlorotic phenotype in seedlings which can be rescued by NO application and an early-senescence phenotype, but expression of the corresponding genes from Arabidopsis*,* maize and rice revealed no NOS activity *in-vitro*, and even the mammalian orthologous dis‐ played no NOS function [132]. Thus *AtNOS1* was renamed to *AtNOA1* (NO associated 1). Nevertheless, there are other enzymatic ways known to produce NO. NR was found to be able to generate NO. It was shown to be involved in NO generation during the transition to flowering Arabidopsis *nr1* and *nr2* mutants display a low endogenous NO content [133, 134]. Additionally, a NR- and NiR-independent pathway of NO production has been pro‐ posed via electron carriers of the mitochondrial respiratory chain [135] and an oxidation-as‐ sociated pathway for NO synthesis has been suggested as hydroxylamines can be oxidized by superoxide and H2O2 generating NO [136].

Despite all the controversy on the topic of NO generation, it seems clear, that there are many ways to generate NO in plant cells and the pivotal role in many regulatory pathways cannot be denied. Involvement in fruit ripening, leaf senescence, flowering and stomatal closure and many other processes has been shown ([129] and references within).

#### **4.2. ROS and RNS: Signaling**

The role of H2O2 and NO during the onset of leaf senescence has been investigated in many studies. Recently, an upstream regulator of the ROS network during ABA-mediated drought-induced leaf senescence has been identified. The drought-responsive NAC tran‐ scription factor AtNTL4 (ANAC053) has been shown to promote ROS production by direct‐ ly binding to promoters of genes encoding ROS biosynthetic enzymes [137]. In guard cells, an ABA-H2O2-NO signaling cascade has been proposed for stomatal closure. H2O2-induced generation of NO in guard cells has been reported for mung bean [138], Arabidopsis [139] and other plant species (see for example [140]). Removal of H2O2 as well as the blocking of calcium channels was able to suppress NO generation [138, 141]. A further interaction of NO and H2O2 was studied in tomato (*Lycopersicon esculentum* Mill. cv. "Perkoz") where the effect of application of exogenous NO scavengers and generators was analyzed in combination with *Botrytis cinerea* inoculation. NO generators specifically reduced H2O2 generation and thus allowed the infection to spread significantly under control conditions and in compari‐ son to NO scavenger pre-treated leaves [142]. Moreover, cytoplasmic H2O2 can also directly activate a specific Arabidopsis MAP triple kinase, AtANP1, which initiates a phosphoryla‐ tion cascade involving two stress AtMAPKs, AtMPK3 and AtMPK6 [143]. A direct interac‐ tion between AtMPK6 and AtNR2 during lateral root development has been shown *in-vitro* and *in-vivo*. During this interaction MPK6 phosphorylates and thus activates NR2 resulting in enhanced NO production [144]. Finally, another point of crosstalk between the NO and H2O2 signaling pathways has been referred to by positional cloning of the rice *NOE1*. This gene codes for a rice catalase, a knock-out leads to increased H2O2 contents which in turn enhance the activity of NR, resulting in elevated NO concentrations. The removal of excess NO ameliorated the cell death symptoms of the *noe1* mutants pointing out a cooperative function of H2O2 and NO during induction of PCD. Here, specifically S-nitrosylated proteins were identified, and overexpression of a rice S-nitrosoglutathione reductase could also alle‐ viate the cell death symptoms [145].

ed via ROS and RNS on these residues. Tryptophane residues have been shown to be spe‐ cifically di-oxygenized in plant mitochondria, thus forming N-formylkynurenine. The proteins found to be specifically oxygenized did, with one exception, all posses redox-ac‐ tivity or were involved in redox-active proteins [150]. Another good example for this spe‐ cificity is Rubisco. Preferential oxidation of certain cysteine residues mediates the binding of Rubisco to the chloroplast envelope, thus causing catalytic inactivation and marking it for degradation [151, 152]. Recently, it has been shown, that chloroplast peroxidases are present in an inactivated form and become activated in part by proteolytic cleavage upon a H2O2 signal; in combination with newly synthesized peroxidases, they regulate plasti‐ dial ROS content in neem (*Azadirachta indica* A. juss) chloroplasts [153]. This displays a specificity of ROS induced processes, rather than undirected, detrimental impacts. How‐ ever, how the cell responds differentially to the variety of H2O2 signals in different signal‐ ing pathways is still unclear. With regard to leaf senescence induction, a dependency of H2O2-mediated effects on the subcellular location was discovered. By using an *in-vivo* H2O2-scavenging system, we manipulated H2O2 contents in the cytosol and peroxisomes in Arabidopsis. While both lines showed lowered H2O2 contents and a delayed leaf senes‐ cence phenotype, the delay of the cytoplasmic line was more pronounced, despite the higher expression of the peroxisomal transgene [65]. Furthermore, lowering mitochondri‐ al H2O2 production by blocking cytochrome *c* dependent respiration with the fungal toxin antimycin A had no effect on induction of leaf senescence [154]. Since senescence is pre‐ dominantly regulated on transcriptional level, the cytoplasmic compartment might have a direct influence on redox regulation of transcription factors. Expression of the MAP triple kinase1 (*MEKK1*) of Arabidopsis can also be induced by H2O2 and shows its expression maximum during onset of leaf senescence [155]. Whether H2O2 induced expression of SAGs is transduced by MAPK signaling or directly by redox-sensitive transcription fac‐

Plant Senescence and Nitrogen Mobilization and Signaling

http://dx.doi.org/10.5772/54392

69

Moreover, the already mentioned evidence of numerous selective oxidation reactions on specific amino acid residues depending on the type of ROS/RNS might lead to the degra‐ dation of the damaged proteins, thus generating distinct peptide patterns. These peptides would contain information being ROS- and source-specific (see [150] and references with‐ in). Spatial control might also be a source of specificity, as for example RBOH proteins are membrane bound and, therefore, localization of the ROS signal could be highly specif‐ ic. Additionally, through the extensive detoxification system, ROS signals also might be spatially confined. In contrast, ROS signal auto-propagation over long distances via RBOHD induced by various stimuli has been shown in Arabidopsis [156]. Interestingly, temporal oscillation of ROS bursts has been observed to modulate root tip growth of Ara‐ bidopsis root hairs [157]. Finally, integration of metabolic reactions also seems to be a convenient way of specific signaling. Local blockage or enhancement of certain pathways would lead to the accumulation of intermediates, which in turn could serve for signaling

tors has yet to be elucidated.

functions (reviewed in [158]).

Senescence-inhibiting features of NO have long been recognized, while H2O2 has often been attributed with senescence-promoting features. Exogenous NO application extends postharvest life of fruits and vegetables and, during leaf maturation in pea, NO contents gradu‐ ally decrease [146, 147]. Furthermore, NO-deficient mutants display an early-senescence phenotype and the heterologues expression of an NO-degrading enzyme in Arabidopsis al‐ so leads to early leaf senescence and SAG up-regulation, which could be inhibited by exter‐ nal supply of NO [148]. Remarkably, the senescence delaying features of NO might be achieved due its ability to scavenge various kinds of ROS. In barley aleuron cells, NO has been shown to act as an antioxidant and thus alleviating GA-mediated PCD induction [149].

#### **4.3. Specificity in ROS and RNS signaling**

Some amino acids are more susceptible for modification by ROS and RNS than others. For example cysteins are often found to be preferentially oxidized. These residues are sen‐ sitive for ROS-derived protein carbonylation and RNS-mediated nitrosylation (-SNO) and glutahionylation. Additionally, sulfenic acid and disulfide formation also can be mediat‐ ed via ROS and RNS on these residues. Tryptophane residues have been shown to be spe‐ cifically di-oxygenized in plant mitochondria, thus forming N-formylkynurenine. The proteins found to be specifically oxygenized did, with one exception, all posses redox-ac‐ tivity or were involved in redox-active proteins [150]. Another good example for this spe‐ cificity is Rubisco. Preferential oxidation of certain cysteine residues mediates the binding of Rubisco to the chloroplast envelope, thus causing catalytic inactivation and marking it for degradation [151, 152]. Recently, it has been shown, that chloroplast peroxidases are present in an inactivated form and become activated in part by proteolytic cleavage upon a H2O2 signal; in combination with newly synthesized peroxidases, they regulate plasti‐ dial ROS content in neem (*Azadirachta indica* A. juss) chloroplasts [153]. This displays a specificity of ROS induced processes, rather than undirected, detrimental impacts. How‐ ever, how the cell responds differentially to the variety of H2O2 signals in different signal‐ ing pathways is still unclear. With regard to leaf senescence induction, a dependency of H2O2-mediated effects on the subcellular location was discovered. By using an *in-vivo* H2O2-scavenging system, we manipulated H2O2 contents in the cytosol and peroxisomes in Arabidopsis. While both lines showed lowered H2O2 contents and a delayed leaf senes‐ cence phenotype, the delay of the cytoplasmic line was more pronounced, despite the higher expression of the peroxisomal transgene [65]. Furthermore, lowering mitochondri‐ al H2O2 production by blocking cytochrome *c* dependent respiration with the fungal toxin antimycin A had no effect on induction of leaf senescence [154]. Since senescence is pre‐ dominantly regulated on transcriptional level, the cytoplasmic compartment might have a direct influence on redox regulation of transcription factors. Expression of the MAP triple kinase1 (*MEKK1*) of Arabidopsis can also be induced by H2O2 and shows its expression maximum during onset of leaf senescence [155]. Whether H2O2 induced expression of SAGs is transduced by MAPK signaling or directly by redox-sensitive transcription fac‐ tors has yet to be elucidated.

**4.2. ROS and RNS: Signaling**

68 Senescence and Senescence-Related Disorders

viate the cell death symptoms [145].

**4.3. Specificity in ROS and RNS signaling**

The role of H2O2 and NO during the onset of leaf senescence has been investigated in many studies. Recently, an upstream regulator of the ROS network during ABA-mediated drought-induced leaf senescence has been identified. The drought-responsive NAC tran‐ scription factor AtNTL4 (ANAC053) has been shown to promote ROS production by direct‐ ly binding to promoters of genes encoding ROS biosynthetic enzymes [137]. In guard cells, an ABA-H2O2-NO signaling cascade has been proposed for stomatal closure. H2O2-induced generation of NO in guard cells has been reported for mung bean [138], Arabidopsis [139] and other plant species (see for example [140]). Removal of H2O2 as well as the blocking of calcium channels was able to suppress NO generation [138, 141]. A further interaction of NO and H2O2 was studied in tomato (*Lycopersicon esculentum* Mill. cv. "Perkoz") where the effect of application of exogenous NO scavengers and generators was analyzed in combination with *Botrytis cinerea* inoculation. NO generators specifically reduced H2O2 generation and thus allowed the infection to spread significantly under control conditions and in compari‐ son to NO scavenger pre-treated leaves [142]. Moreover, cytoplasmic H2O2 can also directly activate a specific Arabidopsis MAP triple kinase, AtANP1, which initiates a phosphoryla‐ tion cascade involving two stress AtMAPKs, AtMPK3 and AtMPK6 [143]. A direct interac‐ tion between AtMPK6 and AtNR2 during lateral root development has been shown *in-vitro* and *in-vivo*. During this interaction MPK6 phosphorylates and thus activates NR2 resulting in enhanced NO production [144]. Finally, another point of crosstalk between the NO and H2O2 signaling pathways has been referred to by positional cloning of the rice *NOE1*. This gene codes for a rice catalase, a knock-out leads to increased H2O2 contents which in turn enhance the activity of NR, resulting in elevated NO concentrations. The removal of excess NO ameliorated the cell death symptoms of the *noe1* mutants pointing out a cooperative function of H2O2 and NO during induction of PCD. Here, specifically S-nitrosylated proteins were identified, and overexpression of a rice S-nitrosoglutathione reductase could also alle‐

Senescence-inhibiting features of NO have long been recognized, while H2O2 has often been attributed with senescence-promoting features. Exogenous NO application extends postharvest life of fruits and vegetables and, during leaf maturation in pea, NO contents gradu‐ ally decrease [146, 147]. Furthermore, NO-deficient mutants display an early-senescence phenotype and the heterologues expression of an NO-degrading enzyme in Arabidopsis al‐ so leads to early leaf senescence and SAG up-regulation, which could be inhibited by exter‐ nal supply of NO [148]. Remarkably, the senescence delaying features of NO might be achieved due its ability to scavenge various kinds of ROS. In barley aleuron cells, NO has been shown to act as an antioxidant and thus alleviating GA-mediated PCD induction [149].

Some amino acids are more susceptible for modification by ROS and RNS than others. For example cysteins are often found to be preferentially oxidized. These residues are sen‐ sitive for ROS-derived protein carbonylation and RNS-mediated nitrosylation (-SNO) and glutahionylation. Additionally, sulfenic acid and disulfide formation also can be mediat‐ Moreover, the already mentioned evidence of numerous selective oxidation reactions on specific amino acid residues depending on the type of ROS/RNS might lead to the degra‐ dation of the damaged proteins, thus generating distinct peptide patterns. These peptides would contain information being ROS- and source-specific (see [150] and references with‐ in). Spatial control might also be a source of specificity, as for example RBOH proteins are membrane bound and, therefore, localization of the ROS signal could be highly specif‐ ic. Additionally, through the extensive detoxification system, ROS signals also might be spatially confined. In contrast, ROS signal auto-propagation over long distances via RBOHD induced by various stimuli has been shown in Arabidopsis [156]. Interestingly, temporal oscillation of ROS bursts has been observed to modulate root tip growth of Ara‐ bidopsis root hairs [157]. Finally, integration of metabolic reactions also seems to be a convenient way of specific signaling. Local blockage or enhancement of certain pathways would lead to the accumulation of intermediates, which in turn could serve for signaling functions (reviewed in [158]).

#### **5. Concluding remarks**

The intriguing connection between efficient nutrient remobilization and progression of leaf senescence is obvious. The correct timing of onset and progression of senescence has great influence on seed and fruit development and viability. Therefore, manipulating leaf senes‐ cence seems to be a promising trait to increase yield in various crop species. Functional stay green traits can prolong carbon assimilation and thus increase yield. However, a too strong delay in leaf senescence might hamper nutrient and especially nitrogen remobilization from the leaves. For various wheat mutants, Derkx et al. (2012) [159] speculated that the stay green phenotype might be associated with a decrease in grain N sink strength. Gpc-B1, a QTL locus in wheat, which is among others associated with increased grain protein content, has been shown to encode a NAC transcription factor (*NAM-B1*). It accelerates senescence and enhances nutrient remobilization from leaves. RNA interference mediated silencing of multiple homologues resulted in a delay of leaf senescence by approximately 3 weeks and decreased grain protein, iron and zinc content by more than 30% [160]. This indicates that the relation between senescence and nitrogen mobilization is very complicated and cannot be modified as easy as expected.

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Besides QTL selection, transgenic approaches to increase nitrogen use efficiency in crop plants have been extensively studied. For example expression of alanine aminotransferase and asparagine synthase often resulted in enhanced seed protein content and higher seed yield. Increased cytokinin biosynthesis almost always resulted in delayed senescence and was sometimes associated with higher seed yield, seed protein content and increased bio‐ mass. Expression of amino acid permease from V*icia faba* under the *LeB4* promoter increased the seed size by 20-30%, as well as the abundance of nitrogen rich AAs and the content of seed storage proteins in the seeds (reviewed in [161]).

Nevertheless, although transgenic approaches have proven to enhance nitrogen use effi‐ ciencies and yield quantity as well as quality, these techniques have to cope with general skepticism on the consumer's side. Although approval for the agricultural use of geneti‐ cally modified organisms has been extensively performed like e.g. in the Swiss National Research program NRP 59 (Benefits and risks of the deliberated release of genetically modified plants) clearly indicating a low risk and a enormously high potential of trans‐ genic crop plants, problems with the acceptance of this technology, especially in Europe, still have to be faced.

#### **Author details**

Stefan Bieker and Ulrike Zentgraf

General Genetics, University of Tuebingen, Tuebingen, Germany

#### **References**

**5. Concluding remarks**

70 Senescence and Senescence-Related Disorders

be modified as easy as expected.

still have to be faced.

**Author details**

Stefan Bieker and Ulrike Zentgraf

seed storage proteins in the seeds (reviewed in [161]).

General Genetics, University of Tuebingen, Tuebingen, Germany

The intriguing connection between efficient nutrient remobilization and progression of leaf senescence is obvious. The correct timing of onset and progression of senescence has great influence on seed and fruit development and viability. Therefore, manipulating leaf senes‐ cence seems to be a promising trait to increase yield in various crop species. Functional stay green traits can prolong carbon assimilation and thus increase yield. However, a too strong delay in leaf senescence might hamper nutrient and especially nitrogen remobilization from the leaves. For various wheat mutants, Derkx et al. (2012) [159] speculated that the stay green phenotype might be associated with a decrease in grain N sink strength. Gpc-B1, a QTL locus in wheat, which is among others associated with increased grain protein content, has been shown to encode a NAC transcription factor (*NAM-B1*). It accelerates senescence and enhances nutrient remobilization from leaves. RNA interference mediated silencing of multiple homologues resulted in a delay of leaf senescence by approximately 3 weeks and decreased grain protein, iron and zinc content by more than 30% [160]. This indicates that the relation between senescence and nitrogen mobilization is very complicated and cannot

Besides QTL selection, transgenic approaches to increase nitrogen use efficiency in crop plants have been extensively studied. For example expression of alanine aminotransferase and asparagine synthase often resulted in enhanced seed protein content and higher seed yield. Increased cytokinin biosynthesis almost always resulted in delayed senescence and was sometimes associated with higher seed yield, seed protein content and increased bio‐ mass. Expression of amino acid permease from V*icia faba* under the *LeB4* promoter increased the seed size by 20-30%, as well as the abundance of nitrogen rich AAs and the content of

Nevertheless, although transgenic approaches have proven to enhance nitrogen use effi‐ ciencies and yield quantity as well as quality, these techniques have to cope with general skepticism on the consumer's side. Although approval for the agricultural use of geneti‐ cally modified organisms has been extensively performed like e.g. in the Swiss National Research program NRP 59 (Benefits and risks of the deliberated release of genetically modified plants) clearly indicating a low risk and a enormously high potential of trans‐ genic crop plants, problems with the acceptance of this technology, especially in Europe,


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82 Senescence and Senescence-Related Disorders

39-52.


**Section 4**

**Immunosenescence and Cancer**

**Immunosenescence and Cancer**

**Chapter 4**

**Immunosenescence and Senescence**

Arnaud Augert and David Bernard

http://dx.doi.org/10.5772/55519

**1. Introduction**

**2. Cellular senescence**

**2.1. Historical discovery of cellular senescence**

Additional information is available at the end of the chapter

**Immunosurveillance: One of the Possible Links**

**Explaining the Cancer Incidence in Ageing Population**

Since the discovery of cellular senescence significant advances were made to understand its moleculardeterminantsanditsphysiologicalroleinbiologicalprocessessuchascancerandaging [1]. Recently, an intimate and complex relationship between senescent cells and the immune system has been highlighted [2]. In addition to their role in senescence immunosurveillance, immune cells display altered functions with age. This process is known as immunosenescence. Althoughimmunosenescenceisaslightlydifferentmechanismthancellularsenescence,itshares some similarities. This review article briefly describes features, markers, triggers and molecular regulators of cellular senescence and focuses on its role during cancer development. We then introduce immunosenescence and highlight what might be its consequences in cancer develop‐ ment. Finally, taking into account that senescence immunosurveillance is crucial for tumor eradication[2],weprovideseveralhypothesestoexplainwhatcouldbetheimpactofimmunose‐

Senescence comes from the Latin word *senex* meaning old age. It was observed approximately half a century ago by Leonard Hayflick while cultivating primary human fibroblast [3, 4]. He observed that primary cells proliferate in culture for approximately 55 population doublings before reaching the "Hayflick limit" which marks the end of their proliferative capacity and

and reproduction in any medium, provided the original work is properly cited.

© 2013 Augert and Bernard; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

nescence on senescence immunosurveillance in a specific cancer context.

## **Immunosenescence and Senescence Immunosurveillance: One of the Possible Links Explaining the Cancer Incidence in Ageing Population**

Arnaud Augert and David Bernard

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55519

### **1. Introduction**

Since the discovery of cellular senescence significant advances were made to understand its moleculardeterminantsanditsphysiologicalroleinbiologicalprocessessuchascancerandaging [1]. Recently, an intimate and complex relationship between senescent cells and the immune system has been highlighted [2]. In addition to their role in senescence immunosurveillance, immune cells display altered functions with age. This process is known as immunosenescence. Althoughimmunosenescenceisaslightlydifferentmechanismthancellularsenescence,itshares some similarities. This review article briefly describes features, markers, triggers and molecular regulators of cellular senescence and focuses on its role during cancer development. We then introduce immunosenescence and highlight what might be its consequences in cancer develop‐ ment. Finally, taking into account that senescence immunosurveillance is crucial for tumor eradication[2],weprovideseveralhypothesestoexplainwhatcouldbetheimpactofimmunose‐ nescence on senescence immunosurveillance in a specific cancer context.

### **2. Cellular senescence**

#### **2.1. Historical discovery of cellular senescence**

Senescence comes from the Latin word *senex* meaning old age. It was observed approximately half a century ago by Leonard Hayflick while cultivating primary human fibroblast [3, 4]. He observed that primary cells proliferate in culture for approximately 55 population doublings before reaching the "Hayflick limit" which marks the end of their proliferative capacity and

© 2013 Augert and Bernard; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the entry into an irreversible growth arrest state. He proposed a theory "that the finite lifetime of diploid cells strains *in vitro* may be an expression of ageing or senescence at the cellular level". Since, the term "replicative senescence" has been used to designate this type of cellular senescence but, as we will see, senescence can also be induced in a replicative independent manner in response to various cellular insults. Senescence is not limited to human primary fibroblast as it has been observed in various primary cells [5-8] including immune cells [9] and also takes place in other species such as Mouse [10], Rat [11], Chicken [12], Caenorhabditis elegans [13], Zebrafish [14] and Yeast [15].

co-factors that interact with and inhibit E2F transcription factors activity required for DNA synthesis. Upon the phosphorylation of RB family members by the CDK-type D and E cyclin complexes, E2F transcription factors are released from their interaction with RB proteins leading to cell cycle progression [33] (Figure 2). The CDK4/6-type D cyclins complexes interact with KIP/CIP inhibitors. However, during cellular senescence, INK4 proteins increase and inhibit CDK4/6-cyclin D formation [32]. This enables the activation of RB family members and the inhibition of E2F transcription factors. Additionally, the bioavailability of KIP and CIP proteins increases and they no longer interact with CDK4/6/Cyclin D complexes. It allows them

Immunosenescence and Senescence Immunosurveillance: One of the Possible Links Explaining the...

Senescent cells

CIP KIP

**G1**

E2F

RB

**S**

Cyclin E CIP CDK2 KIP

http://dx.doi.org/10.5772/55519

89

INK4

CDK4/6

Cyclin D

to interact and inhibit CDK2-type E or A cyclin complexes (Figure 2).

Cyclin E CDK2

P P P P P P P P

RB

E2F

E2F

G1 cell cycle arrest.

*2.2.3. Altered gene expression*

RB

P

P

**G1**

Proliferating cells

**S**

In proliferating cells the CKI levels are low, the CDK/cyclin complexes are functional and RB family members are found hyperphosphorylated leading the E2F family members' activation and G1 progression. In senescent cells, the levels of CKI increase, CDK/Cyclin complexes are inhibited and RB family members are active leading to E2F transcription factors inhibition and

Senescent cells also display a specific gene expression signature. A notable example are senescence-associated-secretory-phenotype (SASP) molecules such as interleukin 6 (IL-6), interleukin 8 (IL-8), plasminogen activator inhibitor 1 (PAI-1), insulin growth factor binding

**Figure 2.** Cell cycle arrest (G1 phase) associated with cellular senescence.

Cyclin D

CDK4/6 CIP KIP

#### **2.2. Features of cellular senescence**

#### *2.2.1. Morphology*

Senescent cells lose their original morphology. Larger than their normal counterparts, they also have a much larger flattened cytoplasm that contain many vacuoles and cytoplasmic filaments [16, 17], a bigger nucleus and nucleoli and are sometimes multinucleated [18, 19]. In some cases, senescent cells display an increase in the number of lysosomes and golgi [20-24].

**Figure 1.** Morphology analysis of primary human fibroblasts (proliferating versus senescent).

#### *2.2.2. Growth arrest*

One of the most obvious features of cellular senescence is growth arrest. Indeed, cells are usually blocked in the G1 phase of the cell cycle [25] and in some cases they display (4n) DNA suggesting that cells are either blocked in the late S, G2 or M phases [26, 27]. Cell cycle progression is regulated by cyclin dependent kinases (CDKs) that bind to cyclins [28]. These complexes are regulated by cyclin dependent kinase inhibitors (CKIs) which are essential for the establishment of the senescent growth arrest state [29]. CKIs are divided into two families. The CIP (CDK-interacting protein) and KIP (cyclin-dependent kinase inhibitor protein) [30] and the INK4, for inhibitors of CDK4 [31]. CDK-cyclin complexes favour G1 cell cycle pro‐ gression by phosphorylating RB family members [32]. RB family proteins are transcriptional co-factors that interact with and inhibit E2F transcription factors activity required for DNA synthesis. Upon the phosphorylation of RB family members by the CDK-type D and E cyclin complexes, E2F transcription factors are released from their interaction with RB proteins leading to cell cycle progression [33] (Figure 2). The CDK4/6-type D cyclins complexes interact with KIP/CIP inhibitors. However, during cellular senescence, INK4 proteins increase and inhibit CDK4/6-cyclin D formation [32]. This enables the activation of RB family members and the inhibition of E2F transcription factors. Additionally, the bioavailability of KIP and CIP proteins increases and they no longer interact with CDK4/6/Cyclin D complexes. It allows them to interact and inhibit CDK2-type E or A cyclin complexes (Figure 2).

**Figure 2.** Cell cycle arrest (G1 phase) associated with cellular senescence.

In proliferating cells the CKI levels are low, the CDK/cyclin complexes are functional and RB family members are found hyperphosphorylated leading the E2F family members' activation and G1 progression. In senescent cells, the levels of CKI increase, CDK/Cyclin complexes are inhibited and RB family members are active leading to E2F transcription factors inhibition and G1 cell cycle arrest.

#### *2.2.3. Altered gene expression*

the entry into an irreversible growth arrest state. He proposed a theory "that the finite lifetime of diploid cells strains *in vitro* may be an expression of ageing or senescence at the cellular level". Since, the term "replicative senescence" has been used to designate this type of cellular senescence but, as we will see, senescence can also be induced in a replicative independent manner in response to various cellular insults. Senescence is not limited to human primary fibroblast as it has been observed in various primary cells [5-8] including immune cells [9] and also takes place in other species such as Mouse [10], Rat [11], Chicken [12], Caenorhabditis

Senescent cells lose their original morphology. Larger than their normal counterparts, they also have a much larger flattened cytoplasm that contain many vacuoles and cytoplasmic filaments [16, 17], a bigger nucleus and nucleoli and are sometimes multinucleated [18, 19]. In some cases, senescent cells display an increase in the number of lysosomes and golgi [20-24].

proliferating senescent

One of the most obvious features of cellular senescence is growth arrest. Indeed, cells are usually blocked in the G1 phase of the cell cycle [25] and in some cases they display (4n) DNA suggesting that cells are either blocked in the late S, G2 or M phases [26, 27]. Cell cycle progression is regulated by cyclin dependent kinases (CDKs) that bind to cyclins [28]. These complexes are regulated by cyclin dependent kinase inhibitors (CKIs) which are essential for the establishment of the senescent growth arrest state [29]. CKIs are divided into two families. The CIP (CDK-interacting protein) and KIP (cyclin-dependent kinase inhibitor protein) [30] and the INK4, for inhibitors of CDK4 [31]. CDK-cyclin complexes favour G1 cell cycle pro‐ gression by phosphorylating RB family members [32]. RB family proteins are transcriptional

**Figure 1.** Morphology analysis of primary human fibroblasts (proliferating versus senescent).

WI38

elegans [13], Zebrafish [14] and Yeast [15].

**2.2. Features of cellular senescence**

88 Senescence and Senescence-Related Disorders

*2.2.1. Morphology*

*2.2.2. Growth arrest*

Senescent cells also display a specific gene expression signature. A notable example are senescence-associated-secretory-phenotype (SASP) molecules such as interleukin 6 (IL-6), interleukin 8 (IL-8), plasminogen activator inhibitor 1 (PAI-1), insulin growth factor binding protein 7 (IGBP7), (ECM) degradation enzymes such as collagenase and metalloprotease (MMPs) but also CKIs, CIP and KIP as previously mentioned [34]. Some genes are also found down-regulated during cellular senescence. It is the case for polycomb complex members such as enhancer of zeste homolog 2 (EZH2) and chromobox homolog 7 (CBX7) [5, 35].

#### *2.2.4. Senescence markers*

Several markers have been used to specifically identify senescent cells. In addition to an altered cell morphology, growth arrest state and a specific gene expression signature, senescent cells are associated with an increased β-galactosidase activity, detectable at pH 6.0 and known as "senescence-associated-beta-galactosidase (SA-βgal) activity" [36] (Figure 3).

TIN2-interacting protein (TPP1) and protection of telomeres 1 (POT1) (Figure 5). The last two components, TPP1 and POT1 regulate the access of the telomeric substrate for the telomerase [40]. Telomere lengths are maintained by telomerase, a ribonucleoprotein complex that includes a RNA template (known as TERC) and the reverse transcriptase catalytic subunit (TERT) (Figure 5) [40]. Telomerase activity is mainly dictated by the TERT expression, as TERC seems to be ubiquitously transcribed [41]. In 1990, it was noticed that the telomeres length decreased during serially passage human primary fibroblasts and it was suggested that telomere size might be responsible for replicative senescence [42]. Eight years later, it was functionally demonstrated that re-introducing telomerase expression in normal primary cells led to elongated telomeres, lifespan extension and abrogation of replicative senescence [43, 44]. Since then, telomerase re-expression alone [45] or in combination with other alterations [46] has been associated with the immortalisation of various human cells types. Although telomere size is a critical trigger for replicative senescence, telomere structure is also a main determinant [47, 48]. In conclusion, dysfunctional telomeres both short or with an altered

**Figure 4.** Senescence-associated-heterochromatin-foci (SAHF) of normal and senescent primary human fibroblasts.

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In 1997, it was observed that in response to an oncogenic form of Ras (H-RasG12V) primary cells entered a premature senescence state [50]. Oncogene-induced senescence (OIS) is not restricted to Ras but can be extended to most of the MAPK pathway actors (Raf, Mek) and other oncogenic pathways. In accordance, the loss of bona fide tumour suppressors genes that restrain the activity of oncogenic pathways, such as phosphatase and tensin homolog (PTEN), Retinoblas‐ toma (Rb), von Hippel-Lindau tumor suppressor (VHL) and neurofibromin 1 (NF1), has also been associated with premature senescence [10]. OIS is suggested to be a replicative senescence independent mechanism as cells expressing the catalytic subunit of the telomerase (TERT) still undergo OIS [51]. Whereas senescence in ageing tissues had been observed since 1995 [36], the

structure trigger replicative senescence.

Dots representing SAHF are visible in senescent cells.

*2.4.1. Oncogene-induced senescence (OIS)*

**2.4. Premature senescence**

**Figure 3.** Senescence-associated-beta-galactosidase (SA-βgal) activity detectable at pH6 and yelling a blue colour in senescent cells. Primary human fibroblasts were used for the analysis.

At a chromatin level, specific facultative heterochromatin structures associated with senescent cells were discovered and termed senescence-associated-heterochromatin-foci (SAHF) [37]. These structures regulated by the Retinoblastoma gene (RB) are involved in E2F target genes repression and maintain the cell cycle arrest. They contain markers of heterochromatin such as hypo-acetylated histones, methylated histones (H3K9Me) and the presence of heterochro‐ matin protein 1 (HP1). The histone variant macroH2A, and HMGA a non histone protein, have been identified as crucial regulators in SAHF formation [38] (Figure 4).

#### **2.3. Replicative senescence**

Although replicative senescence was first observed in 1961, it took more than 30 years to gain insights into the molecular regulators underlying this process. We now know that it is largely due to telomere lengths and structures. Telomeres are protective structures that cap the end of all eukaryotic chromosomes. They are long double stranded DNA sequences composed of TTAGGG repeats, oriented 5'-to-3' towards the end of the chromosome (Figure 5) [39]. Telomeres contain a complex composed of six proteins known as the shelterin complex. It comprises telomeric repeat binding factor 1 (TRF1), telomeric repeat binding factor 2 (TRF2), transcriptional repressor/activator protein (RAP1), TRF1-interacting nuclear factor 2 (TIN2), Immunosenescence and Senescence Immunosurveillance: One of the Possible Links Explaining the... http://dx.doi.org/10.5772/55519 91

**Figure 4.** Senescence-associated-heterochromatin-foci (SAHF) of normal and senescent primary human fibroblasts. Dots representing SAHF are visible in senescent cells.

TIN2-interacting protein (TPP1) and protection of telomeres 1 (POT1) (Figure 5). The last two components, TPP1 and POT1 regulate the access of the telomeric substrate for the telomerase [40]. Telomere lengths are maintained by telomerase, a ribonucleoprotein complex that includes a RNA template (known as TERC) and the reverse transcriptase catalytic subunit (TERT) (Figure 5) [40]. Telomerase activity is mainly dictated by the TERT expression, as TERC seems to be ubiquitously transcribed [41]. In 1990, it was noticed that the telomeres length decreased during serially passage human primary fibroblasts and it was suggested that telomere size might be responsible for replicative senescence [42]. Eight years later, it was functionally demonstrated that re-introducing telomerase expression in normal primary cells led to elongated telomeres, lifespan extension and abrogation of replicative senescence [43, 44]. Since then, telomerase re-expression alone [45] or in combination with other alterations [46] has been associated with the immortalisation of various human cells types. Although telomere size is a critical trigger for replicative senescence, telomere structure is also a main determinant [47, 48]. In conclusion, dysfunctional telomeres both short or with an altered structure trigger replicative senescence.

#### **2.4. Premature senescence**

protein 7 (IGBP7), (ECM) degradation enzymes such as collagenase and metalloprotease (MMPs) but also CKIs, CIP and KIP as previously mentioned [34]. Some genes are also found down-regulated during cellular senescence. It is the case for polycomb complex members such

Several markers have been used to specifically identify senescent cells. In addition to an altered cell morphology, growth arrest state and a specific gene expression signature, senescent cells are associated with an increased β-galactosidase activity, detectable at pH 6.0 and known as

**Figure 3.** Senescence-associated-beta-galactosidase (SA-βgal) activity detectable at pH6 and yelling a blue colour in

At a chromatin level, specific facultative heterochromatin structures associated with senescent cells were discovered and termed senescence-associated-heterochromatin-foci (SAHF) [37]. These structures regulated by the Retinoblastoma gene (RB) are involved in E2F target genes repression and maintain the cell cycle arrest. They contain markers of heterochromatin such as hypo-acetylated histones, methylated histones (H3K9Me) and the presence of heterochro‐ matin protein 1 (HP1). The histone variant macroH2A, and HMGA a non histone protein, have

Although replicative senescence was first observed in 1961, it took more than 30 years to gain insights into the molecular regulators underlying this process. We now know that it is largely due to telomere lengths and structures. Telomeres are protective structures that cap the end of all eukaryotic chromosomes. They are long double stranded DNA sequences composed of TTAGGG repeats, oriented 5'-to-3' towards the end of the chromosome (Figure 5) [39]. Telomeres contain a complex composed of six proteins known as the shelterin complex. It comprises telomeric repeat binding factor 1 (TRF1), telomeric repeat binding factor 2 (TRF2), transcriptional repressor/activator protein (RAP1), TRF1-interacting nuclear factor 2 (TIN2),

senescent cells. Primary human fibroblasts were used for the analysis.

**2.3. Replicative senescence**

been identified as crucial regulators in SAHF formation [38] (Figure 4).

as enhancer of zeste homolog 2 (EZH2) and chromobox homolog 7 (CBX7) [5, 35].

"senescence-associated-beta-galactosidase (SA-βgal) activity" [36] (Figure 3).

*2.2.4. Senescence markers*

90 Senescence and Senescence-Related Disorders

#### *2.4.1. Oncogene-induced senescence (OIS)*

In 1997, it was observed that in response to an oncogenic form of Ras (H-RasG12V) primary cells entered a premature senescence state [50]. Oncogene-induced senescence (OIS) is not restricted to Ras but can be extended to most of the MAPK pathway actors (Raf, Mek) and other oncogenic pathways. In accordance, the loss of bona fide tumour suppressors genes that restrain the activity of oncogenic pathways, such as phosphatase and tensin homolog (PTEN), Retinoblas‐ toma (Rb), von Hippel-Lindau tumor suppressor (VHL) and neurofibromin 1 (NF1), has also been associated with premature senescence [10]. OIS is suggested to be a replicative senescence independent mechanism as cells expressing the catalytic subunit of the telomerase (TERT) still undergo OIS [51]. Whereas senescence in ageing tissues had been observed since 1995 [36], the

determinants of stress-induced-premature-senescence (SIPS). Oxygen singlet (O2) is not toxic for cells however O2 consumption leads to reactive oxygen species (ROS) [21]. ROS can be classified into two groups. The first group which includes reactive species such as superoxide and hydroxyl radicals is composed of molecules with free radical containing one or more unpaired electrons in their outer molecular orbitals. The second group which includes hydrogen peroxide, ozone, peroxinitrate and hydroxide is composed of non-radical ROS that

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Evidences demonstrating that ROS can trigger cellular senescence are now common. For example, cultivating primary human cells in 3% O2 levels which is closer to the physiological conditions (normal physiological conditions vary from 1-2% in some parts of the brain, skin, heart and kidney to 14% in the lungs) [61], allowed cells to undergo 20 supplemental popula‐ tions doublings before reaching replicative senescence [62]. Conversely, raising the oxygen levels over 20% or exposing cells to sublethal doses of ROS such as H202 led to a premature

SIPS is not restrained to oxidative stress. It can be induced in response to additional inadequate culturing conditions such as abnormal growth factors, the absence of neighbour cells and extracellular matrix components and inadequate concentrations of nutrients, [45, 65, 66]. Other physical, chemical and cellular stressors such as mitomycin C and ionizing radiation can also

The INK4B/ARF/INK4A locus encodes three tumour suppressor genes that play critical regulatory roles in cellular senescence [31]. Two of these tumour suppressors, p15INK4B and p16INK4A, are cyclin dependent kinase inhibitors (CKIs) that trigger cell cycle arrest by inhibiting CDK4 and CDK6 complexes [34]. ARF, the third gene encoded by the locus, is a critical regulator of the p53 tumour suppressor pathway [68]. ARF and p16INK4A share common exons but are encoded in alternative reading frames leading to the production of unique proteins [31].

Polycomb repressive group complexes (PRCs) play a crucial role in the regulation of the locus [69]. The polycomb family is composed of two repressive complexes PRC1 and PRC2. PRC2 establishes the repressive mark leading to the recruitment of the maintenance polycomb complex PRC1. Various polycomb proteins such as CBX7, CBX8, TBX2, TBX3 and Bmi1 have been functionally implicated in the repression of cellular senescence by inhibiting the INK4B/ ARF/INK4A locus [5, 70-72]. To counteract the repressive action of the polycomb complexes, several mechanism are activated during cellular senescence. These include the removal or inhibition of polycomb complexes by MAPKAP [73], by the chromatin remodeling SWI/SNF complex [74] and/or the activation of the INK4B/ARF/INK4A locus by JMJD3 [75, 76] (Figure 6).

Additional regulators of the locus include the stress activated p38MAPKinase. Activated in response to various stresses such as high levels of ROS, dysfunctional telomeres and OIS, it regulates cellular senescence in various contexts [77]. It can activate p16INK4A, p15INK4B and ARF

remain chemically reactive and can be converted to radical ROS [60].

senescence like state [63, 64].

*2.5.1. The INK4B/ARF/INK4A*

**2.5. Pathways regulating cellular senescence**

trigger SIPS [67].

**Figure 5.** The telomeres and the telomerase. Telomeres, TTAGGG repeats, oriented 5' to 3' towards the end of the chromosome are regulated by six proteins that make up the shelterin complex. The telomeres lengths are maintained by the telomerase, a ribonucleoprotein complex composed of a RNA template (TERC) and the reverse transcriptase catalytic subunit (TERT). Adapted from [49].

demonstration of OIS in a physiological setting was demonstrated ten years later using various mouse model expressing a hyper-active oncogene form or a deleted tumor suppressor [10].

#### *2.4.2. Oncogene-inactivation-induced senescence (OIIS)*

Cellular senescence is not limited to primary cells but can also be triggered in cancer cells [52]. Cancer cells develop oncogene addiction, a term to describe a cell dependence on an oncogenic pathway to maintain its tumoral properties [53]. Various groups have been trying to identify these oncogene addictions using synthetic lethal screening [53, 54]. Interestingly, targeting oncogene addiction can result in cellular senescence. For example the inhibition of CDK4, a cyclin dependant kinase involved in cell cycle progression, in K-RasG12V driven non small cell lung carcinoma is associated with cellular senescence induction [55]. Moreover, the inhibition of several embryonic factors known to exert oncogenic properties (T-box 2 (TBX2), twist homolog 1/2 (TWIST 1/2)) can also result in cellular senescence [56, 57]. Finally c-Myc inhibi‐ tion, a bHLH-LZ transcription factor involved in several tumoral processes such as prolifera‐ tion, angiogenesis and cell metabolism [58], can leads to senescence induction in cancer cell lines and also in c-Myc transgenic driven lymphomas or osteosarcomas mouse models [59].

#### *2.4.3. Stress-induced premature senescence (SIPS)*

Stresses are major determinants of cellular senescence [21]. Even if replicative senescence, OIS and OIIS can also generate similar stresses and could be classified in the stress-induced cellular senescence category, we decided to write a specific section for SIPS. Oxygen is one of the major determinants of stress-induced-premature-senescence (SIPS). Oxygen singlet (O2) is not toxic for cells however O2 consumption leads to reactive oxygen species (ROS) [21]. ROS can be classified into two groups. The first group which includes reactive species such as superoxide and hydroxyl radicals is composed of molecules with free radical containing one or more unpaired electrons in their outer molecular orbitals. The second group which includes hydrogen peroxide, ozone, peroxinitrate and hydroxide is composed of non-radical ROS that remain chemically reactive and can be converted to radical ROS [60].

Evidences demonstrating that ROS can trigger cellular senescence are now common. For example, cultivating primary human cells in 3% O2 levels which is closer to the physiological conditions (normal physiological conditions vary from 1-2% in some parts of the brain, skin, heart and kidney to 14% in the lungs) [61], allowed cells to undergo 20 supplemental popula‐ tions doublings before reaching replicative senescence [62]. Conversely, raising the oxygen levels over 20% or exposing cells to sublethal doses of ROS such as H202 led to a premature senescence like state [63, 64].

SIPS is not restrained to oxidative stress. It can be induced in response to additional inadequate culturing conditions such as abnormal growth factors, the absence of neighbour cells and extracellular matrix components and inadequate concentrations of nutrients, [45, 65, 66]. Other physical, chemical and cellular stressors such as mitomycin C and ionizing radiation can also trigger SIPS [67].

#### **2.5. Pathways regulating cellular senescence**

#### *2.5.1. The INK4B/ARF/INK4A*

demonstration of OIS in a physiological setting was demonstrated ten years later using various mouse model expressing a hyper-active oncogene form or a deleted tumor suppressor [10].

**Figure 5.** The telomeres and the telomerase. Telomeres, TTAGGG repeats, oriented 5' to 3' towards the end of the chromosome are regulated by six proteins that make up the shelterin complex. The telomeres lengths are maintained by the telomerase, a ribonucleoprotein complex composed of a RNA template (TERC) and the reverse transcriptase

TRF1 TRF2

TIN2

RAP1

TPP1

POT1

telomerase

TERT

TERC

Cellular senescence is not limited to primary cells but can also be triggered in cancer cells [52]. Cancer cells develop oncogene addiction, a term to describe a cell dependence on an oncogenic pathway to maintain its tumoral properties [53]. Various groups have been trying to identify these oncogene addictions using synthetic lethal screening [53, 54]. Interestingly, targeting oncogene addiction can result in cellular senescence. For example the inhibition of CDK4, a cyclin dependant kinase involved in cell cycle progression, in K-RasG12V driven non small cell lung carcinoma is associated with cellular senescence induction [55]. Moreover, the inhibition of several embryonic factors known to exert oncogenic properties (T-box 2 (TBX2), twist homolog 1/2 (TWIST 1/2)) can also result in cellular senescence [56, 57]. Finally c-Myc inhibi‐ tion, a bHLH-LZ transcription factor involved in several tumoral processes such as prolifera‐ tion, angiogenesis and cell metabolism [58], can leads to senescence induction in cancer cell lines and also in c-Myc transgenic driven lymphomas or osteosarcomas mouse models [59].

Stresses are major determinants of cellular senescence [21]. Even if replicative senescence, OIS and OIIS can also generate similar stresses and could be classified in the stress-induced cellular senescence category, we decided to write a specific section for SIPS. Oxygen is one of the major

*2.4.2. Oncogene-inactivation-induced senescence (OIIS)*

catalytic subunit (TERT). Adapted from [49].

Telomeres

92 Senescence and Senescence-Related Disorders

*2.4.3. Stress-induced premature senescence (SIPS)*

The INK4B/ARF/INK4A locus encodes three tumour suppressor genes that play critical regulatory roles in cellular senescence [31]. Two of these tumour suppressors, p15INK4B and p16INK4A, are cyclin dependent kinase inhibitors (CKIs) that trigger cell cycle arrest by inhibiting CDK4 and CDK6 complexes [34]. ARF, the third gene encoded by the locus, is a critical regulator of the p53 tumour suppressor pathway [68]. ARF and p16INK4A share common exons but are encoded in alternative reading frames leading to the production of unique proteins [31].

Polycomb repressive group complexes (PRCs) play a crucial role in the regulation of the locus [69]. The polycomb family is composed of two repressive complexes PRC1 and PRC2. PRC2 establishes the repressive mark leading to the recruitment of the maintenance polycomb complex PRC1. Various polycomb proteins such as CBX7, CBX8, TBX2, TBX3 and Bmi1 have been functionally implicated in the repression of cellular senescence by inhibiting the INK4B/ ARF/INK4A locus [5, 70-72]. To counteract the repressive action of the polycomb complexes, several mechanism are activated during cellular senescence. These include the removal or inhibition of polycomb complexes by MAPKAP [73], by the chromatin remodeling SWI/SNF complex [74] and/or the activation of the INK4B/ARF/INK4A locus by JMJD3 [75, 76] (Figure 6).

Additional regulators of the locus include the stress activated p38MAPKinase. Activated in response to various stresses such as high levels of ROS, dysfunctional telomeres and OIS, it regulates cellular senescence in various contexts [77]. It can activate p16INK4A, p15INK4B and ARF [78]. Although the locus products are mainly regulated by epigenetic mechanism, posttranslational modifications can also play a role in their regulation. For example, ARF stability is regulated by the E3 ubiquitin ligase TRIP12/ULF [68] and TGF-β stabilises p15INK4B [79]. p16INKA4 is activated in response to UV treatment through the inhibition of a SKP2 related degradation [80].

The DNA damage pathway is activated following the activation of two large protein kinases ataxia-telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR). Once recruited at the DNA damage sites, they phosphorylate and activate the histone variant H2AX [85]. The molecular events involved in single stranded breaks (SSBs) and double stranded breaks (DSBs) then differ and will not be described herein. The activation of the DDR kinases cascade, involving DNA damage mediators and diffusible kinases, results in the phosphory‐ lation and activation of p53 which in turn activates p21CIP1, one of its transcriptional targets to regulate growth arrest and cellular senescence [81]. The functional role of DDR in cellular senescence has been demonstrated using various functional approaches. Inactivating DDR proteins as well as p53 and its transcriptional target p21CIP1 is sufficient to abrogate cellular senescence in various settings [81]. The p53 pathway can also be activated in a DDR inde‐ pendent mechanism. For example, ARF plays a crucial role in the activation of p53. Activated in response to oncogenic stimulations, it activates p53 by sequestering the E3 ubiquitin protein ligase mouse double minute 2 (MDM2 or HDM2 in humans), an inhibitor of p53 [86] (Figure 7).

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The DNA damage/p53 pathway is activated in response to dysfunctional telomeres, oncogenic or oxidative stress, ionizing radiations and cytotoxic drugs. A DNA damage response (DDR) is elicited leading to the activation of local apical kinases, DNA-damage mediators, diffusible kinases and ultimately p53 and p21CIP1. The p53 pathway can also be activated by ARF

ROS are critical regulators of OIS [87]. In accordance, ROS regulated proteins such as seladin-1 (modulators of peroxiredoxines, a class of antioxidants) have also been involved in OIS [88]. Enzymes that generate ROS such as 5-lipooxygenase (5LO) mediate Ras induced senescence [89]. ROS are not only involved in Ras induced senescence. Akt was recently identified as a major determinant of various types of cellular senescence by modulating oxygen consumption and down-regulating ROS scavengers [90]. ROS can also mediate replicative senescence. For example, large amount of ROS produced by dysfunctional mitochondria can modulate telomere length and replicative senescence [91]. In accordance, antioxidant proteins can negatively regulate cellular senescence. The extracellular superoxide dismutase (SOD) increases the lifespan of primary cells [92] and over-expressing the antioxidant enzyme

miRNAs are small (approximately 23 nucleotides) RNAs that play a crucial role in gene regulation [94]. miRNAs bind 3'UTR and sometimes 5'UTR of the mRNA to modulate their translation and/or stability. The regulatory role of miRNAs during cellular senescence has recently been addressed [95]. Expression profiling studies indicate an altered expression among various miRNAs during cellular senescence [95]. Functionally, several miRNAs have been identified as repressor [96] or activators of cellular senescence [97] in normal but also

following an oncogenic stress. Adapted from [81].

catalase to the mitochondria increase mice lifespan [93].

*2.5.3. Reactive oxygen species (ROS)*

*2.5.4. Small non coding RNAs*

cancer cells [98].

**Figure 6.** The INK4B/ARF/INK4A locus in cellular senescence.

The locus is mostly regulated by epigenetic mechanism. It involves repressors such as the polycomb proteins (CBX7/8, EZH2, BMI1 and TBX2/3) and activators including histone demethylases (JMJD3), protein kinases (p38MAPK, MAPKAP) and chromatin remodeling complexes (SWI/SNF). Post translational modifications also regulate the locus products.

#### *2.5.2. The DNA damage/p53 pathway*

Activated in response to stimuli that trigger a DDR such as dysfunctional telomeres, OIS, ionising radiation and ROS, the p53 pathway is critical in the regulation of senescence [81] (Figure 7).

The activation of a DNA damage response consists in the recruitment of DNA damage sensors at the sites of damage. Various DNA damage sites associated with cellular senescence have been described. These include telomere-dysfunction-induced-foci (TIFs) [82], senescenceassociated DNA damage foci (SDF) [83] and DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS) [84].

The DNA damage pathway is activated following the activation of two large protein kinases ataxia-telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR). Once recruited at the DNA damage sites, they phosphorylate and activate the histone variant H2AX [85]. The molecular events involved in single stranded breaks (SSBs) and double stranded breaks (DSBs) then differ and will not be described herein. The activation of the DDR kinases cascade, involving DNA damage mediators and diffusible kinases, results in the phosphory‐ lation and activation of p53 which in turn activates p21CIP1, one of its transcriptional targets to regulate growth arrest and cellular senescence [81]. The functional role of DDR in cellular senescence has been demonstrated using various functional approaches. Inactivating DDR proteins as well as p53 and its transcriptional target p21CIP1 is sufficient to abrogate cellular senescence in various settings [81]. The p53 pathway can also be activated in a DDR inde‐ pendent mechanism. For example, ARF plays a crucial role in the activation of p53. Activated in response to oncogenic stimulations, it activates p53 by sequestering the E3 ubiquitin protein ligase mouse double minute 2 (MDM2 or HDM2 in humans), an inhibitor of p53 [86] (Figure 7).

The DNA damage/p53 pathway is activated in response to dysfunctional telomeres, oncogenic or oxidative stress, ionizing radiations and cytotoxic drugs. A DNA damage response (DDR) is elicited leading to the activation of local apical kinases, DNA-damage mediators, diffusible kinases and ultimately p53 and p21CIP1. The p53 pathway can also be activated by ARF following an oncogenic stress. Adapted from [81].

#### *2.5.3. Reactive oxygen species (ROS)*

[78]. Although the locus products are mainly regulated by epigenetic mechanism, posttranslational modifications can also play a role in their regulation. For example, ARF stability is regulated by the E3 ubiquitin ligase TRIP12/ULF [68] and TGF-β stabilises p15INK4B [79]. p16INKA4 is activated in response to UV treatment through the inhibition of a SKP2 related

p15INK4B p14/

modifications TGF- ULF SKP2

The locus is mostly regulated by epigenetic mechanism. It involves repressors such as the polycomb proteins (CBX7/8, EZH2, BMI1 and TBX2/3) and activators including histone demethylases (JMJD3), protein kinases (p38MAPK, MAPKAP) and chromatin remodeling complexes (SWI/SNF). Post translational modifications also regulate the locus products.

Activated in response to stimuli that trigger a DDR such as dysfunctional telomeres, OIS, ionising radiation and ROS, the p53 pathway is critical in the regulation of senescence [81]

The activation of a DNA damage response consists in the recruitment of DNA damage sensors at the sites of damage. Various DNA damage sites associated with cellular senescence have been described. These include telomere-dysfunction-induced-foci (TIFs) [82], senescenceassociated DNA damage foci (SDF) [83] and DNA segments with chromatin alterations

p38MAPK JMJD3 MAPKAP SWI/SNF

p19ARF p16INK4A

CBX7/8 EZH2 BMI1 TBX2/3

Activators Repressors

degradation [80].

INK4B/ARF/INKA4A locus Epigenetic regulators

94 Senescence and Senescence-Related Disorders

INK4B/ARF/INKA4A locus Post translational

**Figure 6.** The INK4B/ARF/INK4A locus in cellular senescence.

*2.5.2. The DNA damage/p53 pathway*

reinforcing senescence (DNA-SCARS) [84].

(Figure 7).

ROS are critical regulators of OIS [87]. In accordance, ROS regulated proteins such as seladin-1 (modulators of peroxiredoxines, a class of antioxidants) have also been involved in OIS [88]. Enzymes that generate ROS such as 5-lipooxygenase (5LO) mediate Ras induced senescence [89]. ROS are not only involved in Ras induced senescence. Akt was recently identified as a major determinant of various types of cellular senescence by modulating oxygen consumption and down-regulating ROS scavengers [90]. ROS can also mediate replicative senescence. For example, large amount of ROS produced by dysfunctional mitochondria can modulate telomere length and replicative senescence [91]. In accordance, antioxidant proteins can negatively regulate cellular senescence. The extracellular superoxide dismutase (SOD) increases the lifespan of primary cells [92] and over-expressing the antioxidant enzyme catalase to the mitochondria increase mice lifespan [93].

#### *2.5.4. Small non coding RNAs*

miRNAs are small (approximately 23 nucleotides) RNAs that play a crucial role in gene regulation [94]. miRNAs bind 3'UTR and sometimes 5'UTR of the mRNA to modulate their translation and/or stability. The regulatory role of miRNAs during cellular senescence has recently been addressed [95]. Expression profiling studies indicate an altered expression among various miRNAs during cellular senescence [95]. Functionally, several miRNAs have been identified as repressor [96] or activators of cellular senescence [97] in normal but also cancer cells [98].

Senescent cells display an increase in autophagic vacuoles, a gradual shift from the proteasome pathway to autophagy for polyubiquitinated protein degradation [100] and an upregulation of autophagyregulatorssuchastheATGrelatedgenes(ULK1andULK3)[101].Modulatingcritical componentsoftheautophagicprocesscanregulatecellularsenescence.InhibitionofATGproteins (ATG-5 and ATG-7) is sufficient to induce an escape from cellular senescence [101]. Conversely,

Immunosenescence and Senescence Immunosurveillance: One of the Possible Links Explaining the...

Ever since the discovery and the term replicative senescence introduced by Hayflick in 1965 [3], it has been proposed that senescence can block tumour progression. Primary normal cells are said to be "mortal", and in contrast, cancer cell lines are immortal. Therefore, an escape from replicative senescence is a critical stage that has to be overcome during tumour progres‐ sion. To physiologically demonstrate this hypothesis, mouse models knockout for the RNA

response to dysfunctional telomeres in these mice was shown to limit tumour progression and lead to tumour regression [102, 103]. In accordance with these results, late generation TERC-

deficient mice have been shown to be resistant to multistage skin carcinogenesis [104] and are more resistant to tumour formation in a subset of cancer mouse models [105, 106]. Oncogeneinduced senescence (OIS) has also been identified as a failsafe program *in vivo,* and this, in response to a physiological aberrant oncogenic activation [10]. Additional inactivation of tumour suppressor genes regulating cellular senescence leads to progression towards malig‐ nant stages and full blown tumours [10]. In accordance, premalignant tumour display high levels of senescence whereas it is absent in later stages of tumorigenesis [10]. Finally, cellular senescence is not only a barrier against early stages of tumor progression. It can be reactivated

In response to various cellular stresses such as dysfunctional telomeres, oncogene activation, oxidative stress and cytotoxic stresses normal cells acquire various alterations. Cellular senescence is activated to restrain neoplastic expansion. The inactivation of critical tumor suppressor genes (p53, p16INK4, ARF…) leads to cellular senescence escape and progression towards more aggressive tumors. Cellular senescence can be reactivated in tumors in response to the activation of tumor suppressors or oncogene inhibition. This reactivation leads to tumor

Cellular senescence can also be detected in immune cells [107]. Immunosenescence is a slightly different process than cellular senescence. Indeed, immunosenescence refers to cellular hypoproliferation in response to mitogenic or antigen stimulation and is often observed in ageing immune cells. In contrast, cellular senescence is associated with a permanent cell cycle arrest underlined with specific molecular markers. Immunosenescence affects both adaptative

) were used. The replicative senescence triggered in

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/ - 97

over-expressing ATG target genes such as of ULK3 reduces cell growth [101].

/ -

**2.6. Cellular senescence and cancer: The tumour point of view**

in established tumors leading to tumor eradication [52].

component of the telomerase (TERC-

regression [52].

**3. Immunosenescence**

**3.1. Features of immunosenescence**

**Figure 7.** The DNA damage/p53 pathway in cellular senescence.

#### *2.5.5. The autophagic pathway*

Autophagy is a recycling process mediated by autophagosomes, which are double membrane vesicules that engulf cytoplasmic contents and then fuse and deliver their content to lysosomes. Lysosomes contain hydrolases that digest the material which ultimately leads to a breakdown of the vesicules and their constituents [99].

Senescent cells display an increase in autophagic vacuoles, a gradual shift from the proteasome pathway to autophagy for polyubiquitinated protein degradation [100] and an upregulation of autophagyregulatorssuchastheATGrelatedgenes(ULK1andULK3)[101].Modulatingcritical componentsoftheautophagicprocesscanregulatecellularsenescence.InhibitionofATGproteins (ATG-5 and ATG-7) is sufficient to induce an escape from cellular senescence [101]. Conversely, over-expressing ATG target genes such as of ULK3 reduces cell growth [101].

#### **2.6. Cellular senescence and cancer: The tumour point of view**

Ever since the discovery and the term replicative senescence introduced by Hayflick in 1965 [3], it has been proposed that senescence can block tumour progression. Primary normal cells are said to be "mortal", and in contrast, cancer cell lines are immortal. Therefore, an escape from replicative senescence is a critical stage that has to be overcome during tumour progres‐ sion. To physiologically demonstrate this hypothesis, mouse models knockout for the RNA component of the telomerase (TERC- / - ) were used. The replicative senescence triggered in response to dysfunctional telomeres in these mice was shown to limit tumour progression and lead to tumour regression [102, 103]. In accordance with these results, late generation TERC- / deficient mice have been shown to be resistant to multistage skin carcinogenesis [104] and are more resistant to tumour formation in a subset of cancer mouse models [105, 106]. Oncogeneinduced senescence (OIS) has also been identified as a failsafe program *in vivo,* and this, in response to a physiological aberrant oncogenic activation [10]. Additional inactivation of tumour suppressor genes regulating cellular senescence leads to progression towards malig‐ nant stages and full blown tumours [10]. In accordance, premalignant tumour display high levels of senescence whereas it is absent in later stages of tumorigenesis [10]. Finally, cellular senescence is not only a barrier against early stages of tumor progression. It can be reactivated in established tumors leading to tumor eradication [52].

In response to various cellular stresses such as dysfunctional telomeres, oncogene activation, oxidative stress and cytotoxic stresses normal cells acquire various alterations. Cellular senescence is activated to restrain neoplastic expansion. The inactivation of critical tumor suppressor genes (p53, p16INK4, ARF…) leads to cellular senescence escape and progression towards more aggressive tumors. Cellular senescence can be reactivated in tumors in response to the activation of tumor suppressors or oncogene inhibition. This reactivation leads to tumor regression [52].

#### **3. Immunosenescence**

*2.5.5. The autophagic pathway*

of the vesicules and their constituents [99].

**Figure 7.** The DNA damage/p53 pathway in cellular senescence.

ATM ATR

Oxidative stress

Ionizing radiation Dysfunctional telomeres

Cytotoxic drugs

H2AX P

DNA-damage mediators

H2AX P

Oncogenic stress

96 Senescence and Senescence-Related Disorders

p53

Diffusible kinases

p21CIP1

Autophagy is a recycling process mediated by autophagosomes, which are double membrane vesicules that engulf cytoplasmic contents and then fuse and deliver their content to lysosomes. Lysosomes contain hydrolases that digest the material which ultimately leads to a breakdown

HDM2

ARF

Oncogenic stress

#### **3.1. Features of immunosenescence**

Cellular senescence can also be detected in immune cells [107]. Immunosenescence is a slightly different process than cellular senescence. Indeed, immunosenescence refers to cellular hypoproliferation in response to mitogenic or antigen stimulation and is often observed in ageing immune cells. In contrast, cellular senescence is associated with a permanent cell cycle arrest underlined with specific molecular markers. Immunosenescence affects both adaptative

high ROS levels, chemical or physical cellular stressors can trigger premature senescence. Additionally, it would be interesting to determine if the autophagic pathway and miRNAs are

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99

Various factors could explain immunosenescence. Thymic involution might play a role. It consists of shrinkage of the thymus with age and results in a decrease output of naïve T lymphocytes [113]. Altered immune signaling also seems to play a role. For example, in the elderly, the composition of lipid rafts is altered and this is associated with a decreased activity of some signaling pathways [114]. Above all, chronic antigen stimulation (e.g. chronic infections, tumor antigens) is the major trigger of immunosenescence. Cytomegalovirus (CMV) but also Epstein-Barr Virus (EBV) infection, to a lesser extent, are determinants of immunosenescence [115]. In accordance, the number of CD8+ cells expressing T cell receptor (TCR) specific CMV antigens in the ageing population increases. This has no effect in the number of T cell in the periphery however the expansion of CMV specific T cells results in a decrease in T cell repertoire [115]. In innate immune cells, an alteration of various receptors activity (e.g. Toll like receptor (TLR)) and in the number of some circulating innate immune

Immunosenescence is associated with decreased immune functions [109]. Neutrophils, the first innate immune cell to be at the site of pathogen entry, display a decreased chemotaxis and in free radical production and a decreased intracellular killing. Monocytes and macrophages both exhibit a decreased in phagocytosis and NK cells a decreased cytotoxicity. Dendritic cells display decreased phagocytosis, chemotaxis but also a reduced ability to activate the adapta‐ tive immune cells via antigen presentation [109]. Additionally to not being efficiently activated by the innate immune cells, adaptative immune cells are also affected by immunosenescence (particularly the T cell compartment). An inhibition of interleukine-2 (IL-2) production, increased DNA damage, telomere shortening but also a decrease in the number of naïve T cell (both CD4+ and CD8+) and an increase in the number of memory T cell leading to a weaker

Escape from immunosurveillance is now considered a cancer hallmark [116]. Immunosurveil‐ lance is a term used to define how immune cells identify and destroy abnormal antigen upon recognition [117]. This immunological process required for clearing tumors is often altered during cancer progression giving place to cancer favoring processes such as immunoselection, immunosubversion and ultimately immunosuppression [115, 117]. During cancer develop‐

also implicated in various immunosenescence settings.

cells seem to favor innate immunosenescence [109].

**3.3. The consequences of immunosenescence**

immunosurveillance are observed [115].

**4. Immunosenescence and cancer**

**4.1. Tumors evade the immune system**

**3.2. The triggers of immunosenescence**

**Figure 8.** Cellular senescence acts as a tumor suppressive mechanism.

(T and B Lymphocytes) and innate immune cells (Natural killer cells (NK), Natural killer T cells (NKT), Macrophages, Neutrophils, Monocytes and Dendritic cells) [108, 109]. To date, the molecular determinants of immunosenescence have not been very well described. However, similarities with senescence in non immune cells are found. For example, immunosenescence of T cells is regulated by p16INK4A and p21CIP1 [110, 111]. Differentiated T cells have shorter telomeres and an inactive telomerase [107]. Functionally, the ectopic telomerase expression maintains telomere size and delays immunosenescence [107]. However, immunosenescence also differs from cellular senescence. For example, inhibiting TNF-α receptor 1 partially reverses T cell immunosenescence in what is suggested to be a caspase-3 dependent mecha‐ nism [112]. Specific immunosenescence markers also exist. For example, cell surface markers such as CD27 and CD28 are lost in differentiated senescent CD4+ and CD8+ T cells and the killer cell lectin-like receptor subfamily G member 1 (KLRG1) is also known as a cell surface marker of immunosenescence [107]. Premature immunosenescence has not been characterized so far but one might expect that similar mechanism could be involved in premature senescence and premature immunosenescence. It could be interesting to assess whether stresses such as high ROS levels, chemical or physical cellular stressors can trigger premature senescence. Additionally, it would be interesting to determine if the autophagic pathway and miRNAs are also implicated in various immunosenescence settings.

#### **3.2. The triggers of immunosenescence**

Various factors could explain immunosenescence. Thymic involution might play a role. It consists of shrinkage of the thymus with age and results in a decrease output of naïve T lymphocytes [113]. Altered immune signaling also seems to play a role. For example, in the elderly, the composition of lipid rafts is altered and this is associated with a decreased activity of some signaling pathways [114]. Above all, chronic antigen stimulation (e.g. chronic infections, tumor antigens) is the major trigger of immunosenescence. Cytomegalovirus (CMV) but also Epstein-Barr Virus (EBV) infection, to a lesser extent, are determinants of immunosenescence [115]. In accordance, the number of CD8+ cells expressing T cell receptor (TCR) specific CMV antigens in the ageing population increases. This has no effect in the number of T cell in the periphery however the expansion of CMV specific T cells results in a decrease in T cell repertoire [115]. In innate immune cells, an alteration of various receptors activity (e.g. Toll like receptor (TLR)) and in the number of some circulating innate immune cells seem to favor innate immunosenescence [109].

#### **3.3. The consequences of immunosenescence**

Immunosenescence is associated with decreased immune functions [109]. Neutrophils, the first innate immune cell to be at the site of pathogen entry, display a decreased chemotaxis and in free radical production and a decreased intracellular killing. Monocytes and macrophages both exhibit a decreased in phagocytosis and NK cells a decreased cytotoxicity. Dendritic cells display decreased phagocytosis, chemotaxis but also a reduced ability to activate the adapta‐ tive immune cells via antigen presentation [109]. Additionally to not being efficiently activated by the innate immune cells, adaptative immune cells are also affected by immunosenescence (particularly the T cell compartment). An inhibition of interleukine-2 (IL-2) production, increased DNA damage, telomere shortening but also a decrease in the number of naïve T cell (both CD4+ and CD8+) and an increase in the number of memory T cell leading to a weaker immunosurveillance are observed [115].

#### **4. Immunosenescence and cancer**

#### **4.1. Tumors evade the immune system**

(T and B Lymphocytes) and innate immune cells (Natural killer cells (NK), Natural killer T cells (NKT), Macrophages, Neutrophils, Monocytes and Dendritic cells) [108, 109]. To date, the molecular determinants of immunosenescence have not been very well described. However, similarities with senescence in non immune cells are found. For example, immunosenescence of T cells is regulated by p16INK4A and p21CIP1 [110, 111]. Differentiated T cells have shorter telomeres and an inactive telomerase [107]. Functionally, the ectopic telomerase expression maintains telomere size and delays immunosenescence [107]. However, immunosenescence also differs from cellular senescence. For example, inhibiting TNF-α receptor 1 partially reverses T cell immunosenescence in what is suggested to be a caspase-3 dependent mecha‐ nism [112]. Specific immunosenescence markers also exist. For example, cell surface markers such as CD27 and CD28 are lost in differentiated senescent CD4+ and CD8+ T cells and the killer cell lectin-like receptor subfamily G member 1 (KLRG1) is also known as a cell surface marker of immunosenescence [107]. Premature immunosenescence has not been characterized so far but one might expect that similar mechanism could be involved in premature senescence and premature immunosenescence. It could be interesting to assess whether stresses such as

**\***

Primary normal cells

Others stresses

Aberrant cells

**alterations**

Oncogenic stress

Dysfunctional telomeres

98 Senescence and Senescence-Related Disorders

**\***

Oncogene inactivation Tumour suppressor reactivation

**Figure 8.** Cellular senescence acts as a tumor suppressive mechanism.

**Cellular senescence**

Inhibition of tumor suppressors genes (e.g. inactivation of p53/p16INK4A/ARF…)

Cancer cells

**Cellular senescence**

Escape from immunosurveillance is now considered a cancer hallmark [116]. Immunosurveil‐ lance is a term used to define how immune cells identify and destroy abnormal antigen upon recognition [117]. This immunological process required for clearing tumors is often altered during cancer progression giving place to cancer favoring processes such as immunoselection, immunosubversion and ultimately immunosuppression [115, 117]. During cancer develop‐ ment, a complex crosstalk between the tumor and the immune microenvironment takes place. Premalignant tumors send stress signals to the immune cells activating both innate and adaptative responses. In this context of immunosurveillance, the immune response is highly functional and eliminates surrounding tumors. However, tumor cells activate various mechanisms that allow immunosurveillance escape. These include the secretion of various cytokines, mechanisms to induce T cell apoptosis, anergy of naïve T cells, the activation of myeloid suppressor cells, the physical interaction of tumor cells with T cells, the suppression of NKT cell activities and downregulation of cell surface markers [115]. Immunosurveillance escape leads to a state of "equilibrium" between the tumor and the immune system, a process known as immunoselection. It is named as such because aggressive tumor cells that are capable of co-existing or suppressing the immune system are selected [115]. These cells eventually increase in number and secrete various immunosuppressive molecules (e.g. prostaglandin E2 (PGE2), nitric oxides) [117]. The immune system is therefore critical in suppressing tumor growth and its alteration is required from tumor progression. Interestingly, virtually of all the immune cells involved in immunosurveillance are known to be altered with age [115].

leads to an alteration in the activity of both naïve and cytotoxic lymphocytes. In summary, various immunosuppressive mechanisms are found up-regulated in the elderly [115]. Con‐ sidering the crucial role of the immune system in cancer eradication, it suggests that immu‐

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101

An intimate relationship between cellular senescence and the immune system has recently been identified [2]. Using various mouse models, it was demonstrated that the reactivation of p53 in established tumours was associated with cellular senescence and tumour regression [119, 120]. However, as senescent cells do not die of apoptosis and can persist in some tissue for many years [121], the fate of senescent cells remained uncertain especially in a cancer context. Since the discovery of a secretory phenotype associated with cellular senescence (SASP), it has been suggested that senescent cells can initiate cross-talks with the microenvir‐ onment [2]. It was therefore hypothesised that senescent cells could be cleared by the immune system. In accordance, it was demonstrated in a mouse model of liver carcinoma that the activation of an innate immune response (NK cells macrophage and neutrophils) was respon‐ sible for the clearance of senescent tumours [120]. In liver cancer, senescence immunosurveil‐ lance thus seems to be required to clear senescent tumors. Interestingly, senescence immunosurveillance is not limited to liver cancer as it was also required for the complete remission of lymphoma and leukemia mouse models [122]. In these models, the adaptative immune response and more specifically CD4+ T cells were necessary for tumour eradication and also for senescence induction suggesting a complex interplay between senescent cells and the immune system [2]. Such interplay has been tackled in a mouse model of lymphomagenesis [123]. In a model of OIS *in viv*o resulting in apoptosis, macrophage were attracted and required to engulf apoptotic reminders. Both cellular senescence and apoptosis were shown to act together via the innate immune response to inhibit tumour progression. In turn, activated macrophages secreted various cytokines such as TGF-β to induce cellular senescence of malignant cells [123]. These results clearly demonstrate a crucial role of immunosurveillance in clearing senescent tumors and amplifying senescence induction. To reinforce the role of senescence immunosurveillance as a barrier against cancer, the inactivation of CD4+ T cell following OIS of hepatocytes in mice led to tumor progression [124].The role CD4+ T cell in preventing tumor progression in this mouse model was dependent upon the activation of monocytes and macrophages [124]. What is surprising is that the inhibition of cellular senescence did not activate the immune system suggesting that during cancer development a functional senescence response might be required for an efficient immunosurveillance and tumor suppression [125]. It is therefore tempting to suggest that cellular senescence plays a significant role during immunosuppression [126]. It will also be interesting to demonstrate if senescence immunosurveillance also takes place in other organs during cancer development

nosenescence could play a role in cancers associated with age.

**5.1. Senescence immunosurveillance**

and whether it is involved in tumor suppression.

**5. Immunosenescence and senescence immunosurveillance**

#### **4.2. Cancer arises with age: A role for immunosenescence?**

Cancer is a pathology associated with age [118]. Indeed, cancer incidence and prevalence increase with age suggesting an important relationship between the two biological processes [118]. During life, an organism is doomed to accumulate mutations favoring cancer develop‐ ment. The triggers of such mutations include environmental factors such as carcinogens, UV lights, viruses and free radicals among others [116]. On the other hand, cancer can no longer be regarded as a cell autonomous process. Recent advances demonstrate that the microenvir‐ onment plays a crucial role in modulating cancer development [116]. Although the role of the immune system in cancer development in ageing population is currently uncertain, it is clear that the immune system is altered with age [115]. As previously mentioned, the immune system is critical for clearing tumors, at least in early stages of tumorigenesis. Additionally, we have seen that immunosenescence is associated with profound immune alterations leading to a decrease in various immune cells activity. The activation of the adaptative immune response by dendritics cells is crucial for the activation of T-cell. Interestingly in the elders, immunosenescence of dendritics cells which impacts various co-receptors leads to a weakened T cell response [115]. The alteration of TLRs could also favor cancer development in ageing populations. Indeed, TLRs are crucial in the activation of innate immune cells. Their alterations leads to a decreased phagocytosis of innate immune cells and renders them less capable of destroying tumors. Ageing populations also have an increase amount of immune suppressive cells such as T regulatory cells that inhibit innate immune cells activity though direct cell-cell interaction. Myeloid derived suppressor cells (MDSC) are composed of a heterogeneous population of innate immune cells that can suppress the activation of CD4+ and CD8+ T cells. Interestingly, MDSC are activated by various anti-inflammatory signals (e.g IL-10, TGF-β) that are found increased in the elderly. Additionally, IDO, an immunosuppressive molecule that inhibits T cells responses, is also found up-regulated in ageing populations. Changes in the immune system that could favor cancer development are not limited to the innate immune cells. With ageing there is a gradual shift of Th1 towards Th2 in the T cells repertoire and this leads to an alteration in the activity of both naïve and cytotoxic lymphocytes. In summary, various immunosuppressive mechanisms are found up-regulated in the elderly [115]. Con‐ sidering the crucial role of the immune system in cancer eradication, it suggests that immu‐ nosenescence could play a role in cancers associated with age.

#### **5. Immunosenescence and senescence immunosurveillance**

#### **5.1. Senescence immunosurveillance**

ment, a complex crosstalk between the tumor and the immune microenvironment takes place. Premalignant tumors send stress signals to the immune cells activating both innate and adaptative responses. In this context of immunosurveillance, the immune response is highly functional and eliminates surrounding tumors. However, tumor cells activate various mechanisms that allow immunosurveillance escape. These include the secretion of various cytokines, mechanisms to induce T cell apoptosis, anergy of naïve T cells, the activation of myeloid suppressor cells, the physical interaction of tumor cells with T cells, the suppression of NKT cell activities and downregulation of cell surface markers [115]. Immunosurveillance escape leads to a state of "equilibrium" between the tumor and the immune system, a process known as immunoselection. It is named as such because aggressive tumor cells that are capable of co-existing or suppressing the immune system are selected [115]. These cells eventually increase in number and secrete various immunosuppressive molecules (e.g. prostaglandin E2 (PGE2), nitric oxides) [117]. The immune system is therefore critical in suppressing tumor growth and its alteration is required from tumor progression. Interestingly, virtually of all the immune cells involved in immunosurveillance are known to be altered with age [115].

Cancer is a pathology associated with age [118]. Indeed, cancer incidence and prevalence increase with age suggesting an important relationship between the two biological processes [118]. During life, an organism is doomed to accumulate mutations favoring cancer develop‐ ment. The triggers of such mutations include environmental factors such as carcinogens, UV lights, viruses and free radicals among others [116]. On the other hand, cancer can no longer be regarded as a cell autonomous process. Recent advances demonstrate that the microenvir‐ onment plays a crucial role in modulating cancer development [116]. Although the role of the immune system in cancer development in ageing population is currently uncertain, it is clear that the immune system is altered with age [115]. As previously mentioned, the immune system is critical for clearing tumors, at least in early stages of tumorigenesis. Additionally, we have seen that immunosenescence is associated with profound immune alterations leading to a decrease in various immune cells activity. The activation of the adaptative immune response by dendritics cells is crucial for the activation of T-cell. Interestingly in the elders, immunosenescence of dendritics cells which impacts various co-receptors leads to a weakened T cell response [115]. The alteration of TLRs could also favor cancer development in ageing populations. Indeed, TLRs are crucial in the activation of innate immune cells. Their alterations leads to a decreased phagocytosis of innate immune cells and renders them less capable of destroying tumors. Ageing populations also have an increase amount of immune suppressive cells such as T regulatory cells that inhibit innate immune cells activity though direct cell-cell interaction. Myeloid derived suppressor cells (MDSC) are composed of a heterogeneous population of innate immune cells that can suppress the activation of CD4+ and CD8+ T cells. Interestingly, MDSC are activated by various anti-inflammatory signals (e.g IL-10, TGF-β) that are found increased in the elderly. Additionally, IDO, an immunosuppressive molecule that inhibits T cells responses, is also found up-regulated in ageing populations. Changes in the immune system that could favor cancer development are not limited to the innate immune cells. With ageing there is a gradual shift of Th1 towards Th2 in the T cells repertoire and this

**4.2. Cancer arises with age: A role for immunosenescence?**

100 Senescence and Senescence-Related Disorders

An intimate relationship between cellular senescence and the immune system has recently been identified [2]. Using various mouse models, it was demonstrated that the reactivation of p53 in established tumours was associated with cellular senescence and tumour regression [119, 120]. However, as senescent cells do not die of apoptosis and can persist in some tissue for many years [121], the fate of senescent cells remained uncertain especially in a cancer context. Since the discovery of a secretory phenotype associated with cellular senescence (SASP), it has been suggested that senescent cells can initiate cross-talks with the microenvir‐ onment [2]. It was therefore hypothesised that senescent cells could be cleared by the immune system. In accordance, it was demonstrated in a mouse model of liver carcinoma that the activation of an innate immune response (NK cells macrophage and neutrophils) was respon‐ sible for the clearance of senescent tumours [120]. In liver cancer, senescence immunosurveil‐ lance thus seems to be required to clear senescent tumors. Interestingly, senescence immunosurveillance is not limited to liver cancer as it was also required for the complete remission of lymphoma and leukemia mouse models [122]. In these models, the adaptative immune response and more specifically CD4+ T cells were necessary for tumour eradication and also for senescence induction suggesting a complex interplay between senescent cells and the immune system [2]. Such interplay has been tackled in a mouse model of lymphomagenesis [123]. In a model of OIS *in viv*o resulting in apoptosis, macrophage were attracted and required to engulf apoptotic reminders. Both cellular senescence and apoptosis were shown to act together via the innate immune response to inhibit tumour progression. In turn, activated macrophages secreted various cytokines such as TGF-β to induce cellular senescence of malignant cells [123]. These results clearly demonstrate a crucial role of immunosurveillance in clearing senescent tumors and amplifying senescence induction. To reinforce the role of senescence immunosurveillance as a barrier against cancer, the inactivation of CD4+ T cell following OIS of hepatocytes in mice led to tumor progression [124].The role CD4+ T cell in preventing tumor progression in this mouse model was dependent upon the activation of monocytes and macrophages [124]. What is surprising is that the inhibition of cellular senescence did not activate the immune system suggesting that during cancer development a functional senescence response might be required for an efficient immunosurveillance and tumor suppression [125]. It is therefore tempting to suggest that cellular senescence plays a significant role during immunosuppression [126]. It will also be interesting to demonstrate if senescence immunosurveillance also takes place in other organs during cancer development and whether it is involved in tumor suppression.

#### **5.2. Defects in senescence immunosurveillance: A role for immunosenescence?**

It appears that a functional immune system is required for efficient senescence clearance and tumor suppression. As immunosenescence affects normal immune homeostasis and senescent cells increase with age [118], it is tempting to speculate that in the elderly a defect in senescence immunosurveillance could contribute to the increased cancer incidence. In line with this hypothesis is the fact that all the immune cells involved in senescence immunosurveillance seem to be altered with age. However, to date no functional experiments have been carried to test such hypothesis. Mouse models of ageing recapitulating immunosenescence in which cellular senescence is induced could be of great interest to determine this possibility.

[5] Gil J, Bernard D, Martinez D, Beach D. Polycomb CBX7 has a unifying role in cellular

Immunosenescence and Senescence Immunosurveillance: One of the Possible Links Explaining the...

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103

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#### **6. Conclusion**

Immunosenescence is a field that holds great promises. To date, some of the molecular determinants of immunosenescence are still to be discovered. Identifying such mechanism could shed light on this cellular process and might help us to find ways to counteract such mechanism. Additionally, assessing the role of immunosenescence during cancer develop‐ ment in ageing population and more specifically its role during senescence immunosurveil‐ lance in the elderly could shed light on the mechanism associated with cancer.

#### **Author details**

Arnaud Augert and David Bernard

\*Address all correspondence to: david.bernard@lyon.unicancer.fr

Centre de Recherche en Cancérologie de Lyon, UMR INSERM U1052/CNRS 5286, Centre Léon Bérard, Université de Lyon, France

#### **References**


[5] Gil J, Bernard D, Martinez D, Beach D. Polycomb CBX7 has a unifying role in cellular lifespan. Nat Cell Biol. 2004 Jan;6(1):67-72.

**5.2. Defects in senescence immunosurveillance: A role for immunosenescence?**

cellular senescence is induced could be of great interest to determine this possibility.

lance in the elderly could shed light on the mechanism associated with cancer.

\*Address all correspondence to: david.bernard@lyon.unicancer.fr

**6. Conclusion**

102 Senescence and Senescence-Related Disorders

**Author details**

**References**

Arnaud Augert and David Bernard

Léon Bérard, Université de Lyon, France

2007 Jul 27;130(2):223-33.

Res. 1965 Mar;37:614-36.

Cell Res. 1961 Dec;25:585-621.

It appears that a functional immune system is required for efficient senescence clearance and tumor suppression. As immunosenescence affects normal immune homeostasis and senescent cells increase with age [118], it is tempting to speculate that in the elderly a defect in senescence immunosurveillance could contribute to the increased cancer incidence. In line with this hypothesis is the fact that all the immune cells involved in senescence immunosurveillance seem to be altered with age. However, to date no functional experiments have been carried to test such hypothesis. Mouse models of ageing recapitulating immunosenescence in which

Immunosenescence is a field that holds great promises. To date, some of the molecular determinants of immunosenescence are still to be discovered. Identifying such mechanism could shed light on this cellular process and might help us to find ways to counteract such mechanism. Additionally, assessing the role of immunosenescence during cancer develop‐ ment in ageing population and more specifically its role during senescence immunosurveil‐

Centre de Recherche en Cancérologie de Lyon, UMR INSERM U1052/CNRS 5286, Centre

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[2] Serrano M. Cancer: final act of senescence. Nature. 2011 Nov 24;479(7374):481-2.


[19] Mitsui Y, Schneider EL. Increased nuclear sizes in senescent human diploid fibro‐ blast cultures. Exp Cell Res. 1976 Jun;100(1):147-52.

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110 Senescence and Senescence-Related Disorders


**Section 5**

**Mastication and Cognition**

## **Mastication and Cognition**

**Chapter 5**

**The Relationship Between Mastication and Cognition**

Although mastication is primarily involved in food intake and digestion, it also promotes and preserves general health, including cognitive function. Functional magnetic resonance imaging (fMRI) and positron emission topography studies recently revealed that mastication leads to increases in cortical blood flow and activates the somatosensory, supplementary motor, and insular cortices, as well as the striatum, thalamus, and cerebellum [1]. Masticating immediately before performing a cognitive task increases blood oxygen levels (BOLD) in the prefrontal cortex and hippocampus, important structures involved in learning and memory, thereby improving task performance [1]. Thus, mastication may be a drug-free and simple method of attenuating the development of senile dementia and stress-related disorders that are often associated with cognitive dysfunction. Previous epidemiologic studies demonstrated that a decreased number of residual teeth, decreased denture use, and a small maximal biting force are directly related to the development of dementia, further supporting the notion that

Here we provide further evidence supporting the interaction between mastication and learning and memory, focusing on the function of the hippocampus, which is essential for the formation of new memories. We first summarize recent progress in understanding how mastication affects learning and memory. We then describe the impaired function and pathology of the hippocampus in an animal model of reduced mastication using senescenceaccelerated prone (SAMP8) mice, and discuss human studies showing that mastication enhances hippocampal-dependent cognitive function. We then describe how occlusal dishar‐ mony is a potential chronic stressor that impedes or suppresses hippocampal-mediated learning and memory, suggesting that normal occlusion is essential for producing the ameliorative effects of mastication on stress-induced changes in the hippocampus. Finally, we focus on the ameliorative effects of mastication on stress-induced suppression of learning and

> © 2013 Kubo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Kin-ya Kubo, Huayue Chen and Minoru Onozuka

Additional information is available at the end of the chapter

mastication contributes to maintain cognitive function [2].

http://dx.doi.org/10.5772/54911

**1. Introduction**

## **The Relationship Between Mastication and Cognition**

Kin-ya Kubo, Huayue Chen and Minoru Onozuka

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54911

### **1. Introduction**

Although mastication is primarily involved in food intake and digestion, it also promotes and preserves general health, including cognitive function. Functional magnetic resonance imaging (fMRI) and positron emission topography studies recently revealed that mastication leads to increases in cortical blood flow and activates the somatosensory, supplementary motor, and insular cortices, as well as the striatum, thalamus, and cerebellum [1]. Masticating immediately before performing a cognitive task increases blood oxygen levels (BOLD) in the prefrontal cortex and hippocampus, important structures involved in learning and memory, thereby improving task performance [1]. Thus, mastication may be a drug-free and simple method of attenuating the development of senile dementia and stress-related disorders that are often associated with cognitive dysfunction. Previous epidemiologic studies demonstrated that a decreased number of residual teeth, decreased denture use, and a small maximal biting force are directly related to the development of dementia, further supporting the notion that mastication contributes to maintain cognitive function [2].

Here we provide further evidence supporting the interaction between mastication and learning and memory, focusing on the function of the hippocampus, which is essential for the formation of new memories. We first summarize recent progress in understanding how mastication affects learning and memory. We then describe the impaired function and pathology of the hippocampus in an animal model of reduced mastication using senescenceaccelerated prone (SAMP8) mice, and discuss human studies showing that mastication enhances hippocampal-dependent cognitive function. We then describe how occlusal dishar‐ mony is a potential chronic stressor that impedes or suppresses hippocampal-mediated learning and memory, suggesting that normal occlusion is essential for producing the ameliorative effects of mastication on stress-induced changes in the hippocampus. Finally, we focus on the ameliorative effects of mastication on stress-induced suppression of learning and

© 2013 Kubo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

memory functions in the hippocampus and on systemic stress responses in both animals and humans.

#### **2. Dysfunctional mastication and cognitive function**

Dysfunctional mastication affects cognitive function, and reduced mastication contributes to senile dementia, Alzheimer's disease, and a declining quality of life in the elderly. In partic‐ ular, the systemic effects of tooth loss are an epidemiologic risk factor for Alzheimer's dis‐ ease [2]. Missing teeth, due to dental caries and periodontitis are common in the elderly, and reduce their ability to masticate. We created a molarless senescence-accelerated prone (SAMP8) mouse model of dysfunctional mastication by extracting or cutting the upper mo‐ lar teeth (molarless). SAMP8 mice mature normally for up to 6 months of age, but then ex‐ hibit accelerated aging (with a median life span of 12 months compared with 2- to 3 years for other strains). SAMP8 mice show clear aging-related impairments in learning and mem‐ ory at 6 months of age [3, 4], and these mice are often used in aging studies. Molarless SAMP8 mice exhibit age-dependent deficits in spatial learning in the Morris water maze [5-10] (Fig. 1). The duration of the molarless condition in aged SAMP8 mice correlates with the level of impaired learning [7], and restoring lost molars with artificial crowns attenuates the molarless-induced increases in the learning and memory deficits [9]. Masticatory stimu‐ lation is also impaired by a soft-food diet [11], which leads to learning impairment [11]. To‐ gether, these findings indicate that masticatory stimulation is closely related to learning and memory.

Several morphologic changes are observed in the hippocampus of molarless mice, including a decreased number of pyramidal cells [6], hypertrophied glial fibrillary acid protein-labeled astrocytes [7, 10] and decreased dendritic spines in the CA1 region [8], suppressed cell pro‐ liferation in the dentate gyrus [12]. These behavioral and morphologic changes are very sim‐ ilar to aging-related changes in the hippocampus [13]. The decreased masticatory stimulation resulting from a soft-food diet results in similar morphologic features [14, 15]. Thus, masticatory dysfunction appears to accelerate the aging process in the hippocampus.

Although the relationship between dysfunctional mastication and these behavioral and morphologic changes in the hippocampus is unclear, there are several possible mechanisms.

the septal nucleus is decreased in molarless mice [24], and decreased acetylcholine concentra‐ tions are observed in the cerebral cortex and hippocampus [24], as well as decreased acetyl‐ choline release [24] in the hippocampus in response to extracellular stimulation. In rodents, spatial memory is associated with acetylcholine levels in the hippocampus [25]. Therefore, the decreased cholinergic activity induced by the molarless condition could contribute learning

**Figure 1.** Effect of molarless condition on spatial learning in the Morris water maze test. The results are expressed as the mean score (mean ± SE, n=6 for each group) of four trials per day. Note that 9-month-old molarless mice required

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a significantly longer time than age-matched controls to reach the platform.

Decreased mastication may also lead to increases in the plasma corticosterone levels. The molarless condition in aged SAMP8 mice increases plasma corticosterone levels [5], and downregulates glucocorticoid receptors (GRs) and GR messenger ribonucleic acid (GR

impairments.

Decreased mastication decreases the information input from the oral area to the central nervous system, which leads to the degeneration of target cells [16], as exercise promotes axonal sprouting and synaptogenesis [17] and enhances neurogenesis in the hippocampus [18]. Tooth loss or extraction causes degenerative changes in the trigeminal ganglion cell bodies of the primary sensory neurons innervating the teeth [19] and transganglionic degeneration in the secondary neurons in the trigeminal spinal tract nucleus [20]. Hence, the impairment in cognitive function due to masticatory dysfunction might be related to the decreased activity of the sensory pathways of the oral areas. Further, dysfunctional mastication leads to increased decreased cholinergic activity. The number of choline acetyltransferase-positive neurons in

memory functions in the hippocampus and on systemic stress responses in both animals and

Dysfunctional mastication affects cognitive function, and reduced mastication contributes to senile dementia, Alzheimer's disease, and a declining quality of life in the elderly. In partic‐ ular, the systemic effects of tooth loss are an epidemiologic risk factor for Alzheimer's dis‐ ease [2]. Missing teeth, due to dental caries and periodontitis are common in the elderly, and reduce their ability to masticate. We created a molarless senescence-accelerated prone (SAMP8) mouse model of dysfunctional mastication by extracting or cutting the upper mo‐ lar teeth (molarless). SAMP8 mice mature normally for up to 6 months of age, but then ex‐ hibit accelerated aging (with a median life span of 12 months compared with 2- to 3 years for other strains). SAMP8 mice show clear aging-related impairments in learning and mem‐ ory at 6 months of age [3, 4], and these mice are often used in aging studies. Molarless SAMP8 mice exhibit age-dependent deficits in spatial learning in the Morris water maze [5-10] (Fig. 1). The duration of the molarless condition in aged SAMP8 mice correlates with the level of impaired learning [7], and restoring lost molars with artificial crowns attenuates the molarless-induced increases in the learning and memory deficits [9]. Masticatory stimu‐ lation is also impaired by a soft-food diet [11], which leads to learning impairment [11]. To‐ gether, these findings indicate that masticatory stimulation is closely related to learning and

Several morphologic changes are observed in the hippocampus of molarless mice, including a decreased number of pyramidal cells [6], hypertrophied glial fibrillary acid protein-labeled astrocytes [7, 10] and decreased dendritic spines in the CA1 region [8], suppressed cell pro‐ liferation in the dentate gyrus [12]. These behavioral and morphologic changes are very sim‐ ilar to aging-related changes in the hippocampus [13]. The decreased masticatory stimulation resulting from a soft-food diet results in similar morphologic features [14, 15]. Thus, masticatory dysfunction appears to accelerate the aging process in the hippocampus. Although the relationship between dysfunctional mastication and these behavioral and morphologic changes in the hippocampus is unclear, there are several possible mechanisms. Decreased mastication decreases the information input from the oral area to the central nervous system, which leads to the degeneration of target cells [16], as exercise promotes axonal sprouting and synaptogenesis [17] and enhances neurogenesis in the hippocampus [18]. Tooth loss or extraction causes degenerative changes in the trigeminal ganglion cell bodies of the primary sensory neurons innervating the teeth [19] and transganglionic degeneration in the secondary neurons in the trigeminal spinal tract nucleus [20]. Hence, the impairment in cognitive function due to masticatory dysfunction might be related to the decreased activity of the sensory pathways of the oral areas. Further, dysfunctional mastication leads to increased decreased cholinergic activity. The number of choline acetyltransferase-positive neurons in

**2. Dysfunctional mastication and cognitive function**

humans.

116 Senescence and Senescence-Related Disorders

memory.

**Figure 1.** Effect of molarless condition on spatial learning in the Morris water maze test. The results are expressed as the mean score (mean ± SE, n=6 for each group) of four trials per day. Note that 9-month-old molarless mice required a significantly longer time than age-matched controls to reach the platform.

the septal nucleus is decreased in molarless mice [24], and decreased acetylcholine concentra‐ tions are observed in the cerebral cortex and hippocampus [24], as well as decreased acetyl‐ choline release [24] in the hippocampus in response to extracellular stimulation. In rodents, spatial memory is associated with acetylcholine levels in the hippocampus [25]. Therefore, the decreased cholinergic activity induced by the molarless condition could contribute learning impairments.

Decreased mastication may also lead to increases in the plasma corticosterone levels. The molarless condition in aged SAMP8 mice increases plasma corticosterone levels [5], and downregulates glucocorticoid receptors (GRs) and GR messenger ribonucleic acid (GR mRNA) in the hippocampus [21]. The hippocampus contains a high density of GR and is in‐ volved in the negative feedback mechanism with the hypothalamo-pituitary-adrenal axis via GR, making it very sensitive to corticosterone [22]. The morphologic and behavioral changes in the hippocampus due to chronic stress or long-term exposure to excessive corti‐ costerone are similar to the changes observed with reduced mastication [23]. In support of this notion, treatment with the corticosterone synthesis inhibitor metyrapone prevents mo‐ larless-induced learning impairments and neuron loss in the hippocampus [5]. Thus, chronic stress induced by masticatory dysfunction could lead to learning and memory impairments.

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**Figure 2.** Effects of aging on brain regional activity during chewing. Significant signal increases associated with gum chewing in a young adult subject (A), middle-aged subject (B), and an aged subject (C). Upper section: activated areas superimposed on a template (P<0.05, corrected for multiple comparisons). Lower section: activated regions superim‐ posed on a T1-weighted MRI scan (P<0.01, uncorrected for multiple comparisons). pfa, prefrontal area; sma, supple‐ mentary motor area; smc, primary sensorimotor cortex; c, cerebellum; i, insula; t, thalamus. Color scale: t value (degree

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Recent fMRI and positron emission tomography studies in humans revealed that several brain regions are activated during mastication [26, 27]. We performed fMRI studies in hu‐ mans to evaluate the areas of the brain that are activated in association with chewing. In these studies, subjects were asked chew gum with no odor or taste components and per‐ form rhythmic chewing at a rate of approximately 1 Hz. Bilateral increases in activity were observed in several brain areas, including the primary somatosensory cortex, pri‐ mary motor cortex, supplementary motor area, premotor area, prefrontal cortex, insula, posterior cortex, thalamus, striatum, and cerebellum [26, 27]. Age-dependent changes in the chewing-induced BOLD signals were observed in the primary sensorimotor cortex, cerebellum, and thalamus [26, 27]. The right prefrontal cortex showed the highest in‐ crease in activity in elderly persons compared to both young adults and young persons [1] (Fig. 2). The prefrontal cortex is involved in cognitive function [28], and neuronal ac‐ tivity between the right prefrontal cortex and hippocampus might contribute to cognitive function. An fMRI evaluation of the effects of chewing on brain activity during a work‐ ing memory task showed an increase in BOLD signals in the right premotor cortex, pre‐ cuneus, thalamus, hippocampus, and inferior parietal lobe [29]. In another fMRI experiment examining the effect of chewing on hippocampal activity in a spatial cogni‐ tion task [1], subjects were shown 16 photographs followed by the same number of pic‐ tures of a plus character (+) on a green background during each cycle. Each picture or photograph was projected every 2 s during the cycle and the subjects were asked to re‐ member as many of the photographs as possible. The hippocampal BOLD signals in young subject were strongly increased but no significant difference was seen before and after chewing, whereas hippocampal activation in aged subject was quite small compared to that in young subject. The activation area and the intensity of the fMRI signals were, however, increased by chewing [1] (Fig. 3 and 4). Memory acquisition in aged subjects is also significantly enhanced by chewing, whereas chewing had no effect in young subject [1] (Fig. 5). These findings in humans support a link between increased hippocampal BOLD signals and enhanced memory acquisition.

Further studies are needed to clarify how the reduced oral input activity to the aging hippo‐ campus resulting from masticatory dysfunction differs from reduced activity of other types sensory pathways.

mRNA) in the hippocampus [21]. The hippocampus contains a high density of GR and is in‐ volved in the negative feedback mechanism with the hypothalamo-pituitary-adrenal axis via GR, making it very sensitive to corticosterone [22]. The morphologic and behavioral changes in the hippocampus due to chronic stress or long-term exposure to excessive corti‐ costerone are similar to the changes observed with reduced mastication [23]. In support of this notion, treatment with the corticosterone synthesis inhibitor metyrapone prevents mo‐ larless-induced learning impairments and neuron loss in the hippocampus [5]. Thus, chronic stress induced by masticatory dysfunction could lead to learning and memory impairments.

118 Senescence and Senescence-Related Disorders

Recent fMRI and positron emission tomography studies in humans revealed that several brain regions are activated during mastication [26, 27]. We performed fMRI studies in hu‐ mans to evaluate the areas of the brain that are activated in association with chewing. In these studies, subjects were asked chew gum with no odor or taste components and per‐ form rhythmic chewing at a rate of approximately 1 Hz. Bilateral increases in activity were observed in several brain areas, including the primary somatosensory cortex, pri‐ mary motor cortex, supplementary motor area, premotor area, prefrontal cortex, insula, posterior cortex, thalamus, striatum, and cerebellum [26, 27]. Age-dependent changes in the chewing-induced BOLD signals were observed in the primary sensorimotor cortex, cerebellum, and thalamus [26, 27]. The right prefrontal cortex showed the highest in‐ crease in activity in elderly persons compared to both young adults and young persons [1] (Fig. 2). The prefrontal cortex is involved in cognitive function [28], and neuronal ac‐ tivity between the right prefrontal cortex and hippocampus might contribute to cognitive function. An fMRI evaluation of the effects of chewing on brain activity during a work‐ ing memory task showed an increase in BOLD signals in the right premotor cortex, pre‐ cuneus, thalamus, hippocampus, and inferior parietal lobe [29]. In another fMRI experiment examining the effect of chewing on hippocampal activity in a spatial cogni‐ tion task [1], subjects were shown 16 photographs followed by the same number of pic‐ tures of a plus character (+) on a green background during each cycle. Each picture or photograph was projected every 2 s during the cycle and the subjects were asked to re‐ member as many of the photographs as possible. The hippocampal BOLD signals in young subject were strongly increased but no significant difference was seen before and after chewing, whereas hippocampal activation in aged subject was quite small compared to that in young subject. The activation area and the intensity of the fMRI signals were, however, increased by chewing [1] (Fig. 3 and 4). Memory acquisition in aged subjects is also significantly enhanced by chewing, whereas chewing had no effect in young subject [1] (Fig. 5). These findings in humans support a link between increased hippocampal

Further studies are needed to clarify how the reduced oral input activity to the aging hippo‐ campus resulting from masticatory dysfunction differs from reduced activity of other types

BOLD signals and enhanced memory acquisition.

sensory pathways.

**Figure 2.** Effects of aging on brain regional activity during chewing. Significant signal increases associated with gum chewing in a young adult subject (A), middle-aged subject (B), and an aged subject (C). Upper section: activated areas superimposed on a template (P<0.05, corrected for multiple comparisons). Lower section: activated regions superim‐ posed on a T1-weighted MRI scan (P<0.01, uncorrected for multiple comparisons). pfa, prefrontal area; sma, supple‐ mentary motor area; smc, primary sensorimotor cortex; c, cerebellum; i, insula; t, thalamus. Color scale: t value (degree of freedom=87.12). (Onuzuka et al., 2008, [1] with permission)

**Figure 3.** Hippocampal activities in young subject. A Task paradigm. B Significant signal increases associated with pho‐ tograph encoding before and after gum chewing. Hip, hippocampus. Color scale: t value. (Onozuka et al., 2008, [1] with permission)

### **3. Occlusal disharmony and cognitive function**

Occlusal disharmony, such as loss of teeth and increases in the vertical dimension of crowns, bridges, or dentures, causes bruxism or pain in the masticatory muscles and temporomandib‐ ular joints, and general malaise [30, 31]. Studies in SAMP8 mice also show that occlusal disharmony impairs learning and memory. Using SAMP8 mice, we created a model of occlusal disharmony by raising the bite by approximately 0.1 mm using dental materials, referred to as the bite-raised condition. Animals in the bite-raised condition show age-dependent deficits in spatial learning in the Morris water maze [32-39] (Fig. 6). Raising the bite in aged SAMP8 mice decreases the number of pyramidal cells [34] as well as the number of their dendritic spines [39], and increases hypertrophy and hyperplasia of grail fibrillary acid protein-labeled astrocytes [38] in the hippocampal CA1and CA3 regions, suggesting that occlusal disharmony

resulting from the bite-raised condition also enhances aging-related changes in the hippo‐

**Figure 4.** Hippocampal activities in an aged subject. A, Significant signal increases are associated with photograph en‐ coding before and after gum chewing. Color scale: t value. B, Changes in signal intensity on an image-by image basis for 64 successive images during four cycles of encoding of photographs: brown (without chewing) and pink (with

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chewing) boxes; plus (+) characters (without boxes) (Onozuka et al., 2008, [1], with permission)

In rodents and monkeys, alterations of the bite alignment induced by attaching acrylic caps to the incisors [40-42] or inserting occlusal splints in the maxilla [43, 44] are associated with increases urinary cortisol levels and plasma corticosterone levels, suggesting that occlusal disharmony is also a source of stress. In support of this notion, aged bite-raised SAMP8 mice with learning deficits exhibit marked increase in plasma corticosterone levels [33, 36, 37] and downregulation of hippocampal GR and GR mRNA [33]. The behavioral and morphologic changes observed in animals with occlusal disharmony closely resemble the changes induced

campus.

**Figure 4.** Hippocampal activities in an aged subject. A, Significant signal increases are associated with photograph en‐ coding before and after gum chewing. Color scale: t value. B, Changes in signal intensity on an image-by image basis for 64 successive images during four cycles of encoding of photographs: brown (without chewing) and pink (with chewing) boxes; plus (+) characters (without boxes) (Onozuka et al., 2008, [1], with permission)

**3. Occlusal disharmony and cognitive function**

with permission)

120 Senescence and Senescence-Related Disorders

Occlusal disharmony, such as loss of teeth and increases in the vertical dimension of crowns, bridges, or dentures, causes bruxism or pain in the masticatory muscles and temporomandib‐ ular joints, and general malaise [30, 31]. Studies in SAMP8 mice also show that occlusal disharmony impairs learning and memory. Using SAMP8 mice, we created a model of occlusal disharmony by raising the bite by approximately 0.1 mm using dental materials, referred to as the bite-raised condition. Animals in the bite-raised condition show age-dependent deficits in spatial learning in the Morris water maze [32-39] (Fig. 6). Raising the bite in aged SAMP8 mice decreases the number of pyramidal cells [34] as well as the number of their dendritic spines [39], and increases hypertrophy and hyperplasia of grail fibrillary acid protein-labeled astrocytes [38] in the hippocampal CA1and CA3 regions, suggesting that occlusal disharmony

**Figure 3.** Hippocampal activities in young subject. A Task paradigm. B Significant signal increases associated with pho‐ tograph encoding before and after gum chewing. Hip, hippocampus. Color scale: t value. (Onozuka et al., 2008, [1]

> resulting from the bite-raised condition also enhances aging-related changes in the hippo‐ campus.

> In rodents and monkeys, alterations of the bite alignment induced by attaching acrylic caps to the incisors [40-42] or inserting occlusal splints in the maxilla [43, 44] are associated with increases urinary cortisol levels and plasma corticosterone levels, suggesting that occlusal disharmony is also a source of stress. In support of this notion, aged bite-raised SAMP8 mice with learning deficits exhibit marked increase in plasma corticosterone levels [33, 36, 37] and downregulation of hippocampal GR and GR mRNA [33]. The behavioral and morphologic changes observed in animals with occlusal disharmony closely resemble the changes induced

**Figure 5.** Memory acquisition before and after gum chewing for 2 min. A recall test was carried out 20 min after the encoding experiments. In the recall test, we used 64 photographs at random: 32 of the photographs were repeated from the encoding test, and the other 32 photographs were newly added. The subject had to judge whether each photograph had been seen before. (Onozuka et al., 2008, [1], with permission)

by chronic stress and/or long-term exposure to glucocorticoid exposure [23, 100]. The hippo‐ campus is very sensitive to corticosterone [22]. These hippocampal behavioral and morpho‐ logic impairments induced by occlusal disharmony are also attenuated by administration of the corticosterone synthesis inhibitor metyrapone [37]. These findings suggest that occlusal disharmony-induced changes in learning behavioral and hippocampal morphology due to increases in the glucocorticoid levels in association with the downregulation of GR and GR mRNA.

substantia nigra, locus coeruleus, and nucleus raphe dorsalis [46], which are similar to the changes induced by chronic stress [47]. These changes in the catecholaminergic and seroto‐ nergic systems induced by occlusal disharmony likely affect the innervations of the hippo‐ campus. The bite-raised condition impairs neurogenesis and leads to apoptosis in the hippocampal dentate gyrus and decreases the expression of hippocampal brain derived neurotrophic factor, all of which could contribute to the learning impairments observed in

cantly longer time to reach the platform than age-matched controls. (Kubo et al., 2007, [34])

**Figure 6.** Effect of bite-raised condition on spatial learning in the Morris water maze test. The results are expressed as the mean score (mean ± SE, n=6 for each group) of four trials per day. 9m BR, 9-month-old bite-raised mice; 9m Cont, 9-month-old control mice; 5m BR, 5-month-old bite-raised mice; 5m Cont, 5-month-old control mice; 3m BR, 3-monthold bite-raised mice; 3m Cont, 3-month-old control mice. Note that 9-month-old bite-raised mice required a signifi‐

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These findings in animal models were extended to humans. In humans, we used a custommade splint that forced the mandible into a retrusive position and a splint without modification as a control in order to measure BOLD signals during clenching in a malocclusal model [48]. Several regions were activated by clenching, including the premotor cortex, prefrontal cortex, sensorimotor cortex, and insula. In the malocclusion model, which also induces psychologic discomfort, clenching further increased BOLD signals in the anterior cingulate cortex and the amygdala [48]. These findings suggest that clenching under malocclusal conditions induces

emotional stress and/or pain-related neuronal processing in the brain.

animals with occlusal disharmony.

Occlusal disharmony, like masticatory dysfunction, decreases hippocampal activity resulting from the activity of masticatory organ pathways. In bite-raised aged SAMP8 mice, both induction of Fos in the hippocampus following a learning task [36] and the number of spines in hippocampal CA1 pyramidal cells are decreased [39]. Further, the bite-raised condition leads to a decreased number of choline acetyltransferase-positive neurons in the septal nucleus, and reduction in extracellular stimulation-induced acetylcholine release [45].

Occlusal disharmony also affects catecholaminergic activity. Altering the bite by placing an acrylic cap on the lower incisors leads to increases in both dopamine and noradrenaline levels in the hypothalamus and/or frontal cortex [40-42], and decreases in tyrosine hydroxylase, GTP cyclohydroxylase I, and serotonin immunoreactivity in the cerebral cortex, caudate nucleus,

**Figure 6.** Effect of bite-raised condition on spatial learning in the Morris water maze test. The results are expressed as the mean score (mean ± SE, n=6 for each group) of four trials per day. 9m BR, 9-month-old bite-raised mice; 9m Cont, 9-month-old control mice; 5m BR, 5-month-old bite-raised mice; 5m Cont, 5-month-old control mice; 3m BR, 3-monthold bite-raised mice; 3m Cont, 3-month-old control mice. Note that 9-month-old bite-raised mice required a signifi‐ cantly longer time to reach the platform than age-matched controls. (Kubo et al., 2007, [34])

by chronic stress and/or long-term exposure to glucocorticoid exposure [23, 100]. The hippo‐ campus is very sensitive to corticosterone [22]. These hippocampal behavioral and morpho‐ logic impairments induced by occlusal disharmony are also attenuated by administration of the corticosterone synthesis inhibitor metyrapone [37]. These findings suggest that occlusal disharmony-induced changes in learning behavioral and hippocampal morphology due to increases in the glucocorticoid levels in association with the downregulation of GR and GR

**Figure 5.** Memory acquisition before and after gum chewing for 2 min. A recall test was carried out 20 min after the encoding experiments. In the recall test, we used 64 photographs at random: 32 of the photographs were repeated from the encoding test, and the other 32 photographs were newly added. The subject had to judge whether each

Occlusal disharmony, like masticatory dysfunction, decreases hippocampal activity resulting from the activity of masticatory organ pathways. In bite-raised aged SAMP8 mice, both induction of Fos in the hippocampus following a learning task [36] and the number of spines in hippocampal CA1 pyramidal cells are decreased [39]. Further, the bite-raised condition leads to a decreased number of choline acetyltransferase-positive neurons in the septal nucleus,

Occlusal disharmony also affects catecholaminergic activity. Altering the bite by placing an acrylic cap on the lower incisors leads to increases in both dopamine and noradrenaline levels in the hypothalamus and/or frontal cortex [40-42], and decreases in tyrosine hydroxylase, GTP cyclohydroxylase I, and serotonin immunoreactivity in the cerebral cortex, caudate nucleus,

and reduction in extracellular stimulation-induced acetylcholine release [45].

photograph had been seen before. (Onozuka et al., 2008, [1], with permission)

122 Senescence and Senescence-Related Disorders

mRNA.

substantia nigra, locus coeruleus, and nucleus raphe dorsalis [46], which are similar to the changes induced by chronic stress [47]. These changes in the catecholaminergic and seroto‐ nergic systems induced by occlusal disharmony likely affect the innervations of the hippo‐ campus. The bite-raised condition impairs neurogenesis and leads to apoptosis in the hippocampal dentate gyrus and decreases the expression of hippocampal brain derived neurotrophic factor, all of which could contribute to the learning impairments observed in animals with occlusal disharmony.

These findings in animal models were extended to humans. In humans, we used a custommade splint that forced the mandible into a retrusive position and a splint without modification as a control in order to measure BOLD signals during clenching in a malocclusal model [48]. Several regions were activated by clenching, including the premotor cortex, prefrontal cortex, sensorimotor cortex, and insula. In the malocclusion model, which also induces psychologic discomfort, clenching further increased BOLD signals in the anterior cingulate cortex and the amygdala [48]. These findings suggest that clenching under malocclusal conditions induces emotional stress and/or pain-related neuronal processing in the brain.

Together these findings suggest that changes in the hippocampus induced by occlusal disharmony result from increased stress. Occlusal disharmony, like masticatory dysfunction leads to alterations in the central nervous system, especially the hippocampus. Further studies are needed to elucidate differences in the effects of dysfunctional mastication and occlusal disharmony.

physiologic processes, and the central nucleus of the amygdala regulates dopamine neuro‐ transmission in the medial prefrontal nucleus [60, 61]. These findings suggest that chewing during stressful conditions modulates catecholaminergic neurotransmission in the central

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Clinical studies have shown that offspring of mothers who suffer social, emotional or hostile experiences displayed enhanced susceptibility to some mental disorders, including depression, schizophrenia and cognitive deficits [68]. Maternal stress is a suggested risk factor for impaired brain developmental and anxiety, depressive-like behavior and learn‐ ing deficits in rodents pups [69-71], and maternal stress model is often used in studies for depression and cognitive deficits in pups. We recently evaluated whether chewing during maternal restraint stress prevents stress-induced anxiety-like behavior and learn‐ ing impairment in pups. Pregnant mice were exposed to restraint stress beginning on day 15 and continuing until delivery. Mice were placed in a ventilated plastic restraint tube in which they were only able to move back and forth, but not turn around, to induce re‐ straint stress. Half of the dams were given a wooden stick (diameter, ~2mm) to chew on during the restraint stress. The pups were raised to adulthood and behavioral and mor‐ phologic changes were assessed. Restraint stress during pregnancy caused anxiety-like, impaired learning and suppressed cell proliferation in the hippocampal dentate gyrus. Chewing during restraint stress, however, attenuated the anxiety-like behavior, impaired learning, and suppressed cell generation induced by restraint stress. These findings sug‐ gest that maternal chewing contributes to prevent stress-induced anxiety-like behavior,

learning impairment, and morphologic changes in hippocampus in pups.

under stress conditions attenuates stress-induced changes to the brain.

**5. Conclusion**

on the brain.

In humans, chewing gum alleviates a negative mood, reduces cortisol levels during acute laboratory-induced psychologic stress [72], and reduces perceived levels of daily stress [73]. These findings indicate that the stress response in human is also ameliorated by chewing.

Additional studies are needed to investigate the mechanisms by which aggressive chewing

Masticatory dysfunction resulting from tooth loss or extraction, feeding on a soft-diet or occlusal disharmony, induces pathologic changes in the hippocampus and deficits in learning and memory. Aggressive biting or chewing activates several regions in the cen‐ tral nervous system, including the right prefrontal cortex, which is strongly involved in learning and memory. Chewing under stressful conditions attenuates stress-induced changes in the brain. These findings together indicate that masticatory function is impor‐ tant for maintaining cognitive function, and chewing during exposure to stress might be a useful method of coping with stress. Chewing gum may thus be a simple method to attenuate or delay the development of dementia and to ameliorate the effects of stress

nervous system, leading to changes in cognitive function.

### **4. Mastication and stress coping**

The act of chewing, or masticatory stimulation, during stressful conditions may attenuate the ef‐ fects of stress on cognitive function. To examine the effect of mastication on stress-induced be‐ havioral and morphologic changes, we placed mice in a ventilated plastic restraint tube in which they were only able to move back and forth, but not turn around, to induce restraint stress. Half of mice were given a wooden stick (diameter, ~2mm) to chew during restraint [12]. As men‐ tioned above, the hippocampus plays a crucial role in memory formation and is highly sensi‐ tive to aging [49, 50] and stress [51]. Increased plasma corticosterone levels suppress synaptic plasticity in the hippocampus [52] and cell proliferation in the hippocampal dentate gyrus [12] (Fig. 7). Chewing during a stressful event, on the other hand, attenuates stress-induced impair‐ ments of plasticity in the hippocampus by activating stress-suppressed N-methyl-D-aspartatereceptor function [53, 54]. Chewing under stress conditions also blocks the stress-induced suppression of cell generation in the hippocampal dentate gyrus [12]. In adults, neurogenesis in the hippocampus is required for hippocampus-dependent learning and memory [55]. Thus, chewing during stress may attenuate stress-induced impairments in cognitive function.

Rodents permitted to chew on a wooden stick during restraint stress showed attenuated restraint-induced increase in plasma corticosterone levels [12] and corticotrophin releasing factor expression [56], c-Fos induction [57], and phosphorylation of extracellular signalregulated protein kinase 1/2 [58], oxidative stress [59], and nitric oxide [60, 61] in the paraven‐ tricular nucleus of the hypothalamus, compared with controls not provided with a wooden stick. Thus, chewing under stressful conditions appears to attenuate stress-induced increase in plasma corticosterone levels by inhibiting the hypothalamo-pituitary-adrenal-axis.

Mastication may also activate histamine neurons through the ventromedial hypothalamus and mesencephalic trigeminal sensory nucleus [62]. The histamine system could modulate the activity of the septohippocampal cholinergic system, which is involved in learning and memory [63]. Chewing under stress conditions increases the release of histamine in the hippocampus by activating H1 receptors [64]. Therefore, chewing may induce changes in the amounts of acetylcholine released, thereby attenuating stress-induced impairments in memory function.

In animals that aggressively chew on a wooden stick during immobilization stress, the stressinduced release of noradrenaline in the amygdala [65] and Fos-immunoreactivity in the right medial prefrontal cortex are increased [66], whereas Fos-immunoreactivity in the right central nucleus of the amygdala [66], and the dopamine response in the medial prefrontal cortex are decreased [67]. The prefrontal cortex has a crucial role in several cognitive, affective, and physiologic processes, and the central nucleus of the amygdala regulates dopamine neuro‐ transmission in the medial prefrontal nucleus [60, 61]. These findings suggest that chewing during stressful conditions modulates catecholaminergic neurotransmission in the central nervous system, leading to changes in cognitive function.

Clinical studies have shown that offspring of mothers who suffer social, emotional or hostile experiences displayed enhanced susceptibility to some mental disorders, including depression, schizophrenia and cognitive deficits [68]. Maternal stress is a suggested risk factor for impaired brain developmental and anxiety, depressive-like behavior and learn‐ ing deficits in rodents pups [69-71], and maternal stress model is often used in studies for depression and cognitive deficits in pups. We recently evaluated whether chewing during maternal restraint stress prevents stress-induced anxiety-like behavior and learn‐ ing impairment in pups. Pregnant mice were exposed to restraint stress beginning on day 15 and continuing until delivery. Mice were placed in a ventilated plastic restraint tube in which they were only able to move back and forth, but not turn around, to induce re‐ straint stress. Half of the dams were given a wooden stick (diameter, ~2mm) to chew on during the restraint stress. The pups were raised to adulthood and behavioral and mor‐ phologic changes were assessed. Restraint stress during pregnancy caused anxiety-like, impaired learning and suppressed cell proliferation in the hippocampal dentate gyrus. Chewing during restraint stress, however, attenuated the anxiety-like behavior, impaired learning, and suppressed cell generation induced by restraint stress. These findings sug‐ gest that maternal chewing contributes to prevent stress-induced anxiety-like behavior, learning impairment, and morphologic changes in hippocampus in pups.

In humans, chewing gum alleviates a negative mood, reduces cortisol levels during acute laboratory-induced psychologic stress [72], and reduces perceived levels of daily stress [73]. These findings indicate that the stress response in human is also ameliorated by chewing.

Additional studies are needed to investigate the mechanisms by which aggressive chewing under stress conditions attenuates stress-induced changes to the brain.

#### **5. Conclusion**

Together these findings suggest that changes in the hippocampus induced by occlusal disharmony result from increased stress. Occlusal disharmony, like masticatory dysfunction leads to alterations in the central nervous system, especially the hippocampus. Further studies are needed to elucidate differences in the effects of dysfunctional mastication and occlusal

The act of chewing, or masticatory stimulation, during stressful conditions may attenuate the ef‐ fects of stress on cognitive function. To examine the effect of mastication on stress-induced be‐ havioral and morphologic changes, we placed mice in a ventilated plastic restraint tube in which they were only able to move back and forth, but not turn around, to induce restraint stress. Half of mice were given a wooden stick (diameter, ~2mm) to chew during restraint [12]. As men‐ tioned above, the hippocampus plays a crucial role in memory formation and is highly sensi‐ tive to aging [49, 50] and stress [51]. Increased plasma corticosterone levels suppress synaptic plasticity in the hippocampus [52] and cell proliferation in the hippocampal dentate gyrus [12] (Fig. 7). Chewing during a stressful event, on the other hand, attenuates stress-induced impair‐ ments of plasticity in the hippocampus by activating stress-suppressed N-methyl-D-aspartatereceptor function [53, 54]. Chewing under stress conditions also blocks the stress-induced suppression of cell generation in the hippocampal dentate gyrus [12]. In adults, neurogenesis in the hippocampus is required for hippocampus-dependent learning and memory [55]. Thus, chewing during stress may attenuate stress-induced impairments in cognitive function.

Rodents permitted to chew on a wooden stick during restraint stress showed attenuated restraint-induced increase in plasma corticosterone levels [12] and corticotrophin releasing factor expression [56], c-Fos induction [57], and phosphorylation of extracellular signalregulated protein kinase 1/2 [58], oxidative stress [59], and nitric oxide [60, 61] in the paraven‐ tricular nucleus of the hypothalamus, compared with controls not provided with a wooden stick. Thus, chewing under stressful conditions appears to attenuate stress-induced increase

Mastication may also activate histamine neurons through the ventromedial hypothalamus and mesencephalic trigeminal sensory nucleus [62]. The histamine system could modulate the activity of the septohippocampal cholinergic system, which is involved in learning and memory [63]. Chewing under stress conditions increases the release of histamine in the hippocampus by activating H1 receptors [64]. Therefore, chewing may induce changes in the amounts of acetylcholine released, thereby attenuating stress-induced impairments in

In animals that aggressively chew on a wooden stick during immobilization stress, the stressinduced release of noradrenaline in the amygdala [65] and Fos-immunoreactivity in the right medial prefrontal cortex are increased [66], whereas Fos-immunoreactivity in the right central nucleus of the amygdala [66], and the dopamine response in the medial prefrontal cortex are decreased [67]. The prefrontal cortex has a crucial role in several cognitive, affective, and

in plasma corticosterone levels by inhibiting the hypothalamo-pituitary-adrenal-axis.

disharmony.

memory function.

**4. Mastication and stress coping**

124 Senescence and Senescence-Related Disorders

Masticatory dysfunction resulting from tooth loss or extraction, feeding on a soft-diet or occlusal disharmony, induces pathologic changes in the hippocampus and deficits in learning and memory. Aggressive biting or chewing activates several regions in the cen‐ tral nervous system, including the right prefrontal cortex, which is strongly involved in learning and memory. Chewing under stressful conditions attenuates stress-induced changes in the brain. These findings together indicate that masticatory function is impor‐ tant for maintaining cognitive function, and chewing during exposure to stress might be a useful method of coping with stress. Chewing gum may thus be a simple method to attenuate or delay the development of dementia and to ameliorate the effects of stress on the brain.

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**Figure 7.** Effect of chewing during prenatal stress on learning ability and cell proliferation in the hippocampal dentate gyrus. Spatial learning in the water maze test (A) and BrdU-positive cells (B). The results are expressed as the mean score (mean ± SD, n=6 for each group) of four trials per day (A). The results are presented as the mean ± SD (n=5 for each group). C, control; S, restraint stress; S/C, restraint/chewing. \**P*<0.05 (B). Note that prenatal stress induced learn‐ ing and memory deficits, and decreased cell proliferation in the hippocampal dentate gyrus. Maternal chewing during stress inhibits the stress-induced learning and memory deficits, and suppression of cell generation.

#### **Acknowledgements**

This work was supported in part by Grant-in Aid Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology-Japan (KAKENHI 22390395).

### **Author details**

Kin-ya Kubo1\*, Huayue Chen2 and Minoru Onozuka3

\*Address all correspondence to: kubo@seijoh-u.ac.jp

1 Seijoh University Graduate School of Health Care Studies, Fukinodai, Tokai, Aichi, Japan

2 Department of Anatomy, Gifu University Graduate School of Medicine, Yanaido, Gifu, Ja‐ pan

3 Nittai Jusei Medical College for Judo Therapeutics, Yoga, Setagaya-ku, Tokyo, Japan

#### **References**

**Acknowledgements**

126 Senescence and Senescence-Related Disorders

**Author details**

pan

Kin-ya Kubo1\*, Huayue Chen2

\*Address all correspondence to: kubo@seijoh-u.ac.jp

This work was supported in part by Grant-in Aid Scientific Research from the Ministry of

**Figure 7.** Effect of chewing during prenatal stress on learning ability and cell proliferation in the hippocampal dentate gyrus. Spatial learning in the water maze test (A) and BrdU-positive cells (B). The results are expressed as the mean score (mean ± SD, n=6 for each group) of four trials per day (A). The results are presented as the mean ± SD (n=5 for each group). C, control; S, restraint stress; S/C, restraint/chewing. \**P*<0.05 (B). Note that prenatal stress induced learn‐ ing and memory deficits, and decreased cell proliferation in the hippocampal dentate gyrus. Maternal chewing during

Education, Culture, Sports, Science, and Technology-Japan (KAKENHI 22390395).

stress inhibits the stress-induced learning and memory deficits, and suppression of cell generation.

and Minoru Onozuka3

1 Seijoh University Graduate School of Health Care Studies, Fukinodai, Tokai, Aichi, Japan

2 Department of Anatomy, Gifu University Graduate School of Medicine, Yanaido, Gifu, Ja‐

3 Nittai Jusei Medical College for Judo Therapeutics, Yoga, Setagaya-ku, Tokyo, Japan


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**Section 6**

**Neurodegenerative Disease**


## **Neurodegenerative Disease**

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**Chapter 6**

**On the Way to Longevity: How to Combat Neuro-**

Aging can be defined as the accumulation of unrepaired, deleterious changes occurring in the molecules, cells, tissues and organs of the body over time generated by internal and external sources. An integral part of the aging process is represented by the adaptive mechanisms that the body sets up to compensate and neutralize the adverse effects of such damage that lead to a progressive change of the body composition and its microenvironments. Among others, the multifaceted dynamic process, known as immunosenescence, encompasses all the complex changes occurring in the immune system during aging. It results from the adaptation process of the body to the continuous challenge of infections and is the basis of the age-associated decrease in immune competence that renders individuals more susceptible to diseases. Immunosenescence is associated with an increase of morbidity and mortality [27, 35, 46]. One of the typical aspects of immunosenescence is the profound modification within the cytokine network leading to the development of a low-grade inflammatory status, known as "inflam‐ maging" [28]. This phenomenon is characterized by a general increase in plasmatic levels and cell capability to produce pro-inflammatory cytokines (Interleukin-6, IL-6, Interleukin-1, IL-1 and Tumour Necrosis Factor-α, TNF-α) and by a subsequent increase of the main inflamma‐ tory markers, such as C-reactive protein (CRP) and serum amyloid A (A-SAA) [29, 31, 32]. This generalized pro-inflammatory status, interacting with the genetic background and environ‐ mental factors, potentially triggers the onset of the most important age-related diseases, such as cardiovascular diseases, atherosclerosis, metabolic syndrome, type 2 diabetes and obesity, neurodegeneration, arthrosis and arthritis, osteoporosis and osteoarthritis, sarcopenia, major

> © 2013 d'Alessio et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Degenerative Disease**

Claudio Franceschi

**1. Introduction**

http://dx.doi.org/10.5772/54914

**1.1. Aging and inflammaging**

Patrizia d'Alessio, Rita Ostan, Miriam Capri and

Additional information is available at the end of the chapter

## **On the Way to Longevity: How to Combat Neuro-Degenerative Disease**

Patrizia d'Alessio, Rita Ostan, Miriam Capri and Claudio Franceschi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54914

#### **1. Introduction**

#### **1.1. Aging and inflammaging**

Aging can be defined as the accumulation of unrepaired, deleterious changes occurring in the molecules, cells, tissues and organs of the body over time generated by internal and external sources. An integral part of the aging process is represented by the adaptive mechanisms that the body sets up to compensate and neutralize the adverse effects of such damage that lead to a progressive change of the body composition and its microenvironments. Among others, the multifaceted dynamic process, known as immunosenescence, encompasses all the complex changes occurring in the immune system during aging. It results from the adaptation process of the body to the continuous challenge of infections and is the basis of the age-associated decrease in immune competence that renders individuals more susceptible to diseases. Immunosenescence is associated with an increase of morbidity and mortality [27, 35, 46]. One of the typical aspects of immunosenescence is the profound modification within the cytokine network leading to the development of a low-grade inflammatory status, known as "inflam‐ maging" [28]. This phenomenon is characterized by a general increase in plasmatic levels and cell capability to produce pro-inflammatory cytokines (Interleukin-6, IL-6, Interleukin-1, IL-1 and Tumour Necrosis Factor-α, TNF-α) and by a subsequent increase of the main inflamma‐ tory markers, such as C-reactive protein (CRP) and serum amyloid A (A-SAA) [29, 31, 32]. This generalized pro-inflammatory status, interacting with the genetic background and environ‐ mental factors, potentially triggers the onset of the most important age-related diseases, such as cardiovascular diseases, atherosclerosis, metabolic syndrome, type 2 diabetes and obesity, neurodegeneration, arthrosis and arthritis, osteoporosis and osteoarthritis, sarcopenia, major

© 2013 d'Alessio et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

depression and frailty [46]. The first evidence of the age-associated modification in the balance of cytokine network was described by [24] who found an increase of IL-6 plasma levels and a corresponding decrease of IL-2 production in healthy elderly subjects [24, 26].

are characterized by inflammaging? Our hypothesis is that their reduced capacity to mount strong inflammatory responses is due to a remarkable genetic pattern (based on anti-inflam‐ matory gene variants) and is able to limit the inflammatory process. A protective genetic component towards the development of age-related pathologies with a strong inflammatory

A Study of the Reversibility of Early Stages of Degenerative Diseases, Dementias and Alzheimer's Disease (AD)

http://dx.doi.org/10.5772/54914

137

Inflammaging can, in turn, undermine the balance between gut microbiota and the gastroin‐ testinal-associated immune system, contributing to the establishment of a vicious inflamma‐ tory cycle [8]. Importantly, recent literature suggests the impact of microbiota inflammatory stimuli on the brain [6]. Several studies even suggest an inflammatory pathogenesis at the basis of activation of microglia in response to injury, illness and aging, as described in the following

The term neuro-inflammation designates chronic, CNS-specific, inflammation-like glial responses that do not reproduce the classic characteristics of inflammation in the periphery but that may provoke neuro-degenerative events, including plaque formation, dystrophic neurite growth and unwarranted tau phosphorylation, among other signs. Aetiology of neuroinflammation is not yet clarified even if many strides forward have been made in this field. In fact, during the last decades important discoveries have been made, particularly on risk factors, genetic-associated variants, pro-inflammatory molecules, cellular and sub-cellular modified processes and, ultimately, the gene expression pathways shared in many neuro-degenerative diseases, such as AD. A recent review summarizes microarray human studies in neurodegenerative diseases showing gene expression profiles shared in these age-associated diseases [15], highlighting the inflammatory component. In addition, RNA splicing and protein turnover are found to be disrupted and mitochondrial dysfunction has been reported.

Franceschi's team is heavily involved in the study of age-related diseases and in particu‐ lar AD, either in terms of nuclear and mitochondrial genetic variants and pro-inflammato‐ ry environments [21, 22, 36-39, 45, 49]. It is well known that AD is a fast growing worldwide pathology: it is a slowly progressive and, after early stage reversible phases, irreversible neuro-degenerative disease. Patients undergo decades of symptomatic pro‐ gression; multiple interacting molecular mechanisms contribute to the development of the early clinical prodromal stages characterized by episodic memory deficits and decline, as well as impairment of general cognitive functioning, particularly during the final syndro‐ mal dementia stage (reviewed in [34]). In the context of AD research, the team studied the role of genetic cytokines variants, such as IL-1, IL-6, TNF-α and interferon-gamma (IFN-γ), in AD patients. The data showed the association between the plasmatic and brain level of IL-6 and IL-6 polymorphisms at 174 position in the promoter region, suggesting a relationship between specific gene variants and circulating levels of a specific inflammato‐ ry cytokine; furthermore, cytokine blood level mirrors the quantity of its level in the brain [38]. Similarly, increased levels of IL-1, another pro-inflammatory cytokine, are observed

pathogenesis would thus be exerted [32].

**2. Neuro-inflammation**

section.

We have provided several contributions on the relevance of the inflammatory reaction at the vascular site for cell senescence in terms of the reversibility of its inflammatory phe‐ notype [16, 17]. These data could be confirmed by *ex vivo* data of Franceschi's laborato‐ ry. A significant increase of IL-6, TNF-α and IL-1β levels were described in mitogenstimulated cultures from aged donors. Indeed, cells from aged people seem able to upregulate the production of these cytokines in response to appropriate stimuli indicating that the cellular machinery for the production of these molecules remains active and effi‐ cient during aging [24]. It has been hypothesized that inflammaging could be due to the antigenic load and its persistence for the entire lifespan. Antigens of common viruses such as human cytomegalovirus (HCMV) or Epstein-Barr virus (EBV) represent a major driving force for the activation of macrophages and expansion of specific T cell clones (megaclones) producing a large amount of inflammatory cytokines [54, 55]. The increase with age of IL-6 plasma levels appears to be unexpectedly present in both those who underwent successful aging and those who suffered pathological aging. Thus, we must question the factors responsible for successful aging. Data obtained on centenarians by the Franceschi laboratory showed that centenarians also are inflamed [3, 4, 26]. Thus, in‐ flammaging *per se* is not incompatible with longevity. But it is likely that many protec‐ tive factors, such as the genetic background, epigenetic markers [33] and antiinflammatory molecules can play a pivotal role in counteracting unfavourable proinflammatory signalling [32].

At present it is not understood whether the alteration in the regulation of inflammatory reac‐ tions could be a cause or rather an effect (or both in a vicious cycle) of the aging process as a whole. A wide range of elements has been claimed to contribute to the development of lowgrade inflammation. In particular, in addition to the main impact of the immune system, a vari‐ ety of tissues (adipose tissue and muscle in particular), organs (liver and brain) and ecosystems (skin, mouth, vagina and gut microbiota) differently contribute to inflammaging onset, pro‐ gression and persistence having specific organ-restricted and/or systemic effects [13].

Gut microbiota and the gastrointestinal-associated immune system coexist in a balanced microenvironment where cytokines and lymphocytes have to cope with the antigenic load, in order to control the enormous variety of bacterial species within the intestinal microflora. During aging, subtle changes in intestinal microbial structure may contribute to the age-related inflammatory status. A reduction of some populations of *Clostridia* in favour of enrichment in facultative anaerobes in centenarians has been described. In addition, the remodelling of centenarians' microbiota was associated with an increased inflammatory state, determined by a series of peripheral inflammatory markers (IL-6, IL-8) [7]. The dysbiosis observed in these extremely long-lived subjects represents an important source of continuous antigenic stimu‐ lation (immune/inflammatory/toxic/metabolic) to other organs and systems, such as the immune system and the liver, contributing to the development and maintenance of inflam‐ maging. So why are healthy centenarians the best example of successful ageing, even if they are characterized by inflammaging? Our hypothesis is that their reduced capacity to mount strong inflammatory responses is due to a remarkable genetic pattern (based on anti-inflam‐ matory gene variants) and is able to limit the inflammatory process. A protective genetic component towards the development of age-related pathologies with a strong inflammatory pathogenesis would thus be exerted [32].

Inflammaging can, in turn, undermine the balance between gut microbiota and the gastroin‐ testinal-associated immune system, contributing to the establishment of a vicious inflamma‐ tory cycle [8]. Importantly, recent literature suggests the impact of microbiota inflammatory stimuli on the brain [6]. Several studies even suggest an inflammatory pathogenesis at the basis of activation of microglia in response to injury, illness and aging, as described in the following section.

### **2. Neuro-inflammation**

depression and frailty [46]. The first evidence of the age-associated modification in the balance of cytokine network was described by [24] who found an increase of IL-6 plasma levels and a

We have provided several contributions on the relevance of the inflammatory reaction at the vascular site for cell senescence in terms of the reversibility of its inflammatory phe‐ notype [16, 17]. These data could be confirmed by *ex vivo* data of Franceschi's laborato‐ ry. A significant increase of IL-6, TNF-α and IL-1β levels were described in mitogenstimulated cultures from aged donors. Indeed, cells from aged people seem able to upregulate the production of these cytokines in response to appropriate stimuli indicating that the cellular machinery for the production of these molecules remains active and effi‐ cient during aging [24]. It has been hypothesized that inflammaging could be due to the antigenic load and its persistence for the entire lifespan. Antigens of common viruses such as human cytomegalovirus (HCMV) or Epstein-Barr virus (EBV) represent a major driving force for the activation of macrophages and expansion of specific T cell clones (megaclones) producing a large amount of inflammatory cytokines [54, 55]. The increase with age of IL-6 plasma levels appears to be unexpectedly present in both those who underwent successful aging and those who suffered pathological aging. Thus, we must question the factors responsible for successful aging. Data obtained on centenarians by the Franceschi laboratory showed that centenarians also are inflamed [3, 4, 26]. Thus, in‐ flammaging *per se* is not incompatible with longevity. But it is likely that many protec‐ tive factors, such as the genetic background, epigenetic markers [33] and antiinflammatory molecules can play a pivotal role in counteracting unfavourable pro-

At present it is not understood whether the alteration in the regulation of inflammatory reac‐ tions could be a cause or rather an effect (or both in a vicious cycle) of the aging process as a whole. A wide range of elements has been claimed to contribute to the development of lowgrade inflammation. In particular, in addition to the main impact of the immune system, a vari‐ ety of tissues (adipose tissue and muscle in particular), organs (liver and brain) and ecosystems (skin, mouth, vagina and gut microbiota) differently contribute to inflammaging onset, pro‐

Gut microbiota and the gastrointestinal-associated immune system coexist in a balanced microenvironment where cytokines and lymphocytes have to cope with the antigenic load, in order to control the enormous variety of bacterial species within the intestinal microflora. During aging, subtle changes in intestinal microbial structure may contribute to the age-related inflammatory status. A reduction of some populations of *Clostridia* in favour of enrichment in facultative anaerobes in centenarians has been described. In addition, the remodelling of centenarians' microbiota was associated with an increased inflammatory state, determined by a series of peripheral inflammatory markers (IL-6, IL-8) [7]. The dysbiosis observed in these extremely long-lived subjects represents an important source of continuous antigenic stimu‐ lation (immune/inflammatory/toxic/metabolic) to other organs and systems, such as the immune system and the liver, contributing to the development and maintenance of inflam‐ maging. So why are healthy centenarians the best example of successful ageing, even if they

gression and persistence having specific organ-restricted and/or systemic effects [13].

corresponding decrease of IL-2 production in healthy elderly subjects [24, 26].

inflammatory signalling [32].

136 Senescence and Senescence-Related Disorders

The term neuro-inflammation designates chronic, CNS-specific, inflammation-like glial responses that do not reproduce the classic characteristics of inflammation in the periphery but that may provoke neuro-degenerative events, including plaque formation, dystrophic neurite growth and unwarranted tau phosphorylation, among other signs. Aetiology of neuroinflammation is not yet clarified even if many strides forward have been made in this field. In fact, during the last decades important discoveries have been made, particularly on risk factors, genetic-associated variants, pro-inflammatory molecules, cellular and sub-cellular modified processes and, ultimately, the gene expression pathways shared in many neuro-degenerative diseases, such as AD. A recent review summarizes microarray human studies in neurodegenerative diseases showing gene expression profiles shared in these age-associated diseases [15], highlighting the inflammatory component. In addition, RNA splicing and protein turnover are found to be disrupted and mitochondrial dysfunction has been reported.

Franceschi's team is heavily involved in the study of age-related diseases and in particu‐ lar AD, either in terms of nuclear and mitochondrial genetic variants and pro-inflammato‐ ry environments [21, 22, 36-39, 45, 49]. It is well known that AD is a fast growing worldwide pathology: it is a slowly progressive and, after early stage reversible phases, irreversible neuro-degenerative disease. Patients undergo decades of symptomatic pro‐ gression; multiple interacting molecular mechanisms contribute to the development of the early clinical prodromal stages characterized by episodic memory deficits and decline, as well as impairment of general cognitive functioning, particularly during the final syndro‐ mal dementia stage (reviewed in [34]). In the context of AD research, the team studied the role of genetic cytokines variants, such as IL-1, IL-6, TNF-α and interferon-gamma (IFN-γ), in AD patients. The data showed the association between the plasmatic and brain level of IL-6 and IL-6 polymorphisms at 174 position in the promoter region, suggesting a relationship between specific gene variants and circulating levels of a specific inflammato‐ ry cytokine; furthermore, cytokine blood level mirrors the quantity of its level in the brain [38]. Similarly, increased levels of IL-1, another pro-inflammatory cytokine, are observed in association with specific IL-1 gene variants [21]. We think that these findings can be in‐ tegrated into the more general vision of the inflammaging process, i.e. the chronic age-re‐ lated pro-inflammatory status together with unfavourable genetic variants can contribute to neuro-inflammation pathogenesis and the onset of AD or neuro-degenerative diseases [30, 11]. Many inflammatory mediators have been detected in regions of the brain of pa‐ tients with AD [45] according to the hypothesis that inflammation might contribute to the neuro-degeneration characterizing this pathology [42].

What are the physiological mechanisms to counteract the pro-inflammatory activation of neuro-inflammation? One of the best characterized is the cholinergic inhibition that con‐ trols microglia activation and thereby limits the severity and duration of delirium. If cholinergic inhibition fails, either because of pre-existing neuro-degeneration or use of drugs with anticholinergic effects, neuro-inflammation could spin out of control, leading to severe prolonged delirium that can become associated with dementia [51]. Thus, the first event, i.e., POD, is often a prodromal event for the development of dementia or AD, i.e., a long-term cognitive decline and also an increased mortality. On this last point limited literature is currently available. It is noteworthy that the inflammatory markers are already abundantly present before the post-operative delirium episode (in particular IL-6, IL-8 and CRP) [5] and sometimes this pro-inflammatory status is accompanied by the decrease of anti-inflammatory molecules such as Acetylcholinesterase enzyme (AchE), inducing an imbalance between pro- and anti-inflammatory responses [12].

A Study of the Reversibility of Early Stages of Degenerative Diseases, Dementias and Alzheimer's Disease (AD)

http://dx.doi.org/10.5772/54914

139

In progress data from the Franceschi laboratory suggest that the assessing of the level of specific plasmatic pro-inflammatory cytokines, together with other parameters [2], before surgery could be the best strategy for early identification of patients who could develop POD and not only for the best management of patients on the ward. This could lead to fast tracking of elderly patients who could develop neuro-degenerative diseases in the

Another model studied by the same laboratory is Down Syndrome (DS), a progeroid syndrome characterized by an accelerated neuro-degenerative process [25, 40]. Ongoing analyses on a cross sectional cohort by means of an ad hoc test battery for cognitive and functional assess‐ ments could be essential in gathering evidence on brain areas that first undergo neuro-

The strategy for counteracting these different age-related neuro-degenerative clinical pic‐ tures and diseases is of primary importance and represents one of most fascinating areas in the field of aging and age-related disease research. In order to slow down and counter‐ act the "destiny" of early identified risk factors in an elderly patient candidate for surgi‐ cal treatment, what could be the most eligible non-invasive and non-toxic therapeutic intervention? Our driving hypothesis is that we can restrain the onset and the progres‐ sion of the age-related neuro-degenerative diseases counteracting immunosenescence [10] and inflammaging by diet intervention, moderate and daily physical exercise and the pos‐ sible use of natural compounds, whose formulation allows specifically reducing inflam‐

Chronic inflammation is an underlying cause of many apparently unrelated, age-related diseases. This fact is often overlooked, yet persuasive scientific evidence exists that correcting a chronic inflammatory disorder will enable many of the infirmities of aging to be prevented or reversed. When we envisage a link between aging and recurrent or chronic inflammation,

future.

degeneration.

matory markers in tissues, cells and blood.

**3. Anti-inflammaging/anti-stress intervention**

The activation of the microglia may be due either to local and/or systemic inflammation. In fact, a strong local inflammatory stimulus, such as a previous head trauma, is a risk factor for AD onset and several epidemiological studies clearly show that blood elevations of acute phase proteins, markers of systemic inflammatory stimuli, may be risk factors for cognitive decline and dementia [21, 52]. Moreover, in AD, astrocytes are involved in the production of neurotoxic substances, such as reactive oxygen and nitrogen species, pro-inflammatory cytokines, complement proteins, and other inflammatory mediators that bring about important neurodegenerative changes [53].

However, the scenario is much more complex than previously thought. The Franceschi group also identified key molecular actors, such as proteasome and immune-proteasome (the molecular complex induced by INFs), as possible motors of protein turnover alteration [43]. The immune-proteasome has been associated with neuro-degenerative and autoimmune diseases as a marker and regulator of inflammatory mechanisms. Its expression in the brain may occur upon neuro-inflammation in different cell types and affect a variety of homeostatic and inflammatory pathways including the oxidized protein clearance and the self-antigen presentation. Recently, its role in epilepsy has been established. In fact the pathology-specific pattern of immune-proteasome expression could provide insight into the complex neuroinflammatory pathogenic components of this disease [44]. The same group is currently working on the circulating proteasome/immuneproteasome, in order to establish its role as a possible early-biomarker in neuro-degenerative and inflammatory diseases. In this regard, the circulating mitochondrial DNA, another systemic biomarker of inflammation and disease, is also being investigated [57]. This type of research could be strategic for the improvement of therapeutic intervention, one of the priorities of the current European and US research. The possibility, as well as the difficulty, of identifying a pro-inflammatory prodromal phenotype which will develop the syndromic stage, is crucial for the prevention, diagnosis and therapy of AD and other age-related neuro-degenerative pathologies [56].

The study of post-operative delirium (POD) in elderly patients [1, 2] has been promoted by the same approach. Recent literature suggests the presence of an inflammatory component in the POD onset, showing again the close relationship between systemic inflammation and CNS, particularly when a stressful event such as surgery (or anaesthesia) or infectious diseases may provoke an acute exacerbation (delirium) interacting with pre-intra and post-operative parameters. One of the main hypotheses related to the delirium onset is that peripherally produced pro-inflammatory cytokines enter the brain and activate microglia. Activated microglia may produce inflammatory mediators affecting neuronal functioning, that may be implicated in the symptomatology of delirium.

What are the physiological mechanisms to counteract the pro-inflammatory activation of neuro-inflammation? One of the best characterized is the cholinergic inhibition that con‐ trols microglia activation and thereby limits the severity and duration of delirium. If cholinergic inhibition fails, either because of pre-existing neuro-degeneration or use of drugs with anticholinergic effects, neuro-inflammation could spin out of control, leading to severe prolonged delirium that can become associated with dementia [51]. Thus, the first event, i.e., POD, is often a prodromal event for the development of dementia or AD, i.e., a long-term cognitive decline and also an increased mortality. On this last point limited literature is currently available. It is noteworthy that the inflammatory markers are already abundantly present before the post-operative delirium episode (in particular IL-6, IL-8 and CRP) [5] and sometimes this pro-inflammatory status is accompanied by the decrease of anti-inflammatory molecules such as Acetylcholinesterase enzyme (AchE), inducing an imbalance between pro- and anti-inflammatory responses [12].

in association with specific IL-1 gene variants [21]. We think that these findings can be in‐ tegrated into the more general vision of the inflammaging process, i.e. the chronic age-re‐ lated pro-inflammatory status together with unfavourable genetic variants can contribute to neuro-inflammation pathogenesis and the onset of AD or neuro-degenerative diseases [30, 11]. Many inflammatory mediators have been detected in regions of the brain of pa‐ tients with AD [45] according to the hypothesis that inflammation might contribute to the

The activation of the microglia may be due either to local and/or systemic inflammation. In fact, a strong local inflammatory stimulus, such as a previous head trauma, is a risk factor for AD onset and several epidemiological studies clearly show that blood elevations of acute phase proteins, markers of systemic inflammatory stimuli, may be risk factors for cognitive decline and dementia [21, 52]. Moreover, in AD, astrocytes are involved in the production of neurotoxic substances, such as reactive oxygen and nitrogen species, pro-inflammatory cytokines, complement proteins, and other inflammatory mediators that bring about important neuro-

However, the scenario is much more complex than previously thought. The Franceschi group also identified key molecular actors, such as proteasome and immune-proteasome (the molecular complex induced by INFs), as possible motors of protein turnover alteration [43]. The immune-proteasome has been associated with neuro-degenerative and autoimmune diseases as a marker and regulator of inflammatory mechanisms. Its expression in the brain may occur upon neuro-inflammation in different cell types and affect a variety of homeostatic and inflammatory pathways including the oxidized protein clearance and the self-antigen presentation. Recently, its role in epilepsy has been established. In fact the pathology-specific pattern of immune-proteasome expression could provide insight into the complex neuroinflammatory pathogenic components of this disease [44]. The same group is currently working on the circulating proteasome/immuneproteasome, in order to establish its role as a possible early-biomarker in neuro-degenerative and inflammatory diseases. In this regard, the circulating mitochondrial DNA, another systemic biomarker of inflammation and disease, is also being investigated [57]. This type of research could be strategic for the improvement of therapeutic intervention, one of the priorities of the current European and US research. The possibility, as well as the difficulty, of identifying a pro-inflammatory prodromal phenotype which will develop the syndromic stage, is crucial for the prevention, diagnosis and therapy

The study of post-operative delirium (POD) in elderly patients [1, 2] has been promoted by the same approach. Recent literature suggests the presence of an inflammatory component in the POD onset, showing again the close relationship between systemic inflammation and CNS, particularly when a stressful event such as surgery (or anaesthesia) or infectious diseases may provoke an acute exacerbation (delirium) interacting with pre-intra and post-operative parameters. One of the main hypotheses related to the delirium onset is that peripherally produced pro-inflammatory cytokines enter the brain and activate microglia. Activated microglia may produce inflammatory mediators affecting neuronal functioning, that may be

neuro-degeneration characterizing this pathology [42].

of AD and other age-related neuro-degenerative pathologies [56].

implicated in the symptomatology of delirium.

degenerative changes [53].

138 Senescence and Senescence-Related Disorders

In progress data from the Franceschi laboratory suggest that the assessing of the level of specific plasmatic pro-inflammatory cytokines, together with other parameters [2], before surgery could be the best strategy for early identification of patients who could develop POD and not only for the best management of patients on the ward. This could lead to fast tracking of elderly patients who could develop neuro-degenerative diseases in the future.

Another model studied by the same laboratory is Down Syndrome (DS), a progeroid syndrome characterized by an accelerated neuro-degenerative process [25, 40]. Ongoing analyses on a cross sectional cohort by means of an ad hoc test battery for cognitive and functional assess‐ ments could be essential in gathering evidence on brain areas that first undergo neurodegeneration.

The strategy for counteracting these different age-related neuro-degenerative clinical pic‐ tures and diseases is of primary importance and represents one of most fascinating areas in the field of aging and age-related disease research. In order to slow down and counter‐ act the "destiny" of early identified risk factors in an elderly patient candidate for surgi‐ cal treatment, what could be the most eligible non-invasive and non-toxic therapeutic intervention? Our driving hypothesis is that we can restrain the onset and the progres‐ sion of the age-related neuro-degenerative diseases counteracting immunosenescence [10] and inflammaging by diet intervention, moderate and daily physical exercise and the pos‐ sible use of natural compounds, whose formulation allows specifically reducing inflam‐ matory markers in tissues, cells and blood.

#### **3. Anti-inflammaging/anti-stress intervention**

Chronic inflammation is an underlying cause of many apparently unrelated, age-related diseases. This fact is often overlooked, yet persuasive scientific evidence exists that correcting a chronic inflammatory disorder will enable many of the infirmities of aging to be prevented or reversed. When we envisage a link between aging and recurrent or chronic inflammation, we refer to the pathological consequences of inflammation in well-documented medical literature. Regrettably, the origins as well as the consequences of systemic inflammation continue to be an unsolved problem. By following specific prevention protocols (such as weight loss), the inflammatory stimulation could be significantly reduced. An important role in preventing the onset of a chronic inflammatory condition has been attributed either to the practice of a physical activity or to the prescription of a personalized diet, or both.

associated as dietary supplementation in addition to the Ristomed diet was validated as an

A Study of the Reversibility of Early Stages of Degenerative Diseases, Dementias and Alzheimer's Disease (AD)

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141

In this article, we will report the results concerning the inflammatory markers and the (concomitant) alterations of the mood, comparing the group receiving the diet without supplementation (14 males, mean age 69.6 ± 4.1 years; 17 females, 71.3 ± 3.8 years) to that receiving a diet supplemented with daily soft gel capsules containing the terpen extract AISA 5203-L (14 males, mean age 70.6 ± 4.4 years; 16 females, 69.6 ± 3.3 years), related to as OPE

The laboratory measurements performed included erythrocyte sedimentation rate (ESR), high-sensitivity C-reactive protein (CRP), white blood cell count (WBC) and fibrinogen measurements. Baseline plasma levels of ESR, CRP, WBC, fibrinogen, IL-6 and TNF-α were used to calculate an inflammation score. This enabled the separation of the patients into two groups of respectively low and high inflammation, so that inflammatory status

Moreover, several self-assessment questionnaires were analysed to investigate quality of life parameters. The SF-36v2 Health Survey was used to evaluate what each subject felt about his/ her health using 36 items covering functional status, wellbeing and an overall evaluation of health, that together are referred to as Quality of Life (QoL). Two summary scores — Physical Component Summary (PCS) and Mental Component Summary (MCS) — were calculated to distinguish a possible physical dysfunction and bodily pain from psychological distress and emotional problems. The State-Trait Anxiety Inventory-X (STAI-X) questionnaire was used to assess the anxiety state and trait, and to describe each subject's feelings at a particular point. The results of this investigation showed that among clinically healthy, aged subjects (i.e., absence of cancer, obesity, metabolic syndrome, diabetes, major cardiovascular complaints, arthritis or dementia), a third of them showed important inflammatory markers' expression. It is precisely these patients that could be at risk of developing delirium in the case of surgical treatment [2]. They would largely be advantaged by a preventive treatment of their inflam‐

Conclusively, the results confirmed the anti-inflammatory action of the terpen extract in an aged matched (65-85) healthy population (figure 1 and [20], www.ristomed.eu).

Moreover, Ristomed results were conclusive also for the capacity to lower anxiety and thus implicitly for the link between inflammation and anxiety. Interestingly, study results obtained for quality of life assessment (PCS, MCS and GHQ-12), mood (STAI-X) and de‐ pression (CES-D) confirmed our findings on mood modulation. We note in particular that OPE treatment was more effective in high-inflamed patients, the anti-depressive effect is more visible in low-inflamed patients (figure 2). These results also confirm previous find‐ ings established by our Functional Observation Battery (FOB) in rodents, where AISA 5203-L supplementation was able to substantially contribute to pain tolerance and mood stabilization. However, the most intriguing result was the fact that the stressed animal (non-pathological stress stimulating anxiety), instead of developing a freezing attitude, following oral administration of the food supplement, developed an activity. These data

could be evaluated according to the scores of these markers.

matory condition, especially if high levels of IL-6 and TNF-α are measured.

anti-inflammatory food complement.

(Orange Peel Extract).

Terpens are a large and varied class of organic components classified as secondary me‐ tabolites. They are produced by a wide variety of plants, particularly conifers, though al‐ so by some insects, such as swallowtail butterflies, which emit terpens from their osmeterium. They are the major components of resin and of turpentine produced from resin. The name terpen is derived from the word "turpentine". The smaller and more volatile terpenoids (C10 and C15) are generally the main constituents of the essential oils obtained from many types of plants and flowers, widely used as natural flavourings for food, as fragrances in perfumes in aromatherapy and in traditional and alternative medicines. Terpenoids possess a common structural feature: they contain an integral number of C5 units (isoprene-like) giving a basic molecular formula (C5H8)n for the hy‐ drocarbons series. They are derived from the metabolism of acetate by the mevalonic acid branch biosynthetic pathways of plants.

Examples of monoterpens (C10) are geraniol and limonene. In particular, *d*-limonene has a pronounced chemotherapeutic activity and minimal toxicity in pre-clinical studies. A phase I clinical trial performed to assess toxicity, maximum tolerated dose (MTD) and pharmacoki‐ netics in patients with advanced cancer was followed by a limited phase II evaluation in breast cancer. We have previously published some *in vitro* results on a tri-terpen [58], implicating a NF-κB dependent anti-inflammatory mechanism of action of the extract of *Trytergium Wolfor‐ dii* hoek, used in traditional Chinese medicine for the prevention of arthritis, rheumatoid arthritis and arthrosis.

In performing the experiments for the assessment of the doses to be administered in an *in vivo* rodent model, an anti-stress effect of the terpen AISA 5203-L was unexpectedly revealed by a functional observation battery (FOB). A plethora of parameters addressing behavioural, physiological and neurological parameters in female rats submitted to several stressful conditions were measured. Results showed important effects leading to the capacity of the animals to tolerate stress and even pain when compared to vehicle-treated animals [9].

To these preliminary pre-clinical data we were recently able to add some clinical data showing the coherence of our anti-inflammatory/anti-stress approach [18, 19]. The European Capacity study "Ristomed" enrolled 125 healthy individuals from three different countries (Italy, France and Germany). They all received an 'optimal diet for the elderly' with the supplementation of some nutraceutic compounds for a period of 56 days. The diet was developed on the basis of the current recommendations for elderly people and personalized individual dietary require‐ ments, with particular attention given to food compounds that can affect inflammation, oxidative stress and gut microbiota, such as polyunsaturated fatty acids (PUFAs), antioxidant vitamins, polyphenols, flavonoids and fibres. The diet was adapted to the dietary habits for each country. AISA Therapeutics treatment (here referred to as OPE, i.e., Orange Peel Extract) associated as dietary supplementation in addition to the Ristomed diet was validated as an anti-inflammatory food complement.

we refer to the pathological consequences of inflammation in well-documented medical literature. Regrettably, the origins as well as the consequences of systemic inflammation continue to be an unsolved problem. By following specific prevention protocols (such as weight loss), the inflammatory stimulation could be significantly reduced. An important role in preventing the onset of a chronic inflammatory condition has been attributed either to the

Terpens are a large and varied class of organic components classified as secondary me‐ tabolites. They are produced by a wide variety of plants, particularly conifers, though al‐ so by some insects, such as swallowtail butterflies, which emit terpens from their osmeterium. They are the major components of resin and of turpentine produced from resin. The name terpen is derived from the word "turpentine". The smaller and more volatile terpenoids (C10 and C15) are generally the main constituents of the essential oils obtained from many types of plants and flowers, widely used as natural flavourings for food, as fragrances in perfumes in aromatherapy and in traditional and alternative medicines. Terpenoids possess a common structural feature: they contain an integral number of C5 units (isoprene-like) giving a basic molecular formula (C5H8)n for the hy‐ drocarbons series. They are derived from the metabolism of acetate by the mevalonic

Examples of monoterpens (C10) are geraniol and limonene. In particular, *d*-limonene has a pronounced chemotherapeutic activity and minimal toxicity in pre-clinical studies. A phase I clinical trial performed to assess toxicity, maximum tolerated dose (MTD) and pharmacoki‐ netics in patients with advanced cancer was followed by a limited phase II evaluation in breast cancer. We have previously published some *in vitro* results on a tri-terpen [58], implicating a NF-κB dependent anti-inflammatory mechanism of action of the extract of *Trytergium Wolfor‐ dii* hoek, used in traditional Chinese medicine for the prevention of arthritis, rheumatoid

In performing the experiments for the assessment of the doses to be administered in an *in vivo* rodent model, an anti-stress effect of the terpen AISA 5203-L was unexpectedly revealed by a functional observation battery (FOB). A plethora of parameters addressing behavioural, physiological and neurological parameters in female rats submitted to several stressful conditions were measured. Results showed important effects leading to the capacity of the animals to tolerate stress and even pain when compared to vehicle-treated animals [9].

To these preliminary pre-clinical data we were recently able to add some clinical data showing the coherence of our anti-inflammatory/anti-stress approach [18, 19]. The European Capacity study "Ristomed" enrolled 125 healthy individuals from three different countries (Italy, France and Germany). They all received an 'optimal diet for the elderly' with the supplementation of some nutraceutic compounds for a period of 56 days. The diet was developed on the basis of the current recommendations for elderly people and personalized individual dietary require‐ ments, with particular attention given to food compounds that can affect inflammation, oxidative stress and gut microbiota, such as polyunsaturated fatty acids (PUFAs), antioxidant vitamins, polyphenols, flavonoids and fibres. The diet was adapted to the dietary habits for each country. AISA Therapeutics treatment (here referred to as OPE, i.e., Orange Peel Extract)

practice of a physical activity or to the prescription of a personalized diet, or both.

acid branch biosynthetic pathways of plants.

140 Senescence and Senescence-Related Disorders

arthritis and arthrosis.

In this article, we will report the results concerning the inflammatory markers and the (concomitant) alterations of the mood, comparing the group receiving the diet without supplementation (14 males, mean age 69.6 ± 4.1 years; 17 females, 71.3 ± 3.8 years) to that receiving a diet supplemented with daily soft gel capsules containing the terpen extract AISA 5203-L (14 males, mean age 70.6 ± 4.4 years; 16 females, 69.6 ± 3.3 years), related to as OPE (Orange Peel Extract).

The laboratory measurements performed included erythrocyte sedimentation rate (ESR), high-sensitivity C-reactive protein (CRP), white blood cell count (WBC) and fibrinogen measurements. Baseline plasma levels of ESR, CRP, WBC, fibrinogen, IL-6 and TNF-α were used to calculate an inflammation score. This enabled the separation of the patients into two groups of respectively low and high inflammation, so that inflammatory status could be evaluated according to the scores of these markers.

Moreover, several self-assessment questionnaires were analysed to investigate quality of life parameters. The SF-36v2 Health Survey was used to evaluate what each subject felt about his/ her health using 36 items covering functional status, wellbeing and an overall evaluation of health, that together are referred to as Quality of Life (QoL). Two summary scores — Physical Component Summary (PCS) and Mental Component Summary (MCS) — were calculated to distinguish a possible physical dysfunction and bodily pain from psychological distress and emotional problems. The State-Trait Anxiety Inventory-X (STAI-X) questionnaire was used to assess the anxiety state and trait, and to describe each subject's feelings at a particular point.

The results of this investigation showed that among clinically healthy, aged subjects (i.e., absence of cancer, obesity, metabolic syndrome, diabetes, major cardiovascular complaints, arthritis or dementia), a third of them showed important inflammatory markers' expression. It is precisely these patients that could be at risk of developing delirium in the case of surgical treatment [2]. They would largely be advantaged by a preventive treatment of their inflam‐ matory condition, especially if high levels of IL-6 and TNF-α are measured.

Conclusively, the results confirmed the anti-inflammatory action of the terpen extract in an aged matched (65-85) healthy population (figure 1 and [20], www.ristomed.eu).

Moreover, Ristomed results were conclusive also for the capacity to lower anxiety and thus implicitly for the link between inflammation and anxiety. Interestingly, study results obtained for quality of life assessment (PCS, MCS and GHQ-12), mood (STAI-X) and de‐ pression (CES-D) confirmed our findings on mood modulation. We note in particular that OPE treatment was more effective in high-inflamed patients, the anti-depressive effect is more visible in low-inflamed patients (figure 2). These results also confirm previous find‐ ings established by our Functional Observation Battery (FOB) in rodents, where AISA 5203-L supplementation was able to substantially contribute to pain tolerance and mood stabilization. However, the most intriguing result was the fact that the stressed animal (non-pathological stress stimulating anxiety), instead of developing a freezing attitude, following oral administration of the food supplement, developed an activity. These data can be useful to answer the question "Is stress relevant for cell senescence and thus ag‐ ing?". The important effects on mood in the presence of stress situations has been docu‐ mented for decades. The mechanism by which a stress is responsible for detrimental organ impairment seems to reside in the complex interconnections between inflammatory and immunosenescence pathways [14, 23, 41, 50].

anti-stress effects. The evidence that post-surgery delirium episodes precipitating dementia are announced by anxiety that in turn is associated to high inflammatory scores, allows us to research efficacious presides to treat these cases. A preventive administration of non-toxic food additives counteracting inflammation and soothing mood alterations could be integrated into the daily diet preceding the surgical intervention. A preventive administration of highly antiinflammatory specific biocomplements should be included in the recommendations to the healthy aged population by medical institutions and supported by healthy aging guidelines

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Finally, in order to counteract inflammatory stimuli and to modulate the impact of the environment on inflammaging, we proposed to intervene with diet and food supplementation.

**Figure 2.** Inflammation subgroups and CES-D variation in diet versus diet plus OPE.

in western countries.

In conclusion, inflammaging is an age-related process arising from the interaction with the genetic/epigenetic/microbioma–specific background and the environment, as shown in figure 3, and this interaction potentially triggers the onset of the most important age-related diseases. In this regard, lessons from the clinical research teach us that inflammation as well as mood alterations seem relevant for the onset of degenerative diseases. The balancing between proand anti-inflammatory agents can be modified by external stimuli both in terms of stress or

**Figure 1.** Results of the Ristomed study: inflammation sub-groups and IL-6 variation in diet versus diet plus terpen extract AISA 5203-L, in the figure mentioned as OPE (i.e., Orange Peel Extract).

anti-stress effects. The evidence that post-surgery delirium episodes precipitating dementia are announced by anxiety that in turn is associated to high inflammatory scores, allows us to research efficacious presides to treat these cases. A preventive administration of non-toxic food additives counteracting inflammation and soothing mood alterations could be integrated into the daily diet preceding the surgical intervention. A preventive administration of highly antiinflammatory specific biocomplements should be included in the recommendations to the healthy aged population by medical institutions and supported by healthy aging guidelines in western countries.

can be useful to answer the question "Is stress relevant for cell senescence and thus ag‐ ing?". The important effects on mood in the presence of stress situations has been docu‐ mented for decades. The mechanism by which a stress is responsible for detrimental organ impairment seems to reside in the complex interconnections between inflammatory

In conclusion, inflammaging is an age-related process arising from the interaction with the genetic/epigenetic/microbioma–specific background and the environment, as shown in figure 3, and this interaction potentially triggers the onset of the most important age-related diseases. In this regard, lessons from the clinical research teach us that inflammation as well as mood alterations seem relevant for the onset of degenerative diseases. The balancing between proand anti-inflammatory agents can be modified by external stimuli both in terms of stress or

**Figure 1.** Results of the Ristomed study: inflammation sub-groups and IL-6 variation in diet versus diet plus terpen

extract AISA 5203-L, in the figure mentioned as OPE (i.e., Orange Peel Extract).

and immunosenescence pathways [14, 23, 41, 50].

142 Senescence and Senescence-Related Disorders

Finally, in order to counteract inflammatory stimuli and to modulate the impact of the environment on inflammaging, we proposed to intervene with diet and food supplementation.

**Figure 2.** Inflammation subgroups and CES-D variation in diet versus diet plus OPE.

[2] Ansaloni L, Catena F, Chattat R, Fortuna,D, Franceschi C, Mascitti P, Melotti R M. Risk factors and incidence of postoperative delirium in elderly patients after elective and

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145

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**Figure 3.** The Inflammaging balance. The low, chronic pro-inflammatory status, characterizing aging interacts with genetic/epigenetic/microbiota background and potentially triggers the onset of the most important age-related dis‐ eases. The balancing between pro- and anti-inflammatory agents can be modified by external stimuli, such as stress or anti-stress (diet and anti-inflammatory compounds). PG: prostaglandins; LT: leukotrienes, LPX: lipoxins

#### **Author details**

Patrizia d'Alessio1 , Rita Ostan2 , Miriam Capri2 and Claudio Franceschi2

1 University Paris Sud-11 and Biopark Cancer Campus 1, Villejuif, France

2 Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Italy

#### **References**

[1] Agnoletti V, Ansaloni L, Catena F, Chattat R, De Cataldis A, Di Nino G, Franceschi C, Gagliardi S, Melotti RM, Potalivo A, Taffurelli M. Postoperative Delirium after elective and emergency surgery: analysis and checking of risk factors. A study protocol. BMC Surg. 2005 5:12.


**Figure 3.** The Inflammaging balance. The low, chronic pro-inflammatory status, characterizing aging interacts with genetic/epigenetic/microbiota background and potentially triggers the onset of the most important age-related dis‐ eases. The balancing between pro- and anti-inflammatory agents can be modified by external stimuli, such as stress or

2 Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna,

[1] Agnoletti V, Ansaloni L, Catena F, Chattat R, De Cataldis A, Di Nino G, Franceschi C, Gagliardi S, Melotti RM, Potalivo A, Taffurelli M. Postoperative Delirium after elective and emergency surgery: analysis and checking of risk factors. A study protocol. BMC

and Claudio Franceschi2

anti-stress (diet and anti-inflammatory compounds). PG: prostaglandins; LT: leukotrienes, LPX: lipoxins

, Miriam Capri2

1 University Paris Sud-11 and Biopark Cancer Campus 1, Villejuif, France

**Author details**

144 Senescence and Senescence-Related Disorders

Patrizia d'Alessio1

Italy

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148 Senescence and Senescence-Related Disorders


## *Edited by Zhiwei Wang and Hiroyuki Inuzuka*

Senescence is a biological process that causes a progressive deterioration of structure and function of all organs chronologically. Recent studies have revealed the detailed molecular mechanisms of senescence using cell culture system and experimental organisms. It is thought that senescence is a potential cause for the development of various age-related disorders such as cancer, cardiovascular and neurodegenerative disorders. This book discusses in detail senescence and its related diseases by distinguished researchers and practicing clinicians. The cumulative knowledge from the studies could lead to developing new approaches for anti-senescence interventions.

Senescence and Senescence-Related Disorders

Senescence and Senescence-

Related Disorders

*Edited by Zhiwei Wang and Hiroyuki Inuzuka*

Photo by egal / iStock