**1. Introduction**

This study reveals the molecular regulation mechanisms of neurosecretory cell apoptosis in physiological and pathological (oncogene human epidermal growth factor receptor (HER)-2/ Neu overexpression) aging. In this chapter, we consider the participation of p53-regulating genes and p53 target genes in activation of this cascade during physiological aging, as well as suppression under HER-2/Neu overexpression. In addition, we consider cytokine-dependent pathways of apoptosis regulation (STAT-signaling pathway and tumor necrosis factor (TNF) dependent pathway).

differentiation, DNA repair, and apoptosis. P53-dependent expression of p21 due to DNA damage causes the arrest of the cell cycle in the late G1 phase [3, 18, 19]. In addition, p21 participates in cell aging [20, 21]. Besides, one of the genes, which play an important role in pathological involution process and take part in p53 regulation, is WRN, gene of Werner's syndrome.

The Apoptosis Regulation Mechanisms in Hypothalamic Neurons in Physiological…

http://dx.doi.org/10.5772/intechopen.72694

101

However, neuron resistance to apoptosis may also be due to the activity of cytokine-dependent STAT-signaling pathway. Various molecules, including hormones, cytokines, and so on, participate in the regulation of apoptosis. As it is known, a significant change in the level of cell death is an important biological problem. Thus, a decrease in the level of apoptosis leads to oncogenesis, and an increase in the proportion of dying cells is the cause of diseases associated with tissue degeneration. Currently, it is considered a proven close connection between apoptosis and aging. To study the mechanisms of apoptosis associated with the aging, we used a

HER2, or ERBB2, belongs to the family of transmembrane tyrosine kinase receptors. After connecting to the ligand, members of this family form homo- or heterodimers and transmit the signal forward for activating a significant amount of cascades. Normally, ERBB receptors are involved in the processes of growth, differentiation, migration, and apoptosis. The extracellular domain of ERBB2 (HER2), unlike the HER1, 3, and 4 domains, has an open conformation and is normally capable of forming functionally active heterodimers with other HER receptors by carrying out and amplifying the signal, without preliminary binding to the ligand.

The HER-2/Neu overexpression in pathology promotes the formation of functionally active homo- and heterodimers, and, so, uncontrolled signal transduction [22] and it is characteristic of a number of tumors. The signal network, initiated by the interaction of ERBB family receptors with ligands, and its key elements regulating the direction and speed of signal transmis-

We have previously shown that a low level of apoptosis of neurons upon aging in HER-2/Neu transgenic mice is associated with the suppression of the p53-dependent cascade. However, cell resistance to apoptosis may also be due to the activity of cytokine-dependent apoptosis-limiting systems (STAT-signaling pathway); these members are synthesized in various cell types, including neurons. At present, there is a report on the presence of a pro-inflammatory component in involutional changes of various tissues, including the brain tissues. It is shown that cellular stress and an inflammatory environment can trigger an immune response and provoke cell aging through epigenetic regulation involving STAT signaling [23]. However, there are almost no data characterizing age-related changes in the expression and activity of cytokines, the main mediators of inflammation. Some authors reported an increased pro-inflammatory reaction accompanying the involution processes, and other ones reported suppression of the inflammatory response at the cellular level, and about a decrease in neuroimmune interactions. STAT proteins (signal transducers and activators of transcription) are transcription factors that conduct a signal from the cytoplasm to the nucleus. For the first time, proteins of this family were described in the 1990s of the twentieth century. Their usual ligands are cytokines, including interferons. In addition to cytokines, STAT are also activated by growth factors and growth factor receptors (a family of tyrosine kinase receptors, including HER-2) that stimulate STAT

line of transgenic mice with overexpression of the oncogene HER-2/Neu.

sion play an important role in the pathogenesis of tumor diseases [22].

factors directly or indirectly through JAK kinases.

As shown at present, the process of apoptosis is an integral part of involutional tissue changes [1]. However, the mechanisms of senile apoptosis differ from the mechanisms of cell death in the early stages of ontogenesis. P53-mediated pathway of apoptosis plays a special role in aging, and its changes (excessive activation or suppression) cause severe pathologies, including neurodegenerative diseases and carcinogenesis. This is especially important for neurons and neurosecretory cells, which, as is known, are not capable of proliferation in the mass, so apoptosis is the only way to regulate their amount. Since the hypothalamic neurosecretory system is one of the main regulatory systems, the decrease in the amount of neurosecretory cells significantly changes the function of the hypothalamus and, consequently, of the target organs. Thus, the study of regulation of the p53-signaling path upon aging has particular importance.

DNA damage leads to the phosphorylation of p53 at the Ser/Thr-Pro sites, so the interaction of p53 with Ser/Thr-Pro sites of peptidyl-prolyl isomerase pin1 becomes possible [2–4]. Several studies have shown the suppressive role of prolyl isomerase pin1 in oxidative stressinduced apoptosis [5]. Pin1 involves in the stabilization of Mcl-1 and, further, in the prevention of apoptosis [6]. However, most authors report a high proapoptotic activity of pin1. Pin1 is necessary for the timely activation of p53, leading to apoptosis or cell arrest [4, 7–9]. It is known that pin1 participates in the realization of the cell cycle and mitosis in mammalian cells, transcription, and differentiation [10, 11]. In addition, overexpression of pin1 in various types of cancer tissue has been demonstrated [12, 13]. Pin1 plays an important role in cellular response to DNA damage [14], by conformational transformations participating in the transfer of the apoptotic stimulus and further initiation of the apoptotic cascade [15]. Pin1 isomerizes the bonds between molecules that are important for a variety of oncogenic and other signaling pathways in the cell, including Bcl-2, p53, c-Jun, beta-catenin, nuclear factor-kappa B (NF-kappaB), cyclin D1, c-Myc, and Raf-1. This can cause conformation changes leading to damage to catalytic activity, protein-protein interactions, subcellular localization, and protein stability. Similar changes have been shown to be associated with cell transformation and cancer development [3, 12, 13]. In addition, the absence of pin1 affects embryonic fibroblasts, leading to rapid genomic instability and the so-called immortality due to inactivation of p53 and further to aggressive transformation and carcinogenesis [16].

After attachment to pin1, p53 changes its transcriptional activity and increases the p21 transcription, which facilitates the cell arrest [3, 17]. Moreover, pin1 regulates the stability and transcriptional activity of p53 in relation to the p21 gene promoter [17]. P21 is known to be an inhibitor of cyclin-dependent kinases. Regulation of the cell cycle is an important part of development, differentiation, DNA repair, and apoptosis. P53-dependent expression of p21 due to DNA damage causes the arrest of the cell cycle in the late G1 phase [3, 18, 19]. In addition, p21 participates in cell aging [20, 21]. Besides, one of the genes, which play an important role in pathological involution process and take part in p53 regulation, is WRN, gene of Werner's syndrome.

**1. Introduction**

100 Hypothalamus in Health and Diseases

dependent pathway).

This study reveals the molecular regulation mechanisms of neurosecretory cell apoptosis in physiological and pathological (oncogene human epidermal growth factor receptor (HER)-2/ Neu overexpression) aging. In this chapter, we consider the participation of p53-regulating genes and p53 target genes in activation of this cascade during physiological aging, as well as suppression under HER-2/Neu overexpression. In addition, we consider cytokine-dependent pathways of apoptosis regulation (STAT-signaling pathway and tumor necrosis factor (TNF)-

As shown at present, the process of apoptosis is an integral part of involutional tissue changes [1]. However, the mechanisms of senile apoptosis differ from the mechanisms of cell death in the early stages of ontogenesis. P53-mediated pathway of apoptosis plays a special role in aging, and its changes (excessive activation or suppression) cause severe pathologies, including neurodegenerative diseases and carcinogenesis. This is especially important for neurons and neurosecretory cells, which, as is known, are not capable of proliferation in the mass, so apoptosis is the only way to regulate their amount. Since the hypothalamic neurosecretory system is one of the main regulatory systems, the decrease in the amount of neurosecretory cells significantly changes the function of the hypothalamus and, consequently, of the target organs. Thus, the

study of regulation of the p53-signaling path upon aging has particular importance.

and further to aggressive transformation and carcinogenesis [16].

DNA damage leads to the phosphorylation of p53 at the Ser/Thr-Pro sites, so the interaction of p53 with Ser/Thr-Pro sites of peptidyl-prolyl isomerase pin1 becomes possible [2–4]. Several studies have shown the suppressive role of prolyl isomerase pin1 in oxidative stressinduced apoptosis [5]. Pin1 involves in the stabilization of Mcl-1 and, further, in the prevention of apoptosis [6]. However, most authors report a high proapoptotic activity of pin1. Pin1 is necessary for the timely activation of p53, leading to apoptosis or cell arrest [4, 7–9]. It is known that pin1 participates in the realization of the cell cycle and mitosis in mammalian cells, transcription, and differentiation [10, 11]. In addition, overexpression of pin1 in various types of cancer tissue has been demonstrated [12, 13]. Pin1 plays an important role in cellular response to DNA damage [14], by conformational transformations participating in the transfer of the apoptotic stimulus and further initiation of the apoptotic cascade [15]. Pin1 isomerizes the bonds between molecules that are important for a variety of oncogenic and other signaling pathways in the cell, including Bcl-2, p53, c-Jun, beta-catenin, nuclear factor-kappa B (NF-kappaB), cyclin D1, c-Myc, and Raf-1. This can cause conformation changes leading to damage to catalytic activity, protein-protein interactions, subcellular localization, and protein stability. Similar changes have been shown to be associated with cell transformation and cancer development [3, 12, 13]. In addition, the absence of pin1 affects embryonic fibroblasts, leading to rapid genomic instability and the so-called immortality due to inactivation of p53

After attachment to pin1, p53 changes its transcriptional activity and increases the p21 transcription, which facilitates the cell arrest [3, 17]. Moreover, pin1 regulates the stability and transcriptional activity of p53 in relation to the p21 gene promoter [17]. P21 is known to be an inhibitor of cyclin-dependent kinases. Regulation of the cell cycle is an important part of development, However, neuron resistance to apoptosis may also be due to the activity of cytokine-dependent STAT-signaling pathway. Various molecules, including hormones, cytokines, and so on, participate in the regulation of apoptosis. As it is known, a significant change in the level of cell death is an important biological problem. Thus, a decrease in the level of apoptosis leads to oncogenesis, and an increase in the proportion of dying cells is the cause of diseases associated with tissue degeneration. Currently, it is considered a proven close connection between apoptosis and aging. To study the mechanisms of apoptosis associated with the aging, we used a line of transgenic mice with overexpression of the oncogene HER-2/Neu.

HER2, or ERBB2, belongs to the family of transmembrane tyrosine kinase receptors. After connecting to the ligand, members of this family form homo- or heterodimers and transmit the signal forward for activating a significant amount of cascades. Normally, ERBB receptors are involved in the processes of growth, differentiation, migration, and apoptosis. The extracellular domain of ERBB2 (HER2), unlike the HER1, 3, and 4 domains, has an open conformation and is normally capable of forming functionally active heterodimers with other HER receptors by carrying out and amplifying the signal, without preliminary binding to the ligand.

The HER-2/Neu overexpression in pathology promotes the formation of functionally active homo- and heterodimers, and, so, uncontrolled signal transduction [22] and it is characteristic of a number of tumors. The signal network, initiated by the interaction of ERBB family receptors with ligands, and its key elements regulating the direction and speed of signal transmission play an important role in the pathogenesis of tumor diseases [22].

We have previously shown that a low level of apoptosis of neurons upon aging in HER-2/Neu transgenic mice is associated with the suppression of the p53-dependent cascade. However, cell resistance to apoptosis may also be due to the activity of cytokine-dependent apoptosis-limiting systems (STAT-signaling pathway); these members are synthesized in various cell types, including neurons. At present, there is a report on the presence of a pro-inflammatory component in involutional changes of various tissues, including the brain tissues. It is shown that cellular stress and an inflammatory environment can trigger an immune response and provoke cell aging through epigenetic regulation involving STAT signaling [23]. However, there are almost no data characterizing age-related changes in the expression and activity of cytokines, the main mediators of inflammation. Some authors reported an increased pro-inflammatory reaction accompanying the involution processes, and other ones reported suppression of the inflammatory response at the cellular level, and about a decrease in neuroimmune interactions.

STAT proteins (signal transducers and activators of transcription) are transcription factors that conduct a signal from the cytoplasm to the nucleus. For the first time, proteins of this family were described in the 1990s of the twentieth century. Their usual ligands are cytokines, including interferons. In addition to cytokines, STAT are also activated by growth factors and growth factor receptors (a family of tyrosine kinase receptors, including HER-2) that stimulate STAT factors directly or indirectly through JAK kinases.

After activation, phosphorylation of the C-terminal domain of the STAT factors occurs, the homo- or heterodimers form, translocate into the nucleus, and activate DNA regions [24]. It is shown that STAT signaling is negatively regulated by two groups of proteins, one of them is suppressors of cytokine signaling (SOCs) and STAT-induced STAT inhibitors [25], or "proteins that inhibit activated STAT" [26]. The STAT family includes at least seven members— STAT1, 2, 3, 4, 5a, 5b, and 6 [27].

processes accompanying aging [37, 38]. So, it is reported that Fas signaling is activated in aged oocytes [39]; the extrinsic apoptotic pathway plays an important role in the development of macular degeneration in accelerated aged OXYS rats while the synthesis of proteins such as Fas, caspase-8, TRAIL increased [40], and in neurodegeneration in ischemia-reperfusion [41]. Apoptosis of the heart cells upon aging is induced via the Fas-FADD pathway, with a significantly suppressed survival signaling pathway which is associated with the insulin-like growth factor receptor (IGF1R), PI3K, and AKT kinases [42]. The senile neurodegeneration is an important clinical pathology, but there are almost no works devoted to the role of Fas-FADD and TNFreceptor-associated death domain (TRADD) pathway in neurons during physiological aging.

The Apoptosis Regulation Mechanisms in Hypothalamic Neurons in Physiological…

http://dx.doi.org/10.5772/intechopen.72694

103

Thus, the aim of the present work is investigation of age-related changes of three signaling cascades, which are involved in both the activation of apoptosis and its suppression p53-dependent (p53, WRN, pin1, p21, caspase-3), STAT-mediated (STAT1, 3, 5, 6, survivin), and TNF-dependent (FAS, FADD, TRADD, caspase-8). This will reveal the general trends of regulation of neurosecretory cell apoptosis during physiological and pathological aging.

We studied a mouse model of accelerated aging, namely transgenic HER-2/Neu female mice, at the age of 2 and 10 months [43], obtained from the Italian National Research Center of Aging; the breeding is maintained at the Petrov Research Institute of Oncology (St. Petersburg, Russia). Outbred FVB/N female mice, descending from mice of the Swiss line (Rappolovo Nursery, Russian Academy of Medical Sciences, St. Petersburg, Russia), were 2 and 18 months of age (four to five mice in each group). The animal room was equipped with a 24-h light-dark cycle

The model of our study is transgenic mice with the overexpression of the transmembrane tyrosine kinase receptor HER-2/Neu, the wild type is the FVB/N line. The FVB/N line mice are often used to produce models of transgenic mice, since this line is characterized by high fertility and good survival of the embryo after injection. Overexpression of activated HER-2/Neu oncogene in transgenic female FVB/N mice leads to malignant transformation of mammary epithelial cells, followed by development of several breast adenocarcinomas, as well as hyperinsulinemia, hyperglycemia, and a decrease in the activity of the antioxidant system, which are biomarkers of the premature aging of transgenic mice (**Figure 1**). The lifespan of these mice is about 11–12 months. Thus, overexpression of HER-2/Neu causes hormone-metabolic changes, which are characteristic of accelerated aging, simultaneously with carcinogenesis, in

**2. Mechanisms of apoptosis regulation in ontogenesis: apoptosis signaling cascades in hypothalamic neurons in physiological and** 

**pathological (overexpression of oncogene HER-2/Neu) aging**

**2.1. Research methods**

*2.1.1. Animals*

with 12:12 period.

this line of mice.

Although all members of the STAT family are structurally similar, they perform various biological roles, participating in such processes as embryonic development, inflammation, organogenesis, cell differentiation and control of cell growth [28, 29], regulation of immune processes, control of proliferation, and apoptosis (STAT3, 5) [30–32]. In many studies, it has been established that STAT proteins play a critical role in the activation of pro-inflammatory and antiproliferative processes (primarily, by the factor STAT1). Members of this family participate in the interferon gamma-induced response [33–35]. Information on the important role of STAT factors in the regulation of cell proliferation, differentiation, and survival suggests the active role of these proteins in malignancy.

However, most studies report about the participation of STAT factors in oncogenesis, but there are almost no data concerning the expression of STAT proteins in neurons with aging and their participation in the regulation of apoptosis. So, the purpose of the next part of the work was to reveal the role and molecular mechanisms of cytokine-dependent signaling in the mechanism of p53-dependent apoptosis suppression in the physiological and pathological (overexpression of the HER-2/Neu oncogene, epidermal growth factor receptor (EGFR)) aging and to investigate the causes of neuronal resistance to apoptosis with aging, possibly due to overexpression of HER-2/Neu.

Also, another cytokine-dependent signaling, an extrinsic apoptosis pathway associated with the family of tumor necrosis factor receptors, has been investigated.

Changes in cytokine expression are observed in various pathological conditions, so, TNFalpha, a protein that plays a role in apoptosis, increases with oxidative stress [36].

As is known, tumor necrosis factor has a high antitumor and pro-inflammatory activity. Expression of the *tnf* gene activates the synthesis of the cytokine TNF-alpha (TNF-α), which regulates the processes of proliferation, apoptosis, immune cell activity, inflammation, embryo-, and carcinogenesis. Binding of TNF-α to cell death receptors leads to the activation of caspase-8, which initiates an apoptosis program. One of the receptors is the Fas-receptor (CD95, APO-1), whose main function is signaling to the development of apoptosis. In the case of Fas-dependent apoptosis, binding of the Fas ligand to the Fas receptor leads to conformational changes in the cytoplasmic domain of Fas receptor. This makes it possible to bind it to FADD-adapter molecule (Fas-associated death domain), and then to the same domain of the RIP protein (receptor-interacting protein). This complex activates caspase-8 (FLICE protease (FADD-like IL-1b-converting enzyme)), which means the development of apoptosis.

We supposed that the activity of a cytokine-dependent pathway associated with a family of tumor necrosis factor—the TNF-signaling pathway—can also be altered by aging. Only a few authors associate the TNF-dependent apoptotic pathway with aging or with the pathological processes accompanying aging [37, 38]. So, it is reported that Fas signaling is activated in aged oocytes [39]; the extrinsic apoptotic pathway plays an important role in the development of macular degeneration in accelerated aged OXYS rats while the synthesis of proteins such as Fas, caspase-8, TRAIL increased [40], and in neurodegeneration in ischemia-reperfusion [41]. Apoptosis of the heart cells upon aging is induced via the Fas-FADD pathway, with a significantly suppressed survival signaling pathway which is associated with the insulin-like growth factor receptor (IGF1R), PI3K, and AKT kinases [42]. The senile neurodegeneration is an important clinical pathology, but there are almost no works devoted to the role of Fas-FADD and TNFreceptor-associated death domain (TRADD) pathway in neurons during physiological aging.

Thus, the aim of the present work is investigation of age-related changes of three signaling cascades, which are involved in both the activation of apoptosis and its suppression p53-dependent (p53, WRN, pin1, p21, caspase-3), STAT-mediated (STAT1, 3, 5, 6, survivin), and TNF-dependent (FAS, FADD, TRADD, caspase-8). This will reveal the general trends of regulation of neurosecretory cell apoptosis during physiological and pathological aging.
