Preface

Motor Neuron Disease (MND) continues to be a progressive loss of structure and function of neurons in the cerebral cortex, brainstem, and the spinal cord. Usually the loss of specific functions precedes the death of affected neurons, and the related clinical features depend on localization and degree of neurodegeneration. Amyotrophic Lateral Sclerosis (ALS) is the more common clinical presentation and MND is also related to mitochondrial dysfunctions, increased oxidative stress and atypical protein assemblies. MND continues to be a serious health problem leading to death in few years' times in most of the patients. Before death, patients suffer from weakness or paralysis, muscle wasting and fasciculation, dysphagia, dysarthria and several complications. There are two forms of this fatal disease: sporadic, with no known genetic component, and familial, which makes up about 5% of all ALS cases. Among the familial cases, approximately 20% are caused by dominantly inherited mutations in the Cu/Zn superoxide dismutase (SOD1) gene, with more than 100 known mutations.

This is the second book that we have edited on ALS/MND. To edit this book, we have provided the best, safest, most confident and novel science information to our readers after following a rigorous process. All chapters were screened and analysed by a strict peer-review process, followed by corrections made by authors and the Academic Editor. Then a second revision was made by authors and editors performed the final review. It is important to highlight that after the peer-review process, all chapters were reviewed twice or even more to be accepted for publication. The chapter written by the Editor was also screened by another peer-review team.

In this book, the readers will find a compilation of state-of-the-art reviews about aetiology, therapies, investigations, the molecular basis of disease progression and clinical manifestations, and the genetics familial ALS, as well as novel diagnostic criteria.

An update aspect on ALS sourced 5 chapters from some of the world's top central nervous system researchers and neurologists to provide a timely review of the latest developments in MND/ALS, covering experimental animal models, genetics, clinical aspects and treatment options, amongst others.

Contributors from different countries have collaborated enthusiastically and efficiently to create this reader-friendly and comprehensive work covering the topics with many explanatory figures, tables and photos to enhance legibility and make the book clinically useful. Countless hours have gone into writing these chapters, precious free time to be dedicated to our family, relatives and friends have been sacrificed but at the end, we all are very proud of this book.

Every effort has been made to check the novel information given in this book but it is important for our readers to scrutinize other information arriving considering it is a dynamic process for learning. We all attempted to include valuable updated information for all issues mentioned in this book. Every effort has been made in the preparation and editing of this book to ensure that the information given is correct, but it is possible that errors have been overlooked. Finally, we would like to highlight that we reviewed all controversial matters and our medical criteria and scientist's opinions have been expressed with modesty, honesty and respect but the reader is advised to refer to other published information from other editorial houses and other reference works to check accuracy.

First of all, we would like to thank IntechOpen, who unconditionally supported us in editing this book. Special thanks should be given to my Author Service Managers, Ms. Dragana Manestar and Natalia Reinic, Ana Pantar, Andrea Koric and Romina Skomersic. They gave me support and encouragement to complete my previous editorial jobs successfully. For this book I had the supervision of Ms Dolores Kuzelj as Author Service Manager.

Currently, some of my previous chapters have reached more than 2000 downloads!

Our family have graciously tolerated the precious time spent on this project.

Fortunately, Mom, Dad and my first daughter Zayra Susana from heaven continue to inspire me. My second daughter, Lorna Maria (36 years old), and Fatima Susana Adolfina (10 years old) encouraged me to continue moving forward with persistence. My son Thabo Humberto Jorge (11 years old) both asked me to play games and to go for bike riding and these helped me to relax and to find new ideas but unfortunately I could not stay with them all times. My wife and the rest of my family contributed to this project and my wife was also my main collaborator - all of them deserve my deep gratitude.

Another token of gratitude must be delivered to all authors and collaborators for their patience and tolerance of the lost evenings, nights, weekends, holidays and free time spent on this project.

Special thanks go to Walter Sisulu University (WSU). Many thanks to Prof. Wilson Akpan: Director of Research & Development of WSU, Prof. AJ Mbokazi: Dean of the Faculty of Health Sciences (WSU), Prof. Thozama Dubula: Head of Department of General Medicine and Therapeutic, Dr. Mdledle and Dr. Nodikida: Acting Governor General: Clinical Governance of Nelson Mandela Central Hospital and Mrs. NP Makwedeni: Chief Executive Officer of Nelson Mandela Central Hospital for their understanding and professional support.

In the end, I extend my deepest sense of appreciation to Dr. Jorge Delgado Bustillo: Head of the National Unit for International Cooperation in Health, for the unconditional support received.

> **Humberto Foyaca Sibat** Professor, Head of Department of Neurology, Faculty of Health Sciences, Walter Sisulu University, Mthatha, South Africa

#### **Dr. Lourdes de Fátima Ibañez-Valdés**

**1**

Section 1

Last Update on Motor

Neuron Disease

Department of Neurology, Nelson Mandela Academic Central Hospital, Faculty of Health Sciences, Walter Sisulu University, Mthatha, South Africa Section 1

## Last Update on Motor Neuron Disease

**3**

**Chapter 1**

**1. Introduction**

outside of the CNS [1].

**2. Comments**

Introductory Chapter:

Motor Neuron Disease

Introduction to Novel Aspects on

*Humberto Foyaca Sibat and Lourdes de Fátima Ibañez Valdés*

Motor neuron disease (MND) represents a wide and heterogeneous expanding group of diseases affecting the upper or lower motor neurons, mainly represented by amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), and progressive bulbar palsy. ALS is a disease of motor neuron degeneration in the cerebral hemisphere, brainstem, and spinal cord with a fatal prognosis in most of the cases due to a progressive paralysis of the diaphragm and other respiratory muscles leading to respiratory dysfunction and failure. Another recently recognized hallmark of ALS pathogenesis is vascular pathology apart from central nervous system capillary injury and microvascular impairment

Our first chapter is about stem cell therapy as a novel and promising modality for the treatment of ALS/MND. Robust safety profiles, low risk to benefit ratio, and ease of access make this approach a strong contender in the race against ALS/ MND. Our authors concluded that this procedure is not a curative treatment, but a combinatorial approach integrating stem cell therapy, intensive neurorehabilitation, and current pharmacotherapeutic agents (e.g., Riluzole, Lithium, etc.) may be the best way forward. This chapter was written early last year (2019), but unfortunately, a prolonged editorial process impeded to publish this information at due time. However, we reviewed the medical literature and found that the abovementioned information has been confirmed recently (March 2020) by other authors [2]. In this book, we discussed about the pathogenic contribution of a subtype of aberrant glial phenotype into the progression and output of the neurodegenerative disease ALS and concluded that aberrant astrocytes or more generally aberrant glial cells are among the most important players in CNS damage causing deleterious effects through many potential pathological mechanisms, mostly sustained on their exacerbated proliferation together with their unprecedented neurotoxicity suggest that controlling these populations seems at least equally important than maintenance or restoration of homeostatic astrocyte functions to achieve CNS protection and repair. Another authors also suggest that aberrant glial cells (AbGC) isolated from the spinal cords of adult paralytic SOD1G93A rats exhibit highly proliferative and neurotoxic properties and may contribute to disease progression [4]. Same authors also established that mitochondrial dysfunction and neurotoxicity that can

#### **Chapter 1**

## Introductory Chapter: Introduction to Novel Aspects on Motor Neuron Disease

*Humberto Foyaca Sibat and Lourdes de Fátima Ibañez Valdés*

#### **1. Introduction**

Motor neuron disease (MND) represents a wide and heterogeneous expanding group of diseases affecting the upper or lower motor neurons, mainly represented by amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), and progressive bulbar palsy. ALS is a disease of motor neuron degeneration in the cerebral hemisphere, brainstem, and spinal cord with a fatal prognosis in most of the cases due to a progressive paralysis of the diaphragm and other respiratory muscles leading to respiratory dysfunction and failure. Another recently recognized hallmark of ALS pathogenesis is vascular pathology apart from central nervous system capillary injury and microvascular impairment outside of the CNS [1].

#### **2. Comments**

Our first chapter is about stem cell therapy as a novel and promising modality for the treatment of ALS/MND. Robust safety profiles, low risk to benefit ratio, and ease of access make this approach a strong contender in the race against ALS/ MND. Our authors concluded that this procedure is not a curative treatment, but a combinatorial approach integrating stem cell therapy, intensive neurorehabilitation, and current pharmacotherapeutic agents (e.g., Riluzole, Lithium, etc.) may be the best way forward. This chapter was written early last year (2019), but unfortunately, a prolonged editorial process impeded to publish this information at due time. However, we reviewed the medical literature and found that the abovementioned information has been confirmed recently (March 2020) by other authors [2].

In this book, we discussed about the pathogenic contribution of a subtype of aberrant glial phenotype into the progression and output of the neurodegenerative disease ALS and concluded that aberrant astrocytes or more generally aberrant glial cells are among the most important players in CNS damage causing deleterious effects through many potential pathological mechanisms, mostly sustained on their exacerbated proliferation together with their unprecedented neurotoxicity suggest that controlling these populations seems at least equally important than maintenance or restoration of homeostatic astrocyte functions to achieve CNS protection and repair. Another authors also suggest that aberrant glial cells (AbGC) isolated from the spinal cords of adult paralytic SOD1G93A rats exhibit highly proliferative and neurotoxic properties and may contribute to disease progression [4]. Same authors also established that mitochondrial dysfunction and neurotoxicity that can

be reduced by dichloroacetate (DCA), a metabolic modulator that has been used in humans, show beneficial effects on disease outcome in SOD1G93A mice. They also highlight that DCA treatment of AbGC reduced extracellular lactate levels indicating that the main recognized DCA action targets the pyruvate dehydrogenase kinase/pyruvate dehydrogenase complex, and the results confirmed that AbGC metabolic phenotype is related to their toxicity to MNs and indicated that its modulation can reduce glial mediated pathology in the spinal cord [3]. At this point, it is important to emphasize that neuronal cell death is the main pathological feature of chronic neurodegenerative diseases (NDs) like ALS. A common hallmark of several NDs is the accumulation and aggregation of proteins; such proteins are thought to be primarily turned over by autophagy. Therefore, autophagy is considered a critical ND-protective pathway, which opens up potential new therapeutic interventions, and some authors have been considering the roles of autophagy and its contribution to neurodegeneration in neurons and concluded that little is known about the functions and disease contribution of the autophagy machinery in glia cells [4].

The next chapter of this book is related to the structural and functional consequences of the SMA-linked missense mutations of the survival motor neuron protein, where the authors deliver a brief update of the structural and functional consequences of the missense mutations of this SMA protein. There is another before published chapter where the same author investigates how SMA-linked mutations of SMN1 lead to structural/functional deficiency of SMN, and a set of computational analysis of SMN-related structures was conducted, described, and highlighted three residues of SMN (Asp44, Glu134, and Gln136), and the electrostatic basis of how the SMA-linked missense mutations of the three residues cause structural/functional deficiency of SMN and also a possibility of SMN's Lys45 and Asp36 acting as two electrostatically stabilizing clips at the SMN-Gemin2 complex structure interface [5].

Mutations to the gene encoding superoxide dismutase-1 (SOD1) were the first genetic elements discovered that cause motor neuron disease (MND). Around 10 years back, the unique way to test ALS-related gene was SOD1 sequencing. Based on this postulate, we approved to include into this project a novel review about the current understanding of ALS-related genes, summarize the worldwide ALS distribution feature by frequency of occurrence in different regions, and outline the genetic testing consideration, within many advances in the field of ALS genetics. In this chapter, the author highlights the recent advances in ALS gene map, genomewide association study on ALS, genetic testing, and gene therapy.

Finally, we made a bibliography research about MND and the most recent advances on treatment and reviewed the most relevant papers published on the first trimester of 2019, but as was before-mentioned, this chapter is going to be published more than 1 year later when some of our information is already oldfashioned. Last year, we reviewed on novel information about edaravone, riluzole (already approved by Food and Drug Administration), nusinersen, EH301, 5Fluoroucil, Tryptophan, RNS60, Rasagiline, Tirasemtiv, Aquaporin, Fasudil, and Lunasil. In order to deliver to our reader community, more novel information about MND/ALS is important to highlight other procedure that has been proposed for treatment such as the multifaceted role of kinases in ALS. The comprehensive regulation of kinases, however, a better understanding of the disturbances in the kinome network in ALS, is needed to properly target specific kinases in the clinic. Different kinases have been recently involved in TDP-43 phosphorylation. Among these, protein casein kinase-1 is the first kinase identified to phosphorylate TDP-43 *in vivo*, followed by tau tubulin kinase 1 and cell division cycle kinase 7. Currently, it is recognized that TDP-43 proteinopathy characterized by truncation, ubiquitination, hyperphosphorylation, and/or nuclear depletion in neurons is the prominent

**5**

**Author details**

in alleviating familial ALS [12].

**Acknowledgements**

development of this project.

patients [7].

Humberto Foyaca Sibat\* and Lourdes de Fátima Ibañez Valdés

\*Address all correspondence to: humbertofoyacasibat@gmail.com

Walter Sisulu University, Mthatha, South Africa

provided the original work is properly cited.

Department of Neurology, Nelson Mandela Academic Central Hospital,

© 2020 The Author(s). Licensee IntechOpen. 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,

*Introductory Chapter: Introduction to Novel Aspects on Motor Neuron Disease*

and common pathological feature of sporadic and familiar ALS [6]. Some authors explored the effects of a chronic treatment with the compound IGS-2.7 in the TDP-43 (A315T) transgenic mouse model and found a significant decrease in the levels of phosphorylation of TDP-43 in sporadic ALS lymphoblast, while no differences were observed in control group, and they arrived to the following conclusion: prolonged treatment with IGS-2.7 prevents the phosphorylation of TDP-43 *in vivo* in the cord of TDP-43 transgenic mice, being this effect associated with an attenuation of most of the events that reflect the worsening of the pathological phenotype, then the inhibition of CK-1δ with the benzothiazole derivative IGS-2.7 may modulate TDP-43 toxicity *in vivo* by limiting TDP-43 phosphorylation, which could explain the benefits obtained with this drug candidate in the preservation of spinal motor neurons. Therefore, benzothiazole IGS-2.7 has neuroprotective properties and not only decreases TDP-43 phosphorylation in cells derived from ALS patients but also corrects the subcellular localization of TDP-43, preventing the abnormal cytosolic

For another hand, other authors reported that 185 miRNAs in serum of affected patients and controls confirmed a downregulation of miR-335-5p in ALS

Because we are under obligation to deliver the most recent information about MND/ALS therapy to our reader's community, then we would like to comment about clinically used ebselen and related analogues to promote thermal stability of A4V SOD1 when binding to Cys111 only [8]. Ebselen is an organoselenium compound with activity similar to glutathione peroxidase [9]. Several studies have demonstrated the neuroprotective activity of ebselen, possibly via its anti-oxidant properties [10, 11]. The capacity of ebselen to decrease mitochondrial cellular toxicity caused by mutant SOD1 confirmed that this compound plays an important role

We thank all authors and their relatives for the support received during the

*DOI: http://dx.doi.org/10.5772/intechopen.92610*

TDP-43 accumulation in ALS lymphoblasts [6].

#### *Introductory Chapter: Introduction to Novel Aspects on Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.92610*

and common pathological feature of sporadic and familiar ALS [6]. Some authors explored the effects of a chronic treatment with the compound IGS-2.7 in the TDP-43 (A315T) transgenic mouse model and found a significant decrease in the levels of phosphorylation of TDP-43 in sporadic ALS lymphoblast, while no differences were observed in control group, and they arrived to the following conclusion: prolonged treatment with IGS-2.7 prevents the phosphorylation of TDP-43 *in vivo* in the cord of TDP-43 transgenic mice, being this effect associated with an attenuation of most of the events that reflect the worsening of the pathological phenotype, then the inhibition of CK-1δ with the benzothiazole derivative IGS-2.7 may modulate TDP-43 toxicity *in vivo* by limiting TDP-43 phosphorylation, which could explain the benefits obtained with this drug candidate in the preservation of spinal motor neurons. Therefore, benzothiazole IGS-2.7 has neuroprotective properties and not only decreases TDP-43 phosphorylation in cells derived from ALS patients but also corrects the subcellular localization of TDP-43, preventing the abnormal cytosolic TDP-43 accumulation in ALS lymphoblasts [6].

For another hand, other authors reported that 185 miRNAs in serum of affected patients and controls confirmed a downregulation of miR-335-5p in ALS patients [7].

Because we are under obligation to deliver the most recent information about MND/ALS therapy to our reader's community, then we would like to comment about clinically used ebselen and related analogues to promote thermal stability of A4V SOD1 when binding to Cys111 only [8]. Ebselen is an organoselenium compound with activity similar to glutathione peroxidase [9]. Several studies have demonstrated the neuroprotective activity of ebselen, possibly via its anti-oxidant properties [10, 11]. The capacity of ebselen to decrease mitochondrial cellular toxicity caused by mutant SOD1 confirmed that this compound plays an important role in alleviating familial ALS [12].

#### **Acknowledgements**

*Novel Aspects on Motor Neuron Disease*

structure interface [5].

be reduced by dichloroacetate (DCA), a metabolic modulator that has been used in humans, show beneficial effects on disease outcome in SOD1G93A mice. They also highlight that DCA treatment of AbGC reduced extracellular lactate levels indicating that the main recognized DCA action targets the pyruvate dehydrogenase kinase/pyruvate dehydrogenase complex, and the results confirmed that AbGC metabolic phenotype is related to their toxicity to MNs and indicated that its modulation can reduce glial mediated pathology in the spinal cord [3]. At this point, it is important to emphasize that neuronal cell death is the main pathological feature of chronic neurodegenerative diseases (NDs) like ALS. A common hallmark of several NDs is the accumulation and aggregation of proteins; such proteins are thought to be primarily turned over by autophagy. Therefore, autophagy is considered a critical ND-protective pathway, which opens up potential new therapeutic interventions, and some authors have been considering the roles of autophagy and its contribution to neurodegeneration in neurons and concluded that little is known about the func-

tions and disease contribution of the autophagy machinery in glia cells [4].

The next chapter of this book is related to the structural and functional consequences of the SMA-linked missense mutations of the survival motor neuron protein, where the authors deliver a brief update of the structural and functional consequences of the missense mutations of this SMA protein. There is another before published chapter where the same author investigates how SMA-linked mutations of SMN1 lead to structural/functional deficiency of SMN, and a set of computational analysis of SMN-related structures was conducted, described, and highlighted three residues of SMN (Asp44, Glu134, and Gln136), and the electrostatic basis of how the SMA-linked missense mutations of the three residues cause structural/functional deficiency of SMN and also a possibility of SMN's Lys45 and Asp36 acting as two electrostatically stabilizing clips at the SMN-Gemin2 complex

Mutations to the gene encoding superoxide dismutase-1 (SOD1) were the first genetic elements discovered that cause motor neuron disease (MND). Around 10 years back, the unique way to test ALS-related gene was SOD1 sequencing. Based on this postulate, we approved to include into this project a novel review about the current understanding of ALS-related genes, summarize the worldwide ALS distribution feature by frequency of occurrence in different regions, and outline the genetic testing consideration, within many advances in the field of ALS genetics. In this chapter, the author highlights the recent advances in ALS gene map, genome-

Finally, we made a bibliography research about MND and the most recent advances on treatment and reviewed the most relevant papers published on the first trimester of 2019, but as was before-mentioned, this chapter is going to be published more than 1 year later when some of our information is already oldfashioned. Last year, we reviewed on novel information about edaravone, riluzole (already approved by Food and Drug Administration), nusinersen, EH301, 5Fluoroucil, Tryptophan, RNS60, Rasagiline, Tirasemtiv, Aquaporin, Fasudil, and Lunasil. In order to deliver to our reader community, more novel information about MND/ALS is important to highlight other procedure that has been proposed for treatment such as the multifaceted role of kinases in ALS. The comprehensive regulation of kinases, however, a better understanding of the disturbances in the kinome network in ALS, is needed to properly target specific kinases in the clinic. Different kinases have been recently involved in TDP-43 phosphorylation. Among these, protein casein kinase-1 is the first kinase identified to phosphorylate TDP-43 *in vivo*, followed by tau tubulin kinase 1 and cell division cycle kinase 7. Currently, it is recognized that TDP-43 proteinopathy characterized by truncation, ubiquitination, hyperphosphorylation, and/or nuclear depletion in neurons is the prominent

wide association study on ALS, genetic testing, and gene therapy.

**4**

We thank all authors and their relatives for the support received during the development of this project.

#### **Author details**

Humberto Foyaca Sibat\* and Lourdes de Fátima Ibañez Valdés Department of Neurology, Nelson Mandela Academic Central Hospital, Walter Sisulu University, Mthatha, South Africa

\*Address all correspondence to: humbertofoyacasibat@gmail.com

© 2020 The Author(s). Licensee IntechOpen. 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.

#### **References**

[1] Foyaca HS, Ibanez-Valdes L de F. Introduction to update in amyotrophic lateral sclerosis and review of this condition in sportsmen. Chapter I. In: Foyaca HS, Ibanez Valdes L, editors. Update on Amyotrophic Lateral Sclerosis. Rijeka: IntechOpen; 2016. DOI: 10.5772/64608. Available from: http://www.intechopen. com/myprofile/index/dashboard. ISBN: 978-953-51-2601-0

[2] Garbuzova-Davis S, Shell R, Mustafa H, Hailu S, Willing AE, Sanberg PR, et al. Advancing stem cell therapy for repair of damaged lung microvasculature in amyotrophic lateral sclerosis. Cell Transplantation. 2020;**29**:963689720913494. DOI: 10.1177/0963689720913494

[3] Martínez-Palma L, Miquel E, Lagos-Rodríguez V, Barbeito L, Cassina A, Cassina P. Mitochondrial modulation by dichloroacetate reduces toxicity of aberrant glial cells and gliosis in the SOD1G93A rat model of amyotrophic lateral sclerosis. Neurotherapeutics. 2019;**16**(1):203-215

[4] Strohm L, Behrends C. Gliaspecific autophagy dysfunction in ALS. Seminars in Cell & Developmental Biology. 2020;**99**:172-182

[5] Li W. How do SMA-linked mutations of SMN1 lead to structural/functional deficiency of the SMA protein? PLOS One. 2017;**12**(6):-e0178519

[6] Martínez-González L, Rodríguez-Cueto C, Cabezudo D, Bartolomé F, Andrés-Benito P, et al. Motor neuron preservation and decrease of *in vivo* TDP-43 phosphorylation by protein CK-1δ kinase inhibitor treatment. Scientific Reports. 2020;**10**:4449. DOI: 10.1038/s41598-020-61265-y

[7] De Luna N, Turon-Sans J, Cortes-Vicente E, Carrasco-Rozas A, et al.

Downregulation of miR-335-5P in amyotrophic lateral sclerosis can contribute to neuronal mitochondrial dysfunction and apoptosis. Scientific Reports. 2020;**10**:4308. DOI: 10.1038/ s41598-020-61246-1

[8] Chantadul V, Wright GSA, Amporndanai K, Shahid M, Antonyuk SV, Washbourn G, et al. Ebselen as template for stabilization of A4V mutant dimer for motor neuron disease therapy. Communications Biology. 2020;**3**:97. DOI: 10.1038/ s42003-020-0826-3

[9] Takasago T, Peters EE, Graham DI, Masayasu H, Macrae IM. Neuroprotective efficacy of ebselen, an anti-oxidant with anti-inflammatory actions, in a rodent model of permanent middle cerebral artery occlusion. British Journal of Pharmacology. 1997;**122**:1251-1256. DOI: 10.1038/ sj.bjp.0701426

[10] Kalayci M et al. Neuroprotective effects of ebselen on experimental spinal cord injury in rats. Neurochemical Research. 2005;**30**:403-410. DOI: 10.1007/s11064-005-2615-2

[11] Martini F et al. A multifunctional compound ebselen reverses memory impairment, apoptosis and oxidative stress in a mouse model of sporadic Alzheimer's disease. Journal of Psychiatric Research. 2019;**109**:107-117. DOI: 10.1016/j.jpsychires.2018.11.021

[12] Wood-Allum CA et al. Impairment of mitochondrial anti-oxidant defence in SOD1-related motor neuron injury and amelioration by ebselen. Brain. 2006;**129**:1693-1709. DOI: 10.1093/ brain/awl118

**7**

**1. Introduction**

**Chapter 2**

**Abstract**

Contribution of Aberrant

Damage and Death in the

Model of ALS

Astrocytes to Motor Neuron

SOD1G93A Rat Experimental

*Gabriel Otero Damianovich, Olga Cristina Parada,* 

*Carmen Isabel Bolatto Pereira and Silvia Olivera-Bravo*

Amyotrophic lateral sclerosis (ALS) is an incurable paralyzing disease characterized by motor neuron death and glial reactivity. Superoxide dismutase 1 (SOD1) are among the most frequent alterations found in around 15–20% of ALS inheritable forms. Mutant SOD1 murine models mimic main human ALS features and allow purposing that pathological mechanisms include defective communication between neural cells together with astrocyte preponderant roles in disease progression. Years ago, a subset of the most neurotoxic aberrant astrocytes (AbAs) was obtained from spinal cords of SOD1G93A rats. AbA cultures show an exponential growing yield since the early symptoms of the disease up to the terminal stages. In cultures, AbAs present unprecedented toxicity to motor neurons, increased proliferation, loss of mature astrocyte markers, as well as extreme ER stress and abundant extracellular matrix components. Strikingly, AbA phenotype seems to be changing along few passages suggesting its signaling and features may accompany disease progression. However, the link between main AbA features and their highest motor neuron toxicity is not yet completely understood. Here, we reviewed ALS underlying pathological mechanisms in association to AbA phenotype, to collaborate with identification of the most relevant processes that seem crucially involved in the

**Keywords:** aberrant astrocytes, motor neuron death, non-cell autonomous disease

This chapter will discuss the pathogenic contribution of a subtype of aberrant glial phenotype into the progression and output of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Complete understanding of neuronal and glial cells roles and communication is necessary to unravel disease processes and

*Pablo Díaz-Amarilla, Eugenia Eloísa Isasi,* 

triggering or maintenance of neurotoxicity.

### **Chapter 2**

## Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat Experimental Model of ALS

*Gabriel Otero Damianovich, Olga Cristina Parada, Pablo Díaz-Amarilla, Eugenia Eloísa Isasi, Carmen Isabel Bolatto Pereira and Silvia Olivera-Bravo*

#### **Abstract**

Amyotrophic lateral sclerosis (ALS) is an incurable paralyzing disease characterized by motor neuron death and glial reactivity. Superoxide dismutase 1 (SOD1) are among the most frequent alterations found in around 15–20% of ALS inheritable forms. Mutant SOD1 murine models mimic main human ALS features and allow purposing that pathological mechanisms include defective communication between neural cells together with astrocyte preponderant roles in disease progression. Years ago, a subset of the most neurotoxic aberrant astrocytes (AbAs) was obtained from spinal cords of SOD1G93A rats. AbA cultures show an exponential growing yield since the early symptoms of the disease up to the terminal stages. In cultures, AbAs present unprecedented toxicity to motor neurons, increased proliferation, loss of mature astrocyte markers, as well as extreme ER stress and abundant extracellular matrix components. Strikingly, AbA phenotype seems to be changing along few passages suggesting its signaling and features may accompany disease progression. However, the link between main AbA features and their highest motor neuron toxicity is not yet completely understood. Here, we reviewed ALS underlying pathological mechanisms in association to AbA phenotype, to collaborate with identification of the most relevant processes that seem crucially involved in the triggering or maintenance of neurotoxicity.

**Keywords:** aberrant astrocytes, motor neuron death, non-cell autonomous disease

#### **1. Introduction**

This chapter will discuss the pathogenic contribution of a subtype of aberrant glial phenotype into the progression and output of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Complete understanding of neuronal and glial cells roles and communication is necessary to unravel disease processes and

**6**

*Novel Aspects on Motor Neuron Disease*

[1] Foyaca HS, Ibanez-Valdes L de F. Introduction to update in amyotrophic lateral sclerosis and review of this condition in sportsmen. Chapter I. In: Foyaca HS, Ibanez Valdes L, editors. Update on Amyotrophic Lateral Sclerosis. Rijeka: IntechOpen; 2016. DOI: 10.5772/64608. Available from: http://www.intechopen. com/myprofile/index/dashboard. ISBN: Downregulation of miR-335-5P in amyotrophic lateral sclerosis can contribute to neuronal mitochondrial dysfunction and apoptosis. Scientific Reports. 2020;**10**:4308. DOI: 10.1038/

s41598-020-61246-1

s42003-020-0826-3

sj.bjp.0701426

brain/awl118

[9] Takasago T, Peters EE,

Graham DI, Masayasu H, Macrae IM. Neuroprotective efficacy of ebselen, an anti-oxidant with anti-inflammatory actions, in a rodent model of permanent middle cerebral artery occlusion. British Journal of Pharmacology. 1997;**122**:1251-1256. DOI: 10.1038/

[10] Kalayci M et al. Neuroprotective effects of ebselen on experimental spinal cord injury in rats. Neurochemical Research. 2005;**30**:403-410. DOI: 10.1007/s11064-005-2615-2

[11] Martini F et al. A multifunctional compound ebselen reverses memory impairment, apoptosis and oxidative stress in a mouse model of sporadic Alzheimer's disease. Journal of

Psychiatric Research. 2019;**109**:107-117. DOI: 10.1016/j.jpsychires.2018.11.021

[12] Wood-Allum CA et al. Impairment of mitochondrial anti-oxidant defence in SOD1-related motor neuron injury and amelioration by ebselen. Brain. 2006;**129**:1693-1709. DOI: 10.1093/

[8] Chantadul V, Wright GSA, Amporndanai K, Shahid M, Antonyuk SV, Washbourn G, et al. Ebselen as template for stabilization of A4V mutant dimer for motor neuron disease therapy. Communications Biology. 2020;**3**:97. DOI: 10.1038/

[2] Garbuzova-Davis S, Shell R, Mustafa H, Hailu S, Willing AE, Sanberg PR, et al. Advancing stem cell therapy for repair of damaged lung microvasculature in amyotrophic lateral sclerosis. Cell Transplantation. 2020;**29**:963689720913494. DOI: 10.1177/0963689720913494

[3] Martínez-Palma L, Miquel E, Lagos-Rodríguez V, Barbeito L, Cassina A, Cassina P. Mitochondrial modulation by dichloroacetate reduces toxicity of aberrant glial cells and gliosis in the SOD1G93A rat model of amyotrophic lateral sclerosis. Neurotherapeutics. 2019;**16**(1):203-215

[4] Strohm L, Behrends C. Gliaspecific autophagy dysfunction in ALS. Seminars in Cell & Developmental

[5] Li W. How do SMA-linked mutations of SMN1 lead to structural/functional deficiency of the SMA protein? PLOS

[6] Martínez-González L, Rodríguez-Cueto C, Cabezudo D, Bartolomé F, Andrés-Benito P, et al. Motor neuron preservation and decrease of *in vivo* TDP-43 phosphorylation by protein CK-1δ kinase inhibitor treatment. Scientific Reports. 2020;**10**:4449. DOI:

Biology. 2020;**99**:172-182

One. 2017;**12**(6):-e0178519

10.1038/s41598-020-61265-y

[7] De Luna N, Turon-Sans J, Cortes-Vicente E, Carrasco-Rozas A, et al.

978-953-51-2601-0

**References**

mechanisms. This will further allow the improvement of more focused therapeutic interventions aimed at reducing disease severity and positively impact on diagnosis, therapeutic management, and patients' care.

#### **2. ALS**

ALS is an adult onset neurodegenerative disease characterized by progressive loss of spinal, brain stem, and cortical motor neurons, leading to fatal paralysis within 1–5 years since the onset of symptoms that include tremor, muscle weakness, and spasticity [1–3]. ALS affects up to 2:100,000 persons per year; has a life risk around 1:500–1:1000; and exhibits a little predominance of men over women affected [4]. Although ALS is a sporadic multifactorial disease resulting from yet unknown interactions among environment, genes, and epigenetic modifications, genetics seemed to be the predominant factor for the risk of developing the disease [5], and more than 10% of ALS patients are linked to inheritable genetic abnormalities. Dominant mutations in the mitochondrial enzyme Cu/Zn superoxide dismutase-1 (SOD1) seem responsible for up to 1% of the total ALS cases and about 20% of the familial types [4, 6, 7]. Missense mutations in the 43 kDa transactive response DNA/RNA-binding protein (TDP-43) [8] and in fused in sarcoma/translocated in liposarcoma (FUS/ TLS) accounted each one for up to 5% of dominantly inherited familial ALS cases [9, 10]. Mutations in the open reading frame 72 on chromosome 9 (C9ORF72) that results in up to thousands of G4C2 hexanucleotide repeats in one allele are found in up to 40 and 7% of the ALS familial and sporadic cases, respectively [11]. There are other genes involved in ALS familial subtypes, but its contribution to the disease is significantly lower in terms of the affected individual number. Regarding to the pathological pathways linking genetic abnormalities to ALS, SOD1 mutations seem to be related to neuronal damage because of abnormal protein folding causes unstable conformations, intracellular inclusion bodies or toxic oligomers, as well as pathological interactions with several proteins [3]. TDP-43 and FUS/TLS mutations are linked to altered RNA processing, transport, and quality control; whereas, G4C2 repeats might sequestrate RNA-binding proteins impairing the regulation of the RNA targets [12, 13] or causing epigenetic changes that decreased C9ORF72 expression [14, 15]. However, up to now, it is not completely understood how single mutations in one protein could elicit the ALS pathological cascades and how these cascades may finally cause a common neuropathological hallmark that is characterized by aggregation and accumulation of neuronal proteinaceous inclusions that in addition, are found in other neurodegenerative conditions including Alzheimer disease.

#### **2.1 Animal models and non-cell autonomous mechanisms in ALS**

To understand the different pathological mechanisms involved in ALS, many experimental models from yeast to rodents have been developed. Whereas, models in lower animals are powerful genetic tools and offer advantages related to short life span and easy handling, distance with mammal nervous systems constitute major limitations when studying human neurodegeneration [16, 17]. Mice and rats are closer to human brain anatomy and complexity, but are not good genetic tools and their lifespan makes necessary the over-expression of mutant human proteins several-fold times to mimic the disease [4], causing the risk that the number of copies over-expressed influence the model by itself. In spite of this, animal models appear as the best approaches to study ALS patho-mechanisms, at least until the employment of inducible pluripotent cells obtained from human patients becomes a well-known and controlled technology.

**9**

ing CNS damage.

*Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat…*

The first successful ALS models, yet under current extensive use, were developed over-expressing different single mutations of human SOD1 (SOD1G93A, SOD1G37R, and SOD1G85R) in mice or rats [18–20]. Most of the models that overexpress SOD1 present an age-dependent progressive motor syndrome that mimics some pathological features of the human disease [4, 20]. In addition, it seems that pathological features elicited do not derive from the loss of SOD1 catalytic activity but from a yet unknown gain-of-function [4, 20]. Among the highest contributions of murine SOD1 models to the ALS knowledge is the introduction of the non-cell autonomous mechanism concept in which the exclusive neuronal presence of mutant SOD1 did not cause motor neuron death. This implies that motor neuron disease results from the involvement of at least two different cell types. Therefore, a defective cell-cell communication between motor neurons and surrounding glial cells seems actively participating in motor neuron death through not completely understood mechanisms. Pioneer works made in LoxSOD1G37R/

mice [21] or specifically excising the mutant SOD1 transgene from dif-

ferent glial cell types in mice [22, 23] showed that astrocytes [21] and microglial cells [22] play active roles in ALS progression. In support of the non-cell autonomous mechanisms in SOD1 models, reactive astrocytes obtained from transgenic rats or mice [24–26], and from patients of sporadic and familial motor neuron diseases [27, 28] caused neurotoxicity to motor neurons even in cases in which

Other ALS models in rodents were not as clear as those over-expressing mutated SOD1. Transgenic animals expressing mutations of TDP-43-, FUS/TLS-, or C9ORF72-linked ALS produced controversial results without a clear association between each mutation and motor neuron disease, in spite of having motor neuron damage, proteinaceous inclusions, and astrogliosis [29, 30]. Despite these drawbacks, ALS models valuably contribute to make the concept that disruption at systemic, cellular, and molecular levels likely result in many different interacting mechanisms and multiple factors, and that a particular combination of factors and mechanisms likely determine the singularity of each case thus explains the hetero-

**3. Contribution of aberrant glial phenotypes to ALS pathogenesis**

SOD1 models support the concept of ALS as a non-cell autonomous disease in which the reactive astrocyte phenotypes that are produced in the injuring environment greatly contribute to motor neuron death. Astrocytes are the most abundant glial cells in the mammal brain and those responsible for the maintenance of CNS homeostasis [31–34]. During injury, CNS homeostasis is lost and astrocytes respond in a process usually called astrogliosis, in which cells became reactive, highly proliferative and with morphological and functional changes that usually result in decreased protection together with activation of injuring cascades to neurons and oligodendrocytes, further affecting the whole CNS [31–37]. Depending on the injury type and context, astrocyte response can become chronic causing a long lasting state characterized by glial scar, structural tissue rearrangement and impeded repair, as well as a permanent imbalance among homeostatic supportive and gain of neurotoxic functions, all potentially participating in the triggering and progression of several neurological diseases [3, 31–35, 37, 38]. Remarkably, astrocytes also contribute to maintain astrogliosis through autocrine and paracrine signaling [35, 38, 39], thus causing a positive feedback that widespread reactivity and dependent injuring cascades perpetuat-

*DOI: http://dx.doi.org/10.5772/intechopen.84695*

GFAP-Cre<sup>+</sup>

SOD1 is not involved [28].

geneity of the human disease.

#### *Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat… DOI: http://dx.doi.org/10.5772/intechopen.84695*

The first successful ALS models, yet under current extensive use, were developed over-expressing different single mutations of human SOD1 (SOD1G93A, SOD1G37R, and SOD1G85R) in mice or rats [18–20]. Most of the models that overexpress SOD1 present an age-dependent progressive motor syndrome that mimics some pathological features of the human disease [4, 20]. In addition, it seems that pathological features elicited do not derive from the loss of SOD1 catalytic activity but from a yet unknown gain-of-function [4, 20]. Among the highest contributions of murine SOD1 models to the ALS knowledge is the introduction of the non-cell autonomous mechanism concept in which the exclusive neuronal presence of mutant SOD1 did not cause motor neuron death. This implies that motor neuron disease results from the involvement of at least two different cell types. Therefore, a defective cell-cell communication between motor neurons and surrounding glial cells seems actively participating in motor neuron death through not completely understood mechanisms. Pioneer works made in LoxSOD1G37R/ GFAP-Cre<sup>+</sup> mice [21] or specifically excising the mutant SOD1 transgene from different glial cell types in mice [22, 23] showed that astrocytes [21] and microglial cells [22] play active roles in ALS progression. In support of the non-cell autonomous mechanisms in SOD1 models, reactive astrocytes obtained from transgenic rats or mice [24–26], and from patients of sporadic and familial motor neuron diseases [27, 28] caused neurotoxicity to motor neurons even in cases in which SOD1 is not involved [28].

Other ALS models in rodents were not as clear as those over-expressing mutated SOD1. Transgenic animals expressing mutations of TDP-43-, FUS/TLS-, or C9ORF72-linked ALS produced controversial results without a clear association between each mutation and motor neuron disease, in spite of having motor neuron damage, proteinaceous inclusions, and astrogliosis [29, 30]. Despite these drawbacks, ALS models valuably contribute to make the concept that disruption at systemic, cellular, and molecular levels likely result in many different interacting mechanisms and multiple factors, and that a particular combination of factors and mechanisms likely determine the singularity of each case thus explains the heterogeneity of the human disease.

#### **3. Contribution of aberrant glial phenotypes to ALS pathogenesis**

SOD1 models support the concept of ALS as a non-cell autonomous disease in which the reactive astrocyte phenotypes that are produced in the injuring environment greatly contribute to motor neuron death. Astrocytes are the most abundant glial cells in the mammal brain and those responsible for the maintenance of CNS homeostasis [31–34]. During injury, CNS homeostasis is lost and astrocytes respond in a process usually called astrogliosis, in which cells became reactive, highly proliferative and with morphological and functional changes that usually result in decreased protection together with activation of injuring cascades to neurons and oligodendrocytes, further affecting the whole CNS [31–37]. Depending on the injury type and context, astrocyte response can become chronic causing a long lasting state characterized by glial scar, structural tissue rearrangement and impeded repair, as well as a permanent imbalance among homeostatic supportive and gain of neurotoxic functions, all potentially participating in the triggering and progression of several neurological diseases [3, 31–35, 37, 38]. Remarkably, astrocytes also contribute to maintain astrogliosis through autocrine and paracrine signaling [35, 38, 39], thus causing a positive feedback that widespread reactivity and dependent injuring cascades perpetuating CNS damage.

*Novel Aspects on Motor Neuron Disease*

**2. ALS**

therapeutic management, and patients' care.

mechanisms. This will further allow the improvement of more focused therapeutic interventions aimed at reducing disease severity and positively impact on diagnosis,

ALS is an adult onset neurodegenerative disease characterized by progressive loss of spinal, brain stem, and cortical motor neurons, leading to fatal paralysis within 1–5 years since the onset of symptoms that include tremor, muscle weakness, and spasticity [1–3]. ALS affects up to 2:100,000 persons per year; has a life risk around 1:500–1:1000; and exhibits a little predominance of men over women affected [4]. Although ALS is a sporadic multifactorial disease resulting from yet unknown interactions among environment, genes, and epigenetic modifications, genetics seemed to be the predominant factor for the risk of developing the disease [5], and more than 10% of ALS patients are linked to inheritable genetic abnormalities. Dominant mutations in the mitochondrial enzyme Cu/Zn superoxide dismutase-1 (SOD1) seem responsible for up to 1% of the total ALS cases and about 20% of the familial types [4, 6, 7]. Missense mutations in the 43 kDa transactive response DNA/RNA-binding protein (TDP-43) [8] and in fused in sarcoma/translocated in liposarcoma (FUS/ TLS) accounted each one for up to 5% of dominantly inherited familial ALS cases [9, 10]. Mutations in the open reading frame 72 on chromosome 9 (C9ORF72) that results in up to thousands of G4C2 hexanucleotide repeats in one allele are found in up to 40 and 7% of the ALS familial and sporadic cases, respectively [11]. There are other genes involved in ALS familial subtypes, but its contribution to the disease is significantly lower in terms of the affected individual number. Regarding to the pathological pathways linking genetic abnormalities to ALS, SOD1 mutations seem to be related to neuronal damage because of abnormal protein folding causes unstable conformations, intracellular inclusion bodies or toxic oligomers, as well as pathological interactions with several proteins [3]. TDP-43 and FUS/TLS mutations are linked to altered RNA processing, transport, and quality control; whereas, G4C2 repeats might sequestrate RNA-binding proteins impairing the regulation of the RNA targets [12, 13] or causing epigenetic changes that decreased C9ORF72 expression [14, 15]. However, up to now, it is not completely understood how single mutations in one protein could elicit the ALS pathological cascades and how these cascades may finally cause a common neuropathological hallmark that is characterized by aggregation and accumulation of neuronal proteinaceous inclusions that in addition, are found in

other neurodegenerative conditions including Alzheimer disease.

**2.1 Animal models and non-cell autonomous mechanisms in ALS**

To understand the different pathological mechanisms involved in ALS, many experimental models from yeast to rodents have been developed. Whereas, models in lower animals are powerful genetic tools and offer advantages related to short life span and easy handling, distance with mammal nervous systems constitute major limitations when studying human neurodegeneration [16, 17]. Mice and rats are closer to human brain anatomy and complexity, but are not good genetic tools and their lifespan makes necessary the over-expression of mutant human proteins several-fold times to mimic the disease [4], causing the risk that the number of copies over-expressed influence the model by itself. In spite of this, animal models appear as the best approaches to study ALS patho-mechanisms, at least until the employment of inducible pluripotent cells obtained from human patients becomes a

**8**

well-known and controlled technology.

A striking question that remained unanswered until recently was to know if all of the astrocytes that share the same injuring environment respond in the same way or if some of them adopt the worse aberrant phenotypes that account for most of the neurotoxic effects. Trying to unravel this question, when we were studying spinal cord astrocyte phenotypes along the symptomatic phase of the rat SOD1G93A ALS experimental model, we isolated a novel type of aberrant astrocyte-like cells (AbAs) from the spinal cord of paralytic animals whose number exponentially increased toward the terminal stages of the disease [40]. AbAs proliferated faster than astrocytes from neonates or adult wild-type rats and were exceptionally toxic to embryonic motor neurons grown in culture, suggesting a link between their emergence and progression of the paralysis that is a characteristic in the SOD1G93A ALS rat model. Moreover, AbAs did not express distinctive markers that clearly allow distinguishing from typical astrocytes; but present peculiar functional and ultrastructural features that suggest a distinctive phenotype. Among the most remarkable features that AbAs possess, Jiménez-Riani et al. [41] describe their permanent absence of contact inhibition that allowed them to grow in multiple layers and arrange in 3D-cell aggregates that adopt a helicoidal pattern with a central core of extracellular matrix surrounded by cells. In addition, AbAs cytoskeleton does not have intermediate filaments but a significant abundance of microtubules and mitochondria, and ER stress have a restricted perinuclear location suggesting disturbed organelle trafficking that may be associated to alteration in microtubule network or Golgi fragmentation [42]. Furthermore, mitochondria from AbAs are small, electron dense matrix and with few crests; all, comparable to what was described early in models and human ALS [43, 44]. AbAs also have prominent ER with extremely swollen cisternae, some of them degenerating, and express high levels of some ER stress markers [45–47], as well as abundant lipid droplets close to ER and to mitochondria. Their cytoplasm is enriched in diverse vesicles with abundant signs of secretion including extracellular vesicles that can be distinguished by MET and SEM in cultures as well as expression of protein that marks secretion granules [41, 48, 49]. AbAs are also highly positive to the autophagy marker LC3B [50] and present cells' autophagic vesicles and residual bodies [51, 52], likely showing signs of increased autophagy that may allow cells coping with ER stress by favoring the clearance of misfolded proteins [53].

Recently, we confirmed that AbAs were not isolated from the cervical spinal cord of paralytic animals. Instead, the cultures obtained from the cervical spinal cord were similar to the age-matched wild type non-transgenic rats, sharing a low rate of proliferation and resembling a phagocytic microglia morphology that persists throughout the cell culture. Similarities also include low survival along few passages together with absence of complete phenotypic transition to flat cells like astrocytes (**Figure 1**). Therefore, AbAs might result as a local lumbar response to damage, acting similar to astrocytes when react stereotypically depending on injury type, location, and signaling [32].

In addition, we have found that some AbAs critical features are changing during few passages, as occurring with their most prominent markers S100β and glial glutamate transporter GLT1. Meanwhile, there were no evident morphological differences between low (LP) (~4–7) and high passages (HP) (~14–18) (**Figure 2A**), since cultured AbAs proliferated without replicative senescence, S100β expression levels decreased ≅98% (**Figure 2B**), suggesting that this aberrant phenotype may exhibit some plasticity along time. S100β is a well-known dangerassociated molecular pattern (DAMP) which downstream trigger the transcription of nuclear factor NFKB that further may elicit increased expression and release of pro-inflammatory cytokines [54, 55]. Given that S100β appears to integrate AbAs

**11**

**Figure 1.**

inhibition [41].

*Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat…*

cytoskeletal elements, we cannot discard that S100β downregulated expression may cause cytoskeleton instability, a characteristic that is linked to exacerbated proliferative capacity as found in AbAs [56]. Thus, decreasing S100β might constitute a reinforcing proliferation feedback that may underlie AbAs invasive properties as disease progressed. We have also found that GLT1 expression levels also decreased strongly along AbAs passages (≅94%, **Figure 2B**), worsening its poor expression

*Morphological appearance of AbAs throughout the cell culture. Light microscopy examination of cells isolated from symptomatic cervical and lumbar spinal cord of transgenic (Tg cervical SC and Tg lumbar SC) or age-matched non-transgenic animals (No Tg lumbar SC) at 48 h, 5 days and 2 weeks after dissection. Note that AbA cells are recovered only from the lumbar spinal cord of transgenic animals whereas the cultures obtained from the cervical spinal cord of the same animals have similar characteristics to the age-matched wild type nontransgenic rats. Adherent cell population is heterogenous with very bright cells that resemble microglial cells and others elongated similar to astrocytes. Scale bar = 150 μm (first line) and 100 μm (second and third lines).*

Concurrence of all of the features makes AbAs a unique aberrant phenotype with unprecedented neurotoxicity, which may rely in the yet unknown combination of ER stress, lipid droplet accumulation, abundant extracellular matrix, secretory granules, and exacerbated proliferation. Likely, all these events causing the active production of proteinaceous or lipidic soluble factors that act by itself or reinforce the defective cell-contact properties produced by loss of contact

which can also aggravated excitotoxic damage [31].

*DOI: http://dx.doi.org/10.5772/intechopen.84695*

*Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat… DOI: http://dx.doi.org/10.5772/intechopen.84695*

#### **Figure 1.**

*Novel Aspects on Motor Neuron Disease*

A striking question that remained unanswered until recently was to know if all of the astrocytes that share the same injuring environment respond in the same way or if some of them adopt the worse aberrant phenotypes that account for most of the neurotoxic effects. Trying to unravel this question, when we were studying spinal cord astrocyte phenotypes along the symptomatic phase of the rat SOD1G93A ALS experimental model, we isolated a novel type of aberrant astrocyte-like cells (AbAs) from the spinal cord of paralytic animals whose number exponentially increased toward the terminal stages of the disease [40]. AbAs proliferated faster than astrocytes from neonates or adult wild-type rats and were exceptionally toxic to embryonic motor neurons grown in culture, suggesting a link between their emergence and progression of the paralysis that is a characteristic in the SOD1G93A ALS rat model. Moreover, AbAs did not express distinctive markers that clearly allow distinguishing from typical astrocytes; but present peculiar functional and ultrastructural features that suggest a distinctive phenotype. Among the most remarkable features that AbAs possess, Jiménez-Riani et al. [41] describe their permanent absence of contact inhibition that allowed them to grow in multiple layers and arrange in 3D-cell aggregates that adopt a helicoidal pattern with a central core of extracellular matrix surrounded by cells. In addition, AbAs cytoskeleton does not have intermediate filaments but a significant abundance of microtubules and mitochondria, and ER stress have a restricted perinuclear location suggesting disturbed organelle trafficking that may be associated to alteration in microtubule network or Golgi fragmentation [42]. Furthermore, mitochondria from AbAs are small, electron dense matrix and with few crests; all, comparable to what was described early in models and human ALS [43, 44]. AbAs also have prominent ER with extremely swollen cisternae, some of them degenerating, and express high levels of some ER stress markers [45–47], as well as abundant lipid droplets close to ER and to mitochondria. Their cytoplasm is enriched in diverse vesicles with abundant signs of secretion including extracellular vesicles that can be distinguished by MET and SEM in cultures as well as expression of protein that marks secretion granules [41, 48, 49]. AbAs are also highly positive to the autophagy marker LC3B [50] and present cells' autophagic vesicles and residual bodies [51, 52], likely showing signs of increased autophagy that may allow cells coping with ER stress by favoring the clearance of misfolded

Recently, we confirmed that AbAs were not isolated from the cervical spinal cord of paralytic animals. Instead, the cultures obtained from the cervical spinal cord were similar to the age-matched wild type non-transgenic rats, sharing a low rate of proliferation and resembling a phagocytic microglia morphology that persists throughout the cell culture. Similarities also include low survival along few passages together with absence of complete phenotypic transition to flat cells like astrocytes (**Figure 1**). Therefore, AbAs might result as a local lumbar response to damage, acting similar to astrocytes when react stereotypically depending on injury

In addition, we have found that some AbAs critical features are changing during few passages, as occurring with their most prominent markers S100β and glial glutamate transporter GLT1. Meanwhile, there were no evident morphological differences between low (LP) (~4–7) and high passages (HP) (~14–18) (**Figure 2A**), since cultured AbAs proliferated without replicative senescence, S100β expression levels decreased ≅98% (**Figure 2B**), suggesting that this aberrant phenotype may exhibit some plasticity along time. S100β is a well-known dangerassociated molecular pattern (DAMP) which downstream trigger the transcription of nuclear factor NFKB that further may elicit increased expression and release of pro-inflammatory cytokines [54, 55]. Given that S100β appears to integrate AbAs

**10**

proteins [53].

type, location, and signaling [32].

*Morphological appearance of AbAs throughout the cell culture. Light microscopy examination of cells isolated from symptomatic cervical and lumbar spinal cord of transgenic (Tg cervical SC and Tg lumbar SC) or age-matched non-transgenic animals (No Tg lumbar SC) at 48 h, 5 days and 2 weeks after dissection. Note that AbA cells are recovered only from the lumbar spinal cord of transgenic animals whereas the cultures obtained from the cervical spinal cord of the same animals have similar characteristics to the age-matched wild type nontransgenic rats. Adherent cell population is heterogenous with very bright cells that resemble microglial cells and others elongated similar to astrocytes. Scale bar = 150 μm (first line) and 100 μm (second and third lines).*

cytoskeletal elements, we cannot discard that S100β downregulated expression may cause cytoskeleton instability, a characteristic that is linked to exacerbated proliferative capacity as found in AbAs [56]. Thus, decreasing S100β might constitute a reinforcing proliferation feedback that may underlie AbAs invasive properties as disease progressed. We have also found that GLT1 expression levels also decreased strongly along AbAs passages (≅94%, **Figure 2B**), worsening its poor expression which can also aggravated excitotoxic damage [31].

Concurrence of all of the features makes AbAs a unique aberrant phenotype with unprecedented neurotoxicity, which may rely in the yet unknown combination of ER stress, lipid droplet accumulation, abundant extracellular matrix, secretory granules, and exacerbated proliferation. Likely, all these events causing the active production of proteinaceous or lipidic soluble factors that act by itself or reinforce the defective cell-contact properties produced by loss of contact inhibition [41].

#### **Figure 2.**

*Morphology and gene expression in AbAs at low and high cell passages. A. Representative light microscopy images of low (LP, left) and high (HP, right) passage cells from transgenic lumbar spinal cords showing their distinctive appearance of flatted elongated cells that appear very similar to astrocytes. Note the low number of bright microglial-like cells. Scale bar = 100 μm. B. Gene expression analysis in AbA cell cultures showing a down-regulation of S100β (left) and glutamate transporter GLT1 (right) in HP cells compared with LP cells (control). The expression levels for each gene were obtained by SYBR green qPCR and normalized to the actin transcripts. Data represent the mean ± the standard deviation for each group.*

#### **3.1 Mechanisms that might link AbA to ALS pathogenesis**

The most important cellular processes implicated in ALS pathophysiology include ER stress and protein clearance, neuron-glia metabolic coupling, and energy homeostasis [57, 58]. Among their most remarkable features, AbAs exhibit a hardly coping extreme ER stress, as well as lipid droplets and disturbed mitochondrial morphology and trafficking [41]. ER stress is produced by the lack of balance between protein synthesis, folding, and degradation rates [59]. To recover ER homeostasis, cells activate the unfolded protein response (UPR) that orchestrates pro-adaptive and pro-death cellular responses that include protein synthesis decrease except for the effectors that mediates UPR [59–61]. ER stress's final outcome depends on stress duration, strength, and cell targets, and if not resolved, it becomes chronic and as one of the earliest perturbations in several neurodegenerative diseases [59]. Interestingly, ER stress is present in ALS

**13**

neurotoxicity.

microgliosis and neuronal death [79].

*Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat…*

experimental models, and is described as a predominant mechanism underlying motor neuron death in patients from sporadic and familial cases [46, 59, 62–66]. Furthermore, active UPR in AbAs may down regulate the expression of peptides and proteins that collaborate with neuron survival such as the most important cellular antioxidant defense glutathione or neurotrophins [3, 32]. Thus, although ER stress in AbAs did not cause their own death, it is highly probable that it affects neuron and oligodendrocyte survival in view of their high dependence on astro-

In close relationship with ER stress, AbAs are also much enriched in lipid droplets that appear near to mitochondria or ER cisternae [41]. Lipid droplets originate from the ER and are described as having a role in ER stress and clearance of protein aggregates as well as in energy homeostasis [67]. Protein turnover is critical for ALS because a number of mutations linked to ALS affect genes directly involved in protein clearance and homeostasis [58]. Lipid droplets appear associated with some of these proteins into the cytoplasm or the nucleus [67, 68], where they appear close and likely associated with the nuclear-naked organelles that control transcriptional activity, cell senescence, and protein degradation named as promyelocytic leukemia nuclear bodies [67, 69, 70], which in addition are found in cell nuclei of ALS patient brains co-localizing with ubiquitin and proteasome components in nuclear inclu-

In brain, lipid droplets are found mainly in glial cells and help to provide fuel for neurons when energy is needed and glucose is scarce. At this time, lipid droplets turned over by cytoplasmic lipases and autophagy, providing fatty acid fuel for ATP production [67], thus playing a crucial role in the anaplerotic support [72]. However, overabundance of lipid droplets as seen in AbAs may suggest a disrupted lipid metabolism in which lipid droplets may not be digested thus decreasing the energy intermediate shuttle to neurons, which can influence motor neuron survival through limited anaplerosis. Lipidic dysfunction could also indirectly impact motor neuron survival as shown in mice over-expressing TDP-43, that beside displaying neurological symptoms and motor deficits, also present increased fat accumulation and adipocyte hypertrophy [73]. Conversely, TDP-43 depletion causes body fat reduction, increased fatty acid consumption, and rapid death [74], likely, because TDP-43 depletion blocks insulin-induced trafficking of glucose transporter Glut4 to the plasma membrane thus impairing glucose uptake and inducing a metabolic switch toward lipids for energy production. This has also been reported in SOD1 mouse models in which spinal cord neurons display decreased glucose usage [75], and a fat-rich diet restores body mass, delays disease onset, and extends life expectancy [57]. Moreover, excessive accumulation of lipid droplets in glial cells is a hallmark in many models of neurodegeneration, and it is usually linked to mitochondrial dysfunction and disease progression [72, 76, 77]. It also seemed enough to promote neurodegeneration by itself [76], therefore indicating that overabundance of lipid droplets in AbAs may have dual functions: for one side not only helping to the clearance of abnormal proteins, but also impairing anaplerotic support to neurons or even having direct

AbAs also show evidences of a high secretory activity, which also is described as being crucial to ALS neuronal damage. Although secretory granules seem a conserved protective response to conserve energy and allow recovery under stress conditions, sustained secretory activity of stress granules seems crucial to ALS pathogenesis [78]. Moreover, it has been demonstrated that chromogranins interact and co-localize with mutated misfolded SOD1 [79]; and can eventually act as chaperones to promote secretion of SOD1 mutants that once released may trigger

*DOI: http://dx.doi.org/10.5772/intechopen.84695*

cyte support.

sions [71].

#### *Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat… DOI: http://dx.doi.org/10.5772/intechopen.84695*

experimental models, and is described as a predominant mechanism underlying motor neuron death in patients from sporadic and familial cases [46, 59, 62–66]. Furthermore, active UPR in AbAs may down regulate the expression of peptides and proteins that collaborate with neuron survival such as the most important cellular antioxidant defense glutathione or neurotrophins [3, 32]. Thus, although ER stress in AbAs did not cause their own death, it is highly probable that it affects neuron and oligodendrocyte survival in view of their high dependence on astrocyte support.

In close relationship with ER stress, AbAs are also much enriched in lipid droplets that appear near to mitochondria or ER cisternae [41]. Lipid droplets originate from the ER and are described as having a role in ER stress and clearance of protein aggregates as well as in energy homeostasis [67]. Protein turnover is critical for ALS because a number of mutations linked to ALS affect genes directly involved in protein clearance and homeostasis [58]. Lipid droplets appear associated with some of these proteins into the cytoplasm or the nucleus [67, 68], where they appear close and likely associated with the nuclear-naked organelles that control transcriptional activity, cell senescence, and protein degradation named as promyelocytic leukemia nuclear bodies [67, 69, 70], which in addition are found in cell nuclei of ALS patient brains co-localizing with ubiquitin and proteasome components in nuclear inclusions [71].

In brain, lipid droplets are found mainly in glial cells and help to provide fuel for neurons when energy is needed and glucose is scarce. At this time, lipid droplets turned over by cytoplasmic lipases and autophagy, providing fatty acid fuel for ATP production [67], thus playing a crucial role in the anaplerotic support [72]. However, overabundance of lipid droplets as seen in AbAs may suggest a disrupted lipid metabolism in which lipid droplets may not be digested thus decreasing the energy intermediate shuttle to neurons, which can influence motor neuron survival through limited anaplerosis. Lipidic dysfunction could also indirectly impact motor neuron survival as shown in mice over-expressing TDP-43, that beside displaying neurological symptoms and motor deficits, also present increased fat accumulation and adipocyte hypertrophy [73]. Conversely, TDP-43 depletion causes body fat reduction, increased fatty acid consumption, and rapid death [74], likely, because TDP-43 depletion blocks insulin-induced trafficking of glucose transporter Glut4 to the plasma membrane thus impairing glucose uptake and inducing a metabolic switch toward lipids for energy production. This has also been reported in SOD1 mouse models in which spinal cord neurons display decreased glucose usage [75], and a fat-rich diet restores body mass, delays disease onset, and extends life expectancy [57]. Moreover, excessive accumulation of lipid droplets in glial cells is a hallmark in many models of neurodegeneration, and it is usually linked to mitochondrial dysfunction and disease progression [72, 76, 77]. It also seemed enough to promote neurodegeneration by itself [76], therefore indicating that overabundance of lipid droplets in AbAs may have dual functions: for one side not only helping to the clearance of abnormal proteins, but also impairing anaplerotic support to neurons or even having direct neurotoxicity.

AbAs also show evidences of a high secretory activity, which also is described as being crucial to ALS neuronal damage. Although secretory granules seem a conserved protective response to conserve energy and allow recovery under stress conditions, sustained secretory activity of stress granules seems crucial to ALS pathogenesis [78]. Moreover, it has been demonstrated that chromogranins interact and co-localize with mutated misfolded SOD1 [79]; and can eventually act as chaperones to promote secretion of SOD1 mutants that once released may trigger microgliosis and neuronal death [79].

*Novel Aspects on Motor Neuron Disease*

**3.1 Mechanisms that might link AbA to ALS pathogenesis**

*transcripts. Data represent the mean ± the standard deviation for each group.*

The most important cellular processes implicated in ALS pathophysiology include ER stress and protein clearance, neuron-glia metabolic coupling, and energy homeostasis [57, 58]. Among their most remarkable features, AbAs exhibit a hardly coping extreme ER stress, as well as lipid droplets and disturbed mitochondrial morphology and trafficking [41]. ER stress is produced by the lack of balance between protein synthesis, folding, and degradation rates [59]. To recover ER homeostasis, cells activate the unfolded protein response (UPR) that orchestrates pro-adaptive and pro-death cellular responses that include protein synthesis decrease except for the effectors that mediates UPR [59–61]. ER stress's final outcome depends on stress duration, strength, and cell targets, and if not resolved, it becomes chronic and as one of the earliest perturbations in several neurodegenerative diseases [59]. Interestingly, ER stress is present in ALS

*Morphology and gene expression in AbAs at low and high cell passages. A. Representative light microscopy images of low (LP, left) and high (HP, right) passage cells from transgenic lumbar spinal cords showing their distinctive appearance of flatted elongated cells that appear very similar to astrocytes. Note the low number of bright microglial-like cells. Scale bar = 100 μm. B. Gene expression analysis in AbA cell cultures showing a down-regulation of S100β (left) and glutamate transporter GLT1 (right) in HP cells compared with LP cells (control). The expression levels for each gene were obtained by SYBR green qPCR and normalized to the actin* 

**12**

**Figure 2.**

Absence of contact inhibition and exacerbated proliferation are other of the relevant features related to AbAs neurotoxic capacity. Contact inhibition that occurs when dividing normal cells contact adjacent ones is crucial to maintain tissue homeostasis [80, 81], thus constituting an important anticancer mechanism which lack unleashes cells to proliferate virtually unchecked. Although underlying mechanisms are mostly unknown, cell contact inhibition seems to occur when injury disrupts intercellular contacts achieving a proliferative status leading to an aggressive state associated with neoplasia [82] and malignant transformation [81]. Thus, AbAs, absence of contact inhibition seemed directly related to their exacerbated proliferation and invasive behavior during the final stages of the disease. In addition, abundance of EM components secreted by AbA cells may create a non-permissive microenvironment that potentiates invasive behavior apart from having a direct neurotoxic influence to motor neurons, as described in ALS astrocytes [83–85]. No one of each proposed mechanisms seem enough to explain AbAs unprecedented neurotoxicity. Instead, it likely results from the concurrence of many pathological pathways. However, it is also possible that one or two underlying mechanisms prevail over the rest and elicit most of AbAs deleterious effects. Identification of these prevalent mechanisms will be a valuable aid to design the best ALS treatment (**Table 1** and **Figure 3**).

(i) Absence of contact inhibition


(ii) Immature phenotype

	- Mitochondrial dysfunction [89, 90]
	- Mitochondrial morphological alterations [41]
	- Defective oxidative phosphorylation [89, 90]
	- Dysfunction in energy homeostasis [24, 89, 91]
	- Dilated ER and degenerating ER cisternae [41]
	- Elevated expression of ER stress markers [41]

(iv) Altered lipid metabolism


(v) Intracellular inclusions


(vi) Aberrant signaling


**Table 1.**

*A summary of AbAs potential pathogenic contribution to the main ALS hallmark. All of ALS main features are listened as (i)–(vi) together with each specific AbAs potential participation.*

**15**

**Figure 3.**

*Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat…*

*DOI: http://dx.doi.org/10.5772/intechopen.84695*

**4. ALS therapeutics focused on aberrant astrocytes**

*energetic support to neurons and oligodendrocytes (light blue).*

search alternative avenues to treat the disease.

ALS is an old disease with a narrow offer of pharmacological approaches. Riluzole, an anti-glutamatergic drug, was the first compound authorized to be used in ALS, providing around of 3-month improvement in survival [92]. Recently, FDA approved the free radical scavenger edaravone, as the second compound to treat ALS, that seemed to have beneficial effects only on patients in an early stages of the disease that in addition satisfy a number of restricted criteria. In that population, edavarone showed a significantly smaller decline of Revised ALS Functional Rating Scale score compared with placebo [93]. A randomized phase III clinical trial that tested the effect of the tyrosine kinase inhibitor masitinib in ALS patients showed an improving in the functioning of ALS patients, and the combination with riluzole caused a delayed disease progression without adverse effects [94]. However, the narrow temporal windows that the two compounds approved offer obligates to

*AbAs: Cytotoxic effects and pathological events. AbAs present mitochondrial dysfunction associated to oxidative stress and ER stress as well as accumulation of lipid droplets, all causing a positive feedback that elicit and perpetuate cell damage (red and black arrows). Mitochondrial abnormalities together with cytoskeletal alterations are also involved in AbAs exacerbated proliferation and cytoskeleton instability both likely underlying the absence of contact inhibition and invasive phenotype as well as S100β-dependent inflammation cascades (green and yellow arrows). Progressive loss of the glutamate transporter GLT1 causes decreased glutamate uptake becoming neurons more expose to glutamate excitoxicity. Altered signaling and disturbed autophagia and proteasome functions caused the release of stress granules and soluble neurotoxins as well as the accumulation of cell detritus and inclusion bodies (violet and blue arrows). Finally, the lack of mitochondrial potential (represented as Ψ) caused increased glycolysis and deficient anaplerosis that alters the trophic and* 

As astrocytes and microglial cells develop both protective and pathological functions its pharmacological targeting must be carefully evaluated. However, in view of the distinctive phenotype of AbAs, it seems rational to direct therapeutic treatments toward the control of this population during disease progression and ideally trying to inhibit their emergence during asymptomatic stages. AbAs expression of S100β at levels higher than wild type astrocytes may imply that they have a role in the amplification of the inflammatory response, therefore new anti-inflammatory drugs targeting the production of pro-inflammatory cytokines *Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat… DOI: http://dx.doi.org/10.5772/intechopen.84695*

#### **Figure 3.**

*Novel Aspects on Motor Neuron Disease*

best ALS treatment (**Table 1** and **Figure 3**).

• Lack of replicative senescence [40, 86] • Exacerbated proliferation [40, 86, 87]

• Lack of gliofilaments [40, 41] • Defective differentiation [40, 41, 88]

• Mitochondrial dysfunction [89, 90]

• Mitochondrial morphological alterations [41] • Defective oxidative phosphorylation [89, 90] • Dysfunction in energy homeostasis [24, 89, 91] • Dilated ER and degenerating ER cisternae [41] • Elevated expression of ER stress markers [41]

• Abundant lipid droplets close to mitochondria & ER [41]

• Intranuclear and intramitochondrial deposits [41]

• Extremely neurotoxic conditioned media [40]

• Abundance of secretory vesicles and secretory body markers [41]

*are listened as (i)–(vi) together with each specific AbAs potential participation.*

*A summary of AbAs potential pathogenic contribution to the main ALS hallmark. All of ALS main features* 

(i) Absence of contact inhibition • Invasive phenotype [40, 41]

(ii) Immature phenotype

(iii) Oxidative and ER stress

(iv) Altered lipid metabolism

(v) Intracellular inclusions

(vi) Aberrant signaling

• Neurotoxic exosomes [90]

• Altered anaplerotic support [88]

• Abundance of autophagic bodies [41]

Absence of contact inhibition and exacerbated proliferation are other of the relevant features related to AbAs neurotoxic capacity. Contact inhibition that occurs when dividing normal cells contact adjacent ones is crucial to maintain tissue homeostasis [80, 81], thus constituting an important anticancer mechanism which lack unleashes cells to proliferate virtually unchecked. Although underlying mechanisms are mostly unknown, cell contact inhibition seems to occur when injury disrupts intercellular contacts achieving a proliferative status leading to an aggressive state associated with neoplasia [82] and malignant transformation [81]. Thus, AbAs, absence of contact inhibition seemed directly related to their exacerbated proliferation and invasive behavior during the final stages of the disease. In addition, abundance of EM components secreted by AbA cells may create a non-permissive microenvironment that potentiates invasive behavior apart from having a direct neurotoxic influence to motor neurons, as described in ALS astrocytes [83–85]. No one of each proposed mechanisms seem enough to explain AbAs unprecedented neurotoxicity. Instead, it likely results from the concurrence of many pathological pathways. However, it is also possible that one or two underlying mechanisms prevail over the rest and elicit most of AbAs deleterious effects. Identification of these prevalent mechanisms will be a valuable aid to design the

**14**

**Table 1.**

*AbAs: Cytotoxic effects and pathological events. AbAs present mitochondrial dysfunction associated to oxidative stress and ER stress as well as accumulation of lipid droplets, all causing a positive feedback that elicit and perpetuate cell damage (red and black arrows). Mitochondrial abnormalities together with cytoskeletal alterations are also involved in AbAs exacerbated proliferation and cytoskeleton instability both likely underlying the absence of contact inhibition and invasive phenotype as well as S100β-dependent inflammation cascades (green and yellow arrows). Progressive loss of the glutamate transporter GLT1 causes decreased glutamate uptake becoming neurons more expose to glutamate excitoxicity. Altered signaling and disturbed autophagia and proteasome functions caused the release of stress granules and soluble neurotoxins as well as the accumulation of cell detritus and inclusion bodies (violet and blue arrows). Finally, the lack of mitochondrial potential (represented as Ψ) caused increased glycolysis and deficient anaplerosis that alters the trophic and energetic support to neurons and oligodendrocytes (light blue).*

#### **4. ALS therapeutics focused on aberrant astrocytes**

ALS is an old disease with a narrow offer of pharmacological approaches. Riluzole, an anti-glutamatergic drug, was the first compound authorized to be used in ALS, providing around of 3-month improvement in survival [92]. Recently, FDA approved the free radical scavenger edaravone, as the second compound to treat ALS, that seemed to have beneficial effects only on patients in an early stages of the disease that in addition satisfy a number of restricted criteria. In that population, edavarone showed a significantly smaller decline of Revised ALS Functional Rating Scale score compared with placebo [93]. A randomized phase III clinical trial that tested the effect of the tyrosine kinase inhibitor masitinib in ALS patients showed an improving in the functioning of ALS patients, and the combination with riluzole caused a delayed disease progression without adverse effects [94]. However, the narrow temporal windows that the two compounds approved offer obligates to search alternative avenues to treat the disease.

As astrocytes and microglial cells develop both protective and pathological functions its pharmacological targeting must be carefully evaluated. However, in view of the distinctive phenotype of AbAs, it seems rational to direct therapeutic treatments toward the control of this population during disease progression and ideally trying to inhibit their emergence during asymptomatic stages. AbAs expression of S100β at levels higher than wild type astrocytes may imply that they have a role in the amplification of the inflammatory response, therefore new anti-inflammatory drugs targeting the production of pro-inflammatory cytokines by a blockade of NFkB activation may have positive results, moreover because NFkB is downstream to S100β [54]. For example, FDA has been approved a drug called mitoxantrone for multiple sclerosis treatment because it inhibited production of IL-12 and IL-23 and suppressed the expression of C-reactive protein by astrocytes in culture and LPS induction of NFkB DNA-binding activity in primary astrocytes, suggesting a novel mechanism that suppresses the expression of astrocytic pro-inflammatory molecules helping to modulate inflammatory diseases [95]. However, as AbAs seem to loss S100β at higher passages, neuroprotection by targeting this via might be successful only during asymptomatic stages. Targeting neuroinflammation, recently, it has been shown that the tyrosine kinase inhibitor (masitinib) that is used to control cancer cell proliferation reduced the emergence of aberrant glial cells in the degenerating spinal cord of SOD1G93A paralytic rats and delay disease progression [87]. Authors proposed that masitinib acts preventing the appearance of aberrant glial phenotypes, likely through the inhibition of CSF-1R kinase which activation potentiates inflammatory phenotypes and glial reactivity, and that are particularly effective on proliferating but not on postmitotic cells [96]. Furthermore, masitinib also prevented astrocyte-induced motor neuron death in cell cultures [97], suggesting that neuroprotection can be achieved through different pathways. In accordance, other report has shown that CSF-1R blockade with the drug GW2580 administered to ALS mice several weeks before paralysis onset decreased both microgliosis and slowed disease progression [98], thus opening a wider avenue to treat aberrant glial phenotypes. Finally, although molecular genetic techniques devoted to switch genes on or off, or to edit their nucleotide sequences, once developed and approved can be effective therapeutic tools to the inherited ALS forms, pharmacological approaches directed against aberrant glial phenotypes may help to control disease progression in a wider range of patients.

#### **5. Conclusions**

All data reviewed here suggest that aberrant astrocytes or more generally, aberrant glial cells, are among the most important players in CNS damage causing deleterious effects through many potential patho-mechanisms, mostly sustained on their exacerbated proliferation together with their unprecedented neurotoxicity. Therefore, controlling these populations seems at least equally important than maintenance or restoration of homeostatic astrocyte functions to achieve CNS protection and repair. Moreover, AbAs seemed a better pharmacological target than astrocytes, since therapeutic approaches focused on astrocytes have to be carefully tailored taking into account their multiple faces, moreover in the context of neurodegeneration where several cell types are involved. Future investigation should aim to elucidate if AbAs can indeed be feasible targets to avoid initiation, progression or outcome of neurodegeneration.

**17**

**Author details**

Gabriel Otero Damianovich1

Montevideo, Uruguay

Estable (IIBCE), Montevideo, Uruguay

Eugenia Eloísa Isasi1,2, Carmen Isabel Bolatto Pereira2

\*Address all correspondence to: solivera@iibce.edu.uy

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. 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,

, Olga Cristina Parada2

1 Cell and Molecular Neurobiology, Institute of Biological Research Clemente

2 Department of Histology and Embriology, School of Medicine (UdelaR),

, Pablo Díaz-Amarilla1

and Silvia Olivera-Bravo1

,

\*

*Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat…*

*DOI: http://dx.doi.org/10.5772/intechopen.84695*

#### **Acknowledgements**

We thank IIBCE (MEC), PEDECIBA, and UdelaR, URUGUAY.

#### **Conflict of interest**

There is no conflict of interest.

*Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat… DOI: http://dx.doi.org/10.5772/intechopen.84695*

### **Author details**

*Novel Aspects on Motor Neuron Disease*

by a blockade of NFkB activation may have positive results, moreover because NFkB is downstream to S100β [54]. For example, FDA has been approved a drug called mitoxantrone for multiple sclerosis treatment because it inhibited production of IL-12 and IL-23 and suppressed the expression of C-reactive protein by astrocytes in culture and LPS induction of NFkB DNA-binding activity in primary astrocytes, suggesting a novel mechanism that suppresses the expression of

astrocytic pro-inflammatory molecules helping to modulate inflammatory diseases [95]. However, as AbAs seem to loss S100β at higher passages, neuroprotection by targeting this via might be successful only during asymptomatic stages. Targeting neuroinflammation, recently, it has been shown that the tyrosine kinase inhibitor (masitinib) that is used to control cancer cell proliferation reduced the emergence of aberrant glial cells in the degenerating spinal cord of SOD1G93A paralytic rats and delay disease progression [87]. Authors proposed that masitinib acts preventing the appearance of aberrant glial phenotypes, likely through the inhibition of CSF-1R kinase which activation potentiates inflammatory phenotypes and glial reactivity, and that are particularly effective on proliferating but not on postmitotic cells [96]. Furthermore, masitinib also prevented astrocyte-induced motor neuron death in cell cultures [97], suggesting that neuroprotection can be achieved through different pathways. In accordance, other report has shown that CSF-1R blockade with the drug GW2580 administered to ALS mice several weeks before paralysis onset decreased both microgliosis and slowed disease progression [98], thus opening a wider avenue to treat aberrant glial phenotypes. Finally, although molecular genetic techniques devoted to switch genes on or off, or to edit their nucleotide sequences, once developed and approved can be effective therapeutic tools to the inherited ALS forms, pharmacological approaches directed against aberrant glial phenotypes may help to control disease progression in a wider range

All data reviewed here suggest that aberrant astrocytes or more generally, aberrant glial cells, are among the most important players in CNS damage causing deleterious effects through many potential patho-mechanisms, mostly sustained on their exacerbated proliferation together with their unprecedented neurotoxicity. Therefore, controlling these populations seems at least equally important than maintenance or restoration of homeostatic astrocyte functions to achieve CNS protection and repair. Moreover, AbAs seemed a better pharmacological target than astrocytes, since therapeutic approaches focused on astrocytes have to be carefully tailored taking into account their multiple faces, moreover in the context of neurodegeneration where several cell types are involved. Future investigation should aim to elucidate if AbAs can indeed be feasible targets to avoid initiation, progression or

We thank IIBCE (MEC), PEDECIBA, and UdelaR, URUGUAY.

**16**

of patients.

**5. Conclusions**

outcome of neurodegeneration.

There is no conflict of interest.

**Acknowledgements**

**Conflict of interest**

Gabriel Otero Damianovich1 , Olga Cristina Parada2 , Pablo Díaz-Amarilla1 , Eugenia Eloísa Isasi1,2, Carmen Isabel Bolatto Pereira2 and Silvia Olivera-Bravo1 \*

1 Cell and Molecular Neurobiology, Institute of Biological Research Clemente Estable (IIBCE), Montevideo, Uruguay

2 Department of Histology and Embriology, School of Medicine (UdelaR), Montevideo, Uruguay

\*Address all correspondence to: solivera@iibce.edu.uy

© 2019 The Author(s). Licensee IntechOpen. 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.

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**19**

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[19] Howland DS, Liu J, She Y, Goad B, Maragakis NJ, Kim B, et al. Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS). Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**:1604-1609. DOI: 10.1073/

[20] Turner BJ, Talbot K. Transgenics, toxicity and therapeutics in rodent

10.1126/science.8209258

pnas.032539299

nature12111

10.1126/science.1232927

*Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat… DOI: http://dx.doi.org/10.5772/intechopen.84695*

[14] Mori K, Weng SM, Arzberger T, May S, Rentzsch K, Kremmer E, et al. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide repeat proteins in FTLD/ALS. Science. 2013;**339**(6125):1335-1338. DOI: 10.1126/science.1232927

[15] Ash PE, Bieniek KF, Gendron TF, Caulfield T, Lin WL, Dejesus-Hernandez M, et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ ALS. Neuron. 2013;**77**(4):639-646. DOI: 10.1016/j.neuron.2013.02.004

[16] Sulston JE, Horvitz HR. Postembryonic cell lineages of the nematode, Caenorhabditis elegans. Developmental Biology. 1977;**56**(1):110-156. DOI: 10.1016/0012-1606(77)90158-0

[17] Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 2013;**496**(7446):498-503. DOI: 10.1038/ nature12111

[18] Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY, Alexander DD, et al. Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation. Science. 1994;**264**:1772-1775. DOI: 10.1126/science.8209258

[19] Howland DS, Liu J, She Y, Goad B, Maragakis NJ, Kim B, et al. Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS). Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**:1604-1609. DOI: 10.1073/ pnas.032539299

[20] Turner BJ, Talbot K. Transgenics, toxicity and therapeutics in rodent

models of mutant SOD1 mediated familial ALS. Progress in Neurobiology. 2008;**85**:94-134. DOI: 10.1016/j. pneurobio.2008.01.001

[21] Yamanaka K, Chun SJ, Boillee S, Fujimori-Tonou N, Yamashita H, Gutmann DH, et al. Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. Nature Neuroscience. 2008;**11**(3): 251-253. DOI: 10.1038/nn2047

[22] Boillée S, Vande Velde C, Cleveland DW. ALS: A disease of motor neurons and their non neuronal neighbors. Neuron. 2006;**52**:39-59. DOI: 10.3109/21678421.2013.778548

[23] Ilieva H, Polymenidou M, Cleveland DW. Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. The Journal of Cell Biology. 2009;**187**:761-772. DOI: 10.1083/ jcb.200908164

[24] Cassina P, Cassina A, Pehar M, Castellanos R, Gandelman M, de León A, et al. Mitochondrial dysfunction in SOD1G93A-bearing astrocytes promotes motor neuron degeneration: Prevention by mitochondrial-targeted antioxidants. The Journal of Neuroscience. 2008;**28**:4115-4122. DOI: 10.1523/ JNEUROSCI.5308-07.2008

[25] Di Giorgio FP, Boulting GL, Bobrowicz S, Eggan KC. Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation. Cell Stem Cell. 2008;**3**:637-648. DOI: 10.1016/j. stem.2008.09.017

[26] Nagai M, Re DB, Nagata T, Chalazonitis A, Jessell TM, Wichterle H, et al. Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nature Neuroscience. 2007;**10**:615-622. DOI: 10.1038/nn1876

**18**

WNL.49.1.213

*Novel Aspects on Motor Neuron Disease*

[1] Cleveland DW, Rothstein JD. From Charcot to Lou Gehrig: Deciphering selective motor neuron death in ALS. Nature Reviews Neuroscience. 2001;**2**:806-819. DOI: lateral sclerosis. Nature. 1993;**364**:362.

[8] Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006;**314**(5796):130-133. DOI: 10.1126/

[9] Kwiatkowski TJ Jr, Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science. 2009;**323**(5918): 1205-1208. DOI: 10.1126/science.

[10] Vance C, Rogelj B, Hortobágyi T, De Vos KJ, Nishimura AL, Sreedharan J, et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009;**323**(5918):1208-1211. DOI: 10.1126/science.1165942

[11] Todd PK, Paulson HL. RNAmediated neurodegeneration in repeat expansion disorders. Annals of Neurology. 2010;**67**(3):291-300. DOI:

[13] van Blitterswijk M, DeJesus-Hernandez M, Rademakers R. How do C9ORF72 repeat expansions cause amyotrophic lateral sclerosis and

Can we learn from other noncoding

frontotemporal dementia:

repeat expansion disorders? Current Opinion in Neurology. 2012;**25**(6):689-700. DOI: 10.1097/

WCO.0b013e32835a3efb

[12] Ling SC, Polymenidou M, Cleveland DW. Converging mechanisms in ALS and FTD: Disrupted RNA and protein homeostasis. Neuron. 2013;**79**(3): 416-438. DOI: 10.1016/j.neuron.

10.1002/ana.21948

2013.07.033

DOI: 10.1038/362059a0

science.1134108

1166066

[2] Kiernan MC, Vucic S, Cheah BC, Turner MR, Eisen A, Hardiman O, et al. Amyotrophic lateral sclerosis. Lancet. 2011;**377**(9769):942-955. DOI: 10.1016/

[3] Pehar M, Harlan BA, Killoy KM, Vargas MR. Role and therapeutic potential of astrocytes in amyotrophic lateral sclerosis. Current Pharmaceutical Design. 2017;**23**(33):5010-5021. DOI: 10.2174/1381612823666170622095802

[4] Ilieva H, Maragakis NJ. Motoneuron disease: Basic science. In: Beart P, Robinson M, Rattray M, Maragakis NJ, editors. Advances in Neurobiology 15; Neurodegenerative Diseases Pathology, Mechanisms, and

Potential Therapeutic Targets. Cham: Springer; 2017. pp. 163-190. DOI: 10.1007/978-3-319-57193-5

[5] Al-Chalabi A, Fang F, Hanby MF, Leigh PN, Shaw CE, Ye W, et al. An estimate of amyotrophic lateral sclerosis heritability using twin data. Journal of Neurology, Neurosurgery, and Psychiatry. 2010;**81**(12):1324-1326. DOI:

[6] Cudkowicz ME, Warren L, Francis JW, Lloyd KJ, Friedlander RM, Borges LF, et al. Intrathecal administration of recombinant human superoxide dismutase 1 in amyotrophic lateral sclerosis: A preliminary safety and pharmacokinetic study. Neurology. 1997;**49**:213-222. DOI: 10.1212/

[7] Rosen DR. Mutations in Cu/ Zn superoxide dismutase gene are associated with familial amyotrophic

10.1136/jnnp.2010.207464

10.1038/35097565

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[77] Yamamoto S, Jaiswal M, Charng WL, Gambin T, Karaca E, Mirzaa G, et al. A drosophila genetic resource of mutants to study mechanisms underlying human genetic diseases. Cell. 2014;**159**:200-214. DOI: 10.1016/j. cell.2014.09.002

[78] Li YR, King OD, Shorter J, Gitler AD. Stress granules as crucibles of ALS pathogenesis. The Journal of Cell Biology. 2013;**201**:361-372. DOI: 10.1083/jcb.201302044

[79] Urushitani M, Ezzi SA, Matsuo A, Tooyama I, Julien JP. The endoplasmic reticulum-Golgi pathway is a target for translocation and aggregation of mutant superoxide dismutase linked to ALS. The FASEB Journal. 2006;**22**:2476-2487. DOI: 10.1096/ fj.07-092783

[80] Eagle H, Levine EM. Growth regulatory effects of cellular interaction. Nature. 1967;**213**:1102-1106. DOI: 10.1038/2131102a0

[81] Choi EH, Dai Y. SIRT1 controls cell proliferation by regulating contact inhibition. Biochemical and Biophysical Research Communications. 2016;**478**(2):868-872. DOI: 10.1016/j. bbrc.2016.08.041

[82] Yang C, Iyer RR, Yu AC, Yong RL, Park DM, Weil RJ, et al. β-Catenin signaling initiates the activation of astrocytes and its dysregulation contributes to the pathogenesis

of astrocytomas. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**:6963-6968. DOI: 10.1073/ pnas.1118754109

[83] Baker DJ, Blackburn DJ, Keatinge M, Sokhi D, Viskaitis P, Heath PR, et al. Lysosomal and phagocytic activity is increased in astrocytes during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis. Frontiers in Cellular Neuroscience. 2015;**9**:410. DOI: 10.3389/ fncel.2015.00410

[84] Das MM, Svendsen CN. Astrocytes show reduced support of motor neurons with aging that is accelerated in a rodent model of ALS. Neurobiology of Aging. 2015;**36**:1130-1139. DOI: 10.1016/j. neurobiolaging.2014.09.020

[85] Song SW, Miranda CJ, Braun L, Meyer K, Frakes AE, Ferraiuolo L, et al. MHC class I protects motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis (ALS). Nature Medicine. 2016;**22**:397-403. DOI: 10.1038/nm.4052

[86] Trias E, Díaz-Amarilla P, Olivera-Bravo S, Isasi E, Drechsel DA, Lopez N, et al. Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS. Frontiers in Cellular Neuroscience. 2013;**7**:274. DOI: 10.3389/ fncel.2013.00274

[87] Trias E, Ibarburu S, Barreto-Núñez R, Babdor J, Maciel TT, Guillo M, et al. Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis. Journal of Neuroinflammation. 2016;**13**(1):177. DOI: 10.1186/s12974-016-0620-9

[88] Lamp J, Keyser B, Koeller DM, Ullrich K, Braulke T, Mühlhausen C. Glutaric aciduria type 1 metabolites impair the succinate transport from astrocytic to neuronal cells. The Journal of Biological Chemistry. 2011;**286**(20):17777-17784. DOI: 10.1074/jbc.M111.232744

[89] Miquel E, Cassina A, Martínez-Palma L, Souza JM, Bolatto C, Rodríguez-Bottero S, et al. Neuroprotective effects of the mitochondria-targeted antioxidant MitoQ in a model of inherited amyotrophic lateral sclerosis. Free Radical Biology & Medicine. 2014;**70**:204-213. DOI: 10.1016/j. freeradbiomed.2014.02.019

[90] Díaz-Amarilla P, Miquel E, Trostchansky A, Trias E, Ferreira AM, Freeman BA, et al. Electrophilic nitro-fatty acids prevent astrocytemediated toxicity to motor neurons in a cell model of familial amyotrophic lateral sclerosis via nuclear factor erythroid 2-related factor activation. Free Radical Biology & Medicine. 2016;**95**:112-120. DOI: 10.1016/j. freeradbiomed.2016.03.013

[91] Martínez-Palma L, Miquel E, Lagos-Rodríguez V, Barbeito L, Cassina A, Cassina P. Mitochondrial modulation by dichloroacetate reduces toxicity of aberrant glial cells and gliosis in the SOD1G93A rat model of amyotrophic lateral sclerosis. Neurotherapeutics. 2018;**16**(1):203-215. DOI: 10.1007/ s13311-018-0659-7

[92] Miller RG, Mitchell JD, Moore DH. Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Cochrane Database of Systematic Reviews. 2012;**14**(3):CD001447. DOI: 10.1002/14651858.CD001447.pub3

[93] Writing Group. Edaravone (MCI-186) ALS 19 Study Group. Safety and efficacy of edaravone in well defined patients with amyotrophic lateral sclerosis: A randomised, doubleblind, placebo-controlled trial. Lancet

**25**

*Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat…*

*DOI: http://dx.doi.org/10.5772/intechopen.84695*

Neurology. 2017;**16**(7):505-512. DOI: 10.1016/S1474-4422(17)30115-1

[94] Scott A. On the treatment trail for ALS. Nature. 2017;**550**(7676):S120-S121.

[95] Burns SA, Lee Archer R, Chavis JA,

[96] Kocic I, Kowianski P, Rusiecka I, Lietzau G, Mansfield C, Moussy A, et al. Neuroprotective effect of masitinib in rats with postischemic stroke. Naunyn-Schmiedeberg's Archives of Pharmacology. 2015;**388**(1):79-86. DOI:

DOI: 10.1038/550S120a

brainres.2012.07.054

Tull CA, Hensley LL, Drew PD. Mitoxantrone repression of astrocyte activation: Relevance to multiple sclerosis. Brain Research. 2012;**1473**:236-241. DOI: 10.1016/j.

10.1007/s00210-014-1061-6

[97] Rojas F, Gonzalez D, Cortes N, Ampuero E, Hernandez DE, Fritz E, et al. Reactive oxygen species trigger motoneuron death in non-cellautonomous models of ALS through activation of c-Abl signaling. Frontiers in Cellular Neuroscience. 2015;**9**:203. DOI: 10.3389/fncel.2015.00203

[98] Martínez-Muriana A, Mancuso R, Francos-Quijorna I, Olmos-Alonso A, Osta R, Perry VH, et al. CSF1R blockade slows the progression of amyotrophic lateral sclerosis by reducing microgliosis and invasion of macrophages into peripheral nerves. Scientific Reports. 2016;**6**:25663. DOI: 10.1038/srep25663

*Contribution of Aberrant Astrocytes to Motor Neuron Damage and Death in the SOD1G93A Rat… DOI: http://dx.doi.org/10.5772/intechopen.84695*

Neurology. 2017;**16**(7):505-512. DOI: 10.1016/S1474-4422(17)30115-1

*Novel Aspects on Motor Neuron Disease*

of astrocytomas. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**:6963-6968. DOI: 10.1073/

[83] Baker DJ, Blackburn DJ, Keatinge M, Sokhi D, Viskaitis P, Heath PR, et al. Lysosomal and phagocytic activity is increased in astrocytes during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis. Frontiers in Cellular

impair the succinate transport from astrocytic to neuronal cells. The Journal of Biological Chemistry. 2011;**286**(20):17777-17784. DOI:

[89] Miquel E, Cassina A, Martínez-

10.1074/jbc.M111.232744

Palma L, Souza JM, Bolatto C, Rodríguez-Bottero S, et al. Neuroprotective effects of the mitochondria-targeted antioxidant MitoQ in a model of inherited amyotrophic lateral sclerosis. Free Radical Biology & Medicine. 2014;**70**:204-213. DOI: 10.1016/j. freeradbiomed.2014.02.019

[90] Díaz-Amarilla P, Miquel E, Trostchansky A, Trias E, Ferreira AM, Freeman BA, et al. Electrophilic nitro-fatty acids prevent astrocytemediated toxicity to motor neurons in a cell model of familial amyotrophic lateral sclerosis via nuclear factor erythroid 2-related factor activation. Free Radical Biology & Medicine. 2016;**95**:112-120. DOI: 10.1016/j. freeradbiomed.2016.03.013

[91] Martínez-Palma L, Miquel E, Lagos-Rodríguez V, Barbeito L, Cassina A, Cassina P. Mitochondrial modulation by dichloroacetate reduces toxicity of aberrant glial cells and gliosis in the SOD1G93A rat model of amyotrophic lateral sclerosis. Neurotherapeutics. 2018;**16**(1):203-215. DOI: 10.1007/

[92] Miller RG, Mitchell JD, Moore DH. Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Cochrane Database of Systematic Reviews. 2012;**14**(3):CD001447. DOI: 10.1002/14651858.CD001447.pub3

[93] Writing Group. Edaravone (MCI-186) ALS 19 Study Group. Safety and efficacy of edaravone in well defined patients with amyotrophic lateral sclerosis: A randomised, doubleblind, placebo-controlled trial. Lancet

s13311-018-0659-7

Neuroscience. 2015;**9**:410. DOI: 10.3389/

[84] Das MM, Svendsen CN. Astrocytes show reduced support of motor neurons with aging that is accelerated in a rodent model of ALS. Neurobiology of Aging. 2015;**36**:1130-1139. DOI: 10.1016/j. neurobiolaging.2014.09.020

[85] Song SW, Miranda CJ, Braun L, Meyer K, Frakes AE, Ferraiuolo L, et al. MHC class I protects motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis (ALS). Nature Medicine. 2016;**22**:397-403. DOI:

[86] Trias E, Díaz-Amarilla P, Olivera-Bravo S, Isasi E, Drechsel DA, Lopez N, et al. Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS. Frontiers in Cellular Neuroscience. 2013;**7**:274. DOI: 10.3389/

[87] Trias E, Ibarburu S, Barreto-Núñez R, Babdor J, Maciel TT, Guillo M, et al. Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis. Journal of Neuroinflammation. 2016;**13**(1):177. DOI: 10.1186/s12974-016-0620-9

[88] Lamp J, Keyser B, Koeller DM, Ullrich K, Braulke T, Mühlhausen C. Glutaric aciduria type 1 metabolites

pnas.1118754109

fncel.2015.00410

10.1038/nm.4052

fncel.2013.00274

**24**

[94] Scott A. On the treatment trail for ALS. Nature. 2017;**550**(7676):S120-S121. DOI: 10.1038/550S120a

[95] Burns SA, Lee Archer R, Chavis JA, Tull CA, Hensley LL, Drew PD. Mitoxantrone repression of astrocyte activation: Relevance to multiple sclerosis. Brain Research. 2012;**1473**:236-241. DOI: 10.1016/j. brainres.2012.07.054

[96] Kocic I, Kowianski P, Rusiecka I, Lietzau G, Mansfield C, Moussy A, et al. Neuroprotective effect of masitinib in rats with postischemic stroke. Naunyn-Schmiedeberg's Archives of Pharmacology. 2015;**388**(1):79-86. DOI: 10.1007/s00210-014-1061-6

[97] Rojas F, Gonzalez D, Cortes N, Ampuero E, Hernandez DE, Fritz E, et al. Reactive oxygen species trigger motoneuron death in non-cellautonomous models of ALS through activation of c-Abl signaling. Frontiers in Cellular Neuroscience. 2015;**9**:203. DOI: 10.3389/fncel.2015.00203

[98] Martínez-Muriana A, Mancuso R, Francos-Quijorna I, Olmos-Alonso A, Osta R, Perry VH, et al. CSF1R blockade slows the progression of amyotrophic lateral sclerosis by reducing microgliosis and invasion of macrophages into peripheral nerves. Scientific Reports. 2016;**6**:25663. DOI: 10.1038/srep25663

**Chapter 3**

**Abstract**

Neuron Disease

*Rohit Das and Hema Biju*

progression in a subset of ALS/MND patients.

mononuclear cells (BMMNCs)

**1. Introduction**

**27**

Stem Cell Therapy in Motor

*Alok Sharma, Hemangi Sane, Nandini Gokulchandran,*

Motor neuron disease (MND) is an insidious, fatal disorder that progresses with the selective loss of anterior horn cells of the spinal column. Over 150 years since it was first described, various therapeutic approaches have been tested in the quest of a cure but with little success. Current standard therapy only improves lifespan by a few months; palliative care is the only option available for patients. Stem cell therapy is a potent approach for the treatment of this devastating disease. A multitude of vitalizing effects, both paracrine and somatic, a robust safety profile, as well as ease of availability make a strong case for using these cells for therapeutic purposes. Coupled with rigorous rehabilitation, this powerful treatment modality has been shown to slow disease progression, improve quality of life, and increase survival, along with being well tolerated by amyotrophic lateral sclerosis (ALS)/ MND patients. Compelling preclinical as well as clinical evidence abounds that stem cells hold great potential as a therapy for ALS/MND. Although not a definitive solution yet, stem cells have been verified to have slowed and/or halted disease

**Keywords:** motor neuron disease (MND), amyotrophic lateral sclerosis (ALS), stem cells, stem cell therapy, neurorehabilitation, neuro-regenerative rehabilitation therapy (NRRT), bone marrow-derived stem cells (BMSCs), bone marrow-derived

Motor neuron disease (MND) is a set of heterogeneous, idiopathic neurodegenerative syndromes characterized by progressive degeneration of anterior horn cells of the spinal cord, clinically characterized by weak and wasting musculature, which is eventually fatal [1]. Diagnosis is confirmed via thorough neuro-electrophysiological investigations [2]. Crude incidence of ALS/MND is 1.75 (1.55–1.96)/100,000 person-years of follow-up [3]. The male/female ratio is reported to be between 1 and 3 but varies with population and age [4]. The pathophysiology is multifarious (see Section 3.1), causing poor prognosis to be the major hurdle faced by clinicians worldwide [5]. Multidisciplinary symptomatic management is the sole option that can be availed by patients [6]. Pharmacological treatment includes riluzole (glutamate inhibition) [7], edaravone (effective only in the early stages) [8], and

*Prerna Badhe, Amruta Paranjape, Radhika Pradhan,*

#### **Chapter 3**

## Stem Cell Therapy in Motor Neuron Disease

*Alok Sharma, Hemangi Sane, Nandini Gokulchandran, Prerna Badhe, Amruta Paranjape, Radhika Pradhan, Rohit Das and Hema Biju*

#### **Abstract**

Motor neuron disease (MND) is an insidious, fatal disorder that progresses with the selective loss of anterior horn cells of the spinal column. Over 150 years since it was first described, various therapeutic approaches have been tested in the quest of a cure but with little success. Current standard therapy only improves lifespan by a few months; palliative care is the only option available for patients. Stem cell therapy is a potent approach for the treatment of this devastating disease. A multitude of vitalizing effects, both paracrine and somatic, a robust safety profile, as well as ease of availability make a strong case for using these cells for therapeutic purposes. Coupled with rigorous rehabilitation, this powerful treatment modality has been shown to slow disease progression, improve quality of life, and increase survival, along with being well tolerated by amyotrophic lateral sclerosis (ALS)/ MND patients. Compelling preclinical as well as clinical evidence abounds that stem cells hold great potential as a therapy for ALS/MND. Although not a definitive solution yet, stem cells have been verified to have slowed and/or halted disease progression in a subset of ALS/MND patients.

**Keywords:** motor neuron disease (MND), amyotrophic lateral sclerosis (ALS), stem cells, stem cell therapy, neurorehabilitation, neuro-regenerative rehabilitation therapy (NRRT), bone marrow-derived stem cells (BMSCs), bone marrow-derived mononuclear cells (BMMNCs)

#### **1. Introduction**

Motor neuron disease (MND) is a set of heterogeneous, idiopathic neurodegenerative syndromes characterized by progressive degeneration of anterior horn cells of the spinal cord, clinically characterized by weak and wasting musculature, which is eventually fatal [1]. Diagnosis is confirmed via thorough neuro-electrophysiological investigations [2]. Crude incidence of ALS/MND is 1.75 (1.55–1.96)/100,000 person-years of follow-up [3]. The male/female ratio is reported to be between 1 and 3 but varies with population and age [4]. The pathophysiology is multifarious (see Section 3.1), causing poor prognosis to be the major hurdle faced by clinicians worldwide [5]. Multidisciplinary symptomatic management is the sole option that can be availed by patients [6]. Pharmacological treatment includes riluzole (glutamate inhibition) [7], edaravone (effective only in the early stages) [8], and

Nuedexta (for treating pseudobulbar affect) [9]. Multidisciplinary rehabilitation is a key in managing secondary complications of the disease [10–12].

these comprise the most widely employed therapeutic strategy [19, 22, 31, 34–36]. UCSCs overcome the ethical concerns faced by the ESCs due to the ease of collection postpartum and minimal processing while posing no risk for the mother or the child. These cells, however, lose their advantage due to slow engraftment, limited single-dose availability, and long-term storage issues. Hereditary disorders further limit the benefits of UCSCs [37]. A minimally invasive subcutaneous accessibility and isolation procedure and a robust, long-term proliferation capacity outline the ADSCs'superiority [38, 39]. However, these cells find their limits in the presence of a highly heterogeneous population [40]. Pluripotent stem cells generated from cultured adult skin fibroblast cells by "inducing" dedifferentiation of unipotent, differentiated adult tissue cells by the addition of only a few defined factors are known as induced pluripotent stem cells [41]. iPSCs circumvent ethical concerns over the use of human embryos for the generation of cells of a desired tissue. However, oncogenic factors are used for induction of iPSCs' phenotype and may risk spontaneous induction of cancerous phenotypes and genomic instability [42].

Grossly, ALS/MND patients exhibit spinal cord atrophy and, in some cases, atrophy of cerebral white and gray matter (**Figure 1**). Some patients who have concomitant frontotemporal dementia show presence of cortical atrophy in frontal and temporal cortex. Microscopically, this is characterized by demyelination and

The clinical outcomes observed in ALS/MND are currently postulated to be due to various paracrine and somatic mechanisms that render a neurotrophic effect in

Stem cells confer neuroprotection through various paracrine mechanisms. Depending on the cellular microenvironment, these cells secrete and regulate a plethora of neurotrophic factors that are essential for the nervous system, like nerve growth factor-β (NGF-β, critical for the development and maintenance of the nervous system [52]), ciliary neurotrophic factor (CNTF, promotes neurogenesis [53]),

*The anterior horn of the spinal cord and the precentral gyrus are selectively affected and atrophy in ALS/MND.*

**3. Stem cells and motor neuron degeneration**

**3.1 Neuropathology of ALS/MND**

*Stem Cell Therapy in Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.87116*

axonal loss (**Figure 2**) [43–51].

*3.2.1 Paracrine effects*

**Figure 1.**

**29**

**3.2 Mechanism of action of stem cells**

various neurodegenerative diseases (**Figure 3**).

Studies worldwide endorse the safety and efficacy of stem cells as a therapeutic intervention, for a variety of neurological disorders [13–15], including ALS/MND [16–22]. Stem cells are a potent weapon in the fight against neurodegeneration. These cells hold the unique capacity to self-renew indefinitely while also giving rise to differentiated progeny under defined physiological conditions, thus repopulating damaged tissue [23]. Exercise has also been shown to enhance the mobilization and recruitment of these cells [24]. Given these properties, harnessing the potential of stem cells as a therapy to attenuate disease progression for neurodegenerative disorders, along with customized rehabilitative regimes, has gained traction in recent years.

#### **2. Stem cells**

The defining characteristics [25] of a stem cell are the unique capabilities of the following:

#### **2.1 Clonogenicity**

Stem cells self-renew throughout life, i.e., the cells undergo symmetric division under defined physiological conditions to produce identical daughter cells and thereby maintain the stem cell pool in the organism.

#### **2.2 Multilineage differentiation**

Under certain physiological conditions, stem cells may differentiate and divide asymmetrically to yield an identical daughter cell and a nonidentical, specialized daughter cell that acquires the properties of a cell type specific to a tissue.

#### **2.3 Tissue regeneration**

Stem cells have the capacity to renew the tissues that they populate. The body contains stem cell "niches," i.e., specific regulatory microenvironments conducive to the maintenance, proliferation, and differentiation of stem cells [26].

Depending on the source, stem cells are classified as embryonic stem cells (ESCs), fetal stem cells (FSCs), adult stem cells (ASCs), and induced pluripotent stem cells (iPSCs). ASCs are further classified into bone marrow stem cells (BMSCs), umbilical cord stem cells (UCSCs), and adipose tissue-derived stem cells (ADSCs). ESCs are pluripotent, self-renewing cells derived from the inner mass of the preimplantation blastocyst [27]. Their most obvious benefit is their pluripotency. However, ESCs tend to be highly tumorigenic, require considerable manipulation, and are in the hotbed of ethical debates [28]. FSCs are multipotent cells obtained from fetal tissues of natural, spontaneous abortuses that undergo in utero death within a specific gestational age range [29]. Limited supply, high degree of heterogeneity in the cell viability and cellular composition, and ethical issues hamper their clinical application [30].

Among ASCs, BMSCs take the lead in stem cell therapy in a wide variety of neurological disorders owing to their robust safety profile and efficient integration into host parenchyma [31–33]. Because these are adult cells, these are easily available and are not tumorigenic. The distinctive advantage of the BMSCs over other cell types is the lack of ethical issues for acquisition and administration. Currently, *Stem Cell Therapy in Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.87116*

Nuedexta (for treating pseudobulbar affect) [9]. Multidisciplinary rehabilitation is

Studies worldwide endorse the safety and efficacy of stem cells as a therapeutic intervention, for a variety of neurological disorders [13–15], including ALS/MND [16–22]. Stem cells are a potent weapon in the fight against neurodegeneration. These cells hold the unique capacity to self-renew indefinitely while also giving rise to differentiated progeny under defined physiological conditions, thus repopulating damaged tissue [23]. Exercise has also been shown to enhance the mobilization and recruitment of these cells [24]. Given these properties, harnessing the potential of stem cells as a therapy to attenuate disease progression for neurodegenerative disorders, along with customized rehabilitative regimes, has gained traction in

The defining characteristics [25] of a stem cell are the unique capabilities of the

Stem cells self-renew throughout life, i.e., the cells undergo symmetric division

Under certain physiological conditions, stem cells may differentiate and divide asymmetrically to yield an identical daughter cell and a nonidentical, specialized daughter cell that acquires the properties of a cell type specific to a tissue.

Stem cells have the capacity to renew the tissues that they populate. The body contains stem cell "niches," i.e., specific regulatory microenvironments conducive

Depending on the source, stem cells are classified as embryonic stem cells (ESCs), fetal stem cells (FSCs), adult stem cells (ASCs), and induced pluripotent stem cells (iPSCs). ASCs are further classified into bone marrow stem cells

(BMSCs), umbilical cord stem cells (UCSCs), and adipose tissue-derived stem cells (ADSCs). ESCs are pluripotent, self-renewing cells derived from the inner mass of

pluripotency. However, ESCs tend to be highly tumorigenic, require considerable manipulation, and are in the hotbed of ethical debates [28]. FSCs are multipotent cells obtained from fetal tissues of natural, spontaneous abortuses that undergo in utero death within a specific gestational age range [29]. Limited supply, high degree of heterogeneity in the cell viability and cellular composition, and ethical issues

Among ASCs, BMSCs take the lead in stem cell therapy in a wide variety of neurological disorders owing to their robust safety profile and efficient integration into host parenchyma [31–33]. Because these are adult cells, these are easily available and are not tumorigenic. The distinctive advantage of the BMSCs over other cell types is the lack of ethical issues for acquisition and administration. Currently,

to the maintenance, proliferation, and differentiation of stem cells [26].

the preimplantation blastocyst [27]. Their most obvious benefit is their

under defined physiological conditions to produce identical daughter cells and

thereby maintain the stem cell pool in the organism.

**2.2 Multilineage differentiation**

*Novel Aspects on Motor Neuron Disease*

hamper their clinical application [30].

**28**

**2.3 Tissue regeneration**

a key in managing secondary complications of the disease [10–12].

recent years.

**2. Stem cells**

**2.1 Clonogenicity**

following:

these comprise the most widely employed therapeutic strategy [19, 22, 31, 34–36]. UCSCs overcome the ethical concerns faced by the ESCs due to the ease of collection postpartum and minimal processing while posing no risk for the mother or the child. These cells, however, lose their advantage due to slow engraftment, limited single-dose availability, and long-term storage issues. Hereditary disorders further limit the benefits of UCSCs [37]. A minimally invasive subcutaneous accessibility and isolation procedure and a robust, long-term proliferation capacity outline the ADSCs'superiority [38, 39]. However, these cells find their limits in the presence of a highly heterogeneous population [40]. Pluripotent stem cells generated from cultured adult skin fibroblast cells by "inducing" dedifferentiation of unipotent, differentiated adult tissue cells by the addition of only a few defined factors are known as induced pluripotent stem cells [41]. iPSCs circumvent ethical concerns over the use of human embryos for the generation of cells of a desired tissue. However, oncogenic factors are used for induction of iPSCs' phenotype and may risk spontaneous induction of cancerous phenotypes and genomic instability [42].

#### **3. Stem cells and motor neuron degeneration**

#### **3.1 Neuropathology of ALS/MND**

Grossly, ALS/MND patients exhibit spinal cord atrophy and, in some cases, atrophy of cerebral white and gray matter (**Figure 1**). Some patients who have concomitant frontotemporal dementia show presence of cortical atrophy in frontal and temporal cortex. Microscopically, this is characterized by demyelination and axonal loss (**Figure 2**) [43–51].

#### **3.2 Mechanism of action of stem cells**

The clinical outcomes observed in ALS/MND are currently postulated to be due to various paracrine and somatic mechanisms that render a neurotrophic effect in various neurodegenerative diseases (**Figure 3**).

#### *3.2.1 Paracrine effects*

Stem cells confer neuroprotection through various paracrine mechanisms. Depending on the cellular microenvironment, these cells secrete and regulate a plethora of neurotrophic factors that are essential for the nervous system, like nerve growth factor-β (NGF-β, critical for the development and maintenance of the nervous system [52]), ciliary neurotrophic factor (CNTF, promotes neurogenesis [53]),

#### **Figure 1.**

*The anterior horn of the spinal cord and the precentral gyrus are selectively affected and atrophy in ALS/MND.*

*3.2.3 Immunomodulation*

*Stem Cell Therapy in Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.87116*

neuroprotective effects [57].

*3.2.4 Neurogenesis*

antigens [58].

*3.2.5 Oligodendrogenesis*

demyelinated CNS [59].

*3.2.6 Astrogliogenesis*

fate decision [61].

*3.2.7 Neoangiogenesis*

**31**

abovementioned mechanisms.

These cells exude various beneficial immunomodulatory effects and are capable of homing onto injured sites, as guided by various chemoattractant pathways [55]. Modification of the exaggerated microglial response by immunomodulatory effects is also observed. Various secreted neurotrophic factors like connective tissue growth factor (CTGF), fibroblast growth factor (FGF) 2 and 7, and various interleukins (ILs) are responsible for cell proliferation and cytoprotection. Stem cells regulate innate and adaptive immune cells through release of soluble factors such as tumor growth factor (TGF)-β and elevation of regulatory T cells (Tregs) and Thelper-2 cells (Th2 cells) [56]. Reduced levels of TNF-α, IL-1β, IL-1α, and IL-6 and increased levels of IL-10 lead to an anti-inflammatory effect on the neural microenvironment [56–58], enhancing neuronal repair. Soluble factors from stem cells have been shown to significantly upregulate the expression of glutamate transporters in ALS astrocytes, resulting in enhanced glutamate uptake function. Stem cells also produce vascular endothelial growth factor (VEGF), hepatic growth factor

Mezey et al. have also shown that in a strain of mice incapable of developing cells

Using cell fate tracking techniques, Sasaki and colleagues show that stem cells can differentiate into an oligodendroglial myelinating phenotype in vivo and repair

Eglitis and Mezey have demonstrated the ability of hematopoietic stem cells (HSCs) to differentiate into both astrocytes and microglia in wild-type adult mice using in situ hybridization [60]. Wislet-Gendebien et al. show that nestin-positive (but not nestin-negative) mesenchymal stem cells are able to favor the astroglial lineage in certain stem cell progenitors. They also demonstrate that mesenchymal stem cells express leukemia inhibitory factor (LIF), CNTF, and BMP2 and BMP4 (bone morphogenic protein) mRNAs-cytokines known to play a role in astroglial

Further secretion of growth factors like vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and brain fibroblast growth factor (bFGF) leads to neoangiogenesis and upregulation of hormones like erythropoietin [62]. The cascade of events triggered due to these leads to formation of new vessels as well as improved blood circulation, thus retrieving lost tissue functions. Stem cells

may thus be instrumental in arresting the disease progression through the

of the myeloid and lymphoid lineages, transplanted adult bone marrow cells migrated into the brain and differentiated into cells that expressed neuron-specific

(HGF), and insulin growth factor (IGF)-1, which are reported to have

#### **Figure 2.**

*Pathophysiology of motor neuron disease is multifaceted. Neuronal and nonneuronal cells like glial cell dysfunction have been postulated to contribute to the pathophysiology. Oxidative stress and subsequent rise in intracellular peroxidation, upregulation of astrocytic glutamate, mitochondrial abnormality, immune dysfunction, excitotoxicity, generalized neuroinflammation due secretion of pro-inflammatory cytokines by microglia, axonal transport system dysfunction, and synaptic failure are some of the mechanisms that have been identified. Apart from these mechanisms, abnormal cytoplasmic protein inclusions in patients with ALS have highlighted genetic causality.*

#### **Figure 3.**

*Stem cells play multifarious roles in mitigating ALS/MND pathology.*

brain-derived neurotrophic factor (BDNF, major role player in neuronal development as well as synaptic plasticity [53]), glial cell-derived neurotrophic factor (GDNF, plays an important role in striatal dopaminergic transport [54]), and angiopoietin 1 (ANG-1, promotes angiogenesis [55]).

#### *3.2.2 Somatic effects*

They also migrate to various tissues by homing strategies and have been shown to integrate into cells of target tissue.

#### *3.2.3 Immunomodulation*

These cells exude various beneficial immunomodulatory effects and are capable of homing onto injured sites, as guided by various chemoattractant pathways [55]. Modification of the exaggerated microglial response by immunomodulatory effects is also observed. Various secreted neurotrophic factors like connective tissue growth factor (CTGF), fibroblast growth factor (FGF) 2 and 7, and various interleukins (ILs) are responsible for cell proliferation and cytoprotection. Stem cells regulate innate and adaptive immune cells through release of soluble factors such as tumor growth factor (TGF)-β and elevation of regulatory T cells (Tregs) and Thelper-2 cells (Th2 cells) [56]. Reduced levels of TNF-α, IL-1β, IL-1α, and IL-6 and increased levels of IL-10 lead to an anti-inflammatory effect on the neural microenvironment [56–58], enhancing neuronal repair. Soluble factors from stem cells have been shown to significantly upregulate the expression of glutamate transporters in ALS astrocytes, resulting in enhanced glutamate uptake function. Stem cells also produce vascular endothelial growth factor (VEGF), hepatic growth factor (HGF), and insulin growth factor (IGF)-1, which are reported to have neuroprotective effects [57].

#### *3.2.4 Neurogenesis*

Mezey et al. have also shown that in a strain of mice incapable of developing cells of the myeloid and lymphoid lineages, transplanted adult bone marrow cells migrated into the brain and differentiated into cells that expressed neuron-specific antigens [58].

#### *3.2.5 Oligodendrogenesis*

Using cell fate tracking techniques, Sasaki and colleagues show that stem cells can differentiate into an oligodendroglial myelinating phenotype in vivo and repair demyelinated CNS [59].

#### *3.2.6 Astrogliogenesis*

Eglitis and Mezey have demonstrated the ability of hematopoietic stem cells (HSCs) to differentiate into both astrocytes and microglia in wild-type adult mice using in situ hybridization [60]. Wislet-Gendebien et al. show that nestin-positive (but not nestin-negative) mesenchymal stem cells are able to favor the astroglial lineage in certain stem cell progenitors. They also demonstrate that mesenchymal stem cells express leukemia inhibitory factor (LIF), CNTF, and BMP2 and BMP4 (bone morphogenic protein) mRNAs-cytokines known to play a role in astroglial fate decision [61].

#### *3.2.7 Neoangiogenesis*

Further secretion of growth factors like vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and brain fibroblast growth factor (bFGF) leads to neoangiogenesis and upregulation of hormones like erythropoietin [62]. The cascade of events triggered due to these leads to formation of new vessels as well as improved blood circulation, thus retrieving lost tissue functions. Stem cells may thus be instrumental in arresting the disease progression through the abovementioned mechanisms.

brain-derived neurotrophic factor (BDNF, major role player in neuronal development as well as synaptic plasticity [53]), glial cell-derived neurotrophic factor (GDNF, plays an important role in striatal dopaminergic transport [54]), and

*Pathophysiology of motor neuron disease is multifaceted. Neuronal and nonneuronal cells like glial cell dysfunction have been postulated to contribute to the pathophysiology. Oxidative stress and subsequent rise in intracellular peroxidation, upregulation of astrocytic glutamate, mitochondrial abnormality, immune dysfunction, excitotoxicity, generalized neuroinflammation due secretion of pro-inflammatory cytokines by microglia, axonal transport system dysfunction, and synaptic failure are some of the mechanisms that have been identified. Apart from these mechanisms, abnormal cytoplasmic protein inclusions in patients with ALS have*

They also migrate to various tissues by homing strategies and have been shown

angiopoietin 1 (ANG-1, promotes angiogenesis [55]).

*Stem cells play multifarious roles in mitigating ALS/MND pathology.*

*3.2.2 Somatic effects*

**Figure 3.**

**30**

**Figure 2.**

*highlighted genetic causality.*

*Novel Aspects on Motor Neuron Disease*

to integrate into cells of target tissue.

#### **4. Literature review**

#### **4.1 Preclinical studies**

A wide variety of preclinical studies show that stem cells migrate to and restore lost function of damaged tissue in ALS/MND. Rodent studies have investigated different cell types such as mouse ES cells differentiated to neurons expressing green fluorescent protein (GFP) under the promoter of the motor neuron (MN) specific gene hb9, mesenchymal stem cells (MSCs), human bone marrow mesenchymal stem cells (hMSCs) obtained from an ALS patient (ALS-hMSCs), human neural stem cells (hNSCs), human cord blood stem cells (HuCB-MNCs), human embryonic stem cell-derived motor neuron progenitors (hMNPs), bone marrow cells (BMCs), mesenchymal stromal (stem) cells (MSCs), human umbilical cord blood (MNC-hUCB), human fetal spinal neural stem cells (hNSCs), human iPSCderived neural progenitors (hiPSNPs), HB1.F3.Olig2 cell (stable immortalized hNSCs encoding the OLIG2 gene)-derived motor neurons, human amniotic mesenchymal stem cells (hAMSCs), glial-rich neural progenitors derived from human iPSCs, enriched population of embryonic stem cell-derived astrocytes (hES-AS), and neural progenitor cells secreting GDNF (hNPCGDNF) [63–79].

4. Autologous bone marrow stem cells (intrathecal) [83]

*Stem Cell Therapy in Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.87116*

(intramuscular, intrathecal, or both) [86]

9. Autologous bone marrow stem cells (intraspinal) [34]

10. Autologous mesenchymal stem cells (intraspinal) [21]

11. Fetal olfactory ensheathing cells (intracerebral) [19]

12. Fetal-derived neural stem cells (intraspinal) [22]

15. Mesenchymal stem cells (intrathecal) [35]

*4.2.2 Our published results*

**33**

*4.2.2.1.1 Pre-intervention procedures*

5. Autologous mesenchymal stem cells (intrathecal and intravenous) [31]

6. Olfactory ensheathing cells (intracerebral) and autologous mesenchymal stromal cells (intrathecal and intravenous or only intrathecal) [84]

13. NSI-566RSC (Neuralstem, Inc.), a human neural stem cell (intrathecal) [87]

Overwhelmingly, results point toward a robust safety profile for stem cell treatment in ALS/MND. Stem cell therapy has also proven to be efficacious in mitigating the hostility of a degenerating prognosis in all these studies (see Appendix). These data collectively advocate for the safety and efficacy of various types of stem cells for the treatment of this disease, although small scale; larger clinical trials with sufficient power are required for clearing the turbid field of ALS/MND therapy.

We use intrathecal autologous bone marrow mononuclear cell (BMMNC) transplantation for the treatment of ALS/MND, chosen according to the World Medical Association Declaration of Helsinki—Ethical Principles for Medical Research Involving Human Subjects. The ethical approval for the intervention is obtained from Institutional Ethics Committee (IEC). Our exclusion criteria include the presence of respiratory distress; thus, the effect of stem cell therapy on such patients cannot be assessed. Our inclusion criteria involve patients diagnosed as definite or probable ALS according to revised El Escorial criteria [90]. The procedure is

7. Neural stem cells derived from a fetal spinal cord (intrathecal) [85]

8. Mesenchymal stem cells induced to secrete neurotrophic factors

14. Autologous bone marrow mononuclear cells (intrathecal) [16]

17. Autologous bone marrow stem cells (intramedullary) [89]

18. Autologous mesenchymal stem cells (intrathecal) [36]

*4.2.2.1 Our published protocol for stem cell therapy in ALS/MND*

16. Autologous mesenchymal stem cells (intravenous, intrathecal) [88]

Primarily, stem cells have been shown to have a vast repertoire of paracrine effects, including release of neurotrophic factors such as GDNF, BDNF, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1, NGF, and neurotrophin (NT)-3. Stem cells also confer neuroprotection by migrating, efficiently engrafting into target tissue, reducing astrogliosis, and differentiating into neuroglial cell types. Further, they improve motor performance as measured on rotarod (test measuring rodent balance, grip strength, endurance, and motor coordination), delay disease pathology, and safely extend survival in ALS rodent models (see Appendix) [20, 63–79].

A limitation of preclinical models of ALS, however, is the inherent inability to replicate the *sporadic* onset of ALS/MND, which constitutes majority of patients in clinical scenario [79]. Additionally, an obvious drawback is the underrepresentation of the genomic, anatomical, and physiological complexity of humans by the disease models, which may preclude the translation of results obtained in preclinical settings to the treatment of ALS patients.

#### **4.2 Clinical studies**

#### *4.2.1 Worldwide published data*

A systematic review and meta-analysis of clinical studies by Moura et al. [80] have suggested that stem cell therapy is a promising therapy and highlighted the need for studies with rigorous methodologies to better understand the efficacy of these therapies. **Table 7** (see Appendix) summarizes the studies reviewed by Moura et al. and other studies that were published using stem cells as therapy in the past decade. Nineteen clinical studies are summarized; a variety of cells have been investigated in clinical settings, such as:


**4. Literature review**

*Novel Aspects on Motor Neuron Disease*

**4.1 Preclinical studies**

(see Appendix) [20, 63–79].

**4.2 Clinical studies**

**32**

*4.2.1 Worldwide published data*

tings to the treatment of ALS patients.

investigated in clinical settings, such as:

1. Autologous mesenchymal stem cells (intraspinal) [81]

3. Autologous peripheral blood stem (intracerebral) [82]

A wide variety of preclinical studies show that stem cells migrate to and restore lost function of damaged tissue in ALS/MND. Rodent studies have investigated different cell types such as mouse ES cells differentiated to neurons expressing green fluorescent protein (GFP) under the promoter of the motor neuron (MN) specific gene hb9, mesenchymal stem cells (MSCs), human bone marrow mesenchymal stem cells (hMSCs) obtained from an ALS patient (ALS-hMSCs), human neural stem cells (hNSCs), human cord blood stem cells (HuCB-MNCs), human embryonic stem cell-derived motor neuron progenitors (hMNPs), bone marrow cells (BMCs), mesenchymal stromal (stem) cells (MSCs), human umbilical cord blood (MNC-hUCB), human fetal spinal neural stem cells (hNSCs), human iPSCderived neural progenitors (hiPSNPs), HB1.F3.Olig2 cell (stable immortalized hNSCs encoding the OLIG2 gene)-derived motor neurons, human amniotic mesenchymal stem cells (hAMSCs), glial-rich neural progenitors derived from human iPSCs, enriched population of embryonic stem cell-derived astrocytes (hES-AS),

and neural progenitor cells secreting GDNF (hNPCGDNF) [63–79].

Primarily, stem cells have been shown to have a vast repertoire of paracrine effects, including release of neurotrophic factors such as GDNF, BDNF, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1, NGF, and neurotrophin (NT)-3. Stem cells also confer neuroprotection by migrating, efficiently engrafting into target tissue, reducing astrogliosis, and differentiating into neuroglial cell types. Further, they improve motor performance as measured on rotarod (test measuring rodent balance, grip strength, endurance, and motor coordination), delay disease pathology, and safely extend survival in ALS rodent models

A limitation of preclinical models of ALS, however, is the inherent inability to replicate the *sporadic* onset of ALS/MND, which constitutes majority of patients in clinical scenario [79]. Additionally, an obvious drawback is the underrepresentation of the genomic, anatomical, and physiological complexity of humans by the disease models, which may preclude the translation of results obtained in preclinical set-

A systematic review and meta-analysis of clinical studies by Moura et al. [80] have suggested that stem cell therapy is a promising therapy and highlighted the need for studies with rigorous methodologies to better understand the efficacy of these therapies. **Table 7** (see Appendix) summarizes the studies reviewed by Moura et al. and other studies that were published using stem cells as therapy in the past decade. Nineteen clinical studies are summarized; a variety of cells have been

2. Bone marrow-derived hematopoietic progenitor stem cells (intraspinal) [18]


Overwhelmingly, results point toward a robust safety profile for stem cell treatment in ALS/MND. Stem cell therapy has also proven to be efficacious in mitigating the hostility of a degenerating prognosis in all these studies (see Appendix). These data collectively advocate for the safety and efficacy of various types of stem cells for the treatment of this disease, although small scale; larger clinical trials with sufficient power are required for clearing the turbid field of ALS/MND therapy.

#### *4.2.2 Our published results*

#### *4.2.2.1 Our published protocol for stem cell therapy in ALS/MND*

#### *4.2.2.1.1 Pre-intervention procedures*

We use intrathecal autologous bone marrow mononuclear cell (BMMNC) transplantation for the treatment of ALS/MND, chosen according to the World Medical Association Declaration of Helsinki—Ethical Principles for Medical Research Involving Human Subjects. The ethical approval for the intervention is obtained from Institutional Ethics Committee (IEC). Our exclusion criteria include the presence of respiratory distress; thus, the effect of stem cell therapy on such patients cannot be assessed. Our inclusion criteria involve patients diagnosed as definite or probable ALS according to revised El Escorial criteria [90]. The procedure is

explained to the patients in detail, and a written informed consent is obtained. Patients are thoroughly examined by an experienced team of doctors and therapists. Pre-surgical routine blood tests, urinalysis, and chest X-ray are carried out for assessing anesthetic and surgical fitness. About 300 μg of granulocyte colonystimulating factor (G-CSF) injections are administrated subcutaneously 48 and 24 hours prior to BMMNC transplantation, as they enhance the mobility of BMMNCs, stimulates CD34+ cells, and increases their survival as well as multiplication rate [91]. The transplant is then carried out in three steps (**Figure 4**).

*4.2.2.2 Case series*

*Stem Cell Therapy in Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.87116*

*4.2.2.3 Case reports*

**Table 2**) [98].

(**Table 3** and **Figure 7**).

neurorehabilitation [99].

**35**

We have published a retrospective controlled cohort study with a total of 57 ALS patients that investigates the effects of stem cell therapy, in addition to standard rehabilitation, lithium, and riluzole [79]. Out of these, 37 patients underwent autologous BMMNC transplantation, while the remaining 20 did not; these served as controls. We saw that there was a clinically significant difference of 30.38 months between the average survival duration of intervention and control groups. Intervention group survived for 87.76 (10.45) months, while controls survived for 57.38 (5.31) months. Patients with the onset of the disease below 50 years of age survived significantly longer (*p* = 0.039), while limb symptom onset and co-administration of lithium improved survival duration in a clinically significant manner [16]. Lithium increases the survival, potency, and target tissue integration of BMSCs [92]. It has been shown to confer neuroprotection in vitro by enhancing cellular BDNF [93]. In vivo, lithium has been shown to activate autophagy, normalize mitochondrial aberration, and suppress reactive astrogliosis. It also reduces ubiquitinated protein aggregates and increases the number of spared motor neurons in transgenic ALS mice [94]. Further, lithium is well tolerated by ALS patients who are on riluzole, even though it may not be effective by itself for treating ALS. This was confirmed by two trials: a phase III multicenter, randomized, double-blind,

placebo-controlled trial (LiCALS) by Al-Chalabi et al. [95] and a phase IIb randomized, double-blind, placebo-controlled, sequential trial by Verstraete et al. [96]. Taken together, these results suggest that a combination strategy of stem cells and lithium may have played a pronounced role in the outcomes of this study, summarizes the prognostic factors that influence survival in the intervention group. Youn-

ger age at symptom onset and spinal symptom onset favors longer survival durations according to our findings. Post intervention, lithium prescription combined with standard riluzole treatment and comprehensive rehabilitation enhances

A 40-year-old female suffering from ALS for 3 years was given intrathecal autologous BMMNC transplantation along with riluzole, lithium, and intensive rehabilitation. The disease progression slowed over 17 months along with improvements in neurological symptoms. de Carvalho et al. have previously reported that the ALSFRS-R score deteriorates about 17% every 6 months [97]; here, the ALSFRS-R score dropped only by 8% over 17 months after cell transplantation (**Figure 6** and

A 41-year-old female suffering from ALS for 3 years was given intrathecal autologous BMMNC therapy combined with riluzole, neuro-rehabilitation, and 6 weeks of lithium. Her ALSFRS-R score increased from 29 to 32, and FIM score increased from 48 to 64. The highlight of this case is halting of disease progression with symptomatic improvements over a period of 12 months after intervention

A 63-year-old man who underwent autologous intrathecal BMMNC transplan-

neurorehabilitation showed improvements in muscle strength, fine motor activities, fasciculation, cramps, and walking (**Table 4**). ALSFRS-R score improved from 33 to 37; Berg's balance score improved from 43 to 50, and 6-minute walk test improved from 283.8 to 303.6 m. His FIM score remained unchanged at 113. These improvements may be attributed to cellular therapy along with standard treatment and

tation as a therapy in a clinical case of MND followed by multidisciplinary

the effect of cellular therapy (**Figure 5** and **Table 1**).

#### *4.2.2.1.2 Bone marrow aspiration*

Performed in the operation theater under aseptic conditions, 100–120 ml of bone marrow is aspirated under local anesthesia from the region of anterior superior iliac spine and collected in the heparinized tubes.

### *4.2.2.1.3 Cell separation*

Using density gradient centrifugation, stem cells are separated. The cell pellet is analyzed under a microscope using trypan blue to check for the viability of the cells. Cell number is counted using Tali cell counter. FACS analysis using CD34 PE antibody is used for identification of CD34+ cells.

#### *4.2.2.1.4 Cell transplantation*

In the operation theater under aseptic conditions, the cells are transplanted intrathecally into the cerebrospinal fluid through lumbar puncture between the level of fourth and fifth lumbar vertebra, using an 18G Touhy needle.

#### *4.2.2.1.5 Posttransplantation*

Cell transplantation is followed by standard multidisciplinary rehabilitation including physiotherapy, occupational therapy, speech therapy, psychological intervention, aquatic therapy, and dietary advice. This approach is termed as neuroregenerative rehabilitative therapy (NRRT). Standard medical treatment was continued with Rilutor. Tablet lithium was prescribed for 6 weeks for its neuroprotective properties. Lithium levels were monitored.

**Figure 4.** *Stem cell therapy protocol at NeuroGen Brain and Spine Institute.*

#### *4.2.2.2 Case series*

explained to the patients in detail, and a written informed consent is obtained. Patients are thoroughly examined by an experienced team of doctors and therapists. Pre-surgical routine blood tests, urinalysis, and chest X-ray are carried out for assessing anesthetic and surgical fitness. About 300 μg of granulocyte colonystimulating factor (G-CSF) injections are administrated subcutaneously 48 and 24 hours prior to BMMNC transplantation, as they enhance the mobility of BMMNCs, stimulates CD34+ cells, and increases their survival as well as multiplication rate [91]. The transplant is then carried out in three steps (**Figure 4**).

Performed in the operation theater under aseptic conditions, 100–120 ml of bone marrow is aspirated under local anesthesia from the region of anterior superior iliac

Using density gradient centrifugation, stem cells are separated. The cell pellet is analyzed under a microscope using trypan blue to check for the viability of the cells. Cell number is counted using Tali cell counter. FACS analysis using CD34 PE

In the operation theater under aseptic conditions, the cells are transplanted intrathecally into the cerebrospinal fluid through lumbar puncture between the level of

Cell transplantation is followed by standard multidisciplinary rehabilitation including physiotherapy, occupational therapy, speech therapy, psychological intervention, aquatic therapy, and dietary advice. This approach is termed as neuroregenerative rehabilitative therapy (NRRT). Standard medical treatment was con-

*4.2.2.1.2 Bone marrow aspiration*

*Novel Aspects on Motor Neuron Disease*

*4.2.2.1.3 Cell separation*

*4.2.2.1.4 Cell transplantation*

*4.2.2.1.5 Posttransplantation*

**Figure 4.**

**34**

spine and collected in the heparinized tubes.

antibody is used for identification of CD34+ cells.

fourth and fifth lumbar vertebra, using an 18G Touhy needle.

tinued with Rilutor. Tablet lithium was prescribed for 6 weeks for its

neuroprotective properties. Lithium levels were monitored.

*Stem cell therapy protocol at NeuroGen Brain and Spine Institute.*

We have published a retrospective controlled cohort study with a total of 57 ALS patients that investigates the effects of stem cell therapy, in addition to standard rehabilitation, lithium, and riluzole [79]. Out of these, 37 patients underwent autologous BMMNC transplantation, while the remaining 20 did not; these served as controls. We saw that there was a clinically significant difference of 30.38 months between the average survival duration of intervention and control groups. Intervention group survived for 87.76 (10.45) months, while controls survived for 57.38 (5.31) months. Patients with the onset of the disease below 50 years of age survived significantly longer (*p* = 0.039), while limb symptom onset and co-administration of lithium improved survival duration in a clinically significant manner [16]. Lithium increases the survival, potency, and target tissue integration of BMSCs [92]. It has been shown to confer neuroprotection in vitro by enhancing cellular BDNF [93]. In vivo, lithium has been shown to activate autophagy, normalize mitochondrial aberration, and suppress reactive astrogliosis. It also reduces ubiquitinated protein aggregates and increases the number of spared motor neurons in transgenic ALS mice [94]. Further, lithium is well tolerated by ALS patients who are on riluzole, even though it may not be effective by itself for treating ALS. This was confirmed by two trials: a phase III multicenter, randomized, double-blind, placebo-controlled trial (LiCALS) by Al-Chalabi et al. [95] and a phase IIb randomized, double-blind, placebo-controlled, sequential trial by Verstraete et al. [96]. Taken together, these results suggest that a combination strategy of stem cells and lithium may have played a pronounced role in the outcomes of this study, summarizes the prognostic factors that influence survival in the intervention group. Younger age at symptom onset and spinal symptom onset favors longer survival durations according to our findings. Post intervention, lithium prescription combined with standard riluzole treatment and comprehensive rehabilitation enhances the effect of cellular therapy (**Figure 5** and **Table 1**).

#### *4.2.2.3 Case reports*

A 40-year-old female suffering from ALS for 3 years was given intrathecal autologous BMMNC transplantation along with riluzole, lithium, and intensive rehabilitation. The disease progression slowed over 17 months along with improvements in neurological symptoms. de Carvalho et al. have previously reported that the ALSFRS-R score deteriorates about 17% every 6 months [97]; here, the ALSFRS-R score dropped only by 8% over 17 months after cell transplantation (**Figure 6** and **Table 2**) [98].

A 41-year-old female suffering from ALS for 3 years was given intrathecal autologous BMMNC therapy combined with riluzole, neuro-rehabilitation, and 6 weeks of lithium. Her ALSFRS-R score increased from 29 to 32, and FIM score increased from 48 to 64. The highlight of this case is halting of disease progression with symptomatic improvements over a period of 12 months after intervention (**Table 3** and **Figure 7**).

A 63-year-old man who underwent autologous intrathecal BMMNC transplantation as a therapy in a clinical case of MND followed by multidisciplinary neurorehabilitation showed improvements in muscle strength, fine motor activities, fasciculation, cramps, and walking (**Table 4**). ALSFRS-R score improved from 33 to 37; Berg's balance score improved from 43 to 50, and 6-minute walk test improved from 283.8 to 303.6 m. His FIM score remained unchanged at 113. These improvements may be attributed to cellular therapy along with standard treatment and neurorehabilitation [99].

**Figure 6.**

**Table 2.**

**Table 3.**

**Figure 7.**

**37**

*over a period of 12 months.*

**Outcome measures**

*0, 5, 15, and 17 months [97].*

*up at 5, 15, and 17 months.*

**Outcome measures**

**At assessment before the first transplant**

*Stem Cell Therapy in Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.87116*

> **At assessment before the first transplant**

*Maintenance of a 40-year-old female ALS patient's condition over the period of 17 months. Disease progression in patient (red) was compared using ALSFRS-R with average values obtained from de Carvalho et al. (blue) at*

> **At 5 months after the first transplant (just before the second transplant)**

ALSFRS-R 36 36 36 33 FIM 113 113 113 113

ALSFRS-R 29 33 34 33 32 FIM 48 72 73 71 64

*ALSFRS-R and FIM scores of a 41-year-old female ALS patient measured at assessment and after intervention (at 2, 6, 9, and 12 months post autologous BMMNC transplantation) depict stark improvement.*

*ALSFRS-R and FIM scores were marked at 2, 4, 6, 9, and 12 months for this patient. ALSFRS-R score was measured across survival duration (in months) to compare the disease progression of this patient with natural disease progression in ALS, as measured by de Carvalho et al. [97]. The patient's condition was maintained*

**At 2 months after the first transplant**

*ALSFRS-R and FIM instrument scores of the patient remain stable as compared to assessment, when followed*

**At 6 months after the first transplant**

**At 15 months after the first transplant**

**At 9 months after the first transplant**

**At 17 months after the first transplant**

**At 12 months after the first transplant**

#### **Figure 5.**

*(a) Kaplan-Meier survival analysis comparing the mean survival duration of the intervention (n = 37) and control group (n = 20) from Sharma et al. [16]. Mean survival duration of patients in the intervention group was higher than the control group by a clinically significant difference of 30.38 months. (b) Subgroup analysis of the effect of age of symptom onset on survival duration within the intervention group, from Sharma et al. [16] shows significantly higher survival of those with an onset of symptoms above 50 years of age (p = 0.039). (c) Subgroup analysis of the effect of the type of symptom onset (limb vs. bulbar) on survival duration in the intervention group shows higher survival of patients with limb onset of symptoms by 12.23 months. (d) Subgroup analysis of the effect of lithium prescription on survival duration within the intervention group shows a clinically higher survival of 106.73 months of the group prescribed with lithium. This is a clinically significant difference of 30.90 months as compared to controls, whose average survival was 66.83 months.*


#### **Table 1.**

*Summary of prognostic factors affecting patient survival in the intervention group, from Sharma et al. [16]. Standard deviation is indicated in parentheses.*

#### **Figure 6.**

*Maintenance of a 40-year-old female ALS patient's condition over the period of 17 months. Disease progression in patient (red) was compared using ALSFRS-R with average values obtained from de Carvalho et al. (blue) at 0, 5, 15, and 17 months [97].*


#### **Table 2.**

*ALSFRS-R and FIM instrument scores of the patient remain stable as compared to assessment, when followed up at 5, 15, and 17 months.*


#### **Table 3.**

**Figure 5.**

*Novel Aspects on Motor Neuron Disease*

*\**

**36**

**Table 1.**

*Statistically significant (p < 0.05).*

*Standard deviation is indicated in parentheses.*

*(a) Kaplan-Meier survival analysis comparing the mean survival duration of the intervention (n = 37) and control group (n = 20) from Sharma et al. [16]. Mean survival duration of patients in the intervention group was higher than the control group by a clinically significant difference of 30.38 months. (b) Subgroup analysis of the effect of age of symptom onset on survival duration within the intervention group, from Sharma et al. [16] shows significantly higher survival of those with an onset of symptoms above 50 years of age (p = 0.039). (c) Subgroup analysis of the effect of the type of symptom onset (limb vs. bulbar) on survival duration in the intervention group shows higher survival of patients with limb onset of symptoms by 12.23 months. (d) Subgroup analysis of the effect of lithium prescription on survival duration within the intervention group shows a clinically higher survival of 106.73 months of the group prescribed with lithium. This is a clinically significant difference of 30.90 months as compared to controls, whose average survival was 66.83 months.*

**Prognostic factor Median survival since symptom onset** *p***-Value** Lithium Given 106.73 (15.69) 0.121 Not given 66.83 (7.52) Age at symptom onset Below 50 years 113.34 (15.45) 0.039\* Above 50 years 63.02 (7.7) Type of symptom onset Limb 78.01 (14.23) 0.902

Bulbar 90.24 (13.27)

*Summary of prognostic factors affecting patient survival in the intervention group, from Sharma et al. [16].*

*ALSFRS-R and FIM scores of a 41-year-old female ALS patient measured at assessment and after intervention (at 2, 6, 9, and 12 months post autologous BMMNC transplantation) depict stark improvement.*

#### **Figure 7.**

*ALSFRS-R and FIM scores were marked at 2, 4, 6, 9, and 12 months for this patient. ALSFRS-R score was measured across survival duration (in months) to compare the disease progression of this patient with natural disease progression in ALS, as measured by de Carvalho et al. [97]. The patient's condition was maintained over a period of 12 months.*


#### **Table 4.**

*Improvements in outcome measures over a period of 6 months in a 63-year-old male ALS patient. ALSFRS-R, FIM, and 6-minute walk test were measured. Although not standard, Berg's balance test was administered to assess his balance.*

A 29-year-old female patient with anterior horn cell involvement suffering from MND for 5 years presented with complaints of sudden onset of weakness in bilateral lower limbs post pregnancy. Her progressive condition was followed by gradual involvement of upper limbs also; EMG studies were also suggestive of MND. Her features were suggestive of pure motor system involvement affecting lower motor neurons. Post NRRT, she immediately showed clinical and functional improvements [17].

*4.2.2.4.2 Correlation of testosterone levels with progression of amyotrophic lateral*

We conducted an open-label nonrandomized cross-sectional study to interrogate the relationship of testosterone (TT) with disease progression. We found that 39 of the total 50 (78%) ALS patients had plasma TT levels lower than the mean levels in healthy, age-matched control males. We also found a decline in TT levels as the disease progressed on King's staging as well as ALSFRS-R (**Figure 8**). There was a statistically significant moderate monotonic correlation between ALSFRS-R scores and King's staging of patients with plasma testosterone levels (ALSFRS-R: *r* = +0.33;

*Kaplan-Meier survival curves for control vs. intervention groups in our open-label study with a total of 157 subjects (intervention = 116; control = 41). On an average, the treated group survived for 32 months longer*

*sclerosis: a cross-sectional study*

*Stem Cell Therapy in Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.87116*

*Relationship of testosterone with (a) King's staging and (b) ALSFRS-R.*

**Figure 8.**

**Figure 9.**

**39**

*than the control group (p = 0.026).*

#### *4.2.2.4 Unpublished data*

#### *4.2.2.4.1 Female hormones enhance the neuroprotective benefits of cellular transplantation in patients with amyotrophic lateral sclerosis (ALS)*

The male/female ratio in ALS/MND worldwide points toward a lower incidence in females as compared to males. We hypothesized that this was due to a neuroprotective effect conferred by female hormones. In order to investigate this hypothesis, we designed a cohort study with 40 sequentially recruited ALS patients (28 males and 12 females) who were treated with stem cell therapy. To study the effect of reproductive hormones, patients were divided into pre- and postmenopausal women and men below and above 50 years of age.

We saw that percentage survival was highest in the premenopausal women (100%) followed by men below the age of 50 years (75%), postmenopausal women (60%), and men above the age of 50 years (45%). The disease progression was also slowest in the premenopausal women, followed by postmenopausal women, and men below 50 years of age; it was fastest in men above the age of 50 years (**Table 5**).


#### **Table 5.**

*Percentage mortalities across the four subgroups in a cohort study.*

*Stem Cell Therapy in Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.87116*

**Figure 8.** *Relationship of testosterone with (a) King's staging and (b) ALSFRS-R.*

#### *4.2.2.4.2 Correlation of testosterone levels with progression of amyotrophic lateral sclerosis: a cross-sectional study*

We conducted an open-label nonrandomized cross-sectional study to interrogate the relationship of testosterone (TT) with disease progression. We found that 39 of the total 50 (78%) ALS patients had plasma TT levels lower than the mean levels in healthy, age-matched control males. We also found a decline in TT levels as the disease progressed on King's staging as well as ALSFRS-R (**Figure 8**). There was a statistically significant moderate monotonic correlation between ALSFRS-R scores and King's staging of patients with plasma testosterone levels (ALSFRS-R: *r* = +0.33;

#### **Figure 9.**

*Kaplan-Meier survival curves for control vs. intervention groups in our open-label study with a total of 157 subjects (intervention = 116; control = 41). On an average, the treated group survived for 32 months longer than the control group (p = 0.026).*

A 29-year-old female patient with anterior horn cell involvement suffering from MND for 5 years presented with complaints of sudden onset of weakness in bilateral lower limbs post pregnancy. Her progressive condition was followed by gradual involvement of upper limbs also; EMG studies were also suggestive of MND. Her features were suggestive of pure motor system involvement affecting lower motor

*Improvements in outcome measures over a period of 6 months in a 63-year-old male ALS patient. ALSFRS-R, FIM, and 6-minute walk test were measured. Although not standard, Berg's balance test was administered to*

**Outcome measures Pre-first SCT At 6 months post the first SCT**

ALSFRS-R 33 37 FIM 113 113 6-minute walk test 283.8 m 303.6 m Berg's balance score 43 50

neurons. Post NRRT, she immediately showed clinical and functional

*4.2.2.4.1 Female hormones enhance the neuroprotective benefits of cellular*

females as compared to males. We hypothesized that this was due to a

pausal women and men below and above 50 years of age.

**ALSFRS-R**

*Percentage mortalities across the four subgroups in a cohort study.*

*transplantation in patients with amyotrophic lateral sclerosis (ALS)*

The male/female ratio in ALS/MND worldwide points toward a lower incidence in

neuroprotective effect conferred by female hormones. In order to investigate this hypothesis, we designed a cohort study with 40 sequentially recruited ALS patients (28 males and 12 females) who were treated with stem cell therapy. To study the effect of reproductive hormones, patients were divided into pre- and postmeno-

We saw that percentage survival was highest in the premenopausal women (100%) followed by men below the age of 50 years (75%), postmenopausal women (60%), and men above the age of 50 years (45%). The disease progression was also slowest in the premenopausal women, followed by postmenopausal women, and men below 50 years of age; it was fastest in men above the age of 50 years

> **Mean post-ALSFRS-R**

**Difference Average follow-**

26 23 3 20 0

21 14 7 14 40

25 20 5 10 25

32 25 7 13 55

**up (months)**

**Percentage mortality**

improvements [17].

**Table 4.**

*assess his balance.*

*Novel Aspects on Motor Neuron Disease*

(**Table 5**).

Premenopausal females (7)

Postmenopausal females (5)

Males below 50 years of age (16)

Males above 50 years of age (12)

**Table 5.**

**38**

**Group Mean pre-**

*4.2.2.4 Unpublished data*

King's staging: *r* = 0.35; *p* = 0.01). Taken together, these results suggest that reduced testosterone may exacerbate motor neuron loss or cause other etiopathological dysfunctions that remain to be elucidated.

#### *4.2.2.4.3 Effect of intrathecal application of autologous bone marrow mononuclear cells on survival duration in ALS*

**5. Conclusion and future directions**

*Stem Cell Therapy in Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.87116*

Stem cell therapy is a novel, promising modality for the treatment of ALS/MND.

Although not a cure yet, a combinatorial approach integrating stem cell therapy,

intensive neurorehabilitation, and current pharmacotherapeutic agents (e.g., riluzole, lithium, etc.) may be the best way forward. Studies that interrogate the genetics of patients and their family are the need of the hour, to enhance response to treatment and develop diagnostics and biomarkers. Also, the role of reproductive hormones such as progesterone, estradiol, and testosterone needs to be further explored. Larger clinical studies with stringent criteria are required to understand the efficacy of these combined methods in the treatment of ALS/MND. The current scenario suggests that autologous stem cell therapy can be considered along with

The authors declare no conflict of interest in the writing of this chapter.

**Type of cells used (route of**

Autologous mesenchymal stem cells (intraspinal)

Bone marrow-derived hematopoietic progenitor stem

Autologous peripheral blood stem (intracerebral)

Autologous bone marrow stem

cells (intraspinal)

cells (intrathecal)

**Results**

Slowing down of the linear decline of muscular strength was evident in four patients and improvement in strength in two patients in proximal lower limb muscles was observed

9/13 (69.23%) patients improved as compared with their preoperative status, as confirmed by EMG

The survival of treated patients was statistically higher (*p* = 0.01) than untreated control patients

There was no significant deterioration in ALSFRS-R composite score from baseline at a 1-year follow-up (*p* = 0.090). The median survival post procedure was 18.0 months and median time to 4 point deterioration was 16.7 months

**administration)**

Robust safety profiles, low risk-to-benefit ratio, and ease of access make this approach a strong contender in the race against ALS/MND. Consistently, autologous BMMNC therapy has been proven to mitigate disease progression; however, this response may be dependent on various factors, like age of symptom onset, gender, hormones, type of onset (limb vs. bulbar), and genetic makeup of the patient, to name a few. Younger patients—especially premenopausal females—may respond better to autologous BMMNC therapy. Stem cells combat tissue degeneration via a host of somatic and paracrine mechanisms, including neurogenesis, astrogliogenesis, neoangiogenesis, and immunomodulation. Multidisciplinary

neurorehabilitation enhances the response to cellular therapy.

standard treatment in carefully selected patients of ALS/MND.

**Conflict of interest**

**Appendix**

See **Table 7**.

**Authors, year, country (type of study, sample size)**

Mazzini et al. [81], Italy (clinical trial, 7)

Deda et al. [18], Turkey (clinical trial, 13)

Martinez et al. [82], Mexico (controlled clinical trial, 10)

Prabhakar et al. [83], India (clinical trial—pilot study, 10)

**41**

We performed a large, open-label cohort study, which included 157 patients diagnosed with ALS from September 2013 to May 2017. About 116 patients who received cell transplantation (autologous BMMNCs) together with standard treatment formed the treatment group, and 41 patients who received only the standard treatment formed the control group (**Figure 9** and **Table 6**). We observed that on an average, the treated group survived for 32 months longer than the control group (*p* = 0.026).

Collectively, these findings were indicative of possible neuroprotective benefits of female reproductive hormones. Prognostic factors that predict improved survival and retarded disease progression include lithium co-administration, limb onset of symptoms, younger age of symptom onset, and, most importantly, presence of female hormones (**Figure 10**).


**Table 6.**

*Demographic data for an open-label study with 157 participants.*

#### **Figure 10.**

*(a) Subgroup analysis: survival duration was 46 months more in patients with limb onset as compared to bulbar onset (p = 0.001). Bulbar onset n = 26 patients and limb onset n = 90. (b) Survival duration was 41 months more in patients with age of symptom onset <50 years as compared to symptom onset at/above 50 years (p < 0.000).*

### **5. Conclusion and future directions**

King's staging: *r* = 0.35; *p* = 0.01). Taken together, these results suggest that reduced testosterone may exacerbate motor neuron loss or cause other

*4.2.2.4.3 Effect of intrathecal application of autologous bone marrow mononuclear cells*

We performed a large, open-label cohort study, which included 157 patients diagnosed with ALS from September 2013 to May 2017. About 116 patients who received cell transplantation (autologous BMMNCs) together with standard treatment formed the treatment group, and 41 patients who received only the standard treatment formed the control group (**Figure 9** and **Table 6**). We observed that on an average, the treated group survived for 32 months longer than the control group

Collectively, these findings were indicative of possible neuroprotective benefits of female reproductive hormones. Prognostic factors that predict improved survival and retarded disease progression include lithium co-administration, limb onset of symptoms, younger age of symptom onset, and, most importantly, presence of

**Characteristics Intervention Control** Total number of patients 157 116 41

Females 35 11

Limb 90 21

Gender Males 81 30

Mean age at symptom onset (years) 51 11 53 9 Type of symptom onset Bulbar 26 6

*(a) Subgroup analysis: survival duration was 46 months more in patients with limb onset as compared to bulbar onset (p = 0.001). Bulbar onset n = 26 patients and limb onset n = 90. (b) Survival duration was 41 months more in patients with age of symptom onset <50 years as compared to symptom onset at/above*

*Demographic data for an open-label study with 157 participants.*

etiopathological dysfunctions that remain to be elucidated.

*on survival duration in ALS*

*Novel Aspects on Motor Neuron Disease*

(*p* = 0.026).

**Table 6.**

**Figure 10.**

**40**

*50 years (p < 0.000).*

female hormones (**Figure 10**).

Stem cell therapy is a novel, promising modality for the treatment of ALS/MND. Robust safety profiles, low risk-to-benefit ratio, and ease of access make this approach a strong contender in the race against ALS/MND. Consistently, autologous BMMNC therapy has been proven to mitigate disease progression; however, this response may be dependent on various factors, like age of symptom onset, gender, hormones, type of onset (limb vs. bulbar), and genetic makeup of the patient, to name a few. Younger patients—especially premenopausal females—may respond better to autologous BMMNC therapy. Stem cells combat tissue degeneration via a host of somatic and paracrine mechanisms, including neurogenesis, astrogliogenesis, neoangiogenesis, and immunomodulation. Multidisciplinary neurorehabilitation enhances the response to cellular therapy.

Although not a cure yet, a combinatorial approach integrating stem cell therapy, intensive neurorehabilitation, and current pharmacotherapeutic agents (e.g., riluzole, lithium, etc.) may be the best way forward. Studies that interrogate the genetics of patients and their family are the need of the hour, to enhance response to treatment and develop diagnostics and biomarkers. Also, the role of reproductive hormones such as progesterone, estradiol, and testosterone needs to be further explored. Larger clinical studies with stringent criteria are required to understand the efficacy of these combined methods in the treatment of ALS/MND. The current scenario suggests that autologous stem cell therapy can be considered along with standard treatment in carefully selected patients of ALS/MND.

#### **Conflict of interest**

The authors declare no conflict of interest in the writing of this chapter.

#### **Appendix**

#### See **Table 7**.



**Author details**

*2009 to February 2019.*

Navi Mumbai, India

Navi Mumbai, India

**43**

Institute, Navi Mumbai, India

**Authors, year, country (type of study, sample size)**

*Stem Cell Therapy in Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.87116*

Oh et al. [35], South Korea (single open-label phase I clinical trial, 8)

Rushkevich et al. [88], Belarus (controlled clinical trial, 10)

Ruiz-López et al. [89], Spain (phase I clinical trial, 11)

Syková et al. [36], Czech Republic (phase I/phase IIa prospective, nonrandomized, open-label clinical trial, 26)

*unrelated to stem cell therapy.*

Institute, Navi Mumbai, India

provided the original work is properly cited.

, Hemangi Sane<sup>2</sup>

1 NeuroGen Brain and Spine Institute, Navi Mumbai, India

\*Address all correspondence to: publications@neurogen.in

Amruta Paranjape2,5\*, Radhika Pradhan2

, Nandini Gokulchandran<sup>3</sup>

**Type of cells used (route of**

Mesenchymal stem cells

Autologous mesenchymal stem cells (intravenous,

Autologous mesenchymal stem cells (intrathecal)

Autologous bone marrow stem cells (intramedullary)

*All studies demonstrate safety of stem cells, except Glass et al. [22]. Safety in this table does not describe events*

*Significant clinical studies employing stem cell therapy for treatment of ALS/MND, primarily from March*

(intrathecal)

intrathecal)

**Results**

period

the onset of the disease below 50 years of age. Limb onset and lithium also showed positive influence on the survival duration

Decline in the ALSFRS-R score was slow during the 6-month follow-up

Evaluation of the 12-month followup revealed slowing down of the disease progression in 10 patients

All 11 patients were 100% stable

A significant reduction/stabilization was found in ALSFRS-R decline at 3, 6, 9, and 12 months after treatment

**administration)**

2 Department of Research and Development, NeuroGen Brain and Spine Institute,

3 Department of Medical Services and Clinical Research, NeuroGen Brain and Spine

4 Department of Regenerative Laboratory Services, NeuroGen Brain and Spine

© 2019 The Author(s). Licensee IntechOpen. 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,

5 Department of Neurorehabilitation, NeuroGen Brain and Spine Institute,

, Prerna Badhe<sup>4</sup>

, Rohit Das5 and Hema Biju<sup>5</sup>

,

Alok Sharma<sup>1</sup>

**Table 7.**


*All studies demonstrate safety of stem cells, except Glass et al. [22]. Safety in this table does not describe events unrelated to stem cell therapy.*

#### **Table 7.**

**Authors, year, country (type of study, sample size)**

*Novel Aspects on Motor Neuron Disease*

Karussis et al. [31], Israel (phase I/phase II clinical

Gamez et al. [84], Spain (observational study, 12)

Riley et al. [85], USA (phase I

Petrou et al. [86], Israel (openlabel proof-of-concept study, phase I/phase II and IIa)

Blanquer et al. [34], Spain (clinical trial, pilot safety

Mazzini et al. [21], Italy (clinical trial, long-term safety

Huang et al. [19], China (controlled pilot study, 35)

Glass et al. [22], USA (phase I

Riley et al. [87], USA (phase I

Sharma et al. [16], India (retrospective controlled cohort study, 57)

clinical trial, 12)

trial, 15)

**42**

study, 11)

study, 9)

safety trial, 12)

trial, 19)

**Type of cells used (route of**

Autologous mesenchymal stem cells (intrathecal and

Olfactory ensheathing cells (intracerebral) and autologous mesenchymal stromal cells (intrathecal + intravenous or

Neural stem cells derived from a fetal spinal cord (intrathecal)

Autologous bone marrow stem

Autologous mesenchymal stem cells (intraspinal)

Fetal olfactory ensheathing cells (intracerebral)

Fetal neural stem cells (intraspinal)

NSI-566RSC-a human neural stem cell (intrathecal)

Autologous bone marrow mononuclear cells (intrathecal)

cells (intraspinal)

Mesenchymal stem cells induced to secrete neurotrophic factors (intramuscular (IM), intrathecal (IT) or (IT+IM))

only intrathecal)

**Results**

intervention

observed

The mean ALSFRS-R score remained stable during the first 6 months of observation. In 80% of the patients, FVC values remained stable or above 70% for a time of 9 months and remained in 60% of patients at 12 months after application. Signs of disease stabilization in some patients during the first 6 months after the

No changes in the decline of FVC and ALSFRS-R compared with the disease's natural history were

Procedural safety of unilateral and bilateral intraspinal lumbar microinjections has been suggested by the results of this trial

Progression rate of the ALSFRS-R score in the IT (or IT+IM)-treated patients was reduced from 1.2 to 0.6 ALSFRS-R points/month (*p* = 0.052), and the progression rate of the forced vital capacity reduced from 5.1% to 1.2%/month during the 6 months follow-up vs. pretreatment period

7/11 (63.63%) patients remained

Brain MRI revealed no structural changes relative to baseline throughout follow-up. No deterioration noted in the psychosocial status as well

7/14 improved; 2/14 remained stable compared to the entry in the treated group, while only 1/17 of the patients remained stable within

Patients remained stable and tolerated the therapy well, as seen in clinical assessments at 6–18 months

tolerated by the patients

Mean survival duration of intervention was 87.76 months, which was higher than the control (57.38 months) or previous epidemiological studies. Survival duration was significantly (*p* = 0.039) higher in people with

Cellular delivery to the cervical or thoracolumbar spinal cord was well

stable post procedure

control

**administration)**

intravenous)

*Significant clinical studies employing stem cell therapy for treatment of ALS/MND, primarily from March 2009 to February 2019.*

### **Author details**

Alok Sharma<sup>1</sup> , Hemangi Sane<sup>2</sup> , Nandini Gokulchandran<sup>3</sup> , Prerna Badhe<sup>4</sup> , Amruta Paranjape2,5\*, Radhika Pradhan2 , Rohit Das5 and Hema Biju<sup>5</sup>

1 NeuroGen Brain and Spine Institute, Navi Mumbai, India

2 Department of Research and Development, NeuroGen Brain and Spine Institute, Navi Mumbai, India

3 Department of Medical Services and Clinical Research, NeuroGen Brain and Spine Institute, Navi Mumbai, India

4 Department of Regenerative Laboratory Services, NeuroGen Brain and Spine Institute, Navi Mumbai, India

5 Department of Neurorehabilitation, NeuroGen Brain and Spine Institute, Navi Mumbai, India

\*Address all correspondence to: publications@neurogen.in

© 2019 The Author(s). Licensee IntechOpen. 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.

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[10] Hardiman O. Multidisciplinary care in ALS: Expanding the team. Amyotrophic Lateral Sclerosis. 2012; **13**(2):165-165

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2017;**46**(1):57-74

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*Novel Aspects on Motor Neuron Disease*

and pathological progression of motor neuron disease in the wobbler mouse. PLoS ONE. 2015;**10**(10):e0140316

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Amyotrophic Lateral Sclerosis. 2012;

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[13] Sharma A, Gokulchandran N, Sane H, Nagrajan A, Paranjape A, Kulkarni P,

mononuclear cell therapy for autism: An open label proof of concept study. Stem Cells International. 2013;**2013**:623875

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et al. Autologous bone marrow

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(1\_suppl):127-138

in ALS: Expanding the team.

**13**(2):165-165

**7**(4):580-585

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[3] Marin B, Boumédiene F, Logroscino

[4] Manjaly ZR, Scott KM, Abhinav K,

[5] Mitchell JD, Callagher P, Gardham J, Mitchell C, Dixon M, Addison-Jones R, et al. Timelines in the diagnostic evaluation of people with suspected amyotrophic lateral sclerosis (ALS)/ motor neuron disease (MND)—A 20 year review: Can we do better?

Amyotrophic Lateral Sclerosis: Official Publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. 2010;**11**(6):537-541

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Oct 2014;**27**(5):524-531

**44**

Wijesekera L, Ganesalingam J, Goldstein LH, et al. The sex ratio in amyotrophic lateral sclerosis: A population based study. Amyotrophic Lateral Sclerosis: Official Publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. 2010;**11**(5):439-442

G, Couratier P, Babron M-C, Leutenegger AL, et al. Variation in worldwide incidence of amyotrophic lateral sclerosis: A meta-analysis. International Journal of Epidemiology. [17] Sharma A, Badhe P, Shetty O, Vijaygopal P, Gokulchandran N, Jacob VC, et al. Autologous bone marrow derived stem cells for motor neuron disease with anterior horn cell involvement. The Bombay Hospital Journal. 2011;**53**(1):71-75

[18] Deda H, Inci MC, Kürekçi AE, Sav A, Kayihan K, Ozgün E, et al. Treatment of amyotrophic lateral sclerosis patients by autologous bone marrow-derived hematopoietic stem cell transplantation: A 1-year follow-up. Cytotherapy. 2009; **11**(1):18-25

[19] Huang H, Chen L, Xi H, Wang H, Zhang J, Zhang F, et al. Fetal olfactory ensheathing cells transplantation in amyotrophic lateral sclerosis patients: A controlled pilot study. Clinical Transplantation. 2008;**22**(6):710-718

[20] Mazzini L, Gelati M, Profico DC, Sgaravizzi G, Projetti Pensi M, Muzi G, et al. Human neural stem cell transplantation in ALS: Initial results from a phase I trial. Journal of Translational Medicine. 2015;**13**:17

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[34] Blanquer M, Moraleda JM, Iniesta F, Gómez-Espuch J, Meca-Lallana J, Villaverde R, et al. Neurotrophic bone marrow cellular nests prevent spinal

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motoneuron degeneration in

Ohio). 2012;**30**(6):1277-1285

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**4**(6):590-597

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2010;**67**(10):1187-1194

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[52] Phruksaniyom C, Dharmasaroja P, Issaragrisil S. Bone marrow nonmesenchymal mononuclear cells induce functional differentiation of neuroblastoma cells. Experimental Hematology & Oncology. 2013;**2**:9

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[54] Pastor D, Viso-León MC, Jones J, Jaramillo-Merchán J, Toledo-Aral JJ, Moraleda JM, et al. Comparative effects between bone marrow and mesenchymal stem cell transplantation in GDNF expression and motor function recovery in a motor neuron

degenerative mouse model. Stem Cell Reviews. 2012;**8**(2):445-458

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[56] Kim SH, Oh K-W, Jin HK, Bae J-S. Immune inflammatory modulation as a potential therapeutic strategy of stem cell therapy for ALS and neurodegenerative diseases. BMB Reports. 2018;**51**(11):545-546

[57] Gu R, Hou X, Pang R, Li L, Chen F, Geng J, et al. Human adipose-derived stem cells enhance the glutamate uptake function of GLT1 in SOD1(G93A) bearing astrocytes. Biochemical and Biophysical Research Communications. 2010;**393**(3):481-486

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**468**(3):190-194

222-226

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after mesenchymal stem cell

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releasing active BMP4. BMC Neuroscience. 2004;**5**:33

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[80] Moura MC, Novaes MRCG, Zago YSSP, Eduardo EJ, Casulari LA. Efficacy of stem cell therapy in amyotrophic lateral sclerosis: A systematic review and meta-analysis. Journal of Clinical Medical Research. 2016;**8**(4):317-324

[81] Mazzini L, Fagioli F, Boccaletti R, Mareschi K, Oliveri G, Olivieri C, et al. Stem cell therapy in amyotrophic lateral sclerosis: A methodological approach in humans. Amyotrophic Lateral Sclerosis: Official Publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. 2003; **4**(3):158-161

[82] Martinez HR, Gonzalez-Garza MT, Moreno-Cuevas JE, Caro E, Gutierrez-Jimenez E, Segura JJ. Stem-cell transplantation into the frontal motor cortex in amyotrophic lateral sclerosis patients. Cytotherapy. 2009;**11**(1):26-34

[83] Prabhakar S, Marwaha N, Lal V, Sharma RR, Rajan R, Khandelwal N. Autologous bone marrow-derived stem cells in amyotrophic lateral sclerosis: A pilot study. Neurology India. 2012; **60**(5):465-469

[84] Gamez J, Carmona F, Raguer N, Ferrer-Sancho J, Martín-Henao GA, Martí-Beltrán S, et al. Cellular transplants in amyotrophic lateral sclerosis patients: An observational study. Cytotherapy. 2010;**12**(5):669-677

[85] Riley J, Federici T, Polak M, Kelly C, Glass J, Raore B, et al. Intraspinal stem cell transplantation in amyotrophic lateral sclerosis: A phase I safety trial, technical note, and lumbar safety outcomes. Neurosurgery. 2012;**71**(2): 405-416; discussion 416

[86] Petrou P, Gothelf Y, Argov Z, Gotkine M, Levy YS, Kassis I, et al. Safety and clinical effects of mesenchymal stem cells secreting neurotrophic factor transplantation in patients with amyotrophic lateral sclerosis: Results of phase 1/2 and 2a clinical trials. JAMA Neurology. 2016; **73**(3):337-344

[87] Riley J, Glass J, Feldman EL, Polak M, Bordeau J, Federici T, et al. Intraspinal stem cell transplantation in amyotrophic lateral sclerosis: A phase I trial, cervical microinjection, and final surgical safety outcomes. Neurosurgery. 2014;**74**(1):77-87

[88] Rushkevich YN, Kosmacheva SM, Zabrodets GV, Ignatenko SI, Goncharova NV, Severin IN, et al. The use of autologous mesenchymal stem cells for cell therapy of patients with amyotrophic lateral sclerosis in Belarus. Bulletin of Experimental Biology and Medicine. 2015;**159**(4):576-581

[89] Ruiz-López FJ, Blanquer M. Autologous bone marrow mononuclear cells as neuroprotective treatment of amyotrophic lateral sclerosis. Neural Regeneration Research. 2016;**11**(4): 568-569

[90] Brooks BR, Miller RG, Swash M, Munsat TL, World Federation of Neurology Research Group on Motor Neuron Diseases. El Escorial revisited: Revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotrophic Lateral Sclerosis: Official Publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. 2000;**1**(5):293-299

[91] Yoon SH, Shim YS, Park YH, Chung JK, Nam JH, Kim MO, et al. Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: Phase I/II clinical trial. Stem Cells. 2007; **25**(8):2066-2073

[92] Richman CM, Kinnealey A, Hoffman PC. Granulopoietic effects of lithium on human bone marrow in vitro. Experimental Hematology. 1981;**9**(4): 449-455

[93] Hashimoto R, Takei N, Shimazu K, Christ L, Lu B, Chuang D-M. Lithium induces brain-derived neurotrophic factor and activates TrkB in rodent cortical neurons: An essential step for

neuroprotection against glutamate excitotoxicity. Neuropharmacology. 2002;**43**(7):1173-1179

[94] Fornai F, Longone P, Cafaro L, Kastsiuchenka O, Ferrucci M, Manca ML, et al. Lithium delays progression of amyotrophic lateral sclerosis. Proceedings of the National Academy of Sciences of the United States of America. 2008;**105**(6):2052-2057

[95] Morrison KE, Dhariwal S, Hornabrook R, Savage L, Burn DJ, Khoo TK, et al. Lithium in patients with amyotrophic lateral sclerosis (LiCALS): A phase 3 multicentre, randomised, double-blind, placebo-controlled trial. Lancet Neurology. 2013;**12**(4):339-345. DOI: 10.1016/S1474-4422(13)70037-1

[96] Verstraete E, Veldink JH, Huisman MHB, Draak T, Uijtendaal EV, van der Kooi AJ, et al. Lithium lacks effect on survival in amyotrophic lateral sclerosis: A phase IIb randomised sequential trial. Journal of Neurology, Neurosurgery, and Psychiatry. 2012;**83**(5):557-564

[97] de Carvalho M, Scotto M, Lopes A, Swash M. Quantitating progression in ALS. Neurology. 2005;**64**(10):1783-1785

[98] Sane H, Sharma A, Gokulchandran N, Kalburgi S, Paranjape A, Badhe P. Neurorestoration in amyotrophic lateral sclerosis—A case report. Journal of Stem cells and Regenerative Medicine. 2016; **2**(1):29-37

[99] Sharma A, Sane H, Paranjape A, Sawant D, Inamdar S, Gokulchandran N, et al. Cellular therapy in motor neuron disease: A case report. International Journal of Recent Advances in Multidisciplinary Research. 2017;**4**(5):2605-2609

**51**

**Chapter 4**

**Abstract**

Neuron Disease

Introduction to Novel Motor

improve motor neuron function by acting on SK channels.

atrophy, riluzole, edaravone

**1. Introduction**

**Keywords:** motor neuron disease, amyotrophic lateral sclerosis, spinal muscular

Motor neuron disease is composed of a group of rare neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), hereditary spastic paraplegia, primary lateral sclerosis, progressive muscular atrophy, pseudobulbar palsy, O'Sullivan-McLeod syndrome, and Madras motor neuron disease, which are fatal in 50% of affected people within 15–20 months after diagnosis. MND is a progressive neuromuscular disease with a fatal outcome; the commonest clinical presentation of MND presentation is ALS, commonly known as Lou Gehrig's disease. Most of ALS patients pass away within 2–5 years of confirmed a diagnosis. Familial ALS (ALSf) is a hereditary presentation of the disease and accounts for 5–10% of affected people. ALS affects persons of all ethnicities worldwide; no cure for ALS has yet been available at any country. Sometimes, ALS is clinically, pathologically, and genetically associated with fronto-temporal dementia, which is the second cause of dementia in elderly people. In our first book, we reviewed all previous chapters published by INTECH, and in the Introductory Chapter, readers could find summarized information about all publications on ALS

*Humberto Foyaca Sibat and Lourdes de Fátima Ibañez Valdés*

Motor neuron disease (MND) is a progressive and fatal neuromuscular disease; the most common and severe form of MND presentation is amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease. The majority of ALS patients die within 2–5 years of receiving a diagnosis. Familial ALS is a hereditary form of the disease and accounts for 5–10% of cases, whereas the remaining cases have no clearly defined etiology. ALS affects persons of all ethnicities and races; currently, no curative treatment for ALS is available worldwide. ALS is also the major adult-onset MND and is clinically, pathologically, and genetically associated with fronto-temporal dementia in some cases, which is the second cause of dementia in elderly people. However, MND does not affect sphincter, sexual function, or eye movements. MND is the most common degenerative disorder affecting the upper and lower motor neurons at the same time. Most of the patients presenting MND in our series complained of muscle weakness, muscle wasting, fasciculation, and spasticity plus lower cranial nerve disturbances. According to our bibliographic studies, apart from nusinersen, it seems to be that riluzole and edaravone also

#### **Chapter 4**

amyotrophic lateral sclerosis: A phase I trial, cervical microinjection, and final surgical safety outcomes. Neurosurgery.

*Novel Aspects on Motor Neuron Disease*

neuroprotection against glutamate excitotoxicity. Neuropharmacology.

[94] Fornai F, Longone P, Cafaro L, Kastsiuchenka O, Ferrucci M, Manca ML, et al. Lithium delays progression of

Proceedings of the National Academy of

Hornabrook R, Savage L, Burn DJ, Khoo TK, et al. Lithium in patients with amyotrophic lateral sclerosis (LiCALS): A phase 3 multicentre, randomised, double-blind, placebo-controlled trial. Lancet Neurology. 2013;**12**(4):339-345. DOI: 10.1016/S1474-4422(13)70037-1

[96] Verstraete E, Veldink JH, Huisman MHB, Draak T, Uijtendaal EV, van der Kooi AJ, et al. Lithium lacks effect on survival in amyotrophic lateral sclerosis: A phase IIb randomised sequential trial. Journal of Neurology, Neurosurgery, and Psychiatry. 2012;**83**(5):557-564

[97] de Carvalho M, Scotto M, Lopes A, Swash M. Quantitating progression in ALS. Neurology. 2005;**64**(10):1783-1785

[98] Sane H, Sharma A, Gokulchandran N, Kalburgi S, Paranjape A, Badhe P. Neurorestoration in amyotrophic lateral sclerosis—A case report. Journal of Stem cells and Regenerative Medicine. 2016;

[99] Sharma A, Sane H, Paranjape A, Sawant D, Inamdar S, Gokulchandran N, et al. Cellular therapy in motor neuron disease: A case report. International Journal of Recent

Advances in Multidisciplinary Research.

**2**(1):29-37

2017;**4**(5):2605-2609

amyotrophic lateral sclerosis.

Sciences of the United States of America. 2008;**105**(6):2052-2057

[95] Morrison KE, Dhariwal S,

2002;**43**(7):1173-1179

[88] Rushkevich YN, Kosmacheva SM,

Goncharova NV, Severin IN, et al. The use of autologous mesenchymal stem cells for cell therapy of patients with amyotrophic lateral sclerosis in Belarus. Bulletin of Experimental Biology and Medicine. 2015;**159**(4):576-581

Zabrodets GV, Ignatenko SI,

[89] Ruiz-López FJ, Blanquer M. Autologous bone marrow mononuclear cells as neuroprotective treatment of amyotrophic lateral sclerosis. Neural Regeneration Research. 2016;**11**(4):

[90] Brooks BR, Miller RG, Swash M, Munsat TL, World Federation of Neurology Research Group on Motor Neuron Diseases. El Escorial revisited: Revised criteria for the diagnosis of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis: Official Publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. 2000;**1**(5):293-299

[91] Yoon SH, Shim YS, Park YH, Chung JK, Nam JH, Kim MO, et al. Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte

macrophage-colony stimulating factor: Phase I/II clinical trial. Stem Cells. 2007;

[93] Hashimoto R, Takei N, Shimazu K, Christ L, Lu B, Chuang D-M. Lithium induces brain-derived neurotrophic factor and activates TrkB in rodent cortical neurons: An essential step for

[92] Richman CM, Kinnealey A, Hoffman PC. Granulopoietic effects of lithium on human bone marrow in vitro. Experimental Hematology. 1981;**9**(4):

**25**(8):2066-2073

449-455

**50**

2014;**74**(1):77-87

568-569

## Introduction to Novel Motor Neuron Disease

*Humberto Foyaca Sibat and Lourdes de Fátima Ibañez Valdés*

#### **Abstract**

Motor neuron disease (MND) is a progressive and fatal neuromuscular disease; the most common and severe form of MND presentation is amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease. The majority of ALS patients die within 2–5 years of receiving a diagnosis. Familial ALS is a hereditary form of the disease and accounts for 5–10% of cases, whereas the remaining cases have no clearly defined etiology. ALS affects persons of all ethnicities and races; currently, no curative treatment for ALS is available worldwide. ALS is also the major adult-onset MND and is clinically, pathologically, and genetically associated with fronto-temporal dementia in some cases, which is the second cause of dementia in elderly people. However, MND does not affect sphincter, sexual function, or eye movements. MND is the most common degenerative disorder affecting the upper and lower motor neurons at the same time. Most of the patients presenting MND in our series complained of muscle weakness, muscle wasting, fasciculation, and spasticity plus lower cranial nerve disturbances. According to our bibliographic studies, apart from nusinersen, it seems to be that riluzole and edaravone also improve motor neuron function by acting on SK channels.

**Keywords:** motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, riluzole, edaravone

#### **1. Introduction**

Motor neuron disease is composed of a group of rare neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), hereditary spastic paraplegia, primary lateral sclerosis, progressive muscular atrophy, pseudobulbar palsy, O'Sullivan-McLeod syndrome, and Madras motor neuron disease, which are fatal in 50% of affected people within 15–20 months after diagnosis. MND is a progressive neuromuscular disease with a fatal outcome; the commonest clinical presentation of MND presentation is ALS, commonly known as Lou Gehrig's disease. Most of ALS patients pass away within 2–5 years of confirmed a diagnosis. Familial ALS (ALSf) is a hereditary presentation of the disease and accounts for 5–10% of affected people. ALS affects persons of all ethnicities worldwide; no cure for ALS has yet been available at any country. Sometimes, ALS is clinically, pathologically, and genetically associated with fronto-temporal dementia, which is the second cause of dementia in elderly people. In our first book, we reviewed all previous chapters published by INTECH, and in the Introductory Chapter, readers could find summarized information about all publications on ALS

made by INTECH. Trying to illustrate the reached progress, we displayed this information grouped by topics and countries in two graphics. As we saw on that book, the number of publications written about ALS increased remarkably for the past 4 years. To have some idea of this phenomenon, be informed that INTECH published more than 40 chapters on ALS in this period of time, and these books "Amyotrophic Lateral Sclerosis," "Current Advances on Amyotrophic Lateral Sclerosis," and others are fully available on line, for free. Therefore, why we are going to publish another chapter? All novel information about MND were not published. Therefore, some aspects published in 2012 need to be update because new ideas, proposals, findings, experiences, and many other's knowledge have been arising despite of this short period of time. Therefore, for the benefit of the readership community, we included update information not reported before, mainly new contribution of aberrant astrocytes to MND damage and death in the SOD1G93A rat experimental model of ALS; novel genetics studies on ALS; an update of the structural and functional consequences of the spinal muscular atrophy-linked mutations of the survival motor neuron protein; stem cell therapy for MND; and the novel treatment for SMA and ALS in the introductory chapter of this book. Compromises have been inevitable to accommodate our visual and factual updated information in a book of his characteristic on top of many chapters about the same issue published recently.

MND does not affect sphincter, sexual function, or eye movements [1]. Although ALS is not associated with thermoregulatory dysfunction, its progression can affect intensively important cerebral regions that control body temperature and affect multiple functions of this homeostatic activity. Nevertheless, experimental ALS animals can display altered thermoregulation as a consequence of affected energy homeostasis. Indirect evidence suggests, performing studies on the body temperature regulatory system, both as a possible modifier of disease progression in ALS and as a potential biomarker [2].

Although edaravone and riluzole do not cure MND/ALS, it seems to be that both medications can slow its progression. The prevalence of ALS in America was 5.2 per 100,000 populations with a total of 16,583 cases identified from January 1 to December 31, 2015 [3].

MND is the most common degenerative disorder, which affects the upper and lower motor neurons at the same time. There are different clinical modalities of MND being ALS the commonest one, and its incidence is around 1–3 patients every 100,000 people [4, 5].

The higher incidence of ALS is in patients with 60 and 70 years of age, but some younger cases (20–30 years of age) have been reported as well [4]. Between 5 and 10% of the patients have a familiar origin due to Mendelian autosomal dominant transmission.

Most of the patients presenting MND in our series complain of muscle weakness, muscle wasting, fasciculation, and spasticity plus cranial nerve disturbances from the lower brainstem.

The most frequent mutation seen in the familial form of ALS (ALSf) occurs on the gene of superoxide dismutase 1 (SOD1) and on the chromosome 9, among others. The decreased endovascular factor and the hereditary hemochromatosis protein are also genetic mutations. Some variations in the number of copies of Genes 1 and 2 that codify the motor neuron survival factors have been reported [6]. No correlation investigations have been done. However, some genome-wide studies in patients presenting ALS show a series of loci confirming a greater susceptibility to develop the disease such as kinase carbohydrate (FGGY), dipeptidyl-peptidase 6 (DPP6), and Type 2 inositol triphosphate receptor [7–9]. Most of these findings were not able to be replicated in further investigations done. At present, there is not specific cure for this deadly disorder as was mentioned before.

**53**

changes [16].

*Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

(in vivo) and human beings [14].

Long time ago, a nitrogenic expansion on the gene C9ORF72 was observed in a number of patients presenting ALS associated with Chromosome 9, which brought more clarity in the ethiopathogenesis of ALS [10, 11], but these findings are also seen in patients presenting fronto-temporal dementia (FTD) and ALS-FTD

Future genetic investigations should be focused on non-European populations in

In the forthcoming years, the exome study that is an emerging field will bring

In 2018, Thompson et al. [12] used a high-throughput proteomic process to distinguish new biomarkers in patient's cerebrospinal fluid (CSF), and they found that three macrophage-derived chitinases had increased concentration in ALS: chitinase-3-like protein 1, chitotriosidase, and chitinase-3-like protein 2. Elevated CHI3L1 was commonly seen in ALS, while CHI3L2 and CHIT1 levels did not. Their results confirmed the important role of macrophage activity in pathogenesis of ALS. Decreased cough capacity is almost always present in respiratory tract infection and is the most important cause of respiratory failure in ALS patients. Other authors determined whether the lung function measurement could identify the cough function in ALS patients with respiratory tract infection. After screening 48 patients presenting ALS, they found only four presenting a remarkable cough with no assistance. The data that identified unassisted cough effectiveness are peak cough flow. These investigators highlighted that the effectiveness of assisted and

unassisted cough function depends on the peak cough flow reached [13].

It is well known that MND does not affect the motor neurons at the oculomotor nucleus in the midbrain. Because it could be remarkably advantageous if neurons of motor system resilience can be modeled in vitro, some authors reached elevated quantities of oculomotor neurons from embryonic stem cells in mouse through transient over expression of PHOX2A in nerve cell progenitors, and they confirmed, using immunocytochemistry techniques, electrophysiology studies, and RNA sequencing, that in vitro-generated neurons are bona fide oculomotor neuron cells based on their neuron properties and similarity to their counterpart in rodent

Increased cortical excitability, thought to reflect pathological changes in the balance of local excitatory and inhibitory neuronal influences that are commonly seen in patients presenting ALS and non-invasive brain stimulation (NIBS), has been shown to modulate cortical activity, with some protocols showing effects that outlast the stimulation by months. Therefore, NIBS has been proposed as a probable candidate to approach therapeutically these disorders associated with pathological

ALS type 8 (ALS8) is a familial presentation of MND, with an important anterior horn cell degeneration, due to mutation of the vesicle-associated membrane protein-associated protein B. Some authors compare the cognitive function of patients with ALS8 and a control group composed by healthy people in order to screen behavioral features in ALS8 patients. These authors found that ALS8 patients showed minimal deficits in executive functions. The total amount of ALS8 patients and the control group have the same scores of facial emotion recognition. They also determined an important clinical expression of psychiatric disorder such as anxiety and depression in 36 and 27% of patients, respectively. However, behavioral disturbances were present in around 30% of participants. They concluded that these patients had mild executive problems and behavioral problems such as apathy, mood disorder, and stereotypic behavior, which suggest that ALS8 is not a motor disorder only, and it is associated with minor cognitive and behavioral

neurophysiology activity, such as ALS, among others [15].

[10, 11]. Below, we will deliver more comments about this topic.

order to bring more clarity on new pathogenic loci.

novel information about some implicated genes in ALSf.

#### *Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

*Novel Aspects on Motor Neuron Disease*

ALS and as a potential biomarker [2].

December 31, 2015 [3].

100,000 people [4, 5].

from the lower brainstem.

cure for this deadly disorder as was mentioned before.

transmission.

made by INTECH. Trying to illustrate the reached progress, we displayed this information grouped by topics and countries in two graphics. As we saw on that book, the number of publications written about ALS increased remarkably for the past 4 years. To have some idea of this phenomenon, be informed that INTECH published more than 40 chapters on ALS in this period of time, and these books "Amyotrophic Lateral Sclerosis," "Current Advances on Amyotrophic Lateral Sclerosis," and others are fully available on line, for free. Therefore, why we are going to publish another chapter? All novel information about MND were not published. Therefore, some aspects published in 2012 need to be update because new ideas, proposals, findings, experiences, and many other's knowledge have been arising despite of this short period of time. Therefore, for the benefit of the readership community, we included update information not reported before, mainly new contribution of aberrant astrocytes to MND damage and death in the SOD1G93A rat experimental model of ALS; novel genetics studies on ALS; an update of the structural and functional consequences of the spinal muscular atrophy-linked mutations of the survival motor neuron protein; stem cell therapy for MND; and the novel treatment for SMA and ALS in the introductory chapter of this book. Compromises have been inevitable to accommodate our visual and factual updated information in a book of his charac-

teristic on top of many chapters about the same issue published recently. MND does not affect sphincter, sexual function, or eye movements [1]. Although ALS is not associated with thermoregulatory dysfunction, its progression can affect intensively important cerebral regions that control body temperature and affect multiple functions of this homeostatic activity. Nevertheless, experimental ALS animals can display altered thermoregulation as a consequence of affected energy homeostasis. Indirect evidence suggests, performing studies on the body temperature regulatory system, both as a possible modifier of disease progression in

Although edaravone and riluzole do not cure MND/ALS, it seems to be that both medications can slow its progression. The prevalence of ALS in America was 5.2 per 100,000 populations with a total of 16,583 cases identified from January 1 to

MND is the most common degenerative disorder, which affects the upper and lower motor neurons at the same time. There are different clinical modalities of MND being ALS the commonest one, and its incidence is around 1–3 patients every

The higher incidence of ALS is in patients with 60 and 70 years of age, but some younger cases (20–30 years of age) have been reported as well [4]. Between 5 and 10% of the patients have a familiar origin due to Mendelian autosomal dominant

Most of the patients presenting MND in our series complain of muscle weakness, muscle wasting, fasciculation, and spasticity plus cranial nerve disturbances

The most frequent mutation seen in the familial form of ALS (ALSf) occurs on the gene of superoxide dismutase 1 (SOD1) and on the chromosome 9, among others. The decreased endovascular factor and the hereditary hemochromatosis protein are also genetic mutations. Some variations in the number of copies of Genes 1 and 2 that codify the motor neuron survival factors have been reported [6]. No correlation investigations have been done. However, some genome-wide studies in patients presenting ALS show a series of loci confirming a greater susceptibility to develop the disease such as kinase carbohydrate (FGGY), dipeptidyl-peptidase 6 (DPP6), and Type 2 inositol triphosphate receptor [7–9]. Most of these findings were not able to be replicated in further investigations done. At present, there is not specific

**52**

Long time ago, a nitrogenic expansion on the gene C9ORF72 was observed in a number of patients presenting ALS associated with Chromosome 9, which brought more clarity in the ethiopathogenesis of ALS [10, 11], but these findings are also seen in patients presenting fronto-temporal dementia (FTD) and ALS-FTD [10, 11]. Below, we will deliver more comments about this topic.

Future genetic investigations should be focused on non-European populations in order to bring more clarity on new pathogenic loci.

In the forthcoming years, the exome study that is an emerging field will bring novel information about some implicated genes in ALSf.

In 2018, Thompson et al. [12] used a high-throughput proteomic process to distinguish new biomarkers in patient's cerebrospinal fluid (CSF), and they found that three macrophage-derived chitinases had increased concentration in ALS: chitinase-3-like protein 1, chitotriosidase, and chitinase-3-like protein 2. Elevated CHI3L1 was commonly seen in ALS, while CHI3L2 and CHIT1 levels did not. Their results confirmed the important role of macrophage activity in pathogenesis of ALS.

Decreased cough capacity is almost always present in respiratory tract infection and is the most important cause of respiratory failure in ALS patients. Other authors determined whether the lung function measurement could identify the cough function in ALS patients with respiratory tract infection. After screening 48 patients presenting ALS, they found only four presenting a remarkable cough with no assistance. The data that identified unassisted cough effectiveness are peak cough flow. These investigators highlighted that the effectiveness of assisted and unassisted cough function depends on the peak cough flow reached [13].

It is well known that MND does not affect the motor neurons at the oculomotor nucleus in the midbrain. Because it could be remarkably advantageous if neurons of motor system resilience can be modeled in vitro, some authors reached elevated quantities of oculomotor neurons from embryonic stem cells in mouse through transient over expression of PHOX2A in nerve cell progenitors, and they confirmed, using immunocytochemistry techniques, electrophysiology studies, and RNA sequencing, that in vitro-generated neurons are bona fide oculomotor neuron cells based on their neuron properties and similarity to their counterpart in rodent (in vivo) and human beings [14].

Increased cortical excitability, thought to reflect pathological changes in the balance of local excitatory and inhibitory neuronal influences that are commonly seen in patients presenting ALS and non-invasive brain stimulation (NIBS), has been shown to modulate cortical activity, with some protocols showing effects that outlast the stimulation by months. Therefore, NIBS has been proposed as a probable candidate to approach therapeutically these disorders associated with pathological neurophysiology activity, such as ALS, among others [15].

ALS type 8 (ALS8) is a familial presentation of MND, with an important anterior horn cell degeneration, due to mutation of the vesicle-associated membrane protein-associated protein B. Some authors compare the cognitive function of patients with ALS8 and a control group composed by healthy people in order to screen behavioral features in ALS8 patients. These authors found that ALS8 patients showed minimal deficits in executive functions. The total amount of ALS8 patients and the control group have the same scores of facial emotion recognition. They also determined an important clinical expression of psychiatric disorder such as anxiety and depression in 36 and 27% of patients, respectively. However, behavioral disturbances were present in around 30% of participants. They concluded that these patients had mild executive problems and behavioral problems such as apathy, mood disorder, and stereotypic behavior, which suggest that ALS8 is not a motor disorder only, and it is associated with minor cognitive and behavioral changes [16].

Because one of the most effective clinical strategies for SMA is to protect the anterior horn cell, apart from nusinersen (that is a very expensive medication), one anti-epileptic medication levetiracetam has been used as well.

Kepra (levetiracetam) provoked neurite elongation in SMA-iPSCs-MNs. TUNEL-positive anterior horn cell was significantly decreased by kepra in SMAiPSCs-MNs. On the other hand, the expression level of cleaved-caspase 3 was diminished by levetiracetam in SMA-iPSCs-MNs. Furthermore, kepra improved impaired mitochondrial function in SMA-iPSCs-MNs. On the other hand, kepra did not modify the expression level of SMN protein in SMA-iPSCs-MNs. These results suggest that kepra has a neuroprotective effect for SMA [17].

For patients presenting SMA (most common reason of inherited infant mortality), the gene therapy seems to be the most effective strategy [18].

Another therapeutic modality to treat ALS is the noninvasive brain stimulation (NIBS), which has been shown to modulate cortical activity, with some protocols leading effects that outlast the stimulation by months. NIBS have been suggested as a potential treatment choice in those processes with associated changes in the cortical neurophysiology [15].

A total of 25 genes associated with ALSf and ALS (sporadic form), mutations in fused-in-sarcoma (FUS) and superoxide dismutase 1 (SOD1) have been intensively studied in the past, focusing on modified excitability of motor neurons. Based on their personal experience, Peikkert et al. [19] proposed that the 4-aminopyridine (4-AP), which is a potassium channel blocker, can be utilized as a probable therapy *for ALS patients* due to its demonstrated hypo excitability and high frequency of apoptosis in a FUS/SOD1-ALS-induced multi-potent stem cell from selected motor neuron; they also found that this process is partly reversible by 4-AP.

One of the clinical presentations of MND is SMA, which encompasses a group of autosomal recessively inherited degenerative neuromuscular diseases. SMA is an inherited disorder that causes progressive lesions on the anterior horn cell leading to weakness or paralysis of the affected limbs, and it is caused by elimination or mutation of survival motor neuron (SMN) 1 gene. It is well known that homozygous damage and loss of functional mutations in the survival motor neuron 1 gene (SMN1) at the chromosome 5q13 are the main cause of SMA, which affect 1 in 11,000 newborn infants.

SMA usually has a very poor prognosis after rapidly progressive weakness and early mortality. However, a new medication named Nusinersen has been released for the treatment of all forms of SMA (not on mechanical ventilation) with very good results. In December 2016, this medication was approved in the United States. Nusinersen, an antisense oligonucleotide (ASO), is administered directly into CSF. It alters *SMN2* pre-RNA splicing, so exon 7 is included, increasing expression of functional SMN protein. *Efficacy assessments for patients receiving nusinersen are based on serial assessments of performance on age-appropriate standardized motor scales. Treatment requires complex financial and logistics because of the very high drug cost, intrathecal administration, and medical fragility of the patients. Treatment implementation also engenders ethical considerations related to cost, insurance coverage, limited clinical data on groups of patients not in clinical trials, and questions of duration of treatment* [20]*.*

One in 50 asymptomatic people carries this autosomal recessive neuromuscular code causing SMA in one over 10,000 live births [21].

Based on age at onset, the highest milestone reached, and phenotypic severity: SMA has been separated into four different subgroups such as "Nonsitters" (Type I), "sitters" (Type II), "walkers" (Type III), and "adult onset" (Type IV) [22].

At the present moment, many patients got confirmation of diagnose very late, or the treatment is administered in advance stages. Therefore, poor response is often obtained.

**55**

*Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

to the cDNA encoded by SMN2.

procedures later on.

died [26].

[24, 25].

Fortunately, some screening programs are available and accurately and then to identify children in pre-symptomatic stages is possible [23]. However, because some children develop their clinical manifestation far from birth then to decide when to

The majority of SMN2 pre-mRNA transcripts undergo alternative splicing due to a nucleotide substitution leading to exclusion of exon 7. Degradation of the resulting truncated SMN protein is very fast, and the overall lack of full-length SMN protein causes permanent damage on anterior horn cell of the spinal cord [23]. In patients with onset of the disease beyond six months of age, large phase 3 trials confirmed improvement in motor activities, very high event-free and remarkable survival in infantile-onset SMA3, also significant improvement in Expanded version of the Hammersmith Functional Motor Scale scores has been recorded

The copy number of the homologous SMN2 gene is inversely correlated with SMA severity and encoded by SMN1 (except for lack of exon 7), which is identical

Currently, for the therapy of SMA, there are pipelines developed by antisense oligonucleotide (ASO), also available for Huntington disease, ALS, spinocerebellar ataxias, Parkinson disease, and Alzheimer disease, among other options, and the pharmaceutical industry on ASO development has been delivering a promising therapeutic approach. The key care concern to MND patients has been developed, and expert consensus guidelines delivered, and best management for lung diseases, nutritional problems, and palliative care has also been reached. However, in this chapter, we will discuss novel aspects related to treatment and other therapeutic

In pre-symptomatic SMA patient's Types I–III released interim results of a phase

Some investigators have been working on stage of improvement after the treatment of SMA and confirmed that the first published data supported important good results on the motor function and quality of life from animal models with early restoration of SMN levels for those studied within the first 3 postnatal days. However, for those treated beyond 5 postnatal days, the level of recovery was low, while delivered treatment after 10 postnatal days, it showed no improvement and

One of the problems found in our preliminary review is the big number of SMA patients diagnosed at late stage. We found Type I patient with 4 months after onset

However, newborn screening programs have been a successful process for identifying affected children at an asymptomatic stage, leading to pre-symptomatic initiation of treatment before irreversible anterior horn cell lesion appears. To perform screening methods before birth are certain, and they available and willing to deliver the possibility of distinguish patients at the beginning of pregnancy, giving

Taiwan and Belgium, also have screening programs for SMA, but in America, the leader and an important number of states, further clinical trials have been implemented to evaluate the applicability and economic advantages. Unfortunately,

Chorionic villus sampling or amniocentesis to identify children with higher risk for SMA with an elevated percentage of accuracy can be done, if it is performed during the 10–14 or 15–20 weeks of pregnancy. These procedures can be dangerous for the mother and the baby to be done in *high-risk pregnancies with proven carrier status of the parents recommended* [23]*.* Nevertheless, isolating circulating fetal trophoblastic cells by noninvasive prenatal diagnosis techniques is also possible [29] or

2 trial evaluating the effects of Nusinersen have been done [23].

and Type III with 10 months or more after onset [27, 28].

a chance to perform a prenatal therapeutic management.

around 5% of mutations in the SMN1 cannot de identified [23].

initiate the treatment and whom qualify for therapy is a dilemma.

#### *Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

*Novel Aspects on Motor Neuron Disease*

cortical neurophysiology [15].

11,000 newborn infants.

Because one of the most effective clinical strategies for SMA is to protect the anterior horn cell, apart from nusinersen (that is a very expensive medication), one

For patients presenting SMA (most common reason of inherited infant mortal-

Another therapeutic modality to treat ALS is the noninvasive brain stimulation (NIBS), which has been shown to modulate cortical activity, with some protocols leading effects that outlast the stimulation by months. NIBS have been suggested as a potential treatment choice in those processes with associated changes in the

A total of 25 genes associated with ALSf and ALS (sporadic form), mutations in fused-in-sarcoma (FUS) and superoxide dismutase 1 (SOD1) have been intensively studied in the past, focusing on modified excitability of motor neurons. Based on their personal experience, Peikkert et al. [19] proposed that the 4-aminopyridine (4-AP), which is a potassium channel blocker, can be utilized as a probable therapy *for ALS patients* due to its demonstrated hypo excitability and high frequency of apoptosis in a FUS/SOD1-ALS-induced multi-potent stem cell from selected motor

One of the clinical presentations of MND is SMA, which encompasses a group of autosomal recessively inherited degenerative neuromuscular diseases. SMA is an inherited disorder that causes progressive lesions on the anterior horn cell leading to weakness or paralysis of the affected limbs, and it is caused by elimination or mutation of survival motor neuron (SMN) 1 gene. It is well known that homozygous damage and loss of functional mutations in the survival motor neuron 1 gene (SMN1) at the chromosome 5q13 are the main cause of SMA, which affect 1 in

SMA usually has a very poor prognosis after rapidly progressive weakness and early mortality. However, a new medication named Nusinersen has been released for the treatment of all forms of SMA (not on mechanical ventilation) with very good results. In December 2016, this medication was approved in the United States. Nusinersen, an antisense oligonucleotide (ASO), is administered directly into CSF. It alters *SMN2* pre-RNA splicing, so exon 7 is included, increasing expression of functional SMN protein. *Efficacy assessments for patients receiving nusinersen are based on serial assessments of performance on age-appropriate standardized motor scales. Treatment requires complex financial and logistics because of the very high drug cost, intrathecal administration, and medical fragility of the patients. Treatment implementation also engenders ethical considerations related to cost, insurance coverage, limited clinical data on groups of patients not in clinical trials, and questions of duration of treatment* [20]*.*

One in 50 asymptomatic people carries this autosomal recessive neuromuscular

Based on age at onset, the highest milestone reached, and phenotypic severity: SMA has been separated into four different subgroups such as "Nonsitters" (Type I),

At the present moment, many patients got confirmation of diagnose very late, or the treatment is administered in advance stages. Therefore, poor response is often

"sitters" (Type II), "walkers" (Type III), and "adult onset" (Type IV) [22].

code causing SMA in one over 10,000 live births [21].

Kepra (levetiracetam) provoked neurite elongation in SMA-iPSCs-MNs. TUNEL-positive anterior horn cell was significantly decreased by kepra in SMAiPSCs-MNs. On the other hand, the expression level of cleaved-caspase 3 was diminished by levetiracetam in SMA-iPSCs-MNs. Furthermore, kepra improved impaired mitochondrial function in SMA-iPSCs-MNs. On the other hand, kepra did not modify the expression level of SMN protein in SMA-iPSCs-MNs. These results

anti-epileptic medication levetiracetam has been used as well.

suggest that kepra has a neuroprotective effect for SMA [17].

ity), the gene therapy seems to be the most effective strategy [18].

neuron; they also found that this process is partly reversible by 4-AP.

**54**

obtained.

Fortunately, some screening programs are available and accurately and then to identify children in pre-symptomatic stages is possible [23]. However, because some children develop their clinical manifestation far from birth then to decide when to initiate the treatment and whom qualify for therapy is a dilemma.

The majority of SMN2 pre-mRNA transcripts undergo alternative splicing due to a nucleotide substitution leading to exclusion of exon 7. Degradation of the resulting truncated SMN protein is very fast, and the overall lack of full-length SMN protein causes permanent damage on anterior horn cell of the spinal cord [23].

In patients with onset of the disease beyond six months of age, large phase 3 trials confirmed improvement in motor activities, very high event-free and remarkable survival in infantile-onset SMA3, also significant improvement in Expanded version of the Hammersmith Functional Motor Scale scores has been recorded [24, 25].

The copy number of the homologous SMN2 gene is inversely correlated with SMA severity and encoded by SMN1 (except for lack of exon 7), which is identical to the cDNA encoded by SMN2.

Currently, for the therapy of SMA, there are pipelines developed by antisense oligonucleotide (ASO), also available for Huntington disease, ALS, spinocerebellar ataxias, Parkinson disease, and Alzheimer disease, among other options, and the pharmaceutical industry on ASO development has been delivering a promising therapeutic approach. The key care concern to MND patients has been developed, and expert consensus guidelines delivered, and best management for lung diseases, nutritional problems, and palliative care has also been reached. However, in this chapter, we will discuss novel aspects related to treatment and other therapeutic procedures later on.

In pre-symptomatic SMA patient's Types I–III released interim results of a phase 2 trial evaluating the effects of Nusinersen have been done [23].

Some investigators have been working on stage of improvement after the treatment of SMA and confirmed that the first published data supported important good results on the motor function and quality of life from animal models with early restoration of SMN levels for those studied within the first 3 postnatal days. However, for those treated beyond 5 postnatal days, the level of recovery was low, while delivered treatment after 10 postnatal days, it showed no improvement and died [26].

One of the problems found in our preliminary review is the big number of SMA patients diagnosed at late stage. We found Type I patient with 4 months after onset and Type III with 10 months or more after onset [27, 28].

However, newborn screening programs have been a successful process for identifying affected children at an asymptomatic stage, leading to pre-symptomatic initiation of treatment before irreversible anterior horn cell lesion appears. To perform screening methods before birth are certain, and they available and willing to deliver the possibility of distinguish patients at the beginning of pregnancy, giving a chance to perform a prenatal therapeutic management.

Taiwan and Belgium, also have screening programs for SMA, but in America, the leader and an important number of states, further clinical trials have been implemented to evaluate the applicability and economic advantages. Unfortunately, around 5% of mutations in the SMN1 cannot de identified [23].

Chorionic villus sampling or amniocentesis to identify children with higher risk for SMA with an elevated percentage of accuracy can be done, if it is performed during the 10–14 or 15–20 weeks of pregnancy. These procedures can be dangerous for the mother and the baby to be done in *high-risk pregnancies with proven carrier status of the parents recommended* [23]*.* Nevertheless, isolating circulating fetal trophoblastic cells by noninvasive prenatal diagnosis techniques is also possible [29] or even getting from maternal blood cell-free fetal DNA [30]. These above-mentioned techniques allow to identify SMA in unborn children with 100% accuracy, promising a better future for SMA patients [23].

At this stage, it is also important to mention that significant ethical issues are involved in this genetic screening methodology and its need to be considered by the medical community before making these procedures fully available [31]. At the present moment, we are not quite sure how to predict disease severity accurately or even its presence because not all patients present clinical manifestation birth or because only few minimal sings are detectable. Treatment algorithm for SMA patients confirmed by newborn screening based on SMN1 deletion analysis in dried blood spots is available since 2005 [28]. Indications of treatment are based on the clinical phenotype of the patients and correlation of SMN2 copy numbers. All patients presenting 2–3 copies should be treated with the immediate effect according to NBS Multidisciplinary Working Group recommendations even if the child is asymptomatic with only one copy, but when four or more copies are present due to milder course of the disease, the treatment must be delayed [28].

In 2017, some authors studied a big number of SMA patients (*n* = 3500) trying to compare SMN2 copy number with their clinical information and found that patients with 1–4 copies have mild to severe phenotype, respectively, while there was an important overlap among patients with 2–3, confirming any possible phenotype [32], but more than 80% of patients in this group cohort carried two to three SMN2 copies, suggesting the similar problem in medical practice. In these cases, presenting two or three SMN2 copies to predict the severity of the disorder is not certain [23].

It is important to take into account that those SMA patients (families and siblings) presenting the same amount of SMN2 copies have another phenotype [33]; in the context, SMN1 (homozygous) mutations, SMN2 copies in people free of symptoms and signs, and even in SMA Type I have been found [34]. SMN2 transcripts and the SMN2 copy number do not show any correlation in a series of investigations done between 2001 and 2017 [35–38].

Following some recommendations delivered by the Phase 1 trial and studies on its pharmacological process, the investigators found that the half-life of nusinersen in the CFS is 163 days, and the ideal way for administration should be intrathecal (at the dosage of 12 mg) every 4 months. After that period of time, patients should receive five intrathecal injections within the first 6 months of treatment. These authors also confirmed that no correlation exits between concentrations of the CSF, age of the patients, and body weight [39, 40].

Based on results published by Luu et al., the doses of nusinersen according to the age of the patients produce more median exposures in the CSF, which suggest that prescribing fixed dosage programs through all age groups is the best choice [40]. For the other hand, Finkel et al. [41] conducted a Phase 2 dose-escalation investigation confirming that a single dose of 12 mg is better than 6 mg. The best way to assess the outcome for these patients is to measure the advantage of motor milestone, depending on ventilator machine, achievement of motor activities, clinical electrophysiological studies, and overall survival. Twenty patients younger than 1 year of age and less than 10 kg of body weight were screened, and these results confirmed the previous postulate [41]. The results from the small group studied in phase 2 clinical trials and the pharmacological studies done can accurately reflect clinical practice, is an interrogation still no responded.

Taking into account the great variability among SMA patients related to the age at disease onset, residual motor function, body weight, and fixed dose at the same intervals for patients, it seems to be remarkably inaccurate [23]. Reliable biomarkers and screening procedures for proper diagnosis at early stage of the disease are

**57**

guidance.

*Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

explanation of the pathophysiology of SMA [23].

need more than ever if we are looking for longer survival and positive modifications of the patient outcome. If reliable biomarkers are not available, then determining the SMN protein level and epigenetic modifiers to provide confident information about the intensity of the process is mandatory. When more than three SMN2 copies are detectable, chance for life-saving treatment is not certain. In summary, novel screening techniques, procedures to predict the intensity of the disease, and reliable biomarkers, which support monitoring of the treatment, have been discovered and recently developed, but unfortunately, none of them provide an unequivocal

To develop more accurate diagnostic procedures including confident biomarkers, better therapeutic approaches, and novel predictors to determine the ideal dosing recommendation, more investigations are required. This is the only way to

Two years ago, the European Medicines Agency finally approved nusinersen an antisense oligonucleotide (ASO) as the treatment of choice for SMA, and later, this medication has been considered as part of the treatment for patients with Type 2 SMA as well [23]. In patients with SMA presenting spinal bone deformities, severe contractures, scoliosis, spine fusion surgeries, and respiratory distress, the adminis-

Recently, with the intention of assess, the accuracy, and feasibility of nusinersen

Nusinersen is not available in Africa as yet but can be found in many European

A few weeks ago, Sansone et al. [43] reported their experience and good results after studied 50 SMA patients treated with intrathecal nusinersen. They concluded that in spite of the severity of the disease and the age of patients, this treatment is feasible, safe, and suitable for SMA patients if they are managed by a good

According to the information provided by Gidaro et al., a few weeks ago, in Australia, the commercial availability of the medication from the transition of expanded access programmed its right in corner. While in New Zealand, a broad access to this program is available, and in Canada, negotiators are discussing about the most convenient price at the present moment. However, some problems such as advanced age, patients with respiratory failure depending ventilator machine, and

As was mentioned before, the traditional LP for intrathecal administration of nusinersen can be impeded due to deformities of the spinal bone and orthopedic surgical procedures among other impediments commonly seen in SMA patients. However, the accumulated experiences from cervical myelograms serve to recommend this procedure as an ideal approach for cervical intrathecal administration of nusinersen, especially if it can be guided by ultrasound [45]. The same investigators studied 14 patients after the administration of nusinersen by cervical punctured guide by ultrasound with local anesthesia and found that all patients presented no major complications. One of the advantages of this technique is that general anesthesia is no required, and patients can be managed in real-time ultrasound

administrated by lumbar puncture (LP) in young patients, Wurster et al. [42] studied in 93 patients in whom the LP is done, highlighting the amount of attempts performed, site of injection, length of the spinal needle, duration of the procedure, medications used for sedative purposes, local anesthesia, level of O2 saturation in blood, appearance of the CFS, and adverse effects. These results confirm that LP is the best way to administer this medication in adolescent and young adults with later-onset SMA even in candidates with spinal bone deformities and respiratory

failure mainly if the patient is managed under a multidisciplinary team.

guarantee a reliable long-term treatment and successful outcome [23].

tration intrathecal of nusinersen could be a great challenge.

patients presenting spinal fusion still need to be solved [44].

countries for all SMA types.

skilled team.

#### *Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

*Novel Aspects on Motor Neuron Disease*

ing a better future for SMA patients [23].

even getting from maternal blood cell-free fetal DNA [30]. These above-mentioned techniques allow to identify SMA in unborn children with 100% accuracy, promis-

At this stage, it is also important to mention that significant ethical issues are involved in this genetic screening methodology and its need to be considered by the medical community before making these procedures fully available [31]. At the present moment, we are not quite sure how to predict disease severity accurately or even its presence because not all patients present clinical manifestation birth or because only few minimal sings are detectable. Treatment algorithm for SMA patients confirmed by newborn screening based on SMN1 deletion analysis in dried blood spots is available since 2005 [28]. Indications of treatment are based on the clinical phenotype of the patients and correlation of SMN2 copy numbers. All patients presenting 2–3 copies should be treated with the immediate effect according to NBS Multidisciplinary Working Group recommendations even if the child is asymptomatic with only one copy, but when four or more copies are present due to

In 2017, some authors studied a big number of SMA patients (*n* = 3500) trying to compare SMN2 copy number with their clinical information and found that patients with 1–4 copies have mild to severe phenotype, respectively, while there was an important overlap among patients with 2–3, confirming any possible phenotype [32], but more than 80% of patients in this group cohort carried two to three SMN2 copies, suggesting the similar problem in medical practice. In these cases, presenting two or three SMN2 copies to predict the severity of the disorder is not

It is important to take into account that those SMA patients (families and siblings) presenting the same amount of SMN2 copies have another phenotype [33]; in the context, SMN1 (homozygous) mutations, SMN2 copies in people free of symptoms and signs, and even in SMA Type I have been found [34]. SMN2 transcripts and the SMN2 copy number do not show any correlation in a series of

Following some recommendations delivered by the Phase 1 trial and studies on its pharmacological process, the investigators found that the half-life of nusinersen in the CFS is 163 days, and the ideal way for administration should be intrathecal (at the dosage of 12 mg) every 4 months. After that period of time, patients should receive five intrathecal injections within the first 6 months of treatment. These authors also confirmed that no correlation exits between concentrations of the CSF,

Based on results published by Luu et al., the doses of nusinersen according to the age of the patients produce more median exposures in the CSF, which suggest that prescribing fixed dosage programs through all age groups is the best choice [40]. For the other hand, Finkel et al. [41] conducted a Phase 2 dose-escalation investigation confirming that a single dose of 12 mg is better than 6 mg. The best way to assess the outcome for these patients is to measure the advantage of motor milestone, depending on ventilator machine, achievement of motor activities, clinical electrophysiological studies, and overall survival. Twenty patients younger than 1 year of age and less than 10 kg of body weight were screened, and these results confirmed the previous postulate [41]. The results from the small group studied in phase 2 clinical trials and the pharmacological studies done can accurately reflect

Taking into account the great variability among SMA patients related to the age at disease onset, residual motor function, body weight, and fixed dose at the same intervals for patients, it seems to be remarkably inaccurate [23]. Reliable biomarkers and screening procedures for proper diagnosis at early stage of the disease are

milder course of the disease, the treatment must be delayed [28].

investigations done between 2001 and 2017 [35–38].

age of the patients, and body weight [39, 40].

clinical practice, is an interrogation still no responded.

**56**

certain [23].

need more than ever if we are looking for longer survival and positive modifications of the patient outcome. If reliable biomarkers are not available, then determining the SMN protein level and epigenetic modifiers to provide confident information about the intensity of the process is mandatory. When more than three SMN2 copies are detectable, chance for life-saving treatment is not certain. In summary, novel screening techniques, procedures to predict the intensity of the disease, and reliable biomarkers, which support monitoring of the treatment, have been discovered and recently developed, but unfortunately, none of them provide an unequivocal explanation of the pathophysiology of SMA [23].

To develop more accurate diagnostic procedures including confident biomarkers, better therapeutic approaches, and novel predictors to determine the ideal dosing recommendation, more investigations are required. This is the only way to guarantee a reliable long-term treatment and successful outcome [23].

Two years ago, the European Medicines Agency finally approved nusinersen an antisense oligonucleotide (ASO) as the treatment of choice for SMA, and later, this medication has been considered as part of the treatment for patients with Type 2 SMA as well [23]. In patients with SMA presenting spinal bone deformities, severe contractures, scoliosis, spine fusion surgeries, and respiratory distress, the administration intrathecal of nusinersen could be a great challenge.

Recently, with the intention of assess, the accuracy, and feasibility of nusinersen administrated by lumbar puncture (LP) in young patients, Wurster et al. [42] studied in 93 patients in whom the LP is done, highlighting the amount of attempts performed, site of injection, length of the spinal needle, duration of the procedure, medications used for sedative purposes, local anesthesia, level of O2 saturation in blood, appearance of the CFS, and adverse effects. These results confirm that LP is the best way to administer this medication in adolescent and young adults with later-onset SMA even in candidates with spinal bone deformities and respiratory failure mainly if the patient is managed under a multidisciplinary team.

Nusinersen is not available in Africa as yet but can be found in many European countries for all SMA types.

A few weeks ago, Sansone et al. [43] reported their experience and good results after studied 50 SMA patients treated with intrathecal nusinersen. They concluded that in spite of the severity of the disease and the age of patients, this treatment is feasible, safe, and suitable for SMA patients if they are managed by a good skilled team.

According to the information provided by Gidaro et al., a few weeks ago, in Australia, the commercial availability of the medication from the transition of expanded access programmed its right in corner. While in New Zealand, a broad access to this program is available, and in Canada, negotiators are discussing about the most convenient price at the present moment. However, some problems such as advanced age, patients with respiratory failure depending ventilator machine, and patients presenting spinal fusion still need to be solved [44].

As was mentioned before, the traditional LP for intrathecal administration of nusinersen can be impeded due to deformities of the spinal bone and orthopedic surgical procedures among other impediments commonly seen in SMA patients. However, the accumulated experiences from cervical myelograms serve to recommend this procedure as an ideal approach for cervical intrathecal administration of nusinersen, especially if it can be guided by ultrasound [45]. The same investigators studied 14 patients after the administration of nusinersen by cervical punctured guide by ultrasound with local anesthesia and found that all patients presented no major complications. One of the advantages of this technique is that general anesthesia is no required, and patients can be managed in real-time ultrasound guidance.

The most significant advantage to antisense oligonucleotide (ASO) therapeutics over other small molecule approaches is that acquisition of the target sequence provides immediate knowledge of putative complementary oligonucleotide therapeutics.

In 2019, Scoles et al. [46] described several therapeutic modalities with ASO and how they can be indicated for medical treatment of SMA, apart from the work done to develop novel ASO therapies looking for better results in the management of neurodegenerative disorders [46]. Novel advances of the genetic studies will allow distinguishing different genetic information for many neurological disorders. The mutated protein found and its chance to be placing into the cellular pathway will support a faster development of way for treatment. For the other hands, new opportunities for reliable treatment have been arising from the new capacities of targeting the disorder gene and RNAs. Among other procedures, to target the expression of RNA, some authors highlighted the utilization of ASOs to treat neurological problems. Treatment based on ASOs varies from 18 to 30 base pairs in length. These investigators changed expression of a target mRNA modifying splicing or by recruiting RNase H (cellular enzyme) that recognizes DNA: RNA hybrids causing target degradation [46].

Apart from nursinersen, other ASO therapeutics approved by FDA are eteplirsen to treat Duchene muscular dystrophy 2 and inotersen for managing patients presenting familial amyloid polyneuropathy. For treatment of Huntington disease, ASOs targeting HTT have been used [47]. As we and other authors reported in other publications, the treatment of choices for ALS is SOD1 and C9ORF72 [48, 49] and MAPT (TAU) in cases affected by Alzheimer disease [50]. Because most of the treatment with ASO does not cross the blood-brain barrier (BBB), it is necessary to administer it by injection into the intraventricular system in mouse and by intrathecal administration in humans.

Some investigators have mentioned that nucleic acids are prompt to nuclease degradation, and its protein binding is weak, leading to inefficient tissue uptake and unreliable use as drugs [26, 51].

Most of these changes modify the pharmacokinetic, pharmacodynamic, or endocytic uptake that controls the specific function of the proteins (cell surface) [51, 52].

Oligonucleotide chemistry, methoxyethyl oligonucleotide, constrained nucleic acids, Stereopure PS ASOs, Peptide nucleic acid, 5′-methylcytosine modification, target fate, mixed chemistry, and gapmers are aspects that are not discussed in this chapter and should be considered by interested readers on this matter. In order to get most complete information about it, we recommend checking the article of Scoles et al. [46].

ASOs are being used for some genetic etiology of ALS. Obviously, these biological pathways affecting the recognized mechanism of production of the disorder also modify the results when ASO is used [53].

#### **2. Therapy with riluzole and edaravone**

In 1994, the idea about the role of the excitatory amino acid neurotransmitter glutamate in the mechanism of production of ALS was prevalent. At that time, some investigators evaluated the safety and accuracy of the antiglutamate agent riluzole, at the dose of 100 mg daily, in patients presenting ALS. They studied 155 outpatients, and after 12 months of treatment, they found that 74% of the patients were still alive (*P* = 0.014), and the decrease of muscle power was remarkable in patients consuming riluzole compared with the control group

**59**

(*P* < 0.001) [70].

*Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

acting on SK channels also causes similar results [55].

for an important number of ALS patients [58].

benzothiazole drug with glutamine antagonist activity [56, 57].

bulbar palsy [54].

Union [59].

results [60].

been performed.

(placebo). Therefore, they concluded that riluzole decreases the speed of progression in ALS patients, and it can prolong the survival period in patients presenting

Without doubt, nusinersen improves motor neuron function, but riluzole by

Since 1996, it is well known that one of the etiological pathogenesis of ALS is caused by neural damage due to glutamate excite-toxicity. Riluzole is a synthetic

The first analyses, and at posteriori meta-analyses done on results obtained from controlled trials by randomization, confirm that riluzole extends survival by 2–3 months and augment the possibilities of an additional 12 months of survival by ~9%. Same authors reported improvements in media survival times over 76 weeks

In 1995, oral riluzole was approved by FDA as part of the treatment of ALS. Riluzole is a well-known presynaptic glutamate release inhibitor, which can provide neural injury and prevent muscle-power worsening. Currently, this medication has been licensed to be prescribed in many places including the European

The dose of 50 mg twice, to be taken 1 hour before meal or 2 hours after it, has been approved by the Institute for Health and Care Excellence since 2001 with good

One good news is that riluzole is now available in oral suspension (Teglutik®)

However, other RCTs have been done for patients with cervical myelopathy,

Real-world evidence confirmed that an important prolongation of median survival times in ALS patients treated with riluzole is certain. Based on retrospective/prospective investigations done on large database, these authors concluded that patients under riluzole therapy had better prognosis than those without treatment,

Brooks et al. studied two series of ALS patients: one group of 51 patients under riluzole treatment and another group of 241 patients without riluzole (before 1996) and a second series of 112 ALS patients' riluzole-treated and 65 nontreated patients (after 1996). These authors found that Cox analysis concluded that patients on treatment got an extension of survival (*P* < 0.0001) and even remarkable improvements in elderly people and patients in advanced stage. Therapy with riluzole provides a median extension of survival in affected patients between 40 and 72 weeks [66]. Survival benefit would be 36 weeks if patients are managed like prospective RCTs according to other researchers [67]. Based on Cox model technique, Mitchell et al. found that survival times are bigger in riluzole-treated ALS patients than nontreated cases (HR 0.20, *P* < 0.001) [68]. While other authors communicated that Cox multivariate analysis of therapy was related to a prolongation of survival at

Retrospective population-based studies on the effect of riluzole on survival of ALS patients done between 1999 and 2008 and made by Lee et al. concluded that Cox multivariate analysis (*n* = 1149) that riluzole provide a longer survival on treated-patients; unadjusted HR 0.32 (*P* < 0.001) and adjusted HR 0.34

Georgoulopoulou et al. and Knibb et al. conducted a prospective populationbased study on the survival of 193 patients (between 2000 and 2009) and 575 cases

which presentation (5 mg/ml) and has been shown its beneficial for patients presenting bulbar palsy with functional dysphagia allowing longer therapy [61, 62]. Not randomized controlled trials (RCTs) with riluzole for ALS patients have

chronic psychosis, and autistic spectrum disorder [63–65].

mainly at the first stage of the pathological process [58].

48 weeks (HR 0.51, *P* = 0.06) [69].

#### *Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

*Novel Aspects on Motor Neuron Disease*

causing target degradation [46].

cal administration in humans.

unreliable use as drugs [26, 51].

modify the results when ASO is used [53].

**2. Therapy with riluzole and edaravone**

[51, 52].

Scoles et al. [46].

therapeutics.

The most significant advantage to antisense oligonucleotide (ASO) therapeutics

over other small molecule approaches is that acquisition of the target sequence provides immediate knowledge of putative complementary oligonucleotide

In 2019, Scoles et al. [46] described several therapeutic modalities with ASO and how they can be indicated for medical treatment of SMA, apart from the work done to develop novel ASO therapies looking for better results in the management of neurodegenerative disorders [46]. Novel advances of the genetic studies will allow distinguishing different genetic information for many neurological disorders. The mutated protein found and its chance to be placing into the cellular pathway will support a faster development of way for treatment. For the other hands, new opportunities for reliable treatment have been arising from the new capacities of targeting the disorder gene and RNAs. Among other procedures, to target the expression of RNA, some authors highlighted the utilization of ASOs to treat neurological problems. Treatment based on ASOs varies from 18 to 30 base pairs in length. These investigators changed expression of a target mRNA modifying splicing or by recruiting RNase H (cellular enzyme) that recognizes DNA: RNA hybrids

Apart from nursinersen, other ASO therapeutics approved by FDA are eteplirsen

to treat Duchene muscular dystrophy 2 and inotersen for managing patients presenting familial amyloid polyneuropathy. For treatment of Huntington disease, ASOs targeting HTT have been used [47]. As we and other authors reported in other publications, the treatment of choices for ALS is SOD1 and C9ORF72 [48, 49] and MAPT (TAU) in cases affected by Alzheimer disease [50]. Because most of the treatment with ASO does not cross the blood-brain barrier (BBB), it is necessary to administer it by injection into the intraventricular system in mouse and by intrathe-

Some investigators have mentioned that nucleic acids are prompt to nuclease degradation, and its protein binding is weak, leading to inefficient tissue uptake and

Most of these changes modify the pharmacokinetic, pharmacodynamic, or endocytic uptake that controls the specific function of the proteins (cell surface)

Oligonucleotide chemistry, methoxyethyl oligonucleotide, constrained nucleic acids, Stereopure PS ASOs, Peptide nucleic acid, 5′-methylcytosine modification, target fate, mixed chemistry, and gapmers are aspects that are not discussed in this chapter and should be considered by interested readers on this matter. In order to get most complete information about it, we recommend checking the article of

ASOs are being used for some genetic etiology of ALS. Obviously, these biological pathways affecting the recognized mechanism of production of the disorder also

In 1994, the idea about the role of the excitatory amino acid neurotransmitter glutamate in the mechanism of production of ALS was prevalent. At that time, some investigators evaluated the safety and accuracy of the antiglutamate agent riluzole, at the dose of 100 mg daily, in patients presenting ALS. They studied 155 outpatients, and after 12 months of treatment, they found that 74% of the patients were still alive (*P* = 0.014), and the decrease of muscle power was remarkable in patients consuming riluzole compared with the control group

**58**

(placebo). Therefore, they concluded that riluzole decreases the speed of progression in ALS patients, and it can prolong the survival period in patients presenting bulbar palsy [54].

Without doubt, nusinersen improves motor neuron function, but riluzole by acting on SK channels also causes similar results [55].

Since 1996, it is well known that one of the etiological pathogenesis of ALS is caused by neural damage due to glutamate excite-toxicity. Riluzole is a synthetic benzothiazole drug with glutamine antagonist activity [56, 57].

The first analyses, and at posteriori meta-analyses done on results obtained from controlled trials by randomization, confirm that riluzole extends survival by 2–3 months and augment the possibilities of an additional 12 months of survival by ~9%. Same authors reported improvements in media survival times over 76 weeks for an important number of ALS patients [58].

In 1995, oral riluzole was approved by FDA as part of the treatment of ALS. Riluzole is a well-known presynaptic glutamate release inhibitor, which can provide neural injury and prevent muscle-power worsening. Currently, this medication has been licensed to be prescribed in many places including the European Union [59].

The dose of 50 mg twice, to be taken 1 hour before meal or 2 hours after it, has been approved by the Institute for Health and Care Excellence since 2001 with good results [60].

One good news is that riluzole is now available in oral suspension (Teglutik®) which presentation (5 mg/ml) and has been shown its beneficial for patients presenting bulbar palsy with functional dysphagia allowing longer therapy [61, 62].

Not randomized controlled trials (RCTs) with riluzole for ALS patients have been performed.

However, other RCTs have been done for patients with cervical myelopathy, chronic psychosis, and autistic spectrum disorder [63–65].

Real-world evidence confirmed that an important prolongation of median survival times in ALS patients treated with riluzole is certain. Based on retrospective/prospective investigations done on large database, these authors concluded that patients under riluzole therapy had better prognosis than those without treatment, mainly at the first stage of the pathological process [58].

Brooks et al. studied two series of ALS patients: one group of 51 patients under riluzole treatment and another group of 241 patients without riluzole (before 1996) and a second series of 112 ALS patients' riluzole-treated and 65 nontreated patients (after 1996). These authors found that Cox analysis concluded that patients on treatment got an extension of survival (*P* < 0.0001) and even remarkable improvements in elderly people and patients in advanced stage. Therapy with riluzole provides a median extension of survival in affected patients between 40 and 72 weeks [66]. Survival benefit would be 36 weeks if patients are managed like prospective RCTs according to other researchers [67]. Based on Cox model technique, Mitchell et al. found that survival times are bigger in riluzole-treated ALS patients than nontreated cases (HR 0.20, *P* < 0.001) [68]. While other authors communicated that Cox multivariate analysis of therapy was related to a prolongation of survival at 48 weeks (HR 0.51, *P* = 0.06) [69].

Retrospective population-based studies on the effect of riluzole on survival of ALS patients done between 1999 and 2008 and made by Lee et al. concluded that Cox multivariate analysis (*n* = 1149) that riluzole provide a longer survival on treated-patients; unadjusted HR 0.32 (*P* < 0.001) and adjusted HR 0.34 (*P* < 0.001) [70].

Georgoulopoulou et al. and Knibb et al. conducted a prospective populationbased study on the survival of 193 patients (between 2000 and 2009) and 575 cases (between 1990 and 2013) consuming riluzole, respectively. According to the Cox multivariate model used during the first series of participants riluzole-treated, they reached a prolonged survival and remarkable delay of pulmonary complications including patients with bulbar palsy and even those with affected four limbs, and the second series of cases riluzole-treated showed a slower progression to pulmonary involvement [71, 72].

Chen et al. also studied a group of ALS patients using the same methodology and concluded that the median survival time in cases riluzole-treated was 268 weeks compared to 256 weeks in nontreated cases (log-rank *P* = 0.780); HR 0.855 (*P* = 0.167) [73].

Based on meta-analysis of RCTs recently done and all data obtained up to date, riluzole prolonged survival in ALS cases by 8–12 weeks and augmented the chance of additional 52 weeks of survival by ~9%.

Other authors reported that riluzole-treated cases can increase their median survival by up to 76 weeks, after reviewing 10 clinical ALS databases with around ~6000 cases [58].

In a series of patients studied by Inoue-Shibui et al., riluzole therapy was interrupted in 20 cases among 92 patients [74]. The most common cause of discontinuation of riluzole was abnormal of liver enzymes (5.4%), followed by interstitial pneumonia, among other causes.

All adverse events happened within 24 weeks of the beginning of riluzole therapy, with 50% of the adverse events occurring within 2 weeks. In almost all patients, adverse events disappeared after stopping the treatment. In the realworld setting, riluzole has been well assimilated for long periods of up to 7years or more [75].

Recently, two patients presenting recurrent pancreatitis were communicated to the medical literature. In both cases, the diagnosis was done within the first 3 months after initiated the treatment with riluzole [76], being another strong reason to highlight our recommendation about a careful observation of adverse events in the first 6months of riluzole administration.

A few weeks ago, Jaiswal et al. also confirmed that riluzole delay progression of ALS in animal model based on their experiences and many experimental drug trials done over the past decades, but riluzole did not show similar results in human beings or results are still nonconcluded under Phase I–III trials, which are quite true, and riluzole is the only available medication with some benefits on survival [77]. Nevertheless, an antioxidant drug (edaravone) has been produced by Mitsubishi Tanabe Pharma, and its effectiveness in halting ALS progression during early stages has been found.

In 2015, edaravone (Ridicut) was launched for the management of patients with ischemic stroke at first and later for the treatment of ALS patients [78]. Edaravone is a drug with a free radical scavenger with no remarkable benefits in ALS patients according to Phase III clinical trial, but edaravone is also a strong antioxidant able to prevent oxidative stress leading to motor neuron fatal damage in ALS. These investigators found another study, which confirmed good therapeutic response to edaravone in diagnosed patients by revised diagnostic criteria (El Escorial) of MND/ALS. Other authors investigated the effect of intraperitoneal administration of edaravone in wobbler's mice and demonstrated that elevated dose (10 mg/kg) of edaravone therapy remarkable attenuated paresis and muscle contracture on the extremities and stopped denervation atrophy in the proximal muscles and degeneration in the cervical anterior horn cell neurons compared to control group. After large waiting period of 22 years, the Mitsubishi Tanabe Pharma America acquired an US FDA approval for edaravone (Radicava) in May 2017 for the therapeutic approach of ALS.

**61**

remarkable side effects [83].

**3. Other medications for MND**

Although there is no available medicine to cure any clinical presentation of MND, during the first semester of this year (2019), a number of medications have been used to treat affected patients. Unfortunately, no remarkable results have been obtained, but some of them still show good action over this disease. In this chapter, we will deliver some comments about the results reached by some authors according to their report to the medical literature. The most common used medications

*Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

To low progression of MND/ALS, edaravone is a good indication according to the previous reports delivered to the medical literature. Recent phase three studies done on ALS patients treated with this medication did not confirm remarkable advantage in the Revised ALS Functional Rating score over the control group [79]. Between November 28, 2011 and September 3, 2014, the Writing Group [80] studied 213 cases and selected 192 candidates. Of these, 137 cases completed the first period for close observation: 69 were selected to be edaravone-treated (randomly), and 68 were assigned to control group to be treated with placebo also randomly, both series were included in the primary efficacy analysis. The results observed from the primary outcome demonstrated that the control group change −7.50 (0.66) in ALSFRS-R score compare with −5.01 (SE 0.64) in the group edaravone-treated. In favor of edaravone, the least-square mean confirmed a difference among two series of 2.49 (SE 0.76, 95% CI 0.99–33: *P* = 0.0013). These researchers concluded that edaravone works in a small subset of ALS patients who met criteria identified in post-hoc analysis of most recent Phase 3 studies, showing a remarkable diminish ALSFRS-R score compared with control group who received placebo. They also highlighted that there is no proof that edaravone might be efficient in a bigger series of ALS cases who do not meet the criteria [80]. The best way to administer edaravone (60 mg) is by slow IV infusion (2 hours duration) every 28 days. Edaravone has demonstrated its capacity to slow down the process of loss of motor function by 33% of ALS cases compared with control group. Being a powerful free radical scavenger, edaravone is able to inhibit nitration of tyrosine in the CSF and to improve the motor neuron cell activity in ALS mouse [81]. Unfortunately, there is no unanimous agreement about the positive effect of riluzole in patients presenting degenerative disorders. At the present moment, most of the neurology community agrees that riluzole is not a remarkable strong effect on the progression of ALS. Because the oxidative stress is considered to be involved in the pathology of ALS, almost all consider that the free radical scavenger edaravone may play a better relevant role for the treatment of cases presenting ALS. Without doubt, the first medication able to provide an efficient inhibition of motor neuron function deterioration in MND/ALS cases is edaravone if it is taken at early stage of this pathological disorder, but the lung function must be well assessed when a deterioration in the respiratory capacity is confirmed [82]. Most reports published in the medical literature show controversial benefits and safety about the edaravone treatment of ALS. Recently, Luo et al. [83] made a meta-analysis research to evaluate the accuracy and safety of edaravone as a therapy of choice for ALS, by searching PubMed, the Cochrane Library, and Embase from the inception of electronic data (April 2018), including randomized, double-blind, placebo-controlled trials reporting ALS cases receiving 60-mg IV edaravone or IV saline solution as placebo for 24 weeks. The study included 367 patients from three randomized controlled trials (183 patients on IV edaravone; 184 receiving IV saline solution). They arrived to the following conclusion: edaravone IV is a good treatment for ALS cases, with no

#### *Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

*Novel Aspects on Motor Neuron Disease*

nary involvement [71, 72].

0.855 (*P* = 0.167) [73].

~6000 cases [58].

more [75].

of additional 52 weeks of survival by ~9%.

the first 6months of riluzole administration.

2017 for the therapeutic approach of ALS.

pneumonia, among other causes.

early stages has been found.

(between 1990 and 2013) consuming riluzole, respectively. According to the Cox multivariate model used during the first series of participants riluzole-treated, they reached a prolonged survival and remarkable delay of pulmonary complications including patients with bulbar palsy and even those with affected four limbs, and the second series of cases riluzole-treated showed a slower progression to pulmo-

Chen et al. also studied a group of ALS patients using the same methodology and concluded that the median survival time in cases riluzole-treated was 268 weeks compared to 256 weeks in nontreated cases (log-rank *P* = 0.780); HR

Based on meta-analysis of RCTs recently done and all data obtained up to date, riluzole prolonged survival in ALS cases by 8–12 weeks and augmented the chance

Other authors reported that riluzole-treated cases can increase their median survival by up to 76 weeks, after reviewing 10 clinical ALS databases with around

In a series of patients studied by Inoue-Shibui et al., riluzole therapy was interrupted in 20 cases among 92 patients [74]. The most common cause of discontinuation of riluzole was abnormal of liver enzymes (5.4%), followed by interstitial

All adverse events happened within 24 weeks of the beginning of riluzole therapy, with 50% of the adverse events occurring within 2 weeks. In almost all patients, adverse events disappeared after stopping the treatment. In the realworld setting, riluzole has been well assimilated for long periods of up to 7years or

Recently, two patients presenting recurrent pancreatitis were communicated to the medical literature. In both cases, the diagnosis was done within the first 3 months after initiated the treatment with riluzole [76], being another strong reason to highlight our recommendation about a careful observation of adverse events in

A few weeks ago, Jaiswal et al. also confirmed that riluzole delay progression of ALS in animal model based on their experiences and many experimental drug trials done over the past decades, but riluzole did not show similar results in human beings or results are still nonconcluded under Phase I–III trials, which are quite true, and riluzole is the only available medication with some benefits on survival [77]. Nevertheless, an antioxidant drug (edaravone) has been produced by Mitsubishi Tanabe Pharma, and its effectiveness in halting ALS progression during

In 2015, edaravone (Ridicut) was launched for the management of patients with ischemic stroke at first and later for the treatment of ALS patients [78]. Edaravone is a drug with a free radical scavenger with no remarkable benefits in ALS patients according to Phase III clinical trial, but edaravone is also a strong antioxidant able to prevent oxidative stress leading to motor neuron fatal damage in ALS. These investigators found another study, which confirmed good therapeutic response to edaravone in diagnosed patients by revised diagnostic criteria (El Escorial) of MND/ALS. Other authors investigated the effect of intraperitoneal administration of edaravone in wobbler's mice and demonstrated that elevated dose (10 mg/kg) of edaravone therapy remarkable attenuated paresis and muscle contracture on the extremities and stopped denervation atrophy in the proximal muscles and degeneration in the cervical anterior horn cell neurons compared to control group. After large waiting period of 22 years, the Mitsubishi Tanabe Pharma America acquired an US FDA approval for edaravone (Radicava) in May

**60**

To low progression of MND/ALS, edaravone is a good indication according to the previous reports delivered to the medical literature. Recent phase three studies done on ALS patients treated with this medication did not confirm remarkable advantage in the Revised ALS Functional Rating score over the control group [79]. Between November 28, 2011 and September 3, 2014, the Writing Group [80] studied 213 cases and selected 192 candidates. Of these, 137 cases completed the first period for close observation: 69 were selected to be edaravone-treated (randomly), and 68 were assigned to control group to be treated with placebo also randomly, both series were included in the primary efficacy analysis. The results observed from the primary outcome demonstrated that the control group change −7.50 (0.66) in ALSFRS-R score compare with −5.01 (SE 0.64) in the group edaravone-treated. In favor of edaravone, the least-square mean confirmed a difference among two series of 2.49 (SE 0.76, 95% CI 0.99–33: *P* = 0.0013). These researchers concluded that edaravone works in a small subset of ALS patients who met criteria identified in post-hoc analysis of most recent Phase 3 studies, showing a remarkable diminish ALSFRS-R score compared with control group who received placebo. They also highlighted that there is no proof that edaravone might be efficient in a bigger series of ALS cases who do not meet the criteria [80]. The best way to administer edaravone (60 mg) is by slow IV infusion (2 hours duration) every 28 days. Edaravone has demonstrated its capacity to slow down the process of loss of motor function by 33% of ALS cases compared with control group. Being a powerful free radical scavenger, edaravone is able to inhibit nitration of tyrosine in the CSF and to improve the motor neuron cell activity in ALS mouse [81]. Unfortunately, there is no unanimous agreement about the positive effect of riluzole in patients presenting degenerative disorders. At the present moment, most of the neurology community agrees that riluzole is not a remarkable strong effect on the progression of ALS. Because the oxidative stress is considered to be involved in the pathology of ALS, almost all consider that the free radical scavenger edaravone may play a better relevant role for the treatment of cases presenting ALS. Without doubt, the first medication able to provide an efficient inhibition of motor neuron function deterioration in MND/ALS cases is edaravone if it is taken at early stage of this pathological disorder, but the lung function must be well assessed when a deterioration in the respiratory capacity is confirmed [82]. Most reports published in the medical literature show controversial benefits and safety about the edaravone treatment of ALS. Recently, Luo et al. [83] made a meta-analysis research to evaluate the accuracy and safety of edaravone as a therapy of choice for ALS, by searching PubMed, the Cochrane Library, and Embase from the inception of electronic data (April 2018), including randomized, double-blind, placebo-controlled trials reporting ALS cases receiving 60-mg IV edaravone or IV saline solution as placebo for 24 weeks. The study included 367 patients from three randomized controlled trials (183 patients on IV edaravone; 184 receiving IV saline solution). They arrived to the following conclusion: edaravone IV is a good treatment for ALS cases, with no remarkable side effects [83].

#### **3. Other medications for MND**

Although there is no available medicine to cure any clinical presentation of MND, during the first semester of this year (2019), a number of medications have been used to treat affected patients. Unfortunately, no remarkable results have been obtained, but some of them still show good action over this disease. In this chapter, we will deliver some comments about the results reached by some authors according to their report to the medical literature. The most common used medications

are EH301, 5Fluoroucil, Tryptophan, RNS60, Rasagiline, Tirasemtiv, Aquaporin, Fasudil, and Lunasil.

de la Rubia et al. [84] evaluated the accuracy and feasibility of Elysium Health's candidate drug EH301 in ALS cases by a single-center, prospective, double-blind, randomized, placebo-controlled pilot study. Thirty-two ALS patients studied underwent for assessment during 4 months. Differences between EH301 and control group were evaluated based on their findings, and EH301 confirmed a remarkable slow progression of ALS compared with nontreated cases and even confirmed clinical benefits in many key outcome measures relative to their baseline.

Searching for drug candidates for ALS, Rando et al. investigated the action of anti-metabolite 5-fluorouracil (5-FU) administered by a single intraperitoneal injection at 150 mg/kg in SOD1G93A model of ALS. Un expectedly, the authors found that 5-FU (anti-cancer drug) increases survival delays of the disorder onset and improves motor function in ALS mice, but they were not able to demonstrate the mechanism of the beneficial 5-FU action in ALS mice. Despite of 5-FU did not improve the modulate motor neuron survival remarkably and did not improve reactive gliosis or change the muscle morphology, their findings recommended that a low dose of 5-FU or its analogs may have good effects on MND/ALS [85].

Other authors postulate that toxic gain of function, spread, and SOD1 misfolding is suggested as part of pathological mechanism of MND/ALS, but the nature of SOD1 toxicity has been hard to describe [86]. Only in SOD1 proteins from humans and other primates, and rarely in other species, a tryptophan residue at position 32 (W32) is predicted to be solvent exposed and to participate in SOD1 misfolding. DuVal et al. considered that W32 is influential in SOD1 acquiring toxicity, as it is known to be important in template-directed misfolding [86].

DuVal et al. highlighted the relevant influence of W32 on cases SOD1 toxicity to upper and lower motor neuron cells morphology and its activities. They assessed pharmaceutical targeting of the W32 residue for rescuing SOD1 toxicity [86]. When RNS60 is administered by IV infusion every 7 days and daily nebulization, it acts as a novel immune-modulator agent able to provide neuroprotective action in MND/ALS preclinical models. Paganoni et al. [87] studied 16 ALS patients during 23 weeks for safety and tolerability. Some investigations were done such as PBR28 positron emission tomography imaging and plasma biomarkers of inflammation. These authors did not find serious reactions, and no participants were removed from the study due to drug-related complications. At the present moment, a large, multicenter, Phase II trial of RNS60 is currently including cases to test the effects of RNS60 on MND/ALS biomarkers and disease deterioration based on the previous findings. They concluded that long-term RNS60 administered by IV infusion (as indicated) is well assimilated by patients and is also accurate [87]. In ALS, the prolong use of immune-modulating therapies has been showing not good results and did not help to understand how the immune system modifies disease outcome [88].

Rasagiline (monoamine oxidase B inhibitor) administered at 2 mg/day has a neuroprotective effect in MNS/ALS cases. In order to verify this postulate, Fernandez et al. performed a trial of 80 ALS patients from 10 hospitals in America. They did not identify any difference between Rasagiline-treated cases and the control group. Therefore, they assumed that Rasagiline did not change disease output compared with control group during 12 months of therapy. Rasagiline was well assimilated, and no serious adverse events were report.

Shefner et al. conducted a multinational clinical trial to study the accuracy of tirasemtiv (125mg twice daily) using an escalating dosage protocol during 4 weeks [89]. Comparisons between two series of cases with ALS were performed. One group constituted by fast skeletal muscle troponin activator and a second control group by placebo. Of 744 candidates, 565 participants assimilated open-label

**63**

chapter.

riluzole or edaravone.

spasticity, among others.

ing future is forthcoming.

tion, and so forth.

*Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

MND/ALS.

tirasemtiv and were treated randomly. As a side effect of tirasemtiv, nausea, weight loss, dizziness, fatigue, and insomnia were more often seen. The frequency of severe side effects was seen equally on both series of cases. Obviously, tirasemtiv did not change the decline of slow vital capacity or remarkable impact secondary outcome assessment and weak tolerability of tirasemtiv may lead to poor effect on

Aquaporin 4 (AQP4) is present in astrocytes in the nervous system as primary water channel and has been postulated to participate in a myriad of acute, chronic brain disorders and the incidence of MND/ALS. Depolarization of AQP4 causes degeneration of the upper and lower motor neurons via GLT-1, and suppressions increase recovery of motor activity in MND/ALS cases probable due to NGF. No

Studies made with Fasudil (30 mg for IV application) have shown good results in cell culture and animal research of MND/ALS. This medication is a Rho kinase (ROCK) inhibitor, which has been used in Japan (1995) for the management of Reynaud's syndrome, pulmonary hypertension, and vasospasm secondary to subarachnoid hemorrhage, angina pectoris, and also to treat complications from high blood pressure. Currently, some authors are looking for efficacy, safety and tolerability of a ROCK-ALS of fasudil in MND/ALS cases that started patient recruitment in 2019 [91]. ROCK (serine/threonine kinase) is a novel medication to target for neurodegenerative brain disorders, and it has two isoforms: ROCK1 is mainly for the peripheral nervous system, and ROCK2 is expressed preferentially in nervous central system [92]. Levels of ROCK augment according to age and tissue of MND/ALS cases. Some authors also confirmed increased levels of ROCK2 and downstream targets LIMK1 and coiling [93]. Fasudil also modifies microglia activity [91]. Main side effect of fasudil is intraparenchymal hemorrhage. Nevertheless, in studied population of cases with subarachnoid hemorrhage, the incidence of bleeding did not differ remarkably from the control group. Therefore, hemorrhage is not an expected complication, and it will not put in risk the life in selected patient. Cases with past medical history of intraparenchymal bleeding, congenital or acquire aneurysms or Moyamoya disease should not be included in the therapeutic group. Lingor et al. confirmed that ROCK-MND/ALS clinical trial provides a welltolerated, safe, and accurate way of treatment. The biomarker collection associated with this study will deliver additional data as indicators of progression. Finally, we comment about Lunasin a soy peptide that modify histone acetylation in vitro joined to single MND/ALS effect but no remarkable activity on histone acetylation or disorder deterioration in 50 participants treated during 5.5 months by Bedlack et al. [94]. Excellent retention and adherence have been found but not better results than

As a result of the great interest showed by investigators and participants on different studies done during the first half of this year (2019), today we can see an important number of other therapeutic procedures also looking for the best management of ALS patients. This modality ranges from physical exercises to acupuncture including other choices such as stem cell therapy, treatment for sialorrhea, and

In our times, many healthy people do physical exercises on regular basis; some do exercise for slimming purposes, others for prophylactic treatment, rehabilita-

Unfortunately, due to lack of space, these topics will not be included in this

After reviewing the most recent studies published in the medical literature, we concluded that we still have no curative treatment for MND patients, but a promis-

clinical trial targeting AQP4 has been done up to date [90].

#### *Introduction to Novel Motor Neuron Disease DOI: http://dx.doi.org/10.5772/intechopen.91921*

*Novel Aspects on Motor Neuron Disease*

Fasudil, and Lunasil.

are EH301, 5Fluoroucil, Tryptophan, RNS60, Rasagiline, Tirasemtiv, Aquaporin,

clinical benefits in many key outcome measures relative to their baseline.

a low dose of 5-FU or its analogs may have good effects on MND/ALS [85].

known to be important in template-directed misfolding [86].

well assimilated, and no serious adverse events were report.

Shefner et al. conducted a multinational clinical trial to study the accuracy of tirasemtiv (125mg twice daily) using an escalating dosage protocol during 4 weeks [89]. Comparisons between two series of cases with ALS were performed. One group constituted by fast skeletal muscle troponin activator and a second control group by placebo. Of 744 candidates, 565 participants assimilated open-label

de la Rubia et al. [84] evaluated the accuracy and feasibility of Elysium Health's candidate drug EH301 in ALS cases by a single-center, prospective, double-blind, randomized, placebo-controlled pilot study. Thirty-two ALS patients studied underwent for assessment during 4 months. Differences between EH301 and control group were evaluated based on their findings, and EH301 confirmed a remarkable slow progression of ALS compared with nontreated cases and even confirmed

Searching for drug candidates for ALS, Rando et al. investigated the action of anti-metabolite 5-fluorouracil (5-FU) administered by a single intraperitoneal injection at 150 mg/kg in SOD1G93A model of ALS. Un expectedly, the authors found that 5-FU (anti-cancer drug) increases survival delays of the disorder onset and improves motor function in ALS mice, but they were not able to demonstrate the mechanism of the beneficial 5-FU action in ALS mice. Despite of 5-FU did not improve the modulate motor neuron survival remarkably and did not improve reactive gliosis or change the muscle morphology, their findings recommended that

Other authors postulate that toxic gain of function, spread, and SOD1 misfolding is suggested as part of pathological mechanism of MND/ALS, but the nature of SOD1 toxicity has been hard to describe [86]. Only in SOD1 proteins from humans and other primates, and rarely in other species, a tryptophan residue at position 32 (W32) is predicted to be solvent exposed and to participate in SOD1 misfolding. DuVal et al. considered that W32 is influential in SOD1 acquiring toxicity, as it is

DuVal et al. highlighted the relevant influence of W32 on cases SOD1 toxicity to upper and lower motor neuron cells morphology and its activities. They assessed pharmaceutical targeting of the W32 residue for rescuing SOD1 toxicity [86]. When RNS60 is administered by IV infusion every 7 days and daily nebulization, it acts as a novel immune-modulator agent able to provide neuroprotective action in MND/ALS preclinical models. Paganoni et al. [87] studied 16 ALS patients during 23 weeks for safety and tolerability. Some investigations were done such as PBR28 positron emission tomography imaging and plasma biomarkers of inflammation. These authors did not find serious reactions, and no participants were removed from the study due to drug-related complications. At the present moment, a large, multicenter, Phase II trial of RNS60 is currently including cases to test the effects of RNS60 on MND/ALS biomarkers and disease deterioration based on the previous findings. They concluded that long-term RNS60 administered by IV infusion (as indicated) is well assimilated by patients and is also accurate [87]. In ALS, the prolong use of immune-modulating therapies has been showing not good results and did not help to understand how the immune system modifies disease outcome [88]. Rasagiline (monoamine oxidase B inhibitor) administered at 2 mg/day has a neuroprotective effect in MNS/ALS cases. In order to verify this postulate, Fernandez et al. performed a trial of 80 ALS patients from 10 hospitals in America. They did not identify any difference between Rasagiline-treated cases and the control group. Therefore, they assumed that Rasagiline did not change disease output compared with control group during 12 months of therapy. Rasagiline was

**62**

tirasemtiv and were treated randomly. As a side effect of tirasemtiv, nausea, weight loss, dizziness, fatigue, and insomnia were more often seen. The frequency of severe side effects was seen equally on both series of cases. Obviously, tirasemtiv did not change the decline of slow vital capacity or remarkable impact secondary outcome assessment and weak tolerability of tirasemtiv may lead to poor effect on MND/ALS.

Aquaporin 4 (AQP4) is present in astrocytes in the nervous system as primary water channel and has been postulated to participate in a myriad of acute, chronic brain disorders and the incidence of MND/ALS. Depolarization of AQP4 causes degeneration of the upper and lower motor neurons via GLT-1, and suppressions increase recovery of motor activity in MND/ALS cases probable due to NGF. No clinical trial targeting AQP4 has been done up to date [90].

Studies made with Fasudil (30 mg for IV application) have shown good results in cell culture and animal research of MND/ALS. This medication is a Rho kinase (ROCK) inhibitor, which has been used in Japan (1995) for the management of Reynaud's syndrome, pulmonary hypertension, and vasospasm secondary to subarachnoid hemorrhage, angina pectoris, and also to treat complications from high blood pressure. Currently, some authors are looking for efficacy, safety and tolerability of a ROCK-ALS of fasudil in MND/ALS cases that started patient recruitment in 2019 [91]. ROCK (serine/threonine kinase) is a novel medication to target for neurodegenerative brain disorders, and it has two isoforms: ROCK1 is mainly for the peripheral nervous system, and ROCK2 is expressed preferentially in nervous central system [92]. Levels of ROCK augment according to age and tissue of MND/ALS cases. Some authors also confirmed increased levels of ROCK2 and downstream targets LIMK1 and coiling [93]. Fasudil also modifies microglia activity [91]. Main side effect of fasudil is intraparenchymal hemorrhage. Nevertheless, in studied population of cases with subarachnoid hemorrhage, the incidence of bleeding did not differ remarkably from the control group. Therefore, hemorrhage is not an expected complication, and it will not put in risk the life in selected patient. Cases with past medical history of intraparenchymal bleeding, congenital or acquire aneurysms or Moyamoya disease should not be included in the therapeutic group.

Lingor et al. confirmed that ROCK-MND/ALS clinical trial provides a welltolerated, safe, and accurate way of treatment. The biomarker collection associated with this study will deliver additional data as indicators of progression. Finally, we comment about Lunasin a soy peptide that modify histone acetylation in vitro joined to single MND/ALS effect but no remarkable activity on histone acetylation or disorder deterioration in 50 participants treated during 5.5 months by Bedlack et al. [94]. Excellent retention and adherence have been found but not better results than riluzole or edaravone.

As a result of the great interest showed by investigators and participants on different studies done during the first half of this year (2019), today we can see an important number of other therapeutic procedures also looking for the best management of ALS patients. This modality ranges from physical exercises to acupuncture including other choices such as stem cell therapy, treatment for sialorrhea, and spasticity, among others.

In our times, many healthy people do physical exercises on regular basis; some do exercise for slimming purposes, others for prophylactic treatment, rehabilitation, and so forth.

Unfortunately, due to lack of space, these topics will not be included in this chapter.

After reviewing the most recent studies published in the medical literature, we concluded that we still have no curative treatment for MND patients, but a promising future is forthcoming.

*Novel Aspects on Motor Neuron Disease*

#### **Author details**

Humberto Foyaca Sibat\* and Lourdes de Fátima Ibañez Valdés Department of Neurology, Faculty of Health Sciences, Nelson Mandela Academic Central Hospital, Walter Sisulu University, Mthatha, South Africa

\*Address all correspondence to: humbertofoyacasibat@gmail.com

© 2020 The Author(s). Licensee IntechOpen. 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.

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**Author details**

Humberto Foyaca Sibat\* and Lourdes de Fátima Ibañez Valdés

Central Hospital, Walter Sisulu University, Mthatha, South Africa

\*Address all correspondence to: humbertofoyacasibat@gmail.com

provided the original work is properly cited.

Department of Neurology, Faculty of Health Sciences, Nelson Mandela Academic

© 2020 The Author(s). Licensee IntechOpen. 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,

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s11064-019-02814-4

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Neurological Sciences. 2019;**40**(2):235- 241. DOI: 10.1007/s10072-018-3653-2

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DOI: 10.1002/mus.26385

10.1002/mus.26288

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10.1371/journal.pone.0140316

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**70**

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**73**

Section 2

Novel Information on

Amyoyrophic Lateral

Sclerosis and Spinal

Muscular Atrophic

### Section 2
