Emerging Etiopathology and Implications

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

disorders. Current Pharmaceutical Design. 2017;**23**:731-752. DOI: 10.2174/1

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in models of Alzheimer's disease. Nature Neuroscience. 2019;**22**:401-412. DOI:

[97] Wang Y, Zhang N, Zhang L, Li R,

[98] Chang C-Y, Liang M-Z, Chen L. Current progress of mitochondrial transplantation that promotes neuronal regeneration. Translational Neurodegeneration. 2019;**8**:17. DOI:

[99] Hayakawa K, Esposito E, Wang X, Terasaki Y, Liu Y, Xing C, et al. Transfer of mitochondria from astrocytes to neurons after stroke. Nature. 2016;**535**:551-555. DOI: 10.1038/

[100] McCully JD, Levitsky S, del Nido PJ, Cowan DB. Mitochondrial transplantation for therapeutic use. Clinical and Translational Medicine. 2016;**5**:16. DOI: 10.1186/

[101] Masuzawa A, Black KM, Pacak CA, Ericsson M, Barnett RJ, Drumm C, et al. Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury. American Journal of Physiology. Heart and Circulatory Physiology. 2013;**304**:H966-H982. DOI: 10.1152/

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**78**

**Chapter 5**

*and David L. Mack*

and cure this inveterate disease.

therapeutic agent

**1. Introduction**

**81**

**Abstract**

The Role of Extracellular Vesicles

in the Progression of ALS and

Their Potential as Biomarkers

and Therapeutic Agents with

Which to Combat the Disease

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that impairs motor neuron function, leading to severe muscular atrophy. The non-cell autonomous and heterogeneous nature of the disease has so far hindered attempts to define ALS etiology, leaving the disease incurable and without effective treatments. Recent studies have focused on the pathologic role of intercellular communication between nerve cells to further our understanding of ALS pathophysiology. In this chapter, we summarize recent works investigating the role of extracellular vesicles (EVs) as a means of cellular crosstalk for ALS disease propagation, diagnosis, and treatment. There is growing evidence that EVs secreted by the majority of mammalian cells serve as effective biomolecule carriers to modulate recipient cell behavior. This underscores the need to understand the EV-mediated interplay that occurs within irreversibly degenerating nervous tissue in ALS patients. Additionally, we highlight current gaps in EV-ALS research, especially in terms of the pathologic role and responsibilities of specific EV cargos in diseased cells, specificity issues associated with the use of EVs in ALS diagnosis, and the efficacy of EVmediated treatments for the restoration of diseased neuromuscular tissue. Finally, we provide suggestions for future EV-ALS research to better understand, diagnose,

**Keywords:** ALS, extracellular vesicle, exosome, propagation, biomarker,

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig's disease, is a heterogeneous neurodegenerative disease that primarily impairs both upper and lower motor neurons [1, 2]. 5000 people in the United States are diagnosed with ALS each year, mostly between the ages of 40 and 70. The irreversibly progressive nature of ALS leads to the death of most patients within 2–5 years of diagnosis,

*Changho Chun, Alec S.T. Smith, Mark Bothwell*

#### **Chapter 5**

## The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers and Therapeutic Agents with Which to Combat the Disease

*Changho Chun, Alec S.T. Smith, Mark Bothwell and David L. Mack*

#### **Abstract**

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that impairs motor neuron function, leading to severe muscular atrophy. The non-cell autonomous and heterogeneous nature of the disease has so far hindered attempts to define ALS etiology, leaving the disease incurable and without effective treatments. Recent studies have focused on the pathologic role of intercellular communication between nerve cells to further our understanding of ALS pathophysiology. In this chapter, we summarize recent works investigating the role of extracellular vesicles (EVs) as a means of cellular crosstalk for ALS disease propagation, diagnosis, and treatment. There is growing evidence that EVs secreted by the majority of mammalian cells serve as effective biomolecule carriers to modulate recipient cell behavior. This underscores the need to understand the EV-mediated interplay that occurs within irreversibly degenerating nervous tissue in ALS patients. Additionally, we highlight current gaps in EV-ALS research, especially in terms of the pathologic role and responsibilities of specific EV cargos in diseased cells, specificity issues associated with the use of EVs in ALS diagnosis, and the efficacy of EVmediated treatments for the restoration of diseased neuromuscular tissue. Finally, we provide suggestions for future EV-ALS research to better understand, diagnose, and cure this inveterate disease.

**Keywords:** ALS, extracellular vesicle, exosome, propagation, biomarker, therapeutic agent

#### **1. Introduction**

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig's disease, is a heterogeneous neurodegenerative disease that primarily impairs both upper and lower motor neurons [1, 2]. 5000 people in the United States are diagnosed with ALS each year, mostly between the ages of 40 and 70. The irreversibly progressive nature of ALS leads to the death of most patients within 2–5 years of diagnosis,

typically due to respiratory failure [1, 3]. In most patients, the cause of ALS is unknown. Only 10% of patients have a familial history of ALS, caused by specific mutations in their genomes, while 90% are exhibit sporadic ALS due to unknown causal factors [1, 4, 5]. The lack of understanding concerning ALS disease mechanisms has led to the development of very few FDA approved drugs. To date, those drugs that have progressed to the US market merely slow down disease progression by a few months and do little to restore patient's neuromuscular function [6]. As progressive degeneration and the non-cell autonomous nature of ALS are known to be major reasons for this stalemate, the aim of much current ALS research is focused on understanding the diverse interplay between neurons and non-neuronal components in neuromuscular tissue. Among the non-neuronal components, glial cells and their crosstalk with neurons have been major targets of study regarding symptomatic development and progression in ALS [7–10]. Accordingly, research into a myriad of proteins and RNAs known to be transmissible between neurons and glial cells has been gaining interest in recent years. It is already well-established that such agents can act as molecular 'messengers' to alter the behavior of nearby cells in ALS, but the detailed mechanisms that underpin specific cargo selection, initiation of transport, and subsequent activation of the internalized biomolecules are not fully understood.

applicability in the early diagnosis and further treatment of the disease. In this review, we will highlight recent research studying the diverse roles of EVs in ALS progression, diagnosis, and treatment. In addition, we will discuss possible solutions for unsolved problems in this area and suggest future directions for ALS-EV research to further our understanding of ALS pathology and help develop advanced

*The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers…*

**2. EVs as dysregulated biomolecule carriers for disease propagation**

in their cytoplasm and these structures often exert detrimental effects on cell viability and function. SOD1 (superoxide dismutase 1) and TDP-43 (transactive DNA binding protein 43 kDa), encoded by the *SOD1* and *TARDBP* genes respectively, are the most well-studied proteins susceptible to ALS-associated aggregation. Aggregation of these proteins, in some cases due to destabilizing mutations, are known to be actively involved in motor neuron degeneration in both familial and sporadic ALS [1, 24, 25]. The SOD1 enzyme resides in the cytoplasm of normal cells to regulate oxidative stress by converting free superoxide radicals into molecular oxygen [2]. There are more than 180 mutations reported in the *SOD1* gene, and oligomerization of the encoded proteins has been shown to cause increased intracellular oxidative stress and anomalous metal binding [2]. However, the pathologic pathway connecting *SOD1* mutation, protein dysregulation, and subsequent

ALS tissues commonly contain cells supporting dysregulated protein aggregates

neurodegeneration have yet to be defined in a comprehensive manner [2, 26]. TDP-43 is a highly conserved nuclear RNA and DNA binding protein, known to be involved in transcriptomic regulation, primarily by RNA splicing but also by effects on mRNA transport and stability, effects on microRNA production, and participating in DNA repair [27–31]. Approximately 97% of ALS patients have abnormally aggregated TDP-43 in their neurons, even without direct mutation of the *TARDBP* gene, leading to ALS proteinopathic characteristics in their pathology [6, 32].

Strikingly, recent ALS studies have demonstrated that abnormally transformed proteins, such as SOD1 and TDP-43, do not merely impair the cells of origin, but migrate to neighboring cells by means of extracellular exosome release, resulting in a spread of their cytotoxic effect to recipient cells [33, 34]. Exosome shuttling has been shown to be a preferred cellular mechanism for removing intracellular toxic molecules to the extracellular space [35]. The exosomal loading of cytotoxic protein aggregates is beneficial to host cells since it minimizes the physiological damage caused by these structures. Such phenomena could be promoted by the host cell recognizing an increase in intracellular protein aggregation or impaired lysosomal autophagy. Interestingly, normal neurons cultured with TDP-43 aggregate-loaded exosomes induce cytoplasmic redistribution of endogenous TDP-43, leading to an exacerbation of the disease phenotype in mice [36]. Moreover, proteins packaged in extracellular vesicles are preferentially taken up by recipient cells and exhibit a greater detrimental physiological effect compared to free protein release, highlighting a crucial role for EV and plasma membrane tethered proteins in regulating protein internalization and functional activation [37]. These studies suggest an important interaction between exogenous TDP-43 transported via extracellular vesicles and endogenous TDP-43 expressed by the recipient cell. Such interactions constitute a pre-requisite for 'prion-like'seeding followed by cytoplasmic protein redistribution and offer a potential mechanism for the rapid propagation of disease phenotypes throughout the motor neuron pool. Indeed, when insoluble TDP-43 aggregates taken from ALS brain were introduced to neuron-like SH-SY5Y cells

diagnosis and treatment methods using EVs.

*DOI: http://dx.doi.org/10.5772/intechopen.91388*

**in ALS**

**83**

Extracellular vesicles (EVs) are lipid bi-layered particles, less than 2 μm in size, secreted by the majority of mammalian cells, including nerve cells, to mediate diverse paracrine signaling pathways [11–13]. These vesicles are typically classified into three categories: microvesicles, apoptotic bodies, and exosomes [14]. Microvesicles and apoptotic bodies are normally 0.1–2 μm in size and bud directly from the plasma membrane. Exosomes, on the other hand, are smaller (50–150 nm) and are generated within cytoplasmic multivesicular bodies (MVBs) before being secreted to the extracellular space through subsequent fusion of MVBs with the plasma membrane [11, 15].

In many neurodegenerative disorders, EVs derived from nerve cells have been proposed to be responsible for spreading neurotoxins to normal cells, accelerating disease progression [16–20]. In the diseased cell, misregulated proteins serve as 'templates' for subsequent protein oligomerization, generating insoluble toxic aggregates. Such aggregates are then either degraded by lysosomes using a 'selfclearance' mechanism, or incorporated into MVBs and/or the plasma membrane facilitating subsequent release into the extracellular space [21]. EV-loaded biomolecules can then be transmitted to recipient cells primarily by endocytosis but also by endosomal fusion of the EV membrane with cell's plasma membrane [22]. For example, exosomes facilitate the intercellular delivery of amyloid beta (Aβ) peptides in Alzheimer's disease, leading to plaque formation in the recipient cells [19, 20]. A similar trend of EV-mediated or free protein spreading in a 'prion-like' manner is observed in Parkinson's disease (PD). Specifically, studies with mice have demonstrated that grafted cells containing aggregated α-synuclein can transfer these protein aggregates to healthy brain tissue [23].

As the contents of EVs reflect the physiological status of the original cell, recent studies have begun to evaluate the possibility of using these structures as biomarkers with which to gauge the onset and progression of a diverse range of neurodegenerative diseases. Issues remain with identifying tissue specificity and cell type of origin for pathogenic EVs found in body fluids. However, convenient sampling and improved understanding of internal cargo molecules' function has led to EV's being seen as one of the strongest candidate classes for next generation prognosis/diagnosis screening. As with other degenerative diseases of the central nervous system (CNS), much recent work on ALS has focused on investigating the pathological role of EVs in diseased neuromuscular tissue, as well as evaluating their *The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.91388*

applicability in the early diagnosis and further treatment of the disease. In this review, we will highlight recent research studying the diverse roles of EVs in ALS progression, diagnosis, and treatment. In addition, we will discuss possible solutions for unsolved problems in this area and suggest future directions for ALS-EV research to further our understanding of ALS pathology and help develop advanced diagnosis and treatment methods using EVs.

#### **2. EVs as dysregulated biomolecule carriers for disease propagation in ALS**

ALS tissues commonly contain cells supporting dysregulated protein aggregates in their cytoplasm and these structures often exert detrimental effects on cell viability and function. SOD1 (superoxide dismutase 1) and TDP-43 (transactive DNA binding protein 43 kDa), encoded by the *SOD1* and *TARDBP* genes respectively, are the most well-studied proteins susceptible to ALS-associated aggregation. Aggregation of these proteins, in some cases due to destabilizing mutations, are known to be actively involved in motor neuron degeneration in both familial and sporadic ALS [1, 24, 25]. The SOD1 enzyme resides in the cytoplasm of normal cells to regulate oxidative stress by converting free superoxide radicals into molecular oxygen [2]. There are more than 180 mutations reported in the *SOD1* gene, and oligomerization of the encoded proteins has been shown to cause increased intracellular oxidative stress and anomalous metal binding [2]. However, the pathologic pathway connecting *SOD1* mutation, protein dysregulation, and subsequent neurodegeneration have yet to be defined in a comprehensive manner [2, 26]. TDP-43 is a highly conserved nuclear RNA and DNA binding protein, known to be involved in transcriptomic regulation, primarily by RNA splicing but also by effects on mRNA transport and stability, effects on microRNA production, and participating in DNA repair [27–31]. Approximately 97% of ALS patients have abnormally aggregated TDP-43 in their neurons, even without direct mutation of the *TARDBP* gene, leading to ALS proteinopathic characteristics in their pathology [6, 32].

Strikingly, recent ALS studies have demonstrated that abnormally transformed proteins, such as SOD1 and TDP-43, do not merely impair the cells of origin, but migrate to neighboring cells by means of extracellular exosome release, resulting in a spread of their cytotoxic effect to recipient cells [33, 34]. Exosome shuttling has been shown to be a preferred cellular mechanism for removing intracellular toxic molecules to the extracellular space [35]. The exosomal loading of cytotoxic protein aggregates is beneficial to host cells since it minimizes the physiological damage caused by these structures. Such phenomena could be promoted by the host cell recognizing an increase in intracellular protein aggregation or impaired lysosomal autophagy. Interestingly, normal neurons cultured with TDP-43 aggregate-loaded exosomes induce cytoplasmic redistribution of endogenous TDP-43, leading to an exacerbation of the disease phenotype in mice [36]. Moreover, proteins packaged in extracellular vesicles are preferentially taken up by recipient cells and exhibit a greater detrimental physiological effect compared to free protein release, highlighting a crucial role for EV and plasma membrane tethered proteins in regulating protein internalization and functional activation [37]. These studies suggest an important interaction between exogenous TDP-43 transported via extracellular vesicles and endogenous TDP-43 expressed by the recipient cell. Such interactions constitute a pre-requisite for 'prion-like'seeding followed by cytoplasmic protein redistribution and offer a potential mechanism for the rapid propagation of disease phenotypes throughout the motor neuron pool. Indeed, when insoluble TDP-43 aggregates taken from ALS brain were introduced to neuron-like SH-SY5Y cells

typically due to respiratory failure [1, 3]. In most patients, the cause of ALS is unknown. Only 10% of patients have a familial history of ALS, caused by specific mutations in their genomes, while 90% are exhibit sporadic ALS due to unknown causal factors [1, 4, 5]. The lack of understanding concerning ALS disease mechanisms has led to the development of very few FDA approved drugs. To date, those drugs that have progressed to the US market merely slow down disease progression by a few months and do little to restore patient's neuromuscular function [6]. As progressive degeneration and the non-cell autonomous nature of ALS are known to be major reasons for this stalemate, the aim of much current ALS research is focused on understanding the diverse interplay between neurons and non-neuronal components in neuromuscular tissue. Among the non-neuronal components, glial cells and their crosstalk with neurons have been major targets of study regarding symptomatic development and progression in ALS [7–10]. Accordingly, research into a myriad of proteins and RNAs known to be transmissible between neurons and glial cells has been gaining interest in recent years. It is already well-established that such agents can act as molecular 'messengers' to alter the behavior of nearby cells in ALS, but the detailed mechanisms that underpin specific cargo selection, initiation of transport, and subsequent activation of the internalized biomolecules

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

Extracellular vesicles (EVs) are lipid bi-layered particles, less than 2 μm in size,

secreted by the majority of mammalian cells, including nerve cells, to mediate diverse paracrine signaling pathways [11–13]. These vesicles are typically classified

Microvesicles and apoptotic bodies are normally 0.1–2 μm in size and bud directly from the plasma membrane. Exosomes, on the other hand, are smaller (50–150 nm) and are generated within cytoplasmic multivesicular bodies (MVBs) before being secreted to the extracellular space through subsequent fusion of MVBs with the

In many neurodegenerative disorders, EVs derived from nerve cells have been proposed to be responsible for spreading neurotoxins to normal cells, accelerating disease progression [16–20]. In the diseased cell, misregulated proteins serve as 'templates' for subsequent protein oligomerization, generating insoluble toxic aggregates. Such aggregates are then either degraded by lysosomes using a 'selfclearance' mechanism, or incorporated into MVBs and/or the plasma membrane facilitating subsequent release into the extracellular space [21]. EV-loaded biomolecules can then be transmitted to recipient cells primarily by endocytosis but also by endosomal fusion of the EV membrane with cell's plasma membrane [22]. For example, exosomes facilitate the intercellular delivery of amyloid beta (Aβ) peptides in Alzheimer's disease, leading to plaque formation in the recipient cells [19, 20]. A similar trend of EV-mediated or free protein spreading in a 'prion-like' manner is observed in Parkinson's disease (PD). Specifically, studies with mice have demonstrated that grafted cells containing aggregated α-synuclein can transfer

As the contents of EVs reflect the physiological status of the original cell, recent

studies have begun to evaluate the possibility of using these structures as biomarkers with which to gauge the onset and progression of a diverse range of neurodegenerative diseases. Issues remain with identifying tissue specificity and cell type of origin for pathogenic EVs found in body fluids. However, convenient sampling and improved understanding of internal cargo molecules' function has led to EV's being seen as one of the strongest candidate classes for next generation prognosis/diagnosis screening. As with other degenerative diseases of the central nervous system (CNS), much recent work on ALS has focused on investigating the pathological role of EVs in diseased neuromuscular tissue, as well as evaluating their

into three categories: microvesicles, apoptotic bodies, and exosomes [14].

are not fully understood.

plasma membrane [11, 15].

**82**

these protein aggregates to healthy brain tissue [23].

endogenously expressing normal TDP-43, the treatment induced significant aggregation of TDP-43 in recipient cells in a self-templating manner [38]. Similarly, SOD1 aggregates transferred via exosomes have been shown to work as self-templating 'seeds' in recipient cells, leading to the propagation of a misfolded protein that persists in culture over multiple passages and population doublings [34].

**Role of EV Study method Source of EV Significance Unknowns Reference**

*The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers…*

Micro-RNAs in ALS astrocytederived extracellular vesicle (EV) caused neuronal network degeneration and growth cone impairment

Brain-derived astrocytes and neurons, but not microglia, were the main EV source in CNS

Microvesicles of ALS patients were enriched with potentially pathological protein (SOD1, TDP-43, FUS), while exosomes did not show any protein changes

EVs derived from ALS myotubes encapsulated H2-AX (neurotoxin) which suggests possible dissemination of various neurotoxic molecules from diseased skeletal muscle to normal neurons

Exosome secretion was beneficial in neuronal clearance of pathological TDP-43, but also it might be responsible for the propagation of the toxic TDP-43 aggregates to the other cells

DPRs can be transmitted to neurons and glial cells with/

The role of C9ORF72 protein for dysregulated miRNA encapsulation in

Detailed cargo modification pattern of EVs in the diseased cells

Contradictory result with other (*in vitro*) studies that suggested huge loading of dysregulated proteins in exosomes of ALS mutant cells

Is H2-AX a major neurotoxin in human ALS skeletal muscle as

Should we inhibit exosome secretion to prevent aggregated TDP-43 propagation or promote the secretion for TDP-43 clearance in neurons?

Difference between propagated DPRs and other

well?

Varcianna et al. [41]

Silverman et al. [7]

Sproviero et al. [59]

Gall et al. [60]

Iguchi et al. [36]

Westergard et al. [61]

EV

Directly differentiated astrocytes from fALS (C9ORF72) patient-derived fibroblast

Brain- and spinal cord tissue from NTg and SOD1 mutant mice

Venous blood of sALS patients

Skeletal muscle cells from SOD1 mutant mouse

Primary neurons and Neuro2a cells from ALS mouse brain

Mutant DPR (dipeptide repeat proteins)

Dysregulated protein and RNA carrier

*In vitro* study with astrocytes and mouse motor neurons

*DOI: http://dx.doi.org/10.5772/intechopen.91388*

*In vitro/ex vivo* study for whole tissue vesicle isolation

Clinical research with sporadic ALS patients' body fluid for EV analysis

*In vitro* study with ALS mouse musclederived exosomes treated to primary/iPSCderived motor neurons

*In vitro* study using Neuro2a and primary neurons to study the role of exosomes in ALS proteinopathy

*In vitro* coculture of NSC-34 with cortical

**85**

Although recent studies have observed intercellular protein transmission via EVs, their specific roles in neuromuscular pathophysiology are poorly understood. In particular, how the transfer of abnormal proteins induces subsequent neuronal damage and a breakdown in their electrophysiological function has yet to be elucidated. Furthermore, by taking into account that ALS exhibits non-neuron autonomous characteristics, it is essential to obtain a more comprehensive understanding of whether and how non-neuronal nerve cells can damage neuromuscular function via the exosomal transfer pathway. **Table 1** provides an overview of the studies performed so far relating to the role of EVs in propagating ALS pathologies to neighboring cells and these studies are discussed in more detail throughout this section.

Among non-neuronal cell types, astrocytes have gained significant interest as carriers of detrimental protein aggregates in ALS tissues. Secretome analysis of astrocytic exosomes from SOD1 (G93A) mutant mice were reported to contain SOD1 aggregates, and the exosomal release of these structures accounted for a larger proportion of SOD1 transport than free SOD1 release [4, 14]. In addition, proteomic analysis has revealed that proteins involved in vesicle trafficking are downregulated in mutant astrocytes, indicating a possible impairment of protein disposal in ALS astrocytes. Moreover, wild-type mice transplanted with astrocyte progenitors expressing mutant SOD1 exhibit motor neuron impairment, raising the reasonable postulation that diseased astrocytes utilize extracellular vesicle-mediated paracrine communication to deliver pathogenic protein aggregates to motor neurons [4, 14]. Another quantitative proteomic analysis of EVs derived from ALS nervous tissue showed a relative absence of the microglial marker (CD11b) but positive expression for the astrocyte marker (GLAST) and the synaptic marker (SNAP25), indicating that astrocytes and neurons may constitute the major cell types involved in EV-mediated communication in the CNS [7]. However, as the main function of microglia in the CNS is immune response regulation under proinflammatory conditions, microglia might also actively secrete EVs within an immune-active environment to modulate the immune response of other nerve cells. Since ALS is reported to have autoimmune disease characteristics as well [39, 40], a pathogenic role for EVs secreted from diseased microglia in ALS tissue could be another important subject to explore.

Do skeletal muscle cells also secrete EVs for neuronal uptake? The breakdown of neuromuscular junctions (NMJs; the synapses connecting lower motor neurons and skeletal muscle fibers) is a critical early indicator of pathological onset in ALS. However, it remains unclear whether NMJ breakdown occurs due to general ill health of the motor neuron or some aberrant signaling between these neurons and their afferent synaptic contacts. If the latter is true, a reasonable hypothesis would be that ALS progression is affected by paracrine communication between skeletal muscle and motor neurons. To this point, an interesting study in 2017 described the involvement of skeletal muscle in EV-based crosstalk in neuromuscular tissues. In ALS muscle biopsies, noticeable accumulation of MVBs containing exosome-like vesicles were measured, with significant increases in vesicular protein concentrations reported when compared with controls. In addition to the denser protein accumulation reported in the EVs, ALS skeletal muscle-derived EVs were shown to exclusively damage neuronal viability *in vitro* [4, 14].


*The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.91388*

endogenously expressing normal TDP-43, the treatment induced significant aggregation of TDP-43 in recipient cells in a self-templating manner [38]. Similarly, SOD1 aggregates transferred via exosomes have been shown to work as self-templating 'seeds' in recipient cells, leading to the propagation of a misfolded protein that persists in culture over multiple passages and population

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

Although recent studies have observed intercellular protein transmission via EVs, their specific roles in neuromuscular pathophysiology are poorly understood. In particular, how the transfer of abnormal proteins induces subsequent neuronal damage and a breakdown in their electrophysiological function has yet to be elucidated. Furthermore, by taking into account that ALS exhibits non-neuron autonomous characteristics, it is essential to obtain a more comprehensive understanding of whether and how non-neuronal nerve cells can damage neuromuscular function via the exosomal transfer pathway. **Table 1** provides an overview of the studies performed so far relating to the role of EVs in propagating ALS pathologies to neighboring cells and these studies are discussed in more detail throughout

Among non-neuronal cell types, astrocytes have gained significant interest as carriers of detrimental protein aggregates in ALS tissues. Secretome analysis of astrocytic exosomes from SOD1 (G93A) mutant mice were reported to contain SOD1 aggregates, and the exosomal release of these structures accounted for a larger proportion of SOD1 transport than free SOD1 release [4, 14]. In addition, proteomic analysis has revealed that proteins involved in vesicle trafficking are downregulated in mutant astrocytes, indicating a possible impairment of protein disposal in ALS astrocytes. Moreover, wild-type mice transplanted with astrocyte progenitors expressing mutant SOD1 exhibit motor neuron impairment, raising the reasonable postulation that diseased astrocytes utilize extracellular vesicle-mediated paracrine communication to deliver pathogenic protein aggregates to motor neurons [4, 14]. Another quantitative proteomic analysis of EVs derived from ALS nervous tissue showed a relative absence of the microglial marker (CD11b) but positive expression for the astrocyte marker (GLAST) and the synaptic marker (SNAP25), indicating that astrocytes and neurons may constitute the major cell types involved in EV-mediated communication in the CNS [7]. However, as the main function of microglia in the CNS is immune response regulation under proinflammatory conditions, microglia might also actively secrete EVs within an immune-active environment to modulate the immune response of other nerve cells. Since ALS is reported to have autoimmune disease characteristics as well [39, 40], a pathogenic role for EVs secreted from diseased microglia in ALS tissue could be

Do skeletal muscle cells also secrete EVs for neuronal uptake? The breakdown of

neuromuscular junctions (NMJs; the synapses connecting lower motor neurons and skeletal muscle fibers) is a critical early indicator of pathological onset in ALS. However, it remains unclear whether NMJ breakdown occurs due to general ill health of the motor neuron or some aberrant signaling between these neurons and their afferent synaptic contacts. If the latter is true, a reasonable hypothesis would be that ALS progression is affected by paracrine communication between skeletal muscle and motor neurons. To this point, an interesting study in 2017 described the involvement of skeletal muscle in EV-based crosstalk in neuromuscular tissues. In ALS muscle biopsies, noticeable accumulation of MVBs containing exosome-like vesicles were measured, with significant increases in vesicular protein concentrations reported when compared with controls. In addition to the denser protein accumulation reported in the EVs, ALS skeletal muscle-derived EVs were shown to

doublings [34].

this section.

**84**

another important subject to explore.

exclusively damage neuronal viability *in vitro* [4, 14].


known to negatively regulate semaphorin-3A expression, which is highly involved in axonal growth and maintenance [41]. Similarly, exosomes secreted by mousederived motor neuron-like cells (NSC-34) transfected with a human SOD1 mutant variant were enriched with miR-124. This miRNA was in turn transmissible to the microglia, where it induced impairment of their phagocytic ability and an increase in pro-inflammatory gene expression [42]. These results indicate the presence of a multi-directional intercellular communication network in ALS that is mediated by miRNAs encapsulated in EVs. Researchers have only begun to scratch the surface of EV-mediated miRNA transfer in ALS and this area requires more comprehensive study to fully disentangle the pathologic milieu that exists among the different types

*The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers…*

Research investigating EV-mediated ALS pathophysiology is in an incipient stage, and impressive results in previous studies are still raising a number of important questions that must be addressed. For example, it is unclear whether the beneficial effect of elimination of intracellular toxic proteins outweighs the deleterious effects of uptake of those proteins by neighboring cells. Understanding this point will be critical when designing therapeutic methods to inhibit the propagation process. Furthermore, to promote extracellular disposal of toxic protein aggregates while inhibiting their uptake by neighboring cells, we need to fully understand the interactions between surface proteins on detrimental EVs and those on the plasma membranes of motor neurons. Alternatively, efforts could be focused on enhancing lysosomal degradation process over EV-mediated protein disposal in diseased cells, but it is unclear whether the majority of detrimental contents in their multivesicular bodies can alternatively be delivered to autophagosomes for degradation. Another gap in EV-ALS research is a lack of studies focused on analyzing motor neuronderived EVs to understand the phenotypic effect of transmitting neuron-originated proteins to other neurons and/or non-neuronal cells within ALS tissues. As motor neurons are the main target in ALS pathogenesis, pathogenic proteins are likely to be enriched in motor neuron-derived EVs and could potentially directly damage glia and/or NMJ structures, or even directly affect skeletal muscle contractility. We believe that answering these questions, in addition to better characterizing nonneuronal EV cargos, should constitute the principle focuses of future studies aimed at improving our understanding of EV-mediated ALS progression. The results of such work would doubtlessly provide invaluable insights into ALS pathophysiology

and help identify suitable targets for future therapeutic development.

There is no reliable biomarker established for ALS, neither for confirming disease onset nor for characterizing disease progression [43]. The lack of a reliable biomarker for ALS makes the correct prognosis challenging, and even limits diagnosis to relatively late stages, after patients recognize their neuromuscular symptoms. As the typical life expectancy for ALS patients is approximately 2–5 years after disease onset, early and accurate diagnosis is crucial not only for developing early-stage applicable therapies, but for improving quality of life for patients during their follow-up period. A new detection method should be robust and convenient for clinical settings, and, critically, have sufficient detection sensitivity, specificity, and reproducibility to ensure confidence in the result. Meeting all these requirements simultaneously is an extremely challenging goal, given the extremely heterogeneous nature of the disease. Recent studies are evaluating ALS patient-derived EVs as potential biomarkers of ALS for prognosis, early diagnosis, and patient stratification. RNAs collected from patient's blood or cerebrospinal fluid (CSF;

**3. Can EVs serve as reliable biomarkers for ALS?**

**87**

of nerve cells in ALS nervous tissue.

*DOI: http://dx.doi.org/10.5772/intechopen.91388*

#### **Table 1.**

*Overview of the recent findings regarding roles for extracellular vesicles in ALS disease propagation.*

Although dysregulated protein transfer is of major interest in ALS-EV research, the role of transmissible miRNAs in facilitating ALS progression should not be overlooked. EVs obtained from astrocytes derived from ALS patient post-mortem tissue (*C9ORF72* mutation) have been shown to display a neurotoxic phenotype, inducing mouse motor neuron degeneration, though fewer EVs were secreted from diseased astrocytes than normal controls [41]. Dysregulated miR-494-3p was identified as a main component of the diseased EVs. This astrocyte-specific miRNA is

#### *The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.91388*

known to negatively regulate semaphorin-3A expression, which is highly involved in axonal growth and maintenance [41]. Similarly, exosomes secreted by mousederived motor neuron-like cells (NSC-34) transfected with a human SOD1 mutant variant were enriched with miR-124. This miRNA was in turn transmissible to the microglia, where it induced impairment of their phagocytic ability and an increase in pro-inflammatory gene expression [42]. These results indicate the presence of a multi-directional intercellular communication network in ALS that is mediated by miRNAs encapsulated in EVs. Researchers have only begun to scratch the surface of EV-mediated miRNA transfer in ALS and this area requires more comprehensive study to fully disentangle the pathologic milieu that exists among the different types of nerve cells in ALS nervous tissue.

Research investigating EV-mediated ALS pathophysiology is in an incipient stage, and impressive results in previous studies are still raising a number of important questions that must be addressed. For example, it is unclear whether the beneficial effect of elimination of intracellular toxic proteins outweighs the deleterious effects of uptake of those proteins by neighboring cells. Understanding this point will be critical when designing therapeutic methods to inhibit the propagation process. Furthermore, to promote extracellular disposal of toxic protein aggregates while inhibiting their uptake by neighboring cells, we need to fully understand the interactions between surface proteins on detrimental EVs and those on the plasma membranes of motor neurons. Alternatively, efforts could be focused on enhancing lysosomal degradation process over EV-mediated protein disposal in diseased cells, but it is unclear whether the majority of detrimental contents in their multivesicular bodies can alternatively be delivered to autophagosomes for degradation. Another gap in EV-ALS research is a lack of studies focused on analyzing motor neuronderived EVs to understand the phenotypic effect of transmitting neuron-originated proteins to other neurons and/or non-neuronal cells within ALS tissues. As motor neurons are the main target in ALS pathogenesis, pathogenic proteins are likely to be enriched in motor neuron-derived EVs and could potentially directly damage glia and/or NMJ structures, or even directly affect skeletal muscle contractility. We believe that answering these questions, in addition to better characterizing nonneuronal EV cargos, should constitute the principle focuses of future studies aimed at improving our understanding of EV-mediated ALS progression. The results of such work would doubtlessly provide invaluable insights into ALS pathophysiology and help identify suitable targets for future therapeutic development.

#### **3. Can EVs serve as reliable biomarkers for ALS?**

There is no reliable biomarker established for ALS, neither for confirming disease onset nor for characterizing disease progression [43]. The lack of a reliable biomarker for ALS makes the correct prognosis challenging, and even limits diagnosis to relatively late stages, after patients recognize their neuromuscular symptoms. As the typical life expectancy for ALS patients is approximately 2–5 years after disease onset, early and accurate diagnosis is crucial not only for developing early-stage applicable therapies, but for improving quality of life for patients during their follow-up period. A new detection method should be robust and convenient for clinical settings, and, critically, have sufficient detection sensitivity, specificity, and reproducibility to ensure confidence in the result. Meeting all these requirements simultaneously is an extremely challenging goal, given the extremely heterogeneous nature of the disease. Recent studies are evaluating ALS patient-derived EVs as potential biomarkers of ALS for prognosis, early diagnosis, and patient stratification. RNAs collected from patient's blood or cerebrospinal fluid (CSF;

Although dysregulated protein transfer is of major interest in ALS-EV research,

**Role of EV Study method Source of EV Significance Unknowns Reference**

without exosome involvement

ALS-CSF incubation with U251 cells increased mislocated TDP-43 in the glioma cells and induced their apoptotic behavior and macro autophagy process

TDP-43 oligomers were present in EVs and showed that microvesicular TDP-43 exerts higher toxicity than free TDP-43

Misfolded wildtype proteins could traverse cell-to-cell as a self-templating 'seed' either as free protein aggregates or loaded on the surface of exosomes

Mutant SOD1 astrocytes released increased number of exosomes, which were toxic for motor neurons

dysregulated proteins (TDP-43, SOD1) in terms of

neurodegeneration

EV encapsulating pathway of neurotoxic TDP-43 oligomers

Which specific receptors control the uptake of misfolded TDP-43/ SOD1 presented on exosomes?

Protein factors which involved in mutant SOD1 astrocytic exosome Zhou et al. [62]

Feiler et al. [37]

Nonaka et al. [38]

Basso et al. [26]

their

effect?

Connection between propagated TDP-43 and autophagy mechanism in recipient cells

transfected NSC-34 cells

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

CSF from 18 sALS patients

ALS postmortem lysate, primary cortical mouse neurons and HEK cells

Neuroblastoma cells expressing TDP-43 and SOD1

Primary astrocytes from mouse expressing human mutant SOD1

neurons and astrocytes

*In vitro* study using ALS patient-derived exosomes and human glioma cell line (U251)

*In vitro* study using primary neurons and TDP-43 transfected HEK cells

*In vitro* study with neuroblastoma cells with brain tissue obtained from ALS/FTD patients

*In vitro* study analyzing neurotoxic effect and amount of SOD1 protein encapsulated in astrocytic exosomes

**Table 1.**

**86**

the role of transmissible miRNAs in facilitating ALS progression should not be overlooked. EVs obtained from astrocytes derived from ALS patient post-mortem tissue (*C9ORF72* mutation) have been shown to display a neurotoxic phenotype, inducing mouse motor neuron degeneration, though fewer EVs were secreted from diseased astrocytes than normal controls [41]. Dysregulated miR-494-3p was identified as a main component of the diseased EVs. This astrocyte-specific miRNA is

*Overview of the recent findings regarding roles for extracellular vesicles in ALS disease propagation.*

often used as a surrogate for nervous tissue sampling) are the main target of analyses so far and advanced RNA-sequencing techniques are being employed to analyze deregulated RNAs exclusively in ALS patient-derived EVs. Recently, Otake et al. reported a new methodology using highly sensitive exoRNA-sequencing for comprehensive analysis of exosomal mRNAs in patient CSF, to identify abnormally expressed mRNAs in exosome samples from ALS patients [43]. The technique identified 543 mRNAs exhibiting statistically different expression patterns compared to normal samples. In particular, this analysis revealed that the gene *CUEDC2* was only detected in ALS patient-derived exosomes. As the gene is known to regulate the ubiquitin-proteasome pathway as part of the inflammatory response, its abnormal expression is postulated to cause potential neuroinflammation in ALS, making *CUEDC2* mRNA a strong biomarker candidate [43]. Follow-up studies are necessary to demonstrate a specific causal relationship between exosomal *CUEDC2* mRNA presence (rather than free intracellular *CUEDC2* expression) and ALS development. Furthermore, work demonstrating an omnipresence of the same RNA mis-regulation in EVs from other ALS patients is also required. However, the described study highlights how exosomal mRNAs could be attractive biomarker candidates, given their stability within body fluids, as well as the convenience of sample collection and ease of subsequent data analysis. Efforts to date to characterize EV cargos as ALS biomarkers, including those discussed in detail above and below, are summarized in **Table 2**.

**Role of EV Study method Source**

*DOI: http://dx.doi.org/10.5772/intechopen.91388*

exosomal mRNAs

Clinical study comparing the expression of miR-27a-3p in serumderived exosomes

Clinical analysis of miRNA profile in 16 ALS patients

*In vitro* study with NSC-34 and N9 microglia to discover ALS specific miRNAs

*In vitro* study using adipose-derived stem cells and human SOD1 overexpressing mouse NSCs

*In vitro* study assessing efficacy of stromal cell-derived exosomes on NSC-34 cells expressing hSOD1 mutants

Proteomic profiling of exosomes derived from mASC

Therapeutic agent

**89**

Biomarker Sequencing

in CSF

**of EV**

CSF of ALS patients and normal donors

*The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers…*

Serum of ALS patients and healthy subjects

Serum of ALS patients and healthy controls

Motor neuron like NSC-34 cells with mSOD1 expression

Adiposederived stem cells

Murine adiposederived stromal cells

Murine adipose**Significance Unknowns Reference**

Specificity level of mRNAs detected in ALS exosome using the technique

Specific role of miR-27-3p in the expression of disease phenotypes in ALS

Are ALSassociated miRNAs actually involved in posttranscriptional regulation of neurons?

miR-124 expression in other health state? (false positive issue)

Major exosomal cargos that exert each of therapeutic effects on recipient neurons

Where does the beneficial effect of ASCexosomes come from?

Exosomal effect on actual restoration of

Otake et al. [43]

Xu et al. [63]

Saucier et al. [45]

Pinto et al. [42]

Lee et al. [48]

Bonafede et al. [50]

Bonafede et al. [47]

A new methodology for comprehensive analysis of exosomal mRNAs in human CSF using newly developed exoRNA-seq, which showed potential applicability to identify specific ALS biomarkers

The expression of miR-27a-3p in patients with ALS was significantly downregulated than that in healthy human serum exosomes

Distinct miRNA expression profile was observed in ALS patient's serum-derived exosomes compared to healthy controls

Increased level of miR-124 in circulating exosomes of NSC-34 may be used as potential biomarker of motor neuron degeneration in

Adipose-derived stem cell exosomes showed paracrine effect to SVG neurons to alleviate their disease phenotypes and mitochondrial dysfunction in ALS

The ASC-derived exosomes were able to protect motor neuron-like NSC-34 (SOD1 mutant) from oxidative stress and increase their viability

Proteomic analysis revealed mASCderived exosomes

ALS

As non-coding RNAs are also reported to be involved in ALS onset and progression, miRNAs loaded in EVs represent another candidate biomarker class for ALS diagnosis. Study of free miRNA for ALS detection has been previously reported. Microarray analyses were performed on ALS mutant mouse-derived cells and patient serum, and the results identified the expression of 10–13 dysregulated miRNAs in diseased samples [44, 45]. In addition to free-miRNA analyses, Saucier et al. tried identifying ALS-associated miRNA signatures in EVs to discriminate blood between healthy and ALS individuals. Extensive exosomal RNA analysis using high-throughput sequencing coupled with droplet digital PCR enabled the identification of a group of dysregulated miRNAs, including miR-183-5p, miR-9-5p, miR-338-3p and miR-1246, which were all remarkably downregulated in ALS patient-derived exosomes [45].

EV-based biomarker studies in ALS have so far given promising results and strong motivation for follow-up studies to further specify molecular candidates with higher disease relevancy. However, limited information on the exosomal RNAs, in terms of the specific signaling pathways they regulate, is currently available. This lack of understanding makes it difficult to determine the relevance of each when attempting to define a diagnostic EV-RNA signature for ALS. Also, as significant difficulty lies in identifying the cellular origin of EVs collected from body fluids, their practical application in diagnostics could be challenging, especially if the same miRNAs secreted from different cells of origin reflect different states of the disease. Furthermore, the RNA profile in different subtypes of EVs, such as exosomes versus microvesicles, that exist in patient's body fluids has not yet been investigated. As such, more comprehensive RNA analysis, using entire EV populations, might give discordant results to those reported from exosomal RNA analysis. Additionally, to date there has not been any analysis of when specific RNAs arise during ALS disease progression and whether certain expression patterns are indicative of certain stages of the pathology. Such an understanding is crucial in determining whether expression of certain RNAs can be employed effectively as diagnostic tools or as methods to chart disease progression. Finally, as hundreds of exosomal RNAs in ALS patient body fluids have been found to exhibit significant differences in their expression levels relative to normal controls, clinically


*The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.91388*

often used as a surrogate for nervous tissue sampling) are the main target of analyses so far and advanced RNA-sequencing techniques are being employed to analyze deregulated RNAs exclusively in ALS patient-derived EVs. Recently, Otake et al. reported a new methodology using highly sensitive exoRNA-sequencing for comprehensive analysis of exosomal mRNAs in patient CSF, to identify abnormally expressed mRNAs in exosome samples from ALS patients [43]. The technique identified 543 mRNAs exhibiting statistically different expression patterns compared to normal samples. In particular, this analysis revealed that the gene *CUEDC2* was only detected in ALS patient-derived exosomes. As the gene is known to regulate the ubiquitin-proteasome pathway as part of the inflammatory response, its abnormal expression is postulated to cause potential neuroinflammation in ALS, making *CUEDC2* mRNA a strong biomarker candidate [43]. Follow-up studies are necessary to demonstrate a specific causal relationship between exosomal *CUEDC2* mRNA presence (rather than free intracellular *CUEDC2* expression) and ALS development. Furthermore, work demonstrating an omnipresence of the same RNA mis-regulation in EVs from other ALS patients is also required. However, the described study highlights how exosomal mRNAs could be attractive biomarker candidates, given their stability within body fluids, as well as the convenience of sample collection and ease of subsequent data analysis. Efforts to date to characterize EV cargos as ALS biomarkers, including those discussed in detail above and

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

As non-coding RNAs are also reported to be involved in ALS onset and progression, miRNAs loaded in EVs represent another candidate biomarker class for ALS diagnosis. Study of free miRNA for ALS detection has been previously reported. Microarray analyses were performed on ALS mutant mouse-derived cells and patient serum, and the results identified the expression of 10–13 dysregulated miRNAs in diseased samples [44, 45]. In addition to free-miRNA analyses, Saucier et al. tried identifying ALS-associated miRNA signatures in EVs to discriminate blood between healthy and ALS individuals. Extensive exosomal RNA analysis using high-throughput sequencing coupled with droplet digital PCR enabled the identification of a group of dysregulated miRNAs, including miR-183-5p, miR-9-5p, miR-338-3p and miR-1246, which were all remarkably downregulated in ALS

EV-based biomarker studies in ALS have so far given promising results and strong motivation for follow-up studies to further specify molecular candidates with higher disease relevancy. However, limited information on the exosomal RNAs, in terms of the specific signaling pathways they regulate, is currently available. This lack of understanding makes it difficult to determine the relevance of each when attempting to define a diagnostic EV-RNA signature for ALS. Also, as significant difficulty lies in identifying the cellular origin of EVs collected from body fluids, their practical application in diagnostics could be challenging, especially if the same miRNAs secreted from different cells of origin reflect different states of the disease. Furthermore, the RNA profile in different subtypes of EVs, such as exosomes versus microvesicles, that exist in patient's body fluids has not yet been investigated. As such, more comprehensive RNA analysis, using entire EV populations, might give discordant results to those reported from exosomal RNA analysis. Additionally, to date there has not been any analysis of when specific RNAs arise during ALS disease progression and whether certain expression patterns are indicative of certain stages of the pathology. Such an understanding is crucial in determining whether expression of certain RNAs can be employed effectively as diagnostic tools or as methods to chart disease progression. Finally, as hundreds of exosomal RNAs in ALS patient body fluids have been found to exhibit significant differences in their expression levels relative to normal controls, clinically

below, are summarized in **Table 2**.

patient-derived exosomes [45].

**88**


rescued normal mitochondrial protein expression in mouse neurons [48]. Another adipose-derived stem cell study performed proteomic analysis of EVs and found 189 exosomal proteins, mainly involved in regulating cell adhesion and negative regulation of apoptosis. Stress response proteins, such as SOD1, were also included in these exosomes and were found to replace the enzymatic function of mutant SOD1 in NSC-34 cells. Additionally, the study reported the presence of ribonuclease RNase 4 in the examined exosomes. This could represent a potential neuroprotective molecule applicable in ALS, as RNase 4 has been reported to have angiogenic, neuroprotective

*The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers…*

The field of EV-mediated therapeutics for ALS treatment is still in its infancy. However, since glial cells and neural stem cells are responsible for regulating neuron differentiation, protection, and synaptic function [13, 51–54], EVs derived from those cells could constitute attractive subcomponents for therapeutic agent development. As mentioned above, EVs possess distinct advantages for therapeutic development, including sustained cargo delivery and avoidance of physiological degradation. Naturally derived EVs, as well as engineered EVs loaded with optimal therapeutic materials, therefore represent a powerful candidate for the future of ALS treatment. However, the following issues should be addressed to facilitate the practical application of EVs in treating this disease. First, since EVs secreted from one type of cell usually do not target a specific cell type for cargo delivery, higher delivery specificity to eliminate potential off-target effects is essential. EV membrane engineering to attach proteins with exclusive affinity to target neuron cell surfaces could be an attractive approach to consider [55–58], if reliable surface markers for diseased cells can be defined. Second, a lack of information regarding the physiological function of specific cargo molecules in glia or stem cell-derived EV limits their applicability in ALS treatment. As disease phenotypes in ALS are extremely heterogeneous, investigating the role of each EV-loadable molecule with more categorized efficacy studies beyond cell viability is also highly required. Production scale of EVs is a critical issue for the actual application of this technology in ALS clinics. Although EV collection technology continues to advance to minimize EV loss and reduce collection time and cost, most EV experiments are still done in small-scale benchtop studies. This is not a huge issue during these early stages of research and development, but will cause a critical problem for scale up and

*Schematic summary of the major role EVs play in ALS propagation, diagnosis, and treatment.*

properties [49, 50].

*DOI: http://dx.doi.org/10.5772/intechopen.91388*

**Figure 1.**

**91**

#### **Table 2.**

*Summary of the applications of extracellular vesicles in ALS diagnosis and treatment.*

applicable diagnosis will be difficult until we better understand the physiological outcomes of those misregulated RNAs in neuromuscular tissue maintenance and function. Future research elucidating the expression patterns of EV cargos across diverse ALS populations at different disease stages, as well as studies of the signaling pathways regulated by EV-RNAs in ALS tissue, are therefore necessary before the value of these molecules in ALS diagnostic medicine can be fully evaluated.

#### **4. EVs for therapeutic agents in ALS treatment**

EVs hold distinct advantages for function as stable biomolecule carriers. Their lipid bilayers, decorated with transmembrane proteins, protect cargo molecules from enzymatic degradation in the extracellular space as well as making them immune-tolerant. Functional transmembrane proteins also promote EV internalization into recipient cells, which naturally occurs through active fusion of EV membranes with cell's plasma membranes. This in turn facilitates release of EV molecular cargo to recipient cell cytoplasms or induces endosome uptake for functional activation [23, 46].

Accordingly, an attractive hypothesis is that therapeutic molecules loaded in vesicles could be delivered to target cells to subsequently modulate phenotypes in neurodegenerative disease. In such a model, cargo molecules could encompass proteins and mRNAs, or non-coding RNAs for post-transcriptional protein regulation, or even specific compounds for sustained activation with avoidance of enzymatic attack. Research efforts to test this hypothesis are summarized in **Table 2** and discussed in detail below. As very few drug candidates with a proven efficacy for treating ALS exist, research on 'drug-loaded EVs' has not yet begun in earnest. However, adipose-derived stromal cells and stem cells have shown a capacity to generate exosomes capable of conferring a therapeutic effect on defective neurons. Exosomes from murine adipose-derived stromal cells alleviated oxidative stress and reduced hydrogen peroxide-induced apoptosis in NSC-34 cells overexpressing a human SOD1 mutant variant [47]. Safe availability and a capacity to migrate to damaged tissues for their reparative processes make stromal cells good candidates for EV sources. Although the study demonstrated that stromal cell-derived EVs can reverse SOD-1 induced cell death, such applications have not yet been specifically targeted to restore electrophysiological phenotypes in ALS neurons. Consequentially, the applicability of stromal cell-derived EVs may be limited if the specific cargo molecule responsible for restoring motor neuron function is not identified.

Retaining the remarkable therapeutic potential of stem cells, while avoiding issues with tumorigenicity, insufficient therapeutic specificity, and substantial cell loss during treatment, stem cell-derived EVs offer an exciting alternative to direct stem cell therapy. Indeed, EVs obtained from undifferentiated stem cells have shown a capacity to alleviate disease phenotypes in ALS mutant neurons. Specifically, exosomes from adipose-derived stem cells reduced SOD1 aggregation and

*The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.91388*

rescued normal mitochondrial protein expression in mouse neurons [48]. Another adipose-derived stem cell study performed proteomic analysis of EVs and found 189 exosomal proteins, mainly involved in regulating cell adhesion and negative regulation of apoptosis. Stress response proteins, such as SOD1, were also included in these exosomes and were found to replace the enzymatic function of mutant SOD1 in NSC-34 cells. Additionally, the study reported the presence of ribonuclease RNase 4 in the examined exosomes. This could represent a potential neuroprotective molecule applicable in ALS, as RNase 4 has been reported to have angiogenic, neuroprotective properties [49, 50].

The field of EV-mediated therapeutics for ALS treatment is still in its infancy. However, since glial cells and neural stem cells are responsible for regulating neuron differentiation, protection, and synaptic function [13, 51–54], EVs derived from those cells could constitute attractive subcomponents for therapeutic agent development. As mentioned above, EVs possess distinct advantages for therapeutic development, including sustained cargo delivery and avoidance of physiological degradation. Naturally derived EVs, as well as engineered EVs loaded with optimal therapeutic materials, therefore represent a powerful candidate for the future of ALS treatment. However, the following issues should be addressed to facilitate the practical application of EVs in treating this disease. First, since EVs secreted from one type of cell usually do not target a specific cell type for cargo delivery, higher delivery specificity to eliminate potential off-target effects is essential. EV membrane engineering to attach proteins with exclusive affinity to target neuron cell surfaces could be an attractive approach to consider [55–58], if reliable surface markers for diseased cells can be defined. Second, a lack of information regarding the physiological function of specific cargo molecules in glia or stem cell-derived EV limits their applicability in ALS treatment. As disease phenotypes in ALS are extremely heterogeneous, investigating the role of each EV-loadable molecule with more categorized efficacy studies beyond cell viability is also highly required. Production scale of EVs is a critical issue for the actual application of this technology in ALS clinics. Although EV collection technology continues to advance to minimize EV loss and reduce collection time and cost, most EV experiments are still done in small-scale benchtop studies. This is not a huge issue during these early stages of research and development, but will cause a critical problem for scale up and

applicable diagnosis will be difficult until we better understand the physiological outcomes of those misregulated RNAs in neuromuscular tissue maintenance and function. Future research elucidating the expression patterns of EV cargos across diverse ALS populations at different disease stages, as well as studies of the signaling pathways regulated by EV-RNAs in ALS tissue, are therefore necessary before the value of these molecules in ALS diagnostic medicine can be fully evaluated.

contain proteins for cell adhesion and negatively regulate cell apoptosis

**Significance Unknowns Reference**

neuronal function

**of EV**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

derived stem cells

*Summary of the applications of extracellular vesicles in ALS diagnosis and treatment.*

EVs hold distinct advantages for function as stable biomolecule carriers. Their lipid bilayers, decorated with transmembrane proteins, protect cargo molecules from enzymatic degradation in the extracellular space as well as making them immune-tolerant. Functional transmembrane proteins also promote EV internalization into recipient cells, which naturally occurs through active fusion of EV membranes with cell's plasma membranes. This in turn facilitates release of EV molecular cargo to recipient cell cytoplasms or induces endosome uptake for func-

Accordingly, an attractive hypothesis is that therapeutic molecules loaded in vesicles could be delivered to target cells to subsequently modulate phenotypes in neurodegenerative disease. In such a model, cargo molecules could encompass proteins and mRNAs, or non-coding RNAs for post-transcriptional protein regulation, or even specific compounds for sustained activation with avoidance of enzymatic attack. Research efforts to test this hypothesis are summarized in **Table 2** and discussed in detail below. As very few drug candidates with a proven efficacy for treating ALS exist, research on 'drug-loaded EVs' has not yet begun in earnest. However, adipose-derived stromal cells and stem cells have shown a capacity to generate exosomes capable of conferring a therapeutic effect on defective neurons. Exosomes from murine adipose-derived stromal cells alleviated oxidative stress and reduced hydrogen peroxide-induced apoptosis in NSC-34 cells overexpressing a human SOD1 mutant variant [47]. Safe availability and a capacity to migrate to damaged tissues for their reparative processes make stromal cells good candidates for EV sources. Although the study demonstrated that stromal cell-derived EVs can reverse SOD-1 induced cell death, such applications have not yet been specifically targeted to restore electrophysiological phenotypes in ALS neurons. Consequentially, the applicability of stromal cell-derived EVs may be limited if the specific cargo molecule responsible for restoring motor neuron function is not identified. Retaining the remarkable therapeutic potential of stem cells, while avoiding issues with tumorigenicity, insufficient therapeutic specificity, and substantial cell loss during treatment, stem cell-derived EVs offer an exciting alternative to direct stem cell therapy. Indeed, EVs obtained from undifferentiated stem cells have shown a capacity to alleviate disease phenotypes in ALS mutant neurons. Specifically, exosomes from adipose-derived stem cells reduced SOD1 aggregation and

**4. EVs for therapeutic agents in ALS treatment**

**Role of EV Study method Source**

**Table 2.**

with *in vitro* assay of exosome treatment on NSC-34 cells

tional activation [23, 46].

**90**

administration as we move closer to clinical trials and subsequent distribution. Lastly, cargo molecule purity is another significant issue. Even with a single cell source, EV cargo composition is likely to be different based on culture conditions, and may fluctuate due to other unknown factors; especially if cell-derived EVs is advanced to large-scale production (**Figure 1**).

#### **5. Conclusions**

Current EV research in relation to ALS is weighted toward investigating the pathogenic role of intercellularly transmissible vesicles, with a particular focus on dysregulated protein propagation. This is likely due to the fact that SOD1 and TDP-43 mutant cells are already known to produce neurotoxic protein aggregates, which makes the hypothesis that their dissemination via EVs contributes to the nature of irreversible degeneration of neuromuscular tissue in ALS a straightforward one. Several studies have demonstrated that EVs collected from ALS patient's body fluids and from mutant non-neuronal cells can be internalized by healthy cells. Their capacity to induce neurodegenerative behavior in these cells supports the notion that these structures contribute to the rapid disease progression characteristic of ALS. However, the effect of collected EV components in specific ALS disease phenotypes is currently quite ambiguous, especially for correlating neuromuscular tissue level abnormalities with defined EV component expression. To address this, EV-mediated interplay, occurring at the diseased NMJ may be a good potential target for future investigations. EVs secreted from skeletal muscle and motor neurons are known to contribute significantly to the development of normal synaptic formation at the NMJ and better understanding how these signaling processes are disrupted in ALS could significantly improve our understanding of early disease etiology. Work in these early stages of ALS and EVs has also highlighted the potential for using EVs as either a biomarker for ALS diagnostics or even a potential therapeutic agent. Although evidence of a causal relationship between misregulated EV-RNAs and functional impairments in ALS neurons is currently sparse, notable differences in ALS EV-RNA expression patterns detected by next generation sequencing does support their potential as future diagnostic tool. EV's exogenous nature and pre-established cellular internalization mechanism also provides substantial motivation to continue research that applies these vesicles in therapy development, either as a molecule carrier or as a naturally-derived drug in and of itself. Further studies addressing the non-specificity of EV delivery and issues with production scale will raise their status as a potent therapeutic means to combat ALS in the future.

**Author details**

Changho Chun<sup>1</sup>

United States

WA, United States

WA, United States

**93**

, Alec S.T. Smith1,2,4, Mark Bothwell2,4 and David L. Mack1,3,4\*

1 Department of Bioengineering, The University of Washington, Seattle, WA,

*The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers…*

*DOI: http://dx.doi.org/10.5772/intechopen.91388*

2 Department of Physiology and Biophysics, The University of Washington, Seattle,

3 Department of Rehabilitation Medicine, The University of Washington, Seattle,

4 The Institute for Stem Cell and Regenerative Medicine, Seattle, WA, United States

© 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,

\*Address all correspondence to: dmack21@uw.edu

provided the original work is properly cited.

*The Role of Extracellular Vesicles in the Progression of ALS and Their Potential as Biomarkers… DOI: http://dx.doi.org/10.5772/intechopen.91388*

#### **Author details**

administration as we move closer to clinical trials and subsequent distribution. Lastly, cargo molecule purity is another significant issue. Even with a single cell source, EV cargo composition is likely to be different based on culture conditions, and may fluctuate due to other unknown factors; especially if cell-derived EVs is

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

Current EV research in relation to ALS is weighted toward investigating the pathogenic role of intercellularly transmissible vesicles, with a particular focus on dysregulated protein propagation. This is likely due to the fact that SOD1 and TDP-43 mutant cells are already known to produce neurotoxic protein aggregates, which makes the hypothesis that their dissemination via EVs contributes to the nature of irreversible degeneration of neuromuscular tissue in ALS a straightforward one. Several studies have demonstrated that EVs collected from ALS patient's body fluids and from mutant non-neuronal cells can be internalized by healthy cells. Their capacity to induce neurodegenerative behavior in these cells supports the notion that these structures contribute to the rapid disease progression characteristic of ALS. However, the effect of collected EV components in specific ALS disease phenotypes is currently quite ambiguous, especially for correlating neuromuscular tissue level abnormalities with defined EV component expression. To address this, EV-mediated interplay, occurring at the diseased NMJ may be a good potential target for future investigations. EVs secreted from skeletal muscle and motor neurons are known to contribute significantly to the development of normal synaptic formation at the NMJ and better understanding how these signaling processes are disrupted in ALS could significantly improve our understanding of early disease etiology. Work in these early stages of ALS and EVs has also highlighted the potential for using EVs as either a biomarker for ALS diagnostics or even a potential therapeutic agent. Although evidence of a causal relationship between misregulated EV-RNAs and functional impairments in ALS neurons is currently sparse, notable differences in ALS EV-RNA expression patterns detected by next generation sequencing does support their potential as future diagnostic tool. EV's exogenous nature and pre-established cellular internalization mechanism also provides substantial motivation to continue research that applies these vesicles in therapy development, either as a molecule carrier or as a naturally-derived drug in and of itself. Further studies addressing the non-specificity of EV delivery and issues with production scale will raise their status as a potent therapeutic means to combat ALS

advanced to large-scale production (**Figure 1**).

**5. Conclusions**

in the future.

**92**

Changho Chun<sup>1</sup> , Alec S.T. Smith1,2,4, Mark Bothwell2,4 and David L. Mack1,3,4\*

1 Department of Bioengineering, The University of Washington, Seattle, WA, United States

2 Department of Physiology and Biophysics, The University of Washington, Seattle, WA, United States

3 Department of Rehabilitation Medicine, The University of Washington, Seattle, WA, United States

4 The Institute for Stem Cell and Regenerative Medicine, Seattle, WA, United States

\*Address all correspondence to: dmack21@uw.edu

© 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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**99**

**Chapter 6**

**Abstract**

**1. Introduction**

*Fatima Pedrosa Domellöf*

sparing of the extraocular muscles in ALS.

p75, neurofilament, Wnt1, Wnt3a, Wnt5a, Wnt7

have been ascribed to supranuclear deficits [2].

The Extraocular Muscles Are

The extraocular muscles differ from other skeletal muscles in many respects but most strikingly in their response to neuromuscular diseases expected to affect the whole body. Oculomotor disturbances are not typical features of ALS. Recent data ascribe the muscle tissue an important role in the pathophysiology of ALS, with early involvement of the neuromuscular junctions and loss of axonal contact. We show that the extraocular muscles of terminal ALS donors and also of mice models of ALS maintain their morphology and well-preserved neuromuscular junctions until the end stages of the disease, whereas the limb muscles are severely affected and their neuromuscular junctions start losing contact with the supplying axons early in the course of ALS. There are intrinsic differences between the extraocular and limb muscles with respect to neurotrophic factors and Wnt isoforms and fundamental differences in their response to ALS that cannot be explained by the aging process. We propose that these differences may be instrumental in the selective

**Keywords:** extraocular muscle, ALS, pathophysiology, neurotrophic factor, neuromuscular junction, Wnt, BDNF, GDNF, NT-3, NT-4, S-100B, synaptophysin,

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder affecting both the upper and lower motor neurons. It is characterized by increasing muscle weakness, paresis, and paralysis, and although the course of the disease may vary considerably, death usually occurs within 3–5 years due to respiratory failure. While almost all skeletal muscles, including the cranially innervated muscles responsible for speech and swallowing, are affected in ALS, oculomotor disturbances are not dominant or typical features of this disease, not even in patients with long-duration ALS, and eye movements may remain the only form of communication available to these patients at the end stages of the disease. The cranial nerve nuclei supplying the eye muscles appear rather normal in terminal ALS patients [1], and abnormalities in eye motility in ALS patients, when present,

The pathophysiology of the neurodegenerative process in ALS has been proposed to be due to a number of different causes. The traditional point of view has been that ALS is a motor neuron disease that progresses to the periphery where it affects the muscles, due to the typical loss of motor neurons in the anterior horns of the spinal cord and sclerosis of the lateral corticospinal tracts. However, a number

Selectively Spared in ALS

#### **Chapter 6**

## The Extraocular Muscles Are Selectively Spared in ALS

*Fatima Pedrosa Domellöf*

#### **Abstract**

The extraocular muscles differ from other skeletal muscles in many respects but most strikingly in their response to neuromuscular diseases expected to affect the whole body. Oculomotor disturbances are not typical features of ALS. Recent data ascribe the muscle tissue an important role in the pathophysiology of ALS, with early involvement of the neuromuscular junctions and loss of axonal contact. We show that the extraocular muscles of terminal ALS donors and also of mice models of ALS maintain their morphology and well-preserved neuromuscular junctions until the end stages of the disease, whereas the limb muscles are severely affected and their neuromuscular junctions start losing contact with the supplying axons early in the course of ALS. There are intrinsic differences between the extraocular and limb muscles with respect to neurotrophic factors and Wnt isoforms and fundamental differences in their response to ALS that cannot be explained by the aging process. We propose that these differences may be instrumental in the selective sparing of the extraocular muscles in ALS.

**Keywords:** extraocular muscle, ALS, pathophysiology, neurotrophic factor, neuromuscular junction, Wnt, BDNF, GDNF, NT-3, NT-4, S-100B, synaptophysin, p75, neurofilament, Wnt1, Wnt3a, Wnt5a, Wnt7

#### **1. Introduction**

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder affecting both the upper and lower motor neurons. It is characterized by increasing muscle weakness, paresis, and paralysis, and although the course of the disease may vary considerably, death usually occurs within 3–5 years due to respiratory failure. While almost all skeletal muscles, including the cranially innervated muscles responsible for speech and swallowing, are affected in ALS, oculomotor disturbances are not dominant or typical features of this disease, not even in patients with long-duration ALS, and eye movements may remain the only form of communication available to these patients at the end stages of the disease. The cranial nerve nuclei supplying the eye muscles appear rather normal in terminal ALS patients [1], and abnormalities in eye motility in ALS patients, when present, have been ascribed to supranuclear deficits [2].

The pathophysiology of the neurodegenerative process in ALS has been proposed to be due to a number of different causes. The traditional point of view has been that ALS is a motor neuron disease that progresses to the periphery where it affects the muscles, due to the typical loss of motor neurons in the anterior horns of the spinal cord and sclerosis of the lateral corticospinal tracts. However, a number

of studies suggest an inverse course of events, with changes starting at the periphery and progressing along the motor neurons toward the CNS, reviewed by [3–5]. In SOD1G93A transgenic mice, it has been shown that peripheral denervation occurs in the limb muscles before the animals become clinically weak or motor neuron loss is detected [6]. Similar results were also reported for a patient with ALS who died unexpectedly, leading the authors to propose that motor neuron pathology in ALS begins at the distal axon end and proceeds in a "dying back" pattern [6]. Later, it has been shown that muscle-restricted expression of three different SOD1 gene variants can initiate the non-autonomous degeneration of motor neurons typical of ALS [7], truly showing that the muscle itself may be a major player in the early stages of ALS.

A unifying model compiling extensive experimental data from SOD1 transgenic models of ALS [3] proposes that although motor neuron degeneration and death are the primary causes of the progressive paralysis, neighboring nonneuronal cells are also involved in the toxicity and damage development. In this model, the earliest event is the retraction of motor axons from their muscle synapses, before any symptoms of the disease are present.

The extraocular muscles (EOMs) are unique in many respects and are considered a separate muscle class, allotype. They are both the fastest and the most fatigue-resistant muscles in the body, and their fiber-type composition is very complex [8, 9]. The unique properties of the EOMs are reflected in their expression profile that differs from that of limb muscle in over 300 genes [10]. However, the most striking property of EOMs is their varied behavior in disease. The EOMs are known to be selectively involved in certain diseases like mitochondrial myopathies, myasthenia gravis, and Miller Fisher syndrome, but preferentially spared in other devastating diseases like Duchenne muscular dystrophy and merosin-deficient muscular dystrophy, that affect all other muscles in the body [11–13].

The extraocular muscles are richly innervated, and their motor units are very small, with 7–25 muscle fibers, in contrast to the motor units of limb muscles, which typically contain hundreds of muscle fibers. The majority of the nerve fibers in the EOMs are large and myelinated and innervate a typical single "en plaque" endplate in the middle portion of each muscle fiber. The remaining 15–20% of the EOM nerve fibers are small and innervate many small, "en grappe" motor endplates along the length of a muscle fiber (the so-called multiply innervated muscle fibers, reviewed in [11]). This traditional description of the innervation of the EOMs has been significantly changed by the recent report of a subpopulation of muscle fibers that exhibit multiple large "en plaque" nerve endings [14]. The NMJs of the EOMs also differ from limb muscle in retaining expression of the fetal gamma subunit of the acetylcholine receptor (AChR), in addition to the adult epsilon subunit, typically present in mature motor endplates [14, 15]. We have shown that the NMJs of the human EOMs have a different immunoreactivity pattern than that seen in limb muscles for antibodies against gangliosides GQ1b, GT1a, and GD1b [16]. Gangliosides are a large family of sialylated glycosphingolipids highly enriched in neuronal and glial plasma membranes and important for the development, function, and maintenance of the nervous system. Complex gangliosides have neurotrophic factor-like activity and may regulate the expression levels of neurotrophic factors and their receptors [17]. Notably, there have been at least two case reports of ALS associated with antibodies against gangliosides [18, 19].

Given that the EOMs differ significantly from the limb and trunk muscles with respect to their response to disease and given the more recent view that the muscle itself may play a key role in the pathophysiology of ALS, we first investigated whether there are any major differences between the EOMs and the limb muscles of terminal ALS donors [20]. Thereafter, we investigated neuromuscular

**101**

*The Extraocular Muscles Are Selectively Spared in ALS DOI: http://dx.doi.org/10.5772/intechopen.89504*

naturally occurring variation among subjects [8].

physiology of the disease.

**muscles at end stages of ALS**

the disease.

**preserved**

junction integrity [21–23], neurotrophic factors (BDNF, NT-3, NT-4, GDNF) and their receptors (p75NTR, TrkB, TrkC) [24, 25], and relevant Wnt isoforms (Wnt1, Wnt3a, Wnt5a, Wnt7a) [26], as possible important puzzle pieces to better understand the pathophysiology of ALS and the selective sparing of the EOMs in

**2. The extraocular muscles of terminal ALS donors are remarkably well** 

We compared the general morphology, fiber-type content, fiber area and myosin heavy chain (MyHC) composition of EOM, biceps brachii, vastus lateralis, and tibialis anterior muscle samples collected from eight terminal ALS donors (age 58–80 years) and from three age-matched (age 72–86 years) and a younger (age 26 years) control, with ethical approval, using immunohistochemistry and SDS-PAGE [20]. There were five cases of sporadic ALS and three cases of familial ALS. The MyHC isoforms are the major determinants of key contractile and biochemical characteristics of the myofibers including shortening velocity and power and therefore very good markers of the physiological properties of myofibers. Our study showed that the morphology of the ALS limb muscle samples is clearly pathological. All limb muscle samples from terminal ALS donors exhibit typical signs of myofiber type grouping, myofiber splitting, necrosis, increased connective tissue, fatty replacement, and ongoing regeneration, with a very wide range of myofiber areas. In contrast, the EOM samples from the same donors show remarkably well-preserved morphology. In 18 out of the 43 EOM samples examined, larger variation in fiber area was apparent than in the control samples and irrespectively of being sporadic or familial ALS cases. In fact, areas that appear well preserved reveal a general atrophy of all myofiber types, whereas areas that appear more affected generally show atrophy of the myofibers containing MyHCslow and hypertrophy of the myofibers containing MyHCfast2A. In 20 EOM samples, increased connective tissue amounts were noted, either around individual myofibers or around groups of myofibers. The changes noticed vary among donors and among different EOM samples from a given donor. Centrally placed nuclei are rarely detected. The MyHC content, determined with SDS-PAGE of whole EOM extracts, varies among ALS donors, whereas it is rather similar among control EOM samples [20]. This variation in MyHC content at the whole muscle level can be interpreted as reflecting different levels of involvement, and, to a lesser degree,

Given that this first study clearly shows that the human EOMs are remarkably well preserved at the end stage of ALS [20] and because the EOMs are known to show a divergent behavior in disease, we decided to further investigate these muscles in ALS, comparing the EOMs and limb muscle samples from human donors and from mouse models of ALS, taking a "muscle perspective" to study the patho-

**3. Maintained neuromuscular junction occupancy in the extraocular** 

shown that loss of contact between the axons and the myofibers, at the neuromuscular junctions, occurs early in the course of the disease and indeed precedes detection of loss of motor neurons in the spinal cord [6]. Similar findings have been

In the SOD1G93A mouse, the most widely used mouse model of ALS, it has been

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

of studies suggest an inverse course of events, with changes starting at the periphery and progressing along the motor neurons toward the CNS, reviewed by [3–5]. In SOD1G93A transgenic mice, it has been shown that peripheral denervation occurs in the limb muscles before the animals become clinically weak or motor neuron loss is detected [6]. Similar results were also reported for a patient with ALS who died unexpectedly, leading the authors to propose that motor neuron pathology in ALS begins at the distal axon end and proceeds in a "dying back" pattern [6]. Later, it has been shown that muscle-restricted expression of three different SOD1 gene variants can initiate the non-autonomous degeneration of motor neurons typical of ALS [7], truly showing that the muscle itself may be a major player in the early

A unifying model compiling extensive experimental data from SOD1 transgenic models of ALS [3] proposes that although motor neuron degeneration and death are the primary causes of the progressive paralysis, neighboring nonneuronal cells are also involved in the toxicity and damage development. In this model, the earliest event is the retraction of motor axons from their muscle synapses, before any

The extraocular muscles (EOMs) are unique in many respects and are considered a separate muscle class, allotype. They are both the fastest and the most fatigue-resistant muscles in the body, and their fiber-type composition is very complex [8, 9]. The unique properties of the EOMs are reflected in their expression profile that differs from that of limb muscle in over 300 genes [10]. However, the most striking property of EOMs is their varied behavior in disease. The EOMs are known to be selectively involved in certain diseases like mitochondrial myopathies, myasthenia gravis, and Miller Fisher syndrome, but preferentially spared in other devastating diseases like Duchenne muscular dystrophy and merosin-deficient

The extraocular muscles are richly innervated, and their motor units are very small, with 7–25 muscle fibers, in contrast to the motor units of limb muscles, which typically contain hundreds of muscle fibers. The majority of the nerve fibers in the EOMs are large and myelinated and innervate a typical single "en plaque" endplate in the middle portion of each muscle fiber. The remaining 15–20% of the EOM nerve fibers are small and innervate many small, "en grappe" motor endplates along the length of a muscle fiber (the so-called multiply innervated muscle fibers, reviewed in [11]). This traditional description of the innervation of the EOMs has been significantly changed by the recent report of a subpopulation of muscle fibers that exhibit multiple large "en plaque" nerve endings [14]. The NMJs of the EOMs also differ from limb muscle in retaining expression of the fetal gamma subunit of the acetylcholine receptor (AChR), in addition to the adult epsilon subunit, typically present in mature motor endplates [14, 15]. We have shown that the NMJs of the human EOMs have a different immunoreactivity pattern than that seen in limb muscles for antibodies against gangliosides GQ1b, GT1a, and GD1b [16]. Gangliosides are a large family of sialylated glycosphingolipids highly enriched in neuronal and glial plasma membranes and important for the development, function, and maintenance of the nervous system. Complex gangliosides have neurotrophic factor-like activity and may regulate the expression levels of neurotrophic factors and their receptors [17]. Notably, there have been at least two case reports of

muscular dystrophy, that affect all other muscles in the body [11–13].

ALS associated with antibodies against gangliosides [18, 19].

Given that the EOMs differ significantly from the limb and trunk muscles with respect to their response to disease and given the more recent view that the muscle itself may play a key role in the pathophysiology of ALS, we first investigated whether there are any major differences between the EOMs and the limb muscles of terminal ALS donors [20]. Thereafter, we investigated neuromuscular

**100**

stages of ALS.

symptoms of the disease are present.

junction integrity [21–23], neurotrophic factors (BDNF, NT-3, NT-4, GDNF) and their receptors (p75NTR, TrkB, TrkC) [24, 25], and relevant Wnt isoforms (Wnt1, Wnt3a, Wnt5a, Wnt7a) [26], as possible important puzzle pieces to better understand the pathophysiology of ALS and the selective sparing of the EOMs in the disease.

#### **2. The extraocular muscles of terminal ALS donors are remarkably well preserved**

We compared the general morphology, fiber-type content, fiber area and myosin heavy chain (MyHC) composition of EOM, biceps brachii, vastus lateralis, and tibialis anterior muscle samples collected from eight terminal ALS donors (age 58–80 years) and from three age-matched (age 72–86 years) and a younger (age 26 years) control, with ethical approval, using immunohistochemistry and SDS-PAGE [20]. There were five cases of sporadic ALS and three cases of familial ALS. The MyHC isoforms are the major determinants of key contractile and biochemical characteristics of the myofibers including shortening velocity and power and therefore very good markers of the physiological properties of myofibers. Our study showed that the morphology of the ALS limb muscle samples is clearly pathological. All limb muscle samples from terminal ALS donors exhibit typical signs of myofiber type grouping, myofiber splitting, necrosis, increased connective tissue, fatty replacement, and ongoing regeneration, with a very wide range of myofiber areas. In contrast, the EOM samples from the same donors show remarkably well-preserved morphology. In 18 out of the 43 EOM samples examined, larger variation in fiber area was apparent than in the control samples and irrespectively of being sporadic or familial ALS cases. In fact, areas that appear well preserved reveal a general atrophy of all myofiber types, whereas areas that appear more affected generally show atrophy of the myofibers containing MyHCslow and hypertrophy of the myofibers containing MyHCfast2A. In 20 EOM samples, increased connective tissue amounts were noted, either around individual myofibers or around groups of myofibers. The changes noticed vary among donors and among different EOM samples from a given donor. Centrally placed nuclei are rarely detected. The MyHC content, determined with SDS-PAGE of whole EOM extracts, varies among ALS donors, whereas it is rather similar among control EOM samples [20]. This variation in MyHC content at the whole muscle level can be interpreted as reflecting different levels of involvement, and, to a lesser degree, naturally occurring variation among subjects [8].

Given that this first study clearly shows that the human EOMs are remarkably well preserved at the end stage of ALS [20] and because the EOMs are known to show a divergent behavior in disease, we decided to further investigate these muscles in ALS, comparing the EOMs and limb muscle samples from human donors and from mouse models of ALS, taking a "muscle perspective" to study the pathophysiology of the disease.

#### **3. Maintained neuromuscular junction occupancy in the extraocular muscles at end stages of ALS**

In the SOD1G93A mouse, the most widely used mouse model of ALS, it has been shown that loss of contact between the axons and the myofibers, at the neuromuscular junctions, occurs early in the course of the disease and indeed precedes detection of loss of motor neurons in the spinal cord [6]. Similar findings have been reported in ALS patients who died of other reasons early in the course of ALS [6]. Furthermore, data show that muscle-specific alterations are sufficient to trigger neuromuscular pathology and distal degeneration of motor neurons [5, 7], suggesting that the muscle itself may play an important role in the pathophysiology of ALS.

We investigated [21] whether there are signs of loss of contact between the axons and the myofibers, at the neuromuscular junctions of the EOMs, extensor digitorum longus, tibialis anterior, and gastrocnemius and soleus muscles from seven SOD1G93A transgenic and six control mice. We used triple labeling immunohistochemistry with antibodies against neurofilament protein and synaptophysin to identify the axonal side of the neuromuscular junctions and their axons and alpha-bungarotoxin, to identify the muscle side of the neuromuscular junctions, in the same muscle section. Our study confirmed that, as described above for the human EOMs of terminal ALS donors, the eye muscles in this animal model of ALS also show rather well-preserved morphological features, in clear contrast to the limb muscles of the same animal, at the end stage of the disease. We could confirm previously reported findings [6] of significant loss of contact between the nerve and myofiber in approximately 33% of the neuromuscular junctions in limb muscles of the ALS mouse model, whereas in the limb muscles of the control animals, only approximately 4.6% of the neuromuscular junctions lack contact with the supplying nerve (p = 0.0071). In contrast, the number of neuromuscular junctions lacking nerve contacts in the EOMs is extremely low and similar between transgenic (1.9%) and control (1.7%) animals (p = 0.659) [21].

Similarly, the neuromuscular junctions of the EOMs of terminal ALS donors show maintenance of their integrity [22, 23]. In a study of 7 terminal ALS donors (age 58–80 years, three cases with SOD1D90A genotype) and 10 controls (adults aged 34–53 years and elderly aged 69–83 years), we compared the EOMs and the limb muscles with respect to the presence and distribution of neurofilament light subunit (marker of the axons in nerves and at the neuromuscular junction), synaptophysin (marker of synaptic vesicles and thereby of functioning neuromuscular junctions), S100B, p75NTR and glial fibrillary acidic protein (GFAP) which are markers of terminal Schwann cells at the neuromuscular junction, and alphabungarotoxin, which labels the muscle side of the synapse [23]. GFAP and p75NTR are upregulated in Schwann cells after injury [27–29]. Two groups of control donors (adult and elderly) were included in this and all our studies of ALS in order to distinguish between the effects of normal aging and those related to ALS. This pioneer study showed that there is a dramatic decrease in the number of neuromuscular junctions and nerve axons containing neurofilament light subunit in limb muscles of terminal ALS donors, whereas the EOMs maintain neurofilament light subunit in their neuromuscular junctions and nerves. There is also a significant decrease in neuromuscular junctions labeled with synaptophysin in the limb muscles but not in the EOMs of terminal ALS donors. Together, these results confirm that there is a marked loss of nerve-muscle contacts in the limb muscles of terminal ALS donors, whereas the EOMs succeed in maintaining their neuromuscular junctions until the end stage of the disease. The neuromuscular junctions of limb muscles show also massive loss of S100B and p75NTR in ALS, whereas the staining pattern of GAFP is maintained. In contrast, the neuromuscular junctions in the EOMs of terminal ALS donors also show loss of S100B but have an unchanged pattern regarding the other two Schwann cell markers, GFAP and p75NTR. The loss of S100B can be interpreted as due to downregulation rather than a sign of loss of Schwann cells, given that the other two markers remain unchanged. It may also indicate a disturbed calcium homeostasis as an early step toward the loss of the neuromuscular junction in ALS. In summary, this study shows fundamental differences between the limb muscles and the EOMs of terminal ALS donors with a significant impact of the

**103**

*The Extraocular Muscles Are Selectively Spared in ALS DOI: http://dx.doi.org/10.5772/intechopen.89504*

in the EOMs are only very mildly affected [23].

junctions of the EOMs of the same animals [30].

to ALS.

**and limb muscles**

disease on important synaptic proteins normally present in the motor axons and terminal Schwann cells in the limb muscles, whereas the neuromuscular junctions

Another study has in addition revealed fragmentation of the postsynaptic membrane, decreased density of acetylcholine receptors, and absence of nerve sprouting in denervated neuromuscular junctions in the limb muscles of the SOD1G93A mouse model of ALS, whereas no such changes are present in the neuromuscular

In the context of motor axon retraction from the muscle synapse being an early event in the pathogenesis of ALS, it is of particular interest to further investigate the cellular and molecular microenvironment of the neuromuscular junctions of the extraocular muscles, as they may provide cues to the unique resistance of the EOMs

**4. Important differences in neurotrophic factors between extraocular** 

Data on the SOD1G93A mouse model of ALS show important differences between the EOMs and the limb muscles over time regarding the levels of expression of neurotrophic factors [24]. The authors compared the expression levels of BDNF, GDNF, NT-3, and NT-4, determined with quantitative RT-PCR, in limb (soleus and gastrocnemius) and extraocular muscles at the early stage (50 days) and at the end stage (150 days) of the disease. Starting with the control animals, the EOMs differ from the limb muscles by having significantly lower levels of BDNF and NT-3 mRNA at age 50 days. At 150 days, the control EOMs have significantly lower levels of BDNF and NT-4 mRNA than the limb muscles from the same animals. In the limb muscles of control animals, mRNA levels of BDNF increase significantly from 50 to 150 days of age, whereas the levels of NT-3 decrease dramatically. In the control EOMs, the mRNA levels of BDNF, GDNF, NT-3, and NT-4

In the transgenic animals, the expression levels of BDNF increase significantly from 50 to 150 days, that is, from the early stage of ALS, when the limb muscle morphology is still normal, to the end stage of the disease, when the limb muscles are extremely affected. Compared to the controls, the limb muscles of the transgenic animals show significantly lower levels of NT-4 at 50 days and significantly higher levels of GDNF at 150 days. The transgenic EOMs show no change in the levels of expression of BDNF or GDNF and show significant decrease in NT-3 and increase in NT-4, over time between 50 and 150 days, and the morphology is comparable to that of the age-matched controls. Compared to the controls, the EOMs of the G93A animals show significantly higher levels of GDNF and NT-3 at 50 days, whereas

whereas NT-3 is of particular importance for sensory neurons.

remain unchanged from 50 to 150 days of age [24].

Alterations in the expression of target-derived neurotrophic factors and in their signaling pathways have been reported in ALS. Neurotrophic factors are important for survival and maintenance of neurons [31–33]. Target-derived neurotrophic factors can be transported to lower motor neurons from other cells, including the muscle cells, by retrograde transport, and from upper motor neurons, by anterograde transport, and act as signaling molecules [31–33]. Among neurotrophic factors, brain-derived neurotrophic factor (BDNF) is of particular interest due to its capacity to promote motor neuron survival and motor axon growth [32–34]. Glial cell line-derived neurotrophic factor (GDNF) is also relevant as it is capable of rescuing motor neurons from injury-induced cell death [35], and neurothrophin-4 (NT-4) plays a role in growth and remodulation of adult motor neurons [36, 37],

*The Extraocular Muscles Are Selectively Spared in ALS DOI: http://dx.doi.org/10.5772/intechopen.89504*

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

and control (1.7%) animals (p = 0.659) [21].

reported in ALS patients who died of other reasons early in the course of ALS [6]. Furthermore, data show that muscle-specific alterations are sufficient to trigger neuromuscular pathology and distal degeneration of motor neurons [5, 7], suggesting that the muscle itself may play an important role in the pathophysiology of ALS. We investigated [21] whether there are signs of loss of contact between the axons and the myofibers, at the neuromuscular junctions of the EOMs, extensor digitorum longus, tibialis anterior, and gastrocnemius and soleus muscles from seven SOD1G93A transgenic and six control mice. We used triple labeling immunohistochemistry with antibodies against neurofilament protein and synaptophysin to identify the axonal side of the neuromuscular junctions and their axons and alpha-bungarotoxin, to identify the muscle side of the neuromuscular junctions, in the same muscle section. Our study confirmed that, as described above for the human EOMs of terminal ALS donors, the eye muscles in this animal model of ALS also show rather well-preserved morphological features, in clear contrast to the limb muscles of the same animal, at the end stage of the disease. We could confirm previously reported findings [6] of significant loss of contact between the nerve and myofiber in approximately 33% of the neuromuscular junctions in limb muscles of the ALS mouse model, whereas in the limb muscles of the control animals, only approximately 4.6% of the neuromuscular junctions lack contact with the supplying nerve (p = 0.0071). In contrast, the number of neuromuscular junctions lacking nerve contacts in the EOMs is extremely low and similar between transgenic (1.9%)

Similarly, the neuromuscular junctions of the EOMs of terminal ALS donors show maintenance of their integrity [22, 23]. In a study of 7 terminal ALS donors (age 58–80 years, three cases with SOD1D90A genotype) and 10 controls (adults aged 34–53 years and elderly aged 69–83 years), we compared the EOMs and the limb muscles with respect to the presence and distribution of neurofilament light subunit (marker of the axons in nerves and at the neuromuscular junction), synaptophysin (marker of synaptic vesicles and thereby of functioning neuromuscular junctions), S100B, p75NTR and glial fibrillary acidic protein (GFAP) which are markers of terminal Schwann cells at the neuromuscular junction, and alphabungarotoxin, which labels the muscle side of the synapse [23]. GFAP and p75NTR are upregulated in Schwann cells after injury [27–29]. Two groups of control donors (adult and elderly) were included in this and all our studies of ALS in order to distinguish between the effects of normal aging and those related to ALS. This pioneer study showed that there is a dramatic decrease in the number of neuromuscular junctions and nerve axons containing neurofilament light subunit in limb muscles of terminal ALS donors, whereas the EOMs maintain neurofilament light subunit in their neuromuscular junctions and nerves. There is also a significant decrease in neuromuscular junctions labeled with synaptophysin in the limb muscles but not in the EOMs of terminal ALS donors. Together, these results confirm that there is a marked loss of nerve-muscle contacts in the limb muscles of terminal ALS donors, whereas the EOMs succeed in maintaining their neuromuscular junctions until the end stage of the disease. The neuromuscular junctions of limb muscles show also massive loss of S100B and p75NTR in ALS, whereas the staining pattern of GAFP is maintained. In contrast, the neuromuscular junctions in the EOMs of terminal ALS donors also show loss of S100B but have an unchanged pattern regarding the other two Schwann cell markers, GFAP and p75NTR. The loss of S100B can be interpreted as due to downregulation rather than a sign of loss of Schwann cells, given that the other two markers remain unchanged. It may also indicate a disturbed calcium homeostasis as an early step toward the loss of the neuromuscular junction in ALS. In summary, this study shows fundamental differences between the limb muscles and the EOMs of terminal ALS donors with a significant impact of the

**102**

disease on important synaptic proteins normally present in the motor axons and terminal Schwann cells in the limb muscles, whereas the neuromuscular junctions in the EOMs are only very mildly affected [23].

Another study has in addition revealed fragmentation of the postsynaptic membrane, decreased density of acetylcholine receptors, and absence of nerve sprouting in denervated neuromuscular junctions in the limb muscles of the SOD1G93A mouse model of ALS, whereas no such changes are present in the neuromuscular junctions of the EOMs of the same animals [30].

In the context of motor axon retraction from the muscle synapse being an early event in the pathogenesis of ALS, it is of particular interest to further investigate the cellular and molecular microenvironment of the neuromuscular junctions of the extraocular muscles, as they may provide cues to the unique resistance of the EOMs to ALS.

#### **4. Important differences in neurotrophic factors between extraocular and limb muscles**

Alterations in the expression of target-derived neurotrophic factors and in their signaling pathways have been reported in ALS. Neurotrophic factors are important for survival and maintenance of neurons [31–33]. Target-derived neurotrophic factors can be transported to lower motor neurons from other cells, including the muscle cells, by retrograde transport, and from upper motor neurons, by anterograde transport, and act as signaling molecules [31–33]. Among neurotrophic factors, brain-derived neurotrophic factor (BDNF) is of particular interest due to its capacity to promote motor neuron survival and motor axon growth [32–34]. Glial cell line-derived neurotrophic factor (GDNF) is also relevant as it is capable of rescuing motor neurons from injury-induced cell death [35], and neurothrophin-4 (NT-4) plays a role in growth and remodulation of adult motor neurons [36, 37], whereas NT-3 is of particular importance for sensory neurons.

Data on the SOD1G93A mouse model of ALS show important differences between the EOMs and the limb muscles over time regarding the levels of expression of neurotrophic factors [24]. The authors compared the expression levels of BDNF, GDNF, NT-3, and NT-4, determined with quantitative RT-PCR, in limb (soleus and gastrocnemius) and extraocular muscles at the early stage (50 days) and at the end stage (150 days) of the disease. Starting with the control animals, the EOMs differ from the limb muscles by having significantly lower levels of BDNF and NT-3 mRNA at age 50 days. At 150 days, the control EOMs have significantly lower levels of BDNF and NT-4 mRNA than the limb muscles from the same animals. In the limb muscles of control animals, mRNA levels of BDNF increase significantly from 50 to 150 days of age, whereas the levels of NT-3 decrease dramatically. In the control EOMs, the mRNA levels of BDNF, GDNF, NT-3, and NT-4 remain unchanged from 50 to 150 days of age [24].

In the transgenic animals, the expression levels of BDNF increase significantly from 50 to 150 days, that is, from the early stage of ALS, when the limb muscle morphology is still normal, to the end stage of the disease, when the limb muscles are extremely affected. Compared to the controls, the limb muscles of the transgenic animals show significantly lower levels of NT-4 at 50 days and significantly higher levels of GDNF at 150 days. The transgenic EOMs show no change in the levels of expression of BDNF or GDNF and show significant decrease in NT-3 and increase in NT-4, over time between 50 and 150 days, and the morphology is comparable to that of the age-matched controls. Compared to the controls, the EOMs of the G93A animals show significantly higher levels of GDNF and NT-3 at 50 days, whereas

no significant differences are detectable at 150 days. Finally, comparison between the EOMs and limb muscles of transgenic animals shows no significant differences in the levels of expression of any of the four neurotrophic factors at 50 days but significantly higher levels of BDNF in the limb muscles at 150 days [24].

Because no significant changes in the levels of expression of BDNF detected with quantitative RT-PCR are apparent in either the limb or EOMs of the transgenic animals at any stage, the authors dismiss a possible role for this neurotrophic factors on ALS. The BDNF results obtained in the SOD1G93A mice are in agreement with a previous report from muscle samples of ALS patients [38]. In contrast, the GDNF mRNA levels are significantly upregulated in the EOMs of the SOD1G93A mice at 50 days, and the authors suggest that this early upregulation is triggered by the disease and seminal for the maintenance of the general morphology of the EOMs and in particular for their capacity of keeping intact neuromuscular junctions. They interpret the upregulation of GDNF in the end stage in the limb muscles as reflecting the advanced motor neuron degeneration. Similarly, the upregulation of NT-3 in the EOMs detected at 50 days is also proposed to be triggered by ALS and to protect the EOMs, whereas early downregulation of NT-4 in the limb muscles is suggested to be detrimental and contribute to the loss of axonal contact at the neuromuscular junctions [24].

At the cellular level, there are also important differences in the patterns of distribution of neurotrophic factors between the EOMs and limb muscles, both among controls and among SOD1G93A animals at 50 and 150 days [25]. In the limb muscles of control animals, GDNF, BDNF, NT-3, and NT-4 are present in the vast majority of the neuromuscular junctions at 50 and 150 days, whereas that is the case in only a little over half of the neuromuscular junctions in the control EOMs, at 50 and 150 days. The proportion of neuromuscular junctions containing BDNF and NT-4 is significantly higher in the limb muscles than in the EOMs of control animals at 50 days, and the same is true for the proportion of neuromuscular junctions containing BDNF and GDNF at 150 days. Particularly noteworthy is that the limb muscles of SOD1G93A animals show a significant decrease in the proportion of neuromuscular junctions containing BDNF, GDNF, and NT-4 at 150 days, whereas the EOMs maintain their pattern. The same is true for the presence of the receptors p75NTR, TrkB, and TrkC, which decline significantly in the neuromuscular junctions of the limb muscles of the SOD1G93A animals at 150 days, but not in their EOMs. TrkB is a high-affinity tyrosine kinase receptor specific for BDNF and NT-4, TrkC is a high-affinity tyrosine kinase receptor specific for NT-3, and p75NTR is a low-affinity receptor for BDNF, NT-3, and NT-4 [39]. There are only discrete differences between the control and the SOD1G93A animals with respect to patterns of neurotrophic factors in the nerve axons and the myofibers of both EOM and limb muscles [25].

Taken together, these studies [24, 25] clearly show both at the mRNA level and immunohistochemically that the EOMs have lower neurotrophic factor levels than the limb muscles, in control mice. One possible explanation resides in the differences in innervation between these muscles, as the EOMs have a rich population of multiply innervated myofibers, in contrast to the exclusively twitch myofibers of limb muscles, and the developmental fates of these different types of axons are dependent upon target-derived neurotrophic factors [40]. They also show that the significant changes in neurotrophic factors in limb muscles of transgenic animals are not related to normal aging.

Studies of neurotrophic factors in muscle tissue pose a technical challenge and clinical trials in ALS have not led to the desired effects, but the early detection of alterations in the levels of expression of neurotrophic factors and the distinct patterns observed in the EOMs versus limb muscles indicate that it is too early to

**105**

*The Extraocular Muscles Are Selectively Spared in ALS DOI: http://dx.doi.org/10.5772/intechopen.89504*

rather unaffected in the disease.

EOMs and limb muscles in ALS [26].

controls or terminal ALS donors.

dismiss their possible roles in the pathophysiology of ALS and in particular in the selective capacity of the EOMs to maintain neuromuscular junctions and remain

**5. Differences in Wnt profiles between extraocular and limb muscles**

Several Wnt proteins, including Wnt1, Wnt3a, Wnt 5a, and Wnt7a, are highly expressed at the neuromuscular junction, in muscle fibers, and in motor neurons, and abnormal Wnt signaling has been reported in ALS and a number of other neuromuscular conditions [41–46]. In particular, Wnt1 has been assigned important roles in muscle regeneration and in synaptic plasticity, acting on both sides of the synapse [41, 47]. Wnt3a modulates the formation of neuromuscular junctions and promotes nerve outgrowth [48–50], whereas Wnt5a is important for motor neuron survival during development [51]. Wnt7a plays an important role in synaptic assembly and plasticity and remodeling of incoming axons [52] and has also been shown to have positive effects on the myofibers [53, 54]. Furthermore, Riluzole, the only medical treatment available for ALS, enhances Wnt/beta catenin signaling. We have performed a comprehensive immunohistochemical study comparing the distribution of Wnt1, Wnt3a, Wnt5a, and Wnt7a in the EOMs and limb muscle samples (biceps brachii, vastus lateralis, and tibialis anterior) from six terminal ALS donors, younger controls (mean age 41 years for EOMs and 33 years for limb muscles, referred to as adult controls), and older controls (mean age 75 years for EOM and 76 years for limb, referred to as elderly controls), and we have reported significant differences and suggested a role for Wnts in the different behaviors of

In the adult control EOMs, roughly 71% of the axons are Wnt1 positive, and in the elderly controls, merely 12% of the axons contain Wnt1, whereas in the EOMs of terminal ALS donors, 43% of axons are labeled for Wnt1, clearly showing that they preferentially maintain Wnt1 in their nerves. The limb muscles of terminal ALS donors contain significantly less Wnt1-positive axons than the EOMs, with extremely high variation among the different donors. No statistically significant differences between the three limb muscle groups are detectable regarding the presence of Wnt1 in the motor nerves. Wnt1 is present in almost 40% of the myofibers in the EOMs of adult controls and almost in all myofibers of terminal ALS donors, whereas it is not present in limb myofibers of adult controls or terminal ALS donors. With regard to Wnt3a, it is present in the vast majority of axons in both the EOMs of adult controls and ALS terminal donors, but again it is significantly lower in the elderly control EOMs. In the limb muscles of all groups, the percentage of axons containing Wnt3a is significantly lower than in the EOMs, with approximately 75% fewer Wnt3a axons. Approximately 17% of the myofibers contain Wnt3a in adult control EOM, whereas almost all myofibers in the EOMs of terminal ALS donors are positive. Higher levels of Wnt3a are present in adult control limb myofibers, but in contrast Wnt3a is practically absent in the limb muscle fibers of terminal ALS donors. Wnt5a is present in practically all EOM axons both in adult controls and terminal ALS donors and is somewhat lower in the EOMs of elderly controls. Similarly, practically all limb muscle axons, irrespective of group, are Wnt5a positive. Wnt5a, seen as weak immunostaining, is present in almost all myofibers of control adult EOMs and terminal ALS donors, whereas it is not present in limb myofibers of adult

A very large proportion of the axons in the EOMs of adult controls and terminal ALS donors are Wnt7a positive, whereas a significantly lower number is found in the EOMs of the elderly controls. A similar proportion of Wnt7a-positive axons

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

no significant differences are detectable at 150 days. Finally, comparison between the EOMs and limb muscles of transgenic animals shows no significant differences in the levels of expression of any of the four neurotrophic factors at 50 days but

Because no significant changes in the levels of expression of BDNF detected with quantitative RT-PCR are apparent in either the limb or EOMs of the transgenic animals at any stage, the authors dismiss a possible role for this neurotrophic factors on ALS. The BDNF results obtained in the SOD1G93A mice are in agreement with a previous report from muscle samples of ALS patients [38]. In contrast, the GDNF mRNA levels are significantly upregulated in the EOMs of the SOD1G93A mice at 50 days, and the authors suggest that this early upregulation is triggered by the disease and seminal for the maintenance of the general morphology of the EOMs and in particular for their capacity of keeping intact neuromuscular junctions. They interpret the upregulation of GDNF in the end stage in the limb muscles as reflecting the advanced motor neuron degeneration. Similarly, the upregulation of NT-3 in the EOMs detected at 50 days is also proposed to be triggered by ALS and to protect the EOMs, whereas early downregulation of NT-4 in the limb muscles is suggested to be detrimental and contribute to the loss of axonal contact at the neuromuscular

At the cellular level, there are also important differences in the patterns of distribution of neurotrophic factors between the EOMs and limb muscles, both among controls and among SOD1G93A animals at 50 and 150 days [25]. In the limb muscles of control animals, GDNF, BDNF, NT-3, and NT-4 are present in the vast majority of the neuromuscular junctions at 50 and 150 days, whereas that is the case in only a little over half of the neuromuscular junctions in the control EOMs, at 50 and 150 days. The proportion of neuromuscular junctions containing BDNF and NT-4 is significantly higher in the limb muscles than in the EOMs of control animals at 50 days, and the same is true for the proportion of neuromuscular junctions containing BDNF and GDNF at 150 days. Particularly noteworthy is that the limb muscles of SOD1G93A animals show a significant decrease in the proportion of neuromuscular junctions containing BDNF, GDNF, and NT-4 at 150 days, whereas the EOMs maintain their pattern. The same is true for the presence of the receptors p75NTR, TrkB, and TrkC, which decline significantly in the neuromuscular junctions of the limb muscles of the SOD1G93A animals at 150 days, but not in their EOMs. TrkB is a high-affinity tyrosine kinase receptor specific for BDNF and NT-4, TrkC is a high-affinity tyrosine kinase receptor specific for NT-3, and p75NTR is a low-affinity receptor for BDNF, NT-3, and NT-4 [39]. There are only discrete differences between the control and the SOD1G93A animals with respect to patterns of neurotrophic factors in the nerve axons and the myofibers of both EOM and limb

Taken together, these studies [24, 25] clearly show both at the mRNA level and immunohistochemically that the EOMs have lower neurotrophic factor levels than the limb muscles, in control mice. One possible explanation resides in the differences in innervation between these muscles, as the EOMs have a rich population of multiply innervated myofibers, in contrast to the exclusively twitch myofibers of limb muscles, and the developmental fates of these different types of axons are dependent upon target-derived neurotrophic factors [40]. They also show that the significant changes in neurotrophic factors in limb muscles of transgenic animals

Studies of neurotrophic factors in muscle tissue pose a technical challenge and clinical trials in ALS have not led to the desired effects, but the early detection of alterations in the levels of expression of neurotrophic factors and the distinct patterns observed in the EOMs versus limb muscles indicate that it is too early to

significantly higher levels of BDNF in the limb muscles at 150 days [24].

**104**

junctions [24].

muscles [25].

are not related to normal aging.

dismiss their possible roles in the pathophysiology of ALS and in particular in the selective capacity of the EOMs to maintain neuromuscular junctions and remain rather unaffected in the disease.

### **5. Differences in Wnt profiles between extraocular and limb muscles**

Several Wnt proteins, including Wnt1, Wnt3a, Wnt 5a, and Wnt7a, are highly expressed at the neuromuscular junction, in muscle fibers, and in motor neurons, and abnormal Wnt signaling has been reported in ALS and a number of other neuromuscular conditions [41–46]. In particular, Wnt1 has been assigned important roles in muscle regeneration and in synaptic plasticity, acting on both sides of the synapse [41, 47]. Wnt3a modulates the formation of neuromuscular junctions and promotes nerve outgrowth [48–50], whereas Wnt5a is important for motor neuron survival during development [51]. Wnt7a plays an important role in synaptic assembly and plasticity and remodeling of incoming axons [52] and has also been shown to have positive effects on the myofibers [53, 54]. Furthermore, Riluzole, the only medical treatment available for ALS, enhances Wnt/beta catenin signaling.

We have performed a comprehensive immunohistochemical study comparing the distribution of Wnt1, Wnt3a, Wnt5a, and Wnt7a in the EOMs and limb muscle samples (biceps brachii, vastus lateralis, and tibialis anterior) from six terminal ALS donors, younger controls (mean age 41 years for EOMs and 33 years for limb muscles, referred to as adult controls), and older controls (mean age 75 years for EOM and 76 years for limb, referred to as elderly controls), and we have reported significant differences and suggested a role for Wnts in the different behaviors of EOMs and limb muscles in ALS [26].

In the adult control EOMs, roughly 71% of the axons are Wnt1 positive, and in the elderly controls, merely 12% of the axons contain Wnt1, whereas in the EOMs of terminal ALS donors, 43% of axons are labeled for Wnt1, clearly showing that they preferentially maintain Wnt1 in their nerves. The limb muscles of terminal ALS donors contain significantly less Wnt1-positive axons than the EOMs, with extremely high variation among the different donors. No statistically significant differences between the three limb muscle groups are detectable regarding the presence of Wnt1 in the motor nerves. Wnt1 is present in almost 40% of the myofibers in the EOMs of adult controls and almost in all myofibers of terminal ALS donors, whereas it is not present in limb myofibers of adult controls or terminal ALS donors.

With regard to Wnt3a, it is present in the vast majority of axons in both the EOMs of adult controls and ALS terminal donors, but again it is significantly lower in the elderly control EOMs. In the limb muscles of all groups, the percentage of axons containing Wnt3a is significantly lower than in the EOMs, with approximately 75% fewer Wnt3a axons. Approximately 17% of the myofibers contain Wnt3a in adult control EOM, whereas almost all myofibers in the EOMs of terminal ALS donors are positive. Higher levels of Wnt3a are present in adult control limb myofibers, but in contrast Wnt3a is practically absent in the limb muscle fibers of terminal ALS donors.

Wnt5a is present in practically all EOM axons both in adult controls and terminal ALS donors and is somewhat lower in the EOMs of elderly controls. Similarly, practically all limb muscle axons, irrespective of group, are Wnt5a positive. Wnt5a, seen as weak immunostaining, is present in almost all myofibers of control adult EOMs and terminal ALS donors, whereas it is not present in limb myofibers of adult controls or terminal ALS donors.

A very large proportion of the axons in the EOMs of adult controls and terminal ALS donors are Wnt7a positive, whereas a significantly lower number is found in the EOMs of the elderly controls. A similar proportion of Wnt7a-positive axons

are present in the limb muscles of both adult controls and terminal ALS donors, but a significantly higher number of positive axons are found in the limb muscles of elderly controls. Only approximately 20% of the myofibers in the adult control EOMs contain Wnt7a, but in the EOMs of terminal ALS donors, this number increases significantly almost 60%. In the limb muscles, Wnt7a is present in more than half of the myofibers of the adult controls and practically in all myofibers in the elderly controls. The differences found between adult controls and terminal ALS donors regarding Wnt7a in limb myofibers are not significant, probably due to large interindividual variation among the donors.

Data collected in controls and in the SOD1G93A mouse model of ALS [26] indicates that practically all neuromuscular junctions in control EOMs and limb muscles express Wnt1, Wnt3a, Wnt5a, and Wnt7 at 50 and 150 days. The same is true for the transgenic EOMs at both 50 and 150 days and the transgenic limb muscles at 50 days, whereas a significantly decrease in the proportion of neuromuscular junctions co-expressing the different Wnt isoforms is clearly apparent in the transgenic limb samples at 150 days.

#### **6. Summary**

In summary, the EOMs remain remarkably well preserved until the end stages of ALS, both in human terminal donors and in mouse models of ALS. They maintain the composition of their neuromuscular junctions and do not loose contact with the supplying axon. Furthermore, important differences exist between the EOMs and the limb muscles with respect to neurotrophic factors and Wnts:


**107**

*The Extraocular Muscles Are Selectively Spared in ALS DOI: http://dx.doi.org/10.5772/intechopen.89504*

ment of new treatment strategies in the future.

There are no conflicts of interest.

**Conflict of interest**

**Author details**

Fatima Pedrosa Domellöf

provided the original work is properly cited.

• Wnt1 and Wnt3a are present in a larger proportion of axons in the human control EOMs than in the limb muscles. The myofibers in the control EOMs contain higher Wnt1 and Wnt5a and lower Wnt3a and Wnt7a than the myofibers of limb muscles, and although the exact implications of these intrinsic differences in Wnt isoform profiles remain to be determined, they may be

• Wnt1 and Wnt3a are present at significantly higher levels in the axons and the muscle fibers of the EOMs than in the axons or muscle fibers of the limb muscles of terminal ALS donors. Because Wnt1 plays a role in synaptic plasticity and muscle regeneration and in neuromuscular junction formation during fetal development and Wnt3a has similar roles, these differences between EOMs and limb muscles are likely to be of importance for the selective sparing of the EOMs, and in particular of their neuromuscular junctions, in ALS.

Altogether, these data show important differences between the EOMs and limb muscles with regard to neurotrophic factors and four different Wnt isoforms, both at base line and in ALS, that likely play a role in the selective sparing of the EOMs in ALS. These data also suggest that muscle itself may provide a door for the develop-

Department of Clinical Science, Ophthalmology, Umeå University, Umeå, Sweden

© 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,

\*Address all correspondence to: fatima.pedrosa-domellof@umu.se

relevant for the selective sparing of the EOMs in ALS.

*The Extraocular Muscles Are Selectively Spared in ALS DOI: http://dx.doi.org/10.5772/intechopen.89504*

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

interindividual variation among the donors.

limb samples at 150 days.

**6. Summary**

are present in the limb muscles of both adult controls and terminal ALS donors, but a significantly higher number of positive axons are found in the limb muscles of elderly controls. Only approximately 20% of the myofibers in the adult control EOMs contain Wnt7a, but in the EOMs of terminal ALS donors, this number increases significantly almost 60%. In the limb muscles, Wnt7a is present in more than half of the myofibers of the adult controls and practically in all myofibers in the elderly controls. The differences found between adult controls and terminal ALS donors regarding Wnt7a in limb myofibers are not significant, probably due to large

Data collected in controls and in the SOD1G93A mouse model of ALS [26] indicates that practically all neuromuscular junctions in control EOMs and limb muscles express Wnt1, Wnt3a, Wnt5a, and Wnt7 at 50 and 150 days. The same is true for the transgenic EOMs at both 50 and 150 days and the transgenic limb muscles at 50 days, whereas a significantly decrease in the proportion of neuromuscular junctions co-expressing the different Wnt isoforms is clearly apparent in the transgenic

In summary, the EOMs remain remarkably well preserved until the end stages of ALS, both in human terminal donors and in mouse models of ALS. They maintain the composition of their neuromuscular junctions and do not loose contact with the supplying axon. Furthermore, important differences exist between the EOMs and

• The mRNA levels of BDNF, NT-4, and NT-3 and the detection of BDNF, NT-4, NT-3, and GDNF at the protein level are lower in the EOMs than in the limb

• NT-3 is upregulated in the EOMs of SOD1G93A mice at 50 days and may be protective. A similar difference in GDNF may also be advantageous for the

• There is early downregulation of NT-4 in the limb muscles of SOD1G93A mice compared to the controls at the same age, which coincides with the timing of early loss of motor axon occupancy at the neuromuscular junctions of limb

• The proportion of neuromuscular junctions in the EOMs positive for BDNF, GDNF, and NT-4 is not changed between the controls and the transgenic mice at 150 days, whereas there is a significant decrease in the limb muscles of the SOD1G93A animals compared to the controls at late stage and there is also a significant decrease over the course of the disease in the limb muscles of the transgenic mice. Again, these changes in neurotrophic factors parallel the loss of axonal occupancy at the neuromuscular junctions of the limb muscles of transgenic animals and these neurotrophic factors are known to play crucial

• There is a significant decrease in the neurotrophic factor receptors p75NTR, TrkB, TrkC, and GFR-alpha1 in the neuromuscular junctions of terminal SOD1G93A mice, whereas they are maintained in the neuromuscular junctions

the limb muscles with respect to neurotrophic factors and Wnts:

muscles of control animals at 50 and/or 150 days.

EOMs and protect their motor neurons.

roles in regulating the synapses [55, 56].

of terminal transgenic EOMs.

muscles in this ALS model [6].

**106**


Altogether, these data show important differences between the EOMs and limb muscles with regard to neurotrophic factors and four different Wnt isoforms, both at base line and in ALS, that likely play a role in the selective sparing of the EOMs in ALS. These data also suggest that muscle itself may provide a door for the development of new treatment strategies in the future.

#### **Conflict of interest**

There are no conflicts of interest.

#### **Author details**

Fatima Pedrosa Domellöf Department of Clinical Science, Ophthalmology, Umeå University, Umeå, Sweden

\*Address all correspondence to: fatima.pedrosa-domellof@umu.se

© 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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[12] Pedrosa-Domellöf F. Extraocular

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Block CH. Expression of acetylcholine receptor isoforms in extraocular muscle endplates. Investigative Ophthalmology & Visual Science. 1996;**37**:345-351

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Section 3

Novel Therapeutics

and Challenges

Section 3
