Exendin-4 ameliorates motor neuron degeneration in cellular and animal models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a progressive loss of lower motor neurons in the spinal cord. The incretin hormone, glucagon-like peptide-1 (GLP-1), facilitates insulin signaling, and the long acting GLP-1 receptor agonist exendin-4 (Ex-4) is currently used as an anti-diabetic drug. GLP-1 receptors are widely expressed in the brain and spinal cord, and our prior studies have shown that Ex-4 is neuroprotective in several neurodegenerative disease rodent models, including stroke, Parkinson's disease and Alzheimer's disease. Here we hypothesized that Ex-4 may provide neuroprotective activity in ALS, and hence characterized Ex-4 actions in both cell culture (NSC-19 neuroblastoma cells) and in vivo (SOD1 G93A mutant mice) models of ALS. Ex-4 proved to be neurotrophic in NSC-19 cells, elevating choline acetyltransferase (ChAT) activity, as well as neuroprotective, protecting cells from hydrogen peroxide-induced oxidative stress and staurosporine-induced apoptosis. Additionally, in both wild-type SOD1 and mutant SOD1 (G37R) stably transfected NSC-19 cell lines, Ex-4 protected against trophic factor withdrawal-induced toxicity. To assess in vivo translation, SOD1 mutant mice were administered vehicle or Ex-4 at 6-weeks of age onwards to end-stage disease via subcutaneous osmotic pump to provide steady-state infusion. ALS mice treated with Ex-4 showed improved glucose tolerance and normalization of behavior, as assessed by running wheel, compared to control ALS mice. Furthermore, Ex-4 treatment attenuated neuronal cell death in the lumbar spinal cord; immunohistochemical analysis demonstrated the rescue of neuronal markers, such as ChAT, associated with motor neurons. Together, our results suggest that GLP-1 receptor agonists warrant further evaluation to assess whether their neuroprotective potential is of therapeutic relevance in ALS.     

Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.     

Rapamycin treatment augments motor neuron degeneration in SOD1G93A mouse model of amyotrophic lateral sclerosis

Aberrant protein misfolding may contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS) but the detailed mechanisms are largely unknown. Our previous study has shown that autophagy is altered in the mouse model of ALS. In the present study, we systematically investigated the correlation of the autophagic alteration with the motor neurons (MNs) degeneration in the ALS mice. We have demonstrated that the autophagic protein marker LC3-II is markedly and specifically increased in the spinal cord MNs of the ALS mice. Electron microscopy and immunochemistry studies have shown that autophagic vacuoles are significantly accumulated in the dystrophic axons of spinal cord MNs of the ALS mice. All these changes in the ALS mice appear at the age of 90 d when the ALS mice display modest clinical symptoms; and they become prominent at the age of 120 d. The clinical symptoms are correlated with the progression of MNs degeneration. Moreover, we have found that p62/SQSTM1 is accumulated progressively in the spinal cord, indicating that the possibility of impaired autophagic flux in the SOD1^G93A mice. Furthermore, to our surprise, we have found that treatment with autophagy enhancer rapamycin accelerates the MNs degeneration, shortens the life span of the ALS mice, and has no obvious effects on the accumulation of SOD1 aggregates. In addition, we have demonstrated that rapamycin treatment in the ALS mice causes more severe mitochondrial impairment, higher Bax levels and greater caspase-3 activation. These findings suggest that selective degeneration of MNs is associated with the impairment of the autophagy pathway and that rapamycin treatment may exacerbate the pathological processing through apoptosis and other mechanisms in the ALS mice.     

Astrocytes in amyotrophic lateral sclerosis: direct effects on motor neuron survival

Selective motor neuron death during amyotrophic lateral sclerosis (ALS) is a non-cell autonomous process in which non-neuronal cells induce and/or contribute to the disease process. The non-neuronal cells that are clearly involved in the pathogenesis of the disease are the surrounding astrocytes. Under normal conditions, astrocytes remove glutamate from the synaptic cleft and release trophic factors. In addition, these cells determine the functional characteristics of motor neurons. Recent evidence suggests that activation of astrocytes in a degenerative disease like ALS disturbs the crosstalk between astrocytes and motor neurons, which could contribute to and/or accelerate selective motor neuron death. These new insights may contribute to the development of therapeutic approaches to slow this fatal neurodegenerative disease.     

PAK4 suppresses motor neuron degeneration in hSOD1G93A‐linked amyotrophic lateral sclerosis cell and rat models

ObjectivesAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons (MN). CREB pathway‐mediated inhibition of apoptosis contributes to neuron protection, and PAK4 activates CREB signalling in diverse cell types. This study aimed to investigate PAK4’s effect and mechanism of action in ALS.MethodsWe analysed RNA levels by qRT‐PCR, protein levels by immunofluorescence and Western blotting, and apoptosis by flow cytometry and TUNEL staining. Cell transfection was performed for in vitro experiment. Mice were injected intraspinally to evaluate PAK4 function in vivo experiment. Rotarod test was performed to measure motor function.ResultsThe expression and activation of PAK4 significantly decreased in the cell and mouse models of ALS as the disease progressed, which was caused by the negative regulation of miR‐9‐5p. Silencing of PAK4 increased the apoptosis of MN by inhibiting CREB‐mediated neuroprotection, whereas overexpression of PAK4 protected MN from hSOD1^G93A‐induced degeneration by activating CREB signalling. The neuroprotective effect of PAK4 was markedly inhibited by CREB inhibitor. In ALS models, the PAK4/CREB pathway was inhibited, and cell apoptosis increased. In vivo experiments revealed that PAK4 overexpression in the spinal neurons of hSOD1^G93A mice suppressed MN degeneration, prolonged survival and promoted the CREB pathway.ConclusionsPAK4 protects MN from degeneration by activating the anti‐apoptotic effects of CREB signalling, suggesting it may be a therapeutic target in ALS.

Schematic representation of the mechanism of PAK4 protecting MN from apoptosis in ALS. PAK4 increases CREB levels and activation, leading to the upregulation of PGC‐1a and Bcl‐2, thereby decreasing cleaved‐caspase3 levels, and inhibiting MN degeneration. miR‐9‐5p is responsible for the decreased expression of PAK4 in ALS.     

Proteomic analysis of muscle affected by motor neuron degeneration: the wobbler mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is the most common form of motor neuron disease in adult patients and characterized by progressive paralysis. The wobbler mouse (phenotype WR, genotype wr/wr) is an established animal model of human motor neuron disease and is characterized by a large variety of cellular abnormalities including muscular atrophy. In analogy to recent proteomic studies of cerebrospinal fluid and spinal cord, we have used here fluorescence difference in-gel electrophoresis to analyze global changes in the skeletal muscle proteome from WR versus normal mice. Relative concentrations of 21 proteins were found to be increased and 3 proteins were decreased. Mass spectrometric analysis identified these proteins to be associated with key metabolic pathways, the contractile apparatus, intermediate filaments and the cellular stress response. Drastically increased levels of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase were confirmed by immunoblotting and this finding agrees with the idea of an oxidative-to-glycolytic shift in disease-related muscular atrophy. The establishment of novel disease-specific biomarkers of motor neuron disease might be helpful in the design of improved diagnostic tools and the identification of novel therapeutic targets.     

Astrocyte activation by fibroblast growth factor-1 and motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.     

Rapamycin treatment augments motor neuron degeneration in SOD1(G93A) mouse model of amyotrophic lateral sclerosis

Aberrant protein misfolding may contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS) but the detailed mechanisms are largely unknown. Our previous study has shown that autophagy is altered in the mouse model of ALS. In the present study, we systematically investigated the correlation of the autophagic alteration with the motor neurons (MNs) degeneration in the ALS mice. We have demonstrated that the autophagic protein marker LC3-II is markedly and specifically increased in the spinal cord MNs of the ALS mice. Electron microscopy and immunochemistry studies have shown that autophagic vacuoles are significantly accumulated in the dystrophic axons of spinal cord MNs of the ALS mice. All these changes in the ALS mice appear at the age of 90 d when the ALS mice display modest clinical symptoms; and they become prominent at the age of 120 d. The clinical symptoms are correlated with the progression of MNs degeneration. Moreover, we have found that p62/SQSTM1 is accumulated progressively in the spinal cord, indicating that the possibility of impaired autophagic flux in the SOD1(G93A) mice. Furthermore, to our surprise, we have found that treatment with autophagy enhancer rapamycin accelerates the MNs degeneration, shortens the life span of the ALS mice, and has no obvious effects on the accumulation of SOD1 aggregates. In addition, we have demonstrated that rapamycin treatment in the ALS mice causes more severe mitochondrial impairment, higher Bax levels and greater caspase-3 activation. These findings suggest that selective degeneration of MNs is associated with the impairment of the autophagy pathway and that rapamycin treatment may exacerbate the pathological processing through apoptosis and other mechanisms in the ALS mice.     

Focal degeneration of astrocytes in amyotrophic lateral sclerosis

Astrocytes emerge as key players in motor neuron degeneration in Amyotrophic Lateral Sclerosis (ALS). Whether astrocytes cause direct damage by releasing toxic factors or contribute indirectly through the loss of physiological functions is unclear. Here we identify in the hSOD1(G93A) transgenic mouse model of ALS a degenerative process of the astrocytes, restricted to those directly surrounding spinal motor neurons. This phenomenon manifests with an early onset and becomes significant concomitant with the loss of motor cells and the appearance of clinical symptoms. Contrary to wild-type astrocytes, mutant hSOD1-expressing astrocytes are highly vulnerable to glutamate and undergo cell death mediated by the metabotropic type-5 receptor (mGluR5). Blocking mGluR5 in vivo slows down astrocytic degeneration, delays the onset of the disease and slightly extends survival in hSOD1(G93A) transgenic mice. We propose that excitotoxicity in ALS affects both motor neurons and astrocytes, favouring their local interactive degeneration. This new mechanistic hypothesis has implications for therapeutic interventions.     

Impaired structural motor connectome in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease selectively affecting upper and lower motor neurons. Patients with ALS suffer from progressive paralysis and eventually die on average after three years. The underlying neurobiology of upper motor neuron degeneration and its effects on the complex network of the brain are, however, largely unknown. Here, we examined the effects of ALS on the structural brain network topology in 35 patients with ALS and 19 healthy controls. Using diffusion tensor imaging (DTI), the brain network was reconstructed for each individual participant. The connectivity of this reconstructed brain network was compared between patients and controls using complexity theory without - a priori selected - regions of interest. Patients with ALS showed an impaired sub-network of regions with reduced white matter connectivity (p = 0.0108, permutation testing). This impaired sub-network was strongly centered around primary motor regions (bilateral precentral gyrus and right paracentral lobule), including secondary motor regions (bilateral caudal middle frontal gyrus and pallidum) as well as high-order hub regions (right posterior cingulate and precuneus). In addition, we found a significant reduction in overall efficiency (p = 0.0095) and clustering (p = 0.0415). From our findings, we conclude that upper motor neuron degeneration in ALS affects both primary motor connections as well as secondary motor connections, together composing an impaired sub-network. The degenerative process in ALS was found to be widespread, but interlinked and targeted to the motor connectome.     

Mapping of the motor neuron degeneration (Mnd) gene, a mouse model of amyotrophic lateral sclerosis (ALS).

The motor neuron degeneration mutation (Mnd) causes a late-onset, progressive degeneration of upper and lower motor neurons in mice. After establishing genetic and environmental conditions that distinguish the phenotypes of Mnd/Mnd from +/Mnd mice, Mnd was mapped to proximal Chr 8, using endogenous retroviruses as markers. The map location was confirmed with additional linked polymorphic markers. The outcross/intercross matings to the strain AKR/J, which were used to follow the segregation of the retroviral markers with respect to Mnd, also revealed the existence of a timing effect. Approximately one-fourth of the affected Mnd/Mnd F2 progeny showed accelerated disease. The Mnd mouse model should allow study of mechanisms affecting onset and progression of specific neuronal degeneration in both animal and human neurological disease.     

A drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease that leads to loss of motor function and early death. About 5% of cases are inherited, with the majority of identified linkages in the gene encoding copper, zinc-superoxide dismutase (SOD1). Strong evidence indicates that the SOD1 mutations confer dominant toxicity on the protein. To provide new insight into mechanisms of ALS, we have generated and characterized a model for familial ALS in Drosophila with transgenic expression of human SOD1. Expression of wild type or disease-linked (A4V, G85R) mutants of human SOD1 selectively in motor neurons induced progressive climbing deficits. These effects were accompanied by defective neural circuit electrophysiology, focal accumulation of human SOD1 protein in motor neurons, and a stress response in surrounding glia. However, toxicity was not associated with oligomerization of SOD1 and did not lead to neuronal loss. These studies uncover cell-autonomous injury by SOD1 to motor neurons in vivo, as well as non-autonomous effects on glia, and provide the foundation for new insight into injury and protection of motor neurons in ALS.     

In vivo optical imaging of motor neuron autophagy in a mouse model of amyotrophic lateral sclerosis

Autophagy is involved in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). We have generated a novel double transgenic (DTg) mouse line by mating a green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (LC3-Tg) mouse and a G93A mutant human Cu/Zn superoxide dismutase (mSOD1) transgenic (mSOD1-Tg) mouse. In vivo imaging of autophagy with these novel DTg mice was conducted at 10 (presymptomatic), 17 (early symptomatic) and 19 (late symptomatic) weeks of age. Fluorescence imaging analysis revealed a strong fluorescent signal in vivo over the T₃-S₁ level at 17 and 19 weeks of age only in the DTg mice. Ex vivo autophagy imaging of spinal cord sections (20 μm) also showed a progressive increase of the fluorescence signal from 17 to 19 weeks in DTg mice in the anterior horn at the L4-5 level, and the fluorescence signals were clearly observed in the gray matter of the spinal cord with a progressive increase of the signal and decreases in large motor neurons. Protein gel blot analysis revealed maximum LC3-I and LC3-II expressions at 19 weeks, consistent with the results from the in vivo autophagy imaging experiment. This method could also be applied as a unique tool for clarifying the role of autophagy, and to monitor the pathologic processes involving autophagy not only in ALS, but also other neurological diseases.     

In vivo optical imaging of motor neuron autophagy in a mouse model of amyotrophic lateral sclerosis

Autophagy is involved in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). We have generated a novel double transgenic (DTg) mouse line by mating a green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (LC3-Tg) mouse and a G93A mutant human Cu/Zn superoxide dismutase (mSOD1) transgenic (mSOD1-Tg) mouse. In vivo imaging of autophagy with these novel DTg mice was conducted at 10 (presymptomatic), 17 (early symptomatic) and 19 (late symptomatic) weeks of age. Fluorescence imaging analysis revealed a strong fluorescent signal in vivo over the T?-S? level at 17 and 19 weeks of age only in the DTg mice. Ex vivo autophagy imaging of spinal cord sections (20 μm) also showed a progressive increase of the fluorescence signal from 17 to 19 weeks in DTg mice in the anterior horn at the L?-? level, and the fluorescence signals were clearly observed in the gray matter of the spinal cord with a progressive increase of the signal and decreases in large motor neurons. Protein gel blot analysis revealed maximum LC3-I and LC3-II expressions at 19 weeks, consistent with the results from the in vivo autophagy imaging experiment. This method could also be applied as a unique tool for clarifying the role of autophagy, and to monitor the pathologic processes involving autophagy not only in ALS, but also other neurological diseases.     

The role of mutant TAR DNA-binding protein 43 in amyotrophic lateral sclerosis and frontotemporal lobar degeneration

TDP-43 (TAR DNA-binding protein 43) has been identified as a key protein of ubiquitinated inclusions in brains of patients with ALS (amyotrophic lateral sclerosis) or FTLD (frontotemporal lobar degeneration), defining a new pathological disease spectrum. Recently, coding mutations have been identified in the TDP-43 gene (TARDBP), which further confirmed the pathogenic nature of the protein. Today, several animal models have been generated to gain more insight into the disease-causing pathways of the FTLD/ALS spectrum. This mini-review summarizes the current status of TDP-43 models, with a focus on mutant TDP-43.     

Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis

Accumulating evidence indicates that abnormal conformation of mutant superoxide dismutase 1 (SOD1) is an essential feature underlying the pathogenesis of mutant SOD1-linked familial amyotrophic lateral sclerosis (ALS). Here we investigated the role of ubiquitin-proteasome pathway in the mutant SOD1-related cell death and the effect of oxidative stress on the misfolding of mutant SOD1. Transient overexpression of ubiquitin with human SOD1 (wild-type, ala4val, gly85arg, gly93ala) in Neuro2A cells decreased the amount of mutant SOD1, but not of wild-type, while only mutants were co-immunoprecipitated with poly-ubiquitin. Proteasome inhibition by lactacystin augmented accumulation of mutant SOD1 in the non-ionic detergent-insoluble fraction. The spinal cord lysates from mutant SOD1 transgenic mice showed multiple carbonylated proteins, including mutant SOD1 with SDS-resistant dimer formation. Furthermore, the treatment of hSOD1-expressing cells with hydrogen peroxide promoted the oligomerization, and detergent-insolubility of mutant SOD1 alone, and the oxidized mutant SOD1 proteins were more heavily poly-ubiquitinated. In Neuro2A cells stably expressing human SOD1 protein, the proteasome function measured by chymotrypsin-like activity, was decreased over time without a quantitative alteration of the 20S proteasomal component. Finally, primary motor neurons from the mouse embryonic spinal cord were more vulnerable to lactacystin than non-motor neurons. These results indicate that the sustained expression of mutant SOD1 leads to proteasomal inhibition and motor neuronal death, which in part explains the pathogenesis of mutant SOD1-linked ALS.     

Failure of protein quality control in amyotrophic lateral sclerosis

The protein chaperoning and ubiquitin-proteasome systems perform many homeostatic functions within cells involving protein folding, transport and degradation. Of paramount importance is ridding cells of mutant or post-translationally modified proteins that otherwise tend to aggregate into insoluble complexes and form inclusions. Such inclusions are characteristic of many neurodegenerative diseases and implicate protein misfolding and aggregation as common aspects of pathogenesis. In the most common familial form of ALS, mutations in SOD1 promote misfolding of the protein and target it for degradation by proteasomes. Although proteasomes can degrade the mutant proteins efficiently, altered solubility and aggregation of mutant SOD1 are features of the disease and occur most prominently in the most vulnerable cells and tissues. Indeed, lumbar spinal cord of mutant SOD1 transgenic mice show early reduction in their capacity for protein chaperoning and proteasome-mediated hydrolysis of substrates, and motor neurons are particularly vulnerable to aggregation of mutant SOD1. A high threshold for upregulating key pathways in response to the stress of added substrate load may contribute to this vulnerability. The broad spectrum neuroprotective capability and efficacy of some chaperone-based therapies in preclinical models makes these pathways attractive as targets for therapy in ALS, as well as other neurodegenerative diseases. A better understanding of the mechanisms governing the regulation of protein chaperones and UPS components would facilitate development of treatments that upregulate these pathways in a coordinated manner in neural tissue without long term toxicity.     

Proteostatic imbalance and protein spreading in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder whose exact causative mechanisms are still under intense investigation. Several lines of evidence suggest that the anatomical and temporal propagation of pathological protein species along the neural axis could be among the main driving mechanisms for the fast and irreversible progression of ALS pathology. Many ALS‐associated proteins form intracellular aggregates as a result of their intrinsic prion‐like properties and/or following impairment of the protein quality control systems. During the disease course, these mutated proteins and aberrant peptides are released in the extracellular milieu as soluble or aggregated forms through a variety of mechanisms. Internalization by recipient cells may seed further aggregation and amplify existing proteostatic imbalances, thus triggering a vicious cycle that propagates pathology in vulnerable cells, such as motor neurons and other susceptible neuronal subtypes. Here, we provide an in‐depth review of ALS pathology with a particular focus on the disease mechanisms of seeding and transmission of the most common ALS‐associated proteins, including SOD1, FUS, TDP‐43, and C9orf72‐linked dipeptide repeats. For each of these proteins, we report historical, biochemical, and pathological evidence of their behaviors in ALS. We further discuss the possibility to harness pathological proteins as biomarkers and reflect on the implications of these findings for future research.     

Regulatory T cells for amyotrophic lateral sclerosis/motor neuron disease: A clinical and preclinical systematic review

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by neuronal degeneration and inflammation in the nerves. The role of the immune system has been concentrated by researchers in the etiopathogenesis of the disease. Given the inhibitory roles of regulatory T cells (Tregs), it is expected that increasing or activating their populations in patients with ALS can have significant therapeutic effects. Here we searched databases, including CENTRAL, MEDLINE, CINAHL Plus, clinicaltrials.gov , and ICTRP for randomized clinical trials (RCTs) and non-RCTs until March 2019. For preclinical studies, we searched PubMed, Scopus, and Google Scholar up to June 2019. We also included preclinical studies, due to the lack of clinical information available, which used Tregs (or directly targeting them) for treating mice models of ALS. We identified 29 records (CENTRAL 7, MEDLINE 4, CINAHL Plus 8, and clinicaltrials.gov 10) and removed 10 duplicated publications. After screening, we identified one RCT which had been published as an abstract, three non-RCTs, and four ongoing studies. We also identified 551 records (PubMed 446, Google Scholar 68, and Scopus 37) for preclinical studies and performed a meta-analysis. Finally, we found three papers that matched our inclusion criteria for preclinical studies. Results indicated the effectiveness of the application of Tregs in the treatment of ALS. Our meta-analysis on preclinical studies revealed that Tregs significantly prolonged survival in mice models of ALS. Overall, our analysis testified that exertion of Tregs in the treatment of ALS is a promising approach, that notwithstanding, requires further evaluations.