Comparative morphometric analysis of microglia in the spinal cord of SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis

It has long been recognized that reactive microglia undergo a series of phenotypic changes accompanying morphological transformation. However, the morphological classification of microglia has not yet been achieved. To address this issue, here we morphometrically analysed three‐dimensionally reconstructed ionized calcium binding adaptor molecule 1‐immunoreactive (Iba1⁺) microglia in the ventral horn of the lumbar spinal cord of SOD1^G93A transgenic mice, a model of amyotrophic lateral sclerosis. The hierarchical cluster analysis revealed that microglia were objectively divided into four groups: type S (named after surveillant microglia) and types R1, R2 and R3 (named after reactive microglia). For the purpose of comparative morphometry, we also analysed two pharmacological disease models using wild‐type mice: 3,3′‐iminodipropionitrile (IDPN)‐induced axonopathy and lipopolysaccharide (LPS)‐induced neuroinflammation. Type S microglia showed a typical ramified morphology of surveillant microglia, and were mostly observed in wild‐type controls. Type R1 microglia were seen at the early stage of disease in SOD1^G93A mice, and also frequently occurred in IDPN‐treated mice. They exhibited small cell bodies with shorter and simple processes. Type R2 microglia were morphologically similar to type R1 microglia, but only transiently occurred in the middle stage of disease in SOD1^G93A mice and in IDPN‐treated mice. Type R3 microglia exhibited a bushy shape, and were observed in the end stage of disease in SOD1^G93A mice and in LPS‐treated mice. These findings indicate that microglia of SOD1^G93A mice can be classified into four types, and also suggest that the phenotypic changes may be induced by the events related to axonopathy and neuroinflammation.     

Extracellular mutant SOD1 induces microglial‐mediated motoneuron injury

Through undefined mechanisms, dominant mutations in (Cu/Zn) superoxide dismutase‐1 (mSOD1) cause the non‐cell‐autonomous death of motoneurons in inherited amyotrophic lateral sclerosis (ALS). Microgliosis at sites of motoneuron injury is a neuropathological hallmark of ALS. Extracellular mutant SOD1 (mSOD1) causes motoneuron injury and triggers microgliosis in spinal cord cultures, but it is unclear whether the injury results from extracellular mSOD1 directly interacting with motoneurons or is mediated through mSOD1‐activated microglia. To dissociate these potential mSOD1‐mediated neurotoxic mechanisms, the effects of extracellular human mSOD1^G93A or mSOD1^G85R were assayed using primary cultures of motoneurons and microglia. The data demonstrate that exogenous mSOD1^G93A did not cause detectable direct killing of motoneurons. In contrast, mSOD1^G93A or mSOD1^G85R did induce the morphological and functional activation of microglia, increasing their release of pro‐inflammatory cytokines and free radicals. Furthermore, only when microglia was co‐cultured with motoneurons did extracellular mSOD1^G93A injure motoneurons. The microglial activation mediated by mSOD1^G93A was attenuated using toll‐like receptors (TLR) 2, TLR4 and CD14 blocking antibodies, or when microglia lacked CD14 expression. These data suggest that extracellular mSOD1^G93A is not directly toxic to motoneurons but requires microglial activation for toxicity, utilizing CD14 and TLR pathways. This link between mSOD1 and innate immunity may offer novel therapeutic targets in ALS. © 2009 Wiley‐Liss, Inc.     

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1β secretion. Both caspase-1 and IL-1β contribute to disease progression in the mouse SOD1^G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1^G93A mice microglia do not express NLRP3, and SOD1^G93A protein generated IL-1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1^G93A mice. We show that both aggregated and soluble SOD1^G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1β cleavage, ASC speck formation, and the secretion of IL-1β in a dose- and time-dependent manner. Importantly, SOD1^G93A was unable to induce IL-1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1β secretion. Microglial NLRP3 upregulation was also observed in the TDP-43^Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1^G93A-mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.     

Rho kinase inhibition modulates microglia activation and improves survival in a model of amyotrophic lateral sclerosis

Disease progression in amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motoneurons (MN) and their axons, but is also influenced by neighboring cells such as astrocytes and microglial cells. The role of microglia in ALS is complex as it switches from an anti‐inflammatory and neuroprotective phenotype in early disease to a proinflammatory and neurotoxic phenotype in later stages. Our previous studies in models of neurodegeneration identified rho kinase (ROCK) as a target, which can be manipulated to beneficially influence disease progression. Here, we examined the neuroprotective potential of the ROCK inhibitor Fasudil to target the central pathogenic features of ALS. Application of Fasudil to kainic acid‐lesioned primary MN in vitro resulted in a strong prosurvival effect. In vivo, SOD1^G93A mice benefited from oral treatment with Fasudil showing prolonged survival and improved motor function. These findings were correlated to an improved survival of motor neurons and a pronounced alteration of astroglial and microglial cell infiltration of the spinal cord under Fasudil treatment. Modeling a proinflammatory microglial phenotype by stimulation with LPS in vitro, Fasudil decreased the release of proinflammatory cytokines and chemokines TNFα, Il6, CCL2, CCL3, and CCL5 while CXCL1 release was only transiently suppressed. In sciatic nerve motor axons, neuromuscular junction remodeling processes were increased. In conclusion, we provide preclinical and neurobiological evidence that inhibition of ROCK by the clinically approved small molecule inhibitor Fasudil may be a novel therapeutic approach in ALS combining both neuroprotection and immunomodulation for the cure of this devastating disease. GLIA 2014;62:217–232     

Immunohistochemical study on the distribution of glycogen synthase kinase 3α in the central nervous system of SOD1G93A transgenic mice

Abstract

Objective: To identify glycogen synthase kinase (GSK) 3α expression in a mouse model of familial amyotrophic lateral sclerosis (ALS), we investigated the changes of GSK3α in the central nervous system of SOD1^G93A transgenic mice by immunohistochemistry.

Methods: We used 12 SOD1^G93A transgenic and ten wild-type (wt) SOD1 transgenic mice bred by 'The Jackson Laboratory' under the strain designations B6SJL-TgN (SOD1^G93A) 1 Gur/J and B6SJL-TgN (SOD1) 2 Gur/J, respectively. Immunohistochemistry was performed in accordance with the free-floating method described earlier.

Results: In symptomatic transgenic mice, GSK3α-immunoreactive astrocytes were detected in the spinal cord, brainstem and cerebellum of symptomatic SOD1^G93A transgenic mice. In contrast to symptomatic mice, no GSK3α-immunoreactive astrocytes were observed in any brain region of wtSOD1 and pre-symptomatic mice, and the number and intensity of stained cells were not different at the age of 8 and 13 weeks.

Discussion: These results provide the first evidence that GSK3α-immunoreactive astrocytes were found in the CNS of SOD1^G93A transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for GSK3α and the functional implications require elucidation.     

Connexin 43 in astrocytes contributes to motor neuron toxicity in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons in the CNS. Astrocytes play a critical role in disease progression of ALS. Astrocytes are interconnected through a family of gap junction proteins known as connexins (Cx). Cx43 is a major astrocyte connexin conducting crucial homeostatic functions in the CNS. Under pathological conditions, connexin expression and functions are altered. Here we report that an abnormal increase in Cx43 expression serves as one of the mechanisms for astrocyte‐mediated toxicity in ALS. We observed a progressive increase in Cx43 expression in the SOD1^G93A mouse model of ALS during the disease course. Notably, this increase in Cx43 was also detected in the motor cortex and spinal cord of ALS patients. Astrocytes isolated from SOD1^G93A mice as well as human induced pluripotent stem cell (iPSC)‐derived astrocytes showed an increase in Cx43 protein, which was found to be an endogenous phenomenon independent of neuronal co‐culture. Increased Cx43 expression led to important functional consequences when tested in SOD1^G93A astrocytes when compared to control astrocytes over‐expressing wild‐type SOD1 (SOD1^WT). We observed SOD1^G93A astrocytes exhibited enhanced gap junction coupling, increased hemichannel‐mediated activity, and elevated intracellular calcium levels. Finally, we tested the impact of increased expression of Cx43 on MN survival and observed that use of both a pan Cx43 blocker and Cx43 hemichannel blocker conferred neuroprotection to MNs cultured with SOD1^G93A astrocytes. These novel findings show a previously unrecognized role of Cx43 in ALS‐related motor neuron loss. GLIA 2016;64:1154–1169     

Altered astrocytic response to activation in SOD1G93A mice and its implications on amyotrophic lateral sclerosis pathogenesis

Amyotrophic Lateral Sclerosis (ALS) is a fatal, rapidly progressive, neurodegenerative disease caused by motor neuron degeneration. Despite extensive efforts, the underlying cause of ALS and the path of neurodegeneration remain elusive. Astrocyte activation occurs in response to central nervous system (CNS) insult and is considered a double edged sword in many pathological conditions. We propose that reduced glutamatergic and trophic response of astrocytes to activation may, over time, lead to accumulative CNS damage, thus facilitating neurodegeneration. We found that astrocytes derived from the SOD1^G93A ALS mouse model exhibit a reduced glutamatergic and trophic response to specific activations compared to their wild‐type counterparts. Wild‐type astrocytes exhibited a robust response when activated with lipopolysaccharide (LPS), G5 or treated with ceftriaxone in many parameters evaluated. These parameters include increased expression of GLT‐1 and GLAST the two major astrocytic glutamate transporters, accompanied by a marked increase in the astrocytic glutamate clearance and up‐regulation of neurtrophic factor expression. However, not only do un‐treated SOD1^G93A astrocytes take up glutamate less efficiently, but in response to activation they show no further increase in any of the glutamatergic parameters evaluated. Furthermore, activation of wild‐type astrocytes, but not SOD1^G93A astrocytes, improved their ability to protect the motor neuron cell line NSC‐34 from glutamate induced excitotoxicity. Our data indicates that altered astrocyte activation may well be pivotal to the pathogenesis of ALS. © 2012 Wiley Periodicals, Inc.     

Appearance of phagocytic microglia adjacent to motoneurons in spinal cord tissue from a presymptomatic transgenic rat model of amyotrophic lateral sclerosis

Microglial activation occurs early during the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent evidence indicates that the expression of mutant Cu²⁺/Zn²⁺ superoxide dismutase 1 (SOD1) in microglia contributes to the late disease progression of ALS. However, the mechanism by which microglia influence the neurodegenerative process and disease progression in ALS remains unclear. In this study, we revealed that activated microglia aggregated in the lumbar spinal cord of presymptomatic mutant SOD1^H46R transgenic rats, an animal model of familial ALS. The aggregated microglia expressed a marker of proliferating cell, Ki67, and phagocytic marker proteins ED1 and major histocompatibility complex (MHC) class II. The motoneurons near the microglial aggregates showed weak choline acetyltransferase (ChAT) immunoreactivity and contained reduced granular endoplasmic reticulum and altered nucleus electron microscopically. Furthermore, immunopositive signals for tumor necrosis factor‐α (TNFα) and monocyte chemoattractant protein‐1 (MCP‐1) were localized in the aggregated microglia. These results suggest that the activated and aggregated microglia represent phagocytic features in response to early changes in motoneurons and possibly play an important role in ALS disease progression during the presymptomatic stage. © 2010 Wiley‐Liss, Inc.     

Lead exposure stimulates VEGF expression in the spinal cord and extends survival in a mouse model of ALS

Exposure to environmental lead (Pb) is a mild risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive degeneration of motor neurons. However, recent evidence has paradoxically linked higher Pb levels in ALS patients with longer survival. We investigated the effects of low-level Pb exposure on survival of mice expressing the ALS-linked superoxide dismutase-1 G93A mutation (SOD1^G93A). SOD1^G93A mice exposed to Pb showed longer survival and increased expression of VEGF in the ventral horn associated with reduced astrocytosis. Pretreatment of cultured SOD1^G93A astrocytes with low, non toxic Pb concentrations upregulated VEGF expression and significantly abrogated motor neuron loss in coculture, an effect prevented by neutralizing antibodies to VEGF. The actions of Pb on astrocytes might explain its paradoxical slowing of disease progression in SOD1^G93A mice and the improved survival of ALS patients. Understanding how Pb stimulates astrocytic VEGF production and reduces neuroinflammation may yield a new therapeutic approach for treating ALS.     

NLRP3 inflammasome is expressed by astrocytes in the SOD1 mouse model of ALS and in human sporadic ALS patients

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motoneurons in the cerebral cortex, brainstem and spinal cord. Neuroinflammation plays an important role in the pathogenesis of ALS and involves the activation of microglia and astrocytes. Intracellular inflammasome complexes are part of the innate immunity as they sense and execute host inflammatory responses. The best characterized component is the NLRP3 inflammasome comprised of the NLR protein NLRP3, the adaptor ASC and pro‐caspase 1. The NLRP3 inflammasome is critical for the activation of caspase 1 and the processing and release of IL1β and IL18. In this study, we investigated the expression, activation and co‐localization of the NLRP3 inflammasome in the spinal cord of male SOD1(G93A) mice carrying a mutant human superoxide dismutase 1 (SOD1) variant and regarded as an animal model for ALS as well as in post‐mortem tissue of ALS patients. NLRP3 and its molecular components as well as IL1β were already detectable in SOD1 mice at a pre‐symptomatic stage after 9 weeks and further increased in 14 week old animals. Spinal cord astrocytes were identified as the major cell type expressing NLRP3 components. In human ALS tissue, we also found increased NLRP3, ASC, IL18 and active caspase 1 levels compared to control patients. Our findings suggest that astroglial NLRP3 inflammasome complexes are critically involved in neuroinflammation in ALS. GLIA 2015;63:2260–2273     

Apoptosis‐Inducing Factor and Cyclophilin A Cotranslocate to the Motor Neuronal Nuclei in Amyotrophic Lateral Sclerosis Model Mice

Aims: Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease whose mechanism is not understood. Recently, it was reported that apoptosis‐inducing factor (AIF) was involved in motor neuronal cell death in ALS model mice, and AIF‐induced neuronal cell death by interacting with cyclophilin A (CypA). However, it is unknown whether the CypA and AIF‐complex induces chromatinolysis in ALS. Therefore, in the present study, we investigated the process of motor neuron degeneration as the disease progresses and to determine whether the CypA‐AIF complex would play a role in inducing motor neuronal cell death in mutant superoxide dismutase 1 (SOD1)^G93A ALS model mice. Methodology: We prepared the nuclear fractions of spinal cords and demonstrated the nuclear translocation of CypA with AIF in SOD1^G93A mice by immunoprecipitation. The localization of CypA and AIF in the spinal cords was assessed by immunohistochemistry. Results: In the spinal cords of SOD1^G93A mice, the expressions of CypA and AIF were detected in the motor neurons, and CypA and AIF cotranslocated to the motor neuronal nuclei with CypA. Furthermore, the expression of CypA was detected in GFAP‐positive astrocytes, but not in CD11b‐positive microglial cells. On the other hand, these findings were not detected in the spinal cords of wild‐type mice. Conclusions: From these results, we suggest that CypA and AIF may play cooperative and pivotal roles in motor neuronal death in the murine ALS model.     

Glycoprotein nonmetastatic melanoma protein B ameliorates skeletal muscle lesions in a SOD1G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons and subsequent muscular atrophy. The quality of life of patients with ALS is significantly improved by ameliorating muscular symptoms. We previously reported that glycoprotein nonmetastatic melanoma protein B (GPNMB; osteoactivin) might serve as a target for ALS therapy. In the present study, superoxide dismutase 1/glycine residue 93 changed to alanine (SOD1^G93A) transgenic mice were used as a model of ALS. Expression of the C‐terminal fragment of GPNMB was increased in the skeletal muscles of SOD1^G93A mice and patients with sporadic ALS. SOD1^G93A/GPNMB transgenic mice were generated to determine whether GPNMB expression ameliorates muscular symptoms. The weight and cross‐sectional area of the gastrocnemius muscle, number and cross‐sectional area of myofibers, and denervation of neuromuscular junctions were ameliorated in SOD1^G93A/GPNMB vs. SOD1^G93A mice. Furthermore, direct injection of a GPNMB expression plasmid into the gastrocnemius muscle of SOD1^G93A mice increased the numbers of myofibers and prevented myofiber atrophy. These findings suggest that GPNMB directly affects skeletal muscle and prevents muscular pathology in SOD1^G93A mice and may therefore serve as a target for therapy of ALS. © 2015 Wiley Periodicals, Inc.     

Sequential activation of microglia and astrocyte cytokine expression precedes increased iba‐1 or GFAP immunoreactivity following systemic immune challenge

Activation of the peripheral immune system elicits a coordinated response from the central nervous system. Key to this immune to brain communication is that glia, microglia, and astrocytes, interpret and propagate inflammatory signals in the brain that influence physiological and behavioral responses. One issue in glial biology is that morphological analysis alone is used to report on glial activation state. Therefore, our objective was to compare behavioral responses after in vivo immune (lipopolysaccharide, LPS) challenge to glial specific mRNA and morphological profiles. Here, LPS challenge induced an immediate but transient sickness response with decreased locomotion and social interaction. Corresponding with active sickness behavior (2–12 h), inflammatory cytokine mRNA expression was elevated in enriched microglia and astrocytes. Although proinflammatory cytokine expression in microglia peaked 2‐4 h after LPS, astrocyte cytokine, and chemokine induction was delayed and peaked at 12 h. Morphological alterations in microglia (Iba‐1⁺) and astrocytes (GFAP⁺), however, were undetected during this 2–12 h timeframe. Increased Iba‐1 immunoreactivity and de‐ramified microglia were evident 24 and 48 h after LPS but corresponded to the resolution phase of activation. Morphological alterations in astrocytes were undetected after LPS. Additionally, glial cytokine expression did not correlate with morphology after four repeated LPS injections. In fact, repeated LPS challenge was associated with immune and behavioral tolerance and a less inflammatory microglial profile compared with acute LPS challenge. Overall, induction of glial cytokine expression was sequential, aligned with active sickness behavior, and preceded increased Iba‐1 or GFAP immunoreactivity after LPS challenge. GLIA 2016;64:300–316     

Degeneration of proprioceptive sensory nerve endings in mice harboring amyotrophic lateral sclerosis–causing mutations

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily targets the motor system. Although much is known about the effects of ALS on motor neurons and glial cells, little is known about its effect on proprioceptive sensory neurons. This study examines proprioceptive sensory neurons in mice harboring mutations associated with ALS, in SOD1^G93A and TDP43^A315T transgenic mice. In both transgenic lines, we found fewer proprioceptive sensory neurons containing fluorescently tagged cholera toxin in their soma five days after injecting this retrograde tracer into the tibialis anterior muscle. We asked whether this is due to neuronal loss or selective degeneration of peripheral nerve endings. We found no difference in the total number and size of proprioceptive sensory neuron soma between symptomatic SOD1^G93A and control mice. However, analysis of proprioceptive nerve endings in muscles revealed early and significant alterations at Ia/II proprioceptive nerve endings in muscle spindles before the symptomatic phase of the disease. Although these changes occur alongside those at α‐motor axons in SOD1^G93A mice, Ia/II sensory nerve endings degenerate in the absence of obvious alterations in α‐motor axons in TDP43^A315T transgenic mice. We next asked whether proprioceptive nerve endings are similarly affected in the spinal cord and found that nerve endings terminating on α‐motor neurons are affected during the symptomatic phase and after peripheral nerve endings begin to degenerate. Overall, we show that Ia/II proprioceptive sensory neurons are affected by ALS‐causing mutations, with pathological changes starting at their peripheral nerve endings. J. Comp. Neurol. 523:2477–2494, 2015. © 2015 Wiley Periodicals, Inc.     

Selective ablation of proliferating astrocytes does not affect disease outcome in either acute or chronic models of motor neuron degeneration

Astrocytes play important roles in normal CNS function; however, following traumatic injury or during neurodegeneration, astrocytes undergo changes in morphology, gene expression and cellular function known as reactive astrogliosis, a process that may also include cell proliferation. At present, the role of astrocyte proliferation is not understood in disease etiology of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder that is characterized by a relatively rapid degeneration of upper and lower motor neurons. Therefore, the role of astrocyte proliferation was assessed in both acute and chronic mouse models of motor neuron degeneration, neuroadapted sindbis virus (NSV)-infected mice and SOD1^G93A mice, respectively. While astrocytes proliferated in the lumbar spinal cord ventral horn of both disease models, they represented only a small percentage of the dividing population in the SOD1^G93A spinal cord. Furthermore, selective ablation of proliferating GFAP⁺ astrocytes in 1) NSV-infected transgenic mice in which herpes simplex virus-thymidine kinase is expressed in GFAP⁺ cells (GFAP-TK) and in 2) SOD1^G93A × GFAP-TK mice did not affect any measures of disease outcome such as animal survival, disease onset, disease duration, hindlimb motor function or motor neuron loss. Ablation of dividing astrocytes also did not alter overall astrogliosis in either model. This was likely due to the finding that proliferation of NG2⁺ glial progenitors were unaffected. These findings demonstrate that while normal astrocyte function is an important factor in the etiology of motor neuron diseases such as ALS, astrocyte proliferation itself does not play a significant role.     

Expression of the axon‐guidance protein receptor Neuropilin 1 is increased in the spinal cord and decreased in muscle of a mouse model of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a degenerative motor neuron disorder. It is supposed that ALS is at least in part an axonopathy. Neuropilin 1 is an important receptor of the axon repellent Semaphorin 3A and a co‐receptor of vascular endothelial growth factor. It is probably involved in neuronal and axonal de‐/regeneration and might be of high relevance for ALS pathogenesis and/or disease progression. To elucidate whether the expression of either Neuropilin1 or Semaphorin3A is altered in ALS we investigated these proteins in human brain, spinal cord and muscle tissue of ALS‐patients and controls as well as transgenic SOD1^G93A and control mice. Neuropilin1 and Semaphorin3A gene and protein expression were assessed by quantitative real‐time PCR (qRT‐PCR), western blot and immunohistochemistry. Groups were compared using either Student t‐test or Mann–Whitney U test. We observed a consistent increase of Neuropilin1 expression in the spinal cord and decrease of Neuropilin1 and Semaphorin3A in muscle tissue of transgenic SOD1^G93A mice at the mRNA and protein level. Previous studies have shown that damage of neurons physiologically causes Neuropilin1 and Semaphorin3A increase in the central nervous system and decrease in the peripheral nervous system. Our results indicate that this also occurs in ALS. Pharmacological modulation of expression and function of axon repellents could be a promising future therapeutic option in ALS.     

Specific Expression of Glial-Derived Neurotrophic Factor in Muscles as Gene Therapy Strategy for Amyotrophic Lateral Sclerosis

Glial cell line–derived neurotrophic factor (GDNF) is a powerful neuroprotective growth factor. However, systemic or intrathecal administration of GDNF is associated with side effects. Here, we aimed to avoid this by restricting the transgene expression to the skeletal muscle by gene therapy. To specifically target most skeletal muscles in the mouse model of amyotrophic lateral sclerosis (ALS), SOD1^G93A transgenic mice were intravenously injected with adeno-associated vectors coding for GDNF under the control of the desmin promoter. Treated and control SOD1^G93A mice were evaluated by rotarod and nerve conduction tests from 8 to 20 weeks of age, and then histological and molecular analyses were performed. Muscle-specific GDNF expression delayed the progression of the disease in SOD1^G93A female and male mice by preserving the neuromuscular function; increasing the number of innervated neuromuscular junctions, the survival of spinal motoneurons; and reducing glial reactivity in treated SOD1^G93A mice. These beneficial actions are attributed to a paracrine protective mechanism from the muscle to the motoneurons by GDNF. Importantly, no adverse secondary effects were detected. These results highlight the potential of muscle GDNF-targeted expression for ALS therapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01025-6.Key Words: GDNF, Amyotrophic lateral sclerosis, Motoneuron, Gene therapy, AAV, Neuromuscular junction