Functional recovery and facial motoneuron survival are influenced by immunodeficiency in crush-axotomized mice

Facial nerve axotomy is a well-described injury paradigm for peripheral nerve regeneration and facial motoneuron (FMN) survival. We have previously shown that CD4⁺ T helper (Th) 1 and 2 effector subsets develop in the draining cervical lymph node, and that the IL-4/STAT-6 pathway of Th2 development is critical for FMN survival after transection axotomy. In addition, delayed behavioral recovery time in immunodeficient mice may be due to the absence of T and B cells. This study utilized a crush axotomy paradigm to evaluate FMN survival and functional recovery in WT, STAT-6 KO (impaired Th2 response), T-Bet KO (impaired Th1 response), and RAG-2 KO (lacking mature T and B cells) mice to elucidate the contributions of specific CD4⁺ T cell subsets in motoneuron survival and recovery mechanisms. STAT-6 KO and RAG-2 KO mice exhibited decreased FMN survival after crush axotomy compared to WT, supporting a critical role for the Th2 effector cell in motoneuron survival before target reconnection. Long term FMN survival was sustained through 10 wpo after crush axotomy in both WT and RAG-2 KO mice, indicating that target derived neurotrophic support maintains FMN survival after target reconnection. In addition, RAG-2 KO mice exhibited delayed functional recovery compared to WT mice. Both STAT-6 and T-Bet KO mice exhibited partially delayed functional recovery compared to WT, though not to the extent of RAG-2 KO mice. Collectively, our findings indicate that both pro- and anti-inflammatory CD4⁺ T cell responses contribute to optimal functional recovery from axotomy-induced facial paralysis, while FMN survival is supported by the anti-inflammatory Th2 response alone.     

Axotomy‐induced target disconnection promotes an additional death mechanism involved in motoneuron degeneration in amyotrophic lateral sclerosis transgenic mice

The target disconnection theory of amyotrophic lateral sclerosis (ALS) pathogenesis suggests that disease onset is initiated by a peripheral pathological event resulting in neuromuscular junction loss and motoneuron (MN) degeneration. Presymptomatic mSOD1^G93A mouse facial MN (FMN) are more susceptible to axotomy‐induced cell death than wild‐type (WT) FMN, which suggests additional CNS pathology. We have previously determined that the mSOD1 molecular response to facial nerve axotomy is phenotypically regenerative and indistinguishable from WT, whereas the surrounding microenvironment shows significant dysregulation in the mSOD1 facial nucleus. To elucidate the mechanisms underlying the enhanced mSOD1 FMN loss after axotomy, we superimposed the facial nerve axotomy model on presymptomatic mSOD1 mice and investigated gene expression for death receptor pathways after target disconnection by axotomy vs. disease progression. We determined that the TNFR1 death receptor pathway is involved in axotomy‐induced FMN death in WT and is partially responsible for the mSOD1 FMN death. In contrast, an inherent mSOD1 CNS pathology resulted in a suppressed glial reaction and an upregulation in the Fas death pathway after target disconnection. We propose that the dysregulated mSOD1 glia fail to provide support the injured MN, leading to Fas‐induced FMN death. Finally, we demonstrate that, during disease progression, the mSOD1 facial nucleus displays target disconnection‐induced gene expression changes that mirror those induced by axotomy. This validates the use of axotomy as an investigative tool in understanding the role of peripheral target disconnection in the pathogenesis of ALS. J. Comp. Neurol. 522:2349–2376, 2014. © 2014 Wiley Periodicals, Inc.     

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     

IL-10 within the CNS is necessary for CD4 T cells to mediate neuroprotection

We have previously shown that immunodeficient mice exhibit significant facial motoneuron (FMN) loss compared to wild-type (WT) mice after a facial nerve axotomy. Interleukin-10 (IL-10) is known as a regulatory cytokine that plays an important role in maintaining the anti-inflammatory environment within the central nervous system (CNS). IL-10 is produced by a number of different cells, including Th2 cells, and may exert an anti-apoptotic action on neurons directly. In the present study, the role of IL-10 in mediating neuroprotection following facial nerve axotomy in Rag-2- and IL-10-deficient mice was investigated. Results indicate that IL-10 is neuroprotective, but CD4⁺ T cells are not the requisite source of IL-10. In addition, using real-time PCR analysis of laser microdissected brainstem sections, results show that IL-10 mRNA is constitutively expressed in the facial nucleus and that a transient, significant reduction of IL-10 mRNA occurs following axotomy under immunodeficient conditions. Dual labeling immunofluorescence data show, unexpectedly, that the IL-10 receptor (IL-10R) is constitutively expressed by facial motoneurons, but is selectively induced in astrocytes within the facial nucleus after axotomy. Thus, a non-CD4⁺ T cell source of IL-10 is necessary for modulating both glial and neuronal events that mediate neuroprotection of injured motoneurons, but only with the cooperation of CD4⁺ T cells, providing an avenue of novel investigation into therapeutic approaches to prevent or reverse motoneuron diseases, such as amyotrophic lateral sclerosis (ALS).     

Endogenous T lymphocytes and microglial reactivity in the axotomized facial motor nucleus of mice: effect of genetic background and the RAG2 gene

Following facial nerve axotomy in mice, peripheral T cells home to the injured facial motor nucleus (FMN) where they may influence the glial response. Interactions between T cells and microglia, which proliferate in response to axotomy, appear to confer neuroprotection to injured motoneurons. The primary objective of this study was to determine whether T lymphocytes could influence the microglial reaction to motoneuron injury. These experiments tested the hypotheses that (1) C57BL/6 (B6) and 129 mice, inbred strains which have high and low levels of astroglial reactivity in the axotomized FMN, respectively, would also exhibit high and low levels of T cell infiltration, and (2) that these differences would correspond with levels of microglial reactivity and neuronal regeneration. Thus, we compared the response to facial nerve axotomy in B6, 129, and immunodeficient RAG2 knockout (RAG2 KO) mice on these two backgrounds at 14 day post-axotomy for differences in levels of 1) CD3+ T cell infiltration; (2) major histocompatibility complex II (MHC2) expression by microglia; (3) perineuronal microglial phagocytic clusters, an indirect measure of neuronal death; and (4) overall microglial activity as assessed by CD11b expression. To examine the inheritance pattern of the abovementioned neuroimmune measures, we also made assessments in B6x129 F1 generation mice. B6 and 129 mice displayed high and low levels of T cell infiltration to the affected FMN and low and high MHC2 expression, respectively. Levels of microglial activity did not differ between the two strains. In immunodeficient RAG2 KO mice on both backgrounds, the number of MHC2+ microglia did not differ from their immunologically normal background controls. Moreover, deletion of either the RAG2 or RAG1 genes in B6 mice was not associated with increased neuronal death at day 14 post-axotomy, as we had previously found in B6 mice with the severe combined immunodeficiency (SCID) mutation. Contrary to our hypothesis, the paucity of T cells in the affected FMN of the 129 mice was associated with less neuronal death when compared to B6 mice, which showed a robust T cell response. Moreover, the data suggest that parameters of the central and peripheral immune responses to axotomy are independently regulated. Assessments in B6x129 F1 generation mice revealed dominant phenotypes for both T cell infiltration and neurodegeneration, whereas both strains contributed significantly to the phenotype for MHC2 expression. Our findings suggest that (1) T cells do not appear to modify measures of microglial reactivity in the axotomized FMN; and (2) the impact of T cells on injured motoneurons in immunologically intact mice and in immunodeficient mice grafted with T cells by adoptive transfer may be different. Further study is required to understand the role of T cells following motoneuron injury in immunologically intact mice and how the seemingly divergent effects of T cells in intact and immunodeficient mice might provide insight into their role in neuronal injury and repair.     

ROCK inhibition improves axonal regeneration in a preclinical model of amyotrophic lateral sclerosis

Alteration of the RhoA/ROCK (Rho kinase) pathway has been shown to be neuroprotective in SOD1^G93A mice, the most commonly used animal model of ALS. Since previous studies indicate that, apart from neuroprotection, ROCK inhibitor Y-27632 can also accelerate regeneration of motor axons, we here assessed the regenerative capability of axons in SOD1^G93A mice with and without treatment with Y-27632. Regeneration of axons was examined after sciatic nerve crush in pre- and symptomatic SOD1^G93A mice. Proregenerative effects of Y-27632 were studied during the disease course in the SOD1^G93A mouse model. In symptomatic SOD1^G93A mice, axonal regeneration was markedly reduced compared to presymptomatic SOD1^G93A mice and wild types. Treatment with Y-27632 improved functional and morphological measures of motor axons after sciatic crush in all tested conditions. Y-27632 treatment did not increase the lifespan of symptomatic SOD1^G93A mice, but did improve axonal (re)innervation of neuromuscular junctions. Our study provides proof of concept that axonal regeneration of motor neurons harboring SOD1^G93A is impaired, but amenable for pharmacological interventions aiming to accelerate axonal regeneration. Given the lack of treatments for ALS, approaches to improve axonal regeneration, including by inhibiting ROCK, should be further explored.     

CD4+CD25+ regulatory T cells and CD1-restricted NKT cells do not mediate facial motoneuron survival after axotomy

CD4+ T cells rescue facial motoneurons (FMN) from axotomy-induced cell death. The objective of this study is to determine if the CD4+ T regulatory subsets, CD4+CD25+ T or CD1d-restricted NKT cells are critical for FMN survival after facial nerve axotomy. Surviving FMN within facial motor nuclei from axotomized and control sides 4 weeks after axotomy were counted to determine percent FMN survival. Data generated by applying this paradigm to recombination activating gene-2-deficient mice reconstituted with CD4+ T cells depleted of CD4+CD25+ T cells and to CD1-/- mice, deficient in CD1d-restricted NKT cells, suggest that neither regulatory CD4+ T subset is critical for FMN survival.     

CD4+ T cell-mediated neuroprotection is independent of T cell-derived BDNF in a mouse facial nerve axotomy model

BackgroundThe production of neurotrophic factors, such as BDNF, has generally been considered an important mechanism of immune-mediated neuroprotection. However, the ability of T cells to produce BDNF remains controversial.MethodsIn the present study, we examined mRNA and protein of BDNF using RT-PCR and western blot, respectively, in purified and reactivated CD4⁺ T cells. In addition, to determine the role of BDNF derived from CD4⁺ T cells, the BDNF gene was specifically deleted in T cells using the Cre-lox mouse model system.ResultsOur results indicate that while both mRNA expression and protein secretion of BDNF in reactivated T cells were detected at 24 h, only protein could be detected at 72 h after reactivation. The results suggest a transient up-regulation of BDNF mRNA in reactivated T cells. Furthermore, in contrast to our hypothesis that the BDNF expression is necessary for CD4⁺ T cells to mediate neuroprotection, mice with CD4⁺ T cells lacking BDNF expression demonstrated a similar level of facial motoneuron survival compared to their littermates that expressed BDNF, and both levels were comparable to wild-type. The results suggest that the deletion of BDNF did not impair CD4⁺ T cell-mediated neuroprotection.ConclusionCollectively, while CD4⁺ T cells are a potential source of BDNF after nerve injury, production of BDNF is not necessary for CD4⁺ T cells to mediate their neuroprotective effects.     

Isoform‐selective as opposed to complete depletion of fibroblast growth factor 2 (FGF‐2) has no major impact on survival and gene expression in SOD1G93A amyotrophic lateral sclerosis mice

We have previously shown that total knockout of fibroblast growth factor‐2 (FGF‐2) results in prolonged survival and improved motor performance in superoxide dismutase 1 (SOD1^G93A) mutant mice, the most widely used animal model of the fatal adult onset motor neuron disease amyotrophic lateral sclerosis (ALS). Moreover, we found differential expression of growth factors in SOD1^G93A mice, with distinct regulation patterns of FGF‐2 in spinal cord and muscle tissue. Within the present study we aimed to characterize FGF‐2‐isoform specific effects on survival, motor performance as well as gene expression patterns predominantly in muscle tissue by generating double mutant SOD1^G93AFGF‐2 high molecular weight‐ and SOD1^G93AFGF‐2 low molecular weight‐knockout mice. While isoform specific depletion was not beneficial regarding survival or motor performance of double mutant mice, we found isoform‐dependent differential gene expression of epidermal growth factor (EGF) in the muscle of SOD1^G93AFGF‐2 low molecular weight knockout mice compared to single mutant SOD1^G93A mice. This significant downregulation of EGF in the muscle tissue of double mutant SOD1^G93AFGF‐2 low molecular weight knockout mice implies that FGF‐2 low molecular weight knockout (or the presence of the FGF‐2 high molecular weight isoform) selectively impacts EGF gene expression in ALS muscle tissue.     

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.     

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.     

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.     

Effects of chronic caffeine intake in a mouse model of amyotrophic lateral sclerosis

Caffeine is a nonselective adenosine receptor antagonist; chronic consumption has proved protective toward neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. The present study was designed to determine whether caffeine intake affected survival and/or motor performance in a transgenic model of amyotrophic lateral sclerosis (ALS). SOD1^G93A mice received caffeine through drinking water from 70 days of age until death. Body weight, motor performance and survival were evaluated. Furthermore, the expression of adenosine A_2A receptors (A_2ARs), glial glutamate transporter (GLT1), and glial fibrillar acidic protein (GFAP) were evaluated by Western blotting. The results showed that caffeine intake significantly shortened the survival of SOD1^G93A mice (log rank test, P = 0.01) and induced a nonsignificant advancing of disease onset. The expression of A_2AR, GLT1, and GFAP was altered in the spinal cords of ALS mice, but caffeine did not influence their expression in either wild‐type or SOD1^G93 mice. These data indicate that adenosine receptors may play an important role in ALS. © 2013 Wiley Periodicals, Inc.     

Early-Stage Treatment with Withaferin A Reduces Levels of Misfolded Superoxide Dismutase 1 and Extends Lifespan in a Mouse Model of Amyotrophic Lateral Sclerosis

Approximately 20 % of cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies have shown that Withaferin A (WA), an inhibitor of nuclear factor-kappa B activity, was efficient in reducing disease phenotype in a TAR DNA binding protein 43 transgenic mouse model of ALS. These findings led us to test WA in mice from 2 transgenic lines expressing different ALS-linked SOD1 mutations, SOD1^G93A and SOD1^G37R. Intraperitoneal administration of WA at a dosage of 4 mg/kg of body weight was initiated from postnatal day 40 until end stage in SOD1^G93A mice, and from 9 months until end stage in SOD1^G37R mice. The beneficial effects of WA in the SOD1^G93A mice model were accompanied by an alleviation of neuroinflammation, a decrease in levels of misfolded SOD1 species in the spinal cord, and a reduction in loss of motor neurons resulting in delayed disease progression and mortality. Interestingly, WA treatment triggered robust induction of heat shock protein 25 (a mouse ortholog of heat shock protein 27), which may explain the reduced level of misfolded SOD1 species in the spinal cord of SOD1^G93A mice and the decrease of neuronal injury responses, as revealed by real-time imaging of biophotonic SOD1^G93A mice expressing a luciferase transgene under the control of the growth-associated protein 43 promoter. These results suggest that WA may represent a potential lead compound for drug development aiming to treat ALS.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0311-0) contains supplementary material, which is available to authorized users.Key Words: ALS, Neuroinflammation, Withaferin A, SOD1^G93A, SOD1^G37R     

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.     

CD4+ T,but not CD8+ or B,lymphocytes mediate facial motoneuron survival after facial nerve transection

The capacity of facial motor neurons (FMN) to survive injury and successfully regenerate is substantially compromised in immunodeficient mice, which lack T and B lymphocytes (). The goal of the present study was to determine which T cell subset (CD4+ and/or CD8+), and whether the B lymphocyte, is involved in FMN survival after nerve injury. All mice were subjected to a right facial nerve axotomy, with the left (uncut) side serving as an internal control. FMN survival, of the right (cut) side, was measured 4 weeks post-operative, and expressed as a percentage of the left (uncut) control side. FMN survival in wild-type mice was 86%+/-1.5. In contrast, FMN survival in CD4 KO mice was 60%+/-2.0. Reconstitution of either CD4 KO mice, or recombinase activating gene-2 knockout (RAG-2 KO) mice (which lack functional T and B cells) with CD4+ T cells alone restored FMN survival to wild-type levels (85%+/-1.2 and 84%+/-2.5, respectively). There was no difference in FMN survival between wild-type, CD8 KO and MmuMT (B cell deficient) mice. Reconstitution of RAG-2 KO mice with CD8+ T cells alone, or B cells alone, failed to restore FMN survival levels (65%+/-1.5 and 63%+/-1.0, respectively). It is concluded that, of the population of FMN that do not survive injury, CD4+ T lymphocytes, but not CD8+ T lymphocytes or B cells, mediate FMN survival after peripheral nerve injury.     

Functional over-load saves motor units in the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis

The fastest, most forceful motor units are lost progressively during asymptomatic disease in the SOD1^G93A transgenic mouse model of amyotrophic lateral sclerosis. As the disease progresses the surviving motor units must increase their levels of activity to sustain posture and movement. If activity-dependent conversion of motor units to more fatigue resistant types increased their resilience and hence survival, we hypothesized that an experimental increase in motor unit activity in the hindlimb muscles of the SOD1^G93A transgenic mouse should “save” those motor units that are normally lost in the first 90 days of age. To test this hypothesis, we partially denervated hindlimb muscles in SOD1^G93A and their corresponding control SOD1^WT transgenic mice by avulsion of either L4 or L5 spinal roots at 40 days of age. Whole muscle and single motor unit isometric twitch forces were recorded and the numbers intact motor units in fast-twitch tibialis anterior, medial gastrocnemius, extensor digitorum longus muscles and the slow-twitch soleus muscle were calculated at 90 days of age. We found that the rapid age-dependent decline in numbers of functional motor units in fast-twitch muscles of the SOD1^G93A transgenic mice was dramatically reduced by the functional hyperactivity in the partially denervated muscles and, that these muscles comprised a significantly higher component of type IIA and type IID/X fibers than those muscles that were innervated by nerves in intact spinal roots. We conclude that the vulnerable motor units are saved by increasing their neuromuscular activity and consequently, converting them to slower, less forceful, fatigue resistant motor units.     

The influence of metallothionein treatment and treadmill running exercise on disease onset and survival in SOD1G93A amyotrophic lateral sclerosis mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by the degeneration of motor neurons innervating skeletal muscle. The mechanisms underlying neurodegeneration in ALS are not yet fully elucidated, and with current therapeutics only able to extend lifespan by a matter of months there is a clear need for novel therapies to increase lifespan and patient quality of life. Here, we evaluated whether moderate‐intensity treadmill exercise and/or treatment with metallothionein‐2 (MT2), a neuroprotective protein, could improve survival, behavioural or neuropathological outcomes in SOD1^G93A familial ALS mice. Six‐week‐old female SOD1^G93A mice were allocated to one of four treatment groups: MT2 injection, i.m.; moderate treadmill exercise; neither MT2 nor exercise; or both MT2 and exercise. MT2‐treated mice survived around 3% longer than vehicle‐treated mice, with this mild effect reaching statistical significance in Cox proportional hazards analysis once adjusted for potential confounders. Mixed model body weight trajectories over time indicated that MT2‐treated mice, with or without exercise, reached maximum body weight at a later age, suggesting a delay in disease onset of around 4% compared to saline‐treated mice. Exercise alone did not significantly increase survival or delay disease onset, and neither exercise nor MT2 substantially ameliorated gait abnormalities or muscle strength loss. We conclude that neither exercise nor MT2 treatment was detrimental in female SOD1^G93A mice, and further study could determine whether the mild effect of peripheral MT2 administration on disease onset and survival could be improved via direct administration of MT2 to the central nervous system.     

Brain-derived neurotrophic factor supports facial motoneuron survival after facial nerve transection in immunodeficient mice

Numerous studies have shown that motoneuron survival can be facilitated by neurotrophic factors (NTF) after injury. However, the ability of specific NTF to rescue facial motoneurons (FMN) from axotomy-induced death in immunodeficient mice has not been tested. Therefore, one goal of this study was to determine if brain-derived neurotrophic factor (BDNF), an NTF with a known ability to rescue FMN from axotomy-induced death, supports FMN from axotomy-induced death in recombinase activating gene-2 knockout (RAG-2 KO) mice that lack functional T and B lymphocytes. Nerve growth factor, which has been shown not to play a role in motoneuron survival, was used as a negative control. Brain derived neurotrophic factor treatment restored FMN survival to wild-type (WT) control levels 4 weeks post-operative (wpo) (80% +/- 1.9, 83% +/- 2.4, respectively). The second goal of this study was to begin to elucidate if CD4+ T cells produce NTF after facial nerve axotomy. Cervical lymph nodes were collected from WT mice 9 days post-operative, re-activated with anti-CD3 and supernatant collected 24 h later. Immediately after injury, the supernatant was administered to RAG-2 KO mice leading to an increase in FMN survival equivalent to WT controls (80% +/- 1.4, 84% +/- 2.1, respectively, 4 wpo). In addition, cervical lymph node supernatant treated with anti-BDNF attenuated FMN rescue in RAG-2 KO mice (62% +/- 3.3) 4 wpo. These data lend support to the hypothesis that CD4+ T cells produce NTF that support motoneuron survival before target reconnection occurs.