Transformation from a neuroprotective to a neurotoxic microglial phenotype in a mouse model of ALS

Neuroinflammation is a prominent pathological feature in the spinal cords of patients with amyotrophic lateral sclerosis (ALS), as well as in transgenic mouse models of inherited ALS, and is characterized by activated microglia. Earlier studies showed that activated microglia play important roles in both motoneuron protection and injury. More recent studies investigating the pathoprogression of disease in ALS mice have demonstrated that the in vivo activation states of microglia, including their anti- versus pro-inflammatory responses, are best characterized as a continuum between two extreme activation states which are represented as a neuroprotective M2 (alternatively-activated) phenotypic state or an injurious/toxic M1 (classically-activated) state; a more complete understanding and determination the temporal transformation of microglia activation states in the ALS disease pathoprogression is therefore warranted. In the current study, we demonstrated a phenotypic and functional transformation of adult ALS mice microglia that overexpress mutant superoxide dismutase (mSOD1). mSOD1 microglia isolated from ALS mice at disease onset expressed higher levels of Ym1, CD163 and BDNF (markers of M2) mRNA and lower levels of Nox2 (a marker of M1) mRNA compared with mSOD1 microglia isolated from ALS mice at end-stage disease. More importantly, when co-cultured with motoneurons, these mSOD1 M2 microglia were neuroprotective and enhanced motoneuron survival than similarly co-cultured mSOD1 M1 microglia; end-stage mSOD1 M1 microglia were toxic to motoneurons. Our study documents that adult microglia isolated from ALS mice at disease onset have an M2 phenotype and protect motoneurons whereas microglia isolated from end-stage disease ALS mice have adopted an M1 phenotype and are neurotoxic supporting the dual phenotypes of microglia and their transformation during disease pathoprogression in these mice. Thus, harnessing the neuroprotective potential of microglia may provide novel avenues for ALS therapies.     

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.     

Regulatory T lymphocytes from ALS mice suppress microglia and effector T lymphocytes through different cytokine-mediated mechanisms

Activated microglia and infiltrating lymphocytes are neuropathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motoneuron disease. Although both cell types play pivotal roles in the ALS pathogenic process, the interactions between microglia and lymphocytes, specifically regulatory CD4(+)CD25(High) T lymphocytes (Tregs) and cytotoxic CD4(+)CD25(-) T lymphocytes (Teffs), have not been addressed. When co-cultured with mSOD1 adult microglia, mSOD1 Tregs suppressed the cytotoxic microglial factors NOX2 and iNOS through an IL-4-mediated mechanism, whereas Teffs were only minimally effective; IL-4 inhibitory antibodies blocked the suppressive function of mSOD1 Tregs, and conditioned media from mSOD1 Tregs or the addition of IL-4 reduced microglial NOX2 expression. During the stable disease phase, the total number of Tregs, specifically the numbers of CD4(+)CD25(High)IL-4(+), CD4(+)CD25(High)IL-10(+) and CD4(+)CD25(High)TGF-β(+) Tregs, were increased in ALS mice compared with WT mice; Tregs isolated during this phase reduced Teff proliferation. In contrast, during the rapidly progressing phase, the number of mSOD1 Tregs decreased while the proliferation of mSOD1 Teffs increased. The combination of IL-4, IL-10, and TGF-β was required to inhibit the proliferation of mSOD1 Teffs by mSOD1 Tregs that were isolated during the slow phase, while inhibition of mSOD1 Teffs by mSOD1 Tregs during the rapid phase, as well as WT Teffs, was not dependent on these factors. Thus, mSOD1 Tregs at the slow phase suppressed microglial toxicity and SOD1 Teff proliferation through different mechanisms; microglial activation was suppressed through IL-4 whereas mSOD1 Teffs were suppressed by IL-4, IL-10 and TGF-β. These data suggest that mSOD1 Tregs contribute to the slowly progressing phase in ALS mice and may offer a novel therapeutic option for ALS patients.     

Reduced calreticulin levels link endoplasmic reticulum stress and Fas-triggered cell death in motoneurons vulnerable to ALS

Cellular responses to protein misfolding are thought to play key roles in triggering neurodegeneration. In the mutant superoxide dismutase (mSOD1) model of amyotrophic lateral sclerosis (ALS), subsets of motoneurons are selectively vulnerable to degeneration. Fast fatigable motoneurons selectively activate an endoplasmic reticulum (ER) stress response that drives their early degeneration while a subset of mSOD1 motoneurons show exacerbated sensitivity to activation of the motoneuron-specific Fas/NO pathway. However, the links between the two mechanisms and the molecular basis of their cellular specificity remained unclear. We show that Fas activation leads, specifically in mSOD1 motoneurons, to reductions in levels of calreticulin (CRT), a calcium-binding ER chaperone. Decreased expression of CRT is both necessary and sufficient to trigger SOD1(G93A) motoneuron death through the Fas/NO pathway. In SOD1(G93A) mice in vivo, reductions in CRT precede muscle denervation and are restricted to vulnerable motor pools. In vitro, both reduced CRT and Fas activation trigger an ER stress response that is restricted to, and required for death of, vulnerable SOD1(G93A) motoneurons. Our data reveal CRT as a critical link between a motoneuron-specific death pathway and the ER stress response and point to a role of CRT levels in modulating motoneuron vulnerability to ALS.     

C-Boutons and Their Influence on Amyotrophic Lateral Sclerosis Disease Progression

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease with progressive motor neuron death, where patients usually die within 5 years of diagnosis. Previously, we showed that the C-boutons, which are large cholinergic synapses to motor neurons that modulate motor neuron activity, are necessary for behavioral compensation in mSOD1^G93A mice, a mouse model for ALS. We reasoned that, since the C-boutons likely increase the excitability of surviving motor neurons to compensate for motor neuron loss during ALS disease progression, then amplitude modulation through the C-boutons likely increases motor neuron stress and worsens disease progression. By comparing male and female mSOD1^G93A mice to mSOD1^G93A mice with genetically silenced C-boutons [mSOD1^G93A; Dbx1::cre; ChAT^fl/fl (mSOD1^G93A/C^off)], we show that the C-boutons do not influence the humane end point of mSOD1^G93A mice; however, our histologic analysis shows that C-bouton silencing significantly improves fast-twitch muscle innervation over time. Using immunohistology, we also show that the C-boutons are active in a task-dependent manner, and that symptomatic mSOD1^G93A mice show significantly higher C-bouton activity than wild-type mice during low-intensity walking. Last, by using behavioral analysis, we provide evidence that C-bouton silencing in combination with swimming is beneficial for the behavioral capabilities of mSOD1^G93A mice. Our observations suggest that manipulating the C-boutons in combination with a modulatory-targeted training program may therefore be beneficial for ALS patients and could result in improved mobility and quality of life.SIGNIFICANCE STATEMENT Despite decades of research on amyotrophic lateral sclerosis (ALS), there have been little improvements in treatments and therapies. We sought to better understand how the activation of C-boutons, which are large cholinergic modulatory synapses on motor neurons, change and affect the disease as it progresses. When these C-boutons are genetically silenced and exercises designed to otherwise activate the C-boutons are frequently performed in ALS model mice, the mice perform better than their untreated counterparts over time. C-bouton-targeted therapies could therefore be beneficial for ALS patients and could result in improved mobility and quality of life.     

Resveratrol Improves Motoneuron Function and Extends Survival in SOD1G93A ALS Mice

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease that causes progressive paralysis and death due to degeneration of motoneurons in spinal cord, brainstem and motor cortex. Nowadays, there is no effective therapy and patients die 2–5 years after diagnosis. Resveratrol (trans-3,4′,5-trihydroxystilbene) is a natural polyphenol found in grapes, with promising neuroprotective effects since it induces expression and activation of several neuroprotective pathways involving Sirtuin1 and AMPK. The objective of this work was to assess the effect of resveratrol administration on SOD1^G93A ALS mice. We determined the onset of symptoms by rotarod test and evaluated upper and lower motoneuron function using electrophysiological tests. We assessed the survival of the animals and determined the number of spinal motoneurons. Finally, we further investigated resveratrol mechanism of action by means of western blot and immunohistochemical analysis. Resveratrol treatment from 8 weeks of age significantly delayed disease onset and preserved lower and upper motoneuron function in female and male animals. Moreover, resveratrol significantly extended SOD1^G93A mice lifespan and promoted survival of spinal motoneurons. Delayed resveratrol administration from 12 weeks of age also improved spinal motoneuron function preservation and survival. Further experiments revealed that resveratrol protective effects were associated with increased expression and activation of Sirtuin 1 and AMPK in the ventral spinal cord. Both mediators promoted normalization of the autophagic flux and, more importantly, increased mitochondrial biogenesis in the SOD1^G93A spinal cord. Taken together, our findings suggest that resveratrol may represent a promising therapy for ALS.     

A Nitroalkene Benzoic Acid Derivative Targets Reactive Microglia and Prolongs Survival in an Inherited Model of ALS via NF-κB Inhibition

Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1^G93A rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1^G93A rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1^G93A rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00953-z.Key Words: ALS, NF-κB-p65, microglia, BANA.     

Mechano-growth factor, an IGF-I splice variant, rescues motoneurons and improves muscle function in SOD1 mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneuron degeneration. Although viral delivery of IGF-I has shown therapeutic efficacy in the SOD1^G93A mouse model of ALS, clinical trials of IGF-I in ALS patients have led to conflicting results. Here we examine the effects of an IGF-I splice variant, mechano-growth factor (MGF) which has previously been shown to have greater neuroprotective effects than IGF-I in a number of models of neurodegeneration. A mammalian expression plasmid containing either MGF or, for comparison, the IGF-I cDNA sequence was delivered to the hindlimb muscles of SOD1^G93A mice at 70 days of age, at symptom onset. Treatment with either IGF-I or MGF resulted in a significant improvement in hindlimb muscle strength, and an increase in motor unit and motoneuron survival. Significantly more motoneurons survived in MGF treated mice.     

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.     

SOD1G93A transgenic mouse CD4+ T cells mediate neuroprotection after facial nerve axotomy when removed from a suppressive peripheral microenvironment

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving motoneuron (MN) axonal withdrawal and cell death. Previously, we established that facial MN (FMN) survival levels in the SOD1^G93A transgenic mouse model of ALS are reduced and nerve regeneration is delayed, similar to immunodeficient RAG2^−/− mice, after facial nerve axotomy. The objective of this study was to examine the functionality of SOD1^G93A splenic microenvironment, focusing on CD4⁺ T cells, with regard to defects in immune-mediated neuroprotection of injured MN. We utilized the RAG2^−/− and SOD1^G93A mouse models, along with the facial nerve axotomy paradigm and a variety of cellular adoptive transfers, to assess immune-mediated neuroprotection of FMN survival levels. We determined that adoptively transferred SOD1^G93A unfractionated splenocytes into RAG2^−/− mice were unable to support FMN survival after axotomy, but that adoptive transfer of isolated SOD1^G93A CD4⁺ T cells could. Although WT unfractionated splenocytes adoptively transferred into SOD1^G93A mice were able to maintain FMN survival levels, WT CD4⁺ T cells alone could not. Importantly, these results suggest that SOD1^G93A CD4⁺ T cells retain neuroprotective functionality when removed from a dysfunctional SOD1^G93A peripheral splenic microenvironment. These results also indicate that the SOD1^G93A central nervous system microenvironment is able to re-activate CD4⁺ T cells for immune-mediated neuroprotection when a permissive peripheral microenvironment exists. We hypothesize that a suppressive SOD1^G93A peripheral splenic microenvironment may compromise neuroprotective CD4⁺ T cell activation and/or differentiation, which, in turn, results in impaired immune-mediated neuroprotection for MN survival after peripheral axotomy in SOD1^G93A mice.     

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.     

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.     

Involvement of activated microglia in increased vulnerability of motoneurons after facial nerve avulsion in presymptomatic amyotrophic lateral sclerosis model rats

Activated microglia are observed in various neurodegenerative diseases and are thought to be involved in the processes of neuronal cell death. Motoneuron damage in the facial nuclei after facial nerve avulsion is accelerated in presymptomatic transgenic rats expressing human mutant Cu(2+) /Zn(2+) superoxide dismutase 1 (SOD1), compared with that in wild-type rats. To reveal the functional role of microglia in motoneuronal death, we investigated the microglial response after facial nerve avulsion in presymptomatic mutant SOD1(H46R) (mSOD1(H46R) ) rats. At 3 days after avulsion, microglial clusters were observed in the facial nuclei of both wild-type and mSOD1(H46R) rats. The numbers of microglial clusters, proliferating microglia, and microglial attachments to motoneurons were significantly higher in mSOD1(H46R) rats, compared with those in wild-type rats. Immunopositive signals for the phagocytic marker ED1 were significantly stronger in mSOD1(H46R) rats, compared with that in wild-type rats, at 2 weeks after avulsion. Furthermore, primary microglia prepared from mSOD1(H46R) rats showed enhanced phagocytic activity, compared with that in wild-type rats. The expression of P2Y(12) mRNA was higher in the facial nuclei of mSOD1(H46R) rats, compared with that in wild-type rats. A laser microdissection system revealed that the expression of ATF3 mRNA was higher in the motoneurons of mSOD1(H46R) rats, compared with that in wild-type rats, at 2 days after avulsion. These results indicate that microglial activation in response to early neuronal damage increased in mSOD1(H46R) rats and suggest that the enhanced activation of microglia may lead to an increase in the vulnerability of motoneurons after avulsion in mSOD1(H46R) rats.     

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     

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     

Treatment with minocycline after disease onset alters astrocyte reactivity and increases microgliosis in SOD1 mutant mice

Several reports have demonstrated that attenuation of microglial activation by minocycline, an antimicrobial drug with anti-inflammatory properties, delays disease progression in a mouse model of ALS. However, the negative results obtained in recent clinical trials raised some questions regarding the role of inflammatory response and glial cells as a therapeutic target in ALS. To investigate this controversy we took advantage of a mouse model for live imaging of neuroinflammatory responses in ALS (GFAP-luc/ SOD1^G93A reporter mouse) and analyzed in real time the effects of minocycline treatment initiated at different stages of the disease. To our surprise, unlike neuroprotection that is conferred when minocycline is administered pre-symptomatically, treatment with minocycline initiated after the disease onset significantly altered glial responses and exaggerated neuroinflammation. Further analysis revealed that the late minocycline treatment was associated with significant induction of the end-stage GFAP-biophotonic signals, expression levels of connexin 43, a major protein of astrocytic gap junction and markers of microglial activation, such as Iba1 and CD68. The results of our study suggest that when administered at later stages of disease, once microglial cells are chronically reactive, minocycline may not have anti-inflammatory properties, and contrary to expectations, may alter astrocyte reactivity and increase microgliosis. Finally, our results further suggest the existence of close interactions/communication between activated microglia and astrocytes in late stage ALS.     

Human mesenchymal stem cell transplantation extends survival, improves motor performance and decreases neuroinflammation in mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a lethal disease affecting motoneurons. In familial ALS, patients bear mutations in the superoxide dismutase gene (SOD1). We transplanted human bone marrow mesenchymal stem cells (hMSCs) into the lumbar spinal cord of asymptomatic SOD1(G93A) mice, an experimental model of ALS. hMSCs were found in the spinal cord 10 weeks after, sometimes close to motoneurons and were rarely GFAP- or MAP2-positive. In females, where progression is slower than in males, astrogliosis and microglial activation were reduced and motoneuron counts with the optical fractionator were higher following transplantation. Motor tests (Rotarod, Paw Grip Endurance, neurological examination) were significantly improved in transplanted males. Therefore hMSCs are a good candidate for ALS cell therapy: they can survive and migrate after transplantation in the lumbar spinal cord, where they prevent astrogliosis and microglial activation and delay ALS-related decrease in the number of motoneurons, thus resulting in amelioration of the motor performance.     

Constitutive downregulation protein kinase C epsilon in hSOD1G93A astrocytes influences mGluR5 signaling and the regulation of glutamate uptake

Accumulating evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is a non‐cell‐autonomous process and that impaired glutamate clearance by astrocytes, leading to excitotoxicity, could participate in progression of the disease. In astrocytes derived from an animal model of ALS (hSOD1^G93A rats), activation of type 5 metabotropic glutamate receptor (mGluR5) fails to increase glutamate uptake, impeding a putative dynamic neuroprotective mechanism involving astrocytes. Using astrocyte cultures from hSOD1^G93A rats, we have demonstrated that the typical Ca²⁺ oscillations associated with mGluR5 activation were reduced, and that the majority of cells responded with a sustained elevation of intracellular Ca²⁺ concentration. Since the expression of protein kinase C epsilon isoform (PKC?) has been found to be considerably reduced in astrocytes from hSOD1^G93A rats, the consequences of manipulating its activity and expression on mGluR5 signaling and on the regulation of glutamate uptake have been examined. Increasing PKC? expression was found to restore Ca²⁺ oscillations induced by mGluR5 activation in hSOD1^G93A‐expressing astrocytes. This was also associated with an increase in glutamate uptake capacity in response to mGluR5 activation. Conversely, reducing PKC? expression in astrocytes from wild‐type animals with specific PKC?‐shRNAs was found to alter the mGluR5 associated oscillatory signaling profile, and consistently reduced the regulation of the glutamate uptake‐mediated by mGluR5 activation. These results suggest that PKC? is required to generate Ca²⁺ oscillations following mGluR5 activation, which support the regulation of astrocytic glutamate uptake. Reduced expression of astrocytic PKC? could impair this neuroprotective process and participate in the progression of ALS.     

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.