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     

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     

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.     

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.     

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.     

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.     

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.     

Epothilone D accelerates disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Aims

Degeneration of the distal neuromuscular circuitry is a hallmark pathology of Amyotrophic Lateral Sclerosis (ALS). The potential for microtubule dysfunction to be a critical pathophysiological mechanism in the destruction of this circuitry is increasingly being appreciated. Stabilization of microtubules to improve neuronal integrity and pathology has been shown to be a particularly favourable approach in other neurodegenerative diseases. We present evidence here that treatment with the microtubule‐targeting compound Epothilone D (EpoD) both positively and negatively affects the spinal neuromuscular circuitry in the SOD1^G93A mouse model of ALS.

Methods

SOD1^G93A mice were treated every 5 days with 2 mg/kg EpoD. Evaluation of motor behaviour, neurological phenotype and survival was completed, with age‐dependent histological characterization also conducted, using the thy1‐YFP mouse. Motor neuron degeneration, axonal integrity, neuromuscular junction (NMJ) health and gliosis were also assessed.

Results

EpoD treatment prevented loss of the spinal motor neuron soma, and distal axon degeneration, early in the disease course. This, however, was not associated with protection of the NMJ synapse and did not improve motor phenotype or clinical progression. EpoD administration was also found to be neurotoxic at later disease stages. This was evidenced by accelerated motor neuron cell body loss, increasing gliosis, and was associated with detrimental outcomes to motor behaviour, clinical assessment and survival.

Conclusions

The results suggest that EpoD accelerates disease progression in the SOD1^G93A mouse model of ALS, and highlights that the pathophysiological involvement of microtubules in ALS is an evolving and underappreciated phenomenon.     

Relationship of microglial and astrocytic activation to disease onset and progression in a transgenic model of familial ALS

Transgenic mice that highly over-express a mutated human CuZn superoxide dismutase (SOD1) gene [gly⁹³→ala; TgN(SOD1-G93A)G1H line] found in some patients with familial ALS (FALS) have been shown to develop motor neuron disease that is characterized by motor neuron loss in the lumbar and cervical spinal regions and a progressive loss of motor activity. The mutant Cu,Zn SOD exhibits essentially normal SOD activity but also generates toxic oxygen radicals as a result of an enhancement of a normally minor peroxidase reaction. Consequently, lipid and protein oxidative damage to the spinal motor neurons occurs and is associated with disease onset and progression. In the present study, we investigated the time course of microglial (major histocompatibility-II antigen immunoreactivity) and astrocytic (glial fibrillary acidic protein immunoreactivity) activation in relation to the course of motor neuron disease in the TgN(SOD1-G93A)G1H FALS mice. Four ages were investigated: 30 days (pre-motor neuron pathology and clinical disease); 60 days (after initiation of pathology, but pre-disease); 100 days (approximately 50% loss of motor neurons and function); and 120 days (near complete hindlimb paralysis). Compared to non-transgenic littermates, the TgN(SOD1-G93A)G1H mice showed significantly increased numbers of activated astrocytes (P < 0.01) at 100 days of age in both the cervical and lumbar spinal cord regions. However, at 120 days of age, the activation lost statistical significance. In contrast, microglial activation was significantly increased several-fold at both 100 and 120 days. We hypothesize that astrocytic activation may exert a trophic influence on the motor neurons that is insufficiently maintained late in the course of the disease. On the other hand, the sustained, intense microglial activation may conceivably contribute to the oxidative stress and damage involved in the disease process. If true, then agents which inhibit microglia may help to limit disease progression. GLIA 23:249–256, 1998. © 1998 Wiley-Liss, 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.     

Relationship between neuropathology and disease progression in the SOD1(G93A) ALS mouse

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. However, recent reports suggest an active role of non-neuronal cells in the pathogenesis of the disease. Here, we examined quantitatively the temporal development of neuropathologic features in the brain and spinal cord of a mouse model of ALS (SOD1^G93A). Four phases of the disease were studied in both male and female SOD1^G93A mice: presymptomatic (PRE-SYM), symptomatic (SYM), endstage (ES) and moribund (MB). Compared to their control littermates, SOD1^G93A mice showed an increase in astrogliosis in the motor cortex, spinal cord and motor trigeminal nucleus in the SYM phase that worsened progressively in ES and MB animals. Associated with this increase in astrogliosis was a concomitant increase in motor neuron cell death in the spinal cord and motor trigeminal nucleus in both ES and MB mice, as well as in the ventrolateral thalamus in MB animals. In contrast, microglial activation was significantly increased in all the same regions but only when the mice were in the MB phase. These results suggest that astrogliosis preceded or occurred concurrently with neuronal degeneration whereas prominent microgliosis was evident later (MB stage), after significant motor neuron degeneration had occurred. Hence, our findings support a role for astrocytes in modulating the progression of non-cell autonomous degeneration of motor neurons, with microglia playing a role in clearing degenerating neurons.     

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.     

Ammonium tetrathiomolybdate delays onset, prolongs survival, and slows progression of disease in a mouse model for amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) cause a form of familial amyotrophic lateral sclerosis (ALS). The pathogenesis of familial ALS may be associated with aberrant copper chemistry through a cysteine residue in mutant SOD1. Ammonium tetrathiomolybdate (TTM) is a copper-chelating drug that is capable of removing a copper ion from copper-thiolate clusters, such as SOD1. We found that TTM exerted therapeutic benefits in a mouse model of familial ALS (SOD1^G93A). TTM treatment significantly delayed disease onset, slowed disease progression and prolonged survival by approximately 20%, 42% and 25%, respectively. TTM also effectively depressed the spinal copper ion level and inhibited lipid peroxidation, with a significant suppression of SOD1 enzymatic activity in SOD1^G93A. These results support the hypothesis that aberrant copper chemistry through a cysteine residue plays a critical role in mutant SOD1 toxicity and that TTM may be a promising therapy for familial ALS with SOD1 mutants.     

A dopamine receptor antagonist L-745,870 suppresses microglia activation in spinal cord and mitigates the progression in ALS model mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brainstem, and spinal cord. It has been shown that oxidative stress plays a pivotal role in the progression of this motor neuron loss. We have previously reported that L-745,870, a dopamine D4 receptor antagonist, selectively inhibits oxidative stress-induced cell death in vitro and exerts a potent neuroprotective effect against ischemia-induced neural cell damage in gerbil. To investigate the efficacy of L-745,870 in the treatment of ALS, we here conducted a chronic administration of L-745,870 to transgenic mice expressing a mutated form of human superoxide dismutase gene (SOD1^H46R); a mouse model of familial ALS, and assessed whether the mice benefit from this treatment. The pre-onset administration of L-745,870 significantly delayed the onset of motor deficits, slowed the disease progression, and extended a life span in transgenic mice. These animals showed a delayed loss of anterior horn cells in the spinal cord concomitant with a reduced level of microglial activation at a late symptomatic stage. Further, the post-onset administration of L-745,870 to the SOD1^H46R transgenic mice remarkably slowed the disease progression and extended their life spans. Taken together, our findings in a rodent model of ALS may have implication that L-745,870 is a possible novel therapeutic means to the treatment of ALS.     

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     

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.     

Presymptomatic electrophysiological tests predict clinical onset and survival in SOD1G93A ALS mice

Introduction: We assessed the predictive value of electrophysiological tests as a marker of clinical disease onset and survival in superoxide‐dismutase 1 (SOD1)^G93A mice. Methods: We evaluated the accuracy of electrophysiological tests in differentiating transgenic versus wild‐type mice. We made a correlation analysis of electrophysiological parameters and the onset of symptoms, survival, and number of spinal motoneurons. Results: Presymptomatic electrophysiological tests show great accuracy in differentiating transgenic versus wild‐type mice, with the most sensitive parameter being the tibialis anterior compound muscle action potential (CMAP) amplitude. The CMAP amplitude at age 10 weeks correlated significantly with clinical disease onset and survival. Electrophysiological tests increased their survival prediction accuracy when evaluated at later stages of the disease and also predicted the amount of lumbar spinal motoneuron preservation. Conclusions: Electrophysiological tests predict clinical disease onset, survival, and spinal motoneuron preservation in SOD1^G93A mice. This is a methodological improvement for preclinical studies. Muscle Nerve 50: 943–949, 2014     

Effects of GM-CSF and ordinary supplements on the ramification of microglia in culture: A morphometrical study

Microglia transform from ameboid to ramified cells during development and display an ameboid appearance again under certain pathological conditions. Some cytokines produced by astrocytes may be responsible for the microglial transformation. In the present study, we compared the effects of cytokines, granulocyte/macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin-3 (IL-3) on the morphology of rat cultured microglia. For quantitative evaluation, we employed “transformation index” as calculated by (perimeter of cell)²/4 π (cell area). GM-CSF facilitated the ramification of cultured rat microglia, which was effectively induced in a serum-free medium. However, M-CSF and IL-3 did not induce the ramification. A certain serum adhesion protein (possibly vitronectin) as well as other high molecular weight substances in fetal calf serum inhibited the GM-CSF-induced microglial ramification. Among ordinary supplements for a chemically defined medium, progesterone, insulin, and a high concentration of glucose suppressed the ramification. These findings suggest that GM-CSF may be involved in microglial ramification and that many kinds of supplements that are added to culture media profoundly affect the morphology of microglial cells. © 1996 Wiley-Liss, 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.     

Sigma-1R Agonist Improves Motor Function and Motoneuron Survival in ALS Mice

Amyotrophic lateral sclerosis is a neurodegenerative disorder characterized by progressive weakness, muscle atrophy, and paralysis due to the loss of upper and lower motoneurons (MNs). Sigma-1 receptor (sigma-1R) activation promotes neuroprotection after ischemic and traumatic injuries to the central nervous system. We recently reported that sigma-1R agonist (PRE-084) improves the survival of MNs after root avulsion injury in rats. Moreover, a mutation of the sigma-1R leading to frontotemporal lobar degeneration/amyotrophic lateral sclerosis (ALS) was recently described in human patients. In the present study, we analyzed the potential therapeutic effect of the sigma-1R agonist (PRE-084) in the SOD1^G93A mouse model of ALS. Mice were daily administered with PRE-084 (0.25?mg/kg) from 8 to 16?weeks of age. Functional outcome was assessed by electrophysiological tests and computerized analysis of locomotion. Histological, immunohistochemical analyses and Western blot of the spinal cord were performed. PRE-084 administration from 8?weeks of age improved the function of MNs, which was manifested by maintenance of the amplitude of muscle action potentials and locomotor behavior, and preserved neuromuscular connections and MNs in the spinal cord. Moreover, it extended survival in both female and male mice by more than 15?%. Delayed administration of PRE-084 from 12?weeks of age also significantly improved functional outcome and preservation of the MNs. There was an induction of protein kinase C-specific phosphorylation of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor in SOD1^G93A animals, and a reduction of the microglial reactivity compared with untreated mice. PRE-084 exerts a dual therapeutic contribution, modulating NMDA Ca²⁺ influx to protect MNs, and the microglial reactivity to ameliorate the MN environment. In conclusion, sigma-1R agonists, such as PRE-084, may be promising candidates for a therapeutical strategy of ALS.