Activation of the Nrf2-ARE pathway in muscle and spinal cord during ALS-like pathology in mice expressing mutant SOD1

Oxidative stress plays a key role in the neuronal loss exhibited in amyotrophic lateral sclerosis (ALS), an event precipitating irreversible muscle atrophy. By crossing ALS mouse models (SOD(G93A) and SOD(H46RH48Q)) with an antioxidant response element (ARE) reporter mouse, we identified activation characteristics of the ARE system throughout the timecourse of motor neuron disease. Surprisingly, the earliest and most significant activation of this genetic sensor of oxidative stress occurred in the distal muscles of mutant SOD mice. The resultant data supports existing hypotheses that the muscle is somehow implicated during the initial pathology of these mice. Subsequently, Nrf2-ARE activation appears to progress in a retrograde fashion along the motor pathway. These data provide timely information concerning the contributions of the Nrf2-ARE pathway in ALS disease progression.     

The lack of effect of specific overexpression of IGF-1 in the central nervous system or skeletal muscle on pathophysiology in the G93A SOD-1 mouse model of ALS

The ability of insulin like growth factor 1 (IGF-1) to prevent the pathophysiology associated with amyotrophic lateral sclerosis (ALS) is currently being explored with animal models and in clinical trials with patients. Several studies have reported positive effects of IGF-1 in reducing motor neuron death, delaying the onset of motor performance decline, and increasing life span, in SOD-1 mouse models of ALS and in one clinical trial. However, a second clinical trial produced no positive results raising questions about the therapeutic efficacy of IGF-1. To investigate the effect of specific and sustained IGF-1 expression in skeletal muscle or central nervous system on motor performance, life span, and motor neuron survival, human-IGF-1 transgenic mice were crossed with the G93A SOD-1 mutant model of ALS. No significant differences were found in onset of motor performance decline, life span, or motor neuron survival in the spinal cord, between SOD+/IGF-1+ and SOD+/IGF-1- hybrid mice. IGF-1 concentration levels, measured by radioimmunoassay, were found to be highly increased throughout life in the central nervous system (CNS) and skeletal muscle of IGF-1 transgenic hybrid mice. Additionally, increased CNS weight in SOD+ mice crossbred with CNS IGF-1 transgenic mice demonstrates that IGF-1 overexpression is biologically active even after the disease is fully developed. Taken together, these results raise questions concerning the therapeutic value of IGF-1 and indicate that further studies are needed to examine the relationship between methods of IGF-1 administration and its potential therapeutic value.     

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.     

Sex-specific behavioural effects of environmental enrichment in a transgenic mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by motor neuron degeneration, muscle wasting and paralysis. While twin studies support a role for both genetic and environmental factors in ALS, the nature of environmental modifiers is unknown. We therefore compared onset and progression of disease symptoms in female and male transgenic ALS mice (expressing the human SOD1^G93A gene mutation) and their wild-type littermates, housed in environmentally enriched versus standard conditions. Environmental enrichment significantly improved motor performance, as measured using the accelerating rotarod, in particular for female mice. This enhanced motor coordination was observed for both SOD1^G93A and wild-type mice, suggesting this effect is independent of genotype. Female SOD1^G93A mice housed with environmental enrichment were found to reach overt end-stage disease sooner than their standard-housed littermates. However, male SOD1^G93A mice did not show significantly accelerated disease progression. This evidence for environmental modulation of ALS pathogenesis in transgenic mice provides insights into activity-dependent aspects of the disease process, and may help identify molecular targets for pharmacological modulators as future therapeutics.     

Lack of changes in the PI3K/AKT survival pathway in the spinal cord motor neurons of a mouse model of familial amyotrophic lateral sclerosis

The vulnerability of motor neurons in transgenic SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), may depend on the failure of these cells to activate survival mechanisms in response to the toxic mutant SOD1. To test this we investigated whether defects in the PI3K/Akt pathway, a survival signal, and of its neuron-specific activator, Rai, were important for motor neuron degeneration in these mice. No substantial changes were found in the levels of Rai, PI3K(p85) or phosphorylated Akt (P-Akt) in the ventral horn of spinal cord of SOD1G93A mice during disease progression. P-Akt immunoreactivity was the same in degenerating and healthy motor neurons. Rai ablation in SOD1G93A mice slightly accelerated the motor dysfunction without affecting their life span. Thus, motor neurons in SOD1G93A mice do not lose the pro-survival PI3K/Akt signal nor increase it in order to suppress the cell death mechanisms.     

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.     

Excitability properties of mouse motor axons in the mutant SOD1G93A model of amyotrophic lateral sclerosis

Non‐invasive excitability studies of motor axons in patients with amyotrophic lateral sclerosis (ALS) have revealed a changing pattern of abnormal membrane properties with disease progression, but the heterogeneity of the changes has made it difficult to relate them to pathophysiology. The SOD1^G93A mouse model of ALS displays more synchronous motoneuron pathology. Multiple excitability measures of caudal and sciatic nerves in mutant and wild‐type mice were compared before onset of signs and during disease progression (4–19 weeks), and they were related to changes in muscle fiber histochemistry. Excitability differences indicated a modest membrane depolarization in SOD1^G93A axons at about the time of symptom onset (8 weeks), possibly due to deficient energy supply. Previously described excitability changes in ALS patients, suggesting altered sodium and potassium conductances, were not seen in the mice. This suggests that those changes relate to features of the human disease that are not well represented in the animal model. Muscle Nerve, 2010     

Loss of synaptophysin-positive boutons on lumbar motor neurons innervating the medial gastrocnemius muscle of the SOD1G93A G1H transgenic mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a common form of motor neuron disease (MND) that involves both upper and lower nervous systems. In the SOD1G93A G1H transgenic mouse, a widely used animal model of human ALS, a significant pathology is linked to the degeneration of lower motor neurons in the lumbar spinal cord and brainstem. In the current study, the number of presynaptic boutons immunoreactive for synaptophysin was estimated on retrogradely labeled soma and proximal dendrites of alpha and gamma motor neurons innervating the medial gastrocnemius muscle. No changes were detected on both soma and proximal dendrites at postnatal day 60 (P60) of alpha and gamma motor neurons. By P90 and P120, however, alpha motor neuron soma had a reduction of 14 and 33% and a dendritic reduction of 19 and 36%, respectively. By P90 and P120, gamma motor neuron soma had a reduction of 17 and 41% and a dendritic reduction of 19 and 35%, respectively. This study shows that levels of afferent innervation significantly decreased on surviving alpha and gamma motor neurons that innervate the medial gastrocnemius muscle. This finding suggests that the loss of motor neurons and the decrease of synaptophysin in the remaining motor neurons could lead to functional motor deficits, which may contribute significantly to the progression of ALS/MND.     

Protective effects of heat shock protein 27 in a model of ALS occur in the early stages of disease progression

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder, characterised by progressive motor neuron degeneration and muscle paralysis. Heat shock proteins (HSPs) have significant cytoprotective properties in several models of neurodegeneration. To investigate the therapeutic potential of heat shock protein 27 (HSP27) in a mouse model of ALS, we conducted an extensive characterisation of transgenic mice generated from a cross between HSP27 overexpressing mice and mice expressing mutant superoxide dismutase (SOD1(G93A)). We report that SOD1(G93A)/HSP27 double transgenic mice showed delayed decline in motor strength, a significant improvement in the number of functional motor units and increased survival of spinal motor neurons compared to SOD1(G93A) single transgenics during the early phase of disease. However, there was no evidence of sustained neuroprotection affecting long-term survival. Marked down-regulation of HSP27 protein occurred during disease progression that was not associated with a reduction in HSP27 mRNA, indicating a translational dysfunction due to the presence of mutant SOD1 protein. This study provides further support for the therapeutic potential of HSPs in ALS and other motor neuron disorders.     

Metabolic progression markers of neurodegeneration in the transgenic G93A-SOD1 mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. Visualizing corresponding metabolic changes in the brain of patients with ALS with proton magnetic resonance spectroscopy ((1)H-MRS) may provide surrogate markers for an early disease detection, for monitoring the progression and for evaluating a treatment response. The primary objective of our study was to evaluate whether modifications in MR metabolite levels occur before clinical disease onset, and whether these changes are directly linked to a distinct spatial progression pattern in the CNS. Therefore, age-dependent alterations in the cerebral and spinal metabolic profile in the mouse model of ALS overexpressing the mutated human G93A-superoxide dismutase 1 (G93A-SOD1) were determined by high-resolution MRS of tissue extracts at 14.1 Tesla. Both non-transgenic mice (control mice) and transgenic mice overexpressing the non-mutated human SOD1 (tg-SOD1) served as controls. In the spinal cord of G93A-SOD1 mice significantly decreased levels of N-acetyl aspartate were already detected 34 days postpartum, i.e. about 60 days before the average disease onset caused by motor neuron decline. In addition, glutamine and gamma-aminobutyric acid concentrations were significantly diminished at Day 75, which is still in the presymptomatic phase of the disease. These metabolic changes were further progressive in the course of the disease and started to involve the brainstem at Day 75. Overall, high-resolution (1)H-MRS allows a sensitive spatial and temporal metabolite profiling in the presymptomatic phase of ALS even before significant neuronal cell loss occurs.     

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.     

Bioenergetic abnormalities in discrete cerebral motor pathways presage spinal cord pathology in the G93A SOD1 mouse model of ALS

Multiple cell death pathways are implicated in the etiology of amyotrophic lateral sclerosis (ALS), but the cause of the characteristic motor neuron degeneration remains unknown. To determine whether CNS metabolic defects are critical for ALS pathogenesis, we examined the temporal evolution of energetic defects in the G93A SOD1 mouse model of familial ALS. [14C]-2-deoxyglucose in vivo autoradiography in G93A mice showed that glucose utilization is impaired in components of the corticospinal and bulbospinal motor tracts prior to either pathologic or bioenergetic changes in the spinal cord. This was accompanied by significant depletions in cortical ATP content in presymptomatic mice, which was partially ameliorated by creatine administration. Findings suggest that bioenergetic defects are involved in the initial stages of mSOD1-induced toxicity in G93A mice and imply that the selective dysfunction and degeneration of spinal cord motor neurons in this model may be secondary to dysfunction within cerebral motor pathways.     

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     

Spinal inhibitory interneuron pathology follows motor neuron degeneration independent of glial mutant superoxide dismutase 1 expression in SOD1-ALS mice

Motor neuron degeneration and skeletal muscle denervation are hallmarks of amyotrophic lateral sclerosis (ALS), but other neuron populations and glial cells are also involved in ALS pathogenesis. We examined changes in inhibitory interneurons in spinal cords of the ALS model low-copy Gurney G93A-SOD1 (G1del) mice and found reduced expression of markers of glycinergic and GABAergic neurons, that is, glycine transporter 2 (GlyT2) and glutamic acid decarboxylase (GAD65/67), specifically in the ventral horns of clinically affected mice. There was also loss of GlyT2 and GAD67 messenger RNA-labeled neurons in the intermediate zone. Ubiquitinated inclusions appeared in interneurons before 20 weeks of age, that is, after their development in motor neurons but before the onset of clinical signs and major motor neuron degeneration, which starts from 25 weeks of age. Because mutant superoxide dismutase 1 (SOD1) in glia might contribute to the pathogenesis, we also examined neuron-specific G93A-SOD1 mice; they also had loss of inhibitory interneuron markers in ventral horns and ubiquitinated interneuron inclusions. These data suggest that, in mutant SOD1-associated ALS, pathological changes may spread from motor neurons to interneuronsin a relatively early phase of the disease, independent of the presence of mutant SOD1 in glia. The degeneration of spinal inhibitory interneurons may in turn facilitate degeneration of motor neurons and contribute to disease progression.     

Muscle contractility dysfunction precedes loss of motor unit connectivity in SOD1(G93A) mice

Introduction: Electrophysiological measurements are used in longitudinal clinical studies to provide insight into the progression of amyotrophic lateral sclerosis (ALS) and the relationship between muscle weakness and motor unit (MU) degeneration. Here, we used a similar longitudinal approach in the Cu/Zn superoxide dismutase (SOD1[G93A]) mouse model of ALS. Methods: In vivo muscle contractility and MU connectivity assays were assessed longitudinally in SOD1(G93A) and wild type mice from postnatal days 35 to 119. Results: In SOD1(G93A) males, muscle contractility was reduced by day 35 and preceded MU loss. Muscle contractility and motor unit reduction were delayed in SOD1(G93A) females compared with males, but, just as with males, muscle contractility reduction preceded MU loss. Discussion: The longitudinal contractility and connectivity paradigm employed here provides additional insight into the SOD1(G93A) mouse model and suggests that loss of muscle contractility is an early finding that may precede loss of MUs and motor neuron death. Muscle Nerve 59 :254–262, 2019     

Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man

The SOD1 mutant mouse is the most widely used model of human amyotrophic lateral sclerosis (ALS). To determine where and when the pathological changes of motor neuron disease begins, we performed a comprehensive spatiotemporal analysis of disease progression in SOD1(G93A) mice. Quantitative pathological analysis was performed in the same mice at multiple ages at neuromuscular junctions (NMJ), ventral roots, and spinal cord. In addition, a patient with sporadic ALS who died unexpectedly was examined at autopsy. Mice became clinically weak at 80 days and died at 131 +/- 5 days. At 47 days, 40% of end-plates were denervated whereas there was no evidence of ventral root or cell body loss. At 80 days, 60% of ventral root axons were lost but there was no loss of motor neurons. Motor neuron loss was well underway by 100 days. Microglial and astrocytic activation around motor neurons was not identified until after the onset of distal axon degeneration. Autopsy of the ALS patient demonstrated denervation and reinnervation changes in muscle but normal appearing motor neurons. We conclude that in this widely studied animal model of human ALS, and in this single human case, motor neuron pathology begins at the distal axon and proceeds in a "dying back" pattern.     

Hind limb muscle atrophy precedes cerebral neuronal degeneration in G93A-SOD1 mouse model of amyotrophic lateral sclerosis: a longitudinal MRI study

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, neurodegenerative disorder caused by the degeneration of motor neurons in the CNS, which results in complete paralysis of skeletal muscles. Recent experimental studies have suggested that the disease could initiate in skeletal muscle, rather than in the motor neurons. To establish the timeframe of motor neuron degeneration in relation to muscle atrophy in motor neuron disease, we have used MRI to monitor changes throughout disease in brain and skeletal muscle of G93A-SOD1 mice, a purported model of ALS. Longitudinal MRI examination of the same animals indicated that muscle volume in the G93A-SOD1 mice was significantly reduced from as early as week 8 of life, 4 weeks prior to clinical onset. Progressive muscle atrophy from week 8 onwards was confirmed by histological analysis. In contrast, brain MRI indicated that neurodegeneration occurs later in G93A-SOD1 mice, with hyperintensity MRI signals detected only at weeks 10–18. Neurodegenerative changes were observed only in the motor nuclei areas of the brainstem; MRI changes indicative of neurodegeneration were not detected in the motor cortex where first motor neurons originate, even at the late disease stage. This longitudinal MRI study establishes unequivocally that, in the experimental murine model of ALS, muscle degeneration occurs before any evidence of neurodegeneration and clinical signs, supporting the postulate that motor neuron disease can initiate from muscle damage and result from retrograde dying-back of the motor neurons.     

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.