Microglial overexpression of fALS-linked mutant SOD1 induces SOD1 processing impairment,activation and neurotoxicity and is counteracted by the autophagy inducer trehalose

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Mutations in the gene encoding copper/zinc superoxide dismutase-1 (SOD1) are responsible for most familiar cases, but the role of mutant SOD1 protein dysfunction in non-cell autonomous neurodegeneration, especially in relation to microglial activation, is still unclear. Here, we focused our study on microglial cells, which release SOD1 also through exosomes. We observed that in rat primary microglia the overexpression of the most-common SOD1 mutations linked to fALS (G93A and A4V) leads to SOD1 intracellular accumulation, which correlates to autophagy dysfunction and microglial activation. In primary contact co-cultures, fALS mutant SOD1 overexpression by microglial cells appears to be neurotoxic by itself. Treatment with the autophagy-inducer trehalose reduced mutant SOD1 accumulation in microglial cells, decreased microglial activation and abrogated neurotoxicity in the co-culture model. These data suggest that i) the alteration of the autophagic pathway due to mutant SOD1 overexpression is involved in microglial activation and neurotoxicity; ii) the induction of autophagy with trehalose reduces microglial SOD1 accumulation through proteasome degradation and activation, leading to neuroprotection. Our results provide a novel contribution towards better understanding key cellular mechanisms in non-cell autonomous ALS neurodegeneration.     

Astroglial inhibition of NF-κB does not ameliorate disease onset and progression in a mouse model for amyotrophic lateral sclerosis (ALS)

Motor neuron death in amyotrophic lateral sclerosis (ALS) is considered a "non-cell autonomous" process, with astrocytes playing a critical role in disease progression. Glial cells are activated early in transgenic mice expressing mutant SOD1, suggesting that neuroinflammation has a relevant role in the cascade of events that trigger the death of motor neurons. An inflammatory cascade including COX2 expression, secretion of cytokines and release of NO from astrocytes may descend from activation of a NF-κB-mediated pathway observed in astrocytes from ALS patients and in experimental models. We have attempted rescue of transgenic mutant SOD1 mice through the inhibition of the NF-κB pathway selectively in astrocytes. Here we show that despite efficient inhibition of this major pathway, double transgenic mice expressing the mutant SOD1(G93A) ubiquitously and the dominant negative form of IκBα (IκBαAA) in astrocytes under control of the GFAP promoter show no benefit in terms of onset and progression of disease. Our data indicate that motor neuron death in ALS cannot be prevented by inhibition of a single inflammatory pathway because alternative pathways are activated in the presence of a persistent toxic stimulus.     

Astrocytes from familial and sporadic ALS patients are toxic to motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for <2% of all ALS cases. Their role in sporadic (S)ALS, which affects >90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.     

Mutant SOD1 protein increases Na<Subscript>v</Subscript>1.3 channel excitability

Amyotrophic lateral sclerosis (ALS) is a lethal paralytic disease caused by the degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) are present in ~20% of familial ALS and ~2% of all ALS cases. The most common SOD1 gene mutation in North America is a missense mutation substituting valine for alanine (A4V). In this study, we analyze sodium channel currents in oocytes expressing either wild-type or mutant (A4V) SOD1 protein. We demonstrate that the A4V mutation confers a propensity to hyperexcitability on a voltage-dependent sodium channel (Na_v1.3) mediated by heightened total Na⁺ conductance and a hyperpolarizing shift in the voltage dependence of Na_v1.3 activation. To estimate the impact of these channel effects on excitability in an intact neuron, we simulated these changes in the program NEURON; this shows that the changes induced by mutant SOD1 increase the spontaneous firing frequency of the simulated neuron. These findings are consistent with the view that excessive excitability of neurons is one component in the pathogenesis of this disease.     

SOD1 and TDP-43 animal models of amyotrophic lateral sclerosis: recent advances in understanding disease toward the development of clinical treatments

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease with no cure. Breakthroughs in understanding ALS pathogenesis came with the discovery of dominant mutations in the superoxide dismutase 1 gene (SOD1) and other genes, including the gene encoding transactivating response element DNA binding protein-43 (TDP-43). This has led to the creation of animal models to further our understanding of the disease and identify a number of ALS-causing mechanisms, including mitochondrial dysfunction, protein misfolding and aggregation, oxidative damage, neuronal excitotoxicity, non-cell autonomous effects and neuroinflammation, axonal transport defects, neurotrophin depletion, effects from extracellular mutant SOD1, and aberrant RNA processing. Here we summarise the SOD1 and TDP-43 animal models created to date, report on recent findings supporting the potential mechanisms of ALS pathogenesis, and correlate this understanding with current developments in the clinic.     

Mutant TDP-43 Expression Triggers TDP-43 Pathology and Cell Autonomous Effects on Primary Astrocytes: Implications for Non-cell Autonomous Pathology in ALS

Neurochemical Research - Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) caused by mutations in superoxide dismutase 1 (SOD1) is partly non-cell autonomous, involving cellular...     

Dexpramipexole Is Ineffective in Two Models of ALS Related Neurodegeneration

Treatment options for people living with amyotrophic lateral sclerosis (ALS) are limited and ineffective. Recently, dexpramipexole (RPPX) was advanced into human ALS clinical trials. In the current studies, we investigated RPPX in two parallel screening systems: 1) appropriately powered, sibling-matched, gender-balanced survival efficacy screening in high-copy B6-SJL-SOD1^G93A/Gur1 mice, and 2) high-content neuronal survival screening in primary rat cortical neurons transfected with wild-type human TDP43 or mutant human TDP43. In both cases, we exposed the test systems to RPPX levels approximating those achieved in human Phase II clinical investigations. In SOD1^G93A mice, no effect was observed on neuromotor disease progression or survival. In primary cortical neurons transfected with either mutant or wild-type human TDP43, a marginally significant improvement in a single indicator of neuronal survival was observed, and only at the 10 µM RPPX treatment. These systems reflect both mutant SOD1- and TDP43-mediated forms of neurodegeneration. The systems also reflect both complex non-cell autonomous and neuronal cell autonomous disease mechanisms. The results of these experiments, taken in context with results produced by other molecules tested in both screening systems, do not argue positively for further study of RPPX in ALS.     

Human adipose-derived stem cells enhance the glutamate uptake function of GLT1 in SOD1-bearing astrocytes

Impaired glutamate uptake function of astrocytes associated with accumulation of extracellular glutamate is a well-documented feature of amyotrophic lateral sclerosis (ALS). Enhancing the uptake function of astrocytic glutamate transport 1 (GLT1) may be a potential treatment for this disease. Human adipose-derived stem cells (hADSCs) are capable of secreting a large number of cytokines which exhibit diverse pharmacological effects. Therefore, we investigate the influence of the soluble factors released by hADSCs on the GLT1 in primary astrocytes cultured from SOD1^G93A mice, a widely studied mutant human SOD1 transgenic model of ALS. Our data indicate that soluble factors from hADSCs significantly upregulate the expression of GLT1 in SOD1^G93A-bearing astrocytes, which result in enhanced glutamate uptake function. The upregulation of GLT1 is accompanied by the inhibition of caspase-3 activation in mutant astrocytes. In addition, we find that hADSCs cocultured with SOD1^G93A-bearing astrocytes produce more VEGF, HGF and IGF-1, which are reported to have neuroprotective effects. Our results suggest that hADSCs may be a potential candidate in cellular therapy for ALS.     

Combined excitotoxic-oxidative stress and the concept of non-cell autonomous pathology of ALS: Insights into motoneuron axonopathy and astrogliosis

Non-cell autonomous pathology is widely accepted to determine the demise of motoneurons (MNs) in amyotrophic lateral sclerosis (ALS) with astrocytes, GFAP and glutamate transport suggested to play roles in reactive astrogliosis. Previously we described actions of excitotoxicity and oxidative stress to produce differential injury of motoneurons and astrocytes, respectively, and our goal here was to define patterns of MN injury and astrogliosis during a combined excitotoxic-oxidative injury since such a paradigm more closely models disease pathology. Using an in vitro neuronal-glial culture of embryonic mouse spinal cord, we demonstrate that glutamate transport activity was maintained or increased initially, despite a loss of cellular viability, induced by exposure to combinations of excitotoxic [(S)-5-fluorowillardiine (FW)] and oxidative [3-morpholinosydnonimine (SIN-1)] insults over 48h. Under these conditions, injury was slow in time course and apoptotic-like as shown by the patterns of annexin V and propidium iodide (PI) labelling. Immunocytochemistry for SMI-32 revealed that injury produced time- and insult-dependent reductions in the size of MN arbours, axonal dieback and appreciable neuritic blebbing. These changes were preceded by early hypertrophy of GFAP-positive astrocytes, and followed by more delayed stellation and eventual gliotoxicity. Alterations to EAAT2 immunolabelling were similar to those found for GFAP being initially maintained and then eventually reduced at 48h. Image analysis of immunocytochemical data confirmed the differential time-dependent changes found with SMI-32, GFAP and EAAT2. Axonopathy and blebbing of MNs was frequently associated with areas of low GFAP immunoreactivity. The exact profile of changes to MNs and astrocytes was context-dependent and sensitive to subtle changes in the mix of excitotoxic-oxidative insults. Overall our findings are consistent with the concepts that the nature, extent and time-course of astrogliosis are insult-dependent, and that discrete pro-survival and destructive components of astrogliosis are likely to determine the precise profile of MN injury in non-cell autonomous pathology of ALS.     

Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1(WT) dimers, increasing the equilibrium dissociation constant K(d) to ~10-20 μM, comparable to that of the aggressive A4V mutant. SOD1(A4V) is further destabilized by glutathionylation, experiencing an ~30-fold increase in K(d). Dissociation kinetics of glutathionylated SOD1(WT) and SOD1(A4V) are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1(I112T) has a modestly increased dissociation rate but no change in K(d) when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.     

Cupric Ions Induce the Oxidation and Trigger the Aggregation of Human Superoxide Dismutase 1

Background

Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1.

Methodology/Principal Findings

As revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn₂-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu²⁺ or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu²⁺: a moderate-affinity site (10⁶ M^-1) and a high-affinity site (10⁸ M^-1). Furthermore, Cu²⁺ binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form.

Conclusions/Significance

We demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu²⁺ plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided.     

Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis

Over 90 different mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause approximately 2% of amyotrophic lateral sclerosis (ALS) cases by an unknown mechanism. We engineered 14 different human ALS-related SOD1 mutants and obtained high yields of biologically metallated proteins from an Sf21 insect cell expression system. Both the wild type and mutant "as isolated" SOD1 variants were deficient in copper and were heterogeneous by native gel electrophoresis. By contrast, although three mutant SOD1s with substitutions near the metal binding sites (H46R, G85R, and D124V) were severely deficient in both copper and zinc ions, zinc deficiency was not a consistent feature shared by the as isolated mutants. Eight mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133 Delta) exhibited normal SOD activity over pH 5.5-10.5, per equivalent of copper, consistent with the presumption that bound copper was in the proper metal-binding site and was fully active. The H48Q variant contained a high copper content yet was 100-fold less active than the wild type enzyme and exhibited a blue shift in the visible absorbance peak of bound Cu(II), indicating rearrangement of the Cu(II) coordination geometry. Further characterization of these as-isolated SOD1 proteins may provide new insights regarding mutant SOD1 enzyme toxicity in ALS.     

A CRMP4‐dependent retrograde axon‐to‐soma death signal in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal non‐cell‐autonomous neurodegenerative disease characterized by the loss of motor neurons (MNs). Mutations in CRMP4 are associated with ALS in patients, and elevated levels of CRMP4 are suggested to affect MN health in the SOD1^G93A‐ALS mouse model. However, the mechanism by which CRMP4 mediates toxicity in ALS MNs is poorly understood. Here, by using tissue from human patients with sporadic ALS, MNs derived from C9orf72‐mutant patients, and the SOD1^G93A‐ALS mouse model, we demonstrate that subcellular changes in CRMP4 levels promote MN loss in ALS. First, we show that while expression of CRMP4 protein is increased in cell bodies of ALS‐affected MN, CRMP4 levels are decreased in the distal axons. Cellular mislocalization of CRMP4 is caused by increased interaction with the retrograde motor protein, dynein, which mediates CRMP4 transport from distal axons to the soma and thereby promotes MN loss. Blocking the CRMP4‐dynein interaction reduces MN loss in human‐derived MNs (C9orf72) and in ALS model mice. Thus, we demonstrate a novel CRMP4‐dependent retrograde death signal that underlies MN loss in ALS.     

The effects of glutaredoxin and copper activation pathways on the disulfide and stability of Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS) through mechanisms proposed to involve SOD1 misfolding, but the intracellular factors that modulate folding and stability of SOD1 are largely unknown. By using yeast and mammalian expression systems, we demonstrate here that SOD1 stability is governed by post-translational modification factors that target the SOD1 disulfide. Oxidation of the human SOD1 disulfide in vivo was found to involve both the copper chaperone for SOD1 (CCS) and the CCS-independent pathway for copper activation. When both copper pathways were blocked, wild type SOD1 stably accumulated in yeast cells with a reduced disulfide, whereas ALS SOD1 mutants A4V, G93A, and G37R were degraded. We describe here an unprecedented role for the thiol oxidoreductase glutaredoxin in reducing the SOD1 disulfide and destabilizing ALS mutants. Specifically, the major cytosolic glutaredoxin of yeast was seen to reduce the intramolecular disulfide of ALS SOD1 mutant A4V SOD1 in vivo and in vitro. By comparison, glutaredoxin was less reactive toward the disulfide of wild type SOD1. The apo-form of A4V SOD1 was highly reactive with glutaredoxin but not SOD1 containing both copper and zinc. Glutaredoxin therefore preferentially targets the immature form of ALS mutant SOD1 lacking metal co-factors. Overall, these studies implicate a critical balance between cellular reductants such as glutaredoxin and copper activation pathways in controlling the disulfide and stability of SOD1 in vivo.     

Astrocytes in amyotrophic lateral sclerosis: direct effects on motor neuron survival

Selective motor neuron death during amyotrophic lateral sclerosis (ALS) is a non-cell autonomous process in which non-neuronal cells induce and/or contribute to the disease process. The non-neuronal cells that are clearly involved in the pathogenesis of the disease are the surrounding astrocytes. Under normal conditions, astrocytes remove glutamate from the synaptic cleft and release trophic factors. In addition, these cells determine the functional characteristics of motor neurons. Recent evidence suggests that activation of astrocytes in a degenerative disease like ALS disturbs the crosstalk between astrocytes and motor neurons, which could contribute to and/or accelerate selective motor neuron death. These new insights may contribute to the development of therapeutic approaches to slow this fatal neurodegenerative disease.     

Expression of mutant SOD1 in astrocytes induces functional deficits in motoneuron mitochondria

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneuron degeneration resulting in paralysis and eventual death. ALS is regarded as a motoneuron-specific disorder but increasing evidence indicates non-neuronal cells play a significant role in disease pathogenesis. Although the precise aetiology of ALS remains unclear, mutations in the superoxide dismutase (SOD1) gene are known to account for approximately 20% of familial ALS. We examined the influence of SOD1(G93A) expression in astrocytes on mitochondrial homeostasis in motoneurons in a primary astrocyte : motoneuron co-culture model. SOD1(G93A) expression in astrocytes induced changes in mitochondrial function of both SOD1(G93A) and wild-type motoneurons. In the presence of SOD1(G93A) astrocytes, mitochondrial redox state of both wild-type and SOD1(G93A) motoneurons was more reduced and mitochondrial membrane potential decreased. While intra-mitochondrial calcium levels [Ca(2+)](m) were elevated in SOD1(G93A) motoneurons, changes in mitochondrial function did not correlate with [Ca(2+)](m). Thus, expression of SOD1(G93A) in astrocytes directly alters mitochondrial function even in embryonic motoneurons, irrespective of genotype. These early deficits in mitochondrial function induced by surrounding astrocytes may increase the vulnerability of motoneurons to other neurotoxic mechanisms involved in ALS pathogenesis.     

Colocalization of 14-3-3 proteins with SOD1 in Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis cases and the animal model

Background and Purpose

Cu/Zn superoxide dismutase (SOD1) is a major component of Lewy body-like hyaline inclusion (LBHI) found in the postmortem tissue of SOD1-linked familial amyotrophic lateral sclerosis (FALS) patients. In our recent studies, 14-3-3 proteins have been found in the ubiquitinated inclusions inside the anterior horn cells of spinal cords with sporadic amyotrophic lateral sclerosis (ALS). To further investigate the role of 14-3-3 proteins in ALS, we performed immunohistochemical analysis of 14-3-3 proteins and compared their distributions with those of SOD1 in FALS patients and SOD1-overexpressing mice.

Methods

We examined the postmortem brains and the spinal cords of three FALS cases (A4V SOD1 mutant). Transgenic mice expressing the G93A mutant human SOD1 (mutant SOD1-Tg mice), transgenic mice expressing the wild-type human SOD1 (wild-type SOD1-Tg mice), and non-Tg wild-type mice were also subjected to the immunohistochemical analysis.

Results

In all the FALS patients, LBHIs were observed in the cytoplasm of the anterior horn cells, and these inclusions were immunopositive intensely for pan 14-3-3, 14-3-3β, and 14-3-3γ. In the mutant SOD1-Tg mice, a high degree of immunoreactivity for misfolded SOD1 (C4F6) was observed in the cytoplasm, with an even greater degree of immunoreactivity present in the cytoplasmic aggregates of the anterior horn cells in the lumbar spinal cord. Furthermore, we have found increased 14-3-3β and 14-3-3γ immunoreactivities in the mutant SOD1-Tg mice. Double immunofluorescent staining showed that C4F6 and 14-3-3 proteins were partially co-localized in the spinal cord with FALS and the mutant SOD1-Tg mice. In comparison, the wild-type SOD1-Tg and non-Tg wild-type mice showed no or faint immunoreactivity for C4F6 and 14-3-3 proteins (pan 14-3-3, 14-3-3β, and 14-3-3γ) in any neuronal compartments.

Discussion

These results suggest that 14-3-3 proteins may be associated with the formation of SOD1-containing inclusions, in FALS patients and the mutant SOD1-Tg mice.     

Increased affinity for copper mediated by cysteine 111 in forms of mutant superoxide dismutase 1 linked to amyotrophic lateral sclerosis

Mutations in Cu,Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (ALS). It has been proposed that neuronal cell death might occur due to inappropriately increased Cu interaction with mutant SOD1. Using Cu immobilized metal-affinity chromatography (IMAC), we showed that mutant SOD1 (A4V, G85R, and G93A) expressed in transfected COS7 cells, transgenic mouse spinal cord tissue, and transformed yeast possessed higher affinity for Cu than wild-type SOD1. Serine substitution for cysteine at the Cys111 residue in mutant SOD1 abolished the Cu interaction on IMAC. C111S substitution reversed the accelerated degradation of mutant SOD1 in transfected cells, suggesting that the Cys111 residue is critical for the stability of mutant SOD1. Aberrant Cu binding at the Cys111 residue may be a significant factor in altering mutant SOD1 behavior and may explain the benefit of controlling Cu access to mutant SOD1 in models of familial ALS.