Soluble misfolded subfractions of mutant superoxide dismutase-1s are enriched in spinal cords throughout life in murine ALS models

273

226

ALS is a fatal motor neuron disease of adult onset. Neuroinflammation contributes to ALS disease progression; however, the inflammatory trigger remains unclear. We report that ALS-linked mutant superoxide dismutase 1 (SOD1) activates caspase-1 and IL-1β in microglia. Cytoplasmic accumulation of mutant SOD1 was sensed by an ASC containing inflammasome and antagonized by autophagy, limiting caspase-1-mediated inflammation. Notably, mutant SOD1 induced IL-1β correlated with amyloid-like misfolding and was independent of dismutase activity. Deficiency in caspase-1 or IL-1β or treatment with recombinant IL-1 receptor antagonist (IL-1RA) extended the lifespan of G93A-SOD1 transgenic mice and attenuated inflammatory pathology. These findings identify microglial IL-1β as a causative event of neuroinflammation and suggest IL-1 as a potential therapeutic target in ALS.A LS is a fatal neurodegenerative disorder characterized by progressive loss of motor neurons causing paralysis and finally death within 1-5 years after diagnosis. Dominant gain of function mutations of SOD1 are the most common genetic cause of ALS and also lead to motor neuron disease in mutant SOD1 transgenic mice (1). Mutations induce toxic misfolding and aggregation of SOD1 and interfere with cellular homeostasis in neurons and glia cells (2, 3). Neurodegeneration in ALS is accompanied by glia mediated neuroinflammation, which accelerates disease progression non-cell-autonomously (2, 4). One of the inflammatory markers found in the CNS of ALS mice is active caspase-1, which is also implicated in amyloid-β-mediated inflammation (5-7). Caspase-1 is activated in response to danger signals by cytosolic protein complexes called inflammasomes and proteolytically matures IL-1β and IL-18 (8). Accordingly, IL-1β levels are elevated in the CNS of mutant SOD1 transgenic mice and ALS patients (7). Extracellular mutant SOD1 has been shown to induce an inflammatory reaction by microglia (9, 10); however, the underlying mechanisms are poorly understood. ResultsMutant SOD1 Activates Caspase-1 in Microglia. To test whether mutant SOD1 can act as a danger signal that activates caspase-1 in resident microglia, we stimulated these cells with purified WT or mutant G93A-SOD1. G93A-SOD1, but not the WT protein, activated caspase-1 in microglia and macrophages in a dose-dependent manner ( Fig. 1 A and B). Consistently, time-and dose-dependent secretion of mature IL-1β was observed specifically upon G93A-SOD1 stimulation of microglia or macrophages (Figs. 1C and S1). G93A-SOD1 induced IL-1β maturation was dependent on caspase-1 and could not be observed in astrocytes (Fig. 1 D and E). Notably, caspase-1-mediated IL-1β release in response to G93A-SOD1 was independent of nonproteinaceous contaminants, LPS priming or transgenic mutant SOD1 expression in microglia or macrophages (Figs. S2 and S3). The inflammasome adaptor protein apoptosisassociated speck-like protein containing a caspase recruitment domain (ASC) was essential for IL-1β release in response to G93A-SOD1, whereas the ...

Section: Discussionsupporting

confidence: 74%

Mutant superoxide dismutase 1-induced IL-1β accelerates ALS pathogenesis

Meissner

Molawi

Zychlinsky

2010

273

226

“…However, in addition to the hypothesis that dimer destabilization causes aggregation, another hypothesis for aggregation is that newly translated fALS SOD1 causing variants never dimerize due to lack of intrasubunit disulfide formation, metal deficiency, etc., resulting in unstable monomers (58,59). Even if fALS variants never dimerize and exist as monomers in vivo (29)(30)(31), and thus C111 residues are rarely in close proximity to each other, our data with G85R suggest that this strategy may be an effective one.…”

Section: Discussionmentioning

confidence: 83%

“…A prevailing hypothesis for the mechanism of the toxicity of fALS-SOD1 variants involves dimer destabilization and dissociation into monomers, which then nucleate the formation of higher-order aggregates (13,(26)(27)(28). Indeed, variant proteins, such as the G85R SOD1 used in this study, are found as monomers in vivo (29)(30)(31). A number of modifications, including loss of Cu or Zn (32), cleavage of the native, intramolecular disulfide (33), oxidation (13,34), and fALS-associated mutation (35), predispose the SOD1 dimer to dissociate.…”

mentioning

confidence: 98%

Strategies for stabilizing superoxide dismutase (SOD1), the protein destabilized in the most common form of familial amyotrophic lateral sclerosis

Auclair

Boggio

Petsko

et al. 2010

Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3-to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches are needed to combat neurodegenerative disease. Some familial forms of ALS (fALS) have been linked to mutations in the Cu/Zn superoxide dismutase (SOD1). The dominant inheritance of mutant SOD1 and lack of symptoms in knockout mice suggest a "gain of toxic function" as opposed to a loss of function. A prevailing hypothesis for the mechanism of the toxicity of fALS-SOD1 variants, or the gain of toxic function, involves dimer destabilization and dissociation as an early step in SOD1 aggregation. Therefore, stabilizing the SOD1 dimer, thus preventing aggregation, is a potential therapeutic strategy. Here, we report a strategy in which we chemically cross-link the SOD1 dimer using two adjacent cysteine residues on each respective monomer (Cys111). Stabilization, measured as an increase in melting temperature, of ∼20°C and ∼45°C was observed for two mutants, G93A and G85R, respectively. This stabilization is the largest for SOD1, and to the best of our knowledge, for any disease-related protein. In addition, chemical cross-linking conferred activity upon G85R, an otherwise inactive mutant. These results demonstrate that targeting these cysteine residues is an important new strategy for development of ALS therapies.mass spectrometry | thiol-disulfide I nnovative approaches are needed to combat neurodegenerative disease, among the most serious of which is amyotrophic lateral sclerosis (ALS), a disorder characterized by the death of both upper and lower motor neurons and by 3-to -5-yr median survival postdiagnosis. The only US Food and Drug Administrationapproved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life (1-3). Although the causes of sporadic neurodegenerative diseases remain a mystery, mutations causing familial forms of many of these diseases (e.g., Alzheimer's, Parkinson, and ALS) are known. For example, mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) are responsible for ∼20% of the familial ALS cases (fALS) and 2% of all ALS (4, 5). Two such mutations are G93A, which maintains wild-type-like enzymatic activity, and the metal-deficient G85R, which is essentially inactive. Posttranslational modifications of proteins involved in familial diseases have been invoked in the etiology of the corresponding sporadic diseases, for example, alpha-synuclein (6) and Parkin (7) modification in Parkinson, Abeta (8) and tau (9) modification in Alzheimer's, and TDP43 (10) and SOD1 (11-14) modification in ALS. The hope, therefore, is that strategies for treating familial diseases may translate to at least a subset of sporadic diseases.Both dominant inheritance of mutant SOD1 (15) and lack of symptoms i...

“…We noted also that basal neutral α-glucosidase activity in WT mice is 1.7-and 4-fold greater in muscle and liver, respectively, vs. spinal cord. Both the basal-and disease-induced differences in neutral α-glucosidase activity within muscle and liver may explain in part why misfolded SOD1 protein was detected at significantly higher levels in CNS than in peripheral tissues in mouse ALS (43,44). Our point of emphasis is that this mouse ALS model displays significant glycogen increases in both CNS and peripheral tissues.…”

Section: Discussionmentioning

confidence: 98%

Metabolic signatures of amyotrophic lateral sclerosis reveal insights into disease pathogenesis

Dodge¹,

Treleaven²,

Fidler³

et al. 2013

110

Metabolic dysfunction is an important modulator of disease course in amyotrophic lateral sclerosis (ALS). We report here that a familial mouse model (transgenic mice over-expressing the G93A mutation of the Cu/Zn superoxide dismutase 1 gene) of ALS enters a progressive state of acidosis that is associated with several metabolic (hormonal) alternations that favor lipolysis. Extensive investigation of the major determinants of H + concentration (i.e., the strong ion difference and the strong ion gap) suggests that acidosis is also due in part to the presence of an unknown anion. Consistent with a compensatory response to avert pathological acidosis, ALS mice harbor increased accumulation of glycogen in CNS and visceral tissues. The altered glycogen is associated with fluctuations in lysosomal and neutral α-glucosidase activities. Disease-related changes in glycogen, glucose, and α-glucosidase activity are also found in spinal cord tissue samples of autopsied patients with ALS. Collectively, these data provide insights into the pathogenesis of ALS as well as potential targets for drug development.