An immunological epitope selective for pathological monomer-misfolded SOD1 in ALS

278

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: 84%

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

Meissner

Molawi

Zychlinsky

2010

278

226

“…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: 89%

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.

“…To test if HuWtSOD1 participates in cell-to-cell transmission of protein misfolding, we make use of previously developed mouse mAb probes for misfolded/oxidized SOD1, recognizing either full-length human mutant or WT SOD1, generated against regions that are antibody-inaccessible in natively folded SOD1 (13)(14)(15). Misfolded SOD1 mAbs used in this work are 10E11C11 and 3H1, directed against an unstructured electrostatic loop [disease-specific epitope-2 (DSE2)], and 10C12, directed against a C-terminal dimer interface peptide in which the cysteine at position 146 is substituted by a cysteic acid residue to mimic oxidation of this residue (DSE1a) (13).…”

mentioning

confidence: 99%

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms

Grad

Yerbury

Turner

et al. 2014

377

397

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWt-SOD1 propagation has been established, misfolding of HuWt-SOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-tocell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.A myotrophic lateral sclerosis (ALS) is a fatal neuromuscular condition that afflicts as many as 1 of 350 males and 420 females over the age of 18 (1). In ALS, degeneration of upper and lower motor neurons causes progressive muscle paralysis and spasticity, affecting mobility, speech, swallowing, and respiration (2). Half of affected individuals die within 3 y, and less than 20% survive for more than 5 y (3); 90-95% of ALS cases are sporadic (SALS) in which some apparently facilitating gene mutations, such as repeat expansions in the gene that encodes ataxin-2 (4), have been identified. The remaining 5-10% of ALS cases are familial (FALS) and predominantly associated with Mendelian-inherited mutations in the genes encoding Cu/Zn superoxide dismutase (SOD1), TAR-DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/ TLS), C9ORF72, and other genes (reviewed in ref. 3).Despite the profusion of functionally diverse genes implicated in FALS and SALS, clinical and pathological similarities between all forms of ALS suggest the existence of a common pathogenic pathway that could be united by a single gene/protein (5). One of the mechanisms by which a mutant or wild-type (WT) protein can dominate pathogenesis of phenotypically diverse diseases is by propagated protein misfolding, such as that underpinning the prion diseases, which has been increa...