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

Abstract

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-1beta 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-1beta correlated with amyloid-like misfolding and was independent of dismutase activity. Deficiency in caspase-1 or IL-1beta 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-1beta as a causative event of neuroinflammation and suggest IL-1 as a potential therapeutic target in ALS.

Fig. 1.

Mutant SOD1 activates caspase-1 in microglia and macrophages. (A–E) Primed cells were stimulated with 10 μM or the depicted concentrations of WT or G93A-SOD1 for 20 h or as indicated. (A) Flow cytometry analysis of caspase-1 activation in primary microglia by a fluorescent cell-permeable inhibitor that binds to active caspase-1 (FLICA). Numbers above bracketed lines indicate percentage of cells with active caspase-1. Unstimulated and ATP-stimulated cells served as controls. (B) Immunoblot analysis of cell lysates and supernatants of stimulated bone marrow–derived macrophages (BMMs) with antibodies to the p10 subunit of caspase-1 and to IL-1β, respectively. Actin shows equal loading of lanes. (C–E) ELISA analysis of secreted, mature IL-1β in the cell supernatant of stimulated primary microglia (C), caspase-1–deficient BMMs (D), or primary astrocytes (E). Data are representative of at least three independent experiments; error bars represent SEM of triplicate wells.

Fig. 2.

IL-1β maturation requires endocytosis of mutant SOD1. (A–D) Primed BMMs were stimulated with WT or G93A-SOD1 as indicated. (A and B) Uptake of fluorescently labeled G93A-SOD1 (5 μM; red) in the presence of Cytochalasin D analyzed by confocal microscopy (A) or flow cytometry (B). Fluorescent cholera toxin B (CTB; green) stains cell membranes. (C) ELISA of SOD1 (10 μM) induced mature IL-1β in the presence of Cytochalasin D. (D) Immunoblot analysis of cytoplasmic and organelle fraction of BMMs stimulated with G93A-SOD1 (2 μM) for indicated times using antibodies against human SOD1, GAPDH (cytoplasmic marker protein), and LAMP1 (organelle marker protein). (E) Confocal microscopy of BMMs stimulated for the indicated time points with labeled G93A-SOD1 (5 mM; green) and Dextran (red) or Dextran only. (Scale bars, 4 μm.) Data are representative of at least three independent experiments; error bars represent SEM of triplicate wells.

Fig. 3.

Mutant SOD1–induced caspase-1 activation is counteracted by autophagy. (A–C) Primed cells were stimulated with WT or G93A-SOD1 as indicated. (A) Immunoblot analysis of cytoplasmic and organelle fraction of BMMs stimulated with G93A-SOD1 (2 μM) for 6 h in the presence of 3-methyladenine (3MA) using antibodies against human SOD1, GAPDH (cytoplasmic marker protein), and LAMP1 (organelle marker protein). (B) ELISA of SOD1 (10 μM) induced mature IL-1β from BMMs in the presence of wortmannin or 3-methyladenine. (C) Caspase-1 activation determined by flow cytometry analysis with caspase-1 FLICA in autophagy-related 5–deficient (ATG5^−/−, ○) and nondeficient ATG5^+/+ (•) mouse embryonic fibroblasts (MEFs) transfected with the indicated concentrations of SOD1. Data are representative of three independent experiments; error bars represent SEM of triplicate wells.

Fig. 4.

SOD1-induced IL-1β maturation correlates with its amyloid-like misfolding. (A) Th-T fluorescence of the indicated SOD1 proteins. (B) IL-1β maturation induced by the indicated SOD1 proteins in primed BMMs determined by ELISA. (C) Correlation of IL-1β maturation and its corresponding Th-T fluorescence at 490 nm. P = 0.0052; Pearson´s correlation coefficient r = 0.8027. AU, arbituary units. Data are representative of two independent experiments.

Fig. 5.

Disease progression in G93A-SOD1 transgenic mice is accelerated by IL-1β and can be delayed by IL-1RA treatment. (A) Survival of G93A-SOD1 transgenic mice (152.2 ± 1.2 d; n = 25), caspase-1–deficient (162.0 ± 1.8 d; n = 24), IL-1β–deficient (159.6 ± 1.7 d; n = 21), and IL-18–deficient (152.2 ± 1.4 ds; n = 24) G93A-SOD1 transgenic mice. Caspase-1– and IL-1β–deficient mice lived significantly longer than G93A-SOD1 transgenic mice (P < 0.001). (B–D) Immunohistochemical analysis of cervical spinal cord sections of 120-d-old G93A-SOD1 transgenic mice, caspase-1–deficient G93A-SOD1 transgenic mice, and IL-1β–deficient G93A-SOD1 transgenic mice using antibodies against the microglial marker protein ionized calcium binding adaptor molecule 1 (IBA1; B), the astrocytic marker glial fibrillary acidic protein (GFAP; C), or the motor neuron marker protein choline acetyltransferase (ChAT; D). Representative images of the ventral horn area of mice with indicated genotypes, and quantification of IBA1-, GFAP-, and ChAT-positive area in the ventral horns of analyzed mice. (E) Survival of G93A-SOD1 transgenic mice treated with indicated dosages of IL-1RA (75 mg/kg: 157.8 ± 1.8 d, n = 23; 150 mg/kg: 159.4 ± 1.8 d; n = 21) or placebo (153.1 ± 1.3 d, n = 19). IL-1RA–treated mice lived significantly longer than placebo-injected mice (75 mg/kg, P = 0.0145; 150 mg/kg, P < 0.005). (F) Motor performance of IL-1RA (150 mg/kg) and placebo-treated G93A-SOD1 transgenic mice determined by the hanging-wire test. (Scale bar, 200 μm.) *P < 0.05. n.s, Not significant. Values are mean ± SEM.