Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model

Abstract

Mutation in superoxide dismutase-1 (SOD1), which is a cause of ALS, alters the folding patterns of this protein. Accumulation of misfolded mutant SOD1 might activate endoplasmic reticulum (ER) stress pathways. Here we show that transgenic mice expressing ALS-linked SOD1 mutants exhibit molecular alterations indicative of a recruitment of ER's signaling machinery. We demonstrate by biochemical and morphological methods that mutant SOD1 accumulates inside the ER, where it forms insoluble high molecular weight species and interacts with the ER chaperone immunoglobulin-binding protein. These alterations are age- and region-specific, because they develop over the course of the disease and occur in the affected spinal cord but not in the nonaffected cerebellum in transgenic mutant SOD1 mice. Our results suggest a toxic mechanism for mutant SOD1 by which this ubiquitously expressed pathogenic protein could affect motor neuron survival and contribute to the selective motor neuronal degeneration in ALS.

Fig. 1.

Activation of UPR transcription factors and apoptosis-associated ER factors in transgenic SOD1^G93A mice. (A) ATF6 immunoblot in spinal cords from transgenic SOD1^G93A mice (G93A) at asymptomatic stage (AS, 1 month old), beginning of symptoms (BS, 3–4 months old), end stage (ES, 5 months old), and from their age-matched nontransgenic littermates (NTG) or from the cerebellum (Cb) of ES-G93A mice. Thapsigargin-treated PC12-cells were used as a positive control (Cont.). Full-length ATF6 is p90, and cleaved ATF6 is p50. (B) Immunolocalization of ATF6 in spinal cord sections: ATF6 (green), neurofilament (red), and DAPI (blue). (Scale bar, 20 μm.) (C) Ratios of IRE1-mediated splicing form of XBP1 (spliced -XBP1) over total XBP1 mRNA. ∗, P < 0.05; Student's t test. (D) Immunoblot for phosphorylated IRE1α (p-IRE1α) and nonphosphorylated IRE1α (IRE1α). (E) ATF4 immunoblot. ∗, P < 0.05; one-way ANOVA, Student–Newman–Keuls test. (F) Immunoblot of procaspase-12 (≈55 kDa) and its cleaved fragment (≈42 kDa). All values are means ± SEM (n = three to six per group). Analyses were performed in ES-G93A and age-matched NTG mouse spinal cords unless indicated otherwise.

Fig. 2.

SOD1^WT and mutant SOD1 are present in the ER. (A) Immunoblot of spinal microsomal fractions of SOD1^G93A mice at different disease stages and 5-month-old transgenic SOD1^WT and nontransgenic mice. The membranes were also probed for the ER resident protein calnexin as internal standard and β-actin. (B) Immunoblot of cerebellar microsomal fractions from mice of the different genotypes. (C) Quantification of microsomal SOD1 content over the course of disease relative to calnexin by optical density analysis (n = three to six per group; one-way ANOVA; ∗, P < 0.01; ∗∗, P < 0.05). (D) Comparison of microsomal SOD1 content in spinal cord and cerebellum. In nontransgenic and transgenic SOD1^WT mice, there is no difference in the SOD1:calnexin ratios between spinal and cerebellar microsomal fractions (P > 0.05). In transgenic SOD1^G93A mice, the microsomal SOD1:calnexin ratios are higher in the spinal cord than in the cerebellum (n = three to five per group, two-way ANOVA, Student–Newman–Keuls test; ∗, P < 0.001). SOD1:calnexin ratios in microsomal fractions from transgenic SOD1^WT and SOD1^G93A mice are higher than in nontransgenic mice in both areas (n = three to five per group; two-way ANOVA; Student—Newman–Keuls method; ∗∗, P < 0.05). (E) SOD1 is protected from surface biotinylation under conditions of intact microsomal membranes. Luminal protein BiP is used as positive control. Lane 1, total input; lane 2, eluate from intact membranes. Little SOD1 and no BiP are biotinylated in the absence of detergent. Lane 3, flow-through from intact membrane. Abundant SOD1 and BiP are found among the nonbiotinylated proteins. Lane 4, eluate from solubilized membranes. Both SOD1 and BiP are now biotinylated. (F) SOD1 is protected from proteinase K proteolysis in intact microsomal membranes and digested after addition of detergent. The mSOD1 is resistant to proteolysis in any condition (lower band).

Fig. 3.

Microscopical demonstration of ER location of SOD1 in spinal motor neurons. (A) Immunoflourescence analysis of SOD1 colocalization with ER markers calnexin and BiP in spinal cord and cerebellar sections. (Scale bar, 20 μm.) (B) Luminal ER labeling of SOD1 in immunoelectron microscopy. (Scale bar, 50 nm.) (C) Quantification of gold particles inside the ER lumen and in the ER periphery. Values represent means ± SEM (n = 23–30 ER profiles per group). ∗, P < 0.05; one-way ANOVA, Student–Newman–Keuls method.

Fig. 4.

High molecular weight aggregates of mutant SOD1 in spinal microsomal fractions. (A) Age-dependent increase of high molecular weight SOD1 species in SOD1^G93A spinal microsomal fractions in overexposed immunoblot of spinal microsomal fractions of SOD1^G93A mice. Asterisks indicate high molecular weight species. (B) Quantification of high molecular weight species over the course of the disease relative to the ER resident protein calnexin by optical density analysis. Values represent means ± SEM (n = three per group). ∗, P < 0.05; one-way ANOVA, Student–Newman–Keuls method.

Fig. 5.

Mutant SOD1, but not hSOD1^WT or mSOD1, interacts with BiP in the microsomal fraction in vivo. (A) Immunoprecipitation with an anti-BiP antibody followed by immunoblot using anti-BiP and anti-SOD1 antibodies. (B) Immunoprecipitation with an anti-BiP antibody using spinal microsomal fractions of SOD1^G93A mice at different disease stages and nontransgenic mice. Input represents 10% of protein from spinal microsomal fraction. (C) Immunoprecipitation with an anti-BiP antibody followed by immunoblot using anti-BiP, -SOD1, and -ATF6 antibodies. (D) High molecular weight (HMW) SOD1 species in microsomal fractions interact with BiP. (E) Immunoprecipitated with/without anti-BiP antibody followed by a size-exclusion filter assay.