SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model
Maged M. Harraz, Jennifer J. Marden, Weihong Zhou, Yulong Zhang, Aislinn Williams, Victor S. Sharov, Kathryn Nelson, Meihui Luo, Henry Paulson, Christian Schöneich, John F. Engelhardt
Maged M. Harraz, Jennifer J. Marden, Weihong Zhou, Yulong Zhang, Aislinn Williams, Victor S. Sharov, Kathryn Nelson, Meihui Luo, Henry Paulson, Christian Schöneich, John F. Engelhardt
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Research Article

SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model

Abstract

Neurodegeneration in familial amyotrophic lateral sclerosis (ALS) is associated with enhanced redox stress caused by dominant mutations in superoxide dismutase–1 (SOD1). SOD1 is a cytosolic enzyme that facilitates the conversion of superoxide (O2•–) to H2O2. Here we demonstrate that SOD1 is not just a catabolic enzyme, but can also directly regulate NADPH oxidase–dependent (Nox-dependent) O2•– production by binding Rac1 and inhibiting its GTPase activity. Oxidation of Rac1 by H2O2 uncoupled SOD1 binding in a reversible fashion, producing a self-regulating redox sensor for Nox-derived O2•– production. This process of redox-sensitive uncoupling of SOD1 from Rac1 was defective in SOD1 ALS mutants, leading to enhanced Rac1/Nox activation in transgenic mouse tissues and cell lines expressing ALS SOD1 mutants. Glial cell toxicity associated with expression of SOD1 mutants in culture was significantly attenuated by treatment with the Nox inhibitor apocynin. Treatment of ALS mice with apocynin also significantly increased their average life span. This redox sensor mechanism may explain the gain-of-function seen with certain SOD1 mutations associated with ALS and defines new therapeutic targets.

Authors

Maged M. Harraz, Jennifer J. Marden, Weihong Zhou, Yulong Zhang, Aislinn Williams, Victor S. Sharov, Kathryn Nelson, Meihui Luo, Henry Paulson, Christian Schöneich, John F. Engelhardt

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Figure 3

SOD1 activates Rac1 and Nox2, a function dysregulated in certain SOD1 ALS mutants.

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SOD1 activates Rac1 and Nox2, a function dysregulated in certain SOD1 AL...
(A and B) Rac1 GTPase assays were performed in the presence or absence of purified (A) bovine SOD1 or (B) E. coli MnSOD and/or p29-GAP. His-tagged Rac1 was preloaded with γP32-GTP, and aliquots of the reaction were analyzed at various time points by TLC for GTP hydrolysis by assessing percent 32Pi released from Rac1. (C) GTPase assay for Rac1 and Cdc42 in the presence or absence of bovine SOD1 and/or p29-GAP. (D) Rac1 GTPase assay in the presence or absence of bovine SOD1 and/or X/XO (100 mU; final concentration 100 μM). (E) Pulldown assays of GTPγS-loaded His-tagged Rac1 in the presence of bovine SOD1 with or without 15-minute exposure to X/XO-derived ROS. Data are representative of at least 3 independent experiments. (F) Endosomes were isolated from PMDFs using Iodixanol density gradient fractionation, and fractions were characterized by Western blot for Nox2gp91phox, Rac1, SOD1, and EEA1. (G) Lucigenin assays were used to assess the rate of NADPH-dependent O2•– production in fraction 10 vesicles from Nox2 WT and KO PMDFs in the presence or absence of 2.5 μM bovine SOD1 and/or 10 μM DPI, a general Nox inhibitor (n = 6). (H) Vesicular fractions from PMEFs heterozygous for a gene disruption of mouse SOD1 were assessed for NADPH-dependent O2•– production in the presence or absence of exogenously added 2.5 μM bovine SOD1 or 2.5 μM E. coli MnSOD (n = 3). (I) Coomassie-stained SDS-PAGE of purified bacterially expressed human SOD1 proteins. Bovine SOD1 was used as a reference control and migrates faster than human SOD1 (12). The Cu/Zn content of each SOD1 protein is shown. SOD activity gel of the bacterial purified SOD1 proteins is shown at bottom. (J) In vitro IP of purified prereduced His-tagged Rac1-GTPγS in the presence of the indicated human SOD1 proteins. Rac1/SOD1 complexes were then divided into 2 parts; 1 half was treated with X/XO-derived ROS for 15 minutes at room temperature prior to IP of the His-tag. Following IP, Western blots for SOD1 and Rac1 were performed. Long and short exposures of the SOD1 blot are shown to demonstrate enhanced binding of each of the mutant forms of SOD1 to Rac1. (K) Rac1 GTPase assays were performed using native bovine SOD1 or the indicated bacterially expressed purified human SOD1 proteins. The molar ratio of Rac1/SOD1 is indicated. (L) Time course of NADPH-dependent O2•– generation by isolated PMEF endosomes in the presence or absence of 1 μM human SOD1WT or SOD1L8Q.