SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model
Maged M. Harraz, … , Christian Schöneich, John F. Engelhardt
Maged M. Harraz, … , Christian Schöneich, John F. Engelhardt
Published January 24, 2008
Citation Information: J Clin Invest. 2008;118(2):659-670. https://doi.org/10.1172/JCI34060.
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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 2

Rac1 binds to SOD1 in a redox-dependent manner.

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Rac1 binds to SOD1 in a redox-dependent manner. (A) Rac1 IP from heart, ...
(A) Rac1 IP from heart, kidney, liver, and/or brain tissue of SOD1+/+ or SOD1–/– mice followed by Western blotting (WB) for SOD1 and Rac1. (B) In vitro IP of purified His-tagged Rac1 and Cdc42 in the presence of purified bovine SOD1 followed by Western blot for SOD1, Rac1, and Cdc42. The His-tagged GTPases were preloaded with the indicated nucleotide analogs prior to incubation with SOD1. (C) In vitro IP of purified His-tagged Rac1 in the presence of purified native, demetalated, or remetalated bovine SOD1 followed by Western blot for SOD1 and Rac1. The His-tagged Rac1 was preloaded with the indicated nucleotide analogs prior to incubation with SOD1. Additionally, untreated His-tagged Rac1 and 300 μM DTT prereduced His-tagged Rac1 were used for in vitro pulldown assays with each of the 3 forms of SOD1. (D) His-tagged Rac1 was prereduced (300 μM DTT), loaded with GTPγS, and treated with the indicated concentrations of H2O2 before performing pulldown assays with SOD1. (E) The indicated concentrations of DTT were added to the 300 pM H2O2-treated His-tagged Rac1 sample in D, and pulldown assays were performed with SOD1. (F) Schematic of GST-Rac1 deletion mutants used to define the SOD1 binding domain and in vitro IP of various GST-tagged Rac1 deletion mutants in the presence of purified bovine SOD1. GST-Rac1 fusion construct numbers above correspond with lane numbers below. Top lanes are Western blot for SOD1 following IP of GST; bottom lanes are Coomassie-stained gel of the purified fusion peptides used for IP.