Giant axonal neuropathy–associated gigaxonin mutations impair intermediate filament protein degradation
Saleemulla Mahammad, … , Puneet Opal, Robert D. Goldman
Saleemulla Mahammad, … , Puneet Opal, Robert D. Goldman
Published April 15, 2013
Citation Information: J Clin Invest. 2013;123(5):1964-1975. https://doi.org/10.1172/JCI66387.
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Research Article Neuroscience

Giant axonal neuropathy–associated gigaxonin mutations impair intermediate filament protein degradation

Abstract

Giant axonal neuropathy (GAN) is an early-onset neurological disorder caused by mutations in the GAN gene (encoding for gigaxonin), which is predicted to be an E3 ligase adaptor. In GAN, aggregates of intermediate filaments (IFs) represent the main pathological feature detected in neurons and other cell types, including patients’ dermal fibroblasts. The molecular mechanism by which these mutations cause IFs to aggregate is unknown. Using fibroblasts from patients and normal individuals, as well as Gan–/– mice, we demonstrated that gigaxonin was responsible for the degradation of vimentin IFs. Gigaxonin was similarly involved in the degradation of peripherin and neurofilament IF proteins in neurons. Furthermore, proteasome inhibition by MG-132 reversed the clearance of IF proteins in cells overexpressing gigaxonin, demonstrating the involvement of the proteasomal degradation pathway. Together, these findings identify gigaxonin as a major factor in the degradation of cytoskeletal IFs and provide an explanation for IF aggregate accumulation, the subcellular hallmark of this devastating human disease.

Authors

Saleemulla Mahammad, S.N. Prasanna Murthy, Alessandro Didonna, Boris Grin, Eitan Israeli, Rodolphe Perrot, Pascale Bomont, Jean-Pierre Julien, Edward Kuczmarski, Puneet Opal, Robert D. Goldman

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Figure 1

VIFs are aggregated in both GAN cells and Gan–/– MEFs.

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VIFs are aggregated in both GAN cells and Gan–/– MEFs. (A) VIF network...
(A) VIF networks in a control (Con) cell and in 2 different GAN cells, the latter of which were aggregated into bundles and large bodies (arrows). HM, higher magnification. Representative images, 5 preparations of 3 GAN patient lines. (B) TEM showing aggregated VIF in GAN cells. Mitochondrion (M) and elements of the endoplasmic reticulum (arrow) are indicated in the right panel. (C) MTs (top) and actin (bottom) in double-stained GAN cells. Representative images, 5 preparations. (D) Western blot analyses of control fibroblasts (AG08470) and 3 GAN cell lines. Fold changes (± SD) in GAN line vimentin levels relative to control were as follows: F07476 (left), 1.623 ± 0.161; 08F699 (middle), 0.687 ± 0.05; F09133 (right), 1.183 ± 0.064 (P = 0.0039). Representative blots, 3 preparations. (E) VIFs were only aggregated in Gan–/– MEFs, not WT MEFs. Representative images, 5 preparations. (F) Western blotting of WT and Gan–/– MEF lysates. Representative blots, 3 preparations. (G) TEM showing VIF aggregates in Gan–/– MEFs. (H) MT (top) and actin (bottom) organization in Gan–/– MEFs. Representative images, 5 preparations. Scale bars: 10 μm (A, C, E, and H); 5.0 μm (B, left, and G, left); 0.5 μm (B, right, and G, right).