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 2

Gigaxonin expression causes vimentin clearance.

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Gigaxonin expression causes vimentin clearance. (A) Immunoblotting of co...
(A) Immunoblotting of control and GAN cells 72 hours after initiating expression of vector control (Vec) or FLAG-tagged WT gigaxonin (Gig). Representative blots, 4 experiments. (B) Immunoblotting of cells expressing vector or FLAG-tagged S52G gigaxonin (S52G Gig). Representative blots, 3 experiments. (C) RT-PCR analysis of VIM mRNA in BJ5ta cells. Average data ± SD, 3 experiments. Fold increases relative to control (normalized to 1) were as follows: mock, 0.92 ± 0.268; WT gigaxonin (P = 0.6459 vs. mock), 1.04 ± 0.321; S52G gigaxonin, 1.1 ± 0.135 (P = 0.3588 vs. mock). (D and E) Immunofluorescence of (D) BJ5ta cells and (E) GAN cells, 72 hours after initiating expression of vector or WT gigaxonin, with anti-FLAG and anti-vimentin or anti-tubulin (MT). Overlays are shown at right (F, FLAG; V, vimentin). Representative images, 5 preparations. (F) BJ5ta (top) and GAN (bottom) cells stained for gigaxonin and actin after 72-hour expression of WT gigaxonin. Overlays are shown at right. Representative images, 4 preparations. Scale bars: 10 μm (D–F).