Giant axonal neuropathy–associated gigaxonin mutations impair intermediate filament protein degradation
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
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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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 5

Mechanism responsible for VIF clearance by gigaxonin.

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Mechanism responsible for VIF clearance by gigaxonin. (A) Immunoblotting...
(A) Immunoblotting of control and WT gigaxonin expressing BJ5ta cells treated with MG-132 using antibodies to vimentin, actin, or FLAG (gigaxonin). MG-132 treatment caused substantial recovery of vimentin in gigaxonin-expressing cells. Representative blots, 4 experiments. (B) In vitro ubiquitination assay showing ubiquitination ladder for the positive control sic 60–barnase–DHFR, but not for vimentin in the same preparation. Representative autoradiogram, 3 experiments. Lanes were run on the same gel but were noncontiguous (black line). (C) Immunoblotting of lysates of BJ5ta cells that had expressed gigaxonin for 72 hours and subsequently were treated with chloroquine (CQ; lysosome inhibitor), MG-132 (proteasome inhibitor), or 3-MA (autophagy inhibitor) for 12 hours. Blots were probed with antibodies to vimentin, actin, tubulin, or gigaxonin. The untreated cell lysate (–) contained very small amounts of vimentin. Vimentin recovery was seen only upon inhibition of proteasomes, not by inhibition of lysosomes or autophagy. Representative immunoblots, 4 experiments. (D) Longer exposure of the anti-vimentin full-length immunoblot in C, showing absence of a typical ubiquitinated protein ladder for vimentin. Representative blots from 4 experiments.