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 4

Gigaxonin interacts with vimentin, peripherin, and NF-L.

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Gigaxonin interacts with vimentin, peripherin, and NF-L. (A) Control and...
(A) Control and GAN cells double labeled with anti-gigaxonin and anti-vimentin. Association between gigaxonin and vimentin was only observed in the GAN cell aggregates. Representative images, 4 preparations for each of 3 GAN cell lines. (B) IPs using anti-gigaxonin were blotted with anti-vimentin. I, input (cell lysate). Rabbit IgG was used as control (M). Representative blot, 3 experiments. (C) IPs using anti-vimentin were blotted with anti-gigaxonin. Mouse IgG was used as control (M). Representative blot, 3 experiments. Lanes were run on the same gel but were noncontiguous (black lines). (D) ELISA demonstrated that gigaxonin bound to purified vimentin and the vimentin rod domain. Average data, 3 experiments. (E) Schematic of the vimentin deletion constructs FLAG-FL-VIM, FLAG-ΔC-VIM, FLAG-ΔN-VIM, FLAG-Rod-VIM, FLAG-Head-VIM, and FLAG-Tail-VIM. (F) Immunoblotting of lysates from 3T3 cell lines with FLAG-ΔC-VIM, FLAG-Rod-VIM, and FLAG-ΔN-VIM after WT gigaxonin expression for 72 hours, using antibodies to vimentin, FLAG (gigaxonin), tubulin, and actin. Representative blots, 3 experiments. (G) Immunoblotting of PC12 cell lysates prepared 72 hours after initiating WT gigaxonin expression using anti-peripherin, tubulin, actin, and gigaxonin. Similar results were obtained in the absence and presence of NGF. Representative blots, 3 experiments. (H) Immunoblotting of SH-SY-5Y cell lysates prepared 72 hours after WT gigaxonin expression using antibodies to NF-L, tubulin, actin, and gigaxonin. NF-L clearance was the same in undifferentiated (-RA-BDNF) and differentiated (+RA+BDNF) cells. Representative blots, 3 experiments. Scale bar: 10 μm (A).