Calpain-mediated proteolysis of vimentin filaments is augmented in Giant Axonal Neuropathy (GAN) fibroblasts exposed to hypotonic stress

Abstract

AbstractGiant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by loss-of-function mutations in the E3 ubiquitin ligase adaptor gigaxonin, which is encoded by the GAN (KLHL16) gene. Gigaxonin regulates the degradation of multiple intermediate filament (IF) proteins, including neurofilaments, GFAP, and vimentin. In the absence of functional gigaxonin, GAN patients display abnormal cytoplasmic IF aggregates in multiple cell types. Understanding how normal IF networks and abnormal IF aggregates respond and are processed under physiologic stress can reveal new GAN disease mechanisms and potential targets for therapy. Here we tested the hypothesis that hypotonic stress-induced vimentin proteolysis is impaired in GAN. In both GAN and control fibroblasts exposed to hypotonic stress, we observed time-dependent vimentin cleavage, resulting in two prominent ~40-45 kDa fragments readily detectable by immunoblot in the total cell lysates and detergent-insoluble fractions. However, vimentin proteolysis occurred more rapidly and extensively in GAN cells compared to unaffected controls. Both fragments were generated earlier in GAN cells and at 4-6-fold higher levels (p<0.0001) compared to control fibroblasts. To test enzymatic involvement, we determined the expression levels and localization of the calcium-sensitive calpain proteases-1 and -2 and their endogenous inhibitor calpastatin. While the latter was not affected, the expression of both calpains was 2-fold higher in GAN cells compared to control cells (p<0.01). Moreover, pharmacologic inhibition of calpains with MDL-28170 or MG-132 attenuated vimentin cleavage, the latter resulting in >95% reduced cleavage (p<0.0001). Imaging analysis revealed striking colocalization between large perinuclear vimentin aggregates and calpain-2 in GAN fibroblasts. This colocalization was dramatically altered by hypotonic stress, where selective breakdown of IF networks with relative sparing of IF aggregates occurred rapidly in GAN cells and coincided with cytoplasmic redistribution of calpain-2. Finally, mass spectrometry-based proteomics revealed that phosphorylation at Ser-412, located at the junction between the central “rod” domain and C-terminal “tail” domain on vimentin, is involved in this stress response. Over-expression studies using phospho-deficient (S412A) and phospho-mimic (S412D) mutants revealed that Ser-412 is important for filament organization, solubility dynamics, and cleavage of vimentin upon hypotonic stress exposure. Collectively, our work reveals that osmotic stress induces calpain- and proteasome-mediated vimentin degradation and IF network breakdown. These effects are significantly augmented in the presence of disease-causing KLHL16 mutations that alter IF spatial distribution and intermediate filament organization. While the specific roles of calpain-generated vimentin IF fragments in GAN cells remain to be defined, this proteolytic pathway is translationally-relevant to GAN because maintaining osmotic homeostasis is critical for nervous system function.