Abstracts Category : Other

Mutant actinin-4 in the pathogenesis of focal segmental glomerulosclerosis

by Albert Yee

Mutations in actinin-4 are associated with heritable focal segmental glomerulosclerosis (FSGS) in humans. We showed that actinin-4 K256E is a misfolded protein that undergoes aggregation and degradation in cultured glomerular epithelial cells (GECs). There is associated proteotoxicity, i.e. induction of endoplasmic reticulum (ER) stress and "choking" of the proteasome. The present study examined whether small molecules could improve the folding and function of mutant actinin-4, and if autophagy is upregulated by actinin-4 K256E and is responsible for its degradation. GFP-tagged actinin-4 wild type (WT) or K256E were expressed in GECs or COS-1 cells by transfection. Interaction of actinin-4 with the cytoskeleton was assessed by detergent-extraction of cells and monitoring actinin-4 by immunoblotting. Autophagy was monitored by immunoblotting of endogenous LC3II or by expression of RFP-LC3 and quantifying formation of LC3II puncta. After extraction of cells with Triton X-100, actinin-4 WT was found mainly in the Triton-soluble fraction (69.8%), whereas K256E was predominantly Triton-insoluble (81.6%), reflecting abnormally tight binding to F-actin. Incubation of cells with the chemical chaperone, 4-phenylbutyrate, decreased the proportion of Triton-insoluble actinin-4 K256E (from 81.6% to 68.6%), but celastrol, a drug that enhances expression of cytosolic and ER chaperones, had no effect. Treatment of actinin-4 K256E transgenic mice with celastrol did not reduce proteinuria. After treatment of cells with chloroquine (to block fusion of autophagosomes with lysosomes), actinin-4 K256E stimulated autophagy, as reflected by a 1.5-fold greater accumulation of endogenous LC3II, compared to WT. Moreover, RFP-LC3II puncta occupied a 45% greater area in cells expressing actinin-4 K256E, compared to WT. Surprisingly, actinin-4 K256E did not co-localize with RFP-LC3II, and was not degraded by autophagy, but rather by the proteasome.In conclusion, a small molecule chemical chaperone improved the function of actinin-4 K256E. Mutant actinin-4 stimulated autophagy, but this process was not responsible for its degradation. The combination of a chemical chaperone and proteasome inhibitor may become a novel therapeutic approach in treating actinin-4-mutant associated FSGS. Les mutations du gne actinine-4 sont associes avec la glomrulosclrose focale et segmentaire (FSGS) hrditaire chez l'humain. Nous avons dmontr que la mutation actinine-4 K256E est mal dplie et entrane une agrgation de la protine et sa dgradation dans les cellules glomrulaires pithliales (GEC) in vitro. La mutation est associe une prototoxicit, i. e. une activation du stress du rticulum endoplasmique (ER) et un tranglement du protasome. Dans cette tude, nous examinons si de petites molcules peuvent amliorer le repliement et les fonctions du mutant actinine-4 K256E, et deuximement si le processus de l'autophagie est induit par cette mutation et serait le responsable de sa dgradation.Les plasmides actinine-4-GFP type sauvage (WT) etAdvisors/Committee Members: Andrey V E Cybulsky (Internal/Supervisor).