The role of small heat shock proteins in mutant superoxide dismutase-linked familial amyotrophic lateral sclerosis

Abstract

The mechanisms by which mutations in the gene encoding superoxide dismutase 1 (S0D1) lead to amyotrophic lateral sclerosis (ALS) remain incompletely understood. Mutant SODI inclusions are observed in both ALS patients and animal models of the disease. Chaperone proteins have been shown to reduce mutant S0D1 inclusion formation in both cell and animal systems and, up-regulation of heat shock proteins (HSPs) in a mouse model of ALS increases their life expectancy. The results presented in this thesis are based on an investigation into the role of small heat shock proteins (sHSPs) in mutant SODI inclusion formation, using a model HEK293 cell system. Over-expression of yellow fluorescent protein (YFP)-tagged G85R mutant SODI in HEK293 cells and subsequent treatment with proteasome inhibitor leads to mutant S0D1-inclusion formation, as shown by immunofluorescence (IMF) microscopy. Using this model of mutant S0D1- inclusion formation, we demonstrate that over-expression of sHSPs decreases the proportion of insoluble mutant SODI present within these cells. Mutations in these sHSPs prevent this function, and further increase the proportion of insoluble mutant S0D1. These mutant sHSPs also cause an increase in the insolubility of normally soluble proteins, such as wild-type SODI. Similar results were observed in Neuro 2a cells, where over-expression of sHSPs caused the phenotype of the mutant SODI inclusions to change, from dense, tight structures to more diffuse ones. We have shown that sHSPs decrease the amount of insoluble mutant SODI in HEK2S3 cells, supporting reports that chaperone proteins prevent mutant SODI-inclusion formation and are beneficial in a mouse model Gf ALS