Alexander disease mutations differentially sensitize glial fibrillary acidic protein (GFAP) to posttranslational modifications and network disruption by oxidants

The type III intermediate filament protein glial fibrillary acidic protein (GFAP) plays a key role in astrocyte and brain homeostasis. Mutations in GFAP can result in Alexander’s disease (AxD), a severe neurodegenerative disease. Studies on AxD models indicate that oxidative stress may be an important pathogenic factor. Cellular expression of certain GFAP AxD mutants can provoke oxidative stress and contribute to a pathogenic cycle, as GFAP itself is an important oxidant target. To understand the molecular mechanisms involved, we have carried out a detailed LC-MS/MS characterization of posttranslational modifications formed on recombinant GFAP wild-type, and AxD-relevant mutants (R79C, R239C and E373K) in response to in vitro treatment with hydrogen peroxide (H 2 O 2 ), hypochlorous acid (HOCl) and peroxynitrous acid (ONOOH). These data indicate that cysteine residues are key targets. AxD mutants show increased susceptibility to modification, with sulfinic and sulfonic acids detected at both the wild-type cysteine (C294), and cysteine residues introduced by disease-related mutations. Mutation-selective variations in nitrations (from ONOOH) and chlorinations (from HOCl) were also detected. Formation of disulfide bonds, some of which involved mutation-introduced cysteine residues, results in GFAP oligomerization in vitro and in cells. Astrocytoma cells transfected with GFP-GFAP wild-type undergo morphologically-distinct remodeling in response to oxidants. In contrast, cells expressing the R239C mutation showed persistent or increased aggregates, particularly after H 2 O 2 treatment. Together, these data show that AxD-associated GFAP mutations favor reversible and irreversible oxidative protein modifications, potentially contributing to impaired protein filament assembly and more severe responses to oxidative stress. • Several recombinant GFAP AxD mutant proteins were generated and exposed to oxidants • Increased oxidative modification was detected by LC-MS/MS on certain AxD mutants • Cys sulfinic/sulfonic acids and disulfides correlated with cellular protein aggregation • Oxidants induce cytoskeletal remodeling and R239C aggregates in astrocytoma cells • Expression of AxD-linked GFAP mutants exacerbate oxidative stress response