Cysteine modifications of glial fibrillary acidic protein and impact in Alexander disease

Resumen

Glial fibrillary acidic protein or GFAP is a member of the class III intermediate filament family, and is mainly expressed in astrocytes. As a key characteristic, GFAP has a conserved single cysteine residue located at position 294 (C294). The main disease associated to GFAP is Alexander disease, a lethal neurodegenerative disease caused by mutations in the GFAP gene, being the R239C the most common substitution. This disease is characterized by the accumulation of mutant GFAP in cytoplasmic aggregates that lead to the dysfunction of astrocytes by a not well-characterized mechanism. In conditions associated with oxidative stress, such as neurodegeneration, transformations of unsaturated lipids lead to the formation of reactive electrophilic lipid species of varied structure, including hydroxynonenal and cyclopentenone prostaglandins (cyPG). These lipids are able to covalently modify nucleophilic residues, mainly cysteines, in a process known as lipoxidation. Lipoxidation of GFAP has been reported in several neurodegenerative diseases although the precise site(s) of modification has not been elucidated. In this work, we show that GFAP C294 is lipoxidized by cyPG and modified by glutathione, in vitro and in cells. Indeed, oxidative and electrophilic damage of GFAP filaments in cells lead to a rearrangement that displays differential features depending on the damaging agent and requires the presence of C294. Additionally, we have confirmed the ability of GFAP to homooligomerize and heterooligomerize with vimentin in vitro and in cells, a process highly dependent on the presence of the cysteine residues in both proteins that seem to be more critical for GFAP than for vimentin. In addition, we have set up several cellular models of Alexander disease by expressing the GFAP R239C mutant. This results in the formation of thick GFAP perinuclear bundles, cytoplasmic aggregates, and putative disulfide-bonded oligomers. Furthermore, GFAP R239C shows a higher susceptibility than GFAP wt to modification and disruption by oxidative and lipoxidative stress mimicked by several agents, including electrophilic lipids. Lysosomes and mitochondria are two of the organelles proposed to be affected in models of Alexander disease. We have observed that expression of GFAP R239C in cells alters the distribution and number of lysosomes, and impairs their acidification capacity. In addition, cells expressing this mutant show several mitochondrial alterations including a decrease in mitochondrial mass, together with increased length and superoxide production, thus suggesting a defective mitochondrial fission