Abstract
Glial fibrillary acidic protein (GFAP) is the major constituent of the glial intermediate filaments that are expressed mainly in mature astrocytes of the central nervous system. Dominant mutations in the gene encoding GFAP cause Alexander disease, a primary genetic disorder of astrocytes that typically affects young children. The expression of mutant GFAP or overexpression of wild-type GFAP promotes the formation of cytoplasmic aggregates, with caspase activation and GFAP proteolysis. In this study, we report that GFAP is cleaved specifically by caspase 6 at VELD225 in its L12 linker domain in vitro. Caspase cleavage of GFAP at Asp225 produces two major cleavage products. While the carboxyl terminal fragment (C-GFAP) is unable to assemble into filaments, the amino-terminal fragment (N-GFAP) forms filamentous structures that are variable in width and prone to aggregation. The effect of N-GFAP is dominant, thus affecting normal filament assembly in a way that promotes filament aggregation. Transient transfection of N-GFAP induces the formation of GFAP-containing aggregates, which also disrupt the endogenous networks of intact GFAP in a human astrocytoma cell line. In addition, we generated a caspase cleavage site-specific antibody that recognizes caspase-cleaved but not intact GFAP, as determine by immunoblotting and immunofluorescence. Using this antibody, we demonstrate the presence of the caspase-generated GFAP fragment in transfected cells expressing a disease-causing mutant GFAP and in two types of AxD models that have previously been shown to have varying levels of GFAP accumulation in different regions of the central nervous system. These results imply that caspase-mediated cleavage of GFAP correlates with elevated GFAP in the context of GFAP mutation and accumulation. Moreover, we provide evidence to suggest that caspase cleavage of GFAP has important functional consequences, decreasing GFAP filament solubility by changing filament–filament interactions in a way that promotes aggregation.