Alexander disease causing mutations in the C-terminal domain of GFAP are deleterious both to assembly and network...

Area of Science:

• Neuroscience
• Cell Biology
• Genetics

Background:

• Alexander disease is a genetic astrocyte disorder caused by mutations in glial fibrillary acidic protein (GFAP).
• Most known mutations affect the GFAP α-helical rod domain, but C-terminal mutations are also implicated.

Purpose of the Study:

• To investigate the impact of five C-terminal GFAP mutations on filament assembly and cellular behavior.
• To compare the pathogenic effects of these C-terminal mutations in vitro and in cultured cells.

Main Methods:

• In vitro filament assembly assays.
• Transient transfection of cultured cells (MCF7, SW13, U343MG) with mutant GFAP.
• Analysis of protein solubility, aggregation, chaperone association (αB-crystallin, HSP27), kinase activation (p38), proteasome activity, caspase 3 activation, and astrocyte viability.

Main Results:

• All five C-terminal GFAP mutations disrupted in vitro filament assembly.
• Mutations decreased GFAP solubility, increased aggregation, and activated p38 kinase and αB-crystallin association.
• The D417M14X mutation showed the most severe effects, including extensive aggregation, HSP27 sequestration, proteasome inhibition, p38 activation, caspase 3 activation, and reduced astrocyte viability.

Conclusions:

• C-terminal GFAP mutations impair filament assembly and cellular integrity.
• These mutations can lead to caspase 3 activation and decreased astrocyte viability, contributing to Alexander disease.
• The D417M14X mutation serves as a potent example of C-terminal GFAP-induced cellular pathology.