Experimental Approaches for Biochemical Analysis of Glial Fibrillary Acidic Protein and Its Disease-associated...

Glial Fibrillary Acidic Protein (GFAP) is a key intermediate filament protein critical for maintaining the structural integrity and function of astrocytes in the central nervous system. Mutations in GFAP are the root cause of Alexander disease (AxD), a rare and often fatal neurodegenerative disorder characterized by elevation of GFAP levels and accumulation of GFAP in the form of Rosenthal fibers. Here, we outline a comprehensive set of experimental approaches for the biochemical characterization of GFAP and its disease-causing variants. Using optimized expression and advanced purification techniques, we achieved high yields and purity of both wild-type and mutant GFAP proteins. Biochemical assays were employed to evaluate the effects of pathogenic mutations on filament assembly, solubility, and aggregation. Additionally, we explored the role of aberrant posttranslational modifications in GFAP aggregation and their impact on filament properties. This work advances our understanding of GFAP's role in AxD and lays a foundation for developing therapeutic strategies targeting GFAP dysfunction. Furthermore, the methodologies presented here serve as valuable tools for investigating the biochemical consequences of GFAP mutations and advancing interventions for GFAP-related disorders.