Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Intermediate filament (IF) proteins, like desmin, can be cleaved by caspases, producing proapoptotic fragments and promoting filament aggregation.
• Mutations in IF proteins are linked to diseases characterized by histopathological aggregates, suggesting a potential role for caspases.
• Glial fibrillary acidic protein (GFAP), an IF protein in astrocytes, is implicated in Alexander disease (AxD) and is a potential caspase substrate.

Purpose of the Study:

• To investigate whether GFAP is a caspase substrate and if its cleavage contributes to Alexander disease pathogenesis.
• To identify the specific caspase responsible for GFAP cleavage and characterize the resulting fragments.
• To determine the in vivo relevance of caspase-mediated GFAP cleavage in disease models.

Main Methods:

• In vitro cleavage assays using purified caspase 6 and GFAP.
• Analysis of GFAP cleavage products (N-GFAP and C-GFAP) for filament assembly and aggregation properties.
• Transient transfection of N-GFAP into human astrocytoma cells to assess its effect on GFAP networks.
• Generation of a neo-epitope antibody specific for caspase-cleaved GFAP.
• Immunohistochemical analysis in transfected cells and mouse models of AxD.

Main Results:

• GFAP is specifically cleaved by caspase 6 at Asp²²⁵ in vitro.
• Caspase cleavage yields N-GFAP, which forms aggregates and disrupts GFAP networks, and C-GFAP, which cannot assemble into filaments.
• Overexpression of N-GFAP in human astrocytoma cells induces cytoplasmic aggregates and disrupts endogenous GFAP.
• Caspase-generated GFAP fragments are detected in cells expressing mutant GFAP and in two mouse models of AxD.

Conclusions:

• Caspase-mediated proteolysis of GFAP occurs at Asp²²⁵, generating aggregation-prone N-GFAP fragments.
• Caspase cleavage of GFAP contributes to cytoplasmic aggregate formation and disruption of astrocyte networks.
• Caspase-generated GFAP fragments are present in disease contexts, suggesting a significant role in Alexander disease pathogenesis.