Abstract
Alexander disease (AxD) is a rare, fatal neurodegenerative disorder caused by dominant mutations in the astrocyte-specific intermediate filament (IF) glial fibrillary acidic protein (GFAP). The pathological feature AxD is the abundant presence of Rosenthal fibers, the ubiquitinated protein inclusions within cytoplasm of astrocytes containing GFAP, the small heat shock proteins (sHSPs) αB-crystallin and HSP27. Although most disease-causing mutations are found in the α-helical rod domain of GFAP, some mutations are also found in the C-terminal tail domain. This study aimed to clarify how C-terminal mutations (N386I, S393I, N398F/Y and D417M14X) affect GFAP filament assembly in vitro and filament network organization in cells. Results showed that these mutations disrupted in vitro assembly, promoted aggregation by solubility alteration, encouraged the association of small heat shock proteins (sHSPs) chaperone, αB-crystalline and phosphorylation of p38. For further investigation, three GFAP α-helical rod domain mutations, R79C, R239H and Y366H were additionally involved for the possible stress-induced mechanism of expressing GFAP mutants. Their abilities of induce stress activated protein kinases (SAPK) Jun N-terminal kinase (JNK) phosphorylation, activation of JNK 3, p53 and caspase 3 have been demonstrated in this study. Collectively, these data confirm that the GFAP mutations affect filament assembly in a way that promotes aggregate formation, chaperone sequestration, p38 and JNK phosphorylation and increasing expression level of JNK, p53 and caspase 3, suggesting these are the key to the mechanism(s) underlying the AxD. Keywords: GFAP, mutation, Alexander disease, intermediate filament, small heat shock proteins, phosphorylation, stress