cells with Triton-X-100-containing buffers showed that the mutant GFAP was more resistant to solubilization at elevated KCl concentrations. Both wild-type and R239C GFAP assembled into 10 nm filaments with similar morphology in vitro. Thus, although the R239C mutation does not appear to affect filament formation per se, the mutation alters the normal solubility and organization of GFAP networks.

Alexander disease (AxD) is a leukodystrophy caused by heterozygous mutations in the gene for glial fibrillary acidic protein, an intermediate filament protein expressed by astrocytes. The mutation causes prominent protein aggregates inside astrocytes; there is also loss of myelin and oligodendrocytes and neuronal degeneration. We show that immunohistochemical staining for glutamate transporter 1, the major brain glutamate transporter expressed primarily in astrocytes suggests decreased levels in the hippocampi of infantile AxD patients. A knock-in mouse model of AxD also shows significant reduction of glutamate transporter 1 in the hippocampus. To explore this phenomenon at the cellular level, wild-type and R239C mutant glial fibrillary acidic proteins (the most common mutation) were overexpressed in astrocytes in culture. Western blotting and whole-cell patch clamp recordings demonstrated that the R239C astrocytes exhibited markedly reduced glutamate transporter 1 protein levels; this resulted in attenuated or abolished glutamate-induced inward transporter current. Neurons cocultured with the R239C astrocytes exhibited increased death after glutamate challenge. These results indicate that aberrant astrocytes have decreased glutamate uptake, which may play an important role in the pathogenesis of neuronal and oligodendrocyte injury and death in AxD.

Alexander disease (AxD) is a rare but fatal neurological disorder caused by mutations in the astrocytespecific intermediate filament protein glial fibrillary acidic protein (GFAP). Histologically, AxD is characterized by cytoplasmic inclusion bodies called Rosenthal fibers (RFs), which contain GFAP, small heat shock proteins, and other undefined components. Here, we describe the expression of the cytoskeletal linker protein plectin in the AxD brain. RFs displayed positive immunostaining for plectin and GFAP, both of which were increased in the AxD brain. Co-localization, co-immunoprecipitation, and in vitro overlay analyses demonstrated direct interaction of plectin and GFAP. GFAP with the most common AxD mutation, R239C (RC GFAP), mainly formed abnormal aggregates in human primary astrocytes and murine plectin-deficient fibroblasts. Transient transfection of full-length plectin cDNA converted these aggregates to thin filaments, which exhibited diffuse cytoplasmic distribution. Compared to wild-type GFAP expression, RC GFAP expression lowered plectin levels in astrocytoma-derived stable transfectants and plectin-positive fibroblasts. A much higher proportion of total GFAP was found in the Triton X-insoluble fraction of plectin-deficient fibroblasts than in wild-type fibroblasts. Taken together, our results suggest that insufficient amounts of plectin, due to RC GFAP expression, promote GFAP aggregation and RF formation in AxD.

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