| The glial fibrillary acidic protein (GFAP), the major intermediate filament (IF) of astrocytes, is a primary component of Rosenthal Fibers (RFs), cytoplasmic aggregates found in Alexander Disease, certain brain tumors, and in glial scars after brain trauma and stroke. We have sought to clarify the role of GFAP overexpression and mutation in RF formation. Previous research showed that overexpression of wild type GFAP for two days led to the formation of RF-like aggregates in primary rat astrocytes and human astrocytoma cell lines. We have found that GFAP aggregates form 9.5 hours after transient transfection into human astrocytoma cells. In addition, GFAP forms areas of increased filament density, a possible intermediate step between a spread IF network and aggregation. Further, GFAP aggregates form in primary rat astrocytes infected with a newly constructed GFAP-expressing adenovirus. We also present studies on the R239C GFAP mutation found in some Alexander Disease patients. Transient transfection into primary rat astrocytes and Cos-7 cells shows that the mutant GFAP is incorporated into the filament network with wild type GFAP and vimentin, respectively. In IF-negative SW13Vim- cells, wild type GFAP forms filament networks. However, the human mutant forms a "diffuse" pattern and an irregular pattern which may represent disorganized filaments. The mouse mutant is able to form short filaments, though the majority appears "diffuse." Co-transfection of wild type and mutant GFAP leads to the incorporation of mutant and wild type GFAP into filaments. At the electron microscopic level, wild type and mutant hGFAP form 10 nm filaments. The majority of GFAP is in the insoluble fraction after extraction of transfected SW13Vim- cells with Triton X-100 and Western blotting. In high salt extractions with KCl and Triton X-100, wild type hGFAP is solubilized, while mutant GFAP remains insoluble. This suggests that the R239C mutation increases GFAP stability, which may contribute to its aggregation. These studies improve the understanding of the effects of overexpressing or mutating GFAP on filament organization, and provide insight to the cellular processes that may precede astrocyte dysfunction in disease. |
