Plectin regulates the organization of glial fibrillary acidic protein in Alexander disease

Abstract

Alexander disease (AxD) is a rare but fatal neurological disorder caused by mutations in the astrocyte-specific 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.

Figure 1

Immunoblot analysis of plectin in AxD and control brains. Equivalent aliquots of white matter homogenates from two control (C1 and C2) and one AxD case (R239C) were resolved on a 4 to 20% gradient gel (A) and 6% SDS-polyacrylamide gel (B). A: Significantly increased total amount of plectin and GFAP were found in AxD brain. B: Abundant plectin (between 200 and 500 kd, arrow) was also detected in the TX-insoluble (P) fraction of AxD brain. Position of the first molecular mass standard is indicated on the right. S, TX soluble.

Figure 2

Plectin is associated with RFs. A: Characteristic pathology of AxD: copious RFs (arrows) located in the perivascular region (*, blood vessel) of the subcortical white matter (H & E). B and C: Immunohistochemical staining of plectin in deparaffinized brain sections of AxD. Immunoreactivity for plectin is found predominantly in the periphery of RFs (arrows) with visible staining of the RFs themselves. Original magnifications: ×200 (A, B); ×400 (C).

Figure 3

Localization of plectin in human astrocytes expressing GFAP. A–C: The localization of endogenous plectin (red) and GFAP (green) in human primary astrocytes. These cells were transiently transfected with Flag-tagged WT (D–F) or RC (G–I) GFAP and stained with anti-FLAG (green) and anti-plectin (red). The merged images (C, F, and I) show the degree of co-localization of plectin with GFAP. Scale bar, 10 μm.

Figure 4

Immunoblot analysis in GFAP stable transfectants of U251 MG astrocytoma cells. Fifteen μg of total (T), 10 μg of high-salt/TX pellet fractions (P), and 30 μg of corresponding supernatant fractions (S) per lane were obtained from nontransfected cells (CON), WT GFAP-GFP (WT), or R239C GFAP-GFP (RC) stable transfectants.

Figure 5

Plectin co-immunoprecipitates with GFAP. WT and RC GFAP were transfected into plectin (+/+) cells and plectin (−/−) cells. After 24 hours, cell lysates (200 μl) were incubated with protein A Sepharose anti-GFAP antibody, followed by Western blotting with anti-plectin antibody. WT was transfected into plectin (+/+) cells, incubated with beads without anti-GFAP antibody as a negative control (Prot A). Ten percent of the total cell lysates (input 1/10, 20 μl) was subjected to SDS-PAGE analysis and probed with anti-GFAP and anti-Ple.

Figure 6

Blot overlay assay of GFAP and plectin. Proteins (2 to 3 mg) were separated on 10% polyacrylamide gel, blotted to the nitrocellulose (A) and overlaid with bacterially expressed plectin recombinant proteins, BN205 and BN192 (B). A: Ponceau S staining of blotted proteins. B: BN205 and BN192 were overlaid onto blotted WT GFAP and GFAP-R239C mutant protein. Bound proteins were detected with myc-tagged monoclonal antibodies. Note binding of BN205 (part of plectin C-terminal domain L3850 to A4687 including IF binding site) and not BN192 (part of a plectin α-helical rod domain E2235 to Q2577) to WT GFAP and R239C mutant. Overlaid proteins did not bind control, BSA. Lines with blotted BN205 serve as control for myc-tag detection. C: Coomassie staining of overlaid proteins.

Figure 7

GFAP organization in plectin (+/+) and plectin (−/−) cells. WT (A) or RC (C) GFAP were transiently transfected into mouse fibroblasts (either plectin-positive or -negative). The WT-transfected cells displayed the following GFAP phenotypes: filamentous bundles (FB), short filaments (SF), and perinuclear bundles (PB). The RC-transfected cells displayed either a diffuse filament (DF) phenotype or a ring-shaped aggregate (RA) phenotype. B and D: The quantitative distributions of each phenotype in plectin-positive and -negative cells. An asterisk indicates the difference compared with other patterns within each group is statistically significant (P < 0.05). Scale bar, 10 μm.

Figure 8

Co-expression of mutant GFAP and full-length plectin in plectin (−/−) cell. A: R239C GFAP in plectin (+/+) cells showed disorganized thin filaments. Green is GFAP, red is Ple. B: R239C GFAP formed ring-shaped aggregates in plectin (−/−) cells. Arrowheads indicate the characteristic ring-shaped aggregates. C: Reorganization of mutant GFAP filaments by co-transfecting a c-myc-tagged full-length plectin (pBN165) with the mutant GFAP in plectin (−/−) cells. Scale bar, 10 μm.

Figure 9

TX fractionation of GFAP in plectin (+/+) and (−/−) cells. A: Immunoblot analysis of detergent-fractionated proteins from plectin (+/+) and (−/−) cells transfected with WT and RC GFAP. Equal amounts of total protein (T, 15 mg), TX-extracted (S, soluble; 50 μg), and nonextracted (P, pellet; 10 μg) fractions were detected by immunoblotting using anti-GFAP and anti-plectin antibodies. B: Comparison of total level of plectin in untransfected (UTxf), WT GFAP (WT), and RC GFAP (RC) transfected plectin (+/+) cells.

Figure 10

RC GFAP organization and plectin levels in human primary astrocytes. A: Detection of endogenous plectin in plectin-positive fibroblasts (+/+), human primary astrocytes (Astro), and plectin-null fibroblasts by immunoblotting. B and C: Human primary astrocyte cells were co-transfected with expression vectors encoding Flag-tagged RC GFAP and a c-myc-tagged full-length plectin (pBN165). At 24 hours after transfection, cells were stained with anti-Flag (green) and anti-c-myc (red). Two examples of co-transfected cells (B, C) display diffuse filamentous GFAP staining in the periphery of the cytoplasm and an intense perinuclear staining. Punctate perinuclear staining was evident in ∼50% of these cells (C, arrowhead). Scale bar, 10 μm.