Elevated GFAP isoform expression promotes protein aggregation and compromises astrocyte function.

Alexander disease (AxD) caused by mutations in the coding region of GFAP is a neurodegenerative disease characterized by astrocyte dysfunction, GFAP aggregation, and Rosenthal fiber accumulation. Although how GFAP mutations cause disease is not fully understood, Rosenthal fibers could be induced by forced overexpression of human GFAP and this could be lethal in mice implicate that an increase in GFAP levels is central to AxD pathogenesis. Our recent studies demonstrated that intronic GFAP mutations cause disease by altering GFAP splicing, suggesting that an increase in GFAP isoform expression could lead to protein aggregation and astrocyte dysfunction that typify AxD. Here we test this hypothesis by establishing primary astrocyte cultures from transgenic mice overexpressing human GFAP. We found that GFAP-δ and GFAP-κ were disproportionately increased in transgenic astrocytes and both were enriched in Rosenthal fibers of human AxD brains. In vitro assembly studies showed that while the major isoform GFAP-α self-assembled into typical 10-nm filaments, minor isoforms including GFAP-δ, -κ, and -λ were assembly-compromised and aggregation prone. Lentiviral transduction showed that expression of these minor GFAP isoforms decreased filament solubility and increased GFAP stability, leading to the formation of Rosenthal fibers-like aggregates that also disrupted the endogenous intermediate filament networks. The aggregate-bearing astrocytes lost their normal morphology and glutamate buffering capacity, which had a toxic effect on neighboring neurons. In conclusion, our findings provide evidence that links elevated GFAP isoform expression with GFAP aggregation and impaired glutamate transport, and suggest a potential non-cell-autonomous mechanism underlying neurodegeneration through astrocyte dysfunction.

Recessively-Inherited Adult-Onset Alexander Disease Caused by a Homozygous Mutation in the GFAP Gene.

BACKGROUND: Alexander disease (AxD) is an autosomal-dominant leukodystrophy caused by heterozygous mutations in the glial fibrillary acidic protein (GFAP) gene. OBJECTIVES: The objective of this report is to characterize the clinical phenotype and identify the genetic mutation associated with adult-onset AxD. METHODS: A man presented with progressive unsteadiness since age 16. Magnetic resonance imaging findings revealed characteristic features of AxD. The GFAP gene was screened, and a candidate variant was functionally tested to evaluate causality. RESULTS: A homozygous c.197G > A (p.Arg66Gln) mutation was found in the proband, and his asymptomatic parents were heterozygous for the same mutation. This mutation affected GFAP solubility and promoted filament aggregation. The presence of the wild-type protein rescued mutational effects, consistent with the recessive nature of this mutation. CONCLUSIONS: This study is the first report of AxD caused by a homozygous mutation in GFAP. The clinical implication is while examining patients with characteristic features on suspicion of AxD, GFAP screening is recommended even without a supportive family history. © 2020 International Parkinson and Movement Disorder Society.

Effects of Alexander disease-associated mutations on the assembly and organization of GFAP intermediate filaments.

Alexander disease is a primary genetic disorder of astrocytes caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP). How single-amino-acid changes can lead to cytoskeletal catastrophe and brain degeneration remains poorly understood. In this study, we have analyzed 14 missense mutations located in the GFAP rod domain to investigate how these mutations affect in vitro filament assembly. Whereas the internal rod mutants assembled into filaments that were shorter than those of wild type, the rod end mutants formed structures with one or more of several atypical characteristics, including short filament length, irregular width, roughness of filament surface, and filament aggregation. When transduced into primary astrocytes, GFAP mutants with in vitro assembly defects usually formed cytoplasmic aggregates, which were more resistant to biochemical extraction. The resistance of GFAP to solubilization was also observed in brain tissues of patients with Alexander disease, in which a significant proportion of insoluble GFAP were accumulated in Rosenthal fiber fractions. These findings provide clinically relevant evidence that link GFAP assembly defects to disease pathology at the tissue level and suggest that altered filament assembly and properties as a result of GFAP mutation are critical initiating factors for the pathogenesis of Alexander disease.

A novel in-frame GFAP p.E138_L148del mutation in Type II Alexander disease with atypical phenotypes.

Alexander disease (AxD) is a neurodegenerative astrogliopathy caused by mutation in the glial fibrillary acidic protein (GFAP) gene. A 42-year-old Korean man presented with temporary gait disturbance and psychiatric regression after a minor head trauma in the absence of bulbar symptoms and signs. Magnetic resonance images of the brain and spinal cord showed significant atrophy of the medulla oblongata and the entire spinal cord as well as contrast-enhanced T2 hypointensity in the basal ganglia. DNA sequencing revealed a novel 33-bp in-frame deletion mutation (p.Glu138_Leu148del) within the 1B rod domain of GFAP, which was predicted to be deleterious by PROVEAN analysis. To test whether the deletion mutant is disease-causing, we performed in vitro GFAP assembly and sedimentation assays, and GFAP aggregation assays in human adrenal carcinoma SW13 (Vim-) cells and rat primary astrocytes. All the assays revealed that GFAP p.Glu138_Leu148del is aggregation prone. Based on these findings, we diagnosed the patient with Type II AxD. This is a report that demonstrates the pathogenicity of InDel mutation of GFAP through functional studies. This patient's atypical presentation as well as the discrepancy between clinical symptoms and radiologic findings may extend the scope of AxD.