Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan.
Department of Medical Science, College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.
FASEB J. 2021 May;35(5):e21614. doi: 10.1096/fj.202100087R.
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.
亚历山大病（AxD）是由 GFAP 编码区突变引起的神经退行性疾病，其特征是星形胶质细胞功能障碍、GFAP 聚集和 Rosenthal 纤维堆积。虽然 GFAP 突变如何导致疾病尚不完全清楚，但强制过表达人 GFAP 可诱导 Rosenthal 纤维形成，这在小鼠中可能是致命的，这表明 GFAP 水平的增加是 AxD 发病机制的核心。我们最近的研究表明，内含子 GFAP 突变通过改变 GFAP 剪接导致疾病，这表明 GFAP 同工型表达的增加可能导致典型的 AxD 蛋白聚集和星形胶质细胞功能障碍。在这里，我们通过建立过表达人 GFAP 的转基因小鼠的原代星形胶质细胞培养来检验这一假说。我们发现，GFAP-δ 和 GFAP-κ 在转基因星形胶质细胞中不成比例地增加，并且都在 AxD 人脑的 Rosenthal 纤维中富集。体外组装研究表明，虽然主要同工型 GFAP-α 自我组装成典型的 10nm 纤维，但包括 GFAP-δ、-κ 和 -λ 在内的次要同工型组装受到损害且易于聚集。慢病毒转导表明，这些次要 GFAP 同工型的表达降低了纤维的溶解性并增加了 GFAP 的稳定性，导致形成类似于 Rosenthal 纤维的聚集体，也破坏了内源性中间丝网络。携带聚集体的星形胶质细胞失去了正常的形态和谷氨酸缓冲能力，这对邻近的神经元产生了毒性作用。总之，我们的研究结果提供了证据，将升高的 GFAP 同工型表达与 GFAP 聚集和谷氨酸转运受损联系起来，并表明星形胶质细胞功能障碍导致神经退行性变的潜在非细胞自主机制。
