Viedma-Poyatos Álvaro, González-Jiménez Patricia, Pajares María A, Pérez-Sala Dolores
Department of Structural and Chemical Biology, Centro de Investigaciones Biológicas Margarita Salas, C.S.I.C., 28040, Madrid, Spain.
Department of Structural and Chemical Biology, Centro de Investigaciones Biológicas Margarita Salas, C.S.I.C., 28040, Madrid, Spain.
Redox Biol. 2022 Sep;55:102415. doi: 10.1016/j.redox.2022.102415. Epub 2022 Jul 30.
Alexander disease is a fatal neurological disorder caused by mutations in the intermediate filament protein Glial Fibrillary Acidic Protein (GFAP), which is key for astrocyte homeostasis. These mutations cause GFAP aggregation, astrocyte dysfunction and neurodegeneration. Remarkably, most of the known GFAP mutations imply a change by more nucleophilic amino acids, mainly cysteine or histidine, which are more susceptible to oxidation and lipoxidation. Therefore, we hypothesized that a higher susceptibility of Alexander disease GFAP mutants to oxidative or electrophilic damage, which frequently occurs during neurodegeneration, could contribute to disease pathogenesis. To address this point, we have expressed GFP-GFAP wild type or the harmful Alexander disease GFP-GFAP R239C mutant in astrocytic cells. Interestingly, GFAP R239C appears more oxidized than the wild type under control conditions, as indicated both by its lower cysteine residue accessibility and increased presence of disulfide-bonded oligomers. Moreover, GFP-GFAP R239C undergoes lipoxidation to a higher extent than GFAP wild type upon treatment with the electrophilic mediator 15-deoxy-Δ-prostaglandin J (15d-PGJ). Importantly, GFAP R239C filament organization is altered in untreated cells and is earlier and more severely disrupted than GFAP wild type upon exposure to oxidants (diamide, HO) or electrophiles (4-hydroxynonenal, 15d-PGJ), which exacerbate GFAP R239C aggregation. Furthermore, HO causes reversible alterations in GFAP wild type, but irreversible damage in GFAP R239C expressing cells. Finally, we show that GFAP R239C expression induces a more oxidized cellular status, with decreased free thiol content and increased mitochondrial superoxide generation. In addition, mitochondria show decreased mass, increased colocalization with GFAP and altered morphology. Notably, a GFP-GFAP R239H mutant recapitulates R239C-elicited alterations whereas an R239G mutant induces a milder phenotype. Together, our results outline a deleterious cycle involving altered GFAP R239C organization, mitochondrial dysfunction, oxidative stress, and further GFAP R239C protein damage and network disruption, which could contribute to astrocyte derangement in Alexander disease.
亚历山大病是一种致命的神经疾病，由中间丝蛋白胶质纤维酸性蛋白（GFAP）的突变引起，GFAP对星形胶质细胞的稳态至关重要。这些突变导致GFAP聚集、星形胶质细胞功能障碍和神经退行性变。值得注意的是，大多数已知的GFAP突变意味着被更多亲核氨基酸取代，主要是半胱氨酸或组氨酸，它们更容易发生氧化和脂氧化。因此，我们推测亚历山大病GFAP突变体对氧化或亲电损伤的更高敏感性（这在神经退行性变过程中经常发生）可能导致疾病的发病机制。为了解决这一问题，我们在星形胶质细胞中表达了绿色荧光蛋白（GFP）标记的野生型GFAP或有害的亚历山大病GFP-GFAP R239C突变体。有趣的是，在对照条件下，GFAP R239C的氧化程度似乎比野生型更高，这既表现为其较低的半胱氨酸残基可及性，也表现为二硫键连接的寡聚体的存在增加。此外，在用亲电介质15-脱氧-Δ-前列腺素J（15d-PGJ）处理后，GFP-GFAP R239C的脂氧化程度比GFAP野生型更高。重要的是，在未处理的细胞中，GFAP R239C的丝状结构发生改变，并且在暴露于氧化剂（二酰胺、过氧化氢）或亲电试剂（4-羟基壬烯醛、15d-PGJ）时，比GFAP野生型更早、更严重地受到破坏，这加剧了GFAP R239C的聚集。此外，过氧化氢对GFAP野生型引起可逆性改变，但对表达GFAP R239C的细胞造成不可逆损伤。最后，我们表明GFAP R239C的表达诱导了更高的氧化细胞状态，游离巯基含量降低，线粒体超氧化物生成增加。此外，线粒体质量减少，与GFAP的共定位增加，形态改变。值得注意的是，GFP-GFAP R239H突变体重现了R239C引起的改变，而R239G突变体诱导的表型较轻。总之，我们的结果勾勒出一个有害的循环，涉及GFAP R239C结构改变、线粒体功能障碍、氧化应激，以及进一步的GFAP R239C蛋白损伤和网络破坏，这可能导致亚历山大病中星形胶质细胞紊乱。
