Department of Biology, Haverford College, Haverford, Pennsylvania, USA.
Translational Neuropathology Research Laboratory, Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, 613A Stellar Chance Laboratories, Philadelphia, Pennsylvania, USA.
J Neuropathol Exp Neurol. 2023 Oct 20;82(11):901-910. doi: 10.1093/jnen/nlad078.
The hexanucleotide G4C2 repeat expansion in C9orf72 is the most frequent genetic cause of familial amyotrophic lateral sclerosis (ALS). Aberrant translation of this hexanucleotide sequence leads to production of 5 dipeptide repeats (DPRs). One of these DPRs is proline-arginine (polyPR), which is found in C9orf72-expanded ALS (C9ALS) patient brain tissue and is neurotoxic across multiple model systems. PolyPR was previously reported to bind and impair proteasomes in vitro. Nevertheless, the clinical relevance of the polyPR-proteasome interaction and its functional consequences in vivo are yet to be established. Here, we aim to confirm and functionally characterize polyPR-induced impairment of proteolysis in C9ALS patient tissue and an in vivo model system. Confocal microscopy and immunofluorescence studies on both human and Drosophila melanogaster brain tissues revealed sequestration of proteasomes by polyPR into inclusion-like bodies. Co-immunoprecipitation in D. melanogaster showed that polyPR strongly binds to the proteasome. In vivo, functional evidence for proteasome impairment is further shown by the accumulation of ubiquitinated proteins along with lysosomal accumulation and hyper-acidification, which can be rescued by a small-molecule proteasomal enhancer. Together, we provide the first clinical report of polyPR-proteasome interactions and offer in vivo evidence proposing polyPR-induced proteolytic dysfunction as a pathogenic mechanism in C9ALS.
C9 orf72 中的六核苷酸 G4C2 重复扩展是家族性肌萎缩侧索硬化症（ALS）最常见的遗传原因。该六核苷酸序列的异常翻译导致产生 5 个二肽重复（DPRs）。这些 DPR 之一是脯氨酸-精氨酸（polyPR），存在于 C9orf72 扩展的 ALS（C9ALS）患者脑组织中，并在多种模型系统中具有神经毒性。先前有报道称 polyPR 在体外结合并损害蛋白酶体。然而，polyPR-蛋白酶体相互作用的临床相关性及其在体内的功能后果尚待确定。在这里，我们旨在确认并在 C9ALS 患者组织和体内模型系统中对 polyPR 诱导的蛋白酶体降解损伤进行功能表征。对人类和黑腹果蝇脑组织的共聚焦显微镜和免疫荧光研究显示，polyPR 将蛋白酶体隔离到包涵体样结构中。在黑腹果蝇中的共免疫沉淀表明，polyPR 与蛋白酶体强烈结合。在体内，通过积累泛素化蛋白以及溶酶体积累和过度酸化来进一步证明蛋白酶体功能受损，这可以通过小分子蛋白酶体增强剂来挽救。综上所述，我们提供了 polyPR-蛋白酶体相互作用的首个临床报告，并提供了体内证据，提出 polyPR 诱导的蛋白水解功能障碍是 C9ALS 的一种致病机制。
