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.

中文翻译：

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C9orf72 脯氨酸-精氨酸二肽重复会破坏蛋白酶体并扰乱蛋白水解活性。

C9orf72 中的六核苷酸 G4C2 重复序列扩增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会导致产生 5 个二肽重复序列 (DPR)。其中一种 DPR 是脯氨酸-精氨酸 (polyPR)，它存在于 C9orf72 扩增的 ALS (C9ALS) 患者脑组织中，并且在多个模型系统中具有神经毒性。此前有报道称，PolyPR 可在体外结合并损害蛋白酶体。然而，聚PR-蛋白酶体相互作用的临床相关性及其体内功能后果仍有待确定。在这里，我们的目的是在 C9ALS 患者组织和体内模型系统中确认和功能表征 polyPR 诱导的蛋白水解损伤。对人和果蝇脑组织的共聚焦显微镜和免疫荧光研究揭示了聚PR将蛋白酶体隔离成包涵体样体。黑腹果蝇的免疫共沉淀表明，polyPR 与蛋白酶体强烈结合。在体内，泛素化蛋白的积累以及溶酶体的积累和过度酸化进一步显示了蛋白酶体损伤的功能证据，这可以通过小分子蛋白酶体增强剂来挽救。我们共同提供了聚 PR-蛋白酶体相互作用的第一份临床报告，并提供了体内证据，表明聚 PR 诱导的蛋白水解功能障碍是 C9ALS 的致病机制。