A hexanucleotide repeat expansion in a noncoding region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reduction of select or total C9orf72 transcript and protein levels is observed in postmortem C9-ALS/FTD tissue, and loss of C9orf72 orthologues in zebrafish and C. elegans results in motor deficits. However, how the reduction in C9orf72 in ALS and FTD might contribute to the disease process remains poorly understood. It has been shown that C9orf72 interacts and forms a complex with SMCR8 and WDR41, acting as a guanine exchange factor for Rab GTPases. Given the known synaptosomal compartmentalization of C9orf72-interacting Rab GTPases, we hypothesized that C9orf72 localization to synaptosomes would be required for the regulation of Rab GTPases and receptor trafficking. This study combined synaptosomal and post-synaptic density preparations together with a knockout-confirmed monoclonal antibody for C9orf72 to assess the localization and role of C9orf72 in the synaptosomes of mouse forebrains. Here, we found C9orf72 to be localized to both the pre- and post-synaptic compartment, as confirmed by both post-synaptic immunoprecipitation and immunofluorescence labelling. In C9orf72 knockout (C9-KO) mice, we demonstrated that pre-synaptic Rab3a, Rab5, and Rab11 protein levels remained stable compared with wild-type littermates (C9-WT). Strikingly, post-synaptic preparations from C9-KO mouse forebrains demonstrated a complete loss of Smcr8 protein levels, together with a significant downregulation of Rab39b and a concomitant upregulation of GluR1 compared with C9-WT mice. We confirmed the localization of Rab39b downregulation and GluR1 upregulation to the dorsal hippocampus of C9-KO mice by immunofluorescence. These results indicate that C9orf72 is essential for the regulation of post-synaptic receptor levels, and implicates loss of C9orf72 in contributing to synaptic dysfunction and related excitotoxicity in ALS and FTD.

中文翻译：

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C9orf72的突触定位可调节突触后谷氨酸受体1的水平。

在C9orf72的非编码区中的六核苷酸重复扩增是肌萎缩性侧索硬化症（ALS）和额颞痴呆（FTD）的最常见遗传原因。在死后的C9-ALS / FTD组织中观察到选择性或全部C9orf72转录本和蛋白质水平的降低，斑马鱼和秀丽隐杆线虫中C9orf72直向同源物的丢失导致运动缺陷。但是，人们对ALS和FTD中C9orf72的减少如何导致疾病进程的认识仍知之甚少。已经显示C9orf72与SMCR8和WDR41相互作用并形成复合物，充当Rab GTPases的鸟嘌呤交换因子。考虑到已知的与C9orf72相互作用的Rab GTPases的突触体间隔，我们假设C9orf72定位于突触体是调控Rab GTPases和受体运输所必需的。这项研究结合了突触体和突触后密度制剂与敲除的C9orf72单克隆抗体，以评估C9orf72在小鼠前脑突触体中的定位和作用。在这里，我们发现C9orf72既位于突触前区又位于突触后区，如通过突触后免疫沉淀和免疫荧光标记所证实的。在C9orf72基因敲除（C9-KO）小鼠中，我们证明了与野生型同窝仔相比（C9-WT），突触前的Rab3a，Rab5和Rab11蛋白水平保持稳定。令人惊讶的是，与C9-WT小鼠相比，C9-KO小鼠前脑的突触后制剂表现出Smcr8蛋白水平的完全丧失，Rab39b的显着下调以及GluR1的上调。我们通过免疫荧光证实了Rab39b下调和GluR1上调在C9-KO小鼠背侧海马中的定位。这些结果表明，C9orf72对于调节突触后受体水平必不可少，并且牵涉到C9orf72的丧失，导致ALS和FTD中的突触功能障碍和相关的兴奋性毒性。