Huntington’s disease is a neurodegenerative disorder caused by an expanded CAG repeat mutation in the Huntingtin (HTT) gene. The mutation impacts neuronal protein homeostasis and cortical/striatal circuitry. SUMOylation is a post-translational modification with broad cellular effects including via modification of synaptic proteins. Here, we used an optimized SUMO protein-enrichment and mass spectrometry method to identify the protein SUMOylation/SUMO interaction proteome in the context of Huntington’s disease using R6/2 transgenic and non-transgenic mice. Significant changes in the enrichment of SUMOylated and SUMO-interacting proteins were observed, including those involved in presynaptic function, cytomatrix at the active zone, cytoskeleton organization and glutamatergic signalling. Mitochondrial and RNA-binding proteins also showed altered enrichment. Modified SUMO-associated pathways in Huntington’s disease tissue include clathrin-mediated endocytosis signalling, synaptogenesis signalling, synaptic long-term potentiation and SNARE signalling. To evaluate how modulation of SUMOylation might influence functional measures of neuronal activity in Huntington’s disease cells in vitro, we used primary neuronal cultures from R6/2 and non-transgenic mice. A receptor internalization assay for the metabotropic glutamate receptor 7 (mGLUR7), a SUMO-enriched protein in the mass spectrometry, showed decreased internalization in R6/2 neurons compared to non-transgenic neurons. SiRNA-mediated knockdown of the E3 SUMO ligase protein inhibitor of activated STAT1 (Pias1), which can SUMO modify mGLUR7, reduced this Huntington’s disease phenotype. In addition, microelectrode array analysis of primary neuronal cultures indicated early hyperactivity in Huntington’s disease cells, while later time points demonstrated deficits in several measurements of neuronal activity within cortical neurons. Huntington’s disease phenotypes were rescued at selected time points following knockdown of Pias1. Collectively, our results provide a mouse brain SUMOome resource and show that significant alterations occur within the post-translational landscape of SUMO-protein interactions of synaptic proteins in Huntington’s disease mice, suggesting that targeting of synaptic SUMO networks may provide a proteostatic systems-based therapeutic approach for Huntington’s disease and other neurological disorders.

中文翻译：

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亨廷顿病 R6/2 小鼠纹状体中蛋白质 SUMOylation 网络失调


亨廷顿病是一种由亨廷顿 （HTT） 基因中 CAG 重复序列扩增突变引起的神经退行性疾病。该突变会影响神经元蛋白稳态和皮层/纹状体回路。SUMOylation 是一种翻译后修饰，具有广泛的细胞效应，包括通过突触蛋白的修饰。在这里，我们使用优化的 SUMO 蛋白富集和质谱方法，使用 R6/2 转基因和非转基因小鼠在亨廷顿病的背景下鉴定蛋白质 SUMOylation/SUMO 相互作用蛋白质组。观察到 SUMO 化和 SUMO 相互作用蛋白富集的显着变化，包括参与突触前功能、活性区细胞基质、细胞骨架组织和谷氨酸能信号传导的蛋白。线粒体和 RNA 结合蛋白也显示出富集改变。亨廷顿病组织中修饰的 SUMO 相关通路包括网格蛋白介导的内吞信号传导、突触发位信号传导、突触长期增强和 SNARE 信号传导。为了评估 SUMOylation 的调节如何影响体外亨廷顿病细胞中神经元活动的功能测量，我们使用了来自 R6/2 和非转基因小鼠的原代神经元培养物。代谢型谷氨酸受体 7 （mGLUR7） 的受体内化测定显示，与非转基因神经元相比，R6/2 神经元的内化降低。SiRNA 介导的激活 STAT1 （Pias1） 的 E3 SUMO 连接酶蛋白抑制剂的敲低，它可以 SUMO 修饰 mGLUR7，从而降低了这种亨廷顿病表型。 此外，原代神经元培养物的微电极阵列分析表明亨廷顿病细胞的早期过度活跃，而后来的时间点表明皮层神经元内神经元活动的几次测量存在缺陷。亨廷顿病表型在敲低 Pias1 后的选定时间点被挽救。总的来说，我们的结果提供了小鼠大脑 SUMOome 资源，并表明亨廷顿病小鼠突触蛋白的 SUMO-蛋白相互作用的翻译后景观发生了显着变化，这表明靶向突触 SUMO 网络可能为亨廷顿病和其他神经系统疾病提供基于蛋白恒流系统的治疗方法。