Rhes-SUMO Pathway in Huntington disease

亨廷顿病中的 Rhes-SUMO 通路

• 批准号: 10785540
• 负责人: Srinivasa Subramaniam
• 金额: $ 6.92万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2023-11-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10785540
• 关键词: Actins; Address; Aging; Animal Diseases; Animal Model; Animals; Area; Autophagocytosis; Award; Behavior; Binding; Brain; Cell Communication; Cell model; Cells; Corpus striatum structure; Cultured Cells; DRD2 gene; Data; Disease; Disease Progression; Disease model; Distant; Etiology; Funding; Genetic Diseases; Glutamine; Homologous Gene; Human; Huntington Disease; Huntington gene; In Vitro; Knowledge; Link; Mass Spectrum Analysis; Mediating; Membrane; Mission; Modification; Molecular; Mus; Nanotubes; Neuroglia; Neurons; Pain; Parents; Pathogenesis; Pathway interactions; Peripheral; Phenotype; Post-Translational Protein Processing; Process; Proteins; Public Health; Reporter; Research; Role; Route; SUMO1 gene; Signal Transduction; Slice; Specificity; Sumoylation Pathway; Testing; Tissues; Toxic effect; Transgenic Animals; Transportation; Ubiquitin; United States National Institutes of Health; Viral Vector; Work; cell type; disability; drug discovery; expression vector; farnesylation; in vivo; inhibition of autophagy; inhibitor; insight; live cell imaging; mutant; nervous system disorder; neuropathology; novel; novel therapeutic intervention; pharmacologic; prevent; transmission process

Project summary of the funded parent award Huntington disease (HD) is a slowly progressing genetic disorder caused by an expansion of glutamine repeats in the huntingtin protein (wtHTT), leading to mutant HTT (mHTT) that is widely expressed throughout the brain and peripheral tissues. Despite this ubiquitous expression, mHTT shows regional effects by promoting degeneration of medium spiny neurons (MSNs) in the striatum and loss of cortical mass. With aging, the effects spread to other brain areas (1-5). The molecular basis for the regional specificity that encompasses many mHTT processes is unclear; thus, etiology-based therapies for this devastating disease remain elusive. To fill this knowledge gap, we will test the hypothesis that Ras-homolog enriched in the striatum (Rhes) and small ubiquitin-like modifier (SUMO)-1 signaling circuitry orchestrate striatal vulnerability and HD progression. This hypothesis is based on our prior finding that Rhes promotes SUMO-1 modification of mHTT (SUMO1– mHTT) and enhances soluble forms SUMO1–mHTT, leading to toxicity in cell and transgenic animal models of HD (6-14). However, the downstream mechanisms of the Rhes–SUMO1–mHTT pathway in HD remain obscure. Serendipitously, we found that Rhes promotes the formation of actin-containing membrane protrusions known as tunneling nanotubes (TNTs) and that Rhes is transported through TNTs to distant cells (15). Rhes also transports mHTT, but not wtHTT, via the TNTs that form between cultured cells. This intercellular transport requires post-translational modifications (PTMs), such as the farnesylation of Rhes and SUMOylation of mHTT, revealing a new role for the Rhes–SUMO1 pathway in mHTT transmission (15). We now demonstrate that Rhes can move between MSNs and spread mHTT in vivo. We tested using cell-type specific reporter mice, Flex (“Cre- On”) and bicistronic viral vectors, and organotypic brain slices and found that Rhes moves from D1R-MSNs to D2R-MSNs and potentiates mHTT spread from the striatum to the cortex in the brain (16). These results indicate that Rhes is a major driver of mHTT transport, both in vitro and in vivo. We also found that SUMO1 deletion diminishes mHTT protein levels and prevents the HD-like phenotype by upregulating autophagic activity in animal (Q175DN) and cellular HD models (17). Taken together, these new results indicate that the Rhes- SUMO1 pathway alters mHTT levels and promotes mHTT spread in the brain. However, the mechanisms of mHTT spread remain unknown. Therefore, uncovering the mechanisms that enable Rhes to spread mHTT and the in vivo neuropathological role of spread remain essential areas to address. Our preliminary data suggest that SUMO1 regulates striatal mTORC1 signaling, a major regulator of autophagy, in Q175DN mice. Defining whether or how SUMO1 contributes to mHTT spread in vivo and its role autophagy dysregulation is therefore critical both for modeling the disease progression and for drug discovery. Our specific aims in this project are: Aim 1. To uncover the role and mechanisms of Rhes-mediated mHTT spreading in the brain. We found that Rhes moves and spreads mHTT between neurons in vivo. We hypothesize that Rhes spreads mHTT and promotes neuropathology involving PTM mechanisms and TNT-like routes. We will employ bicistronic and Cre-On PTM defective mHTT and Rhes expression vectors to investigate mHTT spreading, HD-like behavior, and neuropathology in vivo. We will use MSNs and glial reporter mice to determine if Rhes can transport mHTT from MSNs to the cortex and from MSNs to the glia in the brain. We will use live-cell imaging and organotypic brain slices to establish whether Rhes transportation of mHTT involves TNT-like protrusions ex vivo. These results will uncover novel mechanisms and the role of Rhes-mediated mHTT spread in the brain. Aim 2. To identify the mechanisms of SUMO1-mediated HD pathogenesis. We found that SUMO1 depletion upregulates autophagy, decreases mHTT levels, and prevents HD-like deficits in Q175DN mice. We showed that SUMO1 and mHTT enhance striatal mTORC1 signaling, a known inhibitor of autophagy. Thus, we hypothesize that SUMO1–mHTT inhibits autophagy, thereby allowing accumulation and spread of mHTT from the striatum to the cortex. We will first characterize the SUMO1 role in autophagy flux in cultured cells and HD animals using autophagy reporters. We will then use Cre-On mHTT reporters and WT;Rgs9Cre and SUMO1- KO;Rgs9Cre mice and pharmacological mTORC1 inhibition to corroborate a role for SUMO1 and autophagy signaling in mHTT spread from MSNs to the cortex. Finally, using a mass spectrometry approach, we will identify SUMO1-dependent mHTT binding partners to further unravel SUMO/autophagy regulators in the striatum. Collectively, this study will delineate the mechanisms of the Rhes–SUMO1 pathway in mHTT spread. It will identify the molecular link between autophagy dysregulation and mHTT spread for potential targeting in HD therapy.

资助家长奖项目摘要 亨廷顿病（HD）是一种由谷氨酰胺重复序列扩增引起的缓慢进展的遗传性疾病 亨廷顿蛋白（wtHTT），导致突变HTT（mHTT），广泛表达于整个大脑 和外周组织。尽管这种普遍存在的表达，mHTT显示区域效应，促进 纹状体中的中型多刺神经元（MSN）变性和皮质质量损失。随着年龄的增长， 扩散到其他脑区（1-5）。包括许多mHTT的区域特异性的分子基础 过程尚不清楚;因此，这种毁灭性疾病的基于病因的疗法仍然难以捉摸。填补这一 知识的差距，我们将测试的假设，Ras同源物丰富的纹状体（Rhes）和小 泛素样修饰物（SUMO）-1信号通路协调纹状体脆弱性和HD进展。 这一假设基于我们先前的发现，Rhes促进mHTT的SUMO-1修饰（SUMO 1 - 1修饰）。 mHTT）并增强可溶性形式SUMO 1-mHTT，导致在细胞和转基因动物模型中的毒性。 HD（6-14）。然而，在HD中Rhes-SUMO 1-mHTT通路的下游机制仍然不清楚。 偶然地，我们发现Rhes促进含肌动蛋白的膜突起的形成 已知为隧道纳米管（TNT），并且Rhes通过TNT运输到远处的细胞（15）。Rhes也 通过培养细胞之间形成的TNT转运mHTT，但不转运wtHTT。这种细胞间运输 需要翻译后修饰（PTM），例如Rhes的法尼基化和mHTT的SUMO化， 揭示了Rhes-SUMO 1通路在mHTT传递中的新作用（15）。我们现在证明Rhes 可以在MSN之间移动并在体内传播mHTT。我们使用细胞类型特异性报告小鼠Flex（“Cre-1”）进行了测试。 和双顺反子病毒载体，以及器官型脑切片，发现Rhes从D1 R-MSN移动到 D2 R-MSN和增强mHTT从纹状体扩散到大脑皮层（16）。这些结果表明 Rhes是体外和体内mHTT转运的主要驱动因素。我们还发现SUMO 1缺失 降低mHTT蛋白水平，并通过上调自噬活性来预防HD样表型。 动物（Q175 DN）和细胞HD模型（17）。总之，这些新的结果表明，Rhes- SUMO 1通路改变mHTT水平并促进mHTT在大脑中的扩散。然而，机制 mHTT的传播仍然未知。因此，揭示Rhes传播mHTT的机制 以及扩散的体内神经病理学作用仍然是需要解决的重要领域。我们的初步数据显示 SUMO 1调节Q175 DN小鼠纹状体mTORC 1信号传导，这是自噬的主要调节因子。限定 因此，SUMO 1是否或如何促进mHTT在体内的传播以及其自噬失调的作用是 对于疾病进展的建模和药物发现都至关重要。我们在这个项目中的具体目标是： 目标1.目的：探讨Rh介导的mHTT在脑内的作用及机制。我们 发现Rhes在体内神经元之间移动和传播mHTT。我们假设Rhes传播mHTT 并促进涉及PTM机制和TNT样途径的神经病理学。我们将采用双顺反子和 Cre-On PTM缺陷型mHTT和Rhes表达载体用于研究mHTT扩散、HD样行为， 和体内神经病理学。我们将使用MSNs和神经胶质报告小鼠来确定Rhes是否可以转运mHTT 从MSNs到大脑皮层，从MSNs到大脑中的神经胶质。我们将使用活细胞成像和器官型 脑切片以确定mHTT的Rhes转运是否涉及离体TNT样突起。这些 结果将揭示新的机制和作用的恒河猴介导的mHTT传播在大脑中。 目标二。明确SUMO 1介导的HD发病机制。我们发现相扑1 在Q175 DN小鼠中，消耗上调自噬，降低mHTT水平，并防止HD样缺陷。我们 研究表明，SUMO 1和mHTT增强纹状体mTORC 1信号传导，这是一种已知的自噬抑制剂。因此我们 假设SUMO 1-mHTT抑制自噬，从而允许mHTT从 纹状体到皮层我们将首先描述SUMO 1在培养细胞和HD中自噬通量中的作用。 动物使用自噬报告。然后，我们将使用Cre-On mHTT报告子和WT; Rgs 9 Cre和SUMO 1 - 1。 KO; Rgs 9 Cre小鼠和药理学mTORC 1抑制，以证实SUMO 1和自噬的作用 mHTT中的信号从MSN传播到皮层。最后，使用质谱法，我们将识别 SUMO 1依赖性mHTT结合伴侣进一步阐明纹状体中的SUMO/自噬调节因子。 总的来说，这项研究将描绘mHTT传播中的Rhes-SUMO 1通路的机制。它 将确定自噬失调和mHTT扩散之间的分子联系，以潜在靶向HD 疗法