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.

受资助家长奖项目摘要 亨廷顿病(HD)是一种进展缓慢的遗传性疾病，由谷氨酰胺重复序列的扩张引起 在亨廷顿蛋白(WtHTT)中，导致突变的HTT(MHTT)，它在大脑中广泛表达 和周围组织。尽管这种表达无处不在，但mHTT通过促进 纹状体中棘神经元(MSN)变性和皮质质量丢失。随着年龄的增长，其影响 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 治疗过程尚不清楚；因此，针对这种毁灭性疾病的基于病因学的治疗仍然难以捉摸。为了填满这个 知识鸿沟，我们将检验RAS同源基因在纹状体(RHES)富含和小的假说 泛素样修饰物(SUMO)-1信号通路协调纹状体易损性和HD进展。 这一假说是基于我们先前的发现，即RHES促进mHTT的相扑-1修饰(SUMO1- MHTT)，并增强可溶性形式SUMO1-mHTT，导致对细胞和转基因动物模型的毒性 HD(6-14)。然而，HD中RHES-SUMO1-mHTT通路的下游机制仍不清楚。 偶然的是，我们发现RHES促进了含有肌动蛋白的膜突起的形成 被称为隧道纳米管(TNTs)，并且RHe通过TNTs被传输到远距离的细胞(15)。RHES也是 通过培养细胞之间形成的TNTs运输mHTT，而不是wtHTT。这种细胞间的运输 需要翻译后修饰(PTM)，如RHES的法尼化和mHTT的SUMO化， 揭示了RHES-SUMO1途径在mHTT传播中的新作用(15)。我们现在演示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- On“)和双顺反子病毒载体，以及器官型脑片，发现RHES从D1R-MSN移动到 D2R-MSNS，并增强mHTT从纹状体扩散到大脑皮质(16)。这些结果表明 无论在体外还是在体内，RHES都是mHTT转运的主要驱动力。我们还发现SUMO1的缺失 通过上调自噬活性来降低mHTT蛋白水平并防止HD样表型 动物模型(Q175DN)和细胞HD模型(17)。综上所述，这些新的结果表明，RHE- SUMO1通路改变mHTT水平，促进mHTT在脑内扩散。然而，这些机制 MHTT的传播仍不清楚。因此，揭示使RHE传播mHTT的机制 而弥散在体内的神经病理作用仍然是需要解决的重要领域。我们的初步数据显示 在Q175DN小鼠中，SUMO1调节纹状体mTORC1信号，这是自噬的主要调节因子。定义 因此，SUMO1是否或如何促进mHTT在体内的传播及其在自噬失调中的作用 对于模拟疾病进展和药物发现都是至关重要的。我们在这个项目中的具体目标是： 目的1.揭示RHES介导的mHTT在脑内扩散的作用和机制。我们 研究发现，在活体内，RHES在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样通路的神经病理学。我们将采用双顺反管和 Cre-on PTM缺陷mHTT和RHES表达载体以研究mHTT传播、类HD行为， 以及活体内的神经病理学。我们将使用MSN和神经胶质报告小鼠来确定RHES是否可以运输mHTT 从MSN到皮质，从MSN到大脑中的胶质细胞。我们将使用活细胞成像和器官分型 脑切片以确定mHTT的RHES运输是否涉及体外TNT样突起。这些 结果将揭示RHES介导的mHTT在大脑中传播的新机制和作用。 目的2.探讨SUMO1介导的HD发病机制。我们发现SUMO1 耗竭上调自噬，降低mHTT水平，并防止Q175DN小鼠的HD样缺陷。我们 结果表明，SUMO1和mHTT增强了纹状体mTORC1信号，这是一种已知的自噬抑制因子。因此，我们 假设SUMO1-mHTT抑制自噬，从而允许mHTT从 从纹状体到皮质。我们将首先确定SUMO1在培养细胞和HD自噬通量中的作用 使用自噬记者的动物。然后我们将使用CRE-on mHTT记者和WT；Rgs9Cre和SUMO1- KO；Rgs9Cre小鼠和药理学mTORC1抑制证实SUMO1和自噬的作用 MHTT中的信号从MSN传递到大脑皮层。最后，使用质谱学方法，我们将识别 依赖于SUMO1的mHTT结合伙伴进一步解开纹状体中的相扑/自噬调节因子。 总而言之，这项研究将描绘RHES-SUMO1通路在mHTT传播中的机制。它 将确定自噬失调和mHTT传播之间的分子联系，以实现HD的潜在靶向 心理治疗。