Rhes-SUMO Pathway in Huntington disease

亨廷顿病中的 Rhes-SUMO 通路

基本信息

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
项目类别：
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

项目摘要

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.

资助家长奖项目概要亨廷顿病 (HD) 是一种缓慢进展的遗传性疾病，由谷氨酰胺重复序列扩展引起存在于亨廷顿蛋白 (wtHTT) 中，导致突变 HTT (mHTT) 在整个大脑中广泛表达尽管这种表达无处不在，mHTT 通过促进区域效应。随着年龄的增长，纹状体中多棘神经元（MSN）的退化和皮质质量的损失。扩散到其他大脑区域 (1-5) 的区域特异性的分子基础，涵盖许多 mHTT。其过程尚不清楚；因此，针对这种破坏性疾病的基于病因的治疗方法仍然难以实现。知识差距，我们将检验 Ras 同源物在纹状体 (Rhes) 中富集且较小的假设泛素样修饰剂 (SUMO)-1 信号电路协调纹状体脆弱性和 HD 进展。这一假设基于我们之前的发现，即 Rhes 促进 mHTT 的 SUMO-1 修饰（SUMO1- mHTT）并增强可溶形式 SUMO1–mHTT，导致细胞和转基因动物模型中的毒性 HD (6-14) 然而，HD 中 Rhes-SUMO1-mHTT 通路的下游机制仍不清楚。偶然地，我们发现Rhes促进了含肌动蛋白的膜突起的形成称为隧道纳米管 (TNT)，Rhes 也通过 TNT 运输到远处的细胞 (15)。通过培养细胞之间形成的 TNT 转运 mHTT，但不转运 wtHTT。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-SUMO1 通路在 mHTT 传输中的新作用 (15)。我们使用细胞类型特异性报告小鼠 Flex（“Cre-”）在 MSN 之间移动并在体内传播 mHTT。 On”）和双顺反子病毒载体，以及器官型脑切片，发现Rhes从D1R-MSNs转移到 D2R-MSN 并增强 mHTT 从纹状体到大脑皮层的传播 (16)。在体外和体内，Rhes 都是 mHTT 转运的主要驱动因素。我们还发现 SUMO1 缺失。通过上调自噬活性降低 mHTT 蛋白水平并预防 HD 样表型总而言之，这些新结果表明Rhes-动物（Q175DN）和细胞HD模型（17）。 SUMO1 通路改变 mHTT 水平并促进 mHTT 在大脑中的传播，但其机制尚不清楚。 mHTT 传播的机制仍然未知，因此，揭示 Rhes 传播 mHTT 的机制。我们的初步数据表明，传播的体内神经病理学作用仍然是需要解决的重要领域。 SUMO1 调节纹状体 mTORC1 信号传导，这是 Q175DN 小鼠中自噬的主要调节因子。 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疾病进展建模和药物发现都至关重要，我们在该项目中的具体目标是：目的 1. 揭示 Rhes 介导的 mHTT 在大脑中传播的作用和机制。发现 Rhes 在体内神经元之间移动和传播 mHTT 我们捕获了 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 mHTT 和 Rhes 表达载体，用于研究 mHTT 传播、HD 样行为、我们将使用 MSN 和神经胶质报告小鼠来确定 Rhes 是否可以转运 mHTT。从 MSN 到皮质，从 MSN 到大脑中的神经胶质细胞，我们将使用活细胞成像和器官型。脑切片以确定 mHTT 的 Rhes 运输是否涉及离体 TNT 样突起。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. 确定 SUMO1 介导的 HD 发病机制。耗竭可上调 Q175DN 小鼠的自噬、降低 mHTT 水平并预防 HD 样缺陷。结果表明 SUMO1 和 mHTT 增强纹状体 mTORC1 信号传导，这是一种已知的自噬抑制剂。 SUMO1–mHTT 抑制自噬，允许 mHTT 积累并由此扩散我们将首先描述 SUMO1 在培养细胞和 HD 中自噬流中的作用。然后我们将使用 Cre-On mHTT 生产者和 WT;Rgs9Cre 和 SUMO1- 的动物。 KO;Rgs9Cre 小鼠和药理学 mTORC1 抑制证实了 SUMO1 和自噬的作用最后，我们将使用质谱方法来识别 mHTT 中的信号从 MSN 传播到皮层的情况。 SUMO1 依赖性 mHTT 结合伙伴进一步揭示纹状体中的 SUMO/自噬调节因子。总的来说，这项研究将描述 Rhes-SUMO1 通路在 mHTT 传播中的机制。将确定自噬失调和 mHTT 传播之间的分子联系，以用于 HD 的潜在靶向治疗。