Cellular Models of a Monogenic Small Vessel Disease of the Brain

项目摘要 VCID(血管对认知障碍和痴呆的贡献)越来越被认为既是 痴呆症的主要原因，并作为痴呆症的次要因素，如在阿尔茨海默氏症中 病理学。VCID最常见的进展是由于脑部小血管疾病造成的累积性损伤 (Svd)。虽然有几个危险因素与SVD有关，但推动其发生和发展的机制 进展还没有得到很好的描述。视网膜血管病合并脑白质脑病和 系统性表现(S)是一种罕见的单基因迟发性疾病，具有显著的临床和病理特征 病理上与脑部小血管疾病相似。我们的目标是促进新技术的发展 从RVCL-S调查看散发性SVD的防治方法 RVCL-S是由编码主要致病基因TREX1的常染色体显性突变引起的 胞内3‘DNA核酸外切酶。TREX1蛋白含有两个主要功能区：氨基末端 结构域是核酸外切酶，而羧基末端结构域将蛋白质定位在内质网中 (ER)膜，将DNA酶结构域延伸到胞浆中，在那里它在抑制 对胞浆自身DNA的先天免疫反应。RVCL-S是由全穿透性移码突变引起的 TREX1基因，每个基因产生一种携带有功能核酸外切酶结构域的蛋白质，但缺乏 TREX1核酸外切酶在内质网膜上的非典型定位 遍布胞浆和胞核。重要的是，在核酸外切酶内截短TREX1的突变 编码区，导致蛋白质缺乏DNA酶活性和内质网膜系绳，不是致病的 在杂合子状态下，提示RVCL-S是由非拴系者的毒性功能获得引起的 核酸外切酶。我们认为TREX1核酸外切酶在细胞核中的异常活性干扰了DNA 复制/修复以及密切协调的细胞周期调节信号蛋白的活性。我们的长- 学期目标是确定突变核酸外切酶的关键有害影响并制定策略。 以减轻这些影响。为此，我们提出以下具体目标： 1.检测RVCL-S基因在DNA损伤反应和细胞周期调控中的作用。 2.利用RNAi技术抑制RVCL-S相关TREX1移码突变体的表达。 影响：这项研究的成功完成将提供1)疾病预防的战略；2)生物标志物 疾病监测；3)适用于RVCL-S的新治疗方法，很可能 适用于零星的SVD。

Project Summary VCID (Vascular contributions to cognitive impairment and dementia) is increasingly recognized as both a primary cause of dementia and as a secondary contributor to dementia, as in the setting of Alzheimer’s pathology. VCID most commonly progresses from accumulated injury due to small vessel disease of the brain (SVD). While several risk factors are associated with SVD, the mechanisms that drive its initiation and progression have not been well characterized. Retinal Vasculopathy with Cerebral Leukoencephalopathy and Systemic Manifestations (RVCL-S) is a rare monogenic late onset condition that bears striking clinical and pathological similarities to small vessel disease of the brain. Our goal is to facilitate the development of new methods for the prevention and treatment of sporadic SVD through an investigation of RVCL-S. RVCL-S is caused by dominant mutations in the autosomal gene TREX1 which encodes the major intracellular 3’ DNA exonuclease. TREX1 protein harbors two major functional domains: the amino-terminal domain is the exonuclease while the carboxy-terminal domain positions the protein in the endoplasmic reticulum (ER) membrane, extending the DNAse domain into the cytosol where it plays a critical role in dampening the innate immune response to cytosolic self-DNA. RVCL-S is caused by fully penetrant frameshift mutations in TREX1 gene, each producing a protein that carries a functional exonuclease domain but lacks the domain that anchors the complete protein to the ER membrane resulting in atypical localization of TREX1 exonuclease throughout the cytosol and the nucleus. Importantly, mutations that truncate TREX1 within the exonuclease coding region, resulting in proteins lacking both DNase activity and the ER membrane tether, are not pathogenic in the heterozygous state, suggesting that RVCL-S is caused by a toxic gain-of-function of the untethered exonuclease. We propose that aberrant activity of TREX1 exonuclease in the cell nucleus interferes with DNA replication/repair as well as the activity of closely coordinated cell cycle regulatory signaling proteins. Our long- term objectives are to identify the critical detrimental effects of the mutant exonuclease and to develop strategies to abate these effects. To that end we propose the following specific aims: 1. Examine the effects of the RVCL-S genotype on the DNA damage response and cell cycle regulation. 2. Employ RNA interference to suppress expression of the RVCL-S associated TREX1 frameshift mutant. Impact: Successful completion of this study will provide 1) strategies for disease prevention; 2) biomarkers for disease monitoring; and 3) novel therapeutic approaches that will be applicable to RVCL-S and very likely applicable to sporadic SVD.