Selective neurovascular regulation by a vascular dementia-related noncoding RNA Snord118

项目摘要/摘要 神经血管单位功能障碍(NVU)是神经退行性疾病的重要病理事件，包括 阿尔茨海默病和阿尔茨海默病相关痴呆(AD/ADRD)。细胞背后的机制是- NVU中的类型选择性漏洞知之甚少。这项提议的目标是建立一部小说 NVU细胞(S)因全球核糖体生物发生中断而选择性受损的机制。 我们将关注一个新发现的核糖体疾病基因Snord118，它编码一种非编码的RNA 作为核糖体生物发生因子。这很有趣，因为Snord118突变导致了第一个纯粹的 核糖体病中的神经系统疾病，称为伴有钙化和囊变的白质脑病(LCC)， NVU损害。目前对Snord118和LCC的发病机制知之甚少。这项研究有一个 有机会确定Snord118和LCC病的功能和机制。我们已经组装了 初步数据如下：1)产生了两个病点突变敲入(KI)小鼠，它们表现为早期 周细胞和血脑屏障缺陷。这些结果表明，脑内皮细胞(ECs)和周细胞具有选择性 影响LCC，这证明我们的IPSC研究重点是脑内皮细胞和周细胞；2)产生了五个 具有等基因对照的Snord118突变型IPSC系，建立了将IPSCs引导到脑内的方案 具有中枢神经系统特性的微血管内皮细胞和周细胞，并制备功能分析 对于BMEC、周细胞和血脑屏障(BBB)的特性；3)将Paris方法发展到高 吞吐量映射RNA结构并在单分子和全基因组水平上识别RNA靶标 碱基对分辨。我们的巴黎揭示了Snord118中的动态RNA结构和相互作用网络 核糖体生物发生与LCC。利用这些初步数据和工具，我们建议测试 假设Snord118突变介导的核糖体生物发生中断选择性地影响BMECs和 通过靶向rRNA和非rRNA作用于周细胞。目标1将确定Snord118的细胞功能 神经血管细胞，主要集中于骨髓间充质干细胞和周细胞。AIM 2将识别Snord118靶标及其RNA 结构-功能关系。总体而言，使用我们新的IPSC来源的NVU细胞和最新的PARIS2，这项研究 将生成目前还不存在的SNORD118 LCC的第一个人类细胞模型，识别 SNORD118作用和LCC疾病的机制，揭示了以前未知的特定易损性 NVU细胞破坏了无处不在的核糖体生物发生过程，从而有助于调和 具有全球核糖体生物发生要求的核糖体疾病的神经学表型特异性。

PROJECT SUMMARY/ABSTRACT Dysfunction of neurovascular unit (NVU) is a key pathological event of neurodegenerative diseases, including Alzheimer's disease and Alzheimer's disease-related dementias (AD/ADRD). The mechanism underlying cell- type selective vulnerability in NVU is poorly understood. The goal of this proposal is to establish a novel mechanism by which NVU cell(s) are selectively impaired by the disruption of a global ribosome biogenesis. We will focus on a newly identified ribosomopathy disease gene Snord118, which encodes a noncoding RNA acting as a ribosome biogenesis factor. This is interesting because Snord118 mutations cause the first purely neurological disorder in ribosomopathies, named leukoencephalopathy with calcifications and cysts (LCC), with NVU lesions. There is very little understanding of Snord118 and LCC pathogenesis. This study has an opportunity to determine functions and mechanisms of Snord118 and LCC disease. We have assembled the following preliminary data: 1) generated two disease point mutation knock-in (KI) mice, which display early pericyte and BBB defects. These results suggest that brain endothelial cells (ECs) and pericytes are selectively affected in LCC, which justifies our iPSC research focus on brain ECs and pericytes; 2) generated five Snord118 mutant iPSC lines with isogenic controls, established protocols of directing iPSCs into brain microvascular endothelial cells (BMECs) and pericytes with the CNS identities, and prepared functional assays for BMEC, pericyte, and blood-brain barrier (BBB) properties; 3) developed the PARIS method to high throughput map RNA structures and identify RNA targets at single molecule and genome-wide levels with base-pair resolution. Our PARIS revealed a dynamic RNA structure and interaction network in Snord118 ribosome biogenesis and LCC. Leveraging on these preliminary data and tools, we propose to test the hypothesis that Snord118 mutation-mediated disruption of ribosome biogenesis selectively affects BMECs and pericytes via targeting rRNAs and non-rRNAs. Aim 1 will determine cellular functions of Snord118 in neurovascular cells focusing on BMECs and pericytes. Aim 2 will identify Snord118 targets and its RNA structure-function relationships. Overall, using our new iPSC-derived NVU cells and latest PARIS2, this study will generate the first human cellular models that do not currently exist for SNORD118 LCC, identify mechanisms of SNORD118 action and LCC disease, uncover a previously unknown vulnerability of specific NVU cells to the disruption of a ubiquitous ribosome biogenesis process, and therefore help to reconcile the neurological phenotype specificity of ribosomopathies with the global requirement for ribosome biogenesis.