Contributions of Endothelial RNA-binding Protein Dysregulation to Blood Brain Barrier Defects and Neurodegenerative Disease

内皮 RNA 结合蛋白失调对血脑屏障缺陷和神经退行性疾病的影响

• 批准号: 10037854
• 负责人: Patrick Andries Murphy
• 金额: $ 220.21万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2024-07-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10037854
• 关键词: APP-PS1; Affect; Age; Aging; Alternative Splicing; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease related dementia; Amyotrophic Lateral Sclerosis; Animal Model; Bioinformatics; Blood; Blood - brain barrier anatomy; Blood Platelets; Brain; CRISPR screen; Cell Nucleus; Cells; Cytoplasm; Data; Defect; Dementia; Disease; Disease Progression; Disease model; Endothelial Cells; Endothelium; Event; Fibroblasts; Freezing; Frontotemporal Dementia; Frontotemporal Lobar Degenerations; Functional disorder; Genes; Genetic; Genetic Models; Genetic Transcription; Hemorrhage; Human; Immune; Immune response; Impaired cognition; Impairment; In Vitro; Inflammation; Informatics; Knockout Mice; Link; Maintenance; Mediating; Messenger RNA; Methods; Microglia; Microvascular Dysfunction; Modeling; Mus; Mutate; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurologic Dysfunctions; Neuronal Dysfunction; Neurons; Nuclear; Pancreas; Patients; Play; Post-Transcriptional Regulation; Protein Isoforms; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Role; Sampling; Sterility; Stream; TDP-43 aggregation; Techniques; Temporal Lobe; Testing; Work; blood-brain barrier function; brain cell; brain endothelial cell; brain tissue; cognitive function; frontotemporal lobar dementia-amyotrophic lateral sclerosis; hnRNP A1; human model; immune activation; in vivo; insight; mRNA Precursor; mouse model; neuroinflammation; neurovascular; neurovascular coupling; novel; novel strategies; programs; protein TDP-43; protein function; recruit; response; restoration; screening; tool

Splicing dysregulation, caused by defects in RNA-binding splice factors including TDP-43, is considered a hallmark and potential driver of neuronal dysfunction and cognitive decline in Alzheimer’s (AD) and Related Dementia (ADRD). Although substantial evidence suggests that genetic causes of splicing dysfunction are not limited to neuronal cells, splicing defects in the BBB endothelium in AD and ADRD have not been examined. Defects in the BBB increase with age and early in the progression of AD and ADRD, where they contribute to disease progression. Through in vivo informatics and in vitro CRISPR screening, we identified TDP-43 and several other AD and ADRD associated splice factors as regulators of post-transcriptional splicing in the endothelium in response to sterile inflammation. Using a novel method to isolate endothelial nuclei from frozen banked human brain tissues, we identified reduced nuclear TDP- 43 levels in endothelial cells of the blood brain barrier (BBB) with age and in AD and ADRD patients. Furthermore, in a novel mouse model, we show that specific deletion of TDP-43 from the brain endothelium leads to leak across the BBB, and activation of the microvasculature and microglial cells. Here, we hypothesize that loss of nuclear TDP-43 in the endothelium contributes to defects in the BBB and microvasculature, and to AD and ADRD by affecting the splicing of pre-mRNA required for the maintenance of a quiescent endothelium. We propose to examine TDP-43 expression and splicing activity in the endothelium in human AD and ADRD, and the effect of loss of nuclear TDP-43 on disease progression in mouse models of AD and the ADRD Frontal temporal lobe dementia (FTLD). Furthermore, since our novel techniques will allow us an unprecedented view of the endothelium in AD and ADRD, we propose to extend our work on TDP-43 to broadly examine splicing alterations in the endothelium in these disease states, and use our established bioinformatics and in vitro screening approaches to determine whether defects in other endothelial splice factors also contribute to BBB dysfunction. The completion of this work will provide new insight into the contributions of post-transcriptional regulation by RNA-binding proteins to BBB defects, microvascular dysfunction and the progression of AD and ADRD.

由RNA结合剪接因子（包括TDP-43）缺陷引起的剪接失调， 被认为是神经元功能障碍和认知能力下降的标志和潜在驱动因素， 阿尔茨海默氏症（AD）和相关痴呆症（ADRD）。尽管大量证据表明， 剪接功能障碍的遗传原因不仅限于神经元细胞， AD和ADRD中的BBB内皮尚未检查。BBB中的缺陷随着 年龄和早期AD和ADRD进展，在那里他们有助于疾病 进展通过体内信息学和体外CRISPR筛选，我们鉴定了TDP-43 以及其他几种AD和ADRD相关的剪接因子作为转录后调节因子， 在无菌炎症反应中在内皮中剪接。用一种新的方法分离 从冷冻库人脑组织的内皮细胞核中，我们发现了减少的核TDP- 43随着年龄的增长以及AD和ADRD中血脑屏障（BBB）内皮细胞的水平 患者此外，在一种新的小鼠模型中，我们显示了TDP-43的特异性缺失， 脑内皮导致血脑屏障渗漏，微血管系统激活， 小胶质细胞在此，我们假设内皮细胞核TDP-43的缺失， 导致BBB和微血管缺陷，并通过影响 维持静止内皮所需的前mRNA剪接。我们建议 检测人AD和ADRD内皮中TDP-43的表达和剪接活性， 以及核TDP-43缺失对AD小鼠模型中疾病进展的影响， ADRD额颞叶痴呆（FTLD）。此外，由于我们的新技术将 让我们对AD和ADRD的内皮细胞有了前所未有的认识，我们建议扩大我们的研究范围， 我研究TDP-43，以广泛检查这些疾病中内皮细胞的剪接改变， 国家，并使用我们建立的生物信息学和体外筛选方法，以确定 其他内皮剪接因子的缺陷是否也导致BBB功能障碍。的 这项工作的完成将提供新的见解转录后的贡献， 通过RNA结合蛋白调节BBB缺陷、微血管功能障碍和 AD和ADRD的进展。

项目成果

Targeted inCITE-Seq Analysis Identifies the Loss of Nuclear TDP-43 in Endothelium as a Mediator of Blood Brain Barrier Signaling Pathway Dysfunction in Neurodegeneration.

靶向 inCITE-Seq 分析确定内皮细胞中核 TDP-43 的丢失是神经退行性变中血脑屏障信号通路功能障碍的介质。

• DOI: 10.1101/2023.12.13.571178
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Omar,OmarMF;Kimble,AmyL;Cheemala,Ashok;Tyburski,JordanD;Pandey,Swati;Wu,Qian;Reese,Bo;Jellison,EvanR;Li,Yunfeng;Hao,Bing;Yan,Riqiang;Murphy,PatrickA
• 通讯作者: Murphy,PatrickA

A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue.

• DOI: 10.1038/s41374-021-00698-z
• 发表时间: 2022-03
• 期刊: Laboratory investigation; a journal of technical methods and pathology
• 作者: Kimble AL;Silva J;Omar OM;Murphy M;Hensel JA;Nicholas SE;Jellison ER;Reese B;Murphy PA
• 通讯作者: Murphy PA