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结合剪接因子缺陷（包括TDP-43）的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力 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限于神经元细胞，剪接缺陷 尚未检查AD和ADRD中的BBB内皮。 BBB的缺陷随着 年龄和在AD和ADRD的发展中，它们会导致疾病 进展。通过体内信息和体外CRISPR筛查，我们确定了TDP-43 以及其他几个AD和ADRD相关的剪接因子作为转录后的调节因子 响应无菌炎症的内皮中的剪接。使用一种新颖的方法来隔离 从冷冻的人脑组织中，我们发现核TDP- 随着年龄的年龄和AD和ADRD的血液脑屏障（BBB）的内皮细胞的43个水平 患者。此外，在新型的鼠标模型中，我们显示了TDP-43的特定缺失 脑内皮导致BBB泄漏，并激活微脉管系统和 小胶质细胞。在这里，我们假设内皮中核TDP-43的损失 有助于BBB和微脉管系统中的缺陷，并通过影响AD和ADRD来影响AD和ADRD 维持静态的植物所需的pre-mRNA剪接。我们建议 检查人类AD和ADRD中内皮中TDP-43的表达和剪接活性， 以及核TDP-43损失对AD和AD小鼠模型中疾病进展的影响 Adrd Frontal临时叶痴呆（FTLD）。此外，由于我们的新技术将 允许我们在AD和ADRD中对内皮的前所未有的观点，我们建议扩展我们的 在TDP-43上的工作以广泛检查这些疾病内皮的剪接改变 国家，并使用我们已建立的生物信息学和体外筛查方法来确定 其他内皮剪接因子中的缺陷是否也导致BBB功能障碍。这 这项工作的完成将为转录后的贡献提供新的见解 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