Single Cell Dissection of Cerebrovascular Dysfunction in Parkinson's Disease and Amyotrophic Lateral Sclerosis

帕金森病和肌萎缩侧索硬化症脑血管功能障碍的单细胞解剖

基本信息

批准号：
10508837
负责人：
Raleigh Miller Linville
金额：
$ 6.72万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
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：
2022-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10508837
关键词：
ALS patients Address Affect Age Amyotrophic Lateral Sclerosis Animal Model Astrocytes Atlases Autopsy Benchmarking Blood - brain barrier anatomy Blood Vessels Blood flow Brain Brain Stem Brain imaging Cell Nucleus Cells Cerebrovascular Circulation Cerebrovascular Disorders Cerebrovascular system Cessation of life Chemicals Collaborations Development Disease Disease Progression Dissection Etiology Excision Extravasation Fibroblasts Functional disorder Future Gene Expression Genetic Transcription Goals Histologic Human Immune Immunofluorescence Immunologic In Situ Hybridization In Vitro Induced pluripotent stem cell derived neurons Link Magnetic Resonance Imaging Microglia Modeling Molecular Motor Motor Cortex Motor Neurons Neurodegenerative Disorders Neuroglia Neurologic Neurons Nutrient Paralysed Parkinson Disease Pathologic Pathology Patients Pericytes Phenotype Population Process Protocols documentation Recovery Resolution Role Sampling Site Skeletal Muscle Small Nuclear RNA Smooth Muscle Myocytes Specificity Spinal Cord Stress Substantia nigra structure Supporting Cell Testing Therapeutic Tissues Transgenic Mice Work alpha synuclein angiogenesis base brain endothelial cell brain tissue cell age cell type cerebrovascular computational pipelines disease phenotype dopaminergic neuron familial amyotrophic lateral sclerosis gene regulatory network genome-wide human disease human tissue imaging probe in vitro Model in vivo induced pluripotent stem cell motor neuron degeneration motor symptom new therapeutic target overexpression response single cell technology sporadic amyotrophic lateral sclerosis stem cell model targeted treatment therapeutic target tool trafficking transcription factor transcriptome sequencing transcriptomics wasting whole genome

项目摘要

Project Summary Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS) are irreversible and currently incurable neurodegenerative diseases with more than 65,000 new cases in the USA each year. Their core motor symptoms are respectively caused by dysfunction and death of dopaminergic neurons within the substantia nigra and motor neurons in the cortex, brainstem, and spinal cord. However, non-cell-autonomous contributions to disease progression are widely recognized and include cerebrovascular (CV) dysfunction. The CV is formed by several highly specialized cell populations, including brain endothelial cells (BECs), mural cells, fibroblasts, and glia. Given the CV’s critical role in regulating biomolecule transport into and out of the brain, blood flow, and responses to physical or chemical stress, understanding the molecular underpinnings of early CV changes during PD and ALS may be critical to develop disease-modifying treatments. Prior work indicates that CV changes can occur during the progression of PD and ALS, including leakage of the blood-brain barrier (BBB), angiogenesis, dysfunctional efflux activity, dysregulated blood flow, and increased immune cell trafficking. However, findings from brain imaging (MRI) and histological analysis are not inclusive of all CV functions nor able to identify transcriptional regulators, while studies using animal models are not representative of sporadic human disease which accounts for ~90% of PD and ALS cases. In this proposal, I will characterize cerebrovascular dysfunction during sporadic PD and ALS with cell type-specificity and whole genome-resolution from post-mortem tissue, and will benchmark the degree to which this dysfunction is recapitulated by iPSC-derived in vitro models. This work is grounded in recent application of blood-vessel enrichment (BVE) and single nucleus RNA sequencing (snRNA-seq) approaches to profile gene expression of CV cells, and the development of transcription factor overexpression-based differentiation of BECs from induced pluripotent stem cells (iPSCs). In Aim 1A, I will conduct snRNA-seq on blood vessel enriched substantia nigra from post-mortem PD patients and age-matched healthy controls, and will then validate cell type-specific dysfunction using immunofluorescence and in situ hybridization studies. In Aim 1B, I will differentiate BECs from PD patient iPSCs and age-matched healthy controls and then conduct snRNA-seq to determine how post-mortem hallmarks of dysfunction are reflected in vitro. In Aim 2, I will take a similar approach by conducting snRNA-seq on ALS patients blood vessel enriched motor cortex and iPSC-derived BECs compared to healthy age-matched post-mortem tissue and iPSC controls. By characterizing CV gene expression using cutting-edge single nucleus profiling of PD and ALS post- mortem tissue and iPSC-derived models, this proposal will identity previously unrecognized mechanisms of CV dysfunction and serve as a critical launchpad for future studies to test causality in disease processes and validate therapeutic targets across in vivo and in vitro models.

项目摘要帕金森氏病（PD）和肌萎缩性侧面硬化症（ALS）是不可逆的，目前无法治愈每年在美国有65,000例新病例的神经退行性疾病。他们的核心运动症状分别是由底虫和运动中多巴胺能神经元的功能障碍和死亡引起的皮质，脑干和脊髓中的神经元。但是，对疾病的非细胞自主贡献进展得到广泛认可，包括脑血管（CV）功能障碍。简历由几个高度专业的细胞群，包括脑内皮细胞（BEC），壁画细胞，成纤维细胞和神经胶质。鉴于简历在将生物分子运输转移到大脑中和流动，血流和反应中的关键作用对于物理或化学应力，了解PD期间早期CV变化的分子基础 ALS对于开发疾病改良治疗可能至关重要。先前的工作表明PD和ALS的进展过程中可能发生CV变化，包括泄漏血脑屏障（BBB），血管生成，功能失调的外排活性，血流失调和增加免疫细胞贩运。但是，大脑成像（MRI）和组织学分析的发现并不包括所有简历功能都无法识别转录调节剂，而使用动物模型的研究不是代表零星的人类疾病，占PD和ALS病例的90％。在这个建议中，我将表征具有细胞类型特异性的零星PD和ALS期间脑血管功能障碍全尸体组织的整个基因组分辨率，并将基准以此为基础 IPSC衍生的体外模型概括了功能障碍。这项工作是基于最近的应用血管富集（BVE）和单核RNA测序（SnRNA-Seq）方法用于谱图基因 CV细胞的表达以及基于转录因子过表达的BEC的发展来自诱导的多能干细胞（IPSC）。在AIM 1A中，我将在富含血管上进行SNRNA-SEQ 验尸PD患者和年龄匹配的健康对照的底底尼格拉，然后验证细胞使用免疫荧光和原位杂交研究的类型特异性功能障碍。在AIM 1B中，我会将BEC与PD患者IPSC和年龄匹配的健康对照区分开，然后进行SNRNA-SEQ至确定在体外如何反映验尸后标志。在AIM 2中，我将采取类似的方法通过对ALS患者进行SNRNA-SEQ，富含运动皮层的血管和IPSC衍生的BEC 与健康匹配的年龄匹配后组织和IPSC对照相比。通过使用PD和ALS的尖端单核分析表征CV基因表达 Mortem组织和IPSC衍生的模型，该提案将确定以前未认可的CV机制功能障碍并作为未来研究的关键发射台，以测试疾病过程中的偶然性并验证跨体内和体外模型的治疗靶标。