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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.

项目概要帕金森病 (PD) 和肌萎缩侧索硬化症 (ALS) 是不可逆转的，目前无法治愈美国每年新增神经退行性疾病病例超过 65,000 例。他们的核心运动症状分别由黑质和运动神经元内的多巴胺能神经元功能障碍和死亡引起皮层、脑干和脊髓中的神经元。然而，非细胞自主对疾病的贡献进展已得到广泛认可，包括脑血管（CV）功能障碍。简历由几部分组成高度专业化的细胞群，包括脑内皮细胞 (BEC)、壁细胞、成纤维细胞和神经胶质细胞。鉴于 CV 在调节生物分子进出大脑、血流和反应方面的关键作用物理或化学应激，了解 PD 和早期 CV 变化的分子基础 ALS 可能对于开发疾病缓解疗法至关重要。先前的研究表明，在 PD 和 ALS 的进展过程中可能会发生 CV 变化，包括血脑屏障（BBB）、血管生成、外排活动功能失调、血流失调和增加免疫细胞贩运。然而，脑成像（MRI）和组织学分析的结果并不包括所有 CV 功能也无法识别转录调节因子，而使用动物模型的研究则无法代表散发性人类疾病，约占 PD 和 ALS 病例的 90%。在这个提案中，我将表征散发性 PD 和 ALS 期间的脑血管功能障碍，并具有细胞类型特异性来自死后组织的全基因组分辨率，并将对这种程度进行基准测试 iPSC 衍生的体外模型重现了功能障碍。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) 方法来分析基因 CV 细胞的表达，以及基于转录因子过表达的 BEC 分化的发展来自诱导多能干细胞（iPSC）。在目标 1A 中，我将对富集的血管进行 snRNA-seq 来自死后 PD 患者和年龄匹配的健康对照的黑质，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。在目标 1B 中，我将将 BEC 与 PD 患者 iPSC 和年龄匹配的健康对照区分开来，然后进行 snRNA-seq 确定功能障碍的死后标志如何在体外反映出来。在目标 2 中，我将采取类似的方法通过对 ALS 患者富含血管的运动皮层和 iPSC 衍生的 BEC 进行 snRNA-seq 与健康年龄匹配的死后组织和 iPSC 对照相比。通过使用 PD 和 ALS 后的尖端单核分析来表征 CV 基因表达尸检组织和 iPSC 衍生模型，该提案将识别以前未识别的 CV 机制功能障碍，并作为未来研究的重要启动平台，以测试疾病过程中的因果关系并验证体内和体外模型的治疗靶点。