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

• 批准号: 10677599
• 负责人: Raleigh Miller Linville
• 金额: $ 6.95万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 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/10677599
• 关键词: ALS patients; 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 Disorders; Cerebrovascular system; Cessation of life; Chemicals; Collaborations; Cytoplasm; 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; Microglia; Modeling; Molecular; Motor; Motor Cortex; Motor Neurons; Neurodegenerative Disorders; Neuroglia; Neurologic; Neurons; Nutrient; Paralysed; Parkinson Disease; Pathologic; Pathology; Patients; Pericytes; Phenotype; Pluripotent Stem Cells; Population; Process; Protocols documentation; Recovery; Resolution; Role; Sampling; Site; Skeletal Muscle; Smooth Muscle Myocytes; Specificity; Spinal Cord; Stress; Substantia nigra structure; Supporting Cell; Testing; Therapeutic; Tissues; Transgenic Mice; Work; alpha synuclein; angiogenesis; 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; single nucleus RNA-sequencing; sporadic Parkinson' s Disease; sporadic amyotrophic lateral sclerosis; stem cell model; targeted treatment; therapeutic target; tool; trafficking; transcription factor; transcriptomic profiling; wasting; whole genome

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 帕金森病（PD）和肌萎缩侧索硬化症（ALS）是不可逆转的，目前无法治愈 神经退行性疾病，在美国每年有超过65，000例新发病例。他们的核心运动症状 分别由黑质和运动神经内的多巴胺能神经元的功能障碍和死亡引起 皮层脑干和脊髓的神经元然而，非细胞自主对疾病的贡献 进展被广泛认可，包括脑血管（CV）功能障碍。CV是由几个 高度特化的细胞群，包括脑内皮细胞（BEC）、壁细胞、成纤维细胞和神经胶质。 考虑到CV在调节生物分子进出大脑、血流和反应中的关键作用， 物理或化学应激，了解PD期间早期CV变化的分子基础， ALS可能对开发疾病改善治疗至关重要。 先前的工作表明，在PD和ALS的进展过程中可能发生CV变化，包括 血脑屏障（BBB），血管生成，功能失调的外排活性，失调的血流，并增加 免疫细胞运输。然而，脑成像（MRI）和组织学分析的结果不包括 所有CV功能也不能识别转录调节因子，而使用动物模型的研究则不能 代表散发性人类疾病，占PD和ALS病例的约90%。在这份提案中，我 将以细胞类型特异性表征散发性PD和ALS期间的脑血管功能障碍， 整个基因组分辨率从死后组织，并将基准的程度， iPSC衍生的体外模型再现了功能障碍。这项工作是在最近的应用接地 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BEC基于转录因子过表达的分化的发展 诱导多能干细胞（iPSC）。在目标1A中，我将在血管富集上进行snRNA-seq 来自死后PD患者和年龄匹配的健康对照的黑质，然后将验证细胞 型特异性功能障碍，使用免疫荧光和原位杂交研究。在Aim 1B中，我将 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， 确定死后功能障碍的特征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患者血管富集的运动皮层和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治疗后的尖端单核分析来表征CV基因表达， 死亡组织和iPSC衍生模型，该提案将确定以前未被认识到的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因果关系，并验证 体内和体外模型中的治疗靶点。