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

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，目前是无法治愈的 神经退行性疾病在美国每年有超过65,000例新病例。他们的核心运动症状 分别由黑质和运动区的多巴胺能神经元功能障碍和死亡引起 皮质、脑干和脊髓中的神经元。然而，非细胞自主对疾病的贡献 进展被广泛认识，包括脑血管(CV)功能障碍。简历由以下几部分组成 高度专业化的细胞群，包括脑内皮细胞(BECs)、壁细胞、成纤维细胞和胶质细胞。 鉴于CV在调节生物分子进出大脑、血液流动和反应方面的关键作用 物理或化学应激，了解PD和PD期间早期CV变化的分子基础 肌萎缩侧索硬化症可能对开发改善疾病的治疗方法至关重要。 先前的工作表明，在PD和ALS的进展过程中，可以发生CV的变化，包括渗漏 血脑屏障(BBB)、血管生成、功能紊乱的外排活动、血液流动失调，以及 免疫细胞走私。然而，脑成像(MRI)和组织学分析的结果并不包括 所有的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 散发性人类疾病的代表，约占帕金森病和ALS病例的90%。在这项建议中，我 将表征散发性PD和ALS患者的脑血管功能障碍 全基因组-从死后组织中的分辨率，并将作为基准的程度，这 功能障碍是由IPSC衍生的体外模型概括的。这项工作的基础是最近的应用 血管富集法(BVE)和单核RNA测序(SnRNA-seq)方法检测基因图谱 血管内皮细胞的表达与转录因子过度表达诱导BEC分化的研究进展 来自诱导多能干细胞(IPSCs)。在目标1A中，我将对浓缩的血管进行SnRNA-SEQ 死后帕金森病患者和年龄匹配的健康对照组的黑质，然后将验证细胞 应用免疫荧光和原位杂交研究特定类型的功能障碍。在目标1B中，我将 将BEC与PD患者IPSCs和年龄匹配的健康对照组区分开来，然后进行SnRNA-SEQ 确定功能障碍的死后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患者血管丰富的运动皮质和IPSC来源的BECs进行SnRNA-SEQ 与健康的年龄匹配的死后组织和IPSC对照组进行比较。 通过使用尖端单核图谱来表征PD和ALS患者术后CV基因的表达 死亡组织和IPSC衍生模型，这一建议将识别以前未被认识的心血管机制 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 体内和体外模型的治疗靶点。

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.