A multiplexable in vivo perturbation toolkit to identify genes affecting neurodegeneration in a model of synucleinopathy

一种可多重体内扰动工具包，用于识别影响突触核蛋白病模型中神经变性的基因

• 批准号: 10790626
• 负责人: SERGE E PRZEDBORSKI
• 金额: $ 45.24万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10790626
• 关键词: Affect; Alzheimer' s Disease; Automobile Driving; Biological Markers; Brain; CRISPR library; Candidate Disease Gene; Cell Lineage; Cell Nucleus; Cell Survival; Cells; Cessation of life; Cost of Illness; Cre lox recombination system; Crossbreeding; Cultured Cells; DNA Library; Data; Dementia; Dementia with Lewy Bodies; Development; Disease; Etiology; Experimental Models; Future; Gene Expression; Gene Expression Profiling; Gene Targeting; Genes; Genetic; Goals; Guide RNA; Harvest; Health Care Costs; Human; In Vitro; Individual; Injections; Investigation; Knowledge; Methods; Midbrain structure; Modeling; Molecular; Mouse Strains; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurotransmitters; Parkinson' s Dementia; Pathology; Patients; Pattern; Phenotype; Predisposition; Process; Proteins; Publishing; RNA library; Reporting; Research; Spinocerebellar Ataxias; Stress; Synapses; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Intervention; Time; Transgenic Organisms; Translating; Variant; Virion; Work; alpha synuclein; candidate identification; cell type; cellular resilience; disease phenotype; dopaminergic neuron; in vivo; in vivo Model; indexing; innovation; loss of function; mouse model; nervous system disorder; neuron loss; neuronal survival; neuroprotection; pharmacologic; pre-clinical; preventive intervention; resilience; response; screening; single nucleus RNA-sequencing; stressor; synucleinopathy; therapeutic target; transcriptome sequencing; transcriptomic profiling; transcriptomics; Affect; Alzheimer' s Disease; Automobile Driving; Biological Markers; Brain; CRISPR library; Candidate Disease Gene; Cell Lineage; Cell Nucleus; Cell Survival; Cells; Cessation of life; Cost of Illness; Cre lox recombination system; Crossbreeding; Cultured Cells; DNA Library; Data; Dementia; Dementia with Lewy Bodies; Development; Disease; Etiology; Experimental Models; Future; Gene Expression; Gene Expression Profiling; Gene Targeting; Genes; Genetic; Goals; Guide RNA; Harvest; Health Care Costs; Human; In Vitro; Individual; Injections; Investigation; Knowledge; Methods; Midbrain structure; Modeling; Molecular; Mouse Strains; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurotransmitters; Parkinson' s Dementia; Pathology; Patients; Pattern; Phenotype; Predisposition; Process; Proteins; Publishing; RNA library; Reporting; Research; Spinocerebellar Ataxias; Stress; Synapses; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Intervention; Time; Transgenic Organisms; Translating; Variant; Virion; Work; alpha synuclein; candidate identification; cell type; cellular resilience; disease phenotype; dopaminergic neuron; in vivo; in vivo Model; indexing; innovation; loss of function; mouse model; nervous system disorder; neuron loss; neuronal survival; neuroprotection; pharmacologic; pre-clinical; preventive intervention; resilience; response; screening; single nucleus RNA-sequencing; stressor; synucleinopathy; therapeutic target; transcriptome sequencing; transcriptomic profiling; transcriptomics

Neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB) have no cure, but all three share signs of α-synuclein (α-syn) pathology and a pattern of neurodegeneration in which there is variation in neuronal loss. Our long-term goal is to examine this differential neuronal susceptibility to identify features of neurodegeneration in synucleinopathy, and to use this knowledge for subsequent development of neuroprotective strategies and biomarkers. Our central hypothesis is that CRISPR-Cas9 library screening can be used as a cell-type specific, high- throughput in vivo method to elucidate determinants of neurodegeneration in synucleinopathy. The rationale for the proposed research is that, once susceptibility modifiers are identified, therapeutic strategies can be devised for the treatment of PDD, DLB and even perhaps AD. Thus, in AIM 1, we propose to begin by identifying candidate modifiers of neuronal susceptibility in the transgenic mThy1-α-syn (L61Tg) mouse model of synucleinopathy using cell-type-enriched single-nucleus profiling of dopaminergic (DA) neurons. Given the known differential susceptibility among ventral midbrain DA neurons in synucleinopathies, we posit that transcriptomic profiling of these neurons will not only identify signatures of neuronal resilience to stress, but also identify candidate modifiers for inclusion in our subsequent CRISPR library screen. For this work, our cell-specific nuclei tagging system will allow to selectively profile, by single-nucleus RNA-sequencing, DA neurons in L61Tg and their non-transgenic (NTg) littermates. The resulting sequencing data will then be clustered and investigated using differential composition and gene expression analysis to generate a final set of candidate genes that are expressed in subsets of DA neurons, differentially regulated in L61Tg mice, and relevant to human DA subtypes. These candidate genes, which will be included in the list of perturbation candidates in AIM 2, will also serve as a set of genes for future investigations in neurodegeneration research. Next, in AIM 2, we will perform a targeted in vivo CRISPR library screening of DA-neurons in the same mouse model of synucleinopathy. Here, single guide RNA (sgRNA) libraries will be delivered by systemic AAV injection, before brains are harvested six months later and processed for sgRNA sequencing, to identify which genes from our list, upon being silenced, are functional modifiers of neuronal loss. The candidate gene list will be made of our reported hits (Brichta et al., Nat Neurosci, 2015) and those identified in AIM 1. Successful completion of this project will reveal determinants of neuronal survival in vivo and in response to α-syn, a stressor relevant to human neurological disorders. Thus, our anticipated results will have an important positive impact as they will provide opportunities for preventive and therapeutic interventions and, fundamentally, advance our mechanistic understanding of PDD, DLB and even perhaps AD.

神经退行性疾病，例如阿尔茨海默氏病（AD），患有痴呆症的帕金森氏病（PDD） 和Lewy身体（DLB）的痴呆症无法治愈，但所有三个共享符号的α-突触核蛋白（α-Syn）病理学的迹象 以及神经退行性的模式，其中神经元丧失存在变化。我们的长期目标是 检查这种差异神经元易感性，以识别突触核苷的神经退行性特征， 并将这些知识用于随后发展神经保护策略和生物标志物。我们的 中心假设是CRISPR-CAS9库筛选可以用作细胞类型的特异性，高 - 体内方法阐明了突触核酸中神经退行性的决定剂。理由 对于拟议的研究是，一旦确定了易感性修饰剂，治疗策略就可以是 设计用于治疗PDD，DLB甚至可能的AD。在AIM 1中，我们建议以 识别转基因MTHY1-α-Syn（L61TG）小鼠中神经元敏感性的候选修饰符 使用富含细胞类型的单核分析多巴胺能（DA）神经元的单核谱分析的突触核苷病模型。 鉴于突触核酸中腹中间脑中DA神经元之间已知的差异敏感性，我们定位 这些神经元的转录组学分析不仅会识别出对压力的神经元弹性的特征， 但是，还要确定候选修饰符，以将其包含在我们随后的CRISPR库屏幕中。对于这项工作， 我们的细胞特异性核标记系统将通过单核RNA测序选择性地介绍DA L61TG中的神经元及其非转基因（NTG）窝窝。然后，由此产生的测序数据将是 使用鉴别组成和基因表达分析进行聚类和研究，以生成最终 在DA神经元子集中表达的一组候选基因，在L61TG小鼠中不同的调节， 并且与人类DA亚型有关。这些候选基因将包括在扰动列表中 AIM 2中的候选人还将作为神经退行性研究未来研究的一组基因。 接下来，在AIM 2中，我们将在同一鼠标中对DA-Neurons进行定向的INTAMIN INTAN-CRISPR库筛选 突触核苷的模型。在这里，单个指南RNA（SGRNA）库将通过全身性AAV传递 注射，在六个月后收获大脑之前，并处理了SGRNA测序，以确定哪个 沉默后，我们列表中的基因是神经元损失的功能修饰符。候选基因清单 将由我们报道的热门歌曲（Brichta等人，Nat Neurosci，2015年）制成，并在AIM 1中确定的那些。 该项目的完成将揭示体内神经元存活的确定，并响应α-syn，a 与人类神经系统疾病有关的压力源。那，我们的预期结果将具有重要的 积极影响，因为它们将为预防和治疗干预提供机会，并 从根本上讲，提高了我们对PDD，DLB甚至可能AD的机械理解。