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

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，如阿尔茨海默病(AD)、帕金森氏病伴痴呆(PDD) 和路易体痴呆(DLb)无法治愈，但这三种疾病都有α-突触核蛋白(α-SYN)病理征象 和一种神经退行性变的模式，在这种模式中，神经元的丢失有变化。我们的长期目标是 检查这种不同的神经元敏感性，以确定共核病的神经退行性变的特征， 并将这些知识用于后续神经保护策略和生物标记物的开发。我们的 中心假说是CRISPR-Cas9文库的筛选可以作为细胞类型特异性、高特异性、高选择性的 体内吞吐能力法阐明共核病神经变性的决定因素。其基本原理是 对于拟议的研究来说，一旦确定了易感性调节剂，治疗策略就可以 设计用于治疗PDD、DLB，甚至可能是AD。因此，在目标1中，我们建议从 MThy1-α-syn(L61Tg)转基因小鼠神经元易感性候选修饰物的鉴定 用细胞类型丰富的多巴胺(DA)能神经元单核图谱建立突触核病模型。 鉴于已知的中脑腹侧DA神经元在联核病中的不同易感性，我们假设 这些神经元的转录图谱不仅将识别神经元对应激的韧性信号， 而且还识别候选修饰物以包括在我们随后的CRISPR文库筛选中。对于这项工作， 我们的细胞特异性核标记系统将允许通过单核RNA测序选择性地描述DA L61Tg及其非转基因(NTG)仔鼠的神经元。然后得到的测序数据将是 使用差异成分和基因表达分析进行聚类和调查，以生成最终的 在L61Tg小鼠中差异调控的一组在DA神经元亚群中表达的候选基因， 与人类DA亚型相关。这些候选基因，将被包括在扰动列表中 AIM 2的候选基因也将作为一组基因，用于未来神经变性研究的研究。 接下来，在AIM 2中，我们将对同一小鼠的DA神经元进行有针对性的体内CRISPR文库筛选 联核症模型。在这里，单引导RNA(SgRNA)文库将由系统性AAV提供 在六个月后采集大脑并进行sgRNA测序之前，进行注射，以确定哪种 我们清单中的基因，一旦被沉默，就是神经元丧失的功能修饰者。候选基因列表 将由我们报告的命中率(Brichta等人，Nat Neurosci，2015)和AIM 1中确定的命中率组成。成功 该项目的完成将揭示神经元在体内存活的决定因素，以及对α-SYN的反应 与人类神经紊乱有关的压力源。因此，我们的预期结果将具有重要的 产生积极影响，因为它们将为预防和治疗干预提供机会， 从根本上说，提高我们对PDD、DLB甚至AD的机械性理解。