Elucidating cellular pathways controlling the reactive state of astrocytes in Alzheimer's Disease

阐明控制阿尔茨海默氏病星形胶质细胞反应状态的细胞途径

• 批准号: 10530601
• 负责人: Kun Leng
• 金额: $ 4.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10530601
• 关键词: Affect; Alzheimer disease prevention; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease model; Astrocytes; Autopsy; Binding; Biological Assay; Brain; CRISPR interference; Code; Collaborations; Complement 1q; Data Set; Defect; Disease; Exhibits; Fellowship; Genes; Genetic; Genetic Epistasis; Genetic Transcription; Genome; Glial Fibrillary Acidic Protein; Goals; Human; Hypertrophy; Impaired cognition; Induced pluripotent stem cell derived neurons; Inflammatory; Magnetism; Maps; Mentorship; Modeling; Morphology; Mus; Myosin ATPase; Myosin Type II; Neurologist; Neurology; Operative Surgical Procedures; Outcome; Output; Pathway interactions; Phagocytes; Phagocytosis; Phosphorylation; Physicians; Population; Positioning Attribute; Process; Property; Protein Dephosphorylation; Proteins; Publishing; Regulation; Regulon; Research; Rho-associated kinase; SET gene; Scientist; TNF gene; Technology; Testing; Training; Up-Regulation; Validation; Work; aged; clinical training; cytokine; differential expression; drug development; functional genomics; genome-wide; induced pluripotent stem cell; inflammatory marker; knock-down; loss of function; magnetic beads; mouse model; myosin phosphatase; neuroinflammation; neuropathology; neurotoxic; response; screening; single-cell RNA sequencing; tissue injury; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

PROJECT SUMMARY/ABSTRACT Astrocytes perform essential homeostatic functions in the brain. In response to local tissue injury, astrocytes become “reactive”, a process classically characterized by morphological hypertrophy and upregulation of GFAP. Reactive astrocytes are a defining feature of Alzheimer's disease (AD) neuropathology and are strongly correlated with cognitive decline in AD. However, mechanisms controlling astrocyte reactivity are not well understood. Recent studies have identified a form of reactive astrocyte induced by inflammatory activation (“inflammatory reactivity”, a.k.a. “A1” reactivity) that may play a role in AD. Inflammatory reactive astrocytes are characterized by loss of normal homeostatic functions such as phagocytosis of CNS substrates as well as gain of neurotoxic properties. Importantly, they are found in a mouse model of AD as well as normal aged mice. Furthermore, human post-mortem AD brains are enriched for astrocytes expressing a marker of inflammatory reactivity. To elucidate mechanisms controlling inflammatory reactivity and its functional outputs in human astrocytes, in my preliminary work I implemented pooled CRISPRi loss-of-function screening in human iPSC- derived astrocytes (iAstros). From a preliminary screen, I identified myosin phosphatase as a potential regulator of the decreased phagocytic activity in inflammatory astrocytes. Furthermore, I leveraged existing astrocyte RNA-seq datasets to infer transcriptional regulators of inflammatory reactivity. In my first aim, I propose to determine how regulation of myosin phosphatase leads to decreased phagocytosis in inflammatory reactive astrocytes. In my second aim, I propose integrate genome-wide CRISPRi screening and coexpression analysis of existing astrocyte RNA-seq datasets to discover cellular pathways controlling inflammatory reactivity, followed by connecting these pathways to functional outputs of astrocyte reactivity. My sponsor Dr. Martin Kampmann, who co-developed the CRISPRi screening technology, and my co-sponsor Dr. Sergio Baranzini, an expert in neuroinflammation and the integration and analysis of transcriptomic datasets, are ideally positioned to support my proposed research. Furthermore, my collaborator Dr. Erik Ullian is an expert on the differentiation of high- quality astrocytes from human iPSCs, which further supports the feasibility of the proposed work. In addition to my two sponsors and my collaborator Dr. Ullian, I will also receive mentorship from Dr. Aimee Kao, a physician- scientist and practicing neurologist with whom I will undergo longitudinal clinical training in neurology, and Dr. Bruce Conklin, a global leader in iPSC-based technologies and genome surgery. Overall, the work proposed in this fellowship should contribute towards the development of drugs that can selectively modulate different functional outputs of astrocyte reactivity for the treatment of AD. Furthermore, through this work I will develop expertise in uncovering disease mechanisms with iPSC-derived models and functional genomics which will strengthen my training as a physician-scientist.

项目总结/摘要 星形胶质细胞在大脑中执行基本的稳态功能。星形胶质细胞对局部组织损伤的反应是 变得“反应性”，这是一个典型的特征为形态肥大和GFAP上调的过程。 反应性星形胶质细胞是阿尔茨海默病（AD）神经病理学的一个定义特征，并且与阿尔茨海默病（AD）的神经病理学密切相关。 与AD的认知能力下降相关。然而，控制星形胶质细胞反应性的机制并不清楚， 明白最近的研究已经确定了一种由炎症激活诱导的反应性星形胶质细胞 （“炎性反应性”，也称为“炎症反应性”）。“A1”反应性），可能在AD中发挥作用。炎症反应性星形胶质细胞是 以丧失正常的稳态功能为特征，如CNS底物的吞噬作用以及获得 具有神经毒性重要的是，它们存在于AD小鼠模型以及正常老年小鼠中。 此外，人死后AD脑富含表达炎性细胞标志物的星形胶质细胞。 反应性阐明控制人类炎症反应及其功能输出的机制， 在我的初步工作中，我在人类iPSC中实施了合并的CRISPRi功能丧失筛选， 衍生的星形胶质细胞（iAstros）。通过初步筛选，我确定肌球蛋白磷酸酶是一种潜在的调节剂 炎性星形胶质细胞吞噬活性降低的证据。此外，我利用现有的星形胶质细胞 RNA-seq数据集推断炎症反应性的转录调节因子。我的第一个目标是， 确定肌球蛋白磷酸酶的调节如何导致炎性反应中吞噬作用的降低， 星形胶质细胞在我的第二个目标中，我建议整合全基因组CRISPRi筛选和共表达分析， 现有的星形胶质细胞RNA-seq数据集，以发现控制炎症反应的细胞通路， 通过将这些通路连接到星形胶质细胞反应性的功能输出。我的赞助商马丁·坎普曼博士， 他是CRISPRi筛选技术的共同开发者，我的共同赞助人塞尔吉奥·巴兰齐尼博士是一位专家， 神经炎症和转录组数据集的整合和分析，是理想的定位，以支持 我提议的研究此外，我的合作者Erik Ullian博士是区分高- 高质量的星形胶质细胞，这进一步支持了所提出的工作的可行性。除了 我的两个赞助商和我的合作者尤利安博士，我也将接受来自医生Aimee Kao的指导- 我将和他一起接受神经病学的纵向临床培训。 布鲁斯康克林，在基于iPSC的技术和基因组手术的全球领导者。总体而言， 这项研究应该有助于开发药物，可以选择性地调节不同的 用于治疗AD的星形胶质细胞反应性的功能输出。此外，通过这项工作，我将发展 利用iPSC衍生模型和功能基因组学揭示疾病机制的专业知识， 加强我作为一名物理学家和科学家的训练。

项目成果

Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis.

• DOI: 10.1038/s41593-021-00862-0
• 发表时间: 2021-07
• 期刊: Nature neuroscience
• 影响因子: 25
• 作者: Tian R;Abarientos A;Hong J;Hashemi SH;Yan R;Dräger N;Leng K;Nalls MA;Singleton AB;Xu K;Faghri F;Kampmann M
• 通讯作者: Kampmann M

Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model.

• DOI: 10.1021/acschemneuro.1c00804
• 发表时间: 2022-05-18
• 期刊: ACS chemical neuroscience
• 影响因子: 5
• 作者: Chin MY;Ang KH;Davies J;Alquezar C;Garda VG;Rooney B;Leng K;Kampmann M;Arkin MR;Kao AW
• 通讯作者: Kao AW

Cell type specificity of mosaic chromosome 1q gain resolved by snRNA-seq in a case of epilepsy with hyaline protoplasmic astrocytopathy.

在伴有透明质性星形细胞病的癫痫病例中，通过 snRNA-seq 解析了嵌合染色体 1q 的细胞类型特异性。

• DOI: 10.1101/2023.10.16.562560
• 发表时间: 2024
• 期刊: bioRxiv : the preprint server for biology
• 作者: Leng,Kun;Cadwell,CathrynR;PatrickDevine,W;Tihan,Tarik;Qi,Zhongxia;Singhal,Nilika;Glenn,Orit;Kamiya,Sherry;Wiita,Arun;Berger,Amy;Shieh,JosephT;Titus,ErronW;Paredes,MercedesF;Upadhyay,Vaibhav
• 通讯作者: Upadhyay,Vaibhav