Programmed Cell Death In The Nervous System

神经系统中的程序性细胞死亡

• 批准号: 10688926
• 负责人: Richard James Youle
• 金额: $ 54.7万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10688926
• 关键词: Acanthocytosis; Apoptosis; Artificial Intelligence; BCL2 gene; Biochemical; Biogenesis; Biological; CRISPR screen; Cell Death; Cell membrane; Cells; Cellular biology; Chorea; Cohen syndrome; Cytosol; Development; Disease; Dissection; Drug Targeting; Embryo; Endoplasmic Reticulum; Essential Genes; Event; Family member; Flow Cytometry; Genes; Genetic; Guide RNA; Homologous Gene; Human; Image; Inflammation; Intracellular translocation; Investigation; Knock-out; Light; Link; Lysosomes; Mammals; Mediating; Membrane; Methodology; Microscopy; Mitochondria; Mitochondrial DNA; Morphology; Movement Disorders; Mus; Mutation; Nervous System; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Nuclear; Organelles; Parkinson Disease; Pathologic; Pathway interactions; Patients; Phenotype; Process; Protein Family; Proteins; Role; Site; Spinocerebellar Ataxias; Stimulator of Interferon Genes; Work; Yeasts; artificial intelligence algorithm; autism spectrum disorder; base; constriction; fly; gene product; genome wide association study; genome-wide; interest; lipid transport; loss of function; neurodegenerative phenotype; neuron loss; novel; peroxisome; screening; stem

We have studied programmed cell death in the nervous system and the biochemical mechanisms of apoptosis. The Bcl-2 family of proteins regulate the survival of neurons during development. We previously discovered that one member of this family, Bax, migrates from the cytosol to the cell membranes as a key commitment point of neuron death. Interestingly, Bax coalesces into large aggregates at mitochondrial constriction sites and mitochondrial fission sites. How this is initiated is unknown and identifying novel steps could serve as drug targets to inhibit neuron death. To interrogate this step in more detail we have explored how mitochondria divide in healthy and dying cells and developed a new screen of intracellular translocation events. 1) Mitochondria form contacts with other intracellular organelles including the endoplasmic reticulum (ER), peroxisomes and lysosomes. Mitochondrial ER contact sites are involved in mitochondrial fission, at the precise sites where Bax coalesces. In yeast, Vps13 has been linked to mitochondria ER membrane contact sites and it appears to mediate lipid transport between the two organelles. Mammals have four VPS13 homologues: VPS13A-D. Mutations in VPS13A cause the neurodegenerative disease chorea-acanthocytosis. VPS13B is associated with Cohen syndrome, a neurodevelopmental disorder that shares features with autism. Genome-wide association studies show loss of function VPS13C mutations cause Parkinson's disease. VPS13D mutations are linked to movement disorders classified as a subtype of spinocerebellar ataxia, spinocerebellar ataxia autosomal recessive 4 (SCAR4). In contrast to VPS13A-C, VPS13D is considered an essential gene in human cells and complete loss of VPS13D causes embryonic lethality in mice and flies. We investigated the cellular biology of VPS13A-D knockout (KO) in human cells and found a dual role for VPS13D in mitochondrial morphology and peroxisome biogenesis. This contrasts with the other three Vps13 genes and identifies a new biological role of this SCAR4 gene. Most recent work of ours and others shows that loss of Vps13C or loss of Vps13D is linked to inflammation. We will explore how this may stem from mitochondrial disfunction, release of mitochondrial DNA into the cytosol to activate the cGAS STING inflammation pathway and how inflammation may be associated with the neurodegenerative phenotypes of Vps13C-D patients. 2) To uncover how the apoptosome functions downstream of Bax translocation to mitochondria to mediate cell death we developed a new CRISPR screening platform that allows genetic dissection of Bax translocation and other cell translocation events based on light imaging. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. We are currently using this new platform to screen for genes involved in assembly if the apoptosome relative involved in inflammation, NLRP3, and to screen for genes involved in peroxisome biogenesis and peroxisome biogenesis disorder-Zellweger spectrum disorder (PBD-ZSD) related genes. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.

我们研究了神经系统中的程序性细胞死亡和细胞凋亡的生化机制。Bcl-2蛋白家族在发育过程中调节神经元的存活。我们以前发现，这个家族的一个成员，Bax，从细胞质迁移到细胞膜作为神经元死亡的关键承诺点。有趣的是，Bax在线粒体收缩位点和线粒体分裂位点聚结成大的聚集体。这是如何启动的是未知的，识别新的步骤可以作为抑制神经元死亡的药物靶点。为了更详细地询问这一步骤，我们探索了线粒体如何在健康和垂死细胞中分裂，并开发了一种新的细胞内易位事件筛选方法。 1)线粒体与其他细胞内细胞器，包括内质网（ER），过氧化物酶体和溶酶体形成接触。线粒体ER接触位点参与线粒体分裂，在Bax聚结的精确位点。在酵母中，Vps 13已经连接到线粒体ER膜接触位点，并且似乎介导两个细胞器之间的脂质转运。哺乳动物有四个VPS 13同源物：VPS 13 A-D。VPS 13 A的突变导致神经退行性疾病舞蹈病-棘红细胞增多症。VPS 13 B与科恩综合征有关，科恩综合征是一种神经发育障碍，与自闭症有共同的特征。全基因组关联研究显示功能丧失VPS 13 C突变导致帕金森病VPS 13 D突变与运动障碍有关，运动障碍被分类为脊髓小脑共济失调的一种亚型，脊髓小脑共济失调常染色体隐性遗传4型（SCAR 4）。与VPS 13 A-C相反，VPS 13 D被认为是人类细胞中的必需基因，VPS 13 D的完全缺失导致小鼠和苍蝇的胚胎致死。我们研究了人类细胞中VPS 13 A-D敲除（KO）的细胞生物学，发现VPS 13 D在线粒体形态和过氧化物酶体生物发生中具有双重作用。这与其他三个Vps 13基因形成对比，并确定了该SCAR 4基因的新生物学作用。 我们和其他人最近的工作表明，Vps 13 C或Vps 13 D的丢失与炎症有关。我们将探讨这可能是如何源于线粒体功能障碍，线粒体DNA释放到细胞质中以激活cGAS STING炎症途径，以及炎症如何与Vps 13 C-D患者的神经退行性表型相关。 2)为了揭示凋亡体如何在Bax易位至线粒体下游发挥作用以介导细胞死亡，我们开发了一种新的CRISPR筛选平台，该平台允许基于光成像对Bax易位和其他细胞易位事件进行遗传解剖。细胞通过显微镜进行筛选，并通过人工智能（AI）算法进行分类，精确识别遗传改变的表型。将具有感兴趣表型的细胞光活化并通过流式细胞术分离，并通过测序鉴定gRNA。我们目前正在使用这个新的平台来筛选参与组装的基因，如果与炎症相关的染色体相对，NLRP 3，并筛选参与过氧化物酶体生物合成和过氧化物酶体生物合成障碍-齐薇格谱系障碍（PBD-ZSD）相关基因的基因。这种方法，AI-光开关筛选（AI-PS），提供了一种新的筛选平台，能够分类广泛的哺乳动物亚细胞形态，在基因组范围内的现有方法在很大程度上无法实现的方法。