Programmed Cell Death In The Nervous System

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。Bcl2蛋白家族在发育过程中调节神经元的存活。我们之前发现，这个家族的一个成员Bax，作为神经元死亡的关键承诺点，从胞浆迁移到细胞膜。有趣的是，Bax在线粒体收缩部位和线粒体分裂部位聚集成大团聚体。这是如何启动的尚不清楚，识别新的步骤可以作为抑制神经元死亡的药物靶点。为了更详细地探讨这一步骤，我们探索了线粒体如何在健康和濒临死亡的细胞中分裂，并开发了一种新的细胞内易位事件筛查。 1)线粒体与细胞内其他细胞器，包括内质网、过氧化物体和溶酶体形成接触。线粒体内质网接触部位参与线粒体的分裂，也就是Bax结合的精确部位。在酵母中，Vps13已经连接到线粒体ER膜接触位置，它似乎介导了两个细胞器之间的脂类运输。哺乳动物有四个VPS13同源物：VPS13A-D。VPS13A的突变会导致神经退行性疾病舞蹈病-棘细胞增多症。VPS13B与科恩综合征有关，科恩综合征是一种与自闭症有共同特征的神经发育障碍。全基因组关联研究表明，VPS13C突变功能丧失会导致帕金森氏症。VPS13D突变与运动障碍有关，被归类为脊髓小脑性共济失调的一个亚型，脊髓小脑性共济失调常染色体隐性遗传性4(SCAR4)。与VPS13A-C不同，VPS13D被认为是人类细胞中的一个必不可少的基因，VPS13D的完全缺失会导致小鼠和果蝇的胚胎死亡。我们研究了VPS13A-D基因敲除(KO)在人类细胞中的细胞生物学，发现VPS13D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Vps13基因进行了对比，并确定了该SCAR4基因的一个新的生物学作用。我们和其他人的最新工作表明，Vps13C或Vps13D的丢失与炎症有关。我们将探索这可能是如何源于线粒体功能障碍，线粒体DNA释放到胞浆中以激活cGAS刺痛炎症途径，以及炎症可能如何与Vps13C-D患者的神经退行性表型相关。 2)为了揭示Bax易位到线粒体下游的凋亡体如何调节细胞死亡，我们开发了一个新的CRISPR筛查平台，该平台可以基于光成像对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，从而准确地识别基因改变的表型。具有感兴趣表型的细胞被光激活并通过流式细胞仪分离，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，如果凋亡体相对参与炎症，NLRP3，并筛选参与过氧化物酶体生物发生和过氧化物酶体生物发生障碍-Zellweger谱障碍(PBD-ZSD)相关基因。这种方法，AI-光开关筛选(AI-PS)，提供了一个新的筛选平台，能够对广泛的哺乳动物亚细胞形态进行分类，这一方法在目前的基因组水平上基本上是无法实现的。

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.