Programmed Cell Death In The Nervous System

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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.