Neurodegeneration and peripheral neuropathy are the major clinical consequence of human mutations affecting biosynthesis of the low abundance signaling lipid PI(3,5)P2. We have identified human and mouse mutations in the 3 major proteins of the PI(3,5)P2 biosynthetic complex: the phosphatase FIG4, the phosphokinase PIKFYVE, and the scaffold protein VAC14. The disorders that we have recently identified include hypomyelination in patients with missense mutations of FIG4 and striatonigral degeneration in patients with missense mutations of VAC14. We have extended our focus to a set of functionally related genes that phenocopy the lysosomal impairment associated with PI(3,5)P, deficiency in cultured cells. We used CRISPR/Cas genome-wide screens with Brunello and SAM libraries of sgRNAs that we expressed in the human haploid cell line HAP1. We screened for two classes of genes, genes whose knock-out generates enlarged acidic vesicles, and genes whose overexpression rescued the enlarged acidic vesicles in PI(3,5)P2 deficient cells. We employed FACS sorting to separate cells with enlarged acidic vacuoles from cells lacking vacuoles. The initial screens identified both known genes and novel genes of unknown function. We will focus future efforts on novel genes not previously recognized for their role in lysosome biology. In year 1 we will analyze C4orf32, C10orf35 and SLC12A9, which were positive in both of our assays. Knockout of these genes results in cell vacuolization, and their overexpression rescues vacuoles in FIG4 null cells. We will examine their effects on intracellular PI(3,5)P2 concentration. Protein interactions will be investigated by proximity-directed biotinylation using TURBOID. In vivo phenotypes of mouse knock-out models will inform the search for patient mutations in these unstudied genes. We will test the ability of C4orf32 and C10orf35 to rescue Fig4 null mice in vivo, as a prerequisite for their use in gene therapy. We will carry out new sensitized screens to detect transcription factors and proteins that interact directly with FIG4 and VAC14. We will continue to evaluate patient variants of unknown significance (VUS) in FIG4, VAC14, and novel genes identified here, in order to confirm their role in an increasingly broad spectrum of genetic disorders. The proposed experiments combine genetic analysis of new variants with functional dissection of the PI(3,5)P2 signaling pathway to advance our understanding of known and newly identified human disorders.
We demonstrated previously that mutations of the FIG4 and VAC14 genes are regulators of lysosome turnover and mutations in these genes are responsible for human neurological disorders. We expanded the network of genes contributing to this pathway using genome-wide CRISPR screening. We propose to characterize the newly identified members of this pathway discovered in the previous funding period, extend the gene discovery screening program, and evaluate the contribution of newly identified genes to human disease.
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