Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency.

VPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea-acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumulation of endosomal and lysosomal markers, suggesting a defect in lysosomal degradation capacity leading to partial autophagic dysfunction. Our goal was to determine whether compounds that modulate the endo-lysosomal pathway could be beneficial in the treatment of ChAc. To test this hypothesis, we first generated a KO model using CRISPR/Cas9 to study the consequences of the absence of VPS13A in HeLa cells. We found that inactivation of VPS13A impairs cell growth, which precludes the use of isolated clones due to the undesirable selection of edited clones with residual protein expression. Therefore, we optimized the use of pool cells obtained shortly after transfection with CRISPR/Cas9 components. These cells are a mixture of wild-type and edited cells that allow a comparative analysis of phenotypes and avoids the selection of clones with residual level of VPS13A expression after long-term growth. Consistent with previous observations by siRNA inactivation, VPS13A inactivation by CRISPR/Cas9 resulted in accumulation of the endo-lysosomal markers RAB7A and LAMP1. Notably, we observed that rapamycin partially suppressed the difference in lysosome accumulation between VPS13A KO and WT cells, suggesting that modulation of the autophagic and lysosomal pathway could be a therapeutic target in the treatment of ChAc.