The role of VPS13A in endolysosomal and autophagic pathways: a CRISPR/Cas9-based cellular model of ChAc for phenotype-based compound screening

8Masana et al. Tremor and Other Hyperkinetic Movements DOI: 10.5334/tohm.622 Abstract The evolutionary conserved VPS13A gene is associated with the neurodegenerative disorder Chorea Acanthocytosis. We demonstrated that VPS13A is a peripheral membrane protein, associated with mitochondria, the endoplasmic reticulum, and lipid droplets. VPS13A is localized at sites where the endoplasmic reticulum and mitochondria are in close contact. VPS13A interacts with the ER residing protein VAP-A via its FFAT domain. Interaction with mitochondria is mediated via its C-terminal domain. In VPS13A-depleted cells, ER and mitochondria contact sites are decreased, mitochondria are fragmented and mitophagy is decreased. VPS13A also localizes to lipid droplets and affects lipid droplet motility. We further explored possible cellular functions of VPS13A using Drosophila melanogaster with its large cells and versatile genetic tools as a model organism. We demonstrated the presence of increased numbers of lipid droplets in specific brain areas of Vps13 mutants. We also demonstrated that Drosophila Vps13 is required for timely removal of large cellular remnants. During this process a Vps13-rich and Vps13-dependent membranous structure is being formed that surrounds the to-be-degraded cellular remnants. We will discuss how these new data link impairment of Vps13-related proteins with neurodegeneration. The role of VPS13A in endolysosomal and autophagic pathways: a CRISPR/ Cas9-based cellular model of ChAc for phenotype-based compound screening Alba Tornero-Écija1, María-Ángeles Navas2, Olivier Vincent1 and Ricardo Escalante1 1. Instituto de Investigaciones Biomédicas Alberto Sols; C.S.I.C./U.A.M.; Madrid, Spain. 2. Universidad Complutense de Madrid. Spain. Abstract VPS13A is a lipid transfer protein that localizes to different membrane contact sites between organelles and its mutation causes the rare disease chorea-acanthocytosis (ChAc). Previous work from our laboratory demonstrated that VPS13A localizes at the interface between mitochondria-endoplasmic reticulum and between mitochondria-endosomes in HeLa cells. Inhibition of VPS13A expression by siRNAs results in defects in lysosome function and accumulation of endolysosomal markers such as RAB7A and LAMP1 (Muñoz-Braceras, S; Tornero-Écija, AR; Vincent, O and Escalante, R. (2019). VPS13A is closely associated with mitochondria and is required for efficient lysosomal degradation. Dis Model Mech. 12(2)(PMID: 30709847). Accumulation of endolysosomal markers could be a useful phenotype for testing compounds in preclinical studies. However, the high cellular variability intrinsically linked to the use of siRNAs prevented us from obtaining conclusive results. We have optimized a CRISPR/Cas9 approach to generate a reliable model to compare wild-type and VPS13A Knock-out HeLa cells. The characterization of this model and preliminary results will be presented. Towards understanding the function of VPS13B in vesicular trafficking through the study of spermiogenesis Romain Da Costa1,2*, Morgane Bordessoules1,2, Magali Guilleman3, Virginie Carmignac1,4, Hortense Courot1, Amandine Bataille5, Amandine Chlémaire5, Céline Bruno1,3, Patricia Fauque1,3, Christel Thauvin1,2,6, Laurence Faivre1,2,7 and Laurence Duplomb1,2 1. Inserm, UMR1231, Equipe GAD, Université de Bourgogne Franche Comté, F-21000 Dijon, France. 2. FHU TRANSLAD, CHU Dijon, F-21000 Dijon, France. 3. Laboratoire de Biologie de la Reproduction, Hôpital François Mitterrand, Université de Bourgogne, F-21000 Dijon, France. 4. Centre de Référence Maladies Génétique à Expression Cutanée MAGEC-Mosaique, CHU Dijon, Dijon France. 5. Plateforme d’Imagerie Cellulaire CellImaP/DimaCell, Inserm LNC UMR1231, F-21000 Dijon, France. 6. Centre de Référence Déficiences Intellectuelles de Causes Rares, CHU Dijon, F-21000 Dijon, France. 7. Centre de Référence Anomalies du Développement et Syndromes Malformatifs, CHU Dijon, F-21000 Dijon, France. * [email protected] Abstract Cohen syndrome (CS) is a rare genetic disorder caused by variations affecting the VPS13B gene. It is characterized by a wide variety of clinical features that includes a typical facial dysmorphism, hypotonia, neutropenia, microcephaly, intellectual disability, and severe visual impairments. Understanding the molecular function of VPS13B is a prerequisite to the development of therapeutic approaches to treat chronic and progressive symptoms of CS. So far,