Objective: Since the Membrane contact sites (MCS) between ER and mitochondria are of particular importance for cellular calcium homeostasis, we hypothesize that MCS dysfunction in absence of VPS13A contributes to impaired calcium homeostasis, consequently contributing to neurodegeneration.

Background: VPS13A is a membrane protein located at MCS involved in lipid exchange between the endoplasmic reticulum (ER) and mitochondria and between lipid droplets and mitochondria, respectively. Mutations in the respective gene cause chorea-acanthocytosis/VPS13A disease, a neurodegenerative disorder of the young adulthood.  Furthermore, in VPS13A deficient cells, calcium homeostasis via ORAI1-mediated store operated calcium entry (SOCE) is disturbed, although the exact mechanism has not been completely discovered yet.

Method: We used patient-derived cells (fibroblasts and iPS-derived neurons) with VPS13A deficiency compared to cells from healthy donors. The cells were transfected with a specific marker for mitochondria-ER contact sites (MERCS) and with the ER marker BFP-KDEL. Subsequently, cells were stained with MitoTracker deep red and the calcium dye Rhod2-AM. Using super resolution live cell imaging (LSM900 confocal microscope with Airyscan 2 module, Zeiss), images were acquired over 2 minutes before and after treatments with Thapsigargin or Ru360. Thapsigargin is an inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), preventing calcium uptake by the ER, while Ru360 is a specific inhibitor of the mitochondrial calcium uniporter (MCU), thereby blocking calcium uptake by the mitochondria.

Results: Our analysis show calcium dyshomeostasis in VPS13A-deficient fibroblasts and neurons. Specifically, in  both cell types we observed a MCU-independent mitochondrial calcium influx upon using treatment Thapsigargin and Ru360. Furthermore, VPS13A-deficient cells have an increased baseline calcium level at mitochondria, endoplasmatic reticulum and MERCS.

Conclusion: Our initial findings support the hypothesis that VPS13A is involved in the regulation of cellular, however, little is known about the interplay of lipid metabolism and calcium homeostasis. Our study may contribute to the understanding of these mechanisms and how their disruption in VPS13A deficiency is involved in neurodegeneration.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/calcium-phenotype-in-cellular-models-of-vps13a-disease/