Inactive ryanodine receptors sustain lysosomal availability for autophagy by promoting ER-lysosomal contact site formation

Abstract Lysosomal and endoplasmic reticulum (ER) Ca 2+ release mutually influence each other’s functions. Recent work revealed that ER-located ryanodine receptor (RyR) Ca 2+ release channels suppress autophagosome turnover by the lysosomes. In familial Alzheimer’s disease, in which excessive RyR activity and lysosomal dysfunction are early hallmarks, inhibiting RyR restored autophagic flux by normalizing lysosomal vacuolar H + -ATPase (vATPase) levels. However, the mechanisms by which RyR control lysosomal function and how it involves the vATPase remain unknown. In this work we demonstrate for the first time that, RyR directly interact with the ATP6v0a1 subunit of the vATPase, contributing to ER-lysosomal contact site formation. This interaction suppresses RyR-mediated Ca²⁺ release, which impacts the handling of lysosomal cargo. Pharmacological inhibition of RyR activity was sufficient to mimic these effects on lysosomal cargo handling. Further investigation identified endogenous RyR activity in cortical neurons derived from human induced pluripotent stem cells to promote lysosomal exocytosis. Inhibiting this spontaneous RyR activity limits lysosomal exocytosis, thus retaining lysosomes inside cells. The latter increases ER-lysosomal contact site formation, rendering lysosomes more available for autophagic flux. In summary, these findings establish RyRs/ATP6v0a1 complexes as novel ER-lysosomal tethers that dynamically/Ca 2+ dependently regulate the intracellular availability of lysosomes to participate in autophagic flux.