AbstractBackgroundIt is generally believed that the pathogenesis of PINK1/parkin‐related Parkinson's disease (PD) is due to a disturbance in mitochondrial quality control. However, recent studies have found that PINK1 and Parkin play a significant role in mitochondrial calcium homeostasis and are involved in the regulation of mitochondria–endoplasmic reticulum contact sites (MERCSs).ObjectiveThe aim of our study was to perform an in‐depth analysis of the role of MERCSs and impaired calcium homeostasis in PINK1/Parkin‐linked PD.MethodsIn our study, we used induced pluripotent stem cell–derived dopaminergic neurons from patients with PD with loss‐of‐function mutations in PINK1 or PRKN. We employed a split‐GFP‐based contact site sensor in combination with the calcium‐sensitive dye Rhod‐2 AM and applied Airyscan live‐cell super‐resolution microscopy to determine how MERCSs are involved in the regulation of mitochondrial calcium homeostasis.ResultsOur results showed that thapsigargin‐induced calcium stress leads to an increase of the abundance of narrow MERCSs in wild‐type neurons. Intriguingly, calcium levels at the MERCSs remained stable, whereas the increased net calcium influx resulted in elevated mitochondrial calcium levels. However, PINK1‐PD or PRKN‐PD neurons showed an increased abundance of MERCSs at baseline, accompanied by an inability to further increase MERCSs upon thapsigargin‐induced calcium stress. Consequently, calcium distribution at MERCSs and within mitochondria was disrupted.ConclusionsOur results demonstrated how the endoplasmic reticulum and mitochondria work together to cope with calcium stress in wild‐type neurons. In addition, our results suggests that PRKN deficiency affects the dynamics and composition of MERCSs differently from PINK1 deficiency, resulting in differentially affected calcium homeostasis. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.