Cytoprotection achieved by Ca²⁺ administration requires functional calcineurin signaling.

(A) Schematic of the main Ca²⁺ signaling pathway in yeast. Cytosolic Ca²⁺ binds to Calmodulin, which activates the Ca²⁺/calmodulin-dependent protein kinases Cmk1 and Cmk2 and protein phosphatase calcineurin. Calcineurin consists of a regulatory (Cnb1) and one of two catalytic subunits (Cna1 or Cna2) and dephosphorylates numerous cellular targets, among them the calcineurin-responsive zinc finger transcription factor Crz1, which in response translocates into the nucleus to adapt gene expression. Calcineurin can be inhibited by the immunosuppressant drug FK506. (B) Immunoblot analysis of protein extracts from WT cells and indicated deletion mutants after 24 h of αSyn expression. Blots were probed with antibodies directed against FLAG-epitope to detect FLAG-tagged αSyn and against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. (C) Cell death determined by propidium iodide (PI) staining of WT cells and calcineurin deletion mutants expressing αSyn or harboring the vector control (Ctrl.). Means ± s.e.m; n = 4. (D) Aequorin luminescence-based determination of basal cytosolic Ca²⁺ levels in WT and Δcnb1 cells expressing αSyn for 8 h and 12 h. Data were normalized to WT vector control. Means ± s.e.m; n = 12. (E) Confocal micrograph of WT and Δcnb1 cells expressing αSyn^GFP for 24 h. Cells have been counterstained with PI to visualize dead cells. Scale bar represents 5 μm. Values indicate mean percentages of cells with αSyn^GFP aggregates, with the following s.e.m.: WT: 28.1% ± 1.5% (in total 395 cells were evaluated); Δcnb1: 37.5% ± 3.2% (in total 299 cells were evaluated); n≥4. *p<0.05. (F) Cell death determined by PI staining of WT cells and indicated deletion mutants expressing αSyn for 36 h or harboring the vector control. Cells were supplemented or not with 10 mM Ca²⁺ at the time point of shift to galactose media. Means ± s.e.m; n = 4. (G) Clonogenic death of cells described in (F) grown for 36 h on galactose prior to determination of colony forming units on glucose full media agar plates. Death induced by αSyn was calculated via normalization to isogenic and similarly treated vector control. Means ± s.e.m; n = 8–16. (H) Flow cytometric quantification of cell death via PI staining of WT cells expressing αSyn for 36 h or harboring the vector control upon addition of 10 mM Ca²⁺ and 5 μM FK506. Means ± s.e.m; n = 8–12. (I) Flow cytometric quantification of cell death via PI staining of WT and Δcnb1 cells expressing αSyn or harboring the empty vector upon late addition of Ca²⁺. Cells grown on galactose media were analyzed prior to the addition of 10 mM Ca²⁺ (12 h) and at 36 h. Means ± s.e.m; n = 4. *p<0.05, and ***p<0.001.