Fig. 1: CO-induced calcineurin activity suppresses necroptosis through MLKL Dephosphorylation in a model of neuronal damage caused by MPTP.

a–d SH-SY5Y cells were treated with 20 μM CORM-A1 or 10 μM Nec-1 (necroptosis inhibitor) for 1 h and then treated with 1 mM MPP⁺ for 24 h. a Cell viability was determined by WST-8 assay. Necrotic cell death was detected by LDH secretion (b) and Sytox green staining (c). d MLKL phosphorylation was assessed by western blotting. e, f 8-week-old Mlkl^+/+ and Mlkl^−/− mice (n = 5) were treated with 250 ppm CO gas by inhalation or with Nec-1 (10 mg/kg, intraperitoneal [i.p.]) for 13 days. Starting on day 4, mice were challenged with MPTP (25 mg/kg, i.p.) for 7 days. Dopaminergic neurons of the striatum region in Mlkl^+/+ and Mlkl^−/− mice were determined by tyrosine hydroxylase (TH) staining (e). scale bar: 100 μm. The quantification of TH staining is shown in the middle panel. The protein expression levels of TH in the midbrain were detected by western blotting (f). g, h SH-SY5Y cells were pretreated with 20 μM CORM-A1 for 1 h followed by stimulation with 1 mM MPP⁺ for 24 h. After 12 h of treatment, cells were incubated with calcineurin inhibitors, 20 μM Cyclosporin A (CsA) or 10 μM FK506, for 12 h. Necrotic cell death was monitored by Sytox green staining (g). Sytox green fluorescence was analyzed by flow cytometry (left), and quantification of fluorescence was normalized (right). Harvested cells were subjected to western blotting using antibodies against p-MLKL and MLKL (h, left). Quantification of p-MLKL is shown in the right panel. i, j SH-SY5Y cells were transfected with scRNA or PP2BAα-targeting siRNA (siPP2BAα) and then treated with 20 μM CORM-A1 followed by treatment with 1 mM MPP⁺ for 24 h. MLKL phosphorylation and PP2BAα knockdown were assessed by western blotting (i). The quantification of phosphorylated MLKL was analyzed (j). k, l MLKL overexpressing SH-SY5Y cells were treated with 0.5 and 1 mM MPP⁺ (k) or 10 and 100 ng/ml TNF-α in the presence of 5 μM BV6 and 20 μM zVAD (l) for 24 h or 6 h, respectively. Cell lysates were immunoprecipitated with an anti-MLKL antibody and then incubated with recombinant calcineurin. The reaction mixtures were subjected to western blot analysis with antibodies to p-MLKL and MLKL (lower panel). The relative phosphorylated MLKL/MLKL band density ratio was determined (upper panel). m SH-SY5Y cells were treated with 10 and 100 ng/ml TNF-α in the presence of 5 μM BV6 and 20 μM zVAD (TBZ) for 3 h and then additionally treated with CsA or FK506 for 3 h. The expression of p-MLKL and MLKL was measured by immunoblotting. Quantification of the data is shown in the right panel. Data were expressed as mean ± SD; **p < 0.01, ***p < 0.001, and NS not significant.