Fig. 1

MLKL inhibition or deficiency decreased cell death in response to 6-OHDA plus TNF-α, or toxic α-Syn PFFs treatments. a-b. SH-SY5Y cells (a) or primary MEF cells (b) were treated with or without 6-OHDA plus TNF-α, followed by adding different concentrations of MLKL inhibitor NSA for 24 h. The cell viability was measured by the CCK8 assay and normalized to untreated cells. c. Primary MEF cells were transfected with GFP-tagged A53T synuclein (A53T) or empty vector (Ev). After 24 h, the transfected cells were treated with or without 6-OHDA, TNF-α, and NSA for another 24 h. d. Mlkl^+/+ and Mlkl^−/− MEF cells were transfected with GFP-tagged A53T synuclein. After 24 h, the transfected cells were treated with or without 6-OHDA plus TNF-α for another 24 h. e-f. Cells were treated as described in a and c, respectively. Then western blot analysis was performed for inducible nitric oxide synthase (iNOS) and phosphorylated MLKL (p-MLKL). The quantification results were shown in the lower panels. g-h. Mlkl^+/+ (WT) and Mlkl^−/− MEF cells were treated with GFP-tagged A53T synuclein (A53T) or empty vector (Ev). After 24 h, the transfected cells were treated with or without 6-OHDA plus TNF-α for another 24 h, followed by western blot analysis (g) and cytokine secretion measurement (h). i-k. Assessment of cell viability and protein expression in primary neuronal cells. Primary neuronal cells were treated with or without α-Syn preformed fibrils (PFFs) and NSA for 14 days followed by CCK8 assay and western blotting (i). Mlkl^+/+ (WT) and Mlkl^−/− primary neuronal cells were treated with or without PFFs over a period of 14 days followed by the CCK8 assay and western blotting (k). Quantitative results from the experiments are presented in two panels. The upper panel displays the quantified data from the treatments in i, and the lower panel presents the quantification corresponding to the treatments in k. l-n. Human induced pluripotent stem cell (iPSC)-derived midbrain organoids (hMOs) were subjected to treatments with PFF, both with and without concurrent AAV9-shMLKL (to facilitate MLKL knockdown). Western blot analysis (l) and immunofluorescence staining (m) were employed to assess the treatments. The quantified expression levels of p-α-Syn are presented in the upper section of l, while the quantifications of p-MLKL intensity are illustrated in n. Scale bars, 10 μm. All data are representative of three independent experiments. The error bars represented the standard deviations (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001