Fig. 3
MLKL deficiency protects dopaminergic neuron loss and decreases hyperphosphorylated α-synuclein in the A53T transgenic mice. a. Representative immunostaining for phosphorylated α-Syn (p-α-Syn129S) in the cortex, striatum, and substantia nigra (SN) regions of WT, Tg-Mlkl+/+ and Tg-Mlkl−/− mice. Scale bars, 100 μm. The whole-brain sections were shown in the top right corner. The solid rectangles were zoomed from the selected dashed rectangles in the cortex, striatum, and SN regions, respectively. The quantification results of p-α-Syn129S are shown in b. c. Representative immunofluorescence image of the striatum region in the frozen brain sections of WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice staining with DAPI (blue, denoting the nuclear signal) and anti-p-α-Syn antibody (green). Scale bars, 50 μm. d. Dopaminergic neurons were determined by tyrosine hydroxylase (TH) staining. Representative TH immunostaining images of the cortex and SN sections from WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice were shown in d. The whole-brain sections were shown in the bottom left corner. The solid rectangles were zoomed from the selected dashed rectangles in the striatum and SN regions. Scale bars, 100 μm. e. Quantification of the total number of TH-positive cells in the entire striatum and SN regions, corresponding with d. f-g. Representative Western blot results of the expression levels of TH, α-Syn, p-α-Syn, and iNOS in the cortex, striatum, and SN sections of the WT, Tg-Mlkl+/+ and Tg-Mlkl−/− mice (f). Quantifications of the expressions for TH, α-Syn, p-α-Syn, and iNOS were shown in g. h-i, Western blot results displaying α-Syn and p-α-Syn levels in whole brain tissue (Whole), soluble fractions, and insoluble fractions from WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice. The soluble fraction contains cytoplasmic proteins, while the insoluble fraction includes membrane-bound proteins, organelle-associated proteins, and nuclear proteins. Quantifications of the results for α-Syn and p-α-Syn were shown in i. All data are representative of three independent experiments. The error bars represented the standard deviations (SD). ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance