Disordered motor coordination was evaluated in 13-month-old SNCA^A53T mice using the rotarod test (A, the time latency to drop from the rotarod) and pole climbing (B, the time latency to reach the bottom of the pole). WT mice were used as controls. Significance was determined by 2-way repeated measures ANOVA (n = 5). (C) Immunofluorescence of substantia nigra sections (dotted area) labeled with TH antibody (red) and DAPI (blue). Scale bar: 200 μm. (D) Western blotting (left) and quantitative analysis (right) of 4-HNE expression in midbrain. (E) MDA, (F) iron, and (G) glutathione (GSH) in midbrain were measured (n = 5). The phospholipids in midbrain were isolated and detected by LC-MS/MS (n = 4). Data of phospholipids were extracted and displayed as (H) principal component analysis (PCA) and (I) volcano plots showing the fold changes (X-axis) versus significance (Y-axis). (J) Ferroptosis-related genes were detected by qPCR assay and relative expressions were displayed as a heatmap. Slc7a11, solute carrier family 7 member 11; Gpx4, glutathione peroxidase 4; Pla2g6, phospholipase A2 group VI; Alox5/12/15, arachidonate 5/12/15-lipoxygenase; Tfrc, transferrin receptor; Ncoa4, nuclear receptor coactivator 4; Lpcat3, lyso-PC acyltransferase 3; Ptgs2, prostaglandin-endoperoxide synthase 2; Acsl4, acyl-CoA synthetase long chain family member 4; Dmt1, ferrous ion membrane transport protein DMT1. (K) IHC of midbrain sections labeled with GPX4 and 4-HNE antibodies and hematoxylin. IHC magnification (top) and IHC score (bottom). Scale bar: 50 μm. (L) Western blotting (top) and quantitative analysis (bottom) of GPX4 and TFRC expression in midbrain, cortex, and hippocampus, respectively. (M) MDA level was measured in the cortex and hippocampus, respectively (n = 5). All data represent mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, by independent-samples t-test.