(A) Schematic diagram showing that 8-week-old Gpx4^fl/fl homozygous mice were bilaterally injected with AAV-Cre into the substantia nigra. (B) The strain of conditional Gpx4 knockdown mice acquired from A were verified by Western blotting analysis (n = 5 mice each group). Disordered motor coordination of Cre-injected mice was assessed by behavioral tests, including (C) rotarod and (D) pole climbing. Statistics were determined by 2-way repeated measures ANOVA (n = 7 mice each group). (E and F) Catwalk gait analysis (n = 7 mice each group). (G) IHC of coronal brain sections labeled with TH antibody and hematoxylin (left; scale bar: 2 mm). The substantia nigra (dotted area) were amplified on right; scale bar: 500 μm. (H and I) DA and the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in striatum were quantitated by HPLC-ECD, and the turnover rate of DA and the metabolites were calculated (n = 3 mice each group). (J) α-Synuclein expression and aggregation was unaffected in both midbrain (left) and cortex (right) in the Gpx4 CKD mice as determined by Western blotting analysis (n = 5 mice each group). (K) Western blotting (left) and quantitative analysis (right) of 4-HNE expression in midbrain (n = 5 mice each group). The contents of (L) MDA and (M) GSH were measured in midbrain (n = 5 mice each group). The phospholipids in the midbrain were isolated and detected by LC-MS/MS (n = 4 mice each group). Data of oxidized phospholipids were extracted and displayed as (N) PCA and (O) volcano plots showing the fold changes (X-axis) versus significance (Y-axis) by t test. (P) Ferroptosis-related genes were detected by quantitative real-time PCR assay and the relative expressions were displayed as heatmap (n = 6 mice each group). All data represent mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, by independent-samples t tests.