Fig. 2.

The increasing interaction between hα-syn and ACO2 in mitochondria affects ACO2 activity. (A) Western blots showing the expression of hα-syn and ACO2 in cytosolic (Cyto, left) and mitochondrial (Mito, right) lysates isolated from the striatum of 9-, 12-, and 15-month-old WT and TG mice. VDAC1 served as a mitochondrial marker and β-actin as a loading control. (B) Co-IP experiments showing the interaction between hα-syn and ACO2 using the ACO2 antibody in Mito lysates isolated from the striatum (left) and midbrain (right) of 9-, 12-, and 15-month-old WT and TG mice. The hα-syn/ACO2 interaction increased in the mitochondria of TG mice with age. Homologous IgG was used as the experimental control. (C–F) ACO2 activity (C–D) and mitochondrial complex I activity (E–F) in the striatum and midbrain of 9-, 12-, and 15-month-old WT and TG mice. n = 8. (G–J) Levels of MDA (G), GSH (H), GSSG (I), and Fe²⁺ (J) in the striatum of 9-, 12-, and 15-month-old WT and TG mice. n = 8. MDA: Malondialdehyde; GSH: glutathione; GSSG: oxidized glutathione. (K) With increased hα-syn protein levels and the same amount of GST-ACO2 protein in the system, GST pulldown experiments showing the interaction between GST-ACO2 protein and different levels of hα-syn protein. Right: Quantitation of hα-syn protein-to-GST-ACO2 protein level ratio after GST-pulldown. n = 3. (L) ACO2 protein activity assay. The system was supplemented with ACO2 protein and different amounts of hα-syn protein (0.25, 0.5, 1, and 2 μg) or 2 μg Pgk1 protein. n = 4. (I) ACO2 protein activity assay. The system was supplemented with ACO2 protein, 2 μg of hα-syn protein, and different concentrations of citrate (5, 10, and 20 mM). n = 4. Data are expressed as the mean ± SEM. P values were determined by unpaired t-test (C–J) and one-way ANOVA (K–M). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. WT (C–J), 0.25 μg of hα-syn (K), or ACO2 (L–M), ###P < 0.001 vs. ACO2 + hα-syn (I).