(A) AAV9-PINK1-Flag was injected into the cortex and SN of a 25-year-old WT monkey. The animal was analyzed 9 weeks later. Immunostaining with anti-flag antibody showing that the transgenic PINK1 could be unambiguously detected in the injected brain cortex. (B and C) Representative results of Western blotting (B) and immunostaining (C) showing that PINK1 overexpression in the 25-year-old monkey SN increased pS65-parkin phosphorylation. (D) The accumulation of pS129-α-syn in the old (25 years old) monkey SN was much more abundant than in the young (8 years old) monkey SN and could be reduced by overexpression of PINK1 via AAV9-PINK1-Flag injection. The density of aggregates per image is shown beneath the images (n = 2 animals per group). (E–H) AAV9-WT parkin and AAV9-S65A parkin were injected into the striatum and SN of old WT monkeys aged 22–25 years. The animals were analyzed 9 weeks later. Western blotting of the SN shows that only WT parkin, not mutant parkin (S65A), is phosphorylated (E, arrow). Representative immunostaining images (low magnification in F and high magnification in G) show that the transgenic WT parkin and S65A parkin could be unambiguously detected in the injected striatum tissues, and WT parkin, but not S65A parkin, reduced pS129-α-syn accumulation in the aged monkey brain. WT parkin, but not S65A parkin, was also able to reduce pS129-α-syn accumulation in the SN of the old monkeys (H). (I) Quantification of pS129-α-syn immunostaining density in the monkey striatum and SN injected with AAV WT parkin or AAV S65A parkin. The number of pS129-α-syn aggregates in each image (×40) was counted and color coded for 2 animals (22 and 25 years old). Representative Western blotting results and immunostaining images are from multiple technical replicates of 1 monkey (A–C) or 2 monkeys (D–H) for each group.