Impact of PINK1 knockout on expression and phosphorylation of mitochondrial proteins and dopamine regulation: insight into early-stage Parkinson's Disease in the rat model.

Purpose: Mutations in the gene for PINK1, a mitochondrial protein, causes recessively inherited young-onset Parkinson's Disease (PD). This first report indicated the mutation when mapped to the PARK6 locus in a Spanish family. To model the impact of the PINK1 mutation, a rat model has been developed, but ambiguous results on dopamine regulation and motor function have been reported early in the lifespan. Here we investigated the impact of this mutation on Parkin, a mitochondrial protein that functions with PINK1 in mitophagy and dopamine regulation in early development. We hypothesized that compensatory changes in Parkin expression or its phosphorylation would occur, coinciding with changes in dopamine regulation due to the loss in expression or function of PINK1. Methods: In five-month-old PINK1 KO and age-matched wild-type (WT) rats, we confirmed the genotype by PCR, indicating the 26-base pair deletion as shown in the human mutation. Protein expression of PINK1, Parkin, phosphorylated Parkin, and TOM-20 were evaluated in comparison with dopamine tissue content, dopamine turnover, and tyrosine hydroxylase expression using quantitative Western Blotting. Results: In the PINK1 KO, dopamine tissue content decreased 43% in the striatum and 26% in the substantia nigra as compared to wild-type rats, commensurate with increased dopamine turnover in both regions. Parkin, phosphorylated Parkin, and TOM-20 protein expression were not significantly different in the PINK1 KO rat. Conclusion: This study suggests that compensatory changes in Parkin expression or phosphorylation are not modified in the PINK1 KO rat, though there is increased dopamine turnover and loss in both regions. Our findings indicate that dopamine regulation is affected early in the lifespan of the PINK1 rat, which may portend the overt loss of dopamine that leads to the parkinsonian signs with increasing age. The five-month-old rats used in this study correlate to a human age of approximately 20 years. Given that individuals with the PINK1 knockout mutation begin seeing motor impairments around 34 years of age, targeting mitochondrial dysfunction prior to when symptoms arise could diminish nigrostriatal dopamine loss and be a promising approach to the development of future treatment.