Effect of Parkinson's Disease PINK1 clinical mutants on Mitophagy
Filipa, B. Gonçalves1, Francisco J. Enguita 1, Vanessa, A. Morais1*
1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
Parkinson's disease (PD), the second most common neurodegenerative movement disorder, affects approximately 1% of the population over 65. At present, there is only symptomatic but no causal cure for PD. Mitochondria are double membrane-bound organelles that are essential for energy production and cellular homeostasis in eukaryotic cells. Deficit in Complex I activity and an increase in oxidative damage were identified in multiple brain regions from PD patients, suggesting a connection between mitochondria and PD. This association has been furthered strengthened by the identification of mutations in PINK1, a gene that leads to early-onset recessive PD. PINK1, a mitochondria targeted Serine/Threonine kinase, interacts with several substrates to regulate mitochondrial functions. For example, in a healthy pool of mitochondria, PINK1 regulates ATP production by phosphorylating the Complex I subunit NdufA10. However, when in the presence of damaged mitochondria, PINK1 phosphorylate Parkin and triggers mitochondria for clearance via mitophagy. Therefore, understanding the regulation of PINK1 activity is pivotal.
We aim to understand how the PD-causing clinical mutations of PINK1 cause loss of kinase function and consequent lack of substrate phosphorylation; namely Parkin, NdufA10 and Miro. For this, PINK1 clinical mutant forms were generated and their ability to phosphorylate these substrates will be assessed using an in vitro phosphorylation assay. The (auto)phosphorylation of PINK1 itself will also be determined. Additionally, the ability of these PINK1 clinical mutants to recruit Parkin and trigger mitophagy will be evaluated using immunofluorescence and Western blotting techniques. Moreover, modeling of PINK1 mutants will be performed in order to correlate structure/function of PINK1 with its different substrates. Autophagic receptors, such as p62 and LC3-II, and specific mitophagy receptor signal, as OPTN and NDP52, will also be assessed in mitophagy induced conditions.
At present, our results suggested that PINK1 is needed for Parkin recruitment to mitochondria, however, this process is independent of kinase activity.