Role of Parkin and Its Opponent in Parkinsonís Disease Onset
Rapid Response Innovation Awards, 2014
Parkin is an enzyme that adds molecules to proteins, including one called mitofusin, which bridges mitochondria (cellís powerhouse) to the endoplasmic reticulum (structure essential for intercellular communication). Parkin and its gene also are associated with Parkinsonís disease (PD). Since one of the hallmarks of PD is accumulation of damaged mitochondria, we aim to clarify whether parkin can affect mitochondria activity by regulating mitochondria and endoplasmic reticulum interaction via mitofusin.
This project will clarify the role of parkin in the interaction between mitochondria and endoplasmic reticulum. Balancing parkinís action on mitofusion may provide a mechanism to control this important biological process and interrupt Parkinsonís biology. In this respect, this study also seeks to identify the protein that opposes parkin in the molecular management of mitofusin.
We will measure the degree of interaction between endoplasmic reticulum and mitochondria in cells that lack parkin and compare it to normal cells. We will visualize mitochondria in red and endoplasmic reticulum in green by using fluorescent markers and measure the degree of interaction by looking at the overlap between red and green fluorescence (yellow spots). We will also screen an enzyme library to identify a potential enzyme that is opposing parkin in the modulation of mitofusin. We will use a western blotting approach, which is a technique that separates proteins on special membranes based on the protein size and electrical charge.
Impact on Diagnosis/Treatment of Parkinsonís Disease: † † † † † ††
Inhibitors or activators that affect endoplasmic reticulum-mitochondria interaction may be beneficial in slowing or stopping Parkinsonís progression.
Next Steps for Development:
The identification of a parkin-opposing enzyme in the regulation of endoplasmic reticulum-mitochondria interaction may be instrumental to develop specific inhibitors or activators that can modulate this biological process and widen medical intervention.
We measured the degree of interaction between ER and mitochondria in cells, which lack Parkin and compare it to normal cells. We visualized mitochondria in red and ER in green by using fluorescent markers. We used a confocal microscopy approach to measure the degree of interaction by looking at the overlap between red and green fluorescence (yellow spots). We found that the degree of interaction between ER and mitochondria is decreased in Parkin deficient cells. The functional counterpart of ER-Mitochondria interaction consists of Ca2+ transfer between the two compartments, which regulation is fundamental for cell survival. We measured ER-Mitochondria Ca2+ transfer and found it is decreased. This impairment might impact Ca2+ signaling and affect processing, folding as well as proper export of proteins that are synthetized on the ER and are required for cell survival.
In addition, we screened a de-ubiquitination (DUBs) enzyme library to identify a potential de-ubiquitination enzyme that is opposing Parkin in the ubiquitination of Mitofusin. To do so we used a Western Blotting approach and Mitofusin steady state and ubiquitinated levels as read out for the screening. We identified five potential hits, which expression affects Mitofusin steady state and ubiquitinated levels. Among them, we identified de-ubiquitination enzyme USP8 which downregulation results in decreased Mitofusin protein levels, both in vitro and in vivo, and enhanced mitochondria fission. Overall, this approach identified a new regulator of Mitofusin steady state levels, which opposes Parkin effect on Mitofusin steady state levels and enhances mitochondria fission. In vivo results in flies showed full recue of PINK1 mutant locomotor deficiencies when crossed with USP8 deficient flies. This result strongly suggests that counteracting Parkin activity on its target Mitofusin can be beneficial in PINK1 mutant background.
Professor at University of Padova, Department of Biology
Location: Padova, Italy
Assistant Professor at University of Padova, Department of Biology
Location: Padova, Italy