Mitofusin-PD · Regulation of mitochondria-endoplasmic reticulum tethering by Parkin: implication for Parkinson’s disease

Regulation of mitochondria-endoplasmic reticulum tethering by Parkin: implication for Parkinson’s disease

Parkin is an enzyme that is capable of adding Ubiquitin molecules to target proteins. In several model system, Parkin ubiquitinates Mitofusin, a mitochondria and endoplasmic reticulum (ER) resident protein, that bridges mitochondria to the ER, a structural feature that is essential for inter organelles communication and that affects cell survival. Since one of the hallmark of Parkinson’s Disease, both familiar and sporadic, consists of accumulation of damaged mitochondria, it is important to clarify whether Parkin can affect mitochondria activity by regulating mitochondria and ER interaction via Mitofusin. This project aims at clarify the role of Parkin in the regulation of the interaction between mitochondria and ER. Ubiquitination is a powerful tool to modulate protein activity. Balanced ubiquitination and de-ubiquitination of Mitofusin might provide a reversible mechanism to control the important biological process of ER-mitochondria interaction. In this respect, this study also seeks to identify the protein that opposes Parkin in the ubiquitination of Mitofusin. 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-Mito interaction consists of Ca2+ transfer between the two compartments, which regulation is fundamental for cell survival. We measured ER-Mito Ca2+ transfer and found it 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. Western Blotting is a technique that separates proteins on special membranes based on the protein size and electrical charge. 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 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 but not Parkin 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. De-ubiquitination enzymes are emerging as very attractive druggable candidates. Clinical trials for specific inhibitors of specific de-ubiquitination enzymes have already been approved in cancer therapy. In a similar fashion, de-ubiquitination inhibitors or activators can affect cellular response to stimuli that induce cell death via ER-mitochondria interaction and be beneficial in the treatment of Parkinson’s Disease patients. The identification of a Parkin-opposing de-ubiquitination enzyme in the regulation of ER-mitochondria interaction, might be instrumental to develop specific inhibitors or activators that can modulate the biological process of ER-mitochondria interaction and widen medical intervention.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз