Dundee Research Interest Group

A busy two weeks for me, learning how to transiently transfect my cultured cells and perform various experiments with them to show that my antibody for PPP2R5D works. It was a great relief to find that my antibody for PPP2R5D did indeed work well, allowing me to identify it clearly and assuredly in my future experiments. Obtaining an effective antibody was a critical hurdle to allow me to monitor how the activity of PPP2R5D changes in cells in response to certain treatments, so I can now hit the ground running.

My first main experiment has been to try to implicate PPP2R5D in the dephosphorylation of Ubiquitin following mitochondrial stress. If we recall that mitochondrial stress/damage initiates the PINK1/Parkin pathway - this pathway controls the removal of damaged mitochondria from cells. Components of this pathway are frequently mutated in Parkinson’s disease which results in a disturbance of mitochondrial removal. Activation of PINK1 and Parkin leads to the accumulation of Ubiquitin on the surface of the mitochondria. This Ubiquitin becomes phosphorylated (the addition of a small chemical group PO42-) by PINK1 and it then acts as a strong signal that tells the cell this mitochondria is damaged and it needs to be removed.

It is possible for me to initiate the PINK1/Parkin pathway in cells with the addition of 2 compounds: Antimycin and Oligomycin. After this treatment I can measure the levels of phosphorylated ubiquitin (or pUb) and see that there is a dramatic increase. If I then wash away the Antimycin and Oligomycin, over time we see a steady decrease in the levels of pUb, which indicates there must be an antagonistic mechanism that removes phosphate groups from Ubiquitin. As I have mentioned before, PPP2R5D (my protein of interest) is a component of a protein called PP2A. PP2A is a phosphatase, which means its role is to remove phosphates from certain substrates… but is one of the substrates Ubiquitin? This is the question my first experiment is trying to answer.

Firstly, I treated the cells with Antimycin and Oligomycin to stimulate the PINK1/Parkin pathway. Following this treatment, I added specific inhibitors of PP2A – these will block the phosphate removal activity of PP2A. Hence, if PP2A plays a role in the dephosphorylation of Ubiquitin, when the inhibitor is added, I will not observe the reduction in pUb levels.

Apologies science overload!

As I write, I am awaiting the results of this experiment. Hopefully by Monday I will be able to confirm whether PP2A is involved in the removal of phosphates from Ubiquitin… which will be a very small victory in the grand scheme of things, however I am very excited! So by my next blog I will have an answer for you and will have hopefully made some more headway unpicking the precise role of PPP2R5D in this very complicated equation.

Another quick update… I have recently become a PD Avenger (yes, like the superheroes) and I strongly encourage you to join this global community aiming to end Parkinson’s. PD Avengers was founded by a group of 12 individuals, most of whom live with Parkinson’s, who were inspired by the book ‘Ending Parkinson’s’ (Drs. Ray Dorsey, Michael Okun, Bas Bloem and Todd Sherer). The community has grown to 2168 PD Avengers across 53 countries and is continuing to grow. Their main ideas are that:

“We believe we have been too silent for too long. That we must become a loud, uncomfortable, ever-present voice demanding change in how people view and treat PD.

We believe every person living with PD should have equitable access to the therapies & resources that positively impact Parkinson’s symptoms

We believe by uniting our global voices, we will be impossible to ignore any longer

We believe Parkinson’s can be eliminated through research, and prevention. We must demand that greater resources be made available and greater urgency be placed on stopping the disease and eliminating man-made triggers that cause it.

We believe ending Parkinson’s is an achievable and worthy mission. “

If you agree, then please sign up to become a PD Avenger here: https://www.pdavengers.com/become-a-pd-avenger

Thank you!