Erica,

Increase in SOD2 signs an oxidative stress related to an increase in superoxide anion flux into mitochondria.

This stress is not necessary related to an increase in TCA cycle. This may be due to many other causes.

One exemple is an electron transport chain deficiency. Indeed, in case of complex I or complex III deficiency you have an huge leak of electron, that are liberated every where as well in the inter-membrane space as in the matrix. Due to the présence of oxygen you create immediately a burst in superoxide anion. To circumvent this burt, you have an increase in SOD2. By WB you can’t say if your oxphos protein are functionnal, and if your complex are really well organized. Without complex activities measurement, you can’t know.

An other exemple : superoxide anion may also be formed in cytosol and they may have crossed mitochondrial membrane, inducing SOD2 increase, protecting mitochondria enough to avoid TCA or oxphos perturbation.

Moreover, with CS activity you can't affirm that TCA is not increase. You have many other metabolic entries in TCA, not only oses pathway.

Thus with SOD2 measurement, WB for oxphos, and only CS activity for TCA you can’t interpret what happens

Sorry for bad news.

Marc Conti

Dear Marc,

Thank you a lot for your answer, I really appreciate your comments.

As you said, we need more experiments to interpret the entire situation. We checked CS activity as the first enzyme involved in TCA, as a starting point… As you have many other metabolic entries in TCA, is there any way to measure TCA activity in general? The final outcome of TCA? I can imagine that is so related with OXPHOS….

We are now planning to measure OXPHOS activity and fatty acid oxidation in order to better understand our system.

Thank you again for your time,

Erica

Dear Erica,

First of all, I agree with comments of Marc Conti. I am going to try to clarify some aspects and I hope this can help you.

You have to keep in mind that TCA flux and OXPHOS function are two different things, and as Marc said, with WB results you cannot know anything about function. CS activity is a good marker of mitochondrial content in skeletal muscle because it has a good correlation with TEM. But there are other enzymes in the TCA cycle that are able to produce ROS.

The main site of ROS emission in mitochondria is the electron transfer chain. So you should check OXPHOS coupling and ETC activity (complex I and complex II separately) in your experiments to see if there is something that suggests an alteration in the direction of ROS production. Also, you can test directly ROS emission by using some fluorescent probes (AmplexRed) in the presence of different substrates, with and without ADP.

Sometimes, but not always, an unbalance on OXPHOS coupling is followed by an alteration in ROS emission. And then, this unbalance in ROS emission can further stimulate the increase in SOD-2 protein that you found in your experiments.

But as Marc said, SOD-2 is regulated by other factors and does not need to follow alterations in TCA enzymes activities or OXPHOS proteins. Neither these proteins can explain your results, so far.

I hope my suggestions help you and good luck with your experiments.

Henver

Dear Henver,

Thanks a lot for your explanation! it is very helpful to me in order to interpret my results and to continue with subsequents exeriments that will be focused on the activity of the system.

Thanks again,

Erica