An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS Patients Missailidis et al. 2019

Yes that was really interesting, i read it like a mystery crime thriller set in the body

 

Seahorse, summary of inconsistent results in previous studies.
http://simmaronresearch.com/2019/08/me-cfs-seahorse-energy-production-study-shows-surprises/

My take on Fisher et al; the results show a significant difference between CCC patients and controls, to EBV challenge, which could be significant if replicable.

In this experiment EBV is being used as a means for immortalisation but is often implicated as a contributory factor if not a cause of ME CFS CFIDS in its own right which makes it doubly important not to get ahead of ourselves.

I see this as a pathogen challenge which conveniently immortalises the test cells and reveals a difference in response correlated to ME symptoms, rather than preservation of epigenetic state, so I dont see it as a snapshot of ME in vivo but might reveal something about cell responses of PWME, if replicable...

Another point is the putative blood factor, which is not apparent in this experiment. However I dont think this makes a difference at this point. If I win the lotto I will fund work on this np!

There are a lot of questions to answer before considering how this should be interpreted as a model of ME, not least replication given misgivings about the slackness of CCC criteria.

If you dont have someone sharp on this job you could include other conditions in ME cohorts as is currently often the case IMHO.

What we need is a replicable empirically detectable difference which has the potential to define patient cohorts very concisely. So far the Seahorse results all contradict each other.

 

Only a day to read the paper? Good going. I've now watched the video and taken a look at the paper - and will try to explain, starting with my attempt to explain what mitochondria do, to tee up the paper. Much of this will probably be way too basic for many people here but I usually find it best to explain too much rather than too little.

Mitochondria are the power plants of the cell

Just as electricity is our preferred energy source, powering smartphones to computers to lawnmowers and even cars, ATP is the main power source of life. For humans, it powers nerve impulses (and so the brain) and muscles, the enzymes of digestion, the immune system and just about everything else.

And just as powerplants burn coal, gas and biomass in the air (oxygen) to produce electricity, so mitochondria in our cells combine fat-, protein- and carbohydrate-derived molecules with oxygen to generate ATP.

In fact, two of the primary processes keeping us alive are taking food, and breathing in oxygen (and carbon dioxide out). Mitochondria are behind these core processes at the molecular level, combining digested food molecules with oxygen, and releasing carbon dioxide.

Now for a little biochemistry:

https://www.youtube.com/watch?v=39HTpUG1MwQ

First, in mitochondria, the high energy molecule ATP is made from ADP plus phosphate, and when ATP is consumed by cellular reactions, ATP is broken down back to ADP and phosphate.

We get through roughly our own bodyweight of ATP every day, but, luckily, each molecule is turned over about 100 times a day — and most of the ATP is put back together again in mitochondria. It is a cycle:

Source: Khan academy

There are lots of steps in producing ATP. A fundamental one is the TCA cycle (tricarboxylic acid cycle). Carbohydrates, fats and proteins are broken down in various ways, but all wind up in the TCA cycle. This cycle produces a lot of energy and also, effectively, breaks the fuel molecules down to carbon dioxie and water

Oxidative phosphorylation — the central activity of mitochondria

The TCA cycle drives oxidative phosphorylation, the process that actually makes ATP. Oxidative", because mitochondria are using oxygen to oxidise fuel molecules, and "phosphorylation" because the energy generated from the fuel is then used to add a phosphate to ADP, making ATP.

There are five major protein complexes involved in oxidative phosphorylation, imaginatively named Complexes I-V.



Complexes I to IV do the oxidation (O2 is released at complex IV), and in the process, pump protons (H+) across the inner membrane of the mitochondrion. Pumping protons generates an energy gradient across the membrane, a gradient called the membrane potential - or proton motive force (which sounds like a sci-fi weapon).

Complex V includes ATP synthase, and as protons flow back through the membrane (down the energy gradient), ATP synthase makes ATP from ADP plus phosphate. This flow of protons back through the membrane to make ATP is much like the flow of water from a hydroelectric dam driving a turbine to make electricity.

Paul Fisher's results indicates a problem in complex V, so that ATP is made inefficiently.

And now for a closer look at the paper.

 

Comments on the paper (Part 1)

Overall, the paper looks quite solid, with many different approaches used to reach findings that fit together into a plausible model.

Note that the paper is a pre-print, which means it hasn’t been reviewed. It is increasingly common for scientists to first publish their work as a preprint (physicists have been doing this for decades), but doesn’t have the status of a peer-reviewed journal paper.

The study uses 51 patients, a decent sample, but only has 22 controls, which is a little on the light side.

An important feature of this paper is that the researchers decided not to study PBMC (white blood cells) directly. The reason for this, they say, is that such cells are largely "quiescent", doing little, and substantial numbers are dying (at least after 24 hours). Instead, they used lymphoblasts, which they created from lymphocytes adding EBV (Epstein-Barr virus). The virus turns lymphocytes into immortal lymphoblasts. Not only do these new cell lines conveniently last indefinitely, but they are also much more active. In fact, the study included data showing lymphoblasts used oxygen at about a hundred times the rate of lymphocytes.

The paper argues that:

Key findings

I am going to focus on the findings of the study, for simplicity, and not how they reach the findings, though the methodology looks good to me (I am no expert).

1. Using the Seahorse analyser, they found that most factors of energy metabolism, including glycolysis, were no different between patient and control lymphoblasts.

2. Instead, they found a particular problem with Complex V, which made ATP inefficiently.

3. Curiously, however, they found that's there was no difference in overall ATP production from mitochondria between patients and controls.

4. When they proved this further, they discovered that mitochondria appear to have compensated for the inefficient ATP production by ramping things up elsewhere:

- there was an increase in the maximum potential oxygen consumption (as opposed to ATP production) of mitochondria.

- Increase in the amount of TCA cycle proteins

— Increase in the amount of Complex I and Complex V (increases in other complexes were not statistically significant). They concluded that "electron transport [complexes I-IV] in the ME/CFS cells is functionally normal, but elevated in capacity because of elevated levels of expression of the respiratory complex proteins involved"

— Increase in the amount of proteins transporting fats into the mitochondria.

— Increase in the amount of proteins of beta-oxidation, needed for the pre-processing of fats before they head to the TCA cycle

5. Alongside this, they found that the regulatory protein TOR Complex 1, which senses energy changes in the cell, is chronically upregulated. (AMP kinase plays a similar role, and Julia Newton and her group are very interested in this molecule, but Fisher and colleagues were not able to study this properly in their current study).

(6. They also found that many of theses changes correlated with illness severity, as measured by standing time, albeit the correlation was fairly weak.)

Model

These findings led them to conclude that, for ME/CFS patients, ATP synthesis is inefficient, but mitochondria compensate this so that overall production of ATP is maintained (this kind of compensation mechanism is called homeostasis). However, the authors say that because the mitochondrial activity is already upregulated, they might struggle to cope with increased demand - and this might be behind fatigue:

I do have some significant questions about the real-world relevance of the findings and I contacted Paul Fisher about these. I should have his answers by next week. I will add Part 2 then.

 

I found this NIH site on Mitochondrial complex V deficiency interesting
https://ghr.nlm.nih.gov/condition/mitochondrial-complex-v-deficiency

If I remember right haven't there been some studies showing thickening of the heart wall in ME?

 

I found a study showing heart muscle thickening
Paper : Impaired cardiac function in chronic fatigue syndrome measured using magnetic resonance cardiac tagging
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3627316/

If I understand right by referring to the text of the study, radial thickening is a thickening of the heart wall. This is one outcome of Complex V dysfunction as noted in the post above .
https://ghr.nlm.nih.gov/condition/mitochondrial-complex-v-deficiency

 

Final version now published.

Open access, https://www.mdpi.com/1422-0067/21/3/1074/htm

 

I really don't understand this stuff; however, ---

Yes my immediate thought was I wonder what Karl Morten thinks of this study. I think Karl wanted to look at (other?) options to study mitochondrial functioning in ME. E.g. Karl used muscle cells in a study, and avoided the seahorse, to try to mimic natural conditions. However, I think the key issue in Karl replicating this study would be money --. Also Cara Tomas, and Julia Newton, are other researchers with the knowledge etc. Funding --

They have used immortalised cells in this study; however, there are controls, and they found differences in how the control and ME cells behave.

If all of this stacks up (& I hope it does) what is driving this change --- something in the blood -- & why --- response to a pathogen (altered microbiome/increased translocation).

The authors suggest that potentially these differences, in people with ME & healthy controls, could serve as biomarkers:
"In any case, if this “cellular chronic fatigue” is present in other cell types, it may contribute to the unexplained fatigue experienced by ME/CFS patients, as suggested by the fact that all of the mitochondrial abnormalities we observed were correlated with the severity of patient symptoms measured by the Weighted Standing Time. These correlations also verify that the mitochondrial abnormalities we have found are of clinical relevance to the underlying cytopathological mechanisms and can serve as biomarkers of disease."

 

How easy is it to measure these compounds? You would need a control group and a test group and establish whether the differences are sufficient. Yes it looks like something to be investigated and it doesn't look that difficult (they have done it in this study)/expensive. Also, a biomarker would help to establish whether this change is common to all/most/some people with ME & help to select participants for other studies ---.

 

Two very intriguing papers by this new lab entering ME/CFS research (Fisher). It shows that the small ME/CFS research community desperately needs new groups with greater expertise in particular areas such as mitochondria and energy metabolism, e.g. how did no previous group studying ME mito ever know that ex vivo cells are metabolically quiescent and not representative of actively metabolizing in vivo cells?

What I also very much enjoyed is how this paper spent the time not only introducing all the prior related work but also tying together plausible reasons why some prior work got such inconsistent results in the context of their new results here.

If this is reproduced and also in other cell types and it is an ATP synthase (Complex V) inefficiency problem then it should be straightforward to make animal models with this feature.

 

Well put. Or it’s changing their programming maybe not permanently but for a long enough time.

 

What I also like about the results they found is the plausible connection between ATP synthase inefficiency -> upregulation of mTORC1 to make up for ATP synthesis by other mitochondrial means -> mTORC1 hyperactivity causing many unwanted side effects such as inflammation etc.

So connecting energy metabolism dysregulation and immune dysfunction/inflammation.

 

It sounds like the initial findings were that the mitochondria were producing normal amounts of resting energy, but it turned out they were employing all sorts of compensatory mechanisms to achieve that. They couldn't produce anymore energy beyond that, which would seem like a plausible explanation for the trouble people with ME have with exertion.

If this is the case, I wonder if the really severe cases might show subnormal cellular energy even at rest. I'm not sure if anyone has done such energy tests on the mitochondria of the severely affected, but it might support the hypothesis.

 

It could be due to the reasons laid out in this paper.

Ex vivo lymphocytes are generally metabolically quiescent and respire at such a low rate that they measure at the bottom end of Seahorse detection range regardless of ME or healthy control. They need to be immortalized or stimulated in order to metabolize similarly to in vivo, and as the paper showed then you see the real difference between ME and control.

Also, one main result from the paper is that ex vivo ME lymphocytes die much faster than controls and the Seahorse protocol has you leave them in medium for a day after they are extracted or taken out of cold storage before running the experiment. So in this case you would find metabolic differences between ME and controls but for all the wrong reasons.

 

If mTOR is used as a backup energy system, does that mean that taking mTOR inhibitors (caffeine, green tea, curcumin, etc.) would be a bad idea?

For me personally, taking too much caffeine always brings on some unexpected symptoms, such as dizziness and nausea, maybe there could be a link there.

On the other hand, many sufferers report that taking BCAA's helps them, which actually do the opposite, namely stimulate mTOR.

 

I'm no expert on mitochondria, but I have been reading a "popular" book on mitochondria that was recommended by my nephew, who is an evolutionary biologist working on his PhD.

The book is certainly interesting, but, despite the author's best intentions, it's a little overwhelming for someone without an academic background in molecular biology.

One interesting factoid I picked up is that Complex V (ATpase) is perfectly capable of running backwards. In that mode, it pushes protons back into the intermembrane space, now using ATP as an energy source instead of manufacturing it. Apparently, the strength of the proton pool is critical to transporting molecules in and out of the mitochondria and that job is prioritized over all others, to the point where ATP is consumed rather than created. This kind of thing is no doubt going on all the time, but it does seem like a good way to shut down energy production. I have no idea why the proton pool would lose strength, although protons can apparently be "dissipated" in order to create heat. I've also seen vague references to "proton leak," but that's over my head. Anyway, maybe I'll learn more if I can get through the book.

Just thought it might be of interest to someone smarter than me here, since Complex V is part of the subject of this thread.

 

mTOR isn’t a backup energy system. It’s one of the most important proteins at the center of the mammalian cell stress sensing pathways. Remember too here it’s the cellular biology definition of stress, so not necessarily bad just signals that require the cell the react and alter behavior.

mTOR is a core component of this signaling system which regulates how much the cell should be in growth/proliferation/survival/aging (higher mTOR activity) or autophagy/life extension/house cleaning (lower mTOR activity). So it’s central to cellular metabolism and mitochondria. Think of mTOR as the gas/brake pedal of a car.

What’s also important here is that higher mTOR signals the cell to increase protein and lipid synthesis, i.e. things the cell needs to grow and ramp up function. So as described in this paper mTOR is chronically activated in reaction to a stress signal because of low cell ATP levels due to ATP synthase inefficiency.

More mitochondrial proteins are synthesized in order to increase function and capacity to make up for low ATP levels. So intuitively to me chronically activated mTOR to get ATP synthesis up is going to have a number of side effects all over the cell.

 

It makes sense to me. One of the big questions with ME over the years is how can we be so sick yet no one can find anything wrong when they do standard tests. When someone has cancer whole swathes of biochemistry go out of kilter for instance.

This and other work seems to show that our biochemistry is working overtime trying to compensate for the problems we face and it works for much of the time - we look normal and blood tests are normal - but as soon as any extra demand is made everything falls apart suddenly.

In effect we are running on the emergency system that healthy people use occasionally to cope with a sudden demand. This would explain the way I can be doing fine then .... stop with no warning.

 

Yes, to be so ill and there are no tests to show how sick you are.

Another thing I had a very difficult time with at severe ME was the realisation that any exertion made me even more ill than I already was. I was in a tremendous amount of shock regarding this and how distressed I was that I could no longer do what I wanted to do and how just holding a book in bed made my symptoms worse.