An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS patients 9/2019

[Murph]

An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS Patients

Daniel Missailidis , Sarah Annesley , Claire Allan , Oana Sanislav , Brett Lidbury , Don Lewis , Paul Fisher *


Version 1 : Received: 3 September 2019 / Approved: 4 September 2019 / Online: 4 September 2019 (13:29:14 CEST)

Abstract

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an enigmatic condition characterized by fatigue that is unaided by rest and by exacerbation of symptoms after exertion (post-exertional malaise or “PEM”). There is no definitive molecular marker or known underlying pathological mechanism for the condition. Increasing evidence for aberrant energy metabolism suggests a role for mitochondrial dysfunction in ME/CFS.

Our objective was therefore to measure mitochondrial function and cellular stress sensing in actively metabolising patient blood cells. We immortalized lymphoblasts isolated from 51 ME/CFS patients diagnosed according to the Canadian Consensus Criteria and an age- and gender-matched control group. Parameters of mitochondrial function and energy stress sensing were assessed by Seahorse extracellular flux analysis, proteomics, and an array of additional biochemical assays.

As a proportion of the basal oxygen consumption rate (OCR), the rate of ATP synthesis by Complex V was significantly reduced in ME/CFS lymphoblasts, while significant elevations were observed in Complex I OCR, maximum OCR, spare respiratory capacity, nonmitochondrial OCR and “proton leak” as a proportion of the basal OCR. This was accompanied by an elevation of mitochondrial membrane potential, chronically hyperactivated TOR Complex I stress signalling and upregulated expression of mitochondrial respiratory complexes, fatty acid transporters and enzymes of the β-oxidation and TCA cycles.

By contrast, mitochondrial mass and genome copy number, as well as glycolytic rates and steady state ATP levels were unchanged. Our results suggest a model in which ME/CFS lymphoblasts have a Complex V defect accompanied by compensatory upregulation of their respiratory capacity that includes the mitochondrial respiratory complexes, membrane transporters and enzymes involved in fatty acid β-oxidation. This homeostatically returns ATP synthesis and steady state levels to “normal” in resting cells, but may leave them unable to adequately respond to acute increases in energy demand as the relevant homeostatic pathways are already activated.

https://www.preprints.org/manuscript/201909.0043/v1

 

[Murph]

Video here:

https://mecfsconference.org.au/videos/paulfisher/

 

[Murph]

This research worked on patients from the CFS Discovery clinic, led by Dr Don Lewis. I was a patient there.

Two months ago Lewis died and the clinic shut down, leaving many many patients with nowhere to go, and also presumably stranding researchers who want access to a set of screened patients.

 

[Murph]

The gist of this paper is we have a mitochondrial problem in one of the five steps required to make ATP (the fifth one, known as Complex V), but it is compensated for by other systems when we are at rest.

One of the upregulated systems seems to be mTor. Longstanding residents of phoenixrising may remember this thread!

mTor is also involved in the immune system, which may mean this paper helps provide the necessary bridge between the immune and metabolic systems to fully explain the disease

One implication of all this compensatory upregulation seems to be higher catabolism (breakdown of cells) to provide inputs for these compensatory systems which are running hot. Higher catabolism might be why protein supplementation works for some people.

The paper suggests three hypotheses to explain why the Complex V isn't working:

What might cause such a mitochondrial Complex V inefficiency? Three possibilities are a mutation affecting one of the Complex V subunits or assembly proteins, a dysregulation of Complex V, or an elevation of the relative use of the proton motive force for other purposes (“proton leak”) making less available for ATP synthesis. A mutational defect in Complex V seems unlikely in view of the failure of previous investigations to uncover any single nucleotide polymorphisms in Complex V genes that associate with the disease state [47,48]. Dysregulatory inhibition of Complex V is a second possibility. It is known that mitochondrial ATP synthase activity can be regulated by a variety of proteins, small molecules and signalling pathways, some of them by acting through Complex V’s own inhibitory subunit AIF1 [49,50]. The third possibility, that Complex V is inefficient in ME/CFS cells because of the elevated use of the proton motive force by other processes, is suggested by the elevated proton leak we measured in ME/CFS lymphocytes. However, the ME/CFS mitochondria have excess unused respiratory capacity and an elevated proton motive force. These would suggest that the membrane potential and electron transport capacity in ME /CFS cells are more than sufficient to allow Complex V to operate at normal efficiency. These possible causes for Complex V inefficiency in ME/CFS mitochondria should be investigated in future work.


This is a major paper by Aussie researchers that bridges some previously contradictory seeming observations in other studies.

 

[ScottTriGuy]

Fwiw, I am a Rapamune responder - it is an mTOR inhibitor.

It has lessened my PEM intensity and duration. I can walk 35 mins with Rapamune. Max out at 15 mins without it.

I also seem to need a lot of protein.

 

[nandixon]

Very interesting work that raises more questions for me than any other ME/CFS study by far. Not so much about what was said, but about what wasn't said. Just a few preliminary comments:

The lymphoblast cells that were used are an artificial construct, so we don't know for sure whether they are really representative of what's going on in cells in vivo or not. In fact, it's not clear to me yet whether the behavior of these lymphoblasts is consistent with either of the nanoneedle results or the "something in the blood" findings of the Davis OMF group.

But for purposes of a diagnostic test that wouldn't matter, because the original lymphocyte cells that were immortilized into lymphoblasts appear, at a minimum, to at least be showing a different response than control cells to the immortilization process (even if lymphocyte cells in vivo don't actually have the specific energy problem with respect to Complex V, etc., that was found in the lymphoblasts). Thus, this work may for the very first time finally yield a test that is specific for ME/CFS. (Note that this version of the paper was not peer-reviewed but I like it nonetheless.)

Regarding TORC1 (aka mTORC1) being hyperactivated, I had actually come to the opposite conclusion at the time the pyruvate dehydrogenase findings of Fluge & Mella were published several years ago. But that idea died a quiet death within a short time when I realized that it's actually nonsensical to talk about mTORC1/TORC1 being over- or underactivated in any general sort of way. This is because TORC1 has incredibly complex interactions, more so than any other enzyme I believe, and can not only be overactivated in one cell type while underactivated in another, but it can also be over- and underactivated in different pathways within the same cell. I had thought the low natural killer cell function seemingly found in ME/CFS supported the idea of TORC1 being underactivated, for example. (Interestingly, the anecdotally best supplement treatment on this forum, the use of branched chain amino acids, is actually the single best way known to activate TORC1.)

Anyway, I'm very curious to try to reconcile these findings with Ron Davis' work.

 

[wigglethemouse]

For those in Melbourne Paul Fisher is recruiting for a follow-up study. Recruitment thread on s4me
https://www.s4me.info/threads/australia-healthy-volunteers-needed-for-study-in-melbourne.11113/

 

[wigglethemouse]

Thus, this work may for the very first time finally yield a test that is specific for ME/CFS.

This was a recent quote by Paul Fisher in a La Trobe article. I'm guessing the Torin2 response pictured in a post above could be the simple blood test they are working on.

Here at La Trobe we have discovered a specific defect in the mitochondria – the ‘powerhouse’- of the cells of ME/CFS patients and are working towards a simple diagnostic blood test

https://www.latrobe.edu.au/news/articles/2019/release/chronic-fatigue-syndrome-research-funded

 

[CathyDav]

If a defect in Complex V is replicated in other studies, then this study on the toxic effects of triclosan may be of interest. Triclosan is an antibiotic that has been used as a preservative in many products- everything from make-up to chopping boards.

Experiments with isolated rat liver mitochondria showed that triclosan impaired oxidative phosphorylation, downshifted ATP synthesis, uncoupled respiration and provoked excessive oxygen uptake. These exposure concentrations are 100–1000 fold lower that those permitted in consumer goods.
CharmaineAjao; Maria A.Andersson; Vera V.Teplova et al. (2015)
https://doi.org/10.1016/j.toxrep.2015.03.012

This is part of the abstract and looks a bit similar to the abstract above.
"Experiments with isolated rat liver mitochondria showed that triclosan impaired oxidative phosphorylation, downshifted ATP synthesis, uncoupled respiration and provoked excessive oxygen uptake. These exposure concentrations are 100–1000 fold lower that those permitted in consumer goods."

 

[nandixon]

This was a recent quote by Paul Fisher in a La Trobe article. I'm guessing the Torin2 response pictured in a post above could be the simple blood test they are working on.

The p-value isn't as good but I'm sort of hoping they might be able to use the finding shown in the 3rd bar graph of Figure 2A, ATP as % of basal OCR [oxygen consumption rate]:

If that holds up, then if it's less than 60% you might be said to have ME/CFS.

 

[nandixon]

The first question that came to mind as I read this study was, are the lymphoblast findings consistent with the "something in the blood" finding of Ron Davis (and one or two other groups, I believe)? This unknown serum (or plasma?) factor, which is apparently pretty large (an exosome has been speculated) causes healthy control cells to act like ME/CFS cells when exposed to ME/CFS serum. And likewise, when ME/CFS cells are removed from their own serum and placed in healthy control serum they then appear to act like healthy cells.

The lymphoblasts ultimately are far removed from the original serum their lymphocyte precursors were in, yet they display abnormal behavior (i.e., decreased ATP output by Complex V). This may suggest that an epigenetic modification has taken place that perpetuates the abnormal behavior even in the absence of the unknown serum factor.

So does the epigenetic modification (e.g., something that negatively regulates Complex V) happen when lymphocytes are exposed to the unknown factor, and then this get locked into place when the lymphocytes are immortalized into lymphoblasts?

Or is the epigenetic modification introduced by the immortalization process itself due to an abnormal response of ME/CFS lymphocytes to that immortalization process?

Looking at various studies, either possibility seems like it might be feasible, I think. And both possibilities are consistent (or not inconsistent) with the "something in the blood" finding.

But which is more likely? If an epigenetic modification has happened to the original ME/CFS lymphocytes then why would they return to normal when placed in healthy serum? This makes me think it may be somewhat more likely that the immortalization process itself has introduced the Complex V inefficiency due to a peculiar response of ME/CFS lymphocytes, although it's just a guess. Either way it wouldn't affect the diagnostic utility of a test based on the findings of this study.

 

[Joy&K0$]

I was in a mito study that diagnosed me with a Complex V gene deletion several years ago. No help whatsoever from the mito specialist though. From what I can glean from the research, anti-inflammatory treatment could provide some therapeutic relief. Here's my train of thought:

• I meet all the Canadian criteria for M.E.
• Another part of my Dx is a mitochondrial defect in Complex V, ATP synthase, diagnosed on Deep Gene Sequencing
• One of my classic and most debilitating symptoms is hot, fluey and achey thighs and PENE with pathological post-exertional depletion of energy. AND sweating for hours after I've overdone it.
• Uncoupling of Complex V is classically associated with production of HEAT, instead of ATP, like a plane wastefully jettisoning fuel (survival tactic) when it's coming in for a crash landing – my classic symptom. Many of us have autonomic issues with thermoregulation. Could this be a symptom of a mitochondrial Complex V defect and uncoupling?
• I've been out of the PR loop, so can't recall - how many ME patients experience abnormal heat dissipation as part of PENE?
• In possible treatment, therefore may therapeutically look to INHIBITORS of Uncoupling – the latter which are driven by Thermogenin/UCP1 (read up on uncoupling proteins - IMO this could be significant)
• Norepinephrine inhibits Thermogenin
• Inflammation stimulates norepinephrine
• Therefore need an anti-inflammatory Rx that inhibits norepinephrine????
• May explain why highly-anti inflammatory treatments (eg certain IVIg's, prednisone) have helped me spectacularly, albeit temporarily
• Like most M.E. patients, my docs don't believe me, and funds don't allow Rx
• Nevertheless, this anti-inflammatory Rx avenue could provide an avenue to Rx for our field.

Not well enough to post more, but I'm rather thrilled that Complex V might be an M.E. "thing", as it could provide direction on at least some treatment. Read up on uncoupling proteins, Complex V, and inhibitors of norepinephrine. I think this might provide at least one avenue of Rx, if Complex V defects are found to be endemic in M.E. Note also that mito defects are found in patients with endothelial dysfunction (see Julia Newton's and Fluge/Mella's findings of endothelial dysfunction in M.E.)

The attached excerpt from: The Biology of Mitochondrial Uncoupling Proteins. Diabetes. Vol 53. Supplement 1. Feb 2004.

​

Link: http://diabetes.diabetesjournals.org/content/53/suppl_1/S130.full

"Activation of UCP1 enhances respiration, and the uncoupling process results in a futile cycle and dissipation of oxidation energy as heat."​

 

[nandixon]

@Joy&K0$, are you able to say which Complex V gene that the deletion was found in? Genes that encode the various subunits for that complex are found in both the nuclear DNA and the mitochondrial DNA. I'm wondering which yours was. Thanks!

 

[Joy&K0$]

@nandixon I was promised my full Deep Sequencing results (ostensibly on a thumb drive), and have repeatedly asked for them over the past several years so I might share them with reputable biomedical M.E. researchers, but no luck yet. So I can't answer your question (yet), although it was indeed a mitochondrial DNA deletion. As you know, so many physicians think that M.E. patients, armed with data, are merely psychologizing nuisances. I've asked countless times, and I'm seeing the specialist later this fall, so will report back if they give me the results. Each effort to get this kind of disclosure puts me in PENE, so it has been frustrating and disheartening to say the least. If anyone has legal or reputational clout to get these kind of results from specialists in Canada, please let me know.

 

[nandixon]

This is part of the abstract and looks a bit similar to the abstract above.
"Experiments with isolated rat liver mitochondria showed that triclosan impaired oxidative phosphorylation, downshifted ATP synthesis, uncoupled respiration and provoked excessive oxygen uptake. These exposure concentrations are 100–1000 fold lower that those permitted in consumer goods."

@CathyDav, a big difference with respect to triclosan as compared to the findings of the present study is that triclosan causes a depolarization (a reduction) in mitochondrial membrane potential (MMP), whereas in the present study the MMP was found to be increased. So different mechanisms are at work even though in both cases there is an impairment with respect to Complex V that leads to reduced ATP synthesis.

If I understand correctly, the reduction in MMP that triclosan causes is associated with an "uncoupling" of Complex V which reduces its ability to convert ADP to ATP. Whereas in the present study it was found that there appears to be more than enough MMP for Complex V to operate properly but Complex V is unable to fully take advantage of it for some reason. In both cases though that's correct that there is a reduction in ATP output by Complex V.

 

[Moof]

If an epigenetic modification has happened to the original ME/CFS lymphocytes then why would they return to normal when placed in healthy serum?

This is the really big question. From what I've read, it seems surprising that the modification would be preserved in the immortalised cells anyway; it's an even bigger stretch to suggest that they can not only retain that memory, but revert to a previous state. I'm pretty dubious about it, but of course I hope I'm wrong! If it can be reproduced in a couple of other labs, then maybe we're cooking with gas after all.

ETA: sorry, 'cooking with gas' is an old UK advertising slogan that won't make much sense elsewhere. Just means 'doing it right'.

 

[nandixon]

This is the really big question. From what I've read, it seems surprising that the modification would be preserved in the immortalised cells anyway;

I've seen a number of studies in which epigenetic changes have been shown to end up in the immortalized lymphocytes (i.e., lymphoblasts). But it seems that sometimes the changes are present in the precursor lymphocytes, while other times they might be introduced during the immortalization process, and it seems the changes are often (usually?) not reflective of epigenetic changes that have occurred elsewhere in other cells/cell types from the same individual, meaning they're specific to the particular cells that happen to have been immortalized.

...then maybe we're cooking with gas after all.

I'm hoping they're cooking with gas too, that's for sure.

 

[nandixon]

@nandixonSo I can't answer your question (yet), although it was indeed a mitochondrial DNA deletion.

Thanks. So far it's been found that Complex V consists of 20 (or 17?) subunits and 3 assembly proteins. The mitochondrial genome only has genes for 2 of the subunits. The genes are MT-ATP6 and MT-ATP8. The odds are significantly greater that your deletion is in MT-ATP6 because that gene is several times larger than MT-ATP8 and several times as many mutations have been found there.

A recent study looked at all of the reported cases of mutations/variants in MT-ATP6. From the abstract:

MT-ATP6 mitochondrial disease variants: Phenotypic and biochemical features analysis in 218 published cases and cohort of 14 new cases.

"Pathogenic MT-ATP6 variants resulted in diverse biochemical features. The most common findings were reduced ATP synthesis rate, preserved ATP hydrolysis capacity, and abnormally increased mitochondrial membrane potential. However, no single biochemical feature was universally observed. Extensive heterogeneity exists among both clinical and biochemical features of distinct MT-ATP6 variants."

https://www.ncbi.nlm.nih.gov/m/pubmed/30763462/

From the full text of that same study:

"Among patients with [variants of uncertain significance (VUS)], the recurrent reported symptoms occurring in over 50% of subjects with VUS were largely non-specific, including headaches, fatigue, and exercise intolerance. Muscle weakness, peripheral neuropathy, heart rate or blood pressure variability, and gastrointestinal dysmotility were also recurrent in multiple subjects."

I double-checked my whole genome sequencing results and out of my 40 mitochondrial genome variants there was only one very common variant (G8701A) in MT-ATP6. (It's no telling how many nuclear DNA variants of unknown significance there might be in the genes for the other subunits, though.)

About the possibility of a genetic defect, the authors of the study of this thread said:

"A mutational defect in Complex V seems unlikely in view of the failure of previous investigations to uncover any single nucleotide polymorphisms in Complex V genes that associate with the disease state [47,48]." [Edit: "Disease state" is referring to ME/CFS.]

I guess that's probably correct (although not for @Joy&K0$) provided that the 51 patients recruited for the study from the CFS Discovery Clinic in Melbourne, Australia weren't all cousins or something.

 

[nandixon]

@Janet Dafoe (Rose49), @Ben H

There's a very interesting (and fairly obvious) intersection of Ron Davis's nanoneedle results for SS-31 (elamipretide, aka Bendavia or MTP-131) with Paul Fisher's finding in the study of this thread that mitochondrial Complex V (ATP synthase) is somehow impaired or inefficient in ME/CFS.

I think it would be very interesting if it were possible for Dr Fisher to look at the effect of SS-31 on his lymphoblasts. (It might also be interesting if the lymphoblasts could be tested with the nanoneedle as well.) I'll email him to see if he will look at this after I post it.

Here's my thinking:

SS-31 strongly binds with cardiolipin and has been shown to improve or restore mitochondrial energy production.[1]

Cardiolipin is a critical lipid component of the inner mitochondrial membrane and regulates the formation of various large aggregate structures involving the different mitochondrial complexes. Several of the complexes appear to have an absolute requirement for bound cardiolipin in order to function properly. Complex V itself is capable of binding 4 molecules of cardiolipin. Cardiolipin also serves as a proton trap to maintain pH levels near the mitochondrial membranes.[2]

As just one example of how cardiolipin plays a role with respect to Complex V (and not necessarily the most important one for our purposes):

These data suggest that cardiolipin promotes the ribbonlike assembly of ATP synthase dimers and thus affects lateral organization and morphology of the [mitochondria] crista membrane.

[3]

It's conceivable that if the efficiency of cardiolipin could be improved with respect to its affect on Complex V that it in turn might help to overcome the inefficiency that Dr Fisher found. (And this could be true whether the Complex V inefficiency actually relates to cardiolipin or not.) SS-31 might be able to help in that regard.

I've previously posted that I think Dr Davis's nanoneedle might be measuring mitochondrial membrane potential (MMP), either directly or indirectly as reflected by the cellular plasma membrane potential.

If this is true then the finding that SS-31 normalizes the nanoneedle results might mean that the Complex V inefficiency that Dr Fisher found might be normalized by SS-31 as well.

There is a tricky aspect, however. Because from Dr Fisher's work it was found that MMP is actually already increased in ME/CFS (due to increased capacity of the complexes which come before Complex V, i.e., the electron transport chain), the possibility exists that SS-31 could normalize the nanoneedle results without actually improving the Complex V inefficiency - and therefore not improve patients' symptoms if it were trialed as a drug.

This is because the nanoneedle, instead of showing that a problem with Complex V exists, may instead be showing an inability of the MMP to be increased any further simply because it is already maxed out. Adding SS-31 could lower MMP back to normal (by normalizing the electron transport chain) and make the nanoneedle salt stress test appear normal but possibly without improving things with respect to Complex V. (In that case, a patient taking S-31 as a drug would presumably either feel no difference or would feel worse.)

How the other issue of the "something in the blood" finding of Dr Davis and 2 or 3 other groups might fit into the above speculations I'm not sure.

Note that anticardiolipin autoantibodies are apparently found in a large percentage of ME/CFS patients but this is a very common finding in many illnesses and seemingly the most recent rituximab trial would have seen a better result were they playing an important role in the disease.(?)

A few references:
[1] The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736700/

[2] https://en.wikipedia.org/wiki/Cardiolipin

[3] Cardiolipin Affects the Supramolecular Organization of ATP Synthase in Mitochondria
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150712/

 

[nandixon]

I think it would be very interesting if it were possible for Dr Fisher to look at the effect of SS-31 on his lymphoblasts. (It might also be interesting if the lymphoblasts could be tested with the nanoneedle as well.) I'll email him to see if he will look at this after I post it.

Dr Fisher already replied back. A super nice guy. He read the entire thread and said they were already committed to doing follow-up work, some of which go to issues raised in this thread, but that at some point they want to look at potential treatments and SS-31 would be worth a look.