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

[John Mac]

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

 

[Hoopoe]

So according to this, the cells are still able to operate thanks to compensatory changes but they don't handle additional energy demands well. That sounds similar to ME/CFS.

 

[Andy]

Sigh

characterized by fatigue that is unaided by rest

 

[wigglethemouse]

This is really exciting on first skim. There are lots of test results with big separation between controls and patients (CCC qualified), with low p numbers. Lots of hard data.
Paper was originally presented in this talk
https://www.s4me.info/threads/video...nsatory-changes-in-me-cfs-patient-cells.9177/

And follow-up study planned
https://www.s4me.info/threads/austr...ded-for-study-in-melbourne.11113/#post-198497

Would be fantastic if we could persuade Karl Morten to run a parallel study in real time with Paul Fisher on a spearate cohort and different lab to speed up validation of this work.

I think this is the first time we have seen such detailed differences in the analysis of mitochondria patient vs control!

 

[wigglethemouse]

Paul Fisher is quoted in the press release for the Australian Biobank to be funded by the Mason foundation.
https://www.eqt.com.au/about-us/med...ld-decision-to-back-a-plan-for-a-breakthrough

“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,” Professor Fisher said.

Could this paper possibly describe the meat and potatoes of a diagnostic test. See section 3.7 "Stress-sensing pathways in ME/CFS are perturbed – TORC1 is chronically hyperactivated"

As one of the key substrates of TORC1 involved in regulating protein synthesis, 4E- BP1 is often used as a marker of TORC1 activity [43]. We found that 4E-BP1 phosphorylation levels were significantly elevated in ME/CFS lymphoblasts and were accompanied by a correspondingly increased response to the mTOR inhibitor, Torin2 (Figure 7). This chronic elevation of TORC1 activity could explain the increased expression of mitochondrial proteins and respiratory capacity that we found in ME/CFS lymphoblasts

These p numbers and difference between patients and controls for this test have me rather excited.

 

[Hutan]

Yes, I'm feeling a bit excited by this team's work too.

The team have focused on using lymphoblasts rather than lymphocytes. As I understand it (and that's not saying a lot), they created immortal lines of lymphoblasts from extracted lymphocytes. They have done this by exposing the cells to EpsteinBarr Virus (among other things).

So the lymphoblasts are happily staying alive and growing in their culture and are activated - as opposed to the quiescent lymphocytes. Look how different the lymphoblasts and lymphocytes are with respect to oxygen use (400 to 500 for lymphoblasts compared to less than 10 for lymphocytes).



They found those ME/CFS lymphocytes died more quickly than those from controls.

We therefore assessed the viability over time of ME/CFS lymphocytes versus healthy controls (Figure 1B). There was a dramatically greater death rate in the ME/CFS lymphocytes than in the controls, suggesting that previously reported reductions in ME/CFS lymphocyte mitochondrial function might have resulted from a higher fraction of
dead cells in the assayed population. If it reflects the in vivo life span of unactivated lymphocytes, this result would also suggest that the turnover of unactivated lymphocytes in ME/CFS patients may be dramatically elevated.

The lymphoblasts from the patients and controls are a long way removed from the serum that they would have been in when in vivo.

There are so many variations to be aware of - too easy to try to compare apples with oranges and assume that there's no consistency.

 

[Hoopoe]

@wigglethemouse I also find the increase in carnitine pathway interesting. I think carnitine helps me.

 

[ScottTriGuy]

"As one of the key substrates of TORC1 involved in regulating protein synthesis, 4E- BP1 is often used as a marker of TORC1 activity [43]. We found that 4E-BP1 phosphorylation levels were significantly elevated in ME/CFS lymphoblasts and were accompanied by a correspondingly increased response to the mTOR inhibitor, Torin2 (Figure 7). This chronic elevation of TORC1 activity could explain the increased expression of mitochondrial proteins and respiratory capacity that we found in ME/CFS lymphoblasts"

Fwiw, I am a responder to Rapamune / Rapamycin, an mTOR inhibitor.

(ETA format)

 

[Forbin]

Does the paper address how this impairment in Complex V could happen in enough mitochondria / cells simultaneously to account for the "sudden" onset often seen in the illness? It seems like some sort of exposure to a system wide "trigger" would be needed (or some kind of signaling in response to a "trigger").

 

[beverlyhills]

So what this study tells is that healthy controls are more active.

It is one thing to criticize patient/control selection as a semantical issue (to make results generalizable), but is another to criticize the control selection and knowing why the underlying physiology could nullify it.

 

[borko2100]

The only thing that helps me is fasting for 1-3 days. Coincidentally fasting also reduces mTOR activity.

 

[AliceLily]

The only thing that helps me is fasting for 1-3 days. Coincidentally fasting also reduces mTOR activity.

Yeah, when I am at my very sickest experiencing a PEM that makes me so weak, with retching as if I were poisoned, I cannot bear to eat, even though my stomach is rumbling with hunger I just can't eat at the mouth. I have to nibble every 15 minutes and sip on some liquid. It's a strange thing to be hungry at the stomach but no appetite at the mouth - to eat and to swallow is very difficult.

I dread this severe PEM because I fear I am not going to come out of this kind of PEM and will be left very weak and unable to look after myself. Thankfully I always do improve out of it. But this kind of PEM starves me as if the ME/PEM itself is putting me through a fasting - maybe this is the body just trying to help me out of this severe state by resting completely.

 

[Marky]

Im waiting for the layman version, but was wondering if this could be relevant to the slightly elevated risk of non hodgkins lymphoma for ME-patients?

 

[Marky]

Yeah, when I am at my very sickest experiencing a PEM that makes me so weak, with retching as if I were poisoned, I cannot bear to eat, even though my stomach is rumbling with hunger I just can't eat at the mouth. I have to nibble every 15 minutes and sip on some liquid. It's a strange thing to be hungry at the stomach but no appetite at the mouth - to eat and to swallow is very difficult.

I always feel like my life is going great when I`m lying on my back after making dinner, picking potatoes from the plate with my hand, cus I`m completely devoid of human function

Very few sees just how nightmarish ME can be, and only us feel it too.

Luckily I`m too exhausted to self-pity

 

[Hutan]

From the paper:
"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."

Fwiw, I am a responder to Rapamune / Rapamycin, an mTOR inhibitor.

The impression I got from this paper is that mTOR activation is a response to a lack of capacity to produce ATP in Complex V - it up regulates other parts of the system. So the mTOR activation is a compensatory mechanism. In that case, mTOR inhibition could be expected to make things worse.

I need to read the paper again, would appreciate others thoughts on this.

 

[Hoopoe]

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].

A signalling problem then.

 

[Forbin]

A signalling problem then.

From "something in the blood" ?

 

[Hutan]

Perhaps, but these lymphoblasts were separated from the blood and put through all manner of processes and grown in various clean nutrient fluids. So, if it's something in the blood then it's changing the lymphocytes permanently.

 

[Ravn]

Sigh

characterized by fatigue that is unaided by rest

I sent a message to the authors politely suggesting a more accurate way to phrase this - and I had a reply to the effect that they use the word fatigue because it's used in the CCC but that they will look at rephrasing the 'unaided by rest' bit. It may or may not be too late for this particular paper but they'll keep it in mind for future publications. A big thumbs up to responsive researchers

Oh, and I also thanked them for their interesting work

 

[Jonathan Edwards]

Perhaps, but these lymphoblasts were separated from the blood and put through all manner of processes and grown in various clean nutrient fluids. So, if it's something in the blood then it's changing the lymphocytes permanently.

I think this is the puzzle with results like this.

If a difference survives through to immortalised lymphoblasts in culture it seems unlikely to have anything to do with some systemic signal that arises when the illness arises. That leaves a purely genetic difference as an explanation but then it is hard to understand why people are normal before the illness starts.

It may be that we have to assume that some irreversible signal affected the differentiation of these cells and this is carried over in immortalised cells. This would be unexpected since immortalisation tends to override such differentiation signals.

Another possibility is that it is all due to which subtypes of cells are sampled. If more of the cells in ME blood are lymphocytes of a subgroup p rather than a subgroup q then when cells are immortalised they may appear to behave differently. In that situation we would not be able to draw any conclusions from the difference in culture about what disease mechanism is. The difference in culture wold not be due to cells behaving a different way, just a different proportion of cells behaving a particular way.

This comes back to my main worry about all these studies. Lymphocytes in the blood are not doing anything important there. They are just resting cells moving from A to B for one reason or another. Their function is of no real interest. People with different levels of activity or diet may have different proportions of cells in the blood of different types for reasons nothing to do with the illness.