WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome, 2023 Hwang et al

[Kalliope]

Science A protein that disrupt cell's energy centers may be a culprit in chronic fatigue syndrome

quote:
“It’s extremely encouraging” to see this kind of detailed molecular approach applied to an understudied illness like ME/CFS, says Mady Hornig, a physician-scientist studying the condition at the Columbia University Mailman School of Public Health. Although the NHLBI researchers didn’t study Long Covid directly, their findings “stand to address a very common set of health issues that are very tightly tied to disability in [both] Long Covid and ME/CFS,” she says.

 

[rvallee]

Possibly relevant, from 2011. No thread on S4ME.
New thread on this paper here:
Meta analysis of Chronic Fatigue Syndrome through integration of clinical, gene expression, SNP and proteomic data, 2011, Pihur et al
_____________

Meta analysis of Chronic Fatigue Syndrome through integration of clinical, gene expression, SNP and proteomic data
https://pubmed.ncbi.nlm.nih.gov/21584188/

We start by constructing gene-gene association networks based on about 300 genes whose expression values vary between the groups of CFS patients (plus control). Connected components (modules) from these networks are further inspected for their predictive ability for symptom severity, genotypes of two single nucleotide polymorphisms (SNP) known to be associated with symptom severity, and intensity of the ten most discriminative protein features. We use two different network construction methods and choose the common genes identified in both for added validation. Our analysis identified eleven genes which may play important roles in certain aspects of CFS or related symptoms. In particular, the gene WASF3 (aka WAVE3) possibly regulates brain cytokines involved in the mechanism of fatigue through the p38 MAPK regulatory pathway.

 

[rvallee]

Not sure how it relates, but Prusty's study also flagged p53 as a possible factor: "Subsequent loss of miR-30 and activation of the miR-30–p53–DRP1 axis triggers a profound disruption of mitochondrial architecture.".

Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA, 2022, Prusty et al

 

[B_V]

Possibly relevant, from 2011. No thread on S4ME.


Meta analysis of Chronic Fatigue Syndrome through integration of clinical, gene expression, SNP and proteomic data
https://pubmed.ncbi.nlm.nih.gov/21584188/

We start by constructing gene-gene association networks based on about 300 genes whose expression values vary between the groups of CFS patients (plus control). Connected components (modules) from these networks are further inspected for their predictive ability for symptom severity, genotypes of two single nucleotide polymorphisms (SNP) known to be associated with symptom severity, and intensity of the ten most discriminative protein features. We use two different network construction methods and choose the common genes identified in both for added validation. Our analysis identified eleven genes which may play important roles in certain aspects of CFS or related symptoms. In particular, the gene WASF3 (aka WAVE3) possibly regulates brain cytokines involved in the mechanism of fatigue through the p38 MAPK regulatory pathway.

Paul Hwang used this 2011 paper to ID WASF3 as a gene product of interest.

I've written an article for The Washington Post about how this research came about - it's due to the tenacity of one woman who wanted answers to her own health problems - but August is the worst time of year for science & journalism & due to some factors outside my control it won't be published until next week at the earliest.

 

[B_V]

Not sure how it relates, but Prusty's study also flagged p53 as a possible factor: "Subsequent loss of miR-30 and activation of the miR-30–p53–DRP1 axis triggers a profound disruption of mitochondrial architecture.".

Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA, 2022, Prusty et al

The paper from Hwang et al out today shows that p53 is not involved in the pathway he identified. The patient's Li-Fraumeni Syndrome was unrelated to her chronic fatigue & related health problems.

 

[B_V]

Does anyone have access to the full paper?

 

[Rick Sanchez]

I remember once having hopium that all the massive funding cancer got would some day benefit us. I mean, given the absolutely massive numbers it simply HAD to... right?

Hopefully that day has just occurred!!

 

[Hutan]

Thanks @B_V

S1 (Subject 1) had a history of worsening fatigue after mononucleosis at age 16 and two separate early onset breast cancers, successfully treated. She had frequent cramps in her lower leg muscles at rest and exercise intolerance that required days to recover from exertion.

The first test reported on was phosphorus magnetic resonance spectroscopy (P-MRS) in combination with repeat exercise. They say phosphocreatine is used in exercising skeletal muscle; the length of time it takes to get back to the baseline after exercise is the PCr recovery time. They say that this is a non-invasive measure of mitochondrial ATP synthesis capacity.

S1 was found to have a markedly prolonged PCR recovery time (twice that of healthy controls). The test was repeated several months later, and the same result was found.

The authors say

The delayed PCrTc [the recovery time] of patient S1 was corroborated by previously published P-MRS studies, which also showed slow PCr recovery of the muscle in ME/CFS patients.

I'm thinking to myself - what study was that? And here's the kicker - this is the reference:
Chaudhuri and Behan "In vivo magnetic resonance spectroscopy in chronic fatigue syndrome" 2004.
Two thousand and bloody four.

 

[Hutan]

The researchers compared fibroblasts

A fibroblast is a type of cell that contributes to the formation of connective tissue, a fibrous cellular material that supports and connects other tissues or organs in the body. Fibroblasts secrete collagen proteins that help maintain the structural framework of tissues.

of S1 with her brother S2, who also carried a P38 mutation.

They found that S1's fibroblasts had lower oxygen consumption rate (OCR) and decreased Complex IV (one of the electron transport complexes in the mitochondrial membrane). Complex IV has two proteins MTCO1 and MTCO2. They found a 34% decrease in S1's MTCO1 compared to her brother.

The researchers found that S1's p53 was highly phosphorylated at a specific place, Ser46 (unlike her brother, who did not have the exertion intolerance etc symptoms). They knew that activated P38 MAPK14 could increase that phosphorylation, and indeed they found higher levels of that activated P38 MAPK14. WASF3 was known to activate P38 MAPK14, and there was the report of WASF3 being highly expressed in people with chronic fatigue syndrome. So, they looked at WASF3 levels in S1, and, sure enough, she had higher levels than her brother - 40% higher levels.

Jumping over the mice stuff for the moment, here's the finding on the level of WASF3 protein in 10 controls and 14 people with ME/CFS from the NIH ME/CFS study. So, WASF3 is also higher in the people with ME/CFS. There also seems to be less MTCO1.

 

[DMissa]

Basically, without checking, it's an MRNA that controls the gene ---telling it to make more/less of the protein.

I think of it as: DNA is the blueprint, mRNA is a photocopy of the blueprint sent to the factory floor, protein is what the photocopy tells the assembly line (ribosome) to make.

As for the paper:

"Seahorse XF Metabolic Studies.
The cells were trypsinized, resuspended in Seahorse XF DMEM assay medium (supplemented with 2 mM Glutamax, 1 mM sodium pyruvate and 25 mM glucose) (Agilent), and plated at ~5 × 104 cells/well on XF-24 well plates. The mitochondrial OCR was measured using the Agilent Seahorse XF24 Analyzer according to the manufacturer’s protocol and normalized to the protein content of each well. For measuring ex vivo muscle metabolism, the whole soleus muscle was carefully dissected and embedded on islet capture microplates and its OCR and ECAR were measured."

No description of incubation time, density at time of harvest, evaluation of spatial density in the wells once adherent, passage number. No mention of whether live and dead cells were discriminated and if differences between samples were identified during counting? How long were they trypsinised for? This info is essential for interpreting not only this kind of data in general but particularly fibroblast data because they contact-inhibit each other's metabolism and rapidly progress through senescence with each doubling. I do not agree with normalising to total protein loading when we are working in a context that assumes that protein expression is dysregulated. If your cell counting is accurate, your handling careful, and your replicates sufficient, I don't see this being necessary. It's also usually being done at the end of the assay where your different clinical groups may have responded differently to the pharmacological inhibitors and had resultant changes in protein content that do not reflect initial cell loading. And how was the protein content measured, no detail? Seahorse assays routinely cause cells to detach mostly due to the swelling effects of adding protonophores. This can lead to disruption of the originally desired cell monolayer and cells piling on top of each other which can confound particular plate-based assays (encompassing common protein assays).

For the muscle western blots that looked at WASF3 levels, where is the cohort information? Are the controls sedentary? Activity levels absolutely must be controlled for with muscle study, muscle cells change so much with activity.

There is a paucity of absolutely critical information in this paper that makes it hard for me to draw any conclusions.

 

[Hutan]

Thanks for that @DMissa

For the muscle western blots that looked at WASF3 levels, where is the cohort information? Are the controls sedentary? Activity levels absolutely must be controlled for with muscle study, muscle cells change so much with activity.

I've only read a bit of the paper so far. The sedentary comparison is certainly something I've been thinking about, indeed it could easily be a confounder. There is a reference to the NIH ME/CFS study in the paper, with respect to the WASF3 findings:

Immunoblotting of deidentified skeletal muscle biopsy samples obtained from a comprehensive study exploring the clinical and biological phenotypes of postinfectious ME/CFS patients revealed significantly increased WASF3 levels with concomitant decreases in CIV subunit proteins compared with healthy volunteers (Fig. 5 A and B and SI Appendix, Fig. S11) (ClinicalTrials.govNCT02669212) (30).

I was expecting Reference 30 to be the protocol of the NIH ME/CFS study. It's actually B. Stussman et al., Characterization of post-exertional malaise in patients with myalgic encephalomyelitis/Chronic Fatigue Syndrome. Front. Neurol. 11, 1025 (2020).
Forum thread here:
Characterization of Post–exertional Malaise in Patients With Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (2020) Stussman, Nath et al.
That was the underwhelming sort-of-qualitative study that asked 43 people with ME/CFS about their thoughts about PEM - there was no experimentation; it wasn't the detailed characterisation study. I doubt that it tells us about the healthy controls in the detailed study. I can't recall if we have talked about how the controls were selected, or have seen anything about that?

 

[Hubris]

How can this be squared with the metabolic room showing no differences from controls?

 

[Hutan]

I had to search to find out what the error bars are for Figure 5b (that's the WASF3 levels from the NIH ME/CFS study participants). I eventually found a reference to one of the charts in the Supplementary Information to Standard Error. In my experience, standard errors on charts are not a good sign, and it's even worse when the authors don't clearly say what the error bars are. I really hope this finding turns out to be something useful.

I can't understand why this study, with all of the NIH funding and support that it had, is not open access. I also don't understand why the NIH didn't immediately jump into a study with 100 people with ME/CFS and 50 healthy but sedentary controls and 50 healthy but active controls, looking at levels of WASF3.

 

[Hutan]

How can this be squared with the metabolic room showing no differences from controls?

Were the participants required to exercise in the metabolic room?

 

[Sid]

I can't understand why this study, with all of the NIH funding and support that it had, is not open access. I also don't understand why the NIH didn't immediately jump into a study with 100 people with ME/CFS and 50 healthy but sedentary controls and 50 healthy but active controls, looking at levels of WASF3.

If they believed their own findings, they would. Instead we have this ridiculous paper with 14 ME/CFS patients and controls of no description.

 

[Hubris]

If they believed their own findings, they would. Instead we have this ridiculous paper with 14 ME/CFS patients and controls of no description.

I really hope I'm wrong but this is looking like the half assed studies that have gotten us nowhere in the past 40 years. This is why it was so important for the advocacy orgs to establish a strong narrative. If people aren't motivated they will just do the bare minimum to get their salary, throwing money at the problem isn't going to fix it (see long COVID). At least this study has an animal model and you can tell it has higher standards than the garbage we're used to (since it's coming from the NIH and their reputation is on the line) but it's still lacking that extra conviction of really wanting to figure things out. There is definitely apathy in the mix.

 

[LarsSG]

The WASF3 muscle data alone might not be that convincing, but coupled with the transgenic WASF3 mouse data showing significantly lower running distance compared to controls, plus lower OxPhos/glycosis, lower muscle glucose and higher blood lactate, it looks to me like they might be on to something.

I really hope they have already done some of the same work with the intramural study patients (the P-MRS recovery measurements and the later in vitro work).

 

[Hubris]

Were the participants required to exercise in the metabolic room?

Not as far as I'm aware (another missed opportunity).

 

[RedFox]

I can't understand why this study, with all of the NIH funding and support that it had, is not open access. I also don't understand why the NIH didn't immediately jump into a study with 100 people with ME/CFS and 50 healthy but sedentary controls and 50 healthy but active controls, looking at levels of WASF3.

Pretty sure there's a law that US gov't-funded publications must be open access. It is, however, under a Creative Commons license that permits noncommercial distribution, so it's legal to host a copy here despite the journal's attempt to paywall it.

 

[dreampop]

@B_V

Thanks for keeping us all up to date. It's appreciated.

Is this the publication from Nath you alluded to last month? Will it be the main/only publication from the intramural study?