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

This is very promising. From the paper , we read about the use of TUDCA, a well known inhibitor of ER Stress. According to my case, I believe that TUDCA was a key intervention towards my recovery. I just email Dr Hwang , informing him regarding the use of TUDCA and how I believe it helped me recover :

Screenshot 2023-08-15 at 08.29.18.png



Relevant Twitter thread with the email sent to ME/CFS researchers, discussing the use of TUDCA and ER Stress amelioration :
 
Going a bit further on in the paper
WASF3 Suppresses Mitochondrial Respiration
So, this is still with the cells of patient S1. The expression of the WASF3 gene was reduced (using shRNA - a short hairpinRNA). The authors say that this
improved basal respiration and significantly boosted spare respiratory capacity (SRC), a robust measure of
mitochondrial function, to the level of control S2 cells (Fig. 1D).
Click to expand...

The higher black line is the increased oxygen consumption rate after the WASF3 knock down, the red line is with the high baseline WASF3.
Screen Shot 2023-08-15 at 7.40.26 pm.png

I'm assuming, from the shape of Fig 1D although I expect the later Methods section says, that they measured the basal respiration and spare respiratory capacity using the seahorse machine. We've seen a range of results from ME/CFS cells in the seahorse machine, including no difference with healthy controls, so one person's cells showing a difference is interesting but not convincing in relation to ME/CFS. And there's the points that @DMissa made.

reducing WASF3 increased the subunit levels of CIV, but not of the other complexes, while decreasing the phosphorylations of p53 and p38, but not JNK in S1 cells (Fig. 1E
Click to expand...
They then note that reducing WASF3 increased those components of cytochrome IV, and reduced the downstream phosphorylations of p53 and p38. The immunoblots shown for that look convincing, (although of course this is just for one person). Fig 1 E shows before and after WASF3 knockdown, with the first line, WASF3, then the first and second components of cyctochrome IV.

Screen Shot 2023-08-15 at 7.44.37 pm.png

The researchers did more work to confirm the relationship between WASF3 and mitochondrial spare respiratory capacity, p38 phosphorylation and the complex IV subunits in other cells. The relationships they are suggesting seem to be supported.
As further confirmation of this signaling pathway, knocking down WASF3 in human myoblasts with or without TP53 mutation and control human fibroblasts all showed increased mitochondrial SRC and decreased p38 phosphorylation (SI Appendix, Fig. S3). In contrast, overexpressing WASF3 in mouse C2C12 myoblasts with wild-type p53 induced p38 phosphorylation while decreasing respiration and subunits of complex IV, but not other complexes (Fig. 2 A and B). Notably, some subunits of other respiratory complexes were increased by WASF3 overexpression, suggesting that a feedback signal formitochondrial biogenesis was activated by WASF3 disruption of mitochondria (Fig. 2A).
Click to expand...

Collectively, these results showed that primary overexpression of WASF3 in cells leads to the disruption of respiration, which in turn may activate compensatory responses such as p38 phosphorylation for promoting mitochondrial biogenesis. Our findings also suggest that WASF3 posttranslationally regulates mitochondrial complex IV as evidenced by decreased protein but increased mRNA levels of complex IV subunits in cells overexpressing WASF3 (SI Appendix, Fig. S4). Furthermore, the regulation of mitochondrial respiration by WASF3 was not found to be restricted to a specific cell type or linked to p53 mutation status.
Click to expand...
 
Last edited:
dreampop said:
@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?
Click to expand...
No this is not from the intramural study team.

This was research that arose from a very tenacious lawyer from Albany with Li Fragmeni Syndrome. Its connection to the ME/CFS study was serendipitous. I really wish my article describing how it came about was published today but it'll be at least a week. August is a bad time to get stuff published. But I found it to be a really interesting story.
 
Hutan said:
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.
Click to expand...

I haven't kept up with the federal open access policy but it used to state that NIH funded work had to be made public access within a year of publication. I hope it changes if that's still the case.

Hwang is trying to get a trial going. The paper was just published and he's been working on it...so I'd say that is pretty much immediately in research time terms.
 
Hutan said:
The researchers compared fibroblasts

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 ion 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.

View attachment 20147
Click to expand...

This figure is the bit that really matters for sure. They have taken muscle biopsies from these 10 controls and 14 ME/CFS patients (from the intramural study i think so should be diagnosed well) and done western blots on them for WASF3, MTCO1, COX17, PERK and BiP. Here's the rest of the figure showing the western blots themselves and the quantified bar plots (just quantifying how large the blobs are from the western blot).

upload_2023-8-15_9-45-32.png

They see elevated WASF3, reduced ComplexIV proteins MTCO1 and COX17, increased endoplasmic reticulum (ER) stress marker PERK, and reduced protein-folding chaperone BiP. I really don't like when this type of data is represented in bar plots without showing the individual datapoints as a strip style scatter plot. We have no idea how much the groups overlap and they also don't tell us the effect size. Our only indication of variance is the error bar which as you point out represents the standard error which is the wrong metric to use - it should be standard deviation. The standard error of the mean is always smaller than the standard deviation so it looks better too. They do at least show all of the blots for all these proteins for the controls and patients (some hidden in the supplementary) so theoretically we could make these plots properly ourselves.

They go on to see the same set of proteins showing the same phenotype when they chemically induce ER stress in a separate experiment which is potentially interesting.

Their overall model for what is happening is that ER stress leads to an increase in PERK expression (a marker for ER stress), a reduction in BiP expression (an ER protein which helps proteins fold as they're being made) and in someway therefore an increase in WASF3 expression (being translated directly into the ER co-translationally I think). WASF3 translocates over to the mitochondria and binds to oxidative phosphorylation complex 3 preventing it from forming a complex with complex 4, thereby inhibiting oxidative phosphorylation activity.

According to the text S1 had all sorts of different diseases and that she had chronic fatigue and exercise intolerance - though no specific mention of ME/CFS diagnosis.
authors said:
two separate early-onset breast cancers, as well as extensive evaluation of her fatigue symptoms that resulted in the suspected diagnoses of undifferentiated connective tissue disorder, systemic lupus erythematosus, and most recently, Sjogren’s syndrome by biomarkers.
Click to expand...

So yeah as always it needs to be replicated in a much bigger cohort with sedentary controls carefully selected and with disease controls - possible it could be a broadly applicable phenotype not specific to ME/CFS.
 
Last edited:
DMissa said:
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.
Click to expand...

This is really interesting thankyou it sounds like you're really familiar with this technique. Agree about passage number in particular and didn't know about contact inhibition that's interesting.

Out of interest is there data showing differences in seahorse results with changes in confluence level, trypsinisation time and so on? I can see how trypsinisation could be harming the cells a little bit when you passage them but it would be a while after you trypsinise and seed the cells, let them grow, change culture medium etc before you actually carry out the assay - does it really make that much of a difference?

Also with protein quantification it seems surprising to me that differences in expression would really affect the global amount of protein in a cell so much as to make it a poor proxy for cell mass. I would have thought it would be fairly negligible or otherwise the cells would be visibly different. I'm happy to be wrong just trying to understand.
 
I think the 2011 analysis by Pishur et al. that highlighted the gene WASF3 in CFS, used data from the Wichita CDC study which used a very broad interpretation of the Fukuda criteria and found a prevalence above 2%.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089886/

That isn't necessarily a problem though as this study argues that WASF3 might be involved in fatiguing conditions other than ME/CFS as well.
 
Today's daily email 'newsletter' from Nature features this study as one of five 'headline storys', and links to the article in Science:

"Energy-sapping protein in chronic fatigue

A protein that disrupts cells’ energy production could be one of the reasons why people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) experience extreme exhaustion and cognitive problems. Researchers found higher levels of the protein WASF3 in the muscle cells of 14 people with ME/CFS than in samples from 10 healthy individuals. What exactly causes excessive levels of WASF3, which interferes with energy-generating mitochondria, remains unclear. Treatments that target the protein could help to combat related illnesses, such as long COVID."
 
RedFox said:
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.
Click to expand...
There is, but it's possible it hasn't kicked in yet. There was a delay phase built into it. 2024 actually sounds about right but I'm not sure.
 
You must log in or register to reply here.
Back
Top Bottom