WASF3 – NIH Researchers Find New Mitochondrial Abnormality in ME/CFS

by Cort Johnson | Aug 16, 2023 | Endoplasmic Reticulum, Energy Production, Homepage, ME/CFS, Metabolism, Muscles, Oxidative Stress, Pathogens, Research | 90 comments

The “WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome” study is fraught with possibility. For one, it was published in The Proceedings of the National Academy of Sciences – one of the most prestigious science journals in the world.

The mitochondria are back again in ME/CFS in…this time from NIH.

For another, it was produced by National Institutes of Health (NIH) researchers – a rarity. Usually, when we think of the NIH, we think of it as a funding source but the NIH also contains its own team of researchers. Avindra Nath’s Intramural ME/CFS study, for instance, is all being done “in-house” at the NIH. It’s the exception, though.

Look down the list of the authors of “WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome” and you’ll find they’re all from the NIH – and the vast majority are from an Institute – the National Heart, Lung and Blood Institute (NHLBI) – that hasn’t exactly been falling over itself to study or fund chronic fatigue syndrome (ME/CFS) research.

Lastly, the study boasts a nice Avindra Nath tie-in (Nath and Brian Walitt of the NINDS Institute are co-authors). The study used muscle samples gathered from Nath’s ME/CFS Intramural study and the authors thanked “the many individuals associated with the ME/CFS study … who made it possible for us to obtain de-identified samples of skeletal muscle”

The Chronic Fatigue Syndrome (ME/CFS) Connection

There’s a fascinating backstory. As reported in Science, “A protein that disrupts cells’ energy centers may be a culprit in chronic fatigue syndrome” (thanks to Brandon for the link). Paul Hwang, the senior researcher of the study, was interested in a family with a cancer-promoting mutation in a gene called TP53, one of which – a 38-year-old woman – had experienced increasing fatigue since coming down with infectious mononucleosis at age 16.

Extensive workups left her in a kind of gray area familiar to many. Suspected diagnoses included undifferentiated connective tissue disorder, systemic lupus erythematosus, and most recently, Sjogren’s syndrome (but apparently not chronic fatigue syndrome). Mitochondrial disease testing (mitochondrial genomic DNA sequencing and skeletal muscle biopsy studies) was unrevealing.

Looking for abnormalities in TP53-related pathways, they examined muscle tissue samples – and found very high levels of a protein called WASF3 in her samples but not in her siblings’.

Obscure ME/CFS Paper Provides Link

Suzanne Vernon’s pioneering effort on ME/CFS at the CDC in the mid-2000s ended up providing a key link.

What popped up in a literature search of WASF3 but a 2011 ME/CFS paper, “Meta-analysis of Chronic Fatigue Syndrome through integration of clinical, gene expression, SNP and proteomic data” with its own interesting backstory. The authors, who never published on ME/CFS again, were part of an extraordinary effort put together by Suzanne Vernon at the CDC.

While at the CDC, Vernon (who was later research director for Solve M.E. and is now research director for the Bateman Horne Center) produced one of the first large-scale efforts in any disease to integrate symptom, clinical, laboratory, gene expression, and proteomic data together.

The novel ME/CFS effort, called the C3 Computational Challenge, brought molecular biology, epidemiology, genomics, mathematics, engineering, and physics experts together to analyze the data gathered from 227 ME/CFS patients who’d undergone clinical evaluations, sleep studies, cognitive function, autonomic nervous system function, and extensive blood evaluations during a two-day hospital stay. Vernon said it was:

“a great opportunity to let loose the best and brightest minds in computational science on a really hard biological problem. The return was that we’d get a wealth of information from a first-of-its-kind study and dataset (the Wichita study) to advance our understanding of ME/CFS and maybe get people hooked on trying to solve ME/CFS.”

The results ended up filling an entire issue of the Pharmacogenomics journal in 2006 and prompted CDC leader Julie Gerberding to publicly declare that ME/CFS was a real and serious disorder that deserved more attention.

Next, Vernon enrolled CAMDA (Conference on Critical Assessment of Massive Data Analysis) in taking on the huge ME/CFS dataset. CAMDA, which has been in existence since 2000, invites researchers to probe “complex data sets, often featuring novel technological platforms, exceptionally large cohorts, and heterogeneous data sources and types.)

The short paper that provided the critical link to WASF3 and ME/CFS came out of the ME/CFS CAMDA competition. That paper – which highlighted 11 genes – stated “in particular, the gene WASF3 (aka WAVE3) possibly regulates brain cytokines involved in the mechanism of fatigue through the p38 MAPK regulatory pathway.”

That gene regulates the production of brain cytokines (think neuroinflammation) through the p38 MAPK pathway – which is involved in the central (i.e. brain-produced) mechanisms of fatigue. The paper, which never got much attention even in ME/CFS circles, has been virtually ignored – until now.

NIH Researchers Do Full Court Press on Mitochondrial Findings

It was remarkable watching these (apparently well-funded) researchers digging deeper and deeper into their findings.

Using a foot exercise, the NHLBI researchers twice assessed how well her phosphocreatine (PCr) levels rebounded after exercise and twice found that she had an unusually prolonged recovery period. The authors cited a 2004 ME/CFS study which apparently had similar findings.

Note the high WASF3 and PERK levels and very low MTCO1 and BiP in the ME/CFS patients vs the controls on the left. The findings all fit together.

Next, they found that her muscle tissues demonstrated a lower oxygen consumption rate (read energy production) and a decreased ability to transfer electrons from complex III to complex IV in the electron transport chain of the mitochondria.

Then came a new finding – a 40% increase in WASF3 protein levels that was followed by a 34% decrease in the cytochrome oxidase enzyme that transfers electrons from complex 3 to 4 in the mitochondria.

THE GIST

This study “WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome” stood out in a number of ways. It was published in the Proceedings of the National Academy of Sciences – one of the most prestigious science journals, and was produced by National Institutes of Health (NIH) researchers – a rarity.
It began with a study into a cancer-promoting mutation that featured a 38-year-old woman who had experienced increasing fatigue since coming down with infectious mononucleosis at age 16.
Her muscle tissues were found to have high levels of a protein called WASF3. When the researchers found a link to WASF3 in an obscure 2011 ME/CFS paper they were intrigued given her severe fatigue.
That paper was one of many that resulted from a pioneering ME/CFS effort by Suzanne Vernon at the CDC which gave an enormous dataset derived from hundreds of ME/CFS to a group specializing in computational challenges. The small paper, however, had never attracted any attention until now.
Various tests determined the woman’s cells were producing lower amounts of energy and, in particular, had a defect in their ability to transfer electrons from complex III to complex IV in the electron transport chain in the mitochondria.
A 40% increase in her WASF3 protein levels associated with a 34% in the cytochrome oxidase enzyme sealed the deal: the transition from complex 3 to complex 4 in the mitochondria was toast.
When they knocked down WASF3 levels in her cells her mitochondrial functioning improved. They reported “Collectively, these results showed that primary overexpression of WASF3 in cells leads to the disruption of respiration (energy production)”.
Next, they created high WASF3 mice which produced high blood lactate levels during exercise – which has also been found in ME/CFS. The mice appeared normal but had a “remarkable 50% reduction in maximal running capacity”.
ME/CFS patients from Avindra Nath’s big ME/CFS study were brought in and also found to have significantly increased WASF3 levels as well as evidence of problems in the transition from complex 3 to 4 in the mitochondria.
Next, they went “upstream”: to see if the endoplasmic reticulum which regulate WASF3 production were functioning properly. They found a mess of endoplasmic reticulum problems and concluded that high levels of endoplasmic reticulum stress are present in ME/CFS.
The outcome of all this was that a new mitochondrial abnormality that reduces energy production was found in ME/CFS. The authors stated that their finding provided a molecular explanation for the energy deficiency symptoms of exercise intolerance and postexertional malaise in a patient with chronic fatigue.“
The study was also notable in how deeply this team dug even going to far as to uncover novel ways WASF3 affected the mitochondria and creating genetically altered mice.
The study also involved Avindra Nath’s team. Early on reports suggested that Nath had found mitochondrial abnormalities in ME/CFS. Since then Nath has reported that his 5-year intensive study of ME/CFS was successful and will move the field forward and toward clinical trials. Time will tell but one could envision a major NIH effort with regards to the mitochondria in ME/CFS
ER stress is found in other disorders and supplements and drugs are being assessed. The NIH team is now looking at ER stress-reducing drugs with the hopes of producing a clinical trial in ME/CFS.

Next, using artificial RNA (shRNA) they knocked down WASF3 levels both in her and healthy people’s cells – and saw mitochondrial functioning improve in both. They reported:

“Collectively, these results showed that primary overexpression of WASF3 in cells leads to the disruption of respiration (energy production)”.

They took advantage of the genetic similarity between WASF3 in humans and mice to create transgenic (high WASF3) mice which produced particularly high WASF3 levels in the skeletal muscles. An exercise test indicated the high WASF3 mice produced higher blood lactate levels – which have been found in ME/CFS – and lower glycogen levels.

It got more interesting. As in ME/CFS, the mice appeared normal but showed a “remarkable 50% reduction in maximal running capacity”. Their grip strength, however, was not reduced, nor were the muscles structurally affected. The authors stated that the decrease in endurance appeared to result entirely from the mitochondrial problems found.

Next, they dug deep into WASF3 – which apparently had not been particularly well-studied with regard to mitochondrial functioning – and uncovered a number of new ways it affects the mitochondria. They concluded:

“overexpressed WASF3 can interfere with mitochondrial supercomplex formation…and (decrease) mitochondrial respiration (energy production)”.

Casting a Wider Net

With all that in hand, it was time to cast a wider net and bring in ME/CFS patients from Nath’s study. Not only did they find significantly increased WASF3 levels in the ME/CFS patients vs the healthy controls but also dramatically reduced cytochrome oxidase and MTO1 levels. (MTO1 is a protein expressed in high-energy-demand tissues such as the muscles.)

Because WASF3 is regulated by BiP (GRP78), an endoplasmic reticulum (ER) protein that checks proteins for quality control, the researchers went upstream to see if BiP levels were reduced – and they were even more significantly altered than WASF3. Plus, a marker of the endoplasmic reticulum stress called PERK was high.

With cytochrome oxidase levels negatively correlated with WASF3 and PERK levels, it looked like the researchers had uncovered a nice tight package of dysregulation – and now it was onto stressed out endoplasmic reticulum.

High Endoplasmic Reticulum Stress Response

The endoplasmic reticulum regulates protein folding, as well as protein transport and synthesis. Shape is everything in the body and if the proteins aren’t folded into their very intricate shapes correctly, they are unable to function. When levels of unfolded proteins build up, the endoplasmic reticulum stress response occurs. Viral infections and high levels of oxidative stress, among other things, can trigger an ER response.

ER stress has been found in neurodegenerative diseases, diabetes, metabolic syndromes, and cancer. Given the problem of misfolded proteins in Alzheimer’s Disease, it’s no surprise that ER stress and how to fix it is becoming a major emphasis in that disease. Several natural compounds and drugs (Berberine, Crocin, Bajijiasu, Echinacoside, Ginsenoside-Rg1, Salubrinal, Taurodeoxycholic acid) are being contemplated in AD. Metformin – which has been suggested for fibromyalgia, ME/CFS, and long COVID – is an ER stress response inhibitor.

The outcome of all of this was that the authors showed that “WASF3, induced by ER stress, disrupts the formation of respiratory supercomplexes and reduces mitochondrial oxygen consumption (energy production), providing a molecular explanation for the energy deficiency symptoms of exercise intolerance and postexertional malaise in a patient with chronic fatigue.”

They also showed – in the lab at least – that reducing the endoplasmic reticulum stress in the cells of an ME/CFS patient improved their mitochondrial functioning; i.e. they believe their finding could have “therapeutic implications for relieving fatigue symptoms in ME/CFS.”

They suggested that a similar scenario may be playing out in long COVID and other fatiguing rheumatic diseases such as fibromyalgia and rheumatoid arthritis.

Check out the ME Association’s initial take on the study

Conclusion

There was no holding back on this study. It was a full-court press by some NHLBI researchers who have apparently become very interested in ME/CFS.

The authors were excited enough about the WASF3 finding to dig deeper and ultimately add substantial insights into the role WASF3 plays in the mitochondria. Its ability to disrupt the mitochondrial enzyme supercomplexes in ME/CFS was notable because genetic conditions that inhibit these supercomplexes are characterized by, you guessed it, exercise intolerance. The authors even proposed that high WASF3 levels in the brain may be contributing to mitochondrial problems and brain fog in ME/CFS.

This saga – which came together because of an (undiagnosed) ME/CFS patient in a cancer study and an obscure paper that came out of a novel and exploratory effort 15 years ago – demonstrates how serendipity can strike in the medical world.

Akiko Iwasaki, an immunobiologist at Yale School of Medicine told Science that the study was “very well done”. She cautioned that the suspect protein is likely “a piece of the puzzle, as opposed to explaining the whole disease” and could act as one of several “middlemen” between whatever sparks the illness and symptoms such as fatigue.

She’s probably right given the many other indications (disrupted autophagy, reduced fatty acid metabolism, high levels of intracellular calcium, complex V dysregulation, HHV-6 reactivation/mitochondrial fragmentation, citrate synthase deficiency, inborn errors of metabolism) that the mitochondria are involved in ME/CFS. What we really need is a full-court press on the mitochondria.

Pere Puigserver, a cell biologist at Harvard Medical School, told Science that it wasn’t clear whether ER stress was the problem or if the mitochondria were. Plus, he noted that WASF3 plays so many roles in the body that high levels could be causing other problems as well.

This small study will require further validation, but note that one critical part of the study gives us more hope than usual that these results may be on track. The study used muscle samples from ME/CFS patients in Avindra Nath’s Intramural study who went through an excruciatingly detailed process to ensure they only had ME/CFS. First checked over by a team of ME/CFS experts, they then underwent a week of intensive testing. These patients and the samples they left behind are like gold in a research world where ME/CFS is often considered a heterogenous, difficult-to-study disease.

Paul Hwang, the leader of the NIH effort, told Science he is looking at treatment options and hopes to do a clinical trial.

Avinda Nath and Brian Walitt from the ME/CFS Intramural study are clearly aware of the study. Early reports suggested that Nath might be finding mitochondrial abnormalities in ME/CFS. More recently, Nath reported that he’d found “consistent biological problems” and predicted the study was going to move the ME/CFS field forward “in a big way” and toward clinical drug trials. If both Nath and this team found mitochondrial abnormalities in ME/CFS, perhaps it’s time for the NIH to do the full-court press this issue needs and fund an RFA grant opportunity.

For his part, Paul Hwang, the leader of the effort, is looking at drugs to either reduce ER stress or reduce WASF3 levels to use in a clinical trial, and Director Walter Koroshetz of NINDS reported that Hwang and Nath are continuing to work together.

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