Elevated ATG13 in serum of pwME stimulates oxidative stress response in microglial cells (Gottschalk et al, 2022)

SNT Gatchaman

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Elevated ATG13 in serum of patients with ME/CFS stimulates oxidative stress response in microglial cells via activation of receptor for advanced glycation end products (RAGE)
Gunnar Gottschalk, Daniel Peterson, Konstance Knox, Marco Maynard, Ryan J Whelan, Avik Roy

Myalgic Encephalomyelitis, also known as Chronic Fatigue Syndrome (ME/CFS), is a multisystem illness characterized by extreme muscle fatigue associated with pain, neurocognitive impairment, and chronic inflammation. Despite intense investigation, the molecular mechanism of this disease is still unknown.

Here we demonstrate that autophagy-related protein ATG13 is strongly upregulated in the serum of ME/CFS patients, indicative of impairment in the metabolic events of autophagy. A Thioflavin T-based protein aggregation assay, array screening for autophagy-related factors, densitometric analyses, and confirmation with ELISA revealed that the level of ATG13 was strongly elevated in serum samples of ME/CFS patients compared to age-matched controls.

Moreover, our microglia-based oxidative stress response experiments indicated that serum samples of ME/CFS patients evoke the production of reactive oxygen species (ROS) and nitric oxide in human HMC3 microglial cells, whereas neutralization of ATG13 strongly diminishes the production of ROS and NO, suggesting that ATG13 plays a role in the observed stress response in microglial cells. Finally, an in vitro ligand binding assay provided evidence that ATG13 employs the Receptor for Advanced Glycation End-products (RAGE) to stimulate ROS in microglial cells.

Collectively, our results suggest that an impairment of autophagy following the release of ATG13 into serum could be a pathological signal in ME/CFS.

PubMed | ScienceDirect (paywalled)
 

SNT Gatchaman

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Up to n=12 case/control study. Some selected quotes from the paper, edited for brevity —

  • Taken together, these results indicate there is an increased protein aggregation pattern in the serum of ME/CFS patients compared to healthy controls.
  • Interestingly, we observed that only ATG13, but not ATG5, LC3a, and p62 was found to be strongly elevated in the serum samples in n=10 ME/CFS patients.

  • Collectively, these data suggest that early autophagy marker ATG13 might be dysregulated in ME/CFS patients.
  • Taken together, these results suggest that elevated ATG13 in the serum of ME/CFS patients directly stimulates oxidative stress and iNOS-induced NO production in microglial cells.
  • Taken together, our results indicate that serum samples of ME/CFS patients have elevated ATG13 proteins indicative of a biochemical impairment of autophagy. Our data further demonstrate that the serum-derived ATG13 is phosphorylated and employs the RAGE receptor to induce oxidative stress response in microglial cells.
  • Apart from these neurotoxic implications, serum ATG13 is also correlated with cardiomyopathy. A recent study indicates that during myocardial infarction, the plasma level of ATG13 rises and that selective inhibition of circulating ATG13 is protective against myocardial infarction and other cardiovascular impairments. Interestingly, we observed that a 67-year-old female patient in our present study with significantly high level of serum ATG13 also suffers from cardiovascular abnormalities with exceptionally high blood pressure, which could be related to the high level of circulating ATG13.
  • ATG13 also regulates the expression of antiviral interferon β to restrict viral infection and plays an essential role in innate immune response to provide antiviral immunity.
  • Our In-silico analysis followed by functional binding assay suggest that ATG13 interacts with RAGE receptor in microglial cells to induce ROS and NO production.
  • Interestingly, a recent report suggests that upon phosphorylation, ATG13 aborts the autophagy process and gets released to serum, suggesting that phosphorylated ATG13 in ME/CFS patients might contribute to the impairment of the cellular autophagy process.
 

Pyrrhus

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Well, it has been quite a while since I read up on autophagy and I don't recall reading about ATG13 before, but according to the above information it sounds like un-phosphorylated ATG13 is an autophagy protein, but phosphorylated ATG13 might be a pro-inflammatory cytokine. o_O

It's certainly not unheard of for one protein to have two very different roles in the body, but this is interesting indeed.

I'm not sure which would be the more interesting conclusion:
  • Phosphorylated ATG13 is a pro-inflammatory cytokine.
  • Phosphorylated ATG13 is elevated in the blood of ME patients.
In any case, I do hope someone follows up and tries to replicate at least one of those conclusions!
 

SNT Gatchaman

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So what do you find especially important about these findings?
(Learning as I'm reading. These areas are not familiar to me). For me the idea that a fundamental repair/homeostasis/immune function might be broken is compelling - particularly as it
  1. Relates to recognising and fixing impaired mitochondria
  2. Would be occult, occurring within cells while "all (clinical) tests are normal"
  3. Is a 57 kDa serum protein that seems to induce abnormal behaviour in healthy cells (we're always wondering what the something-in-the-blood is)
Might be a signal of upstream pathology, but sounds definitely worthy of an effort to replicate and see if it's a valid biomarker against healthy controls, with a stretch goal of being unique to ME and related conditions. But perhaps it's a dead end, or valid but more common, and that abnormal phosphorylation of ATG13 happens in many other diseases.
 

Pyrrhus

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For me the idea that a fundamental repair/homeostasis/immune function might be broken is compelling - particularly as it
  1. Relates to recognising and fixing impaired mitochondria
  2. Would be occult, occurring within cells while "all (clinical) tests are normal"
  3. Is a 57 kDa serum protein that seems to induce abnormal behaviour in healthy cells (we're always wondering what the something-in-the-blood is)
Ah, I think I see, so the theory is:
  1. Un-phosphorylated ATG13 is an intracellular protein that is needed for autophagy and repair of mitochondria.
  2. If something abnormally phosphorylates the ATG13, then the phosphorylated ATG13 (p-ATG13) is forced to leave the cell and wander the blood.
  3. The loss of ATG13 from the cell leaves the mitochondrial repair mechanism incomplete, predisposing the cell to energy deficit.
  4. The p-ATG13 circulating in the blood triggers pro-inflammatory activities, such as macrophage/microglia activation.

By the way, here's the graphical abstract for people who like pretty pictures:
1651383132659.png
 

SNT Gatchaman

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While keeping an eye to the "something-in-the-blood" possibility, the other aspect of Prof Ron Davis et al' work to consider would be the IDO-1 metabolic trap theory. Does anyone have good understanding of the kynurenine pathway to suggest how it might predispose to abnormal intracellular phosphorylation (specifically of ATG13)?

I did find a possible link between the tryptophan-kynurenine pathway and β-catenin and autophagy. WNT/β-catenin and autophagy pathways seem to have cross-talk —

  1. Tryptophan-kynurenine pathway attenuates β-catenin-dependent pro-parasitic role of STING-TICAM2-IRF3-IDO1 signalosome in Toxoplasma gondii infection
  2. Interplay Between Autophagy and Wnt/β-Catenin Signaling in Cancer: Therapeutic Potential Through Drug Repositioning
I haven't had a chance to read these through yet, but just briefly skimming suggests there may be a possibility of:

decreased kynurenine -> decreased β-catenin -> inhibition of Beclin-1 -> halt of autophagy.
 

mitoMAN

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While keeping an eye to the "something-in-the-blood" possibility, the other aspect of Prof Ron Davis et al' work to consider would be the IDO-1 metabolic trap theory. Does anyone have good understanding of the kynurenine pathway to suggest how it might predispose to abnormal intracellular phosphorylation (specifically of ATG13)?

I did find a possible link between the tryptophan-kynurenine pathway and β-catenin and autophagy. WNT/β-catenin and autophagy pathways seem to have cross-talk —

  1. Tryptophan-kynurenine pathway attenuates β-catenin-dependent pro-parasitic role of STING-TICAM2-IRF3-IDO1 signalosome in Toxoplasma gondii infection
  2. Interplay Between Autophagy and Wnt/β-Catenin Signaling in Cancer: Therapeutic Potential Through Drug Repositioning
I haven't had a chance to read these through yet, but just briefly skimming suggests there may be a possibility of:

decreased kynurenine -> decreased β-catenin -> inhibition of Beclin-1 -> halt of autophagy.
Is this of value?

Importantly, mTORC1 is incorporated into the ULK1-Atg13-FIP200 complex through ULK1 in a nutrient-dependent manner and mTOR phosphorylates ULK1 and Atg13. ULK1 is dephosphorylated by rapamycin treatment or starvation. These data suggest that mTORC1 suppresses autophagy through direct regulation of the approximately 3-MDa ULK1-Atg13-FIP200 complex.

https://pubmed.ncbi.nlm.nih.gov/19211835/
 

SNT Gatchaman

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SlamDancin

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The strain of mice they used– called B6 – is the most commonly used inbred mouse and was the first to have its entire genome sequenced. They gave the mice a compound , an mTOR activator and autophagy inhibitor that inactivates a protein called ATG13 which activates two mytokines called IL-6 and RANTES. Although, IL-6 is released by the muscles during exercise and is believed to enhance energy production, together with RANTES it can cause molecular changes in muscle tissue causing fatigue

The Simmaron mice, then, were given a drug that impairs energy production and oxygen consumption during exercise – two problems studies have shown are present in ME/CFS.
Interestingly, the mice displayed a dramatic gender split with the female mice much more apt to display fatigue – a sign given the gender split in ME/CFS that they were on the right track. Electromyography (EMG) muscle tests revealed that bicep muscles in the mice given the autophagy inhibitor quickly became fatigued, and their grip strength declined. Likewise, a treadmill test indicated they were less active and displayed increased fatigue. More testing is needed but thus far, they look very much like ME/CFS mice. They’re bringing in more mice to confirm the findings.
I asked the Simmaron team what’s next?
Their ultimate goal is uncovering the molecular factors of fatigue in ME/CFS. Is it neurogenic; i.e. is it caused by a nervous system impairment or is it myogenic; i.e. caused by damage to the muscles?
Roy stated that their next project involves making a transgenic mouse model of ME/CFS; i.e. a mouse model into which human genes have been introduced that they believe will produce some of the cardinal symptoms of ME/CFS such as PEM and OI.
@SNT Gatchaman
 

SNT Gatchaman

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A little bit more cud chewing.

Some LC patients are reporting that inducing autophagy (by intermittent fasting) is helpful. Sometimes these patient reports describe an initial significant deterioration in symptoms which, following a recovery period, lessens on subsequent inductions. Let's not frame these as recovery stories per se, but thinking more about this, it occurs to me that broken autophagy could relate to a couple of our known concepts.

Autophagy happens at a baseline level, especially during sleep. If it's broken, eg arrested and producing deleterious pATG13, that might relate to "unrefreshing sleep". If the autophagy program has failed, it would not be clearing intracellular rubbish, recycling defective mitochondria etc. The cycle would continue.

I have noticed that if my sleep is disturbed (eg cats) I typically have a paradoxically better day. Maybe autophagy attempts were more limited, resulting in less breakage and pATG13 production.

Might this be something of a bistable state, analogous to the metabolic trap? Do some manage to get over the hump to the happy side?

Some people may be able to induce more autophagy, eg by intermittent fasting, which, despite making more pATG13 or similar and making symptoms worse, were still able to improve the intracellular landscape sufficient to have made ground overall. In principle this would be like a Herxheimer effect. Stick with it (to the right degree and with all else being favourable), rinse and repeat to reap the gains of improving returns.

However, people with severe ME are often starving and likely in advanced autophagy already and they certainly don't improve as a matter of course. Is their autophagy system broken in a more comprehensive/unrecoverable way? Are their other things needing to be aligned just right, such as the microbiome or blood glucose levels? Or do they remain in baseline autophagy, unable to induce it further?

If this mechanism were at play, then at the moment we'd be left with a "leap of faith" situation. Fasting induced autophagy would likely worsen symptoms initially, but there's no guidance as to whether this might be "good" or "bad". You might try to push on through and ultimately do more harm and worsen your situation.

I'd definitely like to see more research in this area.