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

[Sly Saint]

For the abstract of the published paper, go to post #9


Preprint

Abstract

Myalgic Encephalomyelitis and chronic fatigue syndrome is a multisystem illness characterized with 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 report two independent case-control studies to demonstrate that autophagy proteins are strongly upregulated in the serum of ME/CFS patients indicative of severe impairment in metabolic events of autophagy.

Serum samples collected from two healthy and two age-matched patients were assayed for protein aggregation, screened for autophagy-related factors via an antibody array, quantified with densitometric analyses, and finally reconfirmed with ELISA analyses. Based on that double-blinded and gender-balanced study, the levels of ATG13, p62, and alpha-synuclein (α-syn) were found to be consistently elevated in the serum samples of these two ME/CFS patients.

Moreover, our microglia-based oxidative stress response study and nitrite analyses indicated that serum samples of ME/CFS patients evoked the production of reactive oxygen species (ROS) and nitrite in human HMC3 microglial cells, whereas neutralization of ATG13 was shown to strongly diminish the production of ROS and nitrite demonstrating the de novo effect bloodborne autophagy factors on inducing the stress response in microglial cells. Collectively, our results indicate that the impairment of autophagy followed by upregulations of autophagy markers especially ATG13 in serum could be a pathological hallmark in ME/CFS.

https://www.researchsquare.com/article/rs-925403/v1

 

[Hoopoe]

@Jonathan Edwards does this look interesting?

 

[Jonathan Edwards]

I am struggling to see how they hope to publish a paper on two patients?

 

[Ravn]

I am struggling to see how they hope to publish a paper on two patients?

Poorly written abstract. The paper actually consists of two parts.

The first is about the 2 case controls mentioned in the abstract.

The second is a follow up with 12 patients and 12 controls - not mentioned in the abstract - to further validate the main findings around ATG13 found in the first part.

Without understanding the biochemistry, together the results look interesting enough to warrant further investigation into ATG13?

 

[Ravn]

Without understanding the biochemistry, together the results look interesting enough to warrant further investigation into ATG13?

Or maybe not just into ATG13 but into possible problems with cell death more generally? ATG13 is linked to autophagy in this paper. A different team* found elevated anti-apoptotic IL9 (in female pwME only, preliminary data from very few patients - again again, sigh).

Autophagy and apoptosis are thought to be linked though nobody seems to be sure how exactly - and that's even before throwing ME into the mix.

*discussed in this thread: https://www.s4me.info/threads/nih-2...work-on-altered-t-cells-in-me-cfs-2021.19726/

 

[Hoopoe]

Physical exercise is one thing that induces autophagy.

 

[SNT Gatchaman]

If anyone is interested in a simplified overview of the mechanisms underlying autophagy (and its potential induction via intermittent fasting), Dr Been released a video today on his channel.



Discusses aspects such as how the immune system can be repaired from dysfunction and how viral proteins that are sequestered in cells but doing harm can be broken down, and subsequently presented for immune recognition and action.

 

[Boba]

The research group extended the study by additional 7 hc matched me/cfs patients and were able to replicate and extend their findings:
https://www.sciencedirect.com/science/article/abs/pii/S1044743122000379

 

[Mij]

Merged thread - published paper

Elevated ATG13 in serum of pwME stimulates oxidative stress response in microglial cells via activation of receptor for advanced glycation end products(RAGE), 2022, GunnarGottschalk et al

Abstract

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.

https://www.sciencedirect.com/science/article/abs/pii/S1044743122000379

 

[Slamdancin]

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714170/

ATG13 has non-autophagic capabilities and it appears to be anti-viral and relate to interferon release

 

[Jonathan Edwards]

Unless people are prepared to put some actual data in the abstract I am unenthusiastic about trying to wade through. I seem to be able to access a half paper with some 'snippets'. It all seems like deliberate obfuscation purely for the benefit of those making money out of the publication process.

If the findings are meaningful they could be very important but if they are why not show them?

 

[Boba]

Here’s some part of the data. Not all patients had elevated ATG13. 2 Controls were elevated as well.

 

[Jonathan Edwards]

Thanks. That seems a pretty bizarre way to present data of this sort. Not sure what negative concentrations are supposed to be!

 

[LarsSG]

There does seem to be some interesting findings in here, though with very small n.

If I'm understanding correctly, they took patient and control serum and added it to microglial cells in vitro and found the ME patient serum caused reactive oxygen species (ROS) to increase significantly more than control serum did (but this was only two patients and two controls). Then they removed as much ATG13 from patient serum as they could and found significantly less ROS, about the same as controls (this part was n=6, plus the initial two subjects).

,
Then they found pretty similar results for patient serum, control serum and patient serum with ATG13 removed for nitric oxide production in microglial cells.


Then they did something to neutralize RAGE and found that it significantly reduced ROS.

But the ATG13 levels were all over the map for controls and patients (and I'm also confused by the negative concentrations). Their initial two patients only had ATG13 around 5ng/ml, while 25% of controls had levels around 4 or 5. I wonder if something is going on with that table though, because Figure 3 seems to be presenting the same data, but it obviously isn't the same.

It's not really clear to me how they got such strong results if ATG13 is not that different in patients versus controls, but then it isn't clear which patients and which controls were used for these charts. This seems strange.

This is all way over my head, but interesting to see more findings on oxidative stress.

 

[LarsSG]

Also, some interesting discussion with one of the authors on Twitter:



He mentions further study in a mouse model.

 

[Hoopoe]

The author says on Twitter that Atg13 in serum is “result” of autophagy impairment, not the “cause”.

Autophagy was initially discovered by studying starvation, then later it was also found to play a major role in homeostasis. Autophagy sounds like the kind of maintenance process that might be involved in PEM.

 

[Hutan]

This interview is a bit relevant - there is a transcript. Norman Swan talking to Professor Luigi Fontana about research on extending human lifespan. Professor Fontana is keen on calorie restriction.
https://www.abc.net.au/radionational/programs/healthreport/the-lowdown-on-longevity/13850872

Luigi Fontana: ... Autophagy is different. So as we get older, we have accumulation of garbage in our cells because our cells are becoming less efficient to remove misfolded proteins, old mitochondria, so mitochondria that get oxidised, they become dysfunctional, and so if you are in energy restriction, the cells, they are trying to say, okay, now where do I get the material to create energy for cell functioning, and they start to digest the garbage, and so that's called autophagy.

I recall that it has been said that ME/CFS looks metabolically like starvation - I don't recall the details or if it's likely to be true. But, Professor Fontana is saying calorie restriction promotes autophagy. Atg13 is important in inducing autophagy. And here, the authors are reporting that Atg13 is upregulated in people with ME/CFS.

But the ATG13 levels were all over the map for controls and patients (and I'm also confused by the negative concentrations).

I don't think 'all over the map' is quite true, if the data above are correct. If you ignore the patient without CFS (just SFN -small fibre neuropathy?), then 10 out of the 11 CFS patients have high values, and 10 of the 12 controls have low values. That's probably as good as we could expect. But yeah, the negative concentrations?

 

[Hutan]

(I'm working with the snippets only.)

So, they tested for 20 autophagy-related proteins in two patients and two controls, and presumably found just ATG13 was different. They then validated the result of elevated ATG13 with 10 or so more patients and controls.

To explore the molecular regulation of autophagy proteins in ME/CFS patients, double-blinded antibody array analyses of 20 autophagy-related proteins were performed in the serum samples of two case-control subjects that demonstrated strong elevation of ATG13. The result was further validated with ELISA analyses in ME/CFS patients (n = 10).

I'm assuming the story they are suggesting is:
1. there's an accumulation of faulty mitochondria (and other debris) in a cell.
2. Levels of ATG13 increase, to start the clean-up process.
3. For some reason, the clean-up process doesn't happen

The author says on Twitter that Atg13 in serum is “result” of autophagy impairment, not the “cause”.

4. Levels of ATG13 increase to keep signalling for the clean-up that never comes

But then, there's also the story of the ATG13 in the patient serum seeming to cause a production of reactive oxygen species, while the ATG13 in the control serum does not. So, is there something different about the ATG13 in ME/CFS patients? Perhaps a faulty ATG13 is in fact the cause of the autophagy not happening, as well as the ROS. (The comment from Twitter seems to suggest the authors have another theory.)

If ATG13 is important in immunity against pathogens as well as in initiating autophagy, then perhaps some pathogens have evolved to hobble the ATG13?

 

[Hutan]

As @Slamdancin suggested above, there's the fact that viral infections cause ATG13 expression to be increased.

See for example:
Antiviral responses of ATG13 to the infection of peste des petits ruminants virus through activation of interferon response, 2020, Ma et al

Here, we demonstrated that ATG13 exerted an obviously antiviral activity against the infection of peste des petits ruminants virus (PPRV) in cell culture model. We found that PPRV infection or the treatment with interferon (IFN) against PPRV infection significantly induced ATG13 expression. Mechanistically, ATG13 stimulated interferon expression and the subsequent activation of the JAK-STAT cascade. These activations triggered the transcription of interferon-stimulated genes (ISGs) to exert antiviral activity.

So, an alternative story might be that the ATG13 is increased in the serum of people with ME/CFS because their cells have been infected by a virus (or at least the cells or some other part of the body think they have been infected).

One thing to think about is that higher levels of ATG13 in the serum is not the same as higher levels of ATG13 in the cell. ATG13 might not be higher in the cells of people with ME/CFS compared to the controls. Maybe ME/CFS cells are self-destructing and releasing their ATG13 into the blood?

 

[SNT Gatchaman]

Not sure what negative concentrations are supposed to be!

I'm also confused by the negative concentrations

I don't know for sure, but I wonder whether it's an offset from a virtual control as they are measuring via optical density.

The density of individual proteins was measured in ImageJ software, subtracted from blank, normalized with the positive control, and then plotted at GraphPad Prism 8 software.