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

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

 

@Jonathan Edwards does this look interesting?

 

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?

 

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/

 

Physical exercise is one thing that induces autophagy.

 

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.

https://www.youtube.com/watch?v=8KzyoEFgQec

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.

 

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

 

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

 

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

 

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?

 

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

 

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

 

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.

 

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

https://twitter.com/user/status/1519097828836102146


He mentions further study in a mouse model.

 

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.

 

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.