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

[SNT Gatchaman]

This paper is very interesting. I'm pretty sure I've read of a number of ME patients who have tried intermittent fasting, but have "failed" with it. Perhaps that might relate to autophagy being broken, so that IF is unable to realise any benefits in the repair of mitochondria and improvement of immune dysfunction.

Thinking about how autophagy might be broken, along with other observations around Long Covid: Bruce Patterson was blocking CCR5 with Maraviroc. In the HIV literature there was comment that "functional CCR5 appears to be involved in autophagy inhibition (during HIV infection with R5-tropic strains)."

Don't know enough to answer if that might apply more generally in other cells and contexts, but it makes me wonder if it's possible that some patients are benefiting from CCR5 blockade disinhibiting autophagy.

See The Interplay of HIV and Autophagy in Early Infection.

 

[Boba]

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.

A quote from a blog post from Cort (https://www.healthrising.org/blog/2...hronic-fatigue-syndrome-starvation-australia/):

„Why is this happening? Armstrong suggested a couple of reasons. He noted that many of the metabolomic anomalies he found in ME/CFS are also found in sepsis and starvation. All show reductions in amino acids and lipids and increased levels of glucose. In both diseases proteins and lipids are used to produce maintain low energy levels while glucose is used for other matters – such as immune cell proliferation in sepsis“

I‘m a long covid patient (bedridden) who had some health problems before contracting covid. My guess is I was managing mild me before coming down with covid. One diagnose I had was Hashimoto’s, therefore my thyroid hormones got checked regularly. After Covid my ratio ft4/ft3 was decreased. Despite having enough ft4 my body was not able to convert it to ft3 as good as before. Later I was diagnosed with low-t3-syndrome. Where else would you have this phenomenon? Starvation and ICU patients.

 

[Hutan]

The autophagic programme is activated by a variety of factors, such as
amino acid limitation,
low cellular energy,
changes in pH or temperature,
hypoxia,
oxidative stress,
pathogen infection and
removal of growth factors

(Burman and Ktistakis, 2010; Yang and Klionsky, 2010). It is well established that autophagy is crucial for the homeostasis of both cells and tissues, and its dysfunction causes disease, with most notable involvement in neurodegeneration, cancer and ageing (Mizushima and Komatsu, 2011; Sridhar et al., 2012).

from Dynamic association of the ULK1 complex with omegasomes during autophagy induction (formatting mine)

I still can't find out how atg13 gets from cells into the blood.

I am aware that I'm getting a bit carried away on the basis of barely understood snippets of a preliminary paper. The complexity of how cells work though - it's amazing stuff.

I'm pretty sure I've read of a number of ME patients who have tried intermittent fasting, but have "failed" with it. Perhaps that might relate to autophagy being broken, so that IF is unable to realise any benefits in the repair of mitochondria and improvement of immune dysfunction.

I felt a bit better while on a ketogenic diet. I was losing weight on the diet, so I assume it was triggering autophagy.

Recent studies point to possible interconnections between ketone body metabolism and autophagy.

Source

When my son (also with ME/CFS) seriously over does things, he sleeps for long periods (e.g. the worst was sleeping 20 hours a day for a month) and then gets back to his baseline. At these times, his already fairly low food intake decreases further. I assume the hypersomnia is protective; perhaps the reduced food intake is as protective as the sleep? Members here have said that they feel better when they don't eat (obviously that can only be taken so far...).

 

[Midnattsol]

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.

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.

Autophagy and calorie restriction is seen in some, not all, animal studies. There's a lot of hype surrounding fasting, sprinkled with the old morality of how much better of a person you are if you can restrict yourself from eating.

Comparing studies on fasting is a nightmare, as protocols differ (alternative day fasting? Eight hour eating window? Four hours? Ten hours? Fasting a set number of days a week? What is allowed in the fasting period? Water? Broth? Food < a set number of calories? Are other changes made to the diet, in addition to the fasting element?). Additionally, many/most studies are weightloss studies and if you have a control group that also loses weight the "magic" behind fasting comes from the weight loss, not something else. A small pet peeve of mine is that fasting studies seldom cares about female hormones. Fasting could potentially mess with the menstrual cycle, but why bother with that, it's not like those hormones do anything important

NTNU in Norway is doing a fasting study on pregnant women right now (10 hour eating window, and participants are encouraged to include high intensity interval training in their daily life), where weight loss is not part of the study design for obvious reasons, will be interesting to see the results in a few years time

This paper is very interesting. I'm pretty sure I've read of a number of ME patients who have tried intermittent fasting, but have "failed" with it. Perhaps that might relate to autophagy being broken, so that IF is unable to realise any benefits in the repair of mitochondria and improvement of immune dysfunction.

Other reasons fasting fails could be that we don't have the metabolic flexibility required The body needs to be able to adapt to the stress of maintaining blood glucose and other functions, that might simply be too much for pwME.

 

[Midnattsol]

When my son (also with ME/CFS) seriously over does things, he sleeps for long periods (e.g. the worst was sleeping 20 hours a day for a month) and then gets back to his baseline. At these times, his already fairly low food intake decreases further. I assume the hypersomnia is protective; perhaps the reduced food intake is as protective as the sleep? Members here have said that they feel better when they don't eat (obviously that can only be taken so far...).

Reduced food intake can be protective in that the body then requires less energy for digestion and dealing with potential post-prandial inflammation (possibly higher in pwME, especially after exertion given the increase seen in LPS in the blood), but at the same time it is very easy to become deficient in nutrients and energy, which is the opposite of protective when ill. There are guidelines for how to tweak the diet to be easier to digest (though these could potentially be low on nutrients/compounds not found in food composition tables which might have other benefits such as improving the integrity of the gut lining etc).

 

[LarsSG]

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?

Confirmed that the negative concentrations mean zero.



But the high/low is just above or below the mean, which isn't particularly meaningful if they are near the mean. The mean is calculated with the negative values for some reason. If you convert all the negative values to zero, then the mean would be significantly higher and those low/high determinations would change. It's definitely true that there's a difference on average between the groups, but there are quite a few patients and controls in that middle range around 5 (and that one very high control).

 

[Slamdancin]

Just wanted to add that I tried Rapamycin for a few months a couple years ago and even though it never helped I stuck with it because some of us here were very hopeful. However it looks like mTor and ATG13 can become uncoupled so perhaps inhibiting mTor might not have been enough to stimulate autophagy?

 

[Jonathan Edwards]

You don't 'simply put a value' because a machine generates it.
You need to know what the machine reading means.

 

[SNT Gatchaman]

I still can't find out how atg13 gets from cells into the blood.

Yes, I wasn't sure whether this indicated failed autophagy -> cell apoptosis -> inc. serum levels; or whether AGT13 was being exported to serum after use (or failed use). Sounds like the latter.

From the paper —

Interestingly, our in-silico study has predicted that a serine-rich N-terminal domain of ATG13 directly binds to the extracellular domain of RAGE and that binding is reinforced once Ser44 of ATG13 is phosphorylated. 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.

The paper comments that intracellular ATG13 is helpful, but extracellular ATG13 is deleterious: in terms of metabolic derangement. I think the suggestion is that the abnormal phosphorylation makes it "bad" and that this may be via enhanced binding to RAGE (Receptor for Advanced Glycated End-products), at least in glial cells under test of their resulting iNOS induction.

There seems to be recent interest in serum (or CSF) concentrations of other autophagy-related proteins, eg Alzheimer's, stroke.

---
This 2014 paper is older but gives an overview of ATG13.
The molecular weight of ATG13 is 56.57 kDa.

 

[LarsSG]

That's what I understood too: The big question is what upstream is causing phosphorylation of ATG13 in ME and subsequent release into serum. It isn't clear to me though if all serum ATG13 is phosphorylated or the degree of phosphorylation in ME serum is abnormal and there's a mix of phosphorylated and un-phosphorylated ATG13 in serum normally.

In any case, I hope they are able to work backwards from this finding and find something significant upstream.

 

[Slamdancin]

From one paper I read in a quick Google search mTorC1 is directly upstream of p-ATG13

 

[LarsSG]

Yes, they mention mTOR in the study:

"ATG13 is heavily phosphorylated by intracellular kinases such as AMPK and mTOR. An immunoblot analysis of ATG13 in protein A-agarose purified and freshly preserved serum (2 ME/CFS and 2 HC) clearly indicated that there was a strong upregulation of ATG13 and that the signal resolved into two bands of different molecular mass. (Fig. 6G and 6H). Further probing with pan phosphoserine antibody clearly identified the higher band as a phosphor ATG13 (Fig. 6G) indicating that some of the ATG13 in ME/CFS serum is phosphorylated."

So that answers my question: There is a mix of phosphorylated and un-phosphorylated ATG13 in ME serum, but it doesn't look like they measured if non-ME serum also had the same mix or if ME serum had a more phosphorylated (which would then bind with RAGE). But it certainly seems to be their hypothesis.

 

[SNT Gatchaman]

The big question is what upstream is causing phosphorylation of ATG13 in ME

Not answering the question in ME, but here's a thread for a paper that discusses phosphorylation of ATG13 in general.

 

[Hutan]

It's annoying not being able to see the paper. But the preprint linked in the first post of this now-merged thread gives some insights.

On the negative values, there's this:

To further confirm the result, we performed ELISA analyses of ATG5, ATG13, p62 and α-syn in serum sample of male ME/CFS patient with commercially available kits as described in method section. The detection sensitivity of these ELISA kits varies from lot to lot and also based on serum concentrations of proteins. Therefore, to demonstrate the accurate detection of these proteins, we adopted a dilution series of serum samples for ELISA-based detection of ATG5, ATG13, p62, and α-syn. Accordingly, a dose responsive non-liner fitting curve analysis followed by measuring X intercept demonstrate that 1:4 dilution of serum provides most efficient detection of these serum-derived factors

So, I imagine the x axis is increasing concentrations of the serum, and the y axis is the concentration of the target protein. And so, for some of the subjects, the proteins might only be detectable at a full strength or half strength serum dilution, whereas for others, there might be identifiable amounts at a 1: 16 dilution. So, they fitted curves to the data points for each subject. And then they used the protein concentration at a specific dilution (perhaps 1:4), even though for those with barely detectable levels at full strength that meant using a negative value estimated by the fitted curve.

 

[Hutan]

Thanks to those who offered to share the paper with me. There are a lot of potentially interesting findings in this paper.

Finding 1: Strong tendency for protein aggregation in the serum of ME/CFS (compared to controls) (Section 3.2)

This was done using a thioflavin T assay. On the face of it, this result looks pretty compelling as a preliminary result to me.

Figure 1 shows the individual graphs of the first two case-control pairs e.g.

This shows the change in protein aggregation over time. The steeper the slope, the faster the aggregation. And the higher the final result on the y axis (i.e. at 100 minutes), the higher the amount of aggregation.

Figure 1C gives the slopes of the aggregation for a further 7 case-control pairs. It's a remarkable difference.



Questions and comments I have about this:

It's not a specific result, this alone doesn't necessarily tell us much about autophagy related proteins.

I don't know about this assay. Is it reliable? Perhaps it hasn't been used in serum samples before for a reason?

Thioflavin-T based fluorometric assay is one of the most reliable strategies to study protein aggregation. Although thioflavin-T based protein aggregation analysis has been utilized in cell-free systems, until now such a method strategy has not previously been brought to bear on serum samples.

There's a sample size calculation to support the use of only 7 case-control pairs. I think it's a bit, I don't know, 'unreasonably applying precision to unknowable things'? to do a calculation like that. I think with preliminary studies, researchers should usually be using sample sizes of at least 20. It's a quibble, I know.

The paper says 'results were confirmed after 3 independent experiments'. I find that the opposite of reassuring. Where's the data? Which experiment produced the data for the chart shown in Figure 1c?

 

[SNT Gatchaman]

Thioflavin-T based fluorometric assay is one of the most reliable strategies to study protein aggregation.

Probably useful to note here for reference, that this is the same assay/agent used in ex vivo acute/long covid microclot detection, where it is described as identifying amyloid fibrin(-ogen). That is not to say that the findings are in any way related.

 

[Hutan]

(please correct anything I get wrong)

Finding 2: increased autophagy proteins in ME/CFS serum (section 3.3)

They used a commercially available autophagy antibody array (i.e. an Elisa method where you have antibodies that bind to the target protein). They started with 1 case control pair, and found a number of autophagy proteins in the serum, and confirmed it using different methods. This is where they adopted that dilution series approach to an Elisa analysis - making protein concentration estimates at different dilutions, which they then applied a curve to (with the result of the artefact of negative concentration estimates). Why not just report a 0 if there is no detectable protein at a particular dilution and do more experiments at different dilutions to finesse things, rather than extrapolate? I suppose, maybe the cost of multiple analyses. But yes, as @LarsSG commented above, calculated means should treat negative values as 0.

They also used a 'novel near- infrared-based Elisa method' which they suggest is much more sensitive to low-abundance proteins. So far, this is all just with one case-control pair. Then they extend the analysis to another case-control pair. And then 12 case-control pairs. As far as I can tell from Table 1, these 12 are different to the first two pairs.

Whereas they had found a few autophagy proteins elevated in the first two ME/CFS patients, only ATG13 was significantly elevated in the larger sample. Table 1 with the data we have already seen (the one with the negative values) gives the results, as does Figure 3a.



But, the results in the chart and in the table look different. Table 1 has 12 patients (although one only has SFN, not ME/CFS) and 12 controls. The table reports a value of 45 for a ME/CFS patient and 15.4 for another - the chart's y axis only goes to 15 and all of the dots are well below it. One of the patients has a value of -9 in the table, but all of the patient dots on the chart are above 0.

The result in the chart is a lot less impressive than the results in the table, but still a bit interesting.

The caption for the chart suggests that 11 case control pairs were analysed - but there are only 10 dots for each of the controls and cases on the chart. The caption does suggest that the values are the mean from 3 experiments, so perhaps that explains the difference with the values in Table 1. However, the paper doesn't explain the difference.

I found the paper confusing when it came to understanding what particular method was used to produce particular results- I think the standard Elisa approach was used for the larger sample.

I still have questions about how and why the autophagy proteins which operate intracellularly might end up in serum. I think there was a couple of references provided on that.

 

[LarsSG]

That's interesting. For LC microclot detection, they are using platelet poor plasma, while this study is in serum with ammonium sulfate. But in theory they could be measuring the same thing, no?

 

[LarsSG]

But, the results in the chart and in the table look different. Table 1 has 12 patients (although one only has SFN, not ME/CFS) and 12 controls. The table has reports a value of 45 for a ME/CFS patient and 15.4 for another - the chart's y axis only goes to 15 and all of the dots are well below it. One of the patients has a value of -9 in the table, but all of the patient dots on the chart are above 0.

I also noticed this inconsistency. I'll try to follow up with Avik Roy about it on Twitter if he gets back to me about the other details I've asked about (trying to figure out which patients and control are shown in Figure 4H) — or you could follow up if you're on Twitter?

There is a lot in there that is hard to connect to particular patients and controls and it sort of seems like every experiment used a slightly different group. All the other data in that figure includes all 12 patients and 12 controls, but the caption says n=11.

 

[Hutan]

I'm not on twitter, please do follow up. Yes, it feels a bit like they give us a blow by blow account of everything they tried, and the detailed disease history of the two initial cases who aren't actually included in the larger sample. Which all gets confusing. It might have been better if they had worked out their approach with a couple of case-control pairs, and then just given us the data for the large sample.