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

On the topic of whether it is normal or good/bad to have autophagy proteins in serum, the paper says:

There's no mention of ATG13 there. For ATG5, a study is referenced that found higher levels in Alzheimers, as well as a study that found lower levels of the same protein, also in Alzheimers. The paper on cerebral palsy found lower levels of ATG5 in cerebral palsy patients:

So, clearly we can't proceed with a certain idea of 'having any autophagy proteins in your serum, or higher levels than average, is definitely bad'.

Another complication is that there are different types of each autophagy protein. Aside from whether they are phosphorylated or not, there are different genetic variants e.g.

source

And for ATG13, the gene might be constant, but there are something called 'splice-variants' producing at least 5 different isoforms.

I don't know if different isoforms can exist in a single human, but it raises the possibility of ATG13 having different forms that perform different functions, some possibly outside the cells. So, potentially, to understand what ATG13 might be doing in ME/CFS, if anything, we might need to know about the isoforms.

I'm wading out of my depth on this. But, potential complications to keep in mind.

 

Back to the paper - Section 3.4 Evaluation of lysosomal function

So, they used a protease array to test for lysosomal proteins in the serum. The particular array covered, matrix metalloproteinases, cathepsins, kallikreins (a cool name, a bit like Kali, the goddess of destruction and change but also love - it occurs to me that a cell biologist could call their daughter Kallikrein; Cathepsin works too, for that matter. I'm heading off-topic, must be time for lunch.)

Anyway, this is short.
They found no difference in the 2 case control pairs with respect to lysosomal proteins. It isn't explained how finding lysosomal proteins in the serum, or not, tells us anything about how the lysosomes are functioning - because surely the lysosomal proteins need to be in the lysosomes, inside the cells, to work, or not work.

They did find a couple of significant differences between the male case control pair for non-lysosomal proteins that happened to be in the test array (MMP-7 and proteinase-3). But they didn't find that in the female case control pair, and they didn't do this analysis on a larger sample.

So, not much we can conclude from this part of the study I think, about lysosome function or much else, except that some proteinases have good names.

 

3.5 The effect of ATG13 in serum on microglia - ROS
No one could accuse these researchers of not covering enough ground in a paper. I think this finding alone would have been worth a paper; I hope the finding is not buried under all the rest.

So, in this study, they put human microglia cells (microglia being the immune cells in the central nervous system) into media (I'm imagining growing media like the agar jelly in a Petri dish) made with ME/CFS serum or control serum. They then measured reactive oxygen species using a fluorescent marker from 30 mins to 2 hours.

The results look convincing, but there was a very small sample size.
Figure 4b - results from one male case-control pair.



Figure 4D - left a female control, right the ME/CFS case



They then neutralised the ATG13 in the serum of the two case-control pairs, and repeated the experiment, finding much less indication of ROS. They went on to repeat the experiment with 6 more case-control pairs (we don't know how they chose them) - results in Figure 4H. The result seems pretty clear, although of course, still a small sample size - serum from ME/CFS cases caused more fluorescence indicative of ROS, but the fluorescence reduced to levels seen in the controls when atg13 was neutralised.



The authors rightly ask if ATG13 inside the microglia might be having the same effect - i.e. creating reactive oxygen species.

That sentence takes some unpicking. siRNA are small interfering RNA! So, ATG13 siRNA prevents atg13 gene expression, but itself causes ROS production, as do other siRNA. I'm not sure that we can necessarily conclude from that that ATG13 inside the cell mitigates microglial ROS production. I'm going to park that one.

 

3.5 The effect of ATG13 in serum on microglia - NO
They found something similar with nitric oxide. It looks like they found that nitric oxide produced by microglial cells was increased by exposure to the serum from the initial male case control pair and 8 other case control pairs (I don't know what happened to the initial female case control pair, or the others.) For the initial male case control pair, they applied a range of dilutions and found that NO production was increased with increasing concentrations in a dose-dependent manner. Serum that had had the atg13 neutralised didn't have that effect.

5A - production of nitrite (?) by microglia treated with the serum of initial male case-control pair, at various concentrations
5B - production of nitrite by microglia treated with the serum from heathy controls, me/cfs patients, me/cfs patients with the atg13 neutralised



Again, that's a remarkable result.

Wikipedia on the iNOS enzyme:

This therefore seems to be a big deal if it holds up - ME/CFS serum causing microglia, and perhaps other immune cells to produce (ROS and) nitric oxide. Production of NO, with its signalling role, could really set the cat among the pigeons.

It will be interesting to look at the findings we have about NO from other papers, to see if the idea is supported.

 

3.6 How might the ATG13 in the serum make microglia produce ROS and Nitric oxide?
Off-the shelf ATG13 (i.e. standard stuff, purchased, not from ME/CFS patients) reacts with a type of receptor called RAGE on the microglia to produce ROS and NO. RAGE= Receptor for Advanced Glycation of End Products. The authors found that ATG13 bound to biotinylated* RAGE in a dose-dependent manner (i.e. the more ATG13, the more RAGE binding). Apparently, it is known that 'activation of RAGE', presumably anything binding to that receptor, produces ROS and NO. (I wonder what normally binds to RAGE - edit - advanced glycated end products?)

*(What is biotinylated? Wikipedia says we can probably just ignore it - "the added biotin is unlikely to disturb the natural function of the molecule". I think it might be part of the protein production process.)

So, then the authors worked with the ME/CFS sera. This statement was unhelpful:

Three different ME/CFS serums displayed strong binding affinity - but how many serums did they try? 12, or just the three? Figure 6B suggests that they just tried 3 samples.



They found that when ME serum had the ATG13 neutralised, there wasn't binding.



"An in-silico homology modeling study" - that's computerised modelling of the structures. They found that it is structurally possible for the ATG13 to be binding to the RAGE.

An immunoblot analysis (yeah, I don't know) and "probing with a pan-phosphoserine antibody" (yeah...) led the authors to conclude that at least some of the ATG13 in the ME/CFS serum is phosphorylated (unlike the control serum it is implied). And the computer modelling of the structures suggested that phosphorylated ATG13 could bind well to RAGE.

Next, (and you have to give the authors credit, they have chased down leads), they tried to neutralise RAGE, to see if ME/CFS serum still caused microglia to produce ROS and NO. RAGE neutralising antibody is fortunately something that you can buy.

7b - binding of ATG13 decreased as the concentration of RAGE neutralising antibody increased.

(Including the quote because 'blocking RAGE in microglia' is quite a vivid image, one that might make the psychosomatic people feel they have something to work with.) But anyway, the authors report that neutralising RAGE resulted in much less ROS production when microglia were exposed to ME/CFS serum and no induction of iNOS expression.

7c - the caption in the paper isn't clear at all. The label says it's a ROS assay
top left is microglia +control serum -> not much ROS
top right is microglia + ME serum - > lots of ROS
bottom left is microglia + neutralised RAGE +ME serum -> not much ROS
bottom right is microglia + IgG + ME serum -> a lot of ROS

I really don't know what is going on in this image. There's no mention of an IgG treatment - I'm not sure if that is the same as the RAGE neutralising antibody. There needs to be more explanation somewhere, including whether this was just for one ME/CFS person's serum. (Edit - SNT Gatchaman's suggestion below that the IgG was a control, expected to be inactive, and therefore have no moderating effect on the ROS-inducing effects of ME/CFS serum, makes sense.)



7d - also isn't clear. There are images labelled with CD11 - that's Integrin aM - I don't think the text explains what that is doing there. Here are the images for iNOS production, with, from left to right, control serum, ME/CFS serum, and ME/CFS serum and neutralised RAGE.

 

I haven't gone through the discussion, but will stop here.

There's certainly very interesting stuff, but I get the feeling that, as the study and paper writing went on, everything just got more hurried and less organised. Basic things like how many samples there were for each analysis, and how they were chosen have been left out. Captions are muddled and very unclear. I'd suggest 'less is more' in terms of a paper's scope.

Still, good on the researchers for what looks to have been a lot of work. edit - and, I should say, possibly, a lot of important work.

Please do correct my misunderstandings.

It will be interesting to poke into this more, and see what eventually comes of it. Top of the S4ME list of threads tagged with nitric oxide is
Decreased NO production in endothelial cells exposed to plasma from ME/CFS patients, Bertinat et al (2022)
That's decreased, not increased, as in this study. Which is a little deflating. The authors mention that study in the discussion. Maybe microglia are different, or something.

 

Different splice variants are found in single humans, and splice variants can also vary by tissue type.

 

I'm not sure that the Gottschalk paper particularly joined the dots between mitochondrial dysfunction and the ATG13 in the serum. (They did do a lot of dot joining from the serum to the microglia though.)

It's possible that the ATG13 is getting into the serum for a reason unrelated to autophagy. For example, we know that ATP is important in energy production in the cell, but when it's outside the cell, it's a signalling molecule to deal with threats. Maybe the ATG13 is like that - maybe there isn't really a problem with autophagy, I'm not sure the evidence for there being a problem with autophagy in ME/CFS is yet bullet-proof, even if it is attractive.

There's this - I think this sub complex formed by ATG13 and FIP200 to deal with certain viruses is inside the cell, but maybe the extracellular ATG13 with its ability to activate microglia has more to do with this anti-viral function.
HSBP1 Is a Novel Interactor of FIP200 and ATG13 That Promotes Autophagy Initiation and Picornavirus Replication, 2021

 

The female metabolome might also be influenced by the menstrual cycle, just to make it even more complicated.