Identification of actin network proteins, talin-1 and filamin-A, in circulating extracellular vesicles as blood biomarkers...ME/CFS 2019, Eguchi et al

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Identification of actin network proteins, talin-1 and filamin-A, in circulating extracellular vesicles as blood biomarkers for human myalgic encephalomyelitis/ chronic fatigue syndrome

AkikoEguchi,SanaeFukuda,HirohikoKuratsunede,JunzoNojimag,YasuhitoNakatomi,YasuyoshiWatanabee,Ariel E.Feldstein
Highlights
•Circulating EV number was increased in ME/CFS patients correlating to CRP and BAP.

•AUROC for circulating EVs was 0.802 allowing correct diagnosis in 90-94% of ME/CFS.

•Proteins in actin skeletal regulation and EB virus infection were identified in ME/CFS patients.

•Talin-1, filamin-A and 14-3-3 proteins were the most abundant proteins representing highly specific ME/CFS.
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Abstract
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious, debilitating disorder with a wide spectrum of symptoms, including pain, depression, and neurocognitive deterioration. Over 17 million people around the world have ME/CFS, predominantly women with peak onset at 30-50 years. Given the wide spectrum of symptoms and unclear etiology, specific biomarkers for diagnosis and stratification of ME/CFS are lacking.

Here we show that actin network proteins in circulating extracellular vesicles (EVs) offer specific non-invasive biomarkers for ME/CFS. We found that circulating EVs were significantly increased in ME/CFS patients correlating to C-reactive protein, as well as biological antioxidant potential. Area under the receiver operating characteristic curve for circulating EVs was 0.80, allowing correct diagnosis in 90-94% of ME/CFS cases.

From two independent proteomic analyses using circulating EVs from ME/CFS, healthy controls, idiopathic chronic fatigue, and depression, proteins identified from ME/CFS patients are involved in focal adhesion, actin skeletal regulation, PI3K-Akt signaling pathway, and Epstein-Barr virus infection. In particular, talin-1, filamin-A, and 14-3-3 family proteins were the most abundant proteins, representing highly specific ME/CFS biomarkers. Our results identified circulating EV number and EV-specific proteins as novel biomarkers for diagnosing ME/CFS, providing important information on the pathogenic mechanisms of ME/CFS.
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https://www.sciencedirect.com/science/article/pii/S0889159119307627
 
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Oh dear, another paper I'm going to have to read... (abstract)

Background:
This is certainly interesting. Initially, I'd thought it must be from Maureen Hanson's group, who are focusing on EVs. But it's from the same Japanese group, including Yasuyoshi Watanabe and Yasuhito Nakatomi who gave us the 2014 PET study with evidence of neuroinflammation. (Note that paper said the authors had a replication study in progress, using improved technology, but no replication paper ever appeared.)
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This is the key point:
From two independent proteomic analyses using circulating EVs from ME/CFS, healthy controls, idiopathic chronic fatigue, and depression, proteins identified from ME/CFS patients are involved in focal adhesion, actin skeletal regulation, PI3K-Akt signaling pathway, and Epstein-Barr virus infection.
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Using sick controls helps to justify the biomarker claim that follows:
In particular, talin-1, filamin-A, and 14-3-3 family proteins were the most abundant proteins, representing highly specific ME/CFS biomarkers. Our results identified circulating EV number and EV-specific proteins as novel biomarkers for diagnosing ME/CFS, providing important information on the pathogenic mechanisms of ME/CFS
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Though, of course, replication will be essential.

And at some point, Maureen Hanson's group will be reporting on their EV findings - so good to have independent groups working on the same issue (co-incidentally, Hanson's group also will be replicating the Japanese groups PET/neuroinflammation work).

Actins are proteins that are the main component of the cell's 'cytoskeleton'. This is a dynamic, 3D web of filaments that pervade the cell and, amongst other things, transport molecules (mitochondria etc) around the cell. It's everywhere but tends not to get much attention.

Video
 
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The full article does not appear to be online with the journal.

I think I will wait to see that.

I am sceptical about a claimed test that identifies 90-94% of PWME. Usually good diagnostic tests looking at potential causal mediators start out with a very low rate (<5%) in normals and maybe 20-40% in the condition of interest, 60% if you are lucky. 90-94% looks like fitting the numbers backwards from measuring lots of things and picking the ones that fit the cohort. The abstract is not written in a way that makes it easy to evaluate.

The correlation with CRP is a bit peculiar since CRP is on average normal in ME.
 
The finding of increased extracellular vesicles in people with ME/CFS looks pretty solid for the population they were sampling in. I can't comment on the methodology and the version of the report I'm looking at doesn't have the figures and tables.

Sample 1 - 33 healthy controls; 39 people with Fukuda ME/CFS.
# of EVs was higher in ME/CFS P=0.001
A first group (ME/CFS 1) included 33 healthy controls (HC) and 39 ME/CFS patients
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Sample 2 - another 30 people with Fukuda ME/CFS.
#of EVs was higher in ME/CFS (presumably compared to Sample 1 HCs). P=0.001
They also note that the ME/CFS #EVs of Sample 1 was 'similar' to that of Sample 2.
To confirm those results, we recruited 30 more ME/CFS patients (ME/CFS 2)
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Sample 3? - 20 healthy controls; 30 people with Fukuda ME/CFS.
Me/CFS # EVs also significantly higher. P=0.001
(The way it is written suggests to me that the 30 ME/CFS were in addition to those of Sample 2)
An increased circulating EV number in ME/CFS was further validated in an additionally recruited 20 HC and 30 ME/CFS patients
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Supplementary Fig 1B hopefully makes it clear. Edit: in the methods they talk about 99 ME/CFS patients in the study, so it seems likely there were those 3 separate samples.

Edit - it is later noted that the number of EVs was not significantly different between people with depression (8), ME/CFS (99) or idiopathic chronic fatigue (6).
 
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They found a correlation between CRP and # of EVs.
Circulating EV number correlated significantly with serum c-reactive protein (CRP) levels ( = 0.442, P = 0.0007)
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They noted that this correlation was true even with data points within the normal range for CRP.

They comment that CRP is a marker known to be increased in ME/CFS, quoting Fukuda 2016
Fukuda, S., Nojima, J., Motoki, Y., Yamaguti, K., Nakatomi, Y., Okawa, N., Fujiwara, K., Watanabe, Y., Kuratsune, H., 2016. A potential biomarker for fatigue: Oxidative stress and anti- oxidative activity. Biological psychology 118, 88-93.

I haven't heard this talked of elsewhere, in fact I thought I had heard the opposite, that CRP is not different. And I keep an eye on that, because my CRP levels have been consistently moderately elevated since becoming ill. I wonder if there is anything different between the Japanese ME/CFS population sampled here and ME/CFS populations elsewhere.
 
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In contrast to the #EV study, the determination of what is in the EVs was much more of a pilot study.

They purified EVs from the plasma of 3 healthy controls and 3 PwME. There were 124 proteins identified overall, with a number of proteins only in PwME or only in HCs. 75 of the proteins were significantly different in PwME compared to healthy controls - which is quite remarkable given the small sample size. I don't know if they accounted for multiple comparisons in that, probably not.

They identified specific pathways that the proteins that were significantly different seemed to be relevant to:
Notably, 63 up-regulated proteins made a cluster in protein-protein interactions (Fig. 2C) relating to several pathways, focal adhesion, regulation of the actin cytoskeletal, phosphoinositide-3-kinase (PI3K)-Akt signaling pathway, and EB virus infection
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Then they analysed the contents of EVs for 4 PwME, 4 with depression and 4 with idiopathic chronic fatigue.
they used a different process for concentrating the EVs I think, I'm not sure what impact this had, but this time a total of 579 proteins were identified.
134 proteins were significantly changed in EVs from ME/CFS patients compared to those from ICF and depression patients (P < 0.05)
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At this point I was despairing slightly, over the size of the EV characterisation samples and so on. But this point was encouraging:
Of the 111 up-regulated proteins, 105 were associated with core protein-protein interactions (Fig. 3C) and involved in focal adhesion, regulation of the actin cytoskeleton, PI3K-Akt signaling pathway, and EB virus infection
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So they seemed to find the same differences between PwME and (depression and ICF) as they had found between PwME and healthy controls.
These quotes are the guts of it:
From the list of significantly changed proteins in EVs from ME/CFS patients, 31 proteins were identified as common proteins in EVs from ME/CFS patients, including 30 up-regulated and 1 down-regulated protein
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The 12 most abundant proteins in EVs were closely matched in the two independent experiments and part of the actin network protein family (talin-1, filamin-A, actin, actinin, vinculin, gelsolin, and integrin) (Fig. 4C and 4D) and 14-3-3 family proteins (Fig. 4C). In particular, the top three proteins in EVs, talin-1, filamin-A, and actin, were exactly the same in two independent experiments (Fig. 4C).
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Where it says '12 most abundant proteins in the EVs' - I assume they mean, of the 31 proteins identified as significantly different. Table 1 has this data, so it would be worth looking to see exactly how they chose those 12.
 
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So, looks like early days to me. I'm not going to spend time googling actinin etc at this point. I think Maureen Hanson's team is working on EVs, it will be interesting to see what they come up with.

Actin network proteins also play important roles in the skeletal muscle as follows: 1) talin 1 regulates the stability of myotendinous junctions through the vinculin-talin-integrin system in skeletal muscle (Conti et al., 2008); and 2) human serum gelsolin is mainly derived from skeletal muscle (Kwiatkowski et al., 1988). Our results suggest that skeletal muscle damage may be involved in ME/CFS pathology, since ME/CFS patients lose mobility with progression of the disease and graded exercise is one of the treatments proven to have an impact on these patients (Pietrangelo et al., 2018).
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Where to start with the suggestion that the fact that GET helps people with ME/CFS indicates there is a muscle damage problem. :banghead:

This evidence led us to hypothesize that EVs may be involved in ME/CFS progression or etiology through EB virus infection and subsequent triggering of autoimmune disease in skeletal muscles, although future studies will need to investigate this hypothesis further.
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Hmm

Before I finish up, I just want to comment on this:
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious, debilitating disorder with a wide spectrum of symptoms, including pain, depression, and neurocognitive deterioration.
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What a weird three symptoms to pick to characterise ME/CFS. Depression? I don't know where they are getting that from.
 
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