Many readers will have seen the paper that just came out on a possible mouse-model demonstration of a connection between autism and the gut microbiome. It’s certainly generated a lot of headlines, and its very title guaranteed that it would: “Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice“. I found that a pretty startling claim, but I’m well aware that GI function is disturbed in many patients with autism, and I’m open to speculative hypotheses. But I don’t think this paper proves its case.
I’m not alone. A number of other observers are raising questions. When you dig into the paper, you find that the statistical power of the study is. . .extremely low. It involves faecal samples from 5 control volunteers and 11 patients diagnosed with autism spectrum disorder, and even those latter donors are divided into three subgroups based on the ADOS severity scale. As many are pointing out, it gets down to the point of a 3-control 5-ASD-patient analysis, which just seems too small to reach a meaningful conclusion in a question this complicated. Especially a question this complicated that’s being addressed by mouse behavior observations. Anyone who’s done CNS drug research will tell you that rodent behavior is complex, difficult to interpret, and can be influenced by a lot of things other than what you’d like to imagine. And before you even get to that, the tiny number of human donors really makes me wonder. It should go without saying that the human microbiome is quite variable, person-to-person, and I have trouble believing that this is (or even can be) a representative sample.
What’s more, the effects were noted in second-generation mice: they took normal C57 rodents and colonized them with donor samples, then bred them, and re-colonized the offspring, and that’s where the effects were seen. The paper says that these mice were “either sampled (feces, serum, brains) or behavior-tested”, which makes me wonder if the animals used for the behavioral conclusions had their microbiomes examined directly at all. The mice and their behavior are correlated to the original donor metadata, but not (from what I can see) to what’s present in their own microbiota – I keep thinking that I’m not reading this correctly, but that’s what I’m getting.
And in those correlations with original donor data, they’re going so far as to mention a connection with donor age, but you’d need an even *larger* set of donors to make any such claim, wouldn’t you? When you look at the S1 supplementary data, those ages range from 3 to 11: how wide is that? Does anyone know?
Another problem I have is with the idea of there being mouse behaviors that can be tied to human autism spectrum behavior in the first place. These doubts creep into one’s consciousness after you’ve done CNS drug discovery work, and they can never be fully expunged. Anyway, here’s a neurogenetics guy, who works on rodent models, who has the same doubts, so it’s not just me. To the best of my knowledge, there are no generally accepted mouse models of autism.
The paper goes further, to identifying metabolites produced by the gut microbiota and testing these separately to see if they’re involved in behavioral effects. That seems like a rather broad jump, right to actionable small molecules. Such cases have of course been suggested, but even those are preliminary and have more statistical power behind them than this paper does. What small molecules do the authors propose? Taurine and 5-aminovaleric acid (5AV). Both of these are characterized in the paper as “weak GABA agonists”, which is true, especially the “weak” part, but note that the earlier work that I linked to is talking about gut production of GABA itself, and even that needs a lot more work. Interestingly, the authors also note changes in the gut levels of genistein and daidzein, which are isoflavones that are coming from the soybeans in the mouse chow (and are notorious frequent hitters in in vitro assays, for what that’s worth).
They go on to administer large amounts of taurine and 5AV to pregnant mice, and do behavioral tests on the offspring. And by this point, I think that things are pretty far out on a limb of loosely connected assumptions, with a great many alternate explanations available. The amounts of these compounds being given are *far* over any reasonable production from gut microbes – and indeed, if the paper tests for blood levels of these in their gut-altered mice, I have missed it. The authors also predict behavioral effects from the genistein and daidzein concentrations, too, and I really don’t know what to say about that.
There’s overall a cavalier attitude towards generating hypotheses in this paper and chaining them end-to-end. You have to believe that these effects are robust enough (large effect size) to show up in very small samples, that the mouse behavioral effects are indeed connected with human autism, that the metabolome changes seen are causative and that the correct ones have been identified, that the high-dose recapitulation experiments are connected to what’s seen in those earlier animal models, and on and on. . .and you also have to believe that (as mentioned) alternate explanations have been excluded, when to my mind, they haven’t been at all. From an outsider’s perspective, the whole paper seems aimed at showing that small-molecule gut metabolites are big players in autism, and that’s a huge conclusion to put on top of such a slender foundation.
And indeed, the end of the paper discloses that its various authors have filed patent applications related to just that hypothesis, and helped to found Axial Biotherapeutics, which is dedicated to that idea. Now, I work in the industry, of course, and I have no problem with industrial scientists publishing their data to make their case. But papers from any source need to back up what they claim. I am also open to the idea that the gut microbiota are involved in autism (and indeed, in other human CNS disorders), but I will accept or reject such ideas based on more evidence than is in this paper. In that context, it should be noted that there are reports of microbiota transplants directly on human patients already, with long-term follow-up and a call for larger, more controlled trials to test these effects. It would seem that such trials are certainly worth running. Taurine and 5-aminovaleric acid, though, have a long way to go.