That 'gut bacteria transplant provokes autistic signs in mice' paper is not perfect but...

The paper by Gil Sharon and colleagues [1] has certainly created headlines and discussion in equal measure (see here and see here and see here and see here). Concluding that: "Mice harboring human ASD [autism spectrum disorder], but not TD [typically developing], microbiomes exhibit ASD-like behaviors", the idea of a gut-brain connection in relation to autism (see here) potentially gains some research traction.

The Sharon study involved transplanting gut bacteria - the gut microbiome - from a small number of participants - "from 5 control volunteers and 11 patients diagnosed with autism spectrum disorder" - into mice lacking a microbiome and breeding said mice. They then analysed the behaviour and other biological parameters of those offspring mice according to whether their mother mice had received a transplant from controls or participants with various 'degrees' of autism. They also looked at 'metabolite profiles' based on "analyses of colon contents from oTD [offspring typically developing] and oASD [offspring autism spectrum disorder] mice."

Results: "colonization with ASD microbiota is sufficient to induce hallmark autistic behaviors." By 'hallmark autistic behaviors' researchers observed that said mice showed "increased repetitive behavior, decreased locomotion, and decreased communication... compared to mice colonized with samples from TD controls (oTD), as tested by marble burying (MB), open-field testing (OFT), and ultrasonic vocalization (USV), respectively." Researchers also observed specific differences across the mouse group gut microbiomes, some of which were consistent with that noted in other independent studies.

Also: "Twenty-seven out of 313 detected metabolites were significantly different in the colon contents of oASD mice, compared to oTD mice." They specifically focused in on two metabolites - taurine and 5-aminovaleric acid (5-AV) - both of which were reported in lower levels in the oASD mice, and how these compounds show a *connection* to GABA, a compound potentially important to autism (see here). Further they showed that supplementation of 5-AV and taurine to another strain of mouse that serves as a 'mouse model of autism' (BTBR T+ tf/J (BTBR) mouse model) resulted in "improved repetitive and social behaviors." I should add the word 'mouse' into the sentence "improved repetitive and social behaviors."

Insofar as the limitations of the Sharon studies and paper, various people have been keen to point out that the results should be viewed cautiously and as preliminary. This on the basis of the number of animals included for study, the reliance on mouse models of autism (and the logical fallacies that can sometimes follow) and some of the generalisations made in the study write-up by the authors. I wouldn't disagree with such cautions, bearing in mind that some mouse models of autism - the valproic acid autism mouse model for example - actually seem to be pretty good at mimicking some facets of (induced) autism. I'd also point out that the metabolomics work undertaken by Sharon and colleagues looks to be pretty wide-ranging (GC-MS and NMR are discussed) and findings related to taurine have also been noted in other independent study (see here). I also observed that there was a research tie-up with Arizona State University in the Sharon study, as the name Dae-Wook Kang is mentioned and 'poo transplants for [some] autism' makes yet another appearance (see here and see here).

"While ours is a limited study, with 16 donor samples from a pediatric cohort, the results support a hypothesis that the human gut microbiota contributes to ASD phenotypes." I'd agree that the Sharon results add a further layer to the idea that the new triad - intestinal permeability, mucosal immunology and intestinal microbiota - could be important to at least some autism. The results offer a road map for further investigation in this area and perhaps eventually, yet another avenue for screening and intervention to complement other recent initiatives (see here); all set with the view of the (plural) 'autisms'.

Finally, I note that another study [2] mentioning the words 'mouse' and 'autism' has been published recently. With some media attention mentioning how: "Exercise reversed autistic behaviors in an animal model of the condition" there didn't seem to be the same 'keenness' to point out the flaws of the Andoh study, despite once again a reliance on 'mouse autism' and all which that entails. It makes me wonder whether the focus on the second brain (gut) and autism detailed in the Sharon study might still have the ability to raise hackles in some quarters?

----------

[1] Sharon G. et al. Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice. Cell. 2019 May 30;177(6):1600-1618.e17.

[2] Andoh M. et al. Exercise Reverses Behavioral and Synaptic Abnormalities after Maternal Inflammation. Cell Reports. 2019; 27: 10. June 4.

----------

On gut bacteria and schizophrenia

Hot on the heels of my discussions about the possible *association* between gut bacteria and depression (see here), the results published by Peng Zheng and colleagues [1] (open-access) entered my Twitter feed recently, extending the gut bacteria 'connection' to schizophrenia.

The Zheng study represents yet more good scientific value for money as researchers initially sought to "compare the gut microbial communities of patients with SCZ [schizophrenia] and healthy controls (HCs) to evaluate whether microbiotal dysbiosis was linked with schizophrenic episodes or the severity of schizophrenic symptoms." They also "transferred gut microbiota from patients with SCZ into GF [germ free] mice to test whether SCZ-relevant behavioral phenotypes were transmissible via their gut microbiome". The study findings have been covered quite a few media outlets (see here).

The results: "seminal evidence that SCZ is associated with changes in gut microbiota composition that are both specific to SCZ and correlated with symptom severity." This translated into:

• reduced (alpha) microbial diversity in those with schizophrenia (n=63) compared with "healthy controls" (authors words not mine),
• the identification of certain bacterial differences between the groups: "the most significant deviations between SCZ and HC subjects occurred for the bacterial families Aerococcaceae, Bifidobacteriaceae, Brucellaceae, Pasteurellaceae, and Rikenellaceae",
• a *correlation* between the presence of some bacterial species and the 'severity' of symptoms of schizophrenia,
• behavioural changes in those mice who received a gut bacterial transplant from participants with schizophrenia,
• "Perturbed gut-brain amino acid and lipid metabolism in SCZ microbiota recipient mice." Gut bacteria produce chemicals (for messaging and the like), and those bacteria transplanted into germ-free mice produced a different cocktail of chemicals that showed up "in the SCZ microbiota compared to the HC microbiota recipient mouse samples." In particular: "lower glutamate and higher glutamine and GABA in the hippocampus."

As I said, the Zheng study was pretty good value for money on the basis of the results obtained. The authors note that their findings "provide a novel framework for understanding the mechanisms of SCZ through the MGB [microbiota-gut-brain] axis and may lead to new diagnostic and treatment strategies."

Caveats? Well, fairly small participant numbers is one thing, as was the potential influence of medication being taken by those with schizophrenia. Authors however mention that: "we found that the distributions of global microbial phenotypes did not vary between medicated and unmedicated patients with SCZ." I'll also point out the inherent 'difficulties' when talking about "SCZ-relevant behaviors in GF recipient mice" similar to that noted with regards to other diagnostic labels (see here). People are given labels like schizophrenia not mice.

But it's a good start and complements other work in 'related' areas talking about the gut-brain axis as being potentially pertinent to 'some' schizophrenia (see here and see here). Whether modifying gut bacteria via use of something like diet, probiotics or the horror that is the gut microbial transplant might relieve some of the signs and symptoms of schizophrenia is an area that requires quite a bit more investigation...

----------

[1] Zheng P. et al. The gut microbiome from patients with schizophrenia modulates the glutamate-glutamine-GABA cycle and schizophrenia-relevant behaviors in mice. Science Advances. 2019; 5: 2.

----------

On gut bacteria and depression

I'm kinda standing on the shoulders of giants with this post talking about the findings reported by Mireia Valles-Colomer and colleagues [1]. An editorial published in Nature [2] to coincide with the publication of the Valles-Colomer paper says just about everything that needs to be said on this research, which observed that: "Gut–brain module analysis of faecal metagenomes identified the microbial synthesis potential of the dopamine metabolite 3,4-dihydroxyphenylacetic acid as correlating positively with mental quality of life and indicated a potential role of microbial γ-aminobutyric acid production in depression." Yes folks, the idea that "microorganisms in the human gut could influence the brain" is moving from 'wild idea' to "wise pursuit".

The sequencing of DNA from donor stool samples as part of the Flemish Gut Flora Project ('You too are a walking bacteria colony' is the strap line) was the starting point for the Valles-Colomer study; stool samples provided by over 1000 participants. Researchers also accessed data on both self-reported and "physician-diagnosed depression" and set to work looking for any potentially important correlates between bacteria and psychology. The words "with validation in independent data sets (ntotal = 1,070)" are also (importantly) mentioned in the Valles-Colomer paper. Oh, and they also "mined the data to generate a catalogue describing the microbiota’s capacity to produce or degrade molecules that can interact with the human nervous system."

Results: "Butyrate-producing Faecalibacterium and Coprococcus bacteria were consistently associated with higher quality of life indicators." There's that word again: butyrate and yet more positive publicity for this compound (see here) and it's standing reaching almost 'bacterial sainthood'. Researchers also observed that two groups of bacteria were also reduced in those with depression: Coprococcus and Dialister alongside observing that this finding held "even after correcting for the confounding effects of antidepressants [use]." And then there was that 3,4-dihydroxyphenylacetic acid, also called DOPAC, finding, a metabolite of the neurotransmitter dopamine. I have actually mentioned DOPAC before on this blog (see here) in relation to what happens when rats are subjected to 'early immune stimulation' [2]. I don't think there is much overlap between that rat study and the Valles-Colomer paper (that rat paper was looking at DOPAC levels in brain tissue for example) but the suggestion from the authors that DOPAC levels were "correlating positively with mental quality of life" requires quite a bit more investigation.

Caveats? Well, out of their initial 1054 participant cohort, only 121 participants had "GP-reported depression." About half of these participants were taking antidepressants for their depression, the others weren't. The participant numbers aren't exactly tremendous for this portion of the study. Similar to something mentioned in other research (see here), I'm also minded to suggest that future research might perhaps consider looking at multiple stool samples taken over different occasions for the same person. This would perhaps establish whether gut bacterial populations are stable and whether that stability translates into stability of something like depressive symptoms too. I am likewise cautious that we don't jump ahead of ourselves here in terms of important issues like cause-and-effect and for example; whether there may be other important 'influencers' of gut bacteria when it comes to depression (see here and see here).

Lots more study is required on the suggestion of a gut bacterial *link* to depression [3], including that focused on the mechanics of any relationship (e.g. any involvement of the vagus nerve). If the link is further established, there are a number of potentially important implications: the possibility of a 'bacterial transferability hypothesis of [some] depression' (see here), intervention options focused on redressing balance in bacterial colonies (see here) and what such findings might do for the whole 'gut-brain axis' idea.

----------

[1] Valles-Colomer M. et al. The neuroactive potential of the human gut microbiota in quality of life and depression. Nature Microbiology. 2019. Feb 4.

[2] Editorial. Links between gut microbes and depression strengthened. Nature. 2019. Feb 4.

[3] Cheung SG. et al. Systematic Review of Gut Microbiota and Major Depression. Front Psychiatry. 2019;10:34.

----------

Blood glutamate levels in autism meta-analysed

"The meta-analysis provided evidence for higher blood glutamate levels in ASD [autism spectrum disorder]."

That was the research bottom-line reported by Zhen Zheng and colleagues [1] (open-access available here) who surveyed the current peer-reviewed science literature in this area and found something to see based on: "Twelve studies involving 880 participants and 446 incident cases."

Drawing on the idea that glutamate is a rather important amino acid that plays a role in various biological processes including that related to the manufacture of GABA (see here), Zheng et al observed higher circulating blood levels of the stuff; a sort-of proxy for what might also be going on with regards to brain levels of glutamate. That "excess glutamate has been shown to be a potent neurotoxin that leads to neuronal cell death and plays a role in the pathophysiology of some neuropsychiatric disorders" is an important point to make as to the potential implications from the Zheng meta-analysis.

Zheng et al do mention how important glutamate is for the purposes of GABA production and in particular, how issues with glutamate decarboxylase (GAD) - a key enzyme that converts glutamate into GABA - described in some cases of autism [2] might account for the elevated levels of glutamate yet the generally lower levels of GABA seen in autism (see here). I'd be inclined to agree that this is perhaps one of the more important implications for glutamate in autism; particularly when added to the whole 'glutamate linked to epilepsy' bit knowing how close a relationship autism and epilepsy seem to share (see here).

Where next with this research area I hear you ask? Well, I'd like to know a little more not just about glutamate but also another linked amino acid called glutamine. It has already been talked about in the autism research literature a while back (see here) but a lot more follow-up work is required on these two important compounds and what their differing ratio might mean. I'd also like to see more work done on the idea that "the mood stabilizer valproic acid, which exerts neuroprotective effects against glutamate-induced excitotoxicity, is effective in ASD [autism spectrum disorder] with seizures." Yes, I know that valproic acid a.k.a valproate is a bit of a double-edged sword when it comes to autism and other offspring developmental issues under certain circumstances (see here) but much like another research story in autism (see here) timing of exposure seems to be a key issue and one wonders whether other unrelated compounds might also exert a similar neuroprotective effect.

As to the idea that "blood glutamate levels may serve as a potential biomarker in the diagnosis of ASD" made by Zheng and colleagues, we'll wait and see...

----------

[1] Zheng Z. et al. Blood Glutamate Levels in Autism Spectrum Disorder: A Systematic Review and Meta-Analysis. PLoS One. 2016 Jul 8;11(7):e0158688.

[2] Yip J. et al. Decreased GAD65 mRNA levels in select subpopulations of neurons in the cerebellar dentate nuclei in autism: an in situ hybridization study. Autism Res. 2009 Feb;2(1):50-9.

----------

Zheng Z, Zhu T, Qu Y, & Mu D (2016). Blood Glutamate Levels in Autism Spectrum Disorder: A Systematic Review and Meta-Analysis. PloS one, 11 (7) PMID: 27390857

Epilepsy begets autism?

"Individuals with epilepsy are at increased risk of ASD [autism spectrum disorder], especially if epilepsy appears in childhood. Further, ASD is more common in the siblings and offspring of individuals with epilepsy, suggesting shared etiology."

That was the research bottom-line from Heléne Sundelin and colleagues [1] reporting results based on examination of the "Swedish Patient Register" with regards to the "risk of autism spectrum disorder (ASD) in individuals with epilepsy and in their first-degree relatives." Including one Jonas F. Ludvigsson, PhD on the authorship list (yes, he of 'gluten and autism: probably not coeliac disease but...' fame), researchers identified some 85,000 individuals diagnosed with epilepsy "as well as all their siblings (n = 80,511) and offspring (n = 98,534)." With some nifty statistical analysis including matching cases 5:1 with control participant data, they were able to conclude that around 1.6% of those with epilepsy were diagnosed with an ASD compared with 0.2% of controls. Those percentages are seemingly quite small both in real terms and also in differences between the groups but given the huge participant numbers included for study came out with a hazard ratio (HR) around 10.49 "confidence interval [CI] 9.55–11.53)." To put that HR of 10.49 in context, other work by Ludvigsson on epilepsy coinciding with coeliac disease for example, with a participant number in the tens of thousands came out with a HR of 1.42.

When also looking at what happened to siblings and offspring of those diagnosed with epilepsy, the authors also observed something of a potentially increased risk of autism being also diagnosed, although quite a bit less than risk to those themselves diagnosed with epilepsy. The results did however suggest that: "The risk in the offspring was particularly high in mothers with epilepsy." And just for good measure, the Sundelin results also noted that risk of epilepsy was "also associated with a prior diagnosis of ASD" confirming what many others have reported over the years (see here).

Although making some headlines I wasn't particularly shocked by the bi-directional associations reported by Sundelin and colleagues. Quite a few times on this blog I've talked about autistic features being potentially over-represented in cases of epilepsy (see here and see here) so to see some of those features crossing diagnostic thresholds into an actual autism diagnosis is perhaps not unsurprising. Continuing that line of thought I do wonder what might happen if the broader autism phenotype (BAP) was also analysed with epilepsy in mind (even the new DSM-5 categorisation of social communication disorder?)

Insofar as the hows and whys of the association between epilepsy and autism, well, we're still in guessing mode at the current time. I've talked about some of the various genetic syndromes that tend to include autism and epilepsy together as a diagnostic package (see here) as evidence for the more plural 'autisms'. Such syndromes suggest that mechanisms linking the two conditions are likely to be multiple and not necessarily the same for everyone. The issue of GABA and autism might also show some connection in some cases as per what is starting to be known about this neurotransmitter (see here) and where it might fit with some autism (see here) on top of epilepsy. Assuming also that epilepsy in pregnant mothers for example is being managed by medication, it is also not outside of the realms of possibility that certain preparations could also exert an effect on offspring autism risk (see here). I say this with no scaremongering intended.

I might also (speculatively) advance the idea that another research area might also be a connecting feature for some autism and some epilepsy: diet. Don't just click away yet as I will first bring your attention to the increasing peer-reviewed literature talking about the use of a ketogenic diet and autism (see here); said dietary intervention more typically indicated in 'some' cases of epilepsy. It's still early days but it strikes me that quite a bit more research is required in this area. Allied to the use of a ketogenic diet (see here) and also perhaps linking back to that other important research area frequently examined by Dr Ludvigsson (coeliac disease) I'm also inclined to ask whether some autism and some epilepsy might show a more specific connection to dietary gluten too. No, I'm not saying that a gluten-free diet nor a ketogenic diet is some sort of 'cure-all' for autism and epilepsy (please don't mess with epilepsy) but rather there may be overlapping genetics or biology potentially linked to facets of gluten metabolism that might be important for some on the autism spectrum with epilepsy. Certainly much more research on this and other less-traditional areas is indicated [2].

There are many questions that remain unanswered in this area of research. With regards to the here and now, well, the Sundelin and other data perhaps suggest that as and when epilepsy is diagnosed, preferential screening for autism could and should be offered particularly in infancy; and perhaps even offered family wide.

----------

[1] Sundelin HEK. et al. Autism and epilepsy: A population-based nationwide cohort study. Neurology. 2016. June 15.

[2] Frye RE. et al. A review of traditional and novel treatments for seizures in autism spectrum disorder: findings from a systematic review and expert panel. Front Public Health. 2013 Sep 13;1:31.

----------

Sundelin, H., Larsson, H., Lichtenstein, P., Almqvist, C., Hultman, C., Tomson, T., & Ludvigsson, J. (2016). Autism and epilepsy Neurology DOI: 10.1212/WNL.0000000000002836

Brain GABA levels and autism meta-analysed

The paper by Remmelt Schür and colleagues [1] provides some (brief) blogging fodder today and the observation that following a "systematic literature review and meta-analysis of 1 H-MRS studies" brain GABA levels were found to be significantly lower in cases of autism spectrum disorder (ASD) than compared to control (not autism) populations.

GABA - gamma-Aminobutyric acid - has been something of interest for quite a few years in autism research circles (see here). It's particular role as an inhibitory neurotransmitter has perhaps been where the lion's share of research has been targeted, bearing in mind it's actions might extend quite a bit further [2]. Indeed, whilst the over-representation of epilepsy in cases of autism (see here) hints at a possible dual role for GABA in relation to autism, I'd be minded to suggest that far more complicated processes might also be at work for some people (see here).

Schür and colleagues surveyed the peer-reviewed research literature for several developmental and psychiatric labels with measured brain levels of GABA in mind. They concluded that outside of autism, there was also some evidence for lower levels of brain GABA in those diagnosed with major depressive disorder (MDD) too (albeit those still presenting with symptoms). Further: "No significant differences in GABA levels were found in bipolar disorder, panic disorder, PTSD [post-traumatic stress disorder], and ADHD [attention-deficit hyperactivity disorder] compared with controls."

Minus any sweeping generalisations about how GABA levels could be a 'uniting' feature of autism and MDD (even though there may be overlap including at a clinical level) I do find the possibility of shared physiology to be an important one. Not least because of discussions about how interventions "aimed at either autism symptoms or symptoms of depression may improve the other" [2] could very much include GABA as one of several potential clinical parameters.

----------

[1] Schür RR. et al. Brain GABA levels across psychiatric disorders: A systematic literature review and meta-analysis of 1 H-MRS studies. Hum Brain Mapp. 2016 May 4.

[2] Andersen PN. et al. Associations Among Symptoms of Autism, Symptoms of Depression and Executive Functions in Children with High-Functioning Autism: A 2 Year Follow-Up Study. J Autism Dev Disord. 2015 Aug;45(8):2497-507.

----------

Schür RR, Draisma LW, Wijnen JP, Boks MP, Koevoets MG, Joëls M, Klomp DW, Kahn RS, & Vinkers CH (2016). Brain GABA levels across psychiatric disorders: A systematic literature review and meta-analysis of 1 H-MRS studies. Human brain mapping PMID: 27145016

Autoantibodies not implicated in cases of autism?

Contrary results are a common feature of the autism peer-reviewed research landscape. No sooner does one group publish the next 'big thing' when it comes to the singular term 'autism' than seemingly opposite results follow suit.

So it is with the paper under discussion today by Simran Kalra and colleagues [1] (open-access) who concluded that: "The idea that autoantibodies represent an underlying cause or are biomarkers for autism pathophysiology is not supported by this report."

Autoantibodies by the way, are part of the process whereby the body's immune system fails to recognise self as 'self' and mounts a response against the body's own tissue. It's a topic that has been discussed quite extensively with the autism spectrum in mind (see here for example) as part of a wider scientific debate about a role for immune function in at least some autism (see here).

The Kalra paper is open-access but a few details might be useful:

• "Serological analysis was performed on typically developing children (n = 55), developmentally delayed children without autism (n = 24) and children diagnosed with autism (n = 104)." I believe this cohort of children were part of a larger study titled: 'Clinical and Immunological Investigations of Subtypes of Autism'.
• Based on an interesting analytical method - Luciferase Immunoprecipitation Systems (LIPS) - used as an alternative to the more traditional ELISA methods, researchers initially set about looking for the presence of "autoantibodies against GAD65." GAD65 by the way, is part and parcel of the mechanism for synthesising GABA (see a previous post on this topic). They then extended the study focus to look for antibodies "against several other autoimmune-associated autoantigens, candidate neurological autoantigens, and viral proteins."
• Results: well, when comparing study samples against samples from three people with diagnosed type 1 diabetes where GAD65 autoantibodies were to be expected to be present (and indeed were): "testing of serum from the typically developed children..., developmentally delayed children... and children with ASD... demonstrated no seropositive autoantibodies to GAD65." 
• Likewise when comparing autism samples with samples from "three positive control samples from subjects with systemic lupus erythematosus" for Ro52 - one of the anti-Ro antibodies found in cases of SLE - there was again nothing of note to see. Collectively the authors conclude that: "These findings rule out the possibility that GAD65 and Ro52 autoantibodies are biomarkers in ASD [autism spectrum disorder]."
• Among the other results reported is an interesting remark when it comes to a retrovirus called XMRV. For those in chronic fatigue syndrome / myalgic encephalomyelitis circles, XMRV will probably be remembered for all the wrong reasons (see here) albeit with not all questions completely answered (see here). Kalra et al found nothing in terms of seropositivity when it came to autism and XMRV (and another target, mouse mammary tumor virus (MMTV)). They do however caution that "additional studies are needed to determine if other infectious agents, or the body's response to such infections agents, might play a role" in some autism.

These results are interesting. As per my opening comment on contrary results being part and parcel of autism research, the lack of GAD65 antibodies detailed is in direct contrast to previous findings such as those produced by Rout and colleagues [2] for example. Whilst there may be various reasons for the difference in findings including a role for the analytical method used, I was drawn to one comment made by Rout et al suggesting that there may be a subgroup of children with autism and/or ADHD (attention-deficit hyperactivity disorder) where further characterisation may be needed. That also reduced levels of GAD65 mRNA levels have been reported [3] in relation to autism (with appropriate caveats regarding tissue used for study) does not mean that GAD65 is off the research menu just yet.

The lack of XMRV antibody findings reported by Kalra et al in relation to their autism group is not necessarily new news. Previous studies such as the one from Satterfield and colleagues [4] basically said as much.

There are of course quite a few other types of autoantibodies and/or antibodies to infective agents that perhaps require more study using the technique utilised by Kalra and colleagues with autism in mind. The various contributions in this research area from the Saudi-Egyptian research tag-team that crop up on this blog every now and again (see here and see here) might be a next port of call. Anti-brain antibodies detailed by other teams might also receive the same treatment (see here). Who knows, researchers might also consider putting a little more flesh on the bones of all that folate receptor autoantibody research that is crying out for independent replication (see here) or even antimitochondrial antibodies (see here). Quite a few areas to consider.

As for the infection side of things and realising the important contribution that at least one of the authors on the Kalra paper has made to another area of research (Swedo and PANDAS/PANS) I can think of quite a few research studies to be done. My growing interest in enterovirus and autism (see here) or even enterovirus and ADHD (see here) is again requiring some further investigation. Perhaps a little more 'out there' are the ways and means that the methods detailed by Kalra might also be transferable to more ancient retroviruses such as the HERVs that have been discussed before on this blog (see here) with autism and various other conditions in mind (see here).

Music: John Newman - Come And Get It.

----------

[1] Kalra S. et al. No evidence of antibodies against GAD65 and other specific antigens in children with autism. BBA Clinical. 2015. August 8.

[2] Rout UK. et al. Presence of GAD65 autoantibodies in the serum of children with autism or ADHD. Eur Child Adolesc Psychiatry. 2012 Mar;21(3):141-7.

[3] Yip J. et al. Decreased GAD65 mRNA levels in select subpopulations of neurons in the cerebellar dentate nuclei in autism: an in situ hybridization study. Autism Res. 2009 Feb;2(1):50-9.

[4] Satterfield BC. et al. PCR and serology find no association between xenotropic murine leukemia virus-related virus (XMRV) and autism. Mol Autism. 2010 Oct 14;1(1):14.

----------

Kalra, S., Burbelo, P., Bayat, A., Ching, K., Thurm, A., Iadarola, M., & Swedo, S. (2015). No evidence of antibodies against GAD65 and other specific antigens in children with autism BBA Clinical DOI: 10.1016/j.bbacli.2015.08.001

Ultrafine particulate matter air pollution, mice and autism

Reading the headline "Study links air pollution to autism, schizophrenia" in a media piece about the study by Joshua Allen and colleagues* (open-access here) made me want to delve a little more into this research. I've talked before about air pollution and autism (see here) on this blog. Although a healthy degree of scepticism is to be expected with any autism correlation, particularly when it comes to something as generalised as air pollution (or pesticide exposure) there is a growing research interest in how this aspect of the environment may have some bearing on autism risk.

Cloudy with a chance of... @ Wikipedia 

A few details about the Allen study might be useful:

• This was a study involving mice. I'll repeat that: this was a study involving mice. It involved exposing a particular strain of mouse, modelled to represent a particular age "during early postnatal development" to "human relevant levels" of air pollution in the form of ultrafine particulates (<100 nm).
• Mouse brains were analysed at different time periods following exposure (24 hours, 40 days and 270 days after) looking at brain morphology, neurotransmitter levels and those all important immune system chemicals involved in processes like inflammation: the cytokines.
• Results: bearing in mind some quite detailed control of the amount of air pollution exposure mimicking ambient doses near roadways, quite a few effects were noted. There was for example, "a persistent dilation of the lateral ventricles" induced by CAPS (concentrated ambient ultrafine particles) "preferentially in male mice". I believe this is called ventriculomegaly.
• "CAPS induces brain region- and sex-dependent alterations in cytokines and neurotransmitters in both males and females". So in male mice, "increased hippocampal glutamate" among other things was observed. In females, "CAPS reduced hippocampal GABA" and more.
• Of the various cytokines included for analysis, an old friend ranked up there when it came to some of the results obtained: IL-6. Again, there seemed to be region and sex specific alterations to this cytokine and some of them were "unanticipated" as per the lower levels of IL-6 and other relations in certain areas. IL-6 shares some features of a pro-inflammatory and anti-inflammatory cytokine [2] although more often than not, it is the pro-inflammatory effects which get the headlines [3]. 
• The word 'microglia' also crops up in the Allen results. "CAPS altered IBA-1 immunostaining in the anterior commissure and hippocampus only in males". IBA-1 is a protein expressed in microglia.
• The authors conclude: "Collectively these data show a dramatic susceptibility of male mice to environmentally relevant levels of early postnatal air pollution exposure, with effects that persist into adulthood and cause permanent neuropathology characterized by ventricular enlargement, a pathology not seen in females".

Reiterating again that this was a study of mice and that mice are mice not humans, these are some intriguing data presented by Allen and colleagues. The focus on male mice slots nicely into the [seemingly] over-representation of autism in boys and men. Elevations in glutamate - hippocampal glutamate [4] in male mice - might also overlap with the growing fascination that autism and schizophrenia research have with this neurotransmitter (see here). Some light reading around the finding of "CAPS-induced ventricular enlargement" observed in males leads down some interesting paths such as a possible relationship with agenesis of the corpus callosum [5] reported to be "a major risk factor for developing autism" according to some authors [6]. In short, there are plenty of correlations seemingly heading back to conditions like autism.

But... there are a few important points to bear in mind before we get too carried away. First and foremost, nothing is reported in the Allen paper around mouse behaviour and how that may or may not have overlapped with other mouse data trying to model autism. One should always be a little cautious when one hears the words 'autistic behaviour' when it comes to a mouse and whether for example, they vocalise or not, or decide to bury their marbles in a particular way as being representative of facets of the condition. It isn't but it's some of the best animal model behaviour that we currently have including the rat models. Allen et al on this occasion reported nothing about behaviour and how it may or may not link to their physiological findings. 

Second is a question already asked by someone in/on the Twittersphere: "Air pollution was so much worse many decades ago yet autism rates staggeringly higher today, not then" (thanks Jill). This is an important point which may have lots of different answers bearing in mind your acceptance that things were worse back in olden times (see here for more news from urban China). Perhaps one of the most relevant issues at the moment was the study by Heather Volk and colleagues [7] discussed in a previous post (see here) talking about gene x environment interactions. If one assumes that genes, gene expression, are being affected by air pollution and that some people might already be more 'at risk' than others, there could be something more to do in this area of investigation.

Finally, Allen and colleagues seemed to have focused all their attention on the brain of their brave mouse participants. They don't talk about whether other organs or biological systems were affected by air pollution. I know that I'm probably going to get some rolling of the eyes for this but harking back to other mouse models of autism, I note some interest in things like the gastrointestinal (GI) tract to be an upcoming area (see here for example on the VPA mouse model). Assuming that the GI tract will also an important exposure point for air pollution [8], could there be merit in looking at this and other organs too all in the name of the gut-brain axis? Also, not forgetting lungs (see here) and skin as important exposure sites too.

----------

[1] Allen JL. et al. Early Postnatal Exposure to Ultrafine Particulate Matter Air Pollution: Persistent Ventriculomegaly, Neurochemical Disruption, and Glial Activation Preferentially in Male Mice. Environ Health Perspect. 2014 Jun 5.

[2] Scheller J. et al. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2011; 1813: 878-888.

[3] Rincon M. Interleukin-6: from an inflammatory marker to a target for inflammatory diseases. Trends in Immunology. 2012; 33: 571-577.

[4] Kraguljac NV. et al. Increased Hippocampal Glutamate and Volumetric Deficits in Unmedicated Patients With Schizophrenia. JAMA Psychiatry. 2013; 70.

[5] Amato M. et al. Fetal ventriculomegaly, agenesis of the corpus callosum and chromosomal translocation--case report. J Perinat Med. 1986;14(4):271-4.

[6] Paul LK. et al. Agenesis of the corpus callosum and autism: a comprehensive comparison. Brain. 2014; April 25.

[7] Volk HE. et al. Autism spectrum disorder: interaction of air pollution with the MET receptor tyrosine kinase gene. Epidemiology. 2014 Jan;25(1):44-7.

[8] Kaplan G. Air pollution and the inflammatory bowel diseases. Inflamm Bowel Dis. 2011 May;17(5):1146-8.

----------

Allen JL, Liu X, Pelkowski S, Palmer B, Conrad K, Oberdörster G, Weston D, Mayer-Pröschel M, & Cory-Slechta DA (2014). Early Postnatal Exposure to Ultrafine Particulate Matter Air Pollution: Persistent Ventriculomegaly, Neurochemical Disruption, and Glial Activation Preferentially in Male Mice. Environmental health perspectives PMID: 24901756

Homocysteine, gut permeability and MMP-9?

A speculative post this one, on the paper by Hao Ding and colleagues [1] (open-access here) looking at how, in a rodent model of colitis, homocysteine (the big 'H') might play some part in aggravating "inflammatory damage" potentially through promotion of some of the matrix metalloproteinases, MMP-2 and MMP-9. The words: "Hcy [homocysteine] can increase intestinal permeability" added to the interest.

If you're used to reading about autism research on this blog, you might be wondering why on earth I've posted about this study. Well, with that pinch of salt at the ready, the Ding study got some of the grey-pinkish matter thinking...

• So, homocysteine and autism. I hope I'm not inflating the collected literature on this topic too much but elevations of Hcy certainly do seem to be quite consistently reported [2] (open-access here) and importantly, spanning various geographies of autism [3] (open-access here). 
• Homocysteine in relation to the MMPs is something new to me but there is quite a body of peer-reviewed work suggesting that Hcy does seem to have an effect on the MMPs, particularly MMP-9 [4] in terms of enhancing MMP-9 production via the ERK (extracellular signal-regulated kinase) pathway. To quote from the Lee paper [4] "MMP-9 expression was concentration- and time-dependently increased by Hcy". MMP-9 is still something which needs exploring when it comes to autism but at least one study has hinted that amniotic fluid levels of MMP-9 when elevated might tie into some elevated risk of subsequent offspring autism [5] (see a previous post on this trial). I might add that MMP-9 levels have also been talked about with ADHD (attention deficit hyperactivity disorder) in mind too (see here).
• Then to the next stage of the speculations... MMP-9 and it's possible relationship with intestinal permeability AKA leaky gut. It seems that MMP-9 might have the ability to affect permeability [6] at least in the laboratory. To quote from Munjal and colleagues: "Hcy instigated HIMEC [human intestinal microvascular endothelial cell] monolayer permeability through activation of MMP-9". Further: "this increased permeability was alleviated by inhibition of MMP-9 activity". Leaky gut and autism y'say? Well, let's just say that there is peer-reviewed science suggesting this phenomenon to be present in at least some cases of autism (see here and see here).

OK, I know there's been speculation a-plenty in this post and by saying all of this I am by no means try to pin everything on homocysteine, MMP-9 or anything else when it comes to autism. I would never be that silly. I would however suggest that there is a study or two to be done based on these speculations, asking questions about whether MMP-9 is truly elevated in some cases of autism, and whether homocysteine levels or gut permeability measures may show some connection to one and another and MMP-9. That also the Ding paper focused on an animal model of acquired colitis, an inflammatory bowel disease, also offers another potential differentiating factor if one is to assume that autism is not protective of any other condition, including those of the inflammatory bowel disease grouping (see here and see here). I could go on further and bring GABA receptors and how use of something like "The GABA-A receptor agonist, muscimol ameliorated the Hcy-mediated MMP-9 activation" [7] (open-access here) but that's perhaps another topic for another day, alongside minocycline [8] or even melatonin [9]. 

I'm quite finished now. Apart, that is, from telling you that You Give Love a Bad Name... (the Bon Jovi song that is, not necessarily you personally).

----------

[1] Ding H. et al. Effect of homocysteine on intestinal permeability in rats with experimental colitis, and its mechanism. Gastroenterol Rep (Oxf). 2014 Apr 27.

[2] Kałużna-Czaplińska J. et al.  A focus on homocysteine in autism. Acta Biochim Pol. 2013;60(2):137-42.

[3] Tu WJ. et al. Serum homocysteine concentrations in Chinese children with autism. Clin Chem Lab Med. 2013 Feb;51(2):e19-22.

[4] Lee SJ. et al. Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways. Toxicol Appl Pharmacol. 2012 Apr 1;260(1):89-94.

[5] Abdallah MW. et al. Amniotic fluid MMP-9 and neurotrophins in autism spectrum disorders: an exploratory study. Autism Res. 2012 Dec;5(6):428-33. 

[6] Munjal C. et al. Matrix metalloproteinase-9 in homocysteine-induced intestinal microvascular endothelial paracellular and transcellular permeability. J Cell Biochem. 2012 Apr;113(4):1159-69.

[7] Tyagi N. et al. Activation of GABA-A receptor ameliorates homocysteine-induced MMP-9 activation by ERK pathway. J Cell Physiol. 2009 Jul;220(1):257-66. 

[8] Dziembowska M. et al. High MMP-9 activity levels in fragile X syndrome are lowered by minocycline. Am J Med Genet A. 2013 Aug;161A(8):1897-903. 

[9] Rudra DS. et al. Melatonin inhibits matrix metalloproteinase-9 activity by binding to its active site. J Pineal Res. 2013 May;54(4):398-405. 

----------

Ding H, Mei Q, Gan HZ, Cao LY, Liu XC, & Xu JM (2014). Effect of homocysteine on intestinal permeability in rats with experimental colitis, and its mechanism. Gastroenterology report PMID: 24787389

Dioxin exposure and autistic traits?

As promised in a previous post, today I'm turning my attention to the paper by Muneko Nishijo and colleagues [1] and their conclusion of "a specific impact of perinatal TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] on autistic traits in childhood, which is different from the neurotoxicity of total dioxins (PCDDs/Fs) [polychlorinated dibenzo-p-dioxins/furans]".

TCDD @ Wikipedia 

With all the recent chatter about [surrogate] environmental markers and the numbers of cases of autism spectrum disorders and environmental toxicants and autism risk it is indeed timely that the Nishijo paper comes to publication now. Environmental factors, however you wish to define this, are certainly no stranger to autism research, and are fast finding a place in the autism research psyche, perhaps in part due to the rise and rise of the science of epigenetics (see here) as a bridge between genetics and environment. Genes, or rather the blueprint that is your genome, might not necessarily be your destiny and all that jazz...

The Nishijo paper in a little more detail:

• Set in Vietnam, which it has to be said, has seen more than its fair share of chemical exposures in recent history, the authors looked at the possibility of perinatal dioxin exposure being linked to the presence of autistic traits based on a sample of 153 infants. This follows other work by this group looking at dioxin exposure and more generalised infant neurodevelopment [2] as part of a wider research agenda. Dioxins for those who might not know, are categorised as environmental pollutants, and because of their biological persistence, are deemed pretty hazardous to human and other animal health (see the WHO fact sheet here). It's accurate that I mentioned Agent Orange in reference to the chemical load witnessed in Vietnam because Agent Orange was contaminated with TCDD - the chemical name for dioxin -  and there are some very scary quotes about TCDD being for example "perhaps the most toxic molecule ever synthesized by man". The US IOM report 'Veterans and Agent Orange' provides some sober reading on the topic.
• Scare tactics aside, the authors assessed the levels of TCDD in breast milk as part of a larger analysis of various other dioxins (PCDDs, PCDFs) based on analysis of samples via GC-MS. Actually quite a good overview of their methods can be found in another paper by this group [3] (open-access). Data from these analyses were transformed to form something called the TEQ (Toxic Equivalent) which basically gives you some idea of the toxicity of these classes of compounds relative to TCDD. TCDD has a value of 1 so setting the gold standard of toxicity. 
• Offspring were followed-up based on the use of the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) and specifically with autism in mind, the Autism Spectrum Rating Scales (ASRS).  
• Results: exposure groups (mild and high) were determined according to a cut-off level of 3.5 pg/g fat of TCDD being detected and results were reported according to gender. So: "The high-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposed groups... showed significantly higher Autism Spectrum Rating Scale (ASRS) scores for both boys and girls than the mild-TCDD exposed groups, without differences in neurodevelopmental scores". This indicates some kind of dose-dependent relationship between TCDD exposure and autistic traits in study participants. When it came to looking at any connection between other PCDDs/Fs and autistic traits, nothing significant was picked up. Ergo, TCDD exposure seemed to have specifically impacted on infant autistic traits.

These are interesting results, of that there is no doubt. I could start to go on about correlation not being the same as causation, or how the ASRS might not necessarily have been the best instrument to use in this particular instance given it being standardised on American children and not officially translated into Vietnamese. That also it is not a professionally-administered instrument is another potential gap. But I'm not going to let all that get too far in the way of the Nishijo findings.

I see from some of the additional data from this paper that there were some other differences noted across the high and mild TCDD exposure groups which may be relevant to the results. When comparing boys and girls in the high and mild exposure groups, I note that mean birth weight was lower in the high exposed group compared to the mild exposed group (average 2920g vs. 3298g respectively) for boys. Realising that low birth weight is not an exclusively autism-correlated phenomena (see here) one might however consider this to be something which could potentially have affected the results obtained.

As per the discussions about the geographical location of this study [4], one of the question which then needs to be asked is whether the possibility of a TCDD exposure link is something applicable to other areas and other cases of autism/autistic traits. I'm no expert on TCDD so cannot readily answer this question aside from directing you to some data from the US Environmental Protection Agency on sources of TCDD. It does appear that there are quite a few potential sources of TCDD; although food seems to be the most widely cited source of exposure in modern times. By saying that I'm not trying to panic anyone, given that many countries do monitor foods for dioxin levels (see here) and act accordingly when high levels are detected.

Still, if we assume that there may be many roads towards a diagnosis of autism or the presentation of autistic traits, and that those roads may not be the same in every part of the world, the Nishijo results make for an interesting addition to the research landscape. As to mechanisms of effect, well take your pick; TCDD is categorised an endocrine disruptor so potentially falling into the same type of effect as that discussed by Rzhetsky and colleagues [5]. I also note some research chatter about TCDD and GABA [6] with specific mention of GAD (see here for some description of what this is). Jumping on the epigenetics bandwagon however, one might also suggest some further inquiry in that area if results like those by Manikkam and colleagues [7] are anything to go by, bearing in mind the continued focus on sperm and eggs and autism risk. That all being said, I don't doubt that it's going to be a complicated effect from TCDD with various factors involved.

----------

[1] Nishijo M. et al. 2,3,7,8-Tetrachlorodibenzo-p-dioxin in breast milk increases autistic traits of 3-year-old children in Vietnam. Mol Psychiatry. 2014 Mar 18.

[2] Tai PT. et al. Dioxin exposure in breast milk and infant neurodevelopment in Vietnam. Occup Environ Med. 2013 Sep;70(9):656-62.

[3] Nishijo M. et al. Impact of perinatal dioxin exposure on infant growth: a cross-sectional and longitudinal studies in dioxin-contaminated areas in Vietnam. PLoS One. 2012;7(7):e40273.

[4] Banout J. et al. Agent orange footprint still visible in rural areas of central Vietnam. J Environ Public Health. 2014;2014:528965.

[5] Rzhetsky A. et al. Environmental and state-level regulatory factors affect the incidence of autism and intellectual disability. PLoS Comput Biol. 2014 Mar 13;10(3):e1003518.

[6] Hays LE. et al. Evidence that GABAergic neurons in the preoptic area of the rat brain are targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin during development. Environ Health Perspect. 2002 Jun;110 Suppl 3:369-76.

[7] Manikkam M. et al. Dioxin (TCDD) Induces Epigenetic Transgenerational Inheritance of Adult Onset Disease and Sperm Epimutations. PLoS ONE. 2012; 7(9): e46249.

----------

Nishijo M, Pham TT, Nguyen AT, Tran NN, Nakagawa H, Hoang LV, Tran AH, Morikawa Y, Ho MD, Kido T, Nguyen MN, Nguyen HM, & Nishijo H (2014). 2,3,7,8-Tetrachlorodibenzo-p-dioxin in breast milk increases autistic traits of 3-year-old children in Vietnam. Molecular psychiatry PMID: 24637425

More bumetanide and autism discussion

For those with their ear to the autism research ground, the paper by Roman Tyzio and colleagues [1] must have sounded like a freight train coming given the volume of headlines that have been generated from this research (see here for example). Circling around the neurotransmitter, GABA (as in GABA dabba doo!), their findings based on two mouse models of autism, or rather autism and Fragile X syndrome - including the very interesting prenatal valproate (VPA) exposure model - suggested "hippocampal neurons in these models have elevated intracellular chloride levels, increased excitatory GABA, enhanced glutamatergic activity, and elevated gamma oscillations".

If that all that sounds like a different language to you, it basically boiled down to GABA doing the opposite of what it is normally supposed to do i.e. primarily act as an inhibitory neurotransmitter, potentially as a result of issues with chloride levels. The fact that the 'cuddle me' hormone, oxytocin is also a suggested trigger to facilitate that excitatory-to-inhibitory transition for GABA [2] in the early days adds to the intrigue. On it's own, this finding would probably have not generated as many media headlines as it did. But when combined with the suggestion that supplementation to mother rats with the diuretic drug bumetanide just before giving birth might help make that transition for GABA to fulfil it's inhibitory destiny ("it is your destiny") in offspring the research starts to take on a slightly different perspective. The Nature news write-up of the paper provides some additional reading on this issue (see here).

I've talked about bumetanide before on this blog (see here) as a consequence of the previous Lemonnier trial [3] which itself generated a fair few headlines at the time of publication. Since then, I've noted the odd mention on the drug in connection to the autism spectrum as per the case report from Grandgeorge and colleagues [4] and another paper from Lemonnier and colleagues [5] with a case of Fragile X syndrome in mind. The Grandgeorge results in particular, are worthy of inspection not least because of the focus on sensory issues and the link I make back to the very intense, intense world theory of autism [6] which has its roots in the VPA rodent model similar to the one used to test bumetanide in the Tyzio paper. I know we should be cautious of sweeping generalised models when it comes to autism (as per some chatter about the model) but lets not throw baby and bathwater out just yet. The scientific puritans out there might also be shaking their heads at the thought of case reports being mentioned here, but just remember the old adage about meeting one person with autism and all that.

There is obviously a degree of step-back caution to take from the Tyzio results insofar as rats being rats not humans. I believe the accompanying editorial from Zimmerman & Connors also raises a few potential issues which need to be resolved; not least how one ascertains who [humans] might be at risk for this process occurring and therefore when bumetanide might be indicated. Bear in mind too, that there are growing moves to look at reducing things like prenatal valproate exposure on the back of some regulatory statements recently being made (see here).

But I have to conclude that I do find the recent Tyzio report and the previous Lemonnier trial very interesting and look forward to seeing more research on this topic including safety studies, long-term follow-up and perhaps more data on who might be more likely to benefit from this research.

----------

[1] Tyzio R. et al. Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring. Science. 2014 Feb 7;343(6171):675-9.

[2] Tyzio R. et al. Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery. Science. 2006 Dec 15;314(5806):1788-92.

[3] Lemonnier E. et al. A randomised controlled trial of bumetanide in the treatment of autism in children. Transl Psychiatry. 2012 Dec 11;2:e202. doi: 10.1038/tp.2012.124.

[4] Grandgeorge M. et al. The effect of bumetanide treatment on the sensory behaviours of a young girl with Asperger syndrome. BMJ Case Rep. 2014 Jan 31;2014.

[5] Lemonnier E. et al. Treating Fragile X syndrome with the diuretic bumetanide: a case report. Acta Paediatr. 2013 Jun;102(6):e288-90. doi: 10.1111/apa.12235.

[6] Markram H. et al. The intense world syndrome--an alternative hypothesis for autism. Front Neurosci. 2007 Oct 15;1(1):77-96.

-----------

Tyzio R, Nardou R, Ferrari DC, Tsintsadze T, Shahrokhi A, Eftekhari S, Khalilov I, Tsintsadze V, Brouchoud C, Chazal G, Lemonnier E, Lozovaya N, Burnashev N, & Ben-Ari Y (2014). Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring. Science (New York, N.Y.), 343 (6171), 675-9 PMID: 24503856