"abnormalities in mitochondrial activity in the lower GI tract of children with ASD"

The findings reported by Shannon Rose and colleagues [1] (open-access) continue a research theme by [some of] this authorship group looking at how mitochondrial dysfunction seems to be part and parcel of at least some autism (see here). Indeed, how when one talks about mitochondrial issues potentially accompanying [some] autism, one really needs to look at it in the context of other issues potentially also 'over-represented' in relation to autism (see here).

This time around, Rose et al set out to "determine whether mitochondrial dysfunction may contribute to GI [gastrointestinal] symptoms in children with ASD [autism spectrum disorder]" on the basis that GI symptoms (whether functional or more pathological) are no stranger to autism (see here). With this in mind, I note the name Tim Buie is included as part of the Rose paper authorship team and so should reference some of the sterling work he and his team have done on the topic of GI issues and autism and its importance down the years.

Researchers analysed mitochondrial function(s) in rectal and cecum mucosal biopsies in a small sample of children diagnosed with ASD (n=10) and compared results with those from "10 children with Crohn’s disease and 10 neurotypical children with nonspecific GI complaints." There are two points for me to make here: first, although it is an invasive procedure to collect them, those biopsies used for study were extracted on a clinical basis as part of "elective diagnostic colonoscopy." This was not a case of 'experimenting' on children for the sake of an experiment; rather that children were already undergoing investigations for their significant bowel issues, save any health inequalities appearing "just 'cos they were autistic" for example. Second, although the authors have chosen to use the term 'neurotypical' to reflect not-autism, I myself still find this terminology to be scientifically problematic (see here) in the context that no brain is seemingly typical or atypical according to current scientific evidence. Not least also on the basis that immune-based conditions such as inflammatory bowel diseases do seem to carry an increased 'risk' of psychiatric issues (see here) and what that might [eventually] mean for those children diagnosed with Crohn's disease (an inflammatory bowel disease) for example. Anyhow, two approaches are described in connection with the study of mitochondria in those biopsy samples looking at both the quantity and activity of various electron transport chain (ETC) complexes. Yet again, I can profess no serious expertise on the various elements of mitochondria but there is some good reading out there in the peer-reviewed science domain on the topic.

Results: "Differences in mitochondrial function were found in children with ASD as compared to the other control groups across several ETC complexes suggesting a difference in overall mitochondrial function rather than a change in one specific mitochondrial enzyme." Accepting the small participant numbers included for study, these are potentially important results. Not least because other work looking at such mitochondrial issues in relation to [some] autism has been predominantly based on activity in muscle; now it appears extending "this observation to altered ETC complex activity in the GI mucosa" too.

Then to some speculation: "The fact that increased ETC complex protein content was primarily seen in the cecum, an area where enteric microbiome fermentation products such as PPA [propionic acid] and BUT [butyrate] are abundant, suggests a role for the enteric microbiome in the evolution of mitochondrial abnormalities in children with ASD." An interesting perspective indeed and in need of some further investigation. Butyrate has, in recent years, been elevated to almost scientific sainthood (see here for example) so one has to perhaps be a little cautious about sweeping statements in the context of autism or any other label. I say this with particular relevance to an 'autism colon' discussed by the authors (see here) which I also think is perhaps a little premature to speculate on.

No mind, the results are what they are and add to the growing literature discussing mitochondria in the context of [some] autism. The implication once again is to screen for such issues within the context that a diagnosis of autism should represent a starting point for further investigations not the finishing line.

And finally, to another author on the Rose paper, I'm still waiting to read about some of your results...

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[1] Rose S. et al. Mitochondrial dysfunction in the gastrointestinal mucosa of children with autism: A blinded case-control study. PLoS One. 2017 Oct 13;12(10):e0186377.

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The prebiotic galactooligosaccharide (B-GOS) and autism: just add to poo(p)

Yes, it is childish but...

With all the continued chatter on a possible role for the collected gut microbiota - those wee beasties that inhabit our deepest, darkest recesses - in relation to some autism (see here for example), the paper by Roberta Grimaldi and colleagues [1] (open-access available here) provides yet more potentially important information.

So, poo(p) samples were the starring material in the paper - "obtained from three non-autistic children and three autistic child donors"- and specifically what happened when something called B-GOS "a prebiotic galactooligosaccharide" was added to samples following their journey through a "Three stage continuous culture gut model system" otherwise known as an artificial gut. Said gut model based at Reading University has already been the topic of other news (see here).

As well as looking at the initial bacterial profile of those stool samples, researchers plotted the changes to the stool's inhabitants (or what was left of the stool) over the course of B-GOS addition, as well as looking at things like the "production of SCFAs [short-chain fatty acids] in the fermentations" and other metabolites via the gold-standard chemical analytical technique called 1H-NMR (see here for more details).

Results: "Consistent with previous studies, the microbiota of ASD [autism spectrum disorder] children contained a higher number of Clostridium spp. and a lower number of bifidobacteria compared to non-autistic children." With the addition of B-GOS to the 'mixture', researchers reported on a significant increase in bifidobacterial populations at the different stages of their gut model and in samples from both those with autism and those without autism. Such "bifidogenic properties of B-GOS" are not unheard of.

As to the metabolites of those bacteria present in the poo(p) samples, there were some interesting knock-on effects noted in both raw and B-GOS supplemented samples. "Our data show a lower concentration of butyrate and propionate in autistic models, compared to non-autistic models, but no
differences in acetate before adding B-GOS into the system." Propionic acid (propionate) has some research history with autism in mind (see here). Butyric acid (butyrate) is something of a rising star in quite a few domains, having also been mentioned in the context of autism too (see here). Indeed it's interesting to note that B-GOS administration "mediated significant production of... butyrate... simulating the transverse and distal colon respectively. There was no effect on propionate." The findings of lower starting levels of butyrate in samples from children with autism were also substantiated by the NMR analyses undertaken. Increases in butyrate and changes to various other metabolites ("increasing ethanol, lactate, acetate and butyrate and decreasing propionate and trimethylamine") were also noted via this analytical method for this group.

A long quote coming up: "This in vitro study showed promising and positive results in that supplementing the microbiota of ASD children with 65%B-GOS may manipulate the gut bacterial population and alter metabolic activity towards a configuration that might represent a health benefit to the host. However, further work will be required to assess such changes in an in vivo human intervention study."

Just before anyone makes a run on B-GOS or any similar product however, I do need to stress a few important points. First, this was a study of poo(p) samples from 3 autistic children compared with samples from 3 non-autistic children. Aside from the small participant numbers, we don't know anything about participants' various comorbidities (although we know they were "free of any metabolic and gastrointestinal diseases") and only limited information on their dietary habits and medication history. Second, poo(p) was the target material included for analysis and what happened when B-GOS was supplemented during the journey through the artificial gut model. This study said nothing about what happens when real people with autism take B-GOS orally for example, and how it might affect gut bacterial populations and metabolites as it progresses down a real gastrointestinal (GI) tract. This also includes a lack of information on any potential side-effects in a real-world situation. We are also assuming that any supplement survives the stomach. There is quite a bit more to do in this area.

But for now, I stick to the idea that the Grimaldi paper provides some potentially important information and certainly, some new routes/methods for further study of the link between prebiotics, probiotics and synbiotics in the context of the gut microbiota and autism...

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[1] Grimaldi R. et al. In vitro fermentation of B-GOS: Impact on faecal bacterial populations and metabolic activity in autistic and non-autistic children. FEMS Microbiol Ecol. 2016 Nov 16. pii: fiw233.

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Grimaldi R, Cela D, Swann JR, Vulevic J, Gibson GR, Tzortzis G, & Costabile A (2016). In vitro fermentation of B-GOS: Impact on faecal bacterial populations and metabolic activity in autistic and non-autistic children. FEMS microbiology ecology PMID: 27856622

Acyl-carnitine profiles and autism

No doubt alongside quite a few others, I was interested to read the latest paper from Richard Frye and colleagues* (open-access) discussing the potential links between an animal model of autism spectrum disorder (ASD) and real-world ASD in a particular cohort of participants.

This is not the first time that I've talked about (a) the work of Dr Frye - as per my [don't panic] post on folate receptor autoantibodies in cases of autism (see here) and (b) some of the difficulties attached to translating rat/mouse model findings in autism research into 'real-world' findings (see here). Indeed this last point might also tie into my recent musings on the use of LPS in autism research (see here).

Daddy o' microbiology @ Wikipedia  

My interest in the latest Frye paper was further piqued upon realising that Derrick MacFabe was also part of the authorship team given his insightful work on how administration of propionic acid - a short chain fatty acid - to rodents might provide some interesting perspectives overlapping with various findings noted in cases of autism (see my previous post here).

The Frye paper is open-access and available to all but here are a few points worth mentioning:

• Based on the animal model work looking at propionic acid (PPA) infusions and some of the collected effects (presentation of autism-type behaviours**, mitochondrial issues***, abnormal measures of glutathione, etc.), the aim of the current study was to map out whether some of the observations in the PPA rodent model were also present in real-life autism.
• Based on a clinic-based sample of children with autism (N=326), acyl-carnitine panels were conducted on about two-thirds of participants (n=213).
• Acyl-carnitines, as their name suggests, are related to carnitine which has previously been tied back to autism (see here) and represent a number of different compounds - complexes of carnitine and various fatty acids - involved in the transport of fatty acids into the mitochondrial matrix.
• The previous PPA rodent model indicated some disturbance in the amount of various acyl-carnitines, particularly with regards to short- and long-chain fatty acids (not so much in the medium-chain fatty acids) reflective of some mitochondrial dysfunction.
• So the authors looked at short-, medium- and long-chain acyl-carnitines in their autism cohort to see if there were any overlaps compared with the PPA treated animals.
• Results: 35% of the autism cohort showed "an increase in three or more acyl-carnitines when initially measured". When further testing were conducted on some of these participants showing elevation, this figure was revised to 17% of the cohort who "demonstrated consistent elevations in short-chain and long-chain, but not medium-chain, acyl-carnitines". You'll note the similarity with PPA animal model in terms of the short- and long-chain acyl-carnitines.
• Furthermore, four participants were also examined with regards to glutathione and oxidative stress markers. As per the quite consistent literature on things like total- and free-reduced glutathione (see this post), compared with controls, there were some familiar trends emerging.

And relax.

There is a lot to take in from this paper both in the protocol and testing undertaken and the possible interpretation of findings. A quote best sums up the results: "This study has demonstrated that ~17% of children with ASD manifest biomarkers of abnormal mitochondrial fatty-acid metabolism that parallel similar biomarkers in the PPA rodent model of ASD".

As perhaps expected, there has been some press attention following the publication of this paper. The headline: Researchers discover link between certain types of autism and gut bacteria has been a common one, reflective of the fact that when it comes to the production of PPA outside of injecting the stuff directly into the rodent brain, the gut and in particular, certain types of gut bacteria, have been suggested as a route to PPA production in cases of autism. Indeed, I'm taken back to the findings by Wang and colleagues from Oz (see this post) on levels of fecal short chain fatty acids (SCFAs) in their cohort of children with autism which included propionic acid (propionate). I'm also reliably informed that there is 'more to come' from the Australian research group in the coming months.

There are a few final aspects to the Frye study which I should have mentioned earlier. This includes the notion that "it is very likely that MD [mitochondrial dysfunction] is acquired" given that in the most part, both nuclear and mitochondrial DNA examinations in their cohort were negative for anything that might genetically account for the results. I'll be coming to DNA and mitochondrial issues in a subsequent 'training' post scheduled quite soon but one can perhaps see how this might strengthen any argument of disruptions to gut bacteria/microflora facilitating the production of elevated PPA onwards to disrupting acyl-carnitine chemistry.

Additionally there is the implication based on the PPA rodent model that affecting PPA production could be a potential therapeutic route for some cases of autism at some point. I don't really want to go too far into the hows and whys of this suggestion at the current time given the current lack of evidence for any effect. Aside that is, from drawing your attention to a related field of inquiry into how disrupting PPA-producing gut bacteria might have some important implications for those suffering from conditions like propionic acidaemia****.

I'm not going to get too carried away with the Frye results as they stand. As per my previous post on PPA, there is still a bit of a stretch from injecting rodent brains with PPA and recording outcomes to suggesting that gut bacteria will be able to produce enough PPA so as to exert a similar effect in cases of autism. At least one group is asking the same question***** (open-access). That being said, I am still interested in the details of this study and how the authors have at least tried to model animal findings into real-life autism. Indeed how we really should be putting a lot more research effort into looking at mitochondria and autism as we potentially also should gut bacteria and autism.

A song to finish... something mellow yet catchy.... Lovefool by the Cardigans.

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* Frye RE. et al. Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder. Translational Psychiatry. 2013; 3: e220.

** MacFabe DF. et al. Effects of the enteric bacterial metabolic product propionic acid on object-directed behavior, social behavior, cognition, and neuroinflammation in adolescent rats: Relevance to autism spectrum disorder. Behav Brain Res. 2011; 217: 47-54.

*** Thomas RH. et al. Altered brain phospholipid and acylcarnitine profiles in propionic acid infused rodents: further development of a potential model of autism spectrum disorders. J Neurochem. 2010; 113: 515-529.

**** Mellon AF. et al. Effect of oral antibiotics on intestinal production of propionic acid. Arch Dis Child 2000; 82: 169-172.

***** El-Ansary AK. et al. Comparative study on the protective effect of carnosine and carnitine against pro-inflammatory/pro-oxidant effects of clindamycin and propionic acid administrations to hamsters. African Journal of Microbiology Research. 2013; 7: 103-114.

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Frye RE, Melnyk S, & Macfabe DF (2013). Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder. Translational psychiatry, 3 PMID: 23340503

Autism and microglia

An interesting paper has appeared by Beumer and colleagues* with the grandiose title: The immune theory of psychiatric diseases: a key role for activated microglia and circulating monocytes. It's an intriguing paper linking specific actions of the immune system on brain areas in connection to various conditions including depression and schizophrenia via a biological entity that seems to be gaining some research interest: microglia.

On the basis of this publication I therefore resisted no longer and decided to set off on the voyage of discovery that is a possible role for microglia in cases of autism. Please understand that I undertake this post with some trepidation as my skill set does not readily encompass the hows and whys of microglia, so be at the ready with that rather large pinch of salt.

Quite a good overview of microglia is here by Kettenmann and colleagues** (full-text) alongside some discussion on their 'constant gardening' effect in this piece by Virginia Hughes*** (full-text). Apparently constituting as much as 10% of the cells of the central nervous system, microglia derive from cells of the immune system and activate in response to injury or inflammation. More than that however, there is some emerging evidence that microglia might also play a role in the pruning process which the developing brain undertakes during the early years and perhaps beyond.

With autism specifically in mind, I've already linked in previous posts to an interesting blog piece from Paul Patterson on microglia potentially eating synapses in autism as a consequence of some interesting research suggesting increased microglial activation in some cases of autism****. I draw your attention also to another interesting blog post on the SFARI website which strengthens a possible connection between microglia and a mouse model of Rett syndrome, noting also the controversial issue of bone marrow transplant as a potential route to "generating healthy microglia". I might add that I am not getting too obsessed with stem cells and the such like, but in light of Prof. Patterson's recent paper, one has to keep an open mind.

Other research on microglia and autism:

• Starting from the early days of the PubMed catalogue on this topic a few familiar names crop up with microglia in mind. Andrew Zimmerman and colleagues***** (he of the 'can you grow out of autism' research) talked about neuroglial activation and neuroinflammation quite a few years ago. They reported "marked activation of microglia" alongside several other inflammatory related findings in the brains of people with autism. Similar follow-up studies from this research group also talked about paradoxical lower levels of quinolinic acid in cerebrospinal fluid which  brings us back to everyone's favourite aromatic amino acid, tryptophan. I'll leave that for now.
• Propionic acid (PPA), the topic of quite a recent blog post, also gets a look-in with microglia in mind as per the paper by Derek MacFabe and colleagues******. They report that during their 'inject PPA into rodent brain' experiments, increases in activated microglia was one of the effects contributing to the neuroinflammatory process described. Whilst I would love to be able to say this might tie into the short chain fatty acid findings also talked about with PPA and autism in mind (here), it is probably not unexpected that PPA administration so directly and at such quantities would provoke a reaction from microglia given their activation at the first sight of any foreign invaders.
• Other research speculates on the role of microglia and autism (here, here and here), in some cases speculating on the potential environmental factors which may exacerbate microglial priming. I was also drawn to this paper by Schwarz & Bilbo******* who discuss how gender differences in the colonisation and function of glia may offer some interesting explanations for the gender differences seen in conditions like autism, acknowledging that glia includes more than just microglia.

There is still some way to go with regards to the microglia-autism story but so far the research base could be described as very interesting. Inflammation, as I have said before, seems to be playing many hands in at least some cases of autism. Whilst the chicken-and-egg debate about immune function and autism continues, there is little doubt that for some, immune involvement - whether mediated/described by microglia activation, circulating cytokines or other markers of inflammation and immune over- or -under activation, seems to be a facet of symptom presentation. The puzzle is still far from complete however.

Finally, there is the question about what can be done about over or chronic activation of microglia. Reiterating my caveat about not giving medical advice or anything approximating such advice and aside from the bone marrow transplant data already touched upon, I was interested to read the various literature on the effect of administration of the tetracycline antibiotic, minocycline on microglial activation as per studies likes this one and this one (both full-text). Accepting that certain antibiotics might be a bit of a double-edged sword for some cases of autism (see this post) as well as such compounds having other, more recognised antimicrobial action - potentially important in some cases of autism - it does make me wonder whether targeting such interventions at activated microglia at a critical time might be a research area worth investing in.

To finish, I'm in the mood for some Ska. So without further ado, the Specials and Too Much Too Young.

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* Beumer W. et al. The immune theory of psychiatric diseases: a key role for activated microglia and circulating monocytes. Journal of Leukocyte Biology. August 2012.

** Kettenmann H. et al. Physiology of microglia. Physiological Reviews. 2011; 91: 461-553.

*** Hughes V. Microglia: the constant gardeners. Nature. 2012; 485: 570-572.

**** Morgan JT. et al. Microglial activation and increased microglial density observed in the dorsolateral prefrontal cortex in autism. Biological Psychiatry. 2010; 68: 368-376.

***** Vargas DL. et al. Neuroglial activation and neuroinflammation in the brain of patients with autism. Annals of Neurology. 2005; 57: 67-81.

****** MacFabe DF. et al. Neurobiological effects of intraventricular propionic acid in rats: possible role of short chain fatty acids on the pathogenesis and characteristics of autism spectrum disorders. Behavioral Brain Research. 2007; 176: 149-169.

******* Schwarz JM. & Bilbo SD. Sex, glia, and development: Interactions in health and disease. Hormones & Behavior. February 2012.

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Beumer W, Gibney SM, & Drexhage RC (2012). The immune theory of psychiatric diseases: a key role for activated microglia and circulating monocytes J Leukoc Biol. DOI: 10.1189/jlb.0212100

Propionic acid and autism

Another post, another organic acid (!) but please don't click away just yet as I attempt once again not to blind you with science. The topic of the day today is propionic acid (PPA) otherwise known as propanoic acid and the very tentative suggestions of some link to autism. 

Search on-line for propionic acid and you're quite likely to get lots of industrial chemical information about it including a materials safety data sheet (MSDS) (here) implying that this stuff is toxic, combustible and corrosive. So why on earth am I talking about it with regards to autism?

Well, like many things in modern life, the chemical industry isn't the only source of 'chemicals' as per our wonderful chemical factory that is the human body and the surprising effects of some of the bacteria which inhabit us. Not for the first time, I might add, has bacteria and biochemistry come together on this blog (see here) and probably not the last time either.

PPA is an organic acid - a short chain fatty acid - which as well as being used as a food additive / preservative and used in the process of polymer production apparently, is also what might be considered a natural product as a result of its endogenous production. Short chain fatty acid you say? Y'mean like the ones discussed not so long ago in relation to autism by Wang and colleagues? Yes, the very same.

Continuing with autism in mind, I am taken back to this news report from 2007 where Dr Derrick MacFabe discussed this piece of research* (followed up by this study** too) suggesting that you are what you eat in terms of foodstuffs and bacteria combining to produce PPA and what effect it had on the rat brain following "intraventricular infusions" (to you and me injecting the stuff directly into rat brains). Dr MacFabe has very definitely taken the lead when it comes to looking at PPA and autism. 

Indeed I was interested to see the effects of PPA administration on rat brains including activated microglia and decreased glutathione alongside the more overt effects on rat behaviour. Understanding that rats (like mice and fish) are not humans, indications of issues with glutathione have been quite strongly shown to be related to cases of autism and indeed the brain has become a recent focus for this area of research. As for microglia, I would once again refer you to this post by Paul Patterson on the suggested dietary habits of microglia in autism with the promise of a post on this research area soon.

Other research groups have also entertained a possible role for PPA in autism. The paper by El-Ansary and colleagues*** replicated the results from the MacFabe group and indeed added a few new aspects also making mention of lactate dehydrogenase and elevations in IL-6 among other things; both factors having been reported in connection to autism (here and here respectively). I think we can, with some degree of confidence, assume from this collected work, that propionic acid injections are probably not good news for rats.

We move onwards then to another piece of research by El-Ansary and colleague**** (full-text) looking at the possibility that supplementation with omega-3 fatty acids might afford some protection against the effects of PPA administration to rats. They reported that omega-3 supplementation did indeed alter some of the parameters associated with PPA administration including some interesting data on caspase-3, an executioner molecule involved in apoptosis previously reported in cases of autism (see here for more information). It seems rats fed a diet rich in omega-3 fatty acids might have some protection against various effects of PPA administration. I assume this ties into this paper by Thomas and colleagues***** (including Dr MacFabe).

So to summarise. An overlap between direct PPA administration and some key biochemical changes noted in cases of autism. Fine. A potential protective effect via use of omega-3 fatty supplementation from PPA administration. OK (fatty acids have some history in cases of autism as per this older post). PPA found to be elevated in cases of autism (based on the analysis of stools). Right.

Reading all that might convince some people of a role for PPA in autism but I'm going to play devil's advocate for a second and suggest why we need more research in this area. First, as mentioned rats aren't human beings. Rats might make good models for human being - as do some mice - but rats are not human beings. Second, administration of PPA was either intracerebroventricularly or at "neurotoxic doses" over 3 days (250mg/kg body weight). Either of these two factors whilst good for studying rats in a lab over a relatively short space of time is probably not going to be representative of chronic PPA exposure in people with autism. Unless of course that is, that something causes a huge acute surge in PPA that is. Finally(!) aside from the Wang findings on fecal PPA levels, the research in this area is still a little bit scant in terms of PPA levels in other biofluids. I haven't for example, seen anything as of yet looking at whether rates of the metabolic disorder propionic acidaemia is any more prevalent in cases of autism than not autism or likewise whether propionic acidaemia is accompanied by an increase in cases of autism accepting the seriousness and poor prognosis of this rare metabolic conditions. A few possible routes to further study perhaps.

That being said, I am still intrigued about propionic acid and the research done so far with regards to autism whether animal model-based or direct measurement. The whole connection back to short chain fatty acids, digestion and gut bacteria is an interesting and very under-researched area with autism in mind -  indeed with lots of other behavioural / cognitive / psychiatric conditions in mind. Could it hurt to put a little more research efforts into this area?

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* MacFabe DF. et al. Neurobiological effects of intraventricular propionic acid in rats: possible role of short chain fatty acids on the pathogenesis and characteristics of autism spectrum disorders. Behavioral Brain Research. 2007; 176: 149-169.

** MacFabe DF. et al. Effects of the enteric bacterial metabolic product propionic acid on object-directed behavior, social behavior, cognition, and neuroinflammation in adolescent rats: Relevance to autism spectrum disorder. Behavioral Brain Research. 2011; 217: 47-54.

*** El-Ansary AK. et al. Etiology of autistic features: the persisting neurotoxic effects of propionic acid. Journal of Neuroinflammation. April 2012.
DOI: 10.1186/1742-2094-9-74

**** El-Ansary AK. et al. On the protective effect of omega-3 against propionic acid-induced neurotoxicity in rat pups. Lipids in Health & Disease. 2011; 10: 142.

***** Thomas RH. et al. The enteric bacterial metabolite propionic acid alters brain and plasma phospholipid molecular species: further development of a rodent model of autism spectrum disorders. Journal of Neuroinflammation. 2012; 9: 153.

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