"serum levels of certain endocannabinoids are substantially decreased in people with ASD"

The quote titling this post - "serum levels of certain endocannabinoids are substantially decreased in people with ASD [autism spectrum disorder]" - comes from the paper published by Adi Aran and colleagues [1]. It adds to previous study on this topic (see here) and continues a research theme from members of this authorship team where the word 'cannabis' is being discussed - in the peer-reviewed science domain - in the context of [some] autism (see here).

Distinct from the last time authors' research appeared on this blog talking about the feasibility of "Cannabidiol-Rich Cannabis" 'for autism' [2], the name of the research game this time around was to assess "the circulating levels of several endocannabinoids and delineate the correlations between their levels and disease characteristics in a large group of children with ASD and their matched controls with typical development." Researchers mention how previous studies in this area "were not designed to comprehensively characterize the involvement of the ECS [endocannabinoid system] in the pathogenesis of ASD" in quite a sweeping blow to some of the other research in this area.

So, endocannabinoids are part of a system that is involved in various important biological processes [3]. As the name suggests there's an overlap between 'endogenous cannabinoids' and some of the chemical components seen in cannabis that provides as good an answer as any as to why cannabis use/misuse is the continuing issue that it is in a population sense. Authors talk about their study focusing on various endocannabinoids: AEA (anandamide), 2-AG (2-arachidonoil-glycerol), AA (arachidonic acid), PEA (N-palmitoylethanolamine), and OEA (N-oleoylethanolamine). They report how said compounds in serum samples were "analyzed by liquid chromatography/tandem mass spectrometry in 93 children with ASD... and 93 age- and gender-matched neurotypical children." Please don't however get me started on the nonsense that is the word 'neurotypical' (see here). Various other behavioural, psychometric and demographic data were also collected and thrown into the statistical mix.

Results: "Serum levels of the main endocannabinoid AEA and its structurally related compounds OEA and PEA were lower in children with ASD versus age-, gender-, and BMI [body mass index]-matched control group of typically developed children." Nothing particularly new there, as the lower levels of anandamide for example, mimic those reported by Karhson and colleagues [4]. Researchers also mentioned how their findings *might* also have some other potential: "circulating AEA, OEA, and PEA might be used to identify a biologically homogeneous subgroup of ASD, predict response to treatments and adverse reactions to medications, and assist in the development of novel drugs that target specific core symptoms of ASD." Interestingly, some of these 'options' have already been explored [5] in humans and also some animal models [6] with autism in mind.

As to the biochemical *links* between the Aran findings and indeed, the ECS more generally with autism, well, there's still a way to go to decipher them all yet. There are clues emerging [7]; clues that intersect with other important autism-relevant concepts like inflammation among other things. I note also the authors mention how their findings "support the rationale in the ongoing and emerging clinical trials of CBD [cannabidiol] in ASD" (see here) and some results to come.

I'm well and truly [cautiously] interested...

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[1] Aran A. et al. Lower circulating endocannabinoid levels in children with autism spectrum disorder. Molecular Autism. 2019; 10:2.

[2] Aran A. et al. Brief Report: Cannabidiol-Rich Cannabis in Children with Autism Spectrum Disorder and Severe Behavioral Problems-A Retrospective Feasibility Study. J Autism Dev Disord. 2018 Oct 31.

[3] Lu HC. & Mackie K. An Introduction to the Endogenous Cannabinoid System. Biol Psychiatry. 2015;79(7):516-25.

[4] Karhson DS. et al. Plasma anandamide concentrations are lower in children with autism spectrum disorder. Mol Autism. 2018 Mar 12;9:18.

[5] Antonucci N. et al. Beneficial Effects of Palmitoylethanolamide on Expressive Language, Cognition, and Behaviors in Autism: A Report of Two Cases. Case Rep Psychiatry. 2015;2015:325061.

[6] Servadio M. et al. Targeting anandamide metabolism rescues core and associated autistic-like symptoms in rats prenatally exposed to valproic acid. Transl Psychiatry. 2016 Sep 27;6(9):e902.

[7] Brigida AL. et al. Endocannabinod Signal Dysregulation in Autism Spectrum Disorders: A Correlation Link between Inflammatory State and Neuro-Immune Alterations. Int J Mol Sci. 2017;18(7):1425. Published 2017 Jul 3.

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Plasma anandamide concentrations are lower in children with autism

The findings reported by Debra Karhson and colleagues [1] piqued my interest for two primary reasons. First, they detail "the first empirical human data to translate preclinical rodent findings to confirm a link between plasma anandamide concentrations in children with ASD [autism spectrum disorder]." Second, authors also report on the use of a gold-standard technique when it came to their analyses: the development and use of "a LC-MS/MS [liquid chromatography-tandem mass spectrometry] method to quantitatively analyze AEA [anandamide] concentrations in small volumes of banked plasma with short sample preparation time and high sample repeatability."

OK, the basis for the Karhson study was the 'increasing interest for ASD' examining the endogenous cannabinoid or endocannabinoid system. This is a system, an internal system, that comprises of quite a few compounds, enzymes and receptors that play "important roles in central nervous system (CNS) development, synaptic plasticity, and the response to endogenous and environmental insults" [2]. Yes, as the name suggests, there is an 'overlap' between some of the workings of the endocannabinoid system (ECS) and components of a certain drug of abuse but that doesn't insinuate anything at the present time.

Anandamide (AEA) is a sort of messenger molecule that is part of the ECS. It shares some chemical characteristics with the active compound found in cannabis, leading quite a few commentators to talk about AEA in terms of being a 'pleasure' or 'bliss' molecule. It does not however, have the 'staying power' of its molecular companion; chemically-speaking being fairly readily degraded in the body. Indeed, of the many biological roles and functions linked to AEA and the ECS more generally, I'd in particular, like to direct your attention to some of the science-so-far literature in relation to pregnancy (see here). And, no, that does NOT mean that smoking marijuana during pregnancy is a good thing...

The authors highlight how the ECS is a research area rising in relation to autism (and associated diagnoses) based, quite extensively, on animal models of autism and all the associated 'issues' that this carries (see here). So: "despite the promise of these preclinical data, no studies to date have investigated AEA concentrations in humans with ASD." They sought to remedy that situation.

Results are reported based on the LC-MS/MS analysis of plasma samples provided by some 59 children with autism and 53 not-autism controls. As per my continued interest in all-things mass spec when specifically applied to autism research, I was encouraged by the use of a "commercially available stable isotope-labeled AEA-d8" being used as an internal standard, and the fact that the lower limit of detection for AEA was in the femtogram range. In short, authors were able to train their system to specifically look for AEA and were able to get down to some really quite low levels of detection.

"Two significant findings were observed: (1) plasma AEA concentrations significantly differentiated ASD cases from controls, such that children with lower AEA concentrations were more likely to have ASD, and (2) AEA concentrations were significantly lower in ASD compared to control children." I don't really need to say much more than that, aside from adding in another quote: "These results, although preliminary, corroborate preclinical evidence that AEA signaling may be impaired in patients with ASD."

The question of what these findings actually mean is still unanswered. It should for example, be noted that the Karhson results in real people were based on the analysis of plasma samples, where previous animal work has tended to be a little more 'invasive' in terms of the tissue types looked at (e.g. in the brain). The authors also mention that further investigations need to give due credit to the idea that autism rarely exists in some sort of diagnostic vacuum (see here), and some of these 'comorbidities' could very well influence the results obtained [3].

Further studies on this topic are very much required.

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[1] Karhson DS. et al. Plasma anandamide concentrations are lower in children with autism spectrum disorder. Molecular Autism. 2018; 9: 18.

[2] Lu H-C. & Mackie K. An introduction to the endogenous cannabinoid system. Biological Psychiatry. 2016;79(7):516-525.

[3] Romigi A. et al. Cerebrospinal fluid levels of the endocannabinoid anandamide are reduced in patients with untreated newly diagnosed temporal lobe epilepsy. Epilepsia. 2010 May;51(5):768-72.

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Paracetamol for fever 'associated' with autism?

"In this study, we again show that acetaminophen use is associated with ASD [autism spectrum disorder]."

That was one of the results reported by Stephen Schultz & Georgianna Gould [1] (open-access available here) as part of their survey of the US "National Database for Autism Research (NDAR) of the National Institute of Mental Health (NIMH)" looking at "whether ASD is associated with acetaminophen use." Acetaminophen by the way, is another name for paracetamol, the over-the-counter pain relief medication that is going through some turbulent times at the moment (see here for example).

Shultz & Gould - one of whom has some research form in this area [2] - eventually relied on information for 118 children diagnosed with an ASD and 79 'non-ASD' children with an average age of about 11 years old. The sorts of data they looked at surrounded the parental choice of medication to treat fevers (I think) including whether paracetamol, ibuprofen or aspirin were used. I have to say that the authors could have made the methodology behind their analysis a little bit clearer in terms of how the questions were posed and to whom rather than just referring to another study with regards to participant selection for example. I had to go fishing for various details which is guaranteed to furrow my brow...

Results: well, I'm slightly puzzled it has to be said. When it came to questions about paracetamol use (I use the term paracetamol 'cos that's what us Limeys are used to) between the ASD and non-ASD groups I didn't see too much difference overall. Take for example the questions about 'only using paracetamol' for fever or 'first choice' use for fever. The percentage figures for the ASD and non-ASD group were 15% and 12% respectively for 'only use this' and 35% and 46% respectively for 'first choice'. Given the participant numbers, I'm not sure that these stats are so wildly different. Yes, I appreciate that when it came to the question about 'rarely or never using' paracetamol to treat fever, 17% of those with ASD reported positive to this question compared with only 3% of controls, but does this really tell us much about very different patterns of paracetamol use?

Further, the authors report results based on "age-adjusted models for levels of fever medication use". They observe that using "acetaminophen as a first choice was 83% less likely in children with ASD... while use of acetaminophen if other medication doesn’t bring down fever was 82% less likely in children with ASD." They interpret this to mean that compared with their previous results [2] findings were reversed in that "older children with ASD compared to control children were significantly less likely to use acetaminophen for fever; whereas, in our 2008 study, younger children with ASD compared to control children were significantly more likely to use acetaminophen at 12-18 months of age and after the MMR vaccination." The mention of immunisation in that last sentence was based on their 2008 paper suggesting that "acetaminophen use after measles-mumps-rubella vaccination was associated with autistic disorder" but I have to say that I'm left a little wanting in terms of these recent findings by Shultz & Gould.

I do think there is a 'where next?' discussion to be had when it comes to the idea that paracetamol use might be linked to 'some' autism. Given the growing research interest in paracetamol use and a 'hyperactive phenotype' of autism (see here), this stalwart of pain relief is deserving of much further inspection in relation to autism. Shultz & Gould do offer one possible research direction based on some speculation about the how the endocannabinoid system might fit into this (something mentioned by other authors too). I am interested in the hypothetical situation they conclude their paper with implicating the endocannabinoid system and how paracetamol might affect 'endocannabinoid tone' but to what extent is perhaps another question.

Just before I finish on this topic I'm minded to bring to your attention another detail from the Shultz / Gould paper with regards to the sentence: "children with ASD vs. non-ASD children are significantly more likely to show an increase in sociability when they have a fever." I've always thought the observations on behaviour and fever when it comes to [some] autism to be quite important (see here). Speculation that "this increase [in sociability] is due to anandamide activation of the endocannabinoid system in ASD children" is also ripe for further scientific investigation...

It's been a while but here is some music to close: Mrs Robinson.

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[1] Schultz ST. & Gould GG. Acetaminophen Use for Fever in Children Associated with Autism Spectrum Disorder. Autism Open Access. 2016 Apr;6(2). pii: 170.

[2] Schultz ST. et al. Acetaminophen (paracetamol) use, measles-mumps-rubella vaccination, and autistic disorder: the results of a parent survey. Autism. 2008 May;12(3):293-307.

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Schultz ST, & Gould GG (2016). Acetaminophen Use for Fever in Children Associated with Autism Spectrum Disorder. Autism-open access, 6 (2) PMID: 27695658

GcMAF and autism continued

"GcMAF treatment was able to normalize the observed differences in the dysregulated gene expression of the endocannabinoid system of the autism group". That is the potentially very important finding from Dario Siniscalco and colleagues* (open-access here) continuing the increasing scientific interest in all-things GcMAF (Gc Macrophage Activating Factor) with autism in mind.

Watson and the shark @ Wikipedia 

A quick recap first: I've talked GcMAF and autism on this blog before (see here and see here) and how activating the 'big eaters' (macrophages) of the immune system might be important for some autism and a variety of other conditions too. The collected literature on GcMAF and autism is small... very small... at the moment comprising one paper by Bradstreet and colleagues [2]. So it is indeed a welcome sight to see some more science being done on this area (with the promise of more to come).

The recent paper by Siniscalco et al is open-access but a few pointers might be useful:

• Blood samples were provided by a small group of participants diagnosed with autism (n=22) and age- and sex-matched asymptomatic controls (n=20). Blood monocyte-derived macrophages (BMDMs) were derived from said samples and dosed with GcMAF.
• At the same time, building on previous work by the authors [3] suggesting involvement of the cannabinoid system (EC) in some cases of autism, authors sought to examine whether the therapeutic effects of GcMAF previously highlighted in autism, might have something to do with the regulation of genes involved with the cannabinoid system. To look at this question, they extracted RNA from the BMDMs to look at the effect of GcMAF on the "transciptional regulation of EC genes". Those genes included CB2R, FAAH, NAPE-PLD and GAPDH.
• The results: quite a few of them but they included: "GcMAF treatment was able to significantly increase gene expressions both NAPE-PLD... and FAAH" in BMDMs from participants with autism. This contrasted with no observed changes in gene expression of any of the EC genes in the control samples.
• Perhaps a little unusually given the meaning of the name GcMAF as a 'macrophage activating factor', "GcMAF was able to trigger overall macrophage deactivation in autistic samples". Based on looking at something called Ki67 involved in cell proliferation, authors reported "a decrease of 23% in GcMAF treated monocyte derived macrophages from autistic children as compared to untreated macrophage cells". This reduction was also noted in the control samples too.

I don't mind telling you that I kinda reached the limits of my very rudimentary knowledge of GcMAF and autism with this paper. I do find that the possibility of involvement of the cannabinoid system to cases of autism to be something really rather interesting as per other results in this area [3] and related research including that potentially impacting on comorbidity such as epilepsy. Linking GcMAF to that system potentially opens up some interesting research avenues.

The fact also that GcMAF seemed to have a deactivating effect on macrophages is also a point of interest. I hope I'm not mis-interpreting the findings or anything but I do wonder if this would reinforce the fact that other biological effects may need further analysis when it comes to GcMAF and autism. It's also interesting that nagalase activity was not discussed in the Siniscalco paper so perhaps further inspection of those EC genes and their expression with nagalase in mind should be indicated in future work too.

And if you want the authors take on this work, look no further...

Here's a little song for everyone out there.... so said Kiss. And the rest is rock history.

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[1] Siniscalco D. et al. The in vitro GcMAF effects on endocannabinoid system transcriptionomics, receptor formation, and cell activity of autism-derived macrophages. J Neuroinflammation. 2014 Apr 17;11(1):78.

[2] Bradstreet JJ. et al. Initial observations of elevated alpha-N-acetylgalactosaminidase activity associated with autism and observed reductions from GC protein—macrophage activating factor injections. Autism Insights. 2012. 4: 31-38.

[3] Kerr DM. et al. Alterations in the endocannabinoid system in the rat valproic acid model of autism. Behav Brain Res. 2013 Jul 15;249:124-32.

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Siniscalco D, Bradstreet JJ, Cirillo A, & Antonucci N (2014). The in vitro GcMAF effects on endocannabinoid system transcriptionomics, receptor formation, and cell activity of autism-derived macrophages. Journal of neuroinflammation, 11 (1) PMID: 24739187

Endocannabinoids and fragile X syndrome

Fragile X syndrome (FXS), one of the known causes of autism or rather autistic behaviours, has been receiving quite a lot of research and media attention lately. Arbaclofen (STX209) with FXS in mind was the topic of the most recent post (see here); now we have the article by Kwang-Mook Jung and colleagues* (open-access) on endocannabinoid signalling. There may be quite a few similarities between this latest endocannabinoid research and the growing interest in glutamate and its receptors with FXS in mind, but more on that shortly.

OK let's get it out of the way. Thinking about cannabinoids, I assume some people might initially make the connection back to cannabis and the chief psychoactive substance, delta-9-tetrahydrocannabinol (Δ9-THC), associated with that (in)famous plant. Indeed with the Jung study in mind, it was perhaps inevitable that a headline like: "Cannabis chemical combats chief genetic cause of autism" complete with the necessary picture of a cannabis plant would appear (all that's missing is Bob Marley and some 'herb' music just in case readers required a little more atmosphere).

Suffice to say that the Jung paper focuses on the functioning of the endogenous (endo)cannabinoid system over and above its external chemical relation.

• In particular how in a mouse model, issues with the fragile X mental retardation protein (FMRP) associated with FXS might have some potentially important effects on endocannabinoid signalling by way of metabotropic glutamate receptor-5 (mGluR5) depression. 
• Mention is made of something called 2-arachidonoyl-sn-glycerol (2-AG), a "retrograde endocannabinoid (eCB) transmitter" but other than saying that this is one of the more abundant endocannabinoids seemingly tied into mGluR5 and related to arachidonic acid (AA), I wouldn't pretend to know much more about it. The suggestion is that loss of FMRP has knock-on effects to mGluR5-dependent release of of 2-AG.
• Aside from the basic science of endocannabinoid signalling in said mouse model of FXS, a particularly interesting part of the Jung study is the suggestion of pharmacological 'rescuing' of 2-AG signalling and the subsequent positive impact on some of the mouse subjects displayed behaviours. 
• The pharmacological agent in question was something called JZL184, an inhibitor of the enzyme which usually degrades 2-AG (monoglycerol lipase, MGL) bearing in mind that said inhibition was irreversible. 

Hopefully I've got the main features of the study and its findings right and not confused you with all the terminology. Jung et al including Daniele Piomelli are no strangers to research on the endocannabinoid system and glutamatergic signalling as per papers like this one (full-text) and this one (full-text).

There are a few very obvious caveats to this latest work; not least that mice were the lucky participants and questions on those all-important extrapolations from mice findings to human findings which still need much greater investigation outside of the range of mouse models available to autism research. Emphasising also that this was a study of fragile X syndrome and the mutations associated with this autism-presenting condition, which may not be directly transferable to the range of other non-FXS autisms.

Having said all that I'm interested in this work for a few other reasons. As mentioned earlier on, fragile X syndrome and its relationship with glutamate and glutamate signalling is fast becoming a hot topic. If I were a betting man, I'd probably put a few quid/bucks/euro on a long-shot for glutamate and its receptors turning up as a commonality in quite a few behaviourally-defined conditions. Not necessarily as a core 'reason' but an important factor nonetheless.

I note also that MGL-inhibitors such as JZL184 have been discussed with concepts like protection from neuroinflammation (as a consequence of altering prostaglandin production) in mind** and indeed with another interesting topic in mind, intestinal permeability as per this study by Alhamoruni and colleagues***. All that outside of the possible inflammatory bowel disease link**** (open-access) too. On the basis of these and other research, I'm wondering about the centrality and specificity of the brain to any effects and indeed whether maintaining 2-AG levels may also be affecting other organs as part and parcel of any therapeutic, or indeed non-therapeutic, effect.

Enough with all this science and stuff. Ladies and gentlemen, welcome to paradise...

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* Jung KM. et al. Uncoupling of the endocannabinoid signalling complex in a mouse model of fragile X syndrome. Nature Communications. 2012; 3: 1080. PMID: 23011134

** Nomura DK. et al. Endocannabinoid hydrolysis generates brain prostaglandins that promote neuroinflammation. Science. 2011; 334: 809-813.

*** Alhamoruni A. et al. Cannabinoids mediate opposing effects on inflammation-induced intestinal permeability. British Journal of Pharmcology. 2012; 165: 2598-2610.

**** Alhouayek M. et al. Increasing endogenous 2-arachidonoylglycerol levels counteracts colitis and related systemic inflammation. FASEB J. 2011; 25: 2711-2712.

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Kwang-Mook Jung, Marja Sepers, Christopher M. Henstridge, Olivier Lassalle, Daniela Neuhofer, Henry Martin, Melanie Ginger, Andreas Frick, Nicholas V. DiPatrizio, Ken Mackie, Istvan Katona, Daniele Piomelli, & Olivier J. Manzoni (2012). Uncoupling of the endocannabinoid signalling complex in a mouse model of fragile X syndrome Nature Communications, 3 DOI: 10.1038/ncomms2045