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.

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[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.

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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

Ear, ear and hyperactivity

The BBC headline - Inner ear disorders 'linked to hyperactivity' - caught my eye recently. Detailing a fascinating piece of research by Michelle Antoine and colleagues* published in the journal Science the suggestion is that issues with the inner ear that affect hearing and balance might also show some relationship with hyperactivity and in mice at least, might be amendable to treatment.

van Gogh @ Wikipedia  

Before going on, I'd like to point out that Science (the publishing journal) has produced some pretty interesting papers at a similar time to the publication of the Antoine research. I'm talking specifically about the gut bacteria from obese discordant twins producing obese mice research (see here) which discusses the 'transmissibility of obesity' among other things (see here for my previous blogging interest in this area of research). There was also that letter on MAR autism which I've covered recently....

Back to the ears - and no, not whale ears either. Apparently this is not the first time that the presence of inner ear disorders have been linked to behavioural issues as per the description from other media on this research (see here). The long-and short of the paper was the study of mice who were bred to carry a mutation in the Slc12a2 gene (see here for more information about the gene) involved with the transportation and reabsorption of sodium and chloride ions. Said mice with genetic mutation in the inner ear were observed to show "increased locomotor activity".

It was then a case of finding out why this genetic glitch produced such effects which eventually led to a part of the brain dealing with motor output: the striatum and the over-production of two proteins (pERK and pCREB) linked to the control of some important neurotransmitters: glutamate and dopamine (and GABA). Confirmation of these proteins as being involved also led to speculation that something could be done about the behaviours noted in their mouse model as per the use of a pERK inhibitor: SL327 wherein "Hyperactivity was remedied by local administration of the pERK inhibitor SL327".

I'll reiterate that as with many experimental studies these days involving knocking out genes and looking at the effects of genetic mutations, this was a study of mice and so one needs to be cautious about making too many sweeping generalisations to humans and the particular complexities that we have. I note that at least one quite prominent ADHD researcher (whose work has appeared previously on this blog) has cautioned against assuming that all ADHD is just a one gene disorder, much in the same light that autism is seemingly getting past that research hurdle too (see here).

A little light background reading on the Slc12a2 gene reveals some other interesting factoids. Schizophrenia for example, has more than one mention when it comes to looking at the expression of the gene; mainly tied back to that GABA link (see the paper by Hyde and colleagues** for example). Panichareon and colleagues*** (open-access) also detailed some potential connection between gene polymorphisms as being associated with cases of schizophrenia.

Taking into account one of the synonyms for the gene - NKCC1 - I note some mention of the drug bumetanide (see this previous post) as being involved (see here****) which 'potentially' suggests some cross-over into cases of autism too. That and the fact that hyperactivity as part of the diagnosis of ADHD may be part of the ESSENCE of at least some autism, although I hasten to add that the Antoine work did not explicitly mention autism. Ears and hearing, have been talked about previously on this blog with autism in mind though (see here and here).

I suppose the thing that really fascinates me about this work is the link between physical presentation and behavioural presentation not necessarily just exclusive to the brain in terms of origin. To me at least, it's of a similar ilk to other research; for example the observation that lower airway abnormalities might be linked with autism (see here) which still requires some follow-up. A real mind-body or body-mind connection you might say?

Some music. How about The Pixies and Here Comes Your Man.

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* Antoine MW. et al. A Causative Link Between Inner Ear Defects and Long-Term Striatal Dysfunction. Science. 2013; 341: 1120-1123.

** Hyde TM. et al. Expression of GABA signaling molecules KCC2, NKCC1, and GAD1 in cortical development and schizophrenia. J Neurosci. 2011 Jul 27;31(30):11088-95.

*** Panichareon B. et al. Association of CTXN3-SLC12A2 polymorphisms and schizophrenia in a Thai population. Behav Brain Funct. 2012; 8: 27.

**** Lemonnier E. & Ben-Ari Y. The diuretic bumetanide decreases autistic behaviour in five infants treated during 3 months with no side effects. Acta Paediatr. 2010 Dec;99(12):1885-8.

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Antoine MW, Hübner CA, Arezzo JC, & Hébert JM (2013). A causative link between inner ear defects and long-term striatal dysfunction. Science (New York, N.Y.), 341 (6150), 1120-3 PMID: 24009395

More sulphate and autism research (at last)

I've mentioned my interest in sulphation (sulfation) and autism in previous posts and how it always seemed like a real shame that the work of Rosemary Waring and others was never really followed up with any great enthusiasm.

To those who might not know about the whole autism-sulphate story, it goes something like this: higher levels of urinary sulphate (and related metabolites) detected accompanied by increased protein excretion in autism compared to controls. Plasma levels of sulphate (sulfate) by contrast tended to be reduced. Ergo, lots of dumping of sulphate in urine and reduced circulating sulphation capacity with some potentially important implications for mucoprotein sulphation and indeed the metabolism of certain drugs.

Enter then a study by Francis Bowling and colleagues* which (thankfully) did look at sulphation with autism in mind and indeed some interesting observations based on genetic sequence variants to a gene involved with renal sulphate transportation, NaS1, disruption to which might have some important implications**. I might add that I think this latest research is an extension to that presented on other occasions by the authors as per this abstract from the 2010 Australian Physiological Society meeting.

The details:

• Based on a sample of 23 people diagnosed with an autism spectrum disorder (ASD) meeting ADOS criteria, several measures were taken or calculated including: (i) levels of creatinine-adjusted sulphate in plasma and urine, (ii) the calculated fractional excretion index (FEI) of sulphate (based on urine and plasma levels) and (iii) the frequency of two separate genetic point mutations affecting NaS1 function based on analysis of the NaS1 gene (SLC13A1) located on an interesting chromosome (7) for autism.
• Results: the normal FEI sulphate values based on other data, was estimated as falling between the range 0.17  - 0.34. Eleven participants presented with elevated FEI sulphate (greater than 0.35). Importantly, no specific clinical features seemed to correlate with FEI sulphate (so things like comorbid gastrointestinal symptoms, self-injurious behaviour, seizures, etc.). That being said they did pick up one child with nephrolithiasis (kidney stones) "due to cystinuria" and another child with "probable mitochondrial disruption".
• Genetic screening for the two variants - one of which (R12X) leads to a complete loss of sulfate transport function - was undertaken and lo and behold, mutation in one or both of the genes seemed to be linked to those elevated FEI sulphate results. Indeed an individual with the highest FEI sulphate reading (0.50) carried both mutations whilst those with no mutation generally showed the lowest FEI sulphate readings.

Quite rightly the authors are pretty buoyed by their results, concluding that their data "may explain the abnormally low sulphonation capacity previously reported in some autistic individuals". I have to say that looking at their data on SLC13A1 genotypes (Table 2 and Figure 1b if you are looking at the full-text), there is a very definite pattern emerging based on variants which is hard to question, bearing in mind the relatively small participant group. Another potential inborn error of metabolism linked to autism?

I suppose the next question is what can be done about this issue and if left as is, what is the long term prognosis of poor sulphate transporter function. With no medical advice given or intended, previously I've talked about efforts to supplement with things like MSM with autism and other conditions in mind, and whether such regimes will actually produce tangible effects on both the supply of sulphate and its usage. I'm going to have do a little bit more reading on the function of NaS1 before commenting any further it has to be said, given that just pumping sulphate in might not necessarily be the best strategy here. I'm also reminded of the emerging work being done on oxalates and autism as also potentially being relevant here.

The Bowling paper is an important one for quite a few reasons outside of just reinvigorating research into sulphation and autism. The results reiterate: (a) that universal genetic (and environmental) findings across all autism(s) are likely to be very few and far between, if any at all, and confirms the move towards studying endophenotypes, and (b) how marrying the disciplines of genetics and biochemistry together using something like a more systems-biology approach, might yet yield some interesting observations regarding the condition(s). Oh and again, that inborn errors of metabolism might actually be quite important to some cases of autism.

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* Bowling FG. et al. Plasma and urinary sulfate determination in a cohort with autism. Biochem Genet. October 2012.

** Lee S. et al. Disruption of NaS1 sulfate transport function in mice leads to enhanced acetaminophen-induced hepatotoxicity. Hepatology. 2006; 43: 1241-1217.

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Bowling, F., Heussler, H., McWhinney, A., & Dawson, P. (2012). Plasma and Urinary Sulfate Determination in a Cohort with Autism Biochemical Genetics DOI: 10.1007/s10528-012-9550-0

Kum-ba-arbaclofen

I assume quite a few people have already read the various reports about STX209* (most definitely not to be confused with ED-209) otherwise known as arbaclofen with Fragile X syndrome in mind. Hailed as 'The First Drug that Could Ease Social Withdrawal in Autism' according to one news source, the headlines are based on the results of this study by Elizabeth Berry-Kravis and colleagues** complemented by this study by Henderson and colleagues*** published on the same day in the same journal.

OK reverse please.

Seaside Therapeutics, yet another pharmaceutical company with autism or autistic behaviours in their sights, had been working for quite a while now on a new formulation, a derivative of baclofen, itself a derivative of everyone's favourite inhibitory neurotransmitter, GABA - gamma aminobutyric acid (see here for a description) as a possible therapeutic for certain behaviours associated with autism. Baclofen is normally indicated for the treatment of spasticity resulting from conditions like multiple sclerosis and cerebral palsy.

GABA is the yin to glutamate - an excitatory neurotransmitter - the yang. The balancing act between excitatory and inhibitory neurotransmitters has, on more than one occasions, been suggested to be slightly skewed in some cases of autism (and other conditions and states) as glutamate takes centre stage.

So then STX209 is, as its chemical relations are, recognised as an agonist for GABAB receptors which in turn inhibits the release of glutamate (and aspartate). You can perhaps see therefore where the interest lies.

I know quite a lot of the press around STX209 has focused on autism, but to be more accurate, Fragile X syndrome  - which presents with autistic features - is the target patient group based on the trials completed so far. The suggestion being that the primary mutation associated with Fragile X syndrome - FMR1 gene - has some knock-on effects**** including "activation of mGluR5, a metabotropic glutamate receptor". Avid watchers of the autism research landscape might remember mGluR5 as being a target for other pharmaceutics with autism/autistic characteristics in mind, including another interesting compound, GRN-529***** (at least in mice).

So:

• The recent trial of STX209** relied on human participants rather than mice, 63 of them in all, mostly male and all carrying "a full mutation in the FMR1 gene". 
• Randomised, double-blind, placebo-controlled (all the things that science really likes to hear about) was the study design and outcome-wise, the Aberrant Behavior Checklist (ABC) and the Vineland Adaptive Behaviour Scales (VABS) are listed.
• The primary endpoint of the study was a focus on irritability, which actually didn't come up trumps over placebo. 
• Having said that, there were some significant positive effects noted on areas of social avoidance in the treatment group acknowledging that the absolute number of participants in the study was relatively small and actually got even smaller following some post-hoc analysis.
• Importantly also, a few side-effects were noted, particularly in the treatment group including URTIs (13%) and headaches (8%).

I note that Seaside Therapeutics have quite recently agreed some terms over STX209 with pharmaceutical giant Roche which suggests to me that things might potentially get quite big for arbaclofen rather quickly. By saying that, I'm not in anyway endorsing the medicine. Quite a bit more data is required on long-term safety, efficacy and best responder characteristics.

With that in mind, I'll be quite interested to see the results of further trials of STX209 as are apparently planned and/or getting underway including a trial specific to children and young adults diagnosed with an autism spectrum disorders (see here). As to the mechanism of STX209 to GABAB receptors to improving social avoidance... more investigation needed I assume.

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* Hopkins CR. ACS Chemical Neuroscience Molecule Spotlight on STX209 (Arbaclofen). ACS Chem Neurosci. 2011; 2: 381.

** Berry-Kravis EM. et al. Effects of STX209 (Arbaclofen) on neurobehavioral function in children and adults with Fragile X Syndrome: a randomized, controlled, phase 2 trial. Science Translational Medicine. 2012; 4: 152ra127

*** Henderson C. Reversal of disease-related pathologies in the Fragile X mouse model by selective activation of GABAB receptors with arbaclofen. Science Translational Medicine. 2012; 4: 152ra128

**** Dölen G. et al. Correction of fragile X syndrome in mice. Neuron. 2007: 56: 955-962.

***** Silverman JL. et al. Negative allosteric modulation of the mGluR5 receptor reduces repetitive behaviors and rescues social deficits in mouse models of autism. Science Translational Medicine. 2012; 4: 131ra51.

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Elizabeth M. Berry-Kravis, David Hessl, Barbara Rathmell, Peter Zarevics, Maryann Cherubini, Karen Walton-Bowen, Yi Mu, Danh V. Nguyen, Joseph Gonzalez-Heydrich, Paul P. Wang, Randall L. Carpenter, Mark F. Bear, & Randi J. Hagerman (2012). Effects of STX209 (Arbaclofen) on neurobehavioral function in children and adults with Fragile X Syndrome: a randomized, controlled, phase 2 trial Science Translational Medicine DOI: 10.1126/scitranslmed.3004214

Forgotten research - sulphation and autism

There is a scene in the film Toy Story 2 where one of the main protagonists, Jessie (the yodelling cowgirl) sings about her unrequited love for her owner as she falls off and slips underneath her owner's bed and is quickly covered in dust and forgotten about. Her normally happy face turns to a sad frown.

Life is like that sometimes. Fashions come and go. Research is like that also; passing phases where concept A or compound B or intervention C wax and wane, wax and wane. Why do they wax and wane? Interest rises and falls; as does funding, as does expertise. For some things the evidence base, whilst initially promising, turns out not to be the heralded thing it was initially suggested to be. Others just fall underneath the bed and gather dust. One area that has gathered quite a bit of dust is the potential role of sulphate (sulfate) in autism spectrum conditions.

Quite a good description of the chemistry of sulphate can be found here. Many people may more readily know about sulphate in one of its more common uses, magnesium sulphate or Epsom salts, where for many thousands of years, people have bathed in Epsom salts in an attempt to 'cure' all kinds of ills.

In autism research, a possible role for sulphate has been discussed for many years. Those in the know will know that when you talk about sulphate and autism, one name comes up time after time: that of Dr Rosemary Waring, now retired, but previously of the University of Birmingham. Rosemary published extensively on a potential role for sulphate in autism (and other things) - some of her main works can be seen here, here and here. I had the previlige of working with her on a few occasions in connection to our creatinine work in autism.

The main findings on sulphate and autism suggested higher levels of urinary sulphate (and related metabolites) accompanied by increased protein excretion in autism compared to controls. Plasma levels of sulphate by contrast tended to be reduced. These issues seemed to be more relevant to those more severely affected by autism. Susan Owens has written a lot about this also.

What does this all mean? Well, our sulphation pathway is used for lots of different things. It helps us to get rid of various 'used' neurotransmitters, it helps maintain gut structural integrity via sulphated glycoproteins and it helps in the release of various digestive enzymes, to name but a few uses. In short, its pretty important. Sulphate is also pretty important when it comes to metabolising certain types of drugs.

Following a period of about 10 years where nothing further really happened with regards to sulphate research in autism, the recent paper by Yap and colleagues reignited some interest with their finding of increased urinary taurine (linked to alteration in cysteine metabolism - note this is not the same as cystine) which I posted about.

I was always struck by the significance and consistency of the original results obtained in the area of sulphate and autism. Adhering to my matra that science is about probability not absolutes, the significance (p) values obtained during some of the studies were impressive (p<0.001) and illustrated potentially how important this area of research could be.

On the basis of such issues with sulphate for some cases of autism, several people have explored the option of trying to replace potentially low plasma sulphate levels. Taking sulphate-complexes orally is not recommended; Epsom salts are quite a good laxative and other sulphate-complexes are used for things like tenderising meat. Given such issues, the use of Epsom salt baths have been suggested to offer an alterative for potentially increasing sulphate levels via transdermal passgae of sulphate across the skin (I am not recommending this by the way). As to the potential effects on the presentation of autism, I am yet to see any results from published clinical trials to corroborate the anecdotal reports of positive changes with some people with autism (of which there are quite a few).

So there we have it. Underneath the bed, a dusty little box of research on sulphate and autism. Going back to our Toy Story character Jessie, viewers of the film will know that at the end, like all good Disney productions, Jessie finds a new home with Woody, Buzz and the other toys complete with the name 'Andy' inscribed on the sole of her boot. Will the same happen to sulphate?