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Friday 6 February 2015

Tuning GABAa receptors, plus Oxytocin

Today’s post will hopefully not get too complicated.

As has been mentioned in this blog, and also at leading institutions like MIT, it does seem possible to fine-tune certain receptors in the brain that have become dysfunctional in autism.  In the case of MIT they were “tuning” a receptor called mGluR5, which they suggested was either hypo or hyper, in other words too much or too little, depending on what the underlying disease variant was.


This was done with something called an allosteric modulator, either a positive one called PAM, or a negative one called NAM.

They found that a particular glumate receptor, called mGluR5, was dysfunction in many autism-like conditions.  But the nature of the dysfunction varied, so different people would require different treatments to return the receptor performance back to normal (top dead center).   So it really becomes like tuning your car engine. 
As I have progressed in my review of the literature it becomes clear that numerous receptors are “out of tune”; so a better analogy is tuning something like a piano.

  



"Tuning" the shape (but not number) of dendritic spines also appears not to be as fanciful as it sounds.


Back to GABAA

Regular readers will know that one of the key dysfunctional receptors in autism is called GABAA.




This subject is very complicated.  In effect what appears to have happened in autism is that the neurons have not matured as they should, and so GABAA receptors continue to function in their “normal” immature state.  The concentration of chloride remains high since the NKCC1 transporter continues to exist, whereas KCC2/3 should have developed.  The result is that when the receptor is stimulated, instead of causing an inhibitory/calming effect it causes an excitatory effect.





This is fortunately treatable by inhibiting the flow of chloride into the cells, through NKCC1, using a drug called Bumetanide.

However this is not the end of the story.


At least 11 binding sites on GABAA receptors

As you can learn from Wikipedia:-


The active site of the GABAA receptor is the binding site for GABA and several drugs such as muscimol, gaboxadol, and bicuculline. The protein also contains a number of different allosteric binding sites which modulate the activity of the receptor indirectly. These allosteric sites are the targets of various other drugs, including the benzodiazepines, nonbenzodiazepines, barbiturates, ethanol, neuroactive steroids, inhaled anaesthetics, and picrotoxin, among others.

We are particularly interested in the allosteric binding sites.
The only one that is usually referred to, in any depth, is the site for benzodiazepines, but there are at least 11 different binding sites.

Abstract
gamma-Aminobutyric acid (GABA)a receptors for the inhibitory neurotransmitter GABA are likely to be found on most, if not all, neurons in the brain and spinal cord. They appear to be the most complicated of the superfamily of ligand-gated ion channels in terms of the large number of receptor subtypes and also the variety of ligands that interact with specific sites on the receptors. There appear to be at least 11 distinct sites on GABAA receptors for these ligands.




These sites include:-

·        GABA Binding Site
·        Benzodiazepine Binding Site
·        Neurosteroid Binding Site
·        Convulsant Binding Site
·        Barbiturate Binding Site
·        b Subunit Binding Site(s)


In an earlier post I highlighted the discovery by Professor Catterall, that tiny doses of a particular Benzodiazepine drug called Clonazepam had a strange effect on the GABAA receptor.

Clonazepam is a known Positive Allosteric Modulator (PAM) of the GABAA site.  In mature neurons it amplifies the calming effect when the GABA binding site is stimulated.  In mouse models of autism (we assume therefore immature neurons)   where GABA is still excitatory, the tiny dose seemed to switch it to inhibitory.

This suggests a new function, rather than a PAM, the effect was to invert the function entirely.

Now it appears that similar things may indeed also be possible at some of the other 9+ binding sites (I exclude GABA Binding Site itself)

As complicated as this subject may sound, it actually gets even more complicated since the GABA receptors are made up of sub-units.  It appears that mutations in these subunits may be a cause of some epilepsies and, I propose, some “oddities” in autism.

Recent studies have again shown that many genetic dysfunctions found in autism relate to GABA, this short article is not so recent, but gives a nice summary:-


GABA is the major inhibitory neurotransmitter in the brain. It essentially acts as a brake for brain activation. Several aspects of GABA regulation have been linked to ASD, from early brain development to adult brain function.
Variations in GABA receptor subunits have been strongly associated with ASD. GABA receptors come in two major forms: fast, “ionotropic” GABAA receptors let negatively charged chloride ions flow into the neuron, and slow, “metabotropic” GABAB receptors produce chemical messages inside the neuron. GABAA receptors, the most common form in the brain, contain five subunits that shape their properties. Genome-wide association studies have linked the GABAA receptor subunit genes GABRA4 (α4 subunit), GABRB1 (β1 subunit), and GABRB3 (β3 subunit) to autism.[1][2] In addition, deletion of a chromosomal region that contains a cluster of a variety of GABA receptor genes (region 15q11-13) causes Angelman Syndrome.[3][4]
Genes controlling the development of GABA-releasing neurons have also been associated with ASD. Autism-linked variations in the ARX and DLX family of transcription factors interfere with proper expression of GABA.[5][6][7] Absence of such GABA-releasing neurons would negatively affect early brain development as well as adult brain stability.

Notably, variations in other ASD-linked genes affect GABA signaling. New evidence shows that the gene MECP2, the mutation of which causes Rett Syndrome, is critical for normal function of GABA-releasing neurons.[8] When MECP2 expression was blocked in GABAergic neurons of mice, GABA expression and release were reduced and the mice exhibited autistic behaviors.

ASD is a complex disorder that is likely to be caused by a combination of mutations in a variety of genes. GABA receptors are a promising therapeutic target because of their important role in monitoring brain excitation. Identification and exploration of autism-linked mutations in other GABA-related genes could shed light on the pathogenesis of autism.


Over to Switzerland

At the University of Bern a small research group is looking  at the world of  GABAA receptors, here is what they say:-

“Many scientists and companies are put off by the complexity of the field of GABAA receptors, but it is exactly this complexity that offers numerous possibilities of fine-tuned pharmacological interventions.” 


Here is one of their recent papers, that shows both what is known and how very much remains unknown.




Ion Conductance
The GABAA receptors are generally GABA-gated anion channels selective for Cl ions, with some permeability for bicarbonate anions (49). Exceptionally, in C. elegans, a cation-selective GABA-gated channel has been discovered (50). Excitatory neurotransmitters increase the cation conductance to depolarize the membrane, whereas inhibitory neurotransmitters increase the anion conductance to tendentially hyperpolarize the membrane. However, if the gradient for Cl ions decreases due to down-regulation of KCC2 chloride ion transporters, opening of GABAA receptors may cause an outward flux of these anions, leading to depolarization of the membrane and thereby to excitation. This phenomenon has been implicated in neuropathic pain (51). During early development (52) and in neuronal subcompartments (53), GABA similarly confers excitation. 
Although it is relatively simple to address questions at the level of individual receptor subunit isoforms, we can only speculate how many GABAA receptors are expressed in our brain and what their subunit composition is, not to mention subunit arrangement.


Conclusions
Many scientists and companies are put off by the complexity of the field of GABAA receptors, but it is exactly this complexity that offers numerous possibilities of fine-tuned pharmacological interventions.

It may be anticipated that genetic alterations of subunits of the GABAA receptor affect any of the above mentioned processes and thereby contribute to inherited human diseases. A start has been made with the analysis of point mutations that cause epilepsy






Why is all this relevant ?

We have in recent posts discovered that at least two anti-convulsants (carbamazepine and phenytoin) appear to modulate GABAA receptors in unexpected ways when given in tiny doses.

We also found out that valproate also seems to possess such qualities.  The exact mode of action of valproate is not known and perhaps it also acts a modulator of one of the many binding sites on the GABAA receptors.

We do think that valproate is working somehow via GABA.



It turns out that Carbamazepine has also been shown to potentiate GABA receptors made up of alpha1, beta2, and gamma2 subunits.

I have already established that the effect of tiny doses of Valproate is not the same as tiny doses of Clonazepam.

The next step would be to look at the effect of tiny doses of carbamazepine, phenytoin and potentially anything else that modulates those mysterious  GABAAsites.  They are clearly all there for a reason.  It seems that their role goes beyond just the allosteric modulation (amplification/reduction) of GABA’s effect.  It is likely much more subtle and they affect emotional behaviour.

Given the difficulty/impossibility of research on human brains, in the end we may need to revert to the medical world’s often used “scientific” discovery methods known as trial and error, and stumbled upon.

For the moment that will be left to Professors Sigel and Catterall and their mice, and Dr Bird, in Australia, with his human subjects.




Oxytocin and Bumetanide share the same mode of action in autism


Whilst on the subject of GABAA, I should come back to Oxytocin.



The conclusion of this Ben-Ari paper from last year is that Oxytocin and Bumetanide share the same effect in autism; they lower the level of chloride within the neurons and help switch GABA back to inhibitory.

It seems that oxytocin from the mother may be the signal to the developing brain to lower Cl levels.  Oxytocin has many other functions in the body.

Small doses of oxytocin/Syntocinon, have been shown to be effective in some people with autism.  One reader from Portugal has written on this blog how effective it has been in his young son.

Oxytocin/Syntocinon is not available everywhere, but is being reintroduced to the US.



I am wondering if in some people, who are not responders, bumetanide/oxytocin lowers the level of chloride, but not enough to show any benefit.  People using Bumetanide, which has a short half-life, comment that the effect fades through the day and that splitting the same daily dose 3 times a day is beneficial over 2 times a day.  This might suggest that combining Oxytocin with Bumetanide might give better results, by maintaining the downward pressure on chloride levels and keeping GABA more inhibitory and for longer.

In the longer term, an analog of Bumetanide is needed without the diuretic effect and with a delayed release, to maintain a constant effective level.  This is known to the researchers, but would require a big financial investment.

Larger doses of oxytocin are likely to produce effects elsewhere in the body.

If anyone tries the combination of Bumetanide + oxytocin, let me know.





4 comments:

  1. Bumetanide for autism: more eye contact, less amygdala activation
    https://www.nature.com/articles/s41598-018-21958-x

    I will be ordering bumetanide soon thats for sure!

    ______

    By the way considering the day after drinking alot of alcohol feeling better, this seems to have a huge impact on GABRA4 and GABRG2 up and downregulation, there are dozens of studies showing that alcohol does this.

    The alpha4 receptor strongly controls neurosteroids (and we all know about the cholesterol problems in autism, including the impaired ability to form pregnenolone from cholesterol and a bunch of others). Im pretty sure progesterone/allopregnenolone therapy could be used in some cases as these affect gaba expression aswell.

    Neurosteroid regulation of GABA(A) receptors: Focus on the alpha4 and delta subunits.
    https://www.ncbi.nlm.nih.gov/pubmed/17512983

    "A number of laboratories have demonstrated that both chronic administration of THP as well as its withdrawal transiently increase expression of the alpha4 subunit of the GABAR in several areas of the central nervous system (CNS) as well as in in vitro neuronal systems. Receptors containing this subunit are insensitive to benzodiazepine (BDZ) modulation and display faster deactivation kinetics, which studies suggest underlie hyperexcitability states. Similar INCREASES IN ALPHA4 EXPRESSION ARE TRIGGERED BY WITHDRAWAL from other GABA-modulatory compounds, such as ETHANOL AND BDZ, suggesting a common mechanism."

    Neurosteroid regulation of oxytocin and vasopressin release from the rat supraoptic nucleus.
    https://www.ncbi.nlm.nih.gov/pubmed/12588901

    Acute alcohol withdrawal upregulates alpha4 receptors, BOOM!:
    https://en.wikipedia.org/wiki/Alcohol_withdrawal_syndrome

    "During acute alcohol withdrawal, changes also occur such as upregulation of alpha4 containing GABAA receptors and downregulation of alpha1 and alpha3 containing GABAA receptors."

    Alpha4-containing GABAA receptors in the nucleus accumbens mediate moderate intake of alcohol. (and thus hedonic tone)
    https://www.ncbi.nlm.nih.gov/pubmed/19144854/

    Association between GABA(A) receptor subunit polymorphisms and autism spectrum disorder (ASD)
    https://www.psy-journal.com/article/S0165-1781(15)00537-5/fulltext

    "Haplotypes: markers rs1912960 (GABRA4) and rs211037 (GABRG2) overrepresented in ASD"

    ReplyDelete
  2. HSF1/HSP70 (Sulforaphane anyone???) upregulates gaba a4 receptors?!!
    Alcohol regulates gene expression in neurons via activation of heat shock factor 1.
    https://www.ncbi.nlm.nih.gov/pubmed/18032669

    "To elucidate these mechanisms, we investigated GABRA4, a neuron-specific gene that is RAPIDLY AND ROBUSTLY ACTIVATED BY ALCOHOL (10-60 mM),both in vitro and IN VIVO. Here we show that alcohol can activate elements of the heat shock pathway in mouse cortical neurons to enhance the expression of Gabra4 and other ARGs"

    "The reduction of HSF1 levels using small interfering RNA prevented the stimulation of Gabra4 and Hsp70 by alcohol and heat shock. Microarray analysis showed that many ARGs contain ARE-like sequences and that some of these genes are also activated by heat shock. We suggest that alcohol activates phylogenetically conserved pathways that involve intermediates in the heat shock cascade and that sequence elements similar to the ARE may mediate some of the changes in gene expression triggered by alcohol intake, which could be important in a variety of pathophysiological responses to alcohol."

    For Peter:

    Another paper showing the overlap between cholesterol/steroid/neurosteroid synthesis and autism/brain function (SLOS also discussed)

    Hypothesis: The Role of Sterols in Autism Spectrum Disorder
    https://www.hindawi.com/journals/aurt/2011/653570/


    I will check my 23andme data later for the gabra4 snp, im 99% sure I have this mutation and that is why acute alcohol withdrawal seems to help me.

    ReplyDelete
  3. I would love to see some studies if bumetanide affects GABRA4, I did an extensive search but could not find anything

    ReplyDelete

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