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Monday 11 January 2016

The GABA Switch, Altered GABAa Receptor subunit expression in Autism and Basmisanil





In today’s post I intended to dig a little deeper into the GABA switch, which appears to underlie much autism, schizophrenia, epilepsy, even Down Syndrome and, not to forget, many mood disorders.  

Once you start digging, it is rather hard to stop.

There is literature on the subject, but very little (almost none, really) looks at the big picture of what is going on.  It is the big picture that matters.





The GABA switch(es); but how many are there?


The post starts out relatively simple, but then it does get complicated, because I discovered a lot interesting avenues exist, that seem to have been completed ignored by autism research.  It seems Down Syndrome researchers are better informed.  

So if you make it to the end of this post, you will have done well.

It seems that there are tens, if not hundreds, of possible ways to repair the faulty GABA switches.  It would very much become a case for personalized medicine, correcting the precise dysfunctions, without disturbing anything else.  Each case will be slightly different.

Back to the simple part.

During very early development certain changes in the brain are expected to occur, that control how the GABA neurotransmitter functions.  If they do not all occur, some of the following may occur:-

·        Autism
·        Epilepsy
·        Mental retardation / Intellectual Disability; including the MR/ID in Down Syndrome
·        Mood disorders (anxiety, depression etc)

If later in life, after brain maturation, these same changes occur the following may occur:-

·        Schizophrenia
·        Bipolar
·        Other mood disorders (anxiety, depression etc)
·        Epilepsy

There are at least two distinct processes involved, both can be considered as part of the GABA switch. It is likely that two further process are involved, but they have not yet been adequately researched.


1.     The lowering of intracellular chloride levels

This has been very deeply documented already in this blog.  If the GABA switch has not been “flipped”, we have overexpression of a cotransporter NKCC1, which overwhelms the effect of another called KCC2 and this results in elevated intracellular chloride levels.  This then prevents GABA signaling switching from excitatory to inhibitory.  This creates an excitatory/inhibitory imbalance and neurons fire when they should not.  This disables cognitive function and creates a tendency towards pre-epilepsy and then epilepsy.

The neurons never reach their expected mature state.


2.     Change in GABAA-receptor subunit expression

We have already seen in this blog, that drugs that positively modulate the α2 and α3 subunits of GABAA receptors, like low-dose clonazepam, rescue some aspects of autism.  This is Professor Catterall’s research.  As far as I can see, he did not really explain the big picture behind this.

We saw in early posts that the composition of the GABA receptor vary over time. Remember there are 5 sub-units in each receptor.  These changes in composition of the receptor directly affect people’s mood and behavior.





Five subunits can combine in different ways to form GABAA channels. The minimal requirement to produce a GABA-gated ion channel is the inclusion of both α and β subunits, but the most common type in the brain is a pentamer comprising two α's, two β's, and a γ (α1β2γ2)
In humans, the units are as follows:
·         six types of α subunits (GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6)
·         three βs (GABRB1, GABRB2, GABRB3)
·         three γs (GABRG1, GABRG2, GABRG3)
·         as well as a δ (GABRD), an ε (GABRE), a π (GABRP), and a θ (GABRQ)


What appears likely to me is that this variation in subunit expression both over time and throughout different parts of the brain is really just another part of the GABA switch.

It appears that in autism the α3 subunit is under-expressed.  In its place it appears we have the α5 subunit over-expressed.  This lowers your IQ.

This under-expression of the α3 subunit has been compensated for by Catterall by using a positive allosteric modulator.

In effect the α5 subunit is dominating, where it should not be and this presents itself as autism.

There is research showing how numerous influences can affect the sub unit expression.  Some are transitory and reversible, but others can be permanent.  It is proposed that, when permanent, the underlying change is epigenetic, when “tags” are placed on perfectly functional genes that either turn them on or off.  One example resulting in permanent change is stress. 

Repeated neonatal handling with maternal separation permanently alters hippocampal GABAA receptors and behavioral stress responses



We will focus on the reversible changes, since that is the purpose of this blog.

After some digging, I did find a nice graphic that does illustrate the first two elements of the GABA switch.  It is based on mouse research and shows the changes that should happen in the first 20 days of life. 

In the first few days NKCC1 is highly expressed, while KCC2 is weakly expressed. This results high levels of intracellular chloride.  As NKCC1 drops, less chloride enters and so intracellular chloride falls.  So depolarizing GABA (excitatory) becomes hyperpolarizing GABA (inhibitory).

In the twenty days while this is happening, the sub unit structure of the GABAA receptors is also changing.

In some autism neurons remain in their immature state, with NKCC1 highly expressed and so high levels of intracellular chloride and depolarizing GABA (excitatory).

My suggestion is that the programmed changes in sub unit expression may also fail to occur.









3.     Density of GABA A receptors ?

In the literature, when they talk about density of GABA A receptors, they are talking about either an increased or reduce number of receptors at a given location.

The density can vary over time and within different parts of the brain.










Source:  http://www.nature.com/articles/srep16347/figures/1


It appears that in autism, there is a reduced number of GABAA receptor, in other words lower density.

GABAA receptor downregulation in brains of subjects with autism


I am suggesting that the under-expression of GABAA receptor, which appears likely to be linked to disturbed calcium channel signaling, could be considered as the third element of the GABA switch.

In the science jargonVGCC (Voltage Gated Calcium Channel) activation is involved in GABA-induced GABAAR down-regulation”. Which begs the question, what is the effect on GABAA receptor density in humans of blocking VGCCs?

Nifedipine is specifically suggested.  My Polypill already includes verapamil, another blocker of VGCCs.




4.     GABAB subunits ?

There probably is a fourth element of the GABA switch.  If there is, it possibly relates to the GABAB receptor.  GABAB receptors are made up of just two subunits, GABAB1 and  GABAB2. This is much less well researched than GABAA, but from what little there is, it is clear that GABAB1 and GABAB2 receptor subunits expression is disturbed in some epilepsy and after TBI (traumatic brain injury).  TBI is interesting because, in many ways, that is what autism is; just it was not a physical trauma, it was a genetic/environmental trauma.

The expression of GABA(B1) and GABA(B2) receptor subunits in the cNS differs from that in peripheral tissues.

Modification of GABA(B1) and GABA(B2) receptor subunits in the somatosensory cerebral cortex and thalamus of rats with absence seizures (GAERS)





The Implication?  Repair the GABA Switch(es)

Before getting involved in the complexities of the research, we can already draw a nice simple conclusion.

Many types of autism are likely associated with a faulty GABA switch.  So if you want to treat someone’s autism, start by repairing the GABA switch(es).  Just realize there is more than one and so therapies will have to vary.

Now to the science, for those who like to go into details:-
                                                         
This is a good paper that was highlighted earlier by Tyler, a reader of this blog:-



The GABAergic neurons of the thalamic reticular nucleus (nRt) provide the primary source of inhibition within the thalamus. Using physiology, pharmacology and immunohistochemistry in mice we characterized post-synaptic developmental changes in these inhibitory projection neurons. First, at postnatal day 3-5 (P3-5), inhibitory postsynaptic currents (IPSCs) decayed very slowly, followed by a biphasic developmental progression, becoming faster at P6-8, then slower again at P9-11 before stabilizing in a mature form around P12. Second, the pharmacological profile of GABAAR mediated IPSCs differed between neonatal and mature nRt neurons and this was accompanied by reciprocal changes in α3 (late) and α5 (early) subunit expression in nRt. Zolpidem, selective for α1- and α3-containing GABAARs, augmented only mature IPSCs, while clonazepam enhanced IPSCs at all stages. This effect was blocked by the α5-specific inverse agonist L-655,708 but only in immature neurons. In α3H126R mice in which α3 subunits were mutated to become BZ insensitive, IPSCs were enhanced compared to wild type animals in early development. Third, tonic GABAAR activation in nRt is age-dependent, and more prominent in immature neurons, which correlates with early expression of α5 containing GABAARs. Thus neonatal nRt neurons show relatively high expression of α5 subunits which contributes to both slow synaptic and tonic extrasynaptic inhibition. The postnatal switch in GABAAR subunits from α5 to α3 could facilitate spontaneous network activity in nRt that occurs at this developmental time point and which is proposed to play a role in early circuit development.



The following paper really covers the first GABA switch very well:-






The switch from excitatory to inhibitory GABAAR-related effects is closely related to the lowering of [Cl−]i during the course of the development. This latter mainly relies on the differential ontogenic expression of the Na+/K+/2Cl− cotransporter isoform 1 (NKCC1), which uptakes chloride ions [76–78], and the neuronal K+/Cl−cotransporter type 2 (KCC2) [79], which extrudes chloride ions [49, 80]. However, other exchangers can control the chloride gradient as the anion (Cl−–HCO3 −) exchangers, either Na+- independent (AE) or Na+-driven (NDCBE also called NDAE) [81] (NCBE) [82]. AE mediates influx of Cl− while exporting HCO3 −, these exchanges being triggered by intracellular alkalinisation. NDCBE, known as an acid extruder (extrudes H+), moves Cl− out in exchange of HCO3 −, driven by the Na+ gradient [83, 84]. NCBE also lowers [Cl−]i (and [H+]i) while importing Na+ and HCO3 − [82, 85].


This is one of Professor Catterall’s papers we looked at previously





Moreover, autistic-like behavioral impairments can be treated effectively in both BTBR and Scn1a+/− mice by enhancement of inhibitory neurotransmission with low doses of subunit-selective positive allosteric modulators of GABAA receptors containing α2 and/or α3 subunits. Together, our results support the hypothesis that reduced GABAergic inhibitory neurotransmission contributes to autism-associated behavioral and cognitive deficits and suggest that enhancement of GABAergic neurotransmission with next-generation subunit-specific pharmacological agents may be beneficial.


Subsynaptic GABAAreceptor subtypes are composed of two α, two β, and one γ subunit (Fritschy and Mohler, 1995). The action of GABA at these ionotropic receptors is increased through positive allosteric modulation by benzodiazepines, which are used to treat anxiety, insomnia, and epilepsy (Rudolph and Knoflach, 2011). In order to determine whether treatment with a benzodiazepine reverses the constitutively decreased GABAergic inhibitory signaling, we treated C57BL/6J and BTBR hippocampal slices with 0.5 μM clonazepam, a broad-acting, traditional benzodiazepine. These recordings revealed increased spontaneous IPSC amplitude (Figures 1E and 1F) and frequency (Figure S1C) in BTBR slices. In contrast, a significant increase of spontaneous IPSC amplitude (Figure S1I), but no change in IPSC frequency (Figure S1J), was observed in C57BL/6J slices. The increased GABAergic signaling after treatment with clonazepam led to a decrease in frequency of spontaneous EPSCs (Figures 1G and 1H), without change in amplitude in BTBR hippocampal slices (Figure S1D). Interestingly, the frequency of spontaneous EPSC was also decreased by clonazepam (Figure S1K), without change in amplitude (Figure S1L) in C57BL/6J slices. These data support the idea that low-dose clonazepam can reverse the underlying deficit in spontaneous GABAergic inhibitory neurotransmission in BTBR mice.

Rescue by α23-Specific Positive Allosteric Modulators of GABAA Receptors

Diversity of GABA receptor function is conferred by more than 20 different subunits, and receptors with different α subunits play distinct roles in the physiological and pharmacological actions of GABA and benzodiazepines (Fritschy and Mohler, 1995, Harmar et al., 2009, Rudolph and Knoflach, 2011,Rudolph and Möhler, 2004, Smith and Olsen, 1995). We tested the effects of subunit-selective positive allosteric modulators of GABAA receptors on social behavior in BTBR mice and C57BL/6J mice. A low dose of the α2,3-subunit-selective positive allosteric modulator L-838,417 (Löw et al., 2000, Mathiasen et al., 2008) increased social interactions in BTBR mice, with maximal effective dose of 0.05 mg/kg, and the beneficial effect was lost when the dose increased (Figures 4I and S4E). In contrast, L-838,417 did not change the social interaction behavior of C57BL/6J mice (Figure S4I). Moreover, the α1-subunit-selective positive GABAA modulator zolpidem (Mathiasen et al., 2008, Sieghart, 1995) failed to show beneficial effects in BTBR mice and actually aggravated their social interaction deficit at high doses (Figures 4J and S4F). Interestingly, a high dose of zolpidem also impaired social behavior in C57BL/6J mice (Figure S4J). Total movement tended to increase at high doses of L-838,417 (Figure S4G; not significant) but significantly decreased at 0.5 mg/kg zolpidem (Figure S4H). These results indicate that different subtypes of GABAA receptors may have opposite roles in social behavior, with activation of GABAA receptors containing α2,3 subunits favoring and activation of GABAA receptors with α1 subunits reducing social interaction, respectively.
Subunit-selective GABAA receptor modulators may also have an important effect on cognitive behaviors. In the context-dependent fear conditioning test, treatment with 0.05 mg/kg L-838,417 improved short-term (30 min) and long-term (24 hr) spatial memory in BTBR mice (Figure 4K), whereas 0.05 mg/kg zolpidem enhanced short-term memory but not long-term memory (Figure 4L). These data show that α2,3-subunit-containing GABAA receptors may also be important for cognitive behaviors in BTBR mice. The bell-shaped dose-response curves observed for both L-838,417 and clonazepam may explain why high-dose benzodiazepine treatment for prevention of anxiety and seizures has not been reported to improve autistic traits in ASD patients. As illustrated in Figures 4N and 4O, treatment with low doses of L-838,417 also improves social interactions in the Scn1a+/− mice, a model of Dravet syndrome with severe autistic-like behaviors (Han et al., 2012), within a narrow dose range. In contrast, similar treatment with zolpidem is not effective. Altogether, these experiments show that treatment with an α2,3-selective positive allosteric modulator of GABAA receptors is sufficient to rescue autistic-like behaviors and cognitive deficit in both a monogenic model of autism-spectrum disorder and the BTBR mouse model of idiopathic autism.

The following paper highlights disrupted subunit expression in one type of epilepsy:-

Altered thalamic GABAA-receptor subunit expression in the stargazer mouse model of absence epilepsy.


Abstract

PURPOSE:

Absence seizures, also known as petit mal seizures, arise from disruptions within the cortico-thalamocortical network. Interconnected circuits within the thalamus consisting of inhibitory neurons of the reticular thalamic nucleus (RTN) and excitatory relay neurons of the ventral posterior (VP) complex, generate normal intrathalamic oscillatory activity. The degree of synchrony in this network determines whether normal (spindle) or pathologic (spike wave) oscillations occur; however, the cellular and molecular mechanisms underlying absence seizures are complex and multifactorial and currently are not fully understood. Recent experimental evidence from rodent models suggests that regional alterations in γ-aminobutyric acid (GABA)ergic inhibition may underlie hypersynchronous oscillations featured in absence seizures. The aim of the current study was to investigate whether region-specific differences in GABAA receptor (GABAAR) subunit expression occur in the VP and RTN thalamic regions in the stargazer mouse model of absence epilepsy where the primary deficit is in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression.

METHODS:

Immunofluorescence confocal microscopy and semiquantitative Western blot analysis were used to investigate region-specific changes in GABAAR subunits in the thalamus of the stargazer mouse model of absence epilepsy to determine whether changes in GABAergic inhibition could contribute to the mechanisms underlying seizures in this model of absence epilepsy.

KEY FINDINGS:

Immunofluorescence confocal microscopy revealed that GABAAR α1 and β2 subunits are predominantly expressed in the VP, whereas α3 and β3 subunits are localized primarily in the RTN. Semiquantitative Western blot analysis of VP and RTN samples from epileptic stargazers and their nonepileptic littermates showed that GABAAR α1 and β2 subunit expression levels in the VP were significantly increased (α1: 33%, β2: 96%) in epileptic stargazers, whereas α3 and β3 subunits in the RTN were unchanged in the epileptic mice compared to nonepileptic control littermates.

SIGNIFICANCE:

These findings suggest that region-specific differences in GABAAR subunits in the thalamus of epileptic mice, specifically up-regulation of GABAARs in the thalamic relay neurons of the VP, may contribute to generation of hypersynchronous thalamocortical activity in absence seizures. Understanding region-specific differences in GABAAR subunit expression could help elucidate some of the cellular and molecular mechanisms underlying absence seizures and thereby identify targets by which drugs can modulate the frequency and severity of epileptic seizures. Ultimately, this information could be crucial for the development of more specific and effective therapeutic drugs for treatment of this form of epilepsy

This is another good paper, this time looking at how fragile X mental retardation protein (FMRP) may disrupt sub-unit expression and how this appears not only in those with Fragile-X but also in schizophrenia, mood disorders, and autism.  mGluR5 is involved not surprisingly and you may recall that mGluR5 is surprisingly also involved in GERD/GORD/reflux, which affects many people with autism.

GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism.



Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in the brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), the absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems.


The logical next step - Regulation of GABAA Receptor Subunit Expression by Pharmacological Agents


The first thing to note is that, if you are using low dose clonazepam, you are already compensating for the lack of α3 subunits. Clonazepam is a so-called positive allosteric modulator of α3.

Are there further options?  Yes there are very many, but are they safe for long term use?  Most fail this test.

For example, ethanol will down-regulate α5 expression and it appears possible that the right benzodiazepine at the right dose might also achieve this, but much mouse research is contradictory.  We are very lucky that the clonazepam dose is so tiny, it appears to have no side effects whatsoever.  At conventional doses, benzodiazepines do have problems.

Initially I looked at research that sets out very generally to look at what I am interested in.  This yielded some interesting studies, some of which we looked at already when looking at Professor Catterall and Clonazepam.

Then I decided to look at very specific things I want to modulate and I found a great deal more.  It looks like we will be able to borrow a drug being developed for Down Syndrome (Basmisanil / RG-1662).

Numerous substances do affect GABAA receptor subunit expression, but the ones available today are generally non-specific.  They will change things, but will it be for the better?

So there are two approaches:-


·        Roll the dice

Some simple substances are known to affect GABAA receptor subunit expression and some of these substances have already been associated with autism.  It is conceivable that a lit bit, more or less, of one of this might just stir things up so that the end result might be better.

·        Clever approach

The clever approach would make sure changes only affect the specific subunits that need to be modulated.  The chance of success is then very high.  The problem is that this requires waiting for a new drug, currently in phase 2 trials, to complete its approval process 


Rolling the Dice

In the following papers there numerous ideas and some of these that have already appeared in previous posts.  Those ideas include:-

·        Calcium channel blockers, like Nifedipine

They may increase GABA receptor density.

·        Zinc

We saw in research from Taiwan, that it appears there is a problem with zinc in autism and schizophrenia.  It is not a lack of zinc, rather it is in the “wrong” place.  They have a drug, Clioquinol, that can move it to the “right” place, but this drug is not regarded as safe in many countries.

Altered Homeostasis in Autism: Cl-, K+, Ca2+, and quite possibly Zn2+


It claimed that immature neurons are more sensitive to zinc than adult neurons.  Even in adults, autistic neurons remain immature.


·        BDNF

We have already seen that growth factors, including BDNF are disrupted in autism.  Some people with autism have too much BDNF and some too little.
BNDF seems to affect GABA receptor density.


·        Progesterone/Pregnenolone

We know that transdermal Progesterone/Pregnenolone helps many people with autism + anxiety.  We know, from Hardan at Stanford, that high dose of oral Pregnenolone seem to help adults with autism or schizophrenia.

We saw in earlier posts that allopregnanolone possesses biphasic, U-shaped actions at the GABAA receptor, meaning that a tiny dose can have the same effect as a large dose. Giving large doses of a female hormone to young boys does not seem a clever idea, presumably this is why the Stanford trial was on adults.

I did previously suggest small doses of oral Pregnenolone might be worthy of a clinical trial. 

Progesterone/Pregnenolone will change GABAA sub unit expression.


·        We have another mushroom-derived substance called muscimol

“The GABAAergic agonist muscimol increases KCC2 mRNA in male neurons, via activation of voltage sensitive calcium channels and calcium signaling [79, 80]; in contrast, muscimol decreases KCC2 mRNA in female SNR neurons”


So mushrooms just for the boys.

Muscimol is a selective agonist for the GABAA receptors that seems to do some clever things.


For those of you who like “natural” substance, here is a paper for you:-



It does actually mention something very interesting.  We know that ethanol would down regulate α5, but clearly you cannot “treat” a child with alcohol.  It appears that the fragrant components of whiskey, wine, sake, brandy may have the same effect as ethanol, but require only tiny concentrations.  So there may be scope for alcohol as a therapy after all.  Note that propolis is usually sold as a solution in alcohol; this widely given to children.

“Volatile components of alcoholic drinks, such as whiskey, wine, sake, brandy, and shochu potentiate GABA responses to varying degrees (Hossain et al., 2002a). Although these fragrant components are present in alcoholic drinks at low concentrations (extremely small quantities compared with ethanol), they may also modulate the mood or consciousness through the potentiation of GABAA responses after absorption into the brain, because these hydrophobic fragrant compounds are easily absorbed into the brain through the blood–brain barrier and are several thousand times as potent as ethanol in the potentiation of GABAA receptor‐mediated responses (Hossain et al., 2002a).”


The following paper is very extensive, but completely omits some extremely important possibilities I later came across.  It should be a must read for those interested in the science.

Regulation of GABAA Receptor Subunit Expression by Pharmacological Agents





F. Mechanisms Regulating GABAA Receptor Subunit Expression

The large number of GABAA receptor genes and the various types of neurons and glial cells in the brain with different patterns of subunit expression suggest a complex system regulating their transcription

Activity-dependent signaling pathways modulate the function of both transcriptional activators and repressors (West et al., 2002). Calcium is a crucial second messenger in the transduction of synaptic activity into gene expression (Carafoli et al., 2001), and it is involved in the mechanisms of GABAA receptor up- and down-regulation
 It was recently shown that the activation of protein kinase C in primary rat neocortical cultures increases transcription of α1 mRNA via phosphorylation of CREB that is bound to the GABRA1 promoter (Hu et al., 2008). In contrast, activation of protein kinase A (PKA) represses α1 mRNA transcription via inducible cAMP early repressor (ICER) that forms inactive heterodimers with CREB (Hu et al., 2008). Brain-derived neurotrophic factor (BDNF) decreases α1 transcription via activation of the Janus kinase/signal transducer and activator of transcription (STAT) pathway (Lund et al., 2008). BDNF-dependent phosphorylation of STAT3 induces the synthesis of ICER that binds with phosphorylated CREB at the GABRA1 promoter CRE site, thereby repressing transcription (Lund et al., 2008).

In cultured CGCs, BDNF induces α6 mRNA expression and enhances the expression of α1 and γ2 mRNA (Bulleit and Hsieh, 2000). These enhancements are mediated via mitogen-activated protein kinase pathway (Bulleit and Hsieh, 2000). In contrast, in cultured hippocampal pyramidal cells, BDNF reduced cell surface expression of α2, β2/3, and γ2 subunits (Brünig et al., 2001). The results suggest that BDNF affects GABAAreceptor expression in a brain region- and cell-specific manner.


Regulation of GABAA Receptor Expression By Pharmacological Agents

A. Benzodiazepines


Read the full paper!


B. Neurosteroids

5. α5 Subunit.

The expression of α5 mRNA was down-regulated by CE in the cerebral cortex (Mhatre and Ticku, 1992), whereas no effect was found by Devaud et al. (1995)(Table 14). CE did not affect cerebral cortical α5 polypeptide expression (Charlton et al., 1997). Withdrawal from CE down-regulated cortical α5 mRNA expression (Mhatre and Ticku, 1992). CE down-regulated α5 polypeptide expression in the cerebellum (Charlton et al., 1997). In the hippocampus, CE up-regulated α5 mRNA expression, although it had no effect on α5 polypeptide expression (Charlton et al., 1997). Withdrawal from CE did not affect hippocampal α5 mRNA expression (Mahmoudi et al., 1997; Petrie et al., 2001). Long-term ethanol treatment or withdrawal from it did not affect α5 mRNA expression in cultured rat hippocampal neurons (Sanna et al., 2003). The results of studies on the CE effect on α5 subunit suggest brain region-specific modulation of the expression.

Here we look at the down/up regulation of the number of receptors, rather than their substructure.  This was shown to vary in nice chart with green spots earlier in this post. In other words this about modulating receptor density.





Changes in GABA receptor (GABAAR) gene expression are detected in animal models of epilepsy, anxiety and in post-mortem schizophrenic brain, suggesting a role for GABAAR regulation in neurological disorders. Persistent (48 h) exposure of brain neurons in culture to GABA results in down-regulation of GABAAR number and uncoupling of GABA and benzodiazepine (BZD) binding sites. Given the central role of GABAARs in fast inhibitory synaptic transmission, GABAAR down-regulation and uncoupling are potentially important mechanisms of regulating neuronal excitability, yet the molecular mechanisms remain unknown. In this report we show that treatment of brain neurons in culture with tetrodotoxin, glutamate receptor antagonists, or depolarization with 25 mm K+ fails to alter GABAAR number or coupling. Changes in neuronal activity or membrane potential are therefore not sufficient to induce either GABAAR down-regulation or uncoupling. Nifedipine, a voltage-gated Ca2+ channel (VGCC) blocker, inhibits both GABA-induced increases in [Ca2+]i and GABAAR down-regulation, suggesting that VGCC activation is required for GABAAR down-regulation. Depolarization with 25 mm K+ produces a sustained increase in intracellular [Ca2+] without causing GABAAR down-regulation, suggesting that activation of VGCCs is not sufficient to produce GABAAR down-regulation. In contrast to GABAAR down-regulation, nifedipine and 25 mm K+ fail to inhibit GABA-induced uncoupling, demonstrating that GABA-induced GABAAR down-regulation and uncoupling are mediated by independent molecular events. Therefore, GABAAR activation initiates at least two distinct signal transduction pathways, one of which involves elevation of intracellular [Ca2+] through VGCCs.

Given that calcium is a ubiquitous signaling molecule, it seems reasonable that increased Ca2+ alone is not sufficient to mediate the effects of signal transduction pathways initiated by activation of a specific receptor. Studies of hippocampal neurons demonstrate that increases in [Ca2+]i by NMDA receptors or VGCCs initiate distinct signal transduction pathways (Bading et al. 1993; Xia et al. 1996). The route of Ca2+influx appears to influence which signal transduction pathway is stimulated. Compartmentalization of molecules involved in second messenger pathways may also account for the observation that Ca2+ influx in dendrites initiates signal transduction cascades distinct from those triggered in the soma (Ghosh and Greenberg 1995). Recent evidence shows that increases in [Ca2+]i initiate different signaling mechanisms depending on whether the Ca2+ increase occurs in the cytoplasm or in the nucleus (Hardingham et al. 1997).
Our results demonstrate that GABA-induced GABAAR down-regulation and uncoupling are mediated by independent molecular events, indicating that GABAAR activation leads to initiation of at least two distinct signal transduction pathways. We present evidence that VGCC activation is involved in GABA-induced GABAAR down-regulation. Understanding molecular mechanisms of GABAAR down-regulation will clarify the role of GABA-induced changes in gene expression in both normal nervous system function and in neurological disease.


This is another paper on epilepsy.  It is a very good one again talks about zinc.  It also talks about using AE3 as well as NKCC1 for therapeutic intervention.

GABAA Receptors in Normal Development and Seizures: Friends or Foes?







Developmental Changes in GABAA Receptor Structure and Pharmacology

Most studies describing developmental changes in GABAAergic signaling have been done in rats. To better understand how might these reflect changes in humans, it is generally thought that brain development in a postnatal day 8-10 (PN8-10) rat is almost equivalent to a newborn human baby. The infantile stage in rats spans from PN7-21 and is followed by the juvenile stage. Puberty onset in rats occurs at approximately P32-37, whereas adulthood is reached at 2 months [230, 342, 343]. GABA is present in the embryonic neural system from the very early days [105, 162]. In the embryonic rat neocortex, GABA is detected diffusely as early as embryonic day 10 (E10) but after E14 its presence is limited to the subplate, cortical plate, marginal and intermediate zones [105]. In parallel, GABAA receptors are expressed, even before the establishment of GABAergic synapses, to permit the autocrine and paracrine actions of GABA on brain development [164,183, 278]. Regional differences in subunit expression have been reported in rats, with α4, β1, γ1 detected in the premigratory neuroblasts of the ventricular zone [164, 183] and α2, α3, β3, γ2 at the cortical or subcortical plate [164, 183, 190]. The spatiotemporal developmental patterns of GABA / GABAA receptor expression are thought to be important in the orchestration of the normal GABA-related regulation of proliferation and migration or neural and glial progenitors [105]. The high levels of GABA in the early stages of development promote the proliferation of ventricular zone progenitors [105], whereas the subsequent decline and restriction of GABAAergic influence within the outer neocortical layers inhibits proliferation [8,105, 177], enhances migration [20], and may therefore permit further neuronal differentiation. GABAAergic signaling is also important for neuronal survival at this stage [128]. In further support of the importance of GABAAergic signaling for brain development, in utero exposure to GABAA receptor inhibitors decreases the number of parvalbumin-immuno-reactive GABAergic neurons in the striatum, by impairing the survival or differentiation of these neurons [182]. Moreover, focal application of GABAAergic agonists in the cortex of newborn rats may induce abnormal migration and heterotopias [107].
Age-related, species, and region-specific changes, gradual or transient, continue through postnatal development, adulthood and ageing for GABAA receptor subunits like α1, α2, α3, α4, α5, γ1, γ2 [138, 171,214, 255, 260, 340]. Fritschy et al. have proposed that during the early postnatal life, a gradual parallel decrease in α2 / α3 and increase in α1 expression occurs in rat brain [74, 120] (Fig. 11). Similar developmental switch from α2 / α3 to α1 subunit predominance has been observed in mouse superior colliculus [111] and visual cortex [37, 109]. Functionally, the postnatal increase in α1 has been linked to increased sensitivity to neurosteroids [214], zolpidem [111] and benzodiazepines [140], and acquisition of mature type postsynaptic IPSCs with shorter duration [29]. The latter may be important for a brain that learns to respond appropriately to novel patterns of neuronal activation. Using α1 knockout mice, Bosman et al. have elegantly shown that lack of α1 subunits leads to preservation of juvenile, long duration IPSCs and impairs spatiotemporal excitation patterns to local high frequency stimulation in the visual cortex [28, 29]. In the dentate granule cells of the rat hippocampus, the developmental switch from α5 to α1, α4, and γ2 subunits correlates with decreasing sensitivity to zinc and increase in the affinity for benzodiazepines [34, 140]. Sensitivity to zinc is important in the functional regulation of GABAAergic transmission, particularly in immature neurons. Large amounts of zinc can be stored in synaptic vesicles of nerve terminals, as in the hippocampal mossy fibers of the immature hippocampus. Stimulation-dependent zinc release in this system may therefore be useful to keep under control the excessive depolarizing effects of GABA, in a subunit-specific pattern [16, 53, 166, 285,331]. This may be less important in adult neurons, which lose their sensitivity to zinc, as GABAA receptor mediated inhibition is more efficient.
There is though regional specificity of the evolution of these changes [56]. Sex differences in GABAAreceptor subunit expression further increase the diversity. These include increased expression of α1 subunit in the female substantia nigra of infantile and juvenile rats [255] and increased γ1 expression in the male rat juvenile medial preoptic area [219]. At the cellular level, GABAA receptor trafficking also evolves. Early in development and before synaptic integration occurs, receptor complexes can be diffusely expressed at the cell membrane and can be tonically activated in the presence of GABA [61, 172, 177, 236, 311]. As the establishment and differentiation of GABAergic synapses begins, they initially occupy both extrasynaptic and synaptic sites; finally targeting and clustering at synaptic sites and dendritic processes increases with maturation and spontaneous IPSCs can be detected [1, 236, 253].
The temporal, regional, sex, and species specific variability in the expression of these subunits in the brain emphasizes that generalization across brain regions, species, genders, and ages is not possible, but one needs to specifically study each structure, age, and condition independently. To further complicate these studies, handling, caloric restriction, and even swim stress regulate GABAA receptor subunit expression, at times with a lasting effect, suggesting that epigenetic influences may be as important in shaping the GABAAreceptor related differentiation and communication patterns [122,170, 202, 238].

Repeated neonatal handling with maternal separation permanently alters hippocampal GABAA receptors and behavioral stress responses



Differential regulation of KCC2 in neurons with depolarizing or hyperpolarizing GABAAergic signaling.GABAA receptor activation and BDNF increase KCC2 in immature neurons with depolarizing GABAA ergic responses, but decrease it in neurons with hyperpolarizing ...


In addition, the intracellular concentrations of Cl-and HCO3- are regulated by anion exchangers (AE). The sodium independent electroneutral AEs exchange HCO3- for extracellular Cl-, lowering intracellular pH and increasing Cl- [112, 300, 336]. Sodium Dependent Anion (Cl- / HCO3-) Exchangers (NDAE), also called sodium-dependent Cl-/HCO3- exchangers (NDCBE or NCBE) function in the opposite direction increasing intracellular pH and lowering intracellular Cl-[87, 92, 151, 287, 288, 315, 321]. The expression of NCBE precedes KCC2 in the embryonic mouse brain and, unlike KCC2, NCBE is expressed in the peripheral nervous system and epithelial non-neuronal tissues [125].

The GABAAergic agonist muscimol increases KCC2 mRNA in male neurons, via activation of voltage sensitive calcium channels and calcium signaling [79, 80]; in contrast, muscimol decreases KCC2 mRNA in female SNR neurons with hyperpolarizing GABAAergic responses [79]. These indicate that the maturational state of a neuron, as it relates to the mode of GABAAergic signaling, is critical in defining its reaction to stimuli that tend to disturb its GABA-related developmental pathway. 

Role of sex hormones in the sexually dimorphic expression of KCC2 in rat substantia nigra.


KCC2 is a neuronal-specific potassium chloride cotransporter. The level of KCC2 expression is a factor determining whether GABA(A) receptor agonists depolarize or hyperpolarize neurons. Substantia nigra reticulata (SNR) neurons of male postnatal day 15 (PN15) rats have low KCC2 mRNA expression and respond to GABA(A) receptor activation with depolarization and activation of calcium-regulated gene expression. Female PN15 SNR neurons have high KCC2 mRNA expression and GABA(A) receptor agonists cannot activate calcium-dependent signaling processes. We investigate whether sex hormones regulate KCC2 mRNA expression in PN15 rat SNR. Using in situ hybridization, we studied the effects of acute (4 h) or prolonged (52 h) subcutaneous (s.c.) administration of testosterone (100 microg), dihydrotestosterone (180 microg) or 17beta-estradiol benzoate (5 microg) on KCC2 mRNA expression in male and female PN15 rat SNR. Different doses of estradiol (1 and 10 microg s.c., 4 h) were also acutely administered in female PN15 rats. Controls received oil injections. Separate groups of PN15 male rats were pretreated with antagonists of L-type voltage-sensitive calcium channels (L-VSCCs) [nifedipine, 100 mg/kg s.c.] or GABA(A) receptors [bicuculline, 2 mg/kg intraperitoneally (i.p.)] or their vehicles, 30 min before estradiol (5 microg s.c., 4 h). Testosterone and dihydrotestosterone upregulated KCC2 mRNA in both sexes. Estradiol downregulated KCC2 mRNA in males but not in females. Both acute and prolonged hormonal administration had similar effects. In male PN15 SNR, nifedipine and bicuculline decreased KCC2 mRNA acutely and prevented further downregulation of KCC2 mRNA by estradiol. Estradiol therefore downregulates KCC2 mRNA in male PN15 SNR, by interacting with the GABA(A) receptor and L-VSCC signaling pathway.

Stimulation of prolactin and growth hormone secretion by muscimol, a gamma-aminobutyric acid agonist.





Conclusion


As we have seen, the normal function of GABAA receptor-mediated inhibition is governed by several factors, including subunit composition and density of the receptors and in by the appropriate ionic gradient of chloride (Cl-) and finally the release of GABA.

From a therapeutic perspective, the options are numerous and include:-


Modify the Chloride gradient

·        Reduce NKCC1 expression and increase KCC2, thereby making mature neurons

·        Block NKCC1 using bumetanide

·        Use a KCC2 agonist to stimulate KCC2

There are no KCC2 agonists currently available, but they are being developed for the treatment of neuropathic pain.



“KCC2 represents a fresh avenue to pain medication because stimulating KCC2 normalizes endogenous pain inhibition,” said De Koninck. “In normal neurons, Cl– levels are kept very low. Therefore, the effect of KCC2 enhancers will mainly touch on troubled neurons with elevated Cl– levels.”

Besides neuropathic pain, other neurological disorders with imbalances in Cl– homeostasis, like epilepsy, migraine or anxiety, could benefit from KCC2 stimulation. De Koninck plans to study KCC2-targeted compounds in an epilepsy model in which epilepsy-related neurological changes develop before they eventually trigger epileptic episodes. The model will provide insights on the effects of KCC2 agonists on seizure-evoking hyperexcited neurons and on changes in neuronal networks.


Recall the link between fibromyalia and autism?  I suggested this is what happened to females who nearly had autism.  Genetically down regulated KCC2 would be the link.

·        Modulate AE3 or NDAE to extrude  Cl 

This is possible using carbonic anhydrase inhibitors, such as Methazolamide and Acetazolamide  (Diamox)



Modulate subunit structure

·        Change the physical sub structure of GABA receptors to increase expression of α3 and perhaps α2

·        Upregulate the existing α3 receptors using a PAM (positive allosteric modulator) such as low dose clonazepam

·        Perhaps down regulate the overexpressed α5 receptors using a negative allosteric modulator

This might sound rather farfetched, but a chance would have it there are studies that show by down regulating α5 receptors you do indeed improve cognitive function.  We need a sub unit selective inverse agonist  or a Negative Allosteric Modulator.

Perhaps more interesting is that researchers trying to reverse cognitive deficit in Down Syndrome have already focused on down regulating α5 receptors.  They even have drugs in the approval pipeline.

But Down Syndrome is not autism, you are thinking.  But recall that in mouse models, bumetanide reverses cognitive dysfunction in Down Syndrome.

So perhaps the GABA switch is key to Down Syndrome, as well as Autism, Schizophrenia?

So keep your eyes out for news from Hoffmann-La Roche regarding RG1662 / Basmisanil currently in Phase 2 trials for Down Syndrome.


Allosteric Modulation of GABAA Receptor Subtypes: Effects on Visual Recognition and Visuospatial Working Memory in Rhesus Monkeys



 in mice and rats, investigational drugs that are negative allosteric modulators (NAMs) at α5GABAARs improved performance 

L-655,708 enhances cognition in rats but is not proconvulsant at a dose selective for alpha5-containing GABAA receptors.

http://www.ncbi.nlm.nih.gov/pubmed/17046030?dopt=Abstract&holding=npg
The in vitro and in vivo properties of L-655,708, a compound with higher affinity for GABA(A) receptors containing an alpha5 compared to an alpha1, alpha2 or alpha3 subunit have been examined further. This compound has weak partial inverse agonist efficacy at each of the four subtypes but, and consistent with the binding data, has higher functional affinity for the alpha5 subtype.
These data further support the potential of alpha5-containing GABA(A) receptors as a target for novel cognition enhancing drugs.



Another α5 NAM is MRK-016, it also demonstrates nootropic effects, but may be developed as an antidepressant.



Inverse agonists of GABAA α5

·         α5IA
·         Basmisanil (RG-1662, RO5186582): derivative of Ro4938581, negative allosteric modulator at GABAA α5, in human trials for treating cognitive deficit in Down syndrome.[3]
·         L-655,708
·         MRK-016
·         PWZ-029: moderate inverse agonist[4]
·         Pyridazines[5]
·         Ro4938581[6]
·         TB-21007[7][8]





Final thoughts


There really is a lot to digest in this post.  I really did not know where to stop; it could have just kept going. 

For example, the amount of GABA itself should start out high in very early life and then should rapidly fall; this may also have been disrupted in autism. This process appears linked to certain growth factors (bFGF/ FGF2) and perhaps physical growth itself, both of which we know are disrupted in some autism. Recently FGF2 was found to be an endogenous inhibitor of anxiety.  Anxiety is mediated through GABA subunit expression.  It turns out some Mexican doctor is injecting FGF2 into kids with autism.  Other recent research shows that FGF2 can promote remyelination.  I think that demyelination is a likely shared feature of severe autism and mitochondrial disease.  There will be a post on demyelination/ remyelination.

 FGF2 and FGFR1 signaling regulate functional recovery following cuprizone demyelination







Sunday 10 January 2016

Barts MS Blog



Multiple Sclerosis (MS) affects about 2.5 million people in the world and nobody understands what causes it; it might even be a virus.  What is odd is that incidence around the equator is extremely low, but the further away you go the higher it gets; Canada and Scotland having very high incidence.

About 75 million people around the world have some degree of autism.

The Bart American readers will be familiar with


The connection between autism and MS is myelin, or rather the lack of it.  This will feature in an upcoming post.

This post is to highlight what one London hospital does for the MS community. 
  
Barts is short for Saint Bartholomew's Hospital. They have a great scientific blog where doctors and researchers interact with people affected by MS.

It is a great site and has had over 5 million page views.

There is no equivalent anywhere for autism.  Insightful doctors and researchers happily answering the, sometimes repetitive, questions of those affected.  A visitor from another planet might find that strange.

Lisa’s Autism Blog, perhaps?











All is not lost.

Simons' Blog is great, but he has a lot of help.  Paul’s Blog is highly informative and all his own work.










Sunday 3 January 2016

Vitamin A (and ATRA) Upregulate Oxytocin via CD38


 A familiar site to Maja, the confluence of the Sava and the Danube


Today’s post is to document an interesting discovery by Maja, one reader of this blog.  She is just ahead of some Korean researchers, who very recently published a paper in Experimental Neurobiology on the same subject.

Maja noticed that giving a small dose of fish oil produced the same benefits as those often claimed for Oxytocin; she then did some investigation and noted that an enzyme called CD38 upregulates oxytocin in the brain.  The level of CD38 is affected by inflammatory cytokines and certain vitamins.  In particular, all-trans retinoic acid (ATRA) increases CD38. All-trans retinoic acid (ATRA) is made in the body from vitamin A.  ATRA is also called vitamin A acid.

Maja suggested this paper:-



Deficits in social behavior in mice lacking the CD38 gene have been attributed to impaired secretion of oxytocin. In humans, similar deficits in social behavior are associated with autistic spectrum disorder (ASD), for which genetic variants of CD38 have been pinpointed as provisional risk factors. We sought to explore, in an in vitro model, the feasibility of the theory that restoring the level of CD38 in ASD patients could be of potential clinical benefit. CD38 transcription is highly sensitive to several cytokines and vitamins. One of these, all-trans retinoic acid (ATRA), a known inducer of CD38, was added during cell culture and tested on a large sample of N = 120 lymphoblastoid cell (LBC) lines from ASD patients and their parents. Analysis of CD38 mRNA levels shows that ATRA has an upmodulatory potential on LBC derived from ASD patients as well as from their parents. The next crucial issue addressed in our study was the relationship between levels of CD38 expression and psychological parameters. The results obtained indicate a positive correlation between CD38 expression levels and patient scores on the Vineland Adaptive Behavior Scale. In addition, analysis of the role of genetic polymorphisms in the dynamics of the molecule revealed that the genotype of a single-nucleotide polymorphism (rs6449182; C>G variation) in the CpG island of intron 1, harboring the retinoic-acid response element, exerts differential roles in CD38 expression in ASD and in parental LBC. In conclusion, our results provide an empirical basis for the development of a pharmacological ASD treatment strategy based on retinoids.


In December some Korean researchers also suggested that ATRA might be used therapeutically to increase Oxytocin.  Maja discovered that vitamin A can also be used, which makes sense.

The Korean paper reviews the existing literature and clinical trials on oxytocin in autism, and I suggest those interested should read it.

Some people clearly benefit from oxytocin, some do not and some suffer side effects.

In those that benefit from oxytocin, it might be simpler to upregulate the body’s own oxytocin via ATRA, or vitamin A.


Is this proof?

Of course there are other explanations possible for what Maja has noted.  She was using fish oil as a source of vitamin A, so it could be related to the other constituents.

However, I for one think it is highly plausible and does fit nicely with the ideas put forward by the Korean researchers and the earlier paper.


Vitamin A for all?

We know that autism genes include many for oxytocin, oxytocin receptors and indeed CD38, so anyone with those genes dysfunctional might benefit.

However, as we saw with biotin, more people may be affected to a lesser degree.

CD38 affects oxytocin secretion in the brain and CD38 is affected by inflammatory cytokines, so at times of elevated cytokine expression, CD38 and oxytocin might be reduced in people with no relevant genetic dysfunction.

You can have too much vitamin A, this is called Hypervitaminosis A.  You cannot suffer this condition by eating fruit and vegetables, but you can by eating too much preformed vitamin A from foods (such as fish or animal liver), supplements, or prescription medications; it can be prevented by ingesting no more than the recommended daily amount.

High intake of provitamin carotenoids (such as beta carotene) from vegetables and fruits does not cause Hypervitaminosis A, as conversion from carotenoids to the active form of vitamin A is regulated by the body to maintain an optimum level of the vitamin. Carotenoids themselves cannot produce toxicity.

So, too much cod liver oil can be bad for you, but you can eat carrots like Bugs Bunny and do no harm.  If you really overdo it, your skin may change colour to orange, something called carotenosis

You can buy vitamin A supplements as the preformed vitamin or as beta carotene.


Too much of a good thing?

In times gone by, children used to be given a tablespoon of cod liver oil daily, as a good source of vitamin D and vitamin A.  These days that amount of both vitamins would be seen as excessive.  Excess of both vitamins is bad for you, but easy to achieve, by accident, while trying to do a good thing.


Maja’s Dose

Maja achieved her positive results with a modest dose of fish oil (using 40% of one capsule) giving 3-4000 IU of vitamin A.

This is actually quite a high dose of vitamin A, if you look at the maximum safe dose.

I think many people are giving kids with autism much larger doses of fish oil and thus far too much vitamin A and D.  This has been raised as an issue by Seth, another reader of this blog.


CD38

CD38 has many other functions other than regulating oxytocin. In people who have an oxytocin dysfunction due to an upstrean CD38 dysfunction, correcting the lack of CD38 might be particularly beneficial.   

CD38 is used as a prognostic biomarker for leukemia.  This is a complex area of science.  In essence, it is an accepted fact that increased CD38 expression is associated with favorable prognosis in adult acute leukemia.

Leukemia is associated with Down Syndrome. 

Not surprisingly, both vitamin A and ATRA can be beneficial in treating leukemia.
ATRA (All Trans-Retinoic Acid) for acute myeloid leukaemia (AML)


CD38 expression is apparently easy to measure.

Perhaps in those numerous oxytocin trials for autism, they might want to bother measuring CD38?


The Recent Korean Paper


Here is what the Koreans have to say about Oxytocin:-




CD38 is a transmembrane antigen that has been studied as a negative prognostic marker for chronic lymphocytic leukemia [72]. CD38 participates in the oxytocin secretion in the brain and affects maternal nurturing and social behavior [73]. Plasma levels of oxytocin are strongly reduced in CD38 knockout mice (CD38-/-mice) and subcutaneous oxytocin injection or lentiviralvector-mediated delivery of human CD38 into the hypothalamus rescued social memory and maternal care in these mice [73].

CD38 transcription is highly sensitive to cytokines and vitamins, including all-trans retinoic acid (ATRA), a known inducer of CD38 [75]. In a study on lymphoblastoid cell lines in patients with ASD and their parents, ATRA exhibited an upmodulatory potential on CD38 mRNA [75]. Although there have been almost no follow up studies on ATRA and ASD treatment, there is a possibility that substances affecting CD38 expression, such as ATRA, may be potential therapeutic candidates














Wednesday 23 December 2015

“More GABA” for Autism and Epilepsy? Not so Simple

Today’s post was prompted by Tyler highlighting a very recent paper from MIT and Harvard, with some interesting research on GABA in autism.  It also provides the occasion to include an interesting epilepsy therapy, which I encountered a while back.  This fits with my suggestion that the onset of much epilepsy in autism could be prevented.

In the MIT/Harvard study, they were looking into the excitatory/ inhibitory (E/I) imbalance found in ASD and schizophrenia. They used a non-invasive optical method to measure E/I imbalance and this did get some media coverage.  However, I am not sure this could be a diagnostic tool in very young children with classic autism, as was suggested; most such children would not cooperate.  It is not just a problem of being non-verbal, as was suggested in the media.

Indeed, due to the nature of the experiment, the researchers involved older subjects, with milder autism and none had MR/ID (IQ<70).  Being a trial done in the US, of the 20 autistic subjects, 11  were being treated with psychiatric medications: antidepressants (n = 8), antipsychotics (n = 2), antiepileptics (n = 4), and anxiolytics (n = 2).

The easy to read version is from the MIT website:-


Study finds altered brain chemistry in people with autism



The full version is here:-




They used something called Binocular Rivalry  as a proxy for  E/I imbalance.

During binocular rivalry, two images, one presented to each eye, vie for perceptual dominance as neuronal populations that are selective for each eye’s input suppress each other in alternation [16, 17]. The strength of perceptual suppression during rivalry is thought to depend on the balance of inhibitory and excitatory cortical dynamics [12–15] and may serve as a non-invasive perceptual marker of the putative perturbation in inhibitory signaling thought to characterize the autistic brain.

We therefore measured the dynamics of binocular rivalry in individuals with and without a diagnosis of autism (41 individuals, 20 with autism). As predicted, individuals with autism demonstrated a slower rate of binocular rivalry (switches per trial: controls = 8.68, autism = 4.19; F(1,37) = 16.52, hp 2 = 0.311, p = 0.001; Figure 1A), which was marked by reduced periods of perceptual suppression (proportion of each trial spent viewing a dominant percept, (dominant percept durations)/(dominant + mixed percept durations): controls = 0.69; autism = 0.55; F(1,36) = 7.27, hp 2 = 0.172, p = 0.011; Figure 1B). The strength of perceptual suppression inversely predicted clinical measures of autistic symptomatology (Autism Diagnostic Observation Schedule [ADOS]: Rs = 0.39, p = 0.027; Figure 1) and showed high test-retest reliability in a control experiment (R = 0.94, p < 0.001; see Supplemental Experimental Procedures and also [18]). These results replicate our previous findings in an independent sample of autistic individuals [11] and confirm rivalry disruptions as a robust behavioral marker of autism.


To test whether altered binocular rivalry dynamics in autism are linked to the reduced action of inhibitory (g-aminobutyric acid [GABA]) or excitatory (glutamate [Glx]) neurotransmitters in the brain, we measured the concentration of these neurotransmitters in visual cortex using magnetic resonance spectroscopy (MRS).


GABA and glutamate are predicted to contribute to different aspects of binocular rivalry dynamics: mutual inhibition between (GABA) and recurrent excitation within (glutamate) populations of neurons coding for the two oscillating percepts [14].

. Critically, reducing either mutual inhibition or recurrent excitation is predicted to reduce the strength of perceptual suppression during rivalry in one implementation of this model [14], mirroring the dynamics we observed in autism. We therefore considered each neurotransmitter separately to test whether inhibitory or excitatory signaling was selectively disrupted in the autistic brain.

As predicted by models of binocular rivalry, GABA concentrations in visual cortex strongly predicted rivalry dynamics in controls, where more GABA corresponded to longer periods of perceptual suppression (Rs = 0.62, p = 0.002; Figure 2B). However, this relationship was strikingly absent in individuals with autism (Rs = 0.02, p = 0.473; Figure 2B). The difference between the two correlations was significant (hp 2 = 0.167, p = 0.013; Figure 2C), indicating a reduced impact of GABA on perceptual suppression in the autistic brain.


GABA was working backwards

Importantly, this finding was specific to GABA: glutamate strongly predicted the dynamics of binocular rivalry in autism (Rs = 0.60, p = 0.004; Figure 2B), to the same degree as that found in controls.


Glumate is working just fine.

These findings suggest that alterations in the GABAergic signaling pathway may characterize autistic neurobiology. Consistent with prior evidence from animal and post-mortem studies, such dysfunction may arise from perturbations in key components of the GABAergic pathway beyond GABA levels, such as receptors [3–9] and inhibitory neuronal density

Together with the pivotal roles of GABA in canonical cortical computations [39] and neurodevelopment [40], these findings point to the GABAergic signaling pathway as a prime suspect in the neurobiology of this pervasive developmental disorder [41]




This study reconfirms what regular readers of this blog already knew.



Epilepsy

I thought it was positive that the MIT researchers suggested that the high level of epilepsy in autism and this E/I imbalance really must be connected.

I have been suggesting for some time that by correcting this E/I imbalance in children with autism, it is likely that the onset of epilepsy could be avoided (in some cases).

I did suggest this to one well known researcher who thought the idea of preventing the onset of epilepsy was not something that the medical community would accept as a concept.

I also raised the novel epilepsy therapy, below, to the same researcher who thought it also would never be considered.

The therapy was to use both bumetanide and potassium bromide to switch GABA back to inhibitory and then give a little boost using a GABA agonist.   

There are many types of epilepsy and some do not respond well to current treatments.  It would seem plausible that the autism-associated type of epilepsy might constitute a specific sub-type.









Potassium Bromide was the original epilepsy therapy over a hundred years ago.  It is still used in Germany as a therapy.  Reports from a century ago suggest it has the same effect in autism as Bumetanide. (we saw this in my post on autism history). 

As you can see on Wikipedia there is a wide range of GABA agonists, but the only ones that would help in epilepsy and autism would be the ones that can cross the blood brain barrier.

GABAA receptor Agonists

·         Bamaluzole
·         GABA
·         Gabamide
·         GABOB
·         Gaboxadol
·         Ibotenic acid
·         Isoguvacine
·         Isonipecotic acid
·         Muscimol
·         Phenibut
·         Picamilon
·         Progabide
·         Quisqualamine
·         SL 75102
·         Thiomuscimol


In an earlier post, we looked at the possible use of small doses of AEDs (anti-epileptic drugs).  One reader found that tiny dose of Valproate (known to raise GABA) had a positive effect when combines with Bumetanide.

In a recent comment one reader showed the same result by combing picamilon with bumetanide.

Both Picamilon and Valproate are having the effect proposed by the epilepsy researchers.

Potassium Bromide does have known side effects, but the idea of further boosting the effect of Bumetanide is interesting.  I have suggested before that this should also be possible using Diamox (Acetazolamide).  Diamox does not affect NKCC1 or EGABA,  it affects the  Cl-/HCO3-exchanger AE3  to further affect Cl- levels.  

I did suggest this a long time ago in my posts on the GABAa receptor.  I am not the only one to realize this.

NKCC1 and AE3 Appear to Accumulate Chloride in Embryonic Motoneurons

   

Picamilon is well researched Russian drug, sold in other countries as a supplement.  It is a modified version of GABA that includes niacin; together it can cross the blood brain barrier (BBB).



So I think a better version of what the epilepsy researchers suggest might be:-

                           Bumetanide  +  Diamox  +  a touch of Picamilon



What would be the effect in autism?











Wednesday 16 December 2015

Long Term use of Low Dose Clonazepam and More Science on the Excitatory/Inhibitory Imbalance in Schizophrenia and ASD


   
A small number of readers of this blog have followed Professor Catterall’s ideas and trialed low dose clonazepam for autism.  

This post summarizes my findings from using it long-term; it would be a good place to collect the findings of other people.


The science part of this blog is courtesy of a reader who highlighted the full-text version of a paper I mentioned.  Perhaps it was the author?

For information on Catterall’s clonazepam research, go to the “Index by Subject” tab and click on Clonazepam.






Before getting to that, I do get asked how I know, for sure, these therapies really do work for Monty, aged 12, with classic autism.  As I told Ben-Ari, the Bumetanide researcher, the best way to convince the doubting public will be to measure IQ, not autism.  If you can add 30 to 50 points to your IQ result, even the sceptics would pay attention.

I am not measuring IQ directly, but I do note things like spelling tests, math tests and handwriting.  The first pleasant surprise was actually reaching the point of sitting the same tests as the NT kids. Piano playing is another interesting proxy.

Monty’s one to one Assistant (and pal) from age 3 to 9 came to visit the other day and could not believe what his handwriting now looks like.  She had spent hundreds of hours with him practicing fine motor skills, like pencil control.  The end result was handwriting, but even then not like that of his peers. 

Cursive handwriting is now great.  Spelling tests and “quick-fire” math tests are also great.

As we now know, 20% of people diagnosed very young with quite severe autism seem to make wonderful progress.  This has happened by 5 or 6 years old, while the brain is still highly plastic.  Spontaneous accelerated development thereafter rarely seems to happen.  Monty started his Polypill therapy at the age of 9 years, in December 2012.

This is a spelling test from school, given to NT (neurotypical) ten year olds and 12 year old Monty (on paper without lines).  It is not rocket science and big brother could probably have got 20/20 in this test when he was eight years old.  But when Monty was eight years old, he was trying to break the windows of my car with his head and his handwriting did not look like this.





I have all the proof I need that modulating the excitatory/inhibitory imbalance in Monty’s autism is well worth the effort.  The effects are reversible if you stop the therapy, as should be the case.


Clonazepam

Here I am repurposing an existing drug for a different use, at a dosage so low it is highly unlikely to cause side effects.  This is mirroring the use of the same drug, at similar low doses, in mouse models of autism by Professor Catterall.

Clonazepam at “high” doses is widely used already in people with autism, to treat seizures and extreme anxiety.  

Catterall showed that the drug has a totally different effect at very low doses (less than 10% of normal), via a specific mechanism which he has identified, the positive modulation of the α 2,3  subunits of GABAA receptors. 

GABAA receptors are made up of five sub-units, the strict composition does indeed vary over time, just to make things even more complicated.  The most common GABAA receptors have two αs, two βs, and one γ 2β2γ). For each subunit, many subtypes exist (α1–6, β1–3, and γ1–3). It is these subtypes of the subunits that Catterall showed to be key.  Clonazepam was one of the substances that he showed to be effective (in mice).

At “high” doses Clonazepam does have side effects, people build up tolerance to it and so take ever higher doses, and then they get hooked on it.

At very low doses the reverse seems to occur.  Over time you become more sensitive to it and need lower and lower doses.  This was a surprise to me.

The other surprise was that slightly above the effective “low dose” you get some anxiety and irritability.  When I first wrote about this I did wonder if this was just a coincidence, but it is not.

My chart from back then:-




Another interesting point was that some other readers found the effective dose was even less than mine.

When you read about the use of Clonazepam at regular, much higher doses, it is clear that there are wide variations in people’s sensitivity to this drug.  So much so that there is standard lab test to measure blood concentration of this drug, so that the clinician can vary the dose to achieve the desired level in blood.



It is not an expensive test and I did wonder if this could be used by clinicians to find the effective low dose in their patients with autism.

It did sound a clever idea, but then I read that even the same blood concentration of clonazepam (at high doses) can have markedly different effects in different people.  Still it is better than doing nothing and would reduce some of the guesswork with dosage.



The effective dose

In my n=1 example, the effective dose started out at 40mcg a day.  The half-life is very long and so you need three days to reach a stable level.

Other people contacted me to say that in their case 25mcg a day was effective and in one case, dosage once every two days was optimal.

In my case 40mcg, now gives the negative effects I has originally discovered at higher doses.

Currently the effective dose is 20 to 25 mcg.

This is a tiny dose, technically sub-clinical, but it really is better than giving none.  I have discontinued on several occasions.  There is cognitive loss, which is then regained when re-starting. 

The incremental cognitive effect is not as great in magnitude as I found with Bumetanide, but in people not using Bumetanide, the effect seems to be much greater.  Put more simply, Clonazepam plus Bumetanide is more beneficial than Bumetanide alone, at least in my case.

At this dose the annual cost of the therapy is one dollar/euro/pound. So it will not break the bank.

Tablets are available as 0.5mg  (giving 20 days of use) and 2mg (giving 80 days of use).  A bottle of 2mg tablets will last someone a few years.

I wish they made 0.025 mg (25 mcg) tablets.

I see no reason why, in ten to twenty years’ time, low dose clonazepam will not be a mainstream therapy for some autism; the only problem is the variability of the effective dosage.



Science

For those diehards who have made it this far, now I move from the Peter-reviewed science to the Peer-reviewed science, but from yet another Peter, Peter Penzes from Northwestern University, close by the Windy City.




Abstract: Autism Spectrum Disorders (ASD) and Schizophrenia (SCZ) are cognitive disorders with complex genetic architectures but overlapping behavioral phenotypes, which suggests common pathway perturbations. Multiple lines of evidence implicate imbalances in excitatory and inhibitory activity (E/I imbalance) as a shared pathophysiological mechanism.
Thus, understanding the molecular underpinnings of E/I imbalance may provide essential insight into the etiology of these disorders and may uncover novel targets for future drug discovery. Here, we review key genetic, physiological, neuropathological, functional, and pathway studies that suggest alterations to excitatory/inhibitory circuits are keys to ASD and SCZ pathogenesis.


This study really shows how the common genetic dysfunctions in both schizophrenia and autism come together to produce the Excitatory/Inhibitory (E/I) imbalance.  Numerous different dysfunctions result in the same imbalance, some relate to GABA and some to NMDAR, but the end result is the same.

It is a really good paper, mentioning many of the genes we have encountered in this blog, plus many of the pathways like mTOR and even PAK inhibitors.

The study does not cover any therapeutic methods to correct the E/I imbalance, but this blog has those in spades.  They relate to modulating GABAA, GABAB and NMDA receptors.

Low dose clonazepam is modulating GABAA , as does Bumetanide and as should Acetazolamide (Diamox).  More of that in 2016.