Showing posts with label Down Syndrome. Show all posts
Showing posts with label Down Syndrome. Show all posts

Thursday 24 June 2021

Betaine (TMG) and Gene Therapy as potential alternatives to Bumetanide Treatment in Autism?

Betaine (also known as TMG, or trimethylglycine) is a methyl derivative of glycine, first isolated from sugar beet and hence its name.

Today’s post was prompted by our reader, and Covid home-school instructor, AJ.  He raised the question of whether betaine can be used like Bumetanide to normalize chloride levels in neurons.

I am combing this idea with news from Genoa in Italy, where they have developed gene therapy as an alternative to Bumetanide and in their words :-

“This sets the stage for the development of a gene therapy approach to overcome the shortcomings of bumetanide treatment.”

The interesting thing is that neither of these ideas come from autism research.  The idea to use Betaine was stumbled upon and was then written up in a Norwegian case study about Creatine transporter deficiency.  The Italians are trying to improve cognition in brain disorders and their model of choice was Down syndrome. 

As we have seen time and again, elevated chloride within neurons is a common feature of many types of brain disorders from some idiopathic autism, to Down syndrome, to adult conditions such as Parkinson’s disease.  Today we learn that it is may well be a feature of Creatine Transporter Deficiency.

I have been rather wary of writing about any kind of gene therapy, because it seemed either too far ahead of its time, or just absurdly expensive.  There are some new $1+ million treatments.

This may be about to change given that the Biontech (AKA Pfizer vaccine), Moderna, Janssen (Johnson & Johnson) and Oxford AstraZeneca vaccines for Covid 19 are all based on gene therapy.

The Biontech people are really clever and were already trying to treat various kinds of cancer and other condition using gene therapy, before they developed their highly successful Covid vaccine.

The Italians in Genoa used an adeno-associated virus (AAV)-mediated RNA interference (RNAi) to target and reduce neuronal NKCC1 expression, rescue neuronal Cl-  homeostasis, GABAergic transmission, and cognitive deficits.   The benefit was still there 6 months after the injection.

Don’t worry if the above paragraph makes little sense. Just read on.

The same type of adeno-associated virus (AAV) vector is the platform for gene therapy delivery used in the Astra Zeneca, Janssen and the Russian Sputnik covid vaccines.

The virus is just the delivery system (vector) to get some genetic code into cells.

The Oxford-AstraZeneca COVID-19 vaccine uses a chimpanzee adenoviral vector. It delivers the gene that encodes the SARS-CoV-2 spike protein, to our cells.  Our cells then transcribe this gene into messenger RNA, or mRNA, which in turn prompts our cellular machine to make the spike protein in the main body of the cell. The mRNA molecule behaves essentially like a recipe.  Then our cells present the spike protein on the cell surface, prompting our immune system to make antibodies and mount T cell responses.

Biontech and Moderna are pioneers of mRNA vaccines, which bypass one step in the above process. They do not require our cells to make the messenger RNA, or mRNA.  They have already made it for you.


Gene therapy for autism?

Single gene autisms are all potential candidates for gene therapy.

The problem is that most autism and all Down syndrome is polygenic, there can be hundreds of miss-expressed genes.

But the researchers in Italy show us that even polygenic autism and Down syndrome can benefit from therapy targeting a single gene.  You just have to select the right one.

The problem is the price. Covid vaccines are made in huge quantities and are cheap.

Customized gene therapy is ultra expensive, in part because each therapy has to be approved individually.


An NKCC1 Gene Therapy?

The Italians have already made the NKCC1 Gene Therapy.  The question is will it ever going be available to humans with Down Syndrome, Autism or even Parkinson’s disease?

Restoring neuronal chloride homeostasis with anti-NKCC1 gene therapy rescues cognitive deficits in a mouse model of Down syndrome

A common feature of diverse brain disorders, is the alteration of GABA-mediated inhibition due to aberrant intracellular chloride homeostasis induced by changes in the expression and/or function of chloride transporters. Notably, pharmacological inhibition of the chloride importer NKCC1 is able to rescue brain-related core deficits in animal models of these pathologies and some human clinical studies. Here, we show that reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down syndrome (DS) restores intracellular chloride concentration, efficacy of GABA-mediated inhibition and neuronal network dynamics in vitro and ex vivo. Importantly, AAV-mediated neuron-specific NKCC1 knockdown in vivo rescues cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a mechanistic link between NKCC1 expression and behavioral abnormalities in DS mice, and establish a molecular target for new therapeutic approaches, including gene therapy, to treat brain disorders characterized by neuronal chloride imbalance.


This sets the stage for the development of a gene therapy approach to overcome the shortcomings of bumetanide treatment.

This highlights a causative role of NKCC1 upregulation in learning and memory deficits in adult Ts65Dn mice, thus also validating brain NKCC1 as a target for ameliorating cognitive disabilities in DS. Furthermore, our neuro-specific knockdown approach points to neurons as major players in the NKCC1- dependent cognitive impairment in DS mice. Nevertheless, we cannot exclude that other cell types which also express NKCC1 (e.g. glial cells) could still play a role in the overall cognitive impairment that characterizes DS.

Despite the very large and fast-increasing literature both on animal models and patients indicating positive outcomes upon bumetanide treatment, there is not yet a strong demonstrated direct link between NKCC1 inhibition, restoration of Cl- homeostasis and full GABAergic inhibitory signaling, and rescue of brain deficits.  Moreover, bumetanide has strong diuretic activity, triggering ionic imbalance, and potential ototoxicity 25,26.  This hampers its use for clinical applications in lifelong treatments4,27 and may strongly jeopardize treatment compliance along years of treatment.  Moreover, bumetanide was given systemically in most studies, and the suboptimal brain pharmacokinetic profile of the drug28 raises questions on its mechanism of action29.  Here, we demonstrate that adeno-associated virus (AAV)-mediated RNA interference (RNAi) to target (and reduce) neuronal NKCC1 expression rescues neuronal Cl- homeostasis, GABAergic transmission, and cognitive deficits in the Ts65Dn mouse model of Down syndrome. This sets the stage for the development of a gene therapy approach to overcome the shortcomings of bumetanide treatment.


“Thus, our results indicate the efficacy of long-term AAV9-mediated neuro-specific NKCC1 knockdown in rescuing cognitive deficits in Ts65Dn mice.”


“Besides establishing a causal link between NKCC1 upregulation and cognitive impairment in DS, our data also provide a proof-of-concept for a neuro-specific RNAi gene therapy approach to restore hippocampus-dependent cognitive behaviors in adult animals specifically in the brain, and without affecting peripheral organs (e.g., the kidney). This is particularly relevant in the context of the current clinical trials repurposing the strong diuretic bumetanide to treat brain disorders with impaired chloride homeostasis3.  Importantly, we achieved a comparable degree of long-term cognitive rescue with two different amiR sequences against NKCC1, underlining the specificity of our approach.”


Gone Fishing

If a trip to Italy for gene therapy is not realistic, this takes us back to AJ’s idea, which is to use Betaine.  The correct version is TMG or glycine betaine, and confusingly not Betaine HCl.

Fish love the taste of betaine.

Betaine was first isolated from sugar beet.

I recall from my time at the sugar factory, when I was 18, that once you have sliced up the sugar beet and extracted as much sugar as possible you are left with the pulp.  This pulp is dried, molasses is added back and then it is made into pellets.  The pellets are fed to cattle and horses.  They taste pretty bad in my opinion.

To humans it tastes bad because of the beet molasses by-product.

The molasses by-product from sugar cane tastes great to humans.  That is why they make rum in the Caribbean, and not in England or Canada.

Brown sugar from a sugar beet factory is made by adding sugar cane molasses to white sugar from beet.  It is a cheat really.

Cows love sugar beet by-products.

It turns out that fish love betaine HCl.

Betaine HCl is an excellent natural attractor that stimulates a strong, prolonged feeding response from carp and many other coarse fish.

Betaine HCl is now used to induce feeding in the fish farming industry

As our reader Tyler has highlighted, Betaine HCl, that fish like and is available is a cheap supplement is not the same as the Betaine used in the medical case study. Confusingly, the original Betaine (TMG, or called glycine betaine) gave way to a class of compounds all called betaines. One of these betaines is betaine HCL.

In most cases, in the medical literature when they refer to Betaine, they mean glycine betaine, also known as TMG.

Betaine HCl is used to increase acidity in your stomach. The effect of betaine compounds other than glycine betaine/TMG on NKCC1 is unknown.

Glycine Betaine (TMG) and NKCC1

It seems that betaine reduces your level of NKCC1 RNA. 

In your DNA are the instructions to make the NKCC1 transporter. To go from these instructions to actually making the transporters you need RNA.

In some autism there are too many NKCC1 transporters, so put simply there was too much NKCC1 RNA. So, if you can find a substance that reduces NKCC1 RNA, you might well solve the problem.

The caveat is that the substance must not also increase KCC2 RNA.  This appears to be what taurine does.

Here, finally, is AJ’s paper:

Treatment experience in two adults with creatine transporter deficiency


Creatine transporter deficiency (CTD) is an X-linked form of intellectual disability (ID) caused by SCL6A8 mutations. Limited information exists on the adult course of CTD, and there are no treatment studies in adults.


We report two half-brothers with CTD, 36 and 31 years at intervention start. Their clinical phenotypes were consistent with CTD, and intervention was indicated because of progressive disease course, with increased difficulties speaking, walking and eating, resulting in fatigue, and malnutrition. We therefore performed treatment trials with arginine, glycine and a proprietary product containing creatine and betaine, and then a trial supplementing with betaine alone. Results In the older patient, glycine and arginine were accompanied by adverse effects, while betaine containing proprietary product gave improved balance, speech and feeding. When supplementation stopped, his condition deteriorated, and improved again after starting betaine supplement. Betaine supplementation was also beneficial in the younger patient, reducing his exhaustion, feeding difficulties and weight loss, making him able to resume his protected work.

Discussion & conclusion

We report for the first time that betaine supplement was well tolerated and efficient in adults with CTD, while arginine and/or glycine were accompanied by side effects. Thus, betaine is potentially a new useful treatment for CTD patients. We discuss possible underlying treatment mechanisms. Betaine has been reported to have antagonistic effect on NKCC1 channels, a mechanism shared with bumetanide, a medication with promising results in both in autism and epilepsy. Further studies of betaine's effects in well-designed studies are warranted.


The mechanism of betaine’s assumed favorable effect is unknown. We do not know whether betaine influences the cell creatine content in itself or its effects are more aspesific. However, we would like to present some hypotheses. First, betaine may have effect in CTD by modulating GABA-transmission. Betaine has been reported to have an antagonistic effect on NKCC1 channels, which also influences GABAergic neurotransmission. Inhibiting NKCC1 is a mechanism shared with bumetanide, a well-known diuretic medication that in recent years has been found to influence GABAergic transmission, and thereby it has been found promising in treatment of several brain conditions, including autism, and epilepsy. NKCC1 inhibition by bumetanide has also been tried with success in other rare neurodevelopmental disorders fragile X syndrome and tuberous sclerosis. Second, betaine’s properties as an osmolyte may be of importance, as betaine has similarities with creatine in being an osmolyte. Osmotic properties are thought to be one of the central mechanism behind bumetanide’s efficacy in treating brain disorders. Thus, it could be speculated that the lack of intracellular creatine in CTD may result in inefficient osmolyte regulation, and that betaine supplementation replaces the lacking creatine and thereby improves the neuronal adaption to salinity changes, edema or cellular dehydration. Betaine has osmolyte properties that even makes it act as a “chemical chaperone” increasing the stability of cell and membrane proteins. Fourth, it is possible that betaine has some effect through modifying methylation. Methylation of GAA by GAMT to form creatine is a rate-limiting step in the creatine synthesis by neurons. Betaine could stimulate this by donating methyl groups to SAMe, which donates a methyl group to GAA to form creatine. This might reduce the burden when body demands more methyl groups for creatine synthesis. Similar mechanisms may be responsible for a beneficial effect of both betaine and s-adenosyl methionine (SAMe). However, as creatine and GAA share the same transporter, one would not expect GAA to enter the GAMTexpressing cells in patients suffering from CTD. Still, it cannot be excluded that there is some rest function in the creatine transporter, and that increased endogenous synthesis improves the condition slightly. Furthermore, it is possible that CTD increases the need for methylation agents in general, as creatine supplementation has been found to reduce the need for other methylation agents [34]. Thus, it is likely that betaine may have a positive effect in CTD by improving methylation capacity for other reactions than those directly involved in creatine production. Betaine’s effect on muscle may be also of importance, as animal studies have shown that muscles growth improves with betaine [35], which potentially could have had a positive impact on our patients fatigue and weight loss. To summarize, betaine has several properties that make it likely that it will have a beneficial effect in CTD, especially the properties as an osmolyte, a down regulator of the NKCC1 channel and an influencer of GABAergic transmission. These properties are similar to the properties of bumetanide, a promising new medication for treatment of autism and epilepsy, which are common symptoms of CTD. Further research is needed, however, to elucidate the role of betaine in CTD.

If you read the detail of the old paper that is referred to in the above paper, you see that betaine is not blocking the NKCC1 channels as suggested, but it seems to be reducing the number of them.  The net effect may be the same, but the process is very different.


Expression and regulation of the Na+/K+/2Cl− cotransporter NKCC1 in rat liver and human HuH-7 hepatoma cells

The expression of sodium potassium chloride cotransporter 1 (NKCC1) was studied in different liver cell types. NKCC1 was found in rat liver parenchymal and sinusoidal endothelial cells and in human HuH-7 hepatoma cells. NKCC1 expression in rat hepatic stellate cells increased during culture-induced transformation in the myofibroblast-like phenotype. NKCC1 inhibition by bumetanide increased α1-smooth muscle actin expression in 2-day-cultured hepatic stellate cells but was without effect on basal and platelet-derived-growth-factor-induced proliferation of the 14-day-old cells. In perfused rat liver the NKCC1 made a major contribution to volume-regulatory K+ uptake induced by hyperosmolarity. Long-term hyperosmotic treatment of HuH-7 cells by elevation of extracellular NaCl or raffinose concentration but not hyperosmotic urea or mannitol profoundly induced NKCC1 mRNA and protein expression. This was antagonized by the compatible organic osmolytes betaine or taurine. The data suggest a role of NKCC1 in stellate cell transformation, hepatic volume regulation, and long-term adaption to dehydrating conditions.


Aha!  Glycine Betaine and Taurine – not so fast 

You have to check the effect on both NKCC1 and KCC2.  One lets chloride into neurons and the lets it out.  You want to block NKCC1 and not KCC2, otherwise you undo all the good you have done.

Both glycine betaine (TMG) and taurine are already used as autism supplements at low doses.  The paper below suggest that Taurine is not a good idea for people with high levels of chloride within neurons.


Taurine inhibits K+-Cl- cotransporter KCC2 to regulate embryonic Cl- homeostasis via with-no-lysine (WNK) protein kinase signaling pathway

GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.


So, it is likely only Glycine Betaine (TMG) may be of potential benefit, in the case of lowering chloride.


Glycine Betaine in the broader research


Betaine in Inflammation: Mechanistic Aspects and Applications

Betaine is known as trimethylglycine and is widely distributed in animals, plants, and microorganisms. Betaine is known to function physiologically as an important osmoprotectant and methyl group donor. Accumulating evidence has shown that betaine has anti-inflammatory functions in numerous diseases. Mechanistically, betaine ameliorates sulfur amino acid metabolism against oxidative stress, inhibits nuclear factor-κB activity and NLRP3 inflammasome activation, regulates energy metabolism, and mitigates endoplasmic reticulum stress and apoptosis. Consequently, betaine has beneficial actions in several human diseases, such as obesity, diabetes, cancer, and Alzheimer’s disease.


Betaine is a stable and nontoxic natural substance. Because it looks like a glycine with three extra methyl groups, betaine is also called trimethylglycine . In addition, betaine has a zwitterionic quaternary ammonium form [(CH3)3N+ CH2COO−] (Figure 1). In the nineteenth century, betaine was first identified in the plant Beta vulgaris. It was then found at high concentrations in several other organisms, including wheat bran, wheat germ, spinach, beets, microorganisms, and aquatic invertebrates. Dietary betaine intake plays a decisive role in the betaine content of the body. Betaine is safe at a daily intake of 9–15 g for human and distributes primarily to the kidneys, liver, and brain. The accurate amount of betaine intake generally relies on its various sources and cooking methods. Besides dietary intake, betaine can be synthesized from choline in the body. Studies report that high concentrations of betaine in human and animal neonates indicate the effectiveness of this synthetic mechanism.


Boosting amino acid derivative may be a treatment for schizophrenia

Many psychiatric drugs act on the receptors or transporters of certain neurotransmitters in the brain. However, there is a great need for alternatives, and research is looking at other targets along the brain's metabolic pathways. Lack of glycine betaine contributes to brain pathology in schizophrenia, and new research shows that betaine supplementation can counteract psychiatric symptoms in mice.



Supplement treats schizophrenia in mice, restores healthy “dance” and structure of neurons Repurposed drug works by building cells’ skeleton and transportation network




Early on in the Covid saga, I saw interviews with both the Moderna researchers and the Oxford (AstraZeneca) researchers. Both claimed that they designed their vaccines over a weekend.  This was made possible by the Chinese releasing the DNA code of the virus.

When you think about gene therapy for autism and Down syndrome, the same likely applies; much could be achieved over a weekend.

The expensive and time-consuming part is the testing and approval process.

In the Covid pandemic the approval process was modified to allow for emergency use.  Perhaps this should also be the case for all gene therapies?

What use is a $2 million therapy for autism or Down syndrome?

In theory, if you gave your gene therapy prior to birth or shortly thereafter, it might be fully curative.  Realistically, by the time you get the therapy it is just going to be beneficial and you will still need other ongoing therapies.

Note that gene therapy normally applies to just one gene.  In Down syndrome people have a third copy of all, or just part, of Chromosome 21.  This results directly in the miss-expression of hundreds of genes from that chromosome.

The gene that encodes NKCC1 is on Chromosome 5, which has nothing directly to do with Down syndrome.

The NKCC1 transporter is over-expressed in Down syndrome as a down stream consequence of the disorder. It is caused by the “faulty GABA switch”, referred to in earlier posts.

The Italian gene therapy to lower chloride in neurons and so raise cognition, has numerous applications, in people currently of all ages, so there is a big potential market.

Why not gene therapy for all single gene autisms?  It could be a highly productive use of the researcher’s weekends, for a year or two.

The issue is who would pay for the $20 to $30 million approval process, for each gene?

Maybe some of the billions in profit from clever Covid vaccines could be used for pro bono gene therapy?  Highly unlikely.

Biontech, who are the brains behind the Pfizer vaccine, do have plans to develop gene therapy for other medical conditions.  I think these will be ultra expensive,

That brings me back to Glycine Betaine (TMG), is 10g a day of this supplement really going to reduce the expression of NKCC1 transporters in neurons and so lower chloride within neurons?  It seems to work in creatine transporter deficiency, is all we can say.  

Glycine betaine, at much lower doses, has been used by DAN and now MAPS doctors for decades. They use it as a “methyl-donor”.  There is a combination of real science and hocus-pocus surrounding DNA methylation. 

 DNA Methylation and Susceptibility to Autism Spectrum Disorder




Tuesday 6 April 2021

GABRA5 - Too much, or too little in Autism and Down Syndrome?


It is easy to get things the wrong way round.

This applies to science and to some people getting dressed


Today’s post was prompted by a reader updating me about Roche’s autism drug RO7017773, which targets the alpha 5 sub-unit of GABAA receptors, encoded by the gene GABRA5.

Enrollment opens in phase II study of RO-7017773 for autism spectrum disorder

A 12-Week Placebo-Controlled Study to Investigate the Efficacy, Safety, and Tolerability of RO7017773 in Participants Aged 15-45 Years With Autism Spectrum Disorder (ASD)


Some people with severe autism, or just plain old ID/MR, which has gone out of fashion as a diagnosis these days, struggle to dress themselves because they do not notice what is inside out, or back to front.  I recall reading a few years ago about one autism parent who started a clothing company to get round this problem.


ADAPTIVE CLOTHING with no front/back and reversible so cannot be inside out.


I must say that writing this blog I am often left wondering which way round things are.  Do we want an agonist or an antagonist, a positive allosteric modulator or a negative one.  Many times things do seem to work backwards.

If you follow the research you will see that researchers often get things mixed up, with one group trying one strategy and yet another group of Ivy league bright-sparks doing exactly the opposite. The Vasopressin research is a good example.

Are they dyslexic? Perhaps dyspraxic?

Today it is the turn of GABRA5: do we want to upregulate it, or downregulate it?

GABRA5 is the gene that encodes the alpha 5 sub-unit of GABAa receptors.

A few years ago, the drug firm Roche spent a lot of money developing a negative modulator of these receptors.  That did not work and Basmisanil  (developmental codes RG-1662 and RO5186582) was abandoned as a treatment to raise cognition in Down syndrome.

Roche are now trialing the opposite therapy, a positive allosteric modulator of alpha 5 sub-unit of GABAa receptors, this time to treat autism.


Targeting GABA to treat autism

GABA is an important neurotransmitter and it seems to be dysfunctional in many types of autism, as well as other neurological conditions.

Both the A-type and the B-type of GABA receptors can respond to treatment.

When it comes to the A-type, we can be very clever and target specific sub-units of the receptor to achieve different goals.


Each receptor is made up of two α subunits, two βs and one γ.

In humans, the possibilities are made up of :

·         six types of α subunits (GABRA1GABRA2GABRA3GABRA4GABRA5GABRA6)

·         three βs (GABRB1GABRB2GABRB3)

·         three γs (GABRG1GABRG2GABRG3)


What is particularly interesting is that the make up these receptors is not fixed, it is changing all the time and you can influence it with therapy.

It looks like you might even be able to treat alcohol addiction by targeting one of the sub-units.

In the world of autism it is more anxiety and cognition that we are targeting, but some types of seizure may also be targeted.

In previous posts I identified alpha 3 (GABRA3)  and alpha 5 (GABRA5) as subunits that I felt were the interesting ones to improve cognition in autism.  Alpha 3 is the target of the low dose clonazepam therapy.

Alpha 5 also fits in with my experience of inflammation-induced reduction in cognitive function.


α5 GABAA Receptors Regulate Inflammation-Induced Impairment of Long-Term Potentiation 

these results show that α5GABAA receptor activity increases during inflammation and that this increase is critical for inflammation-induced memory deficits.


We know that female hormones modulate subunit expression, today we see that oxytocin also does this. So, yet another possible effect of a little more oxytocin.


Oxytocin modulates GABAAR subunits to confer neuroprotection in stroke in vitro

Before I forget, I should add that that the nootropic herb Bacopa affects GABRA5 (in rats):-

“BME (Bacopa monnieri) significantly reversed the down-regulated Gabra1Gabra4Gabra5 gene expression of GABAA receptors subunits”


The following paper has been published since I wrote my earlier posts on GABRA5 and is very thorough.


Neurobiology and Therapeutic Potential of α5-GABA Type A Receptors

α5 subunit containing GABA type A receptors (GABAARs) have long been an enigmatic receptor subtype of interest due to their specific brain distribution, unusual surface localization and key role in synaptic plasticity, cognition and memory. These receptors are uniquely positioned to sculpt both the developing and mature hippocampal circuitry due to high overall expression and a distinct peak within the critical synapse formation period during the second postnatal week. Unlike the majority of other GABAARs, they exhibit both receptor clustering at extrasynaptic sites via interactions with the radixin scaffold as well as synaptic sites via gephyrin, thus contributing respectively to tonic currents and synaptic GABAergic neurotransmission. α5 GABAAR signaling can be altered in neurodevelopmental disorders including autism and mental retardation and by inflammation in CNS injury and disease. Due to the unique physiology and pharmacology of α5 GABAARs, drugs targeting these receptors are being developed and tested as treatments for neurodevelopmental disorders, depression, schizophrenia, and mild cognitive impairment. This review article focuses on advances in understanding how the α5 subunit contributes to GABAAR neurobiology. In particular, I discuss both recent insights and remaining knowledge gaps for the functional role of these receptors, pathologies associated with α5 GABAAR dysfunction, and the effects and potential therapeutic uses of α5 receptor subtype targeted drugs.


Genetic Disorders with Altered α5 GABAAR Neurotransmission

While acute reduction in α5 GABAARs has shown potential for improving cognition and memory, further studies both in mouse models and human patients link long term reduction with significant pathologies. Reduced α5 GABAAR levels, function or protein interactions have been observed in patients with neurodevelopmental disorders including intellectual disability, epilepsy and autism. Common conditions among these disorders include cognitive impairments, increased anxiety, autism-related behaviors, sleep disorders and epilepsy susceptibility


α5 GABAAR Therapeutics

NAMs that selectively reduce α5 GABAAR function have been heavily pursued for the potential development of cognitive enhancing or “smart” drugs. The following are a selection of α5 GABAAR NAMs: L-655,708, α5IA, Ro15-4513, MRK-016, RO4938581, and RY-80 Importantly, α5 NAMs did not exhibit the convulsant or pro-convulsant activity of more general alpha subunit NAMs, had good oral bioavailability and easily crossed the blood brain barrierIn contrast to NAMs which act via the GABAAR benzodiazepine binding site, S44819 was recently identified as a competitive antagonist of GABA at α5 GABAAR and showed similar pro-cognitive effects as NAMs: blocking α5-GABAAR tonic current, enhancing LTP, reversing scopolamine-induced impairment of spatial working memory and enhancing object recognition memory). Finally, recent evidence for beneficial effects of positive allosteric modulators (PAMs) in aged brain cognition, autism, depression and schizophrenia has bolstered α5 PAM drug development. A selection of α5 preferring PAMs includes SH-053-R-CH3-2′F, MP-III-022, and GL-II-73. Potential therapeutic applications for α5 preferring NAMs and PAMs are discussed below with a focus on CNS specific uses (Table 1).


NAM α5 GABAAR Therapeutic Applications


Developmental Disorders

Although these pharmacological successes led to a Phase II clinical trial for a related compound RG1662 (Hoffman-La Roche) in Down syndrome patients, the trial did not meet the primary and secondary endpoints of improved cognition and function.

Inflammation Induced Mild Cognitive Impairment and Post Anesthesia Memory Blockade



PAM α5 GABAAR Therapeutic Applications

Neurodevelopmental Disorders

Mouse models of neurodevelopmental disorders that present with insufficient inhibitory tone show improvement with positive modulators of GABAAR signaling. In the Scn1a+/− mouse model of Dravet syndrome, a severe childhood epileptic encephalopathy syndrome with hyperactivity and autism behaviors, abnormal social behaviors and fear memory deficits were rescued following treatment with a benzodiazepine, clonazepam. In an ASD mouse model with reduced GABAAR-mediated inhibition, the BTBR T+tf/J mouse, the α2,3 and 5 PAM L-838,417, improved deficits in social interaction, repetitive behaviors, and spatial learning.


Mild Cognitive Impairment in Aging

Although α5 GABAAR NAMs enhance memory in young rodents, it appears positive modulation may be more therapeutic in aging brains impaired by excess activity. Particularly in disorders such as Alzheimer’s which are hallmarked by overexcitation 


Depression and Schizophrenia

Another important unmet need where α5 GABAARs PAM pharmacotherapy may be applicable is in the development of new fast-acting anti-depressant drugs




Roche did develop a (NAM) drug to target the alpha 5 sub-unit in order to improve cognition in Down Syndrome. 


The GABA A α5-selective Modulator, RO4938581, Rescues Protein Anomalies in the Ts65Dn Mouse Model of Down Syndrome


RG1662, a Selective GABAA α5 Receptor Negative Allosteric Modulator, Increases Gamma Power in Young Adults with Down Syndrome.


Basmisanil ( RG-1662 and RO5186582) is a highly selective inverse agonist/negative allosteric modulator of α5 subunit-containing GABAA receptors which is under development by Roche for the treatment of cognitive impairment associated with Down syndrome. As of June 2016, it is no longer studied.


Then came the opposite strategy, a PAM (positive allosteric modulator):-


RG 7816

Alternative Names: RG-7816; RO-7017773


Mechanism of Action  GABA A alpha 5 receptor modulators

Orphan Drug Status  No

New Molecular Entity  Yes

Highest Development Phases

Phase II  Pervasive child development disorders

Most Recent Events

·         23 Feb 2021Phase-II clinical trials in Pervasive child development disorders (In adolescents, In adults) in Canada (PO) (NCT04299464)

·         12 Mar 2020Hoffmann-La Roche plans a phase II trial for Pervasive child development disorders (Autism Spectrum Disorder) in USA (PO) (NCT04299464) (EudraCT2019-003524-20)

·         22 Apr 2019Roche completes a phase I trial in Pervasive child development disorders (In volunteers) in USA (PO, Capsule, Tablet) (NCT03847987)



autism spectrum disorder

4. Phase 1


RG7816 is a small molecule highly selective positive allosteric modulator of the GABAA α5 receptor, which is expressed in key brain regions for autism spectrum disorder. Two phase I clinical trial is evaluating RG7816 for the treatment of patients with autism spectrum disorder.



Modifying the response specific to sub-units of GABAA receptors is a really nuanced therapy.

In a way I am not surprised that there is, as yet, no one size fits all therapy.

Will Roche’s trial of a drug to increase the effect of GABRA5 (a PAM) be more successful than their drug to reduce the effect of GABRA5 a (NAM)?

I do not know, but in the perfect world you would have both drugs and then see if fine-tuning GABRA5 ( + or -), on a case by case basis, was therapeutic. That would be personalized medicine.

At least we can modify GABRA3 extremely cheaply with Professor Catterall’s low dose clonazepam.

Note that we saw in my original posts that the Japanese attribute the benefit of low dose clonazepam to the γ2 subunit of GABAa receptors, which is encoded by GABRG2, for those who don’t speak Greek.


PX-RICS-deficient mice mimic autism spectrum disorder in Jacobsen syndrome through impaired GABAA receptor trafficking  

A curative effect of clonazepam on autistic-like behaviour


These results demonstrate that ASD-like behaviour in PX-RICS−/− mice is caused by impaired postsynaptic GABA signalling and that GABAAR agonists have the potential to treat ASD-like behaviour in JBS patients and possibly non-syndromic ASD individuals.


Jacobsen syndrome is a condition mainly found in girls and it is one of those more rare small-headed conditions (microcephaly). It features MR/ID and often an autism diagnosis.  It is caused by missing part of chromosome 11, apparently one of the most disease-rich chromosomes.

The fact that low-dose (sub-anxiolytic) clonazepam rescued the autistic behavior in mice does not mean that anyone has tried it in little girls with Jacobsen syndrome; that would require too much common sense.