Piperine/Resveratrol/Sunitinib for Rett’s and indeed much Autism? Or, R-Baclofen to raise KCC2 expression in Bumetanide-responsive autism.

Piperine/Pepper             Resveratrol/Red wine          Sunitinib/Sutent

  

This post is all about lowering chloride within neurons, by increasing the expression of the transporter that lets it leave, called KCC2.

Today’s post is one I never finished writing from last year; I looked up the price of Sutent/Sunitinib and then I remembered why. It does again highlight how cancer drugs, when they become cheap generics, will provide interesting options for autism treatment. It also shows again how Rett Syndrome is getting attention from researchers.

It also highlights that really clever Americans are looking for bumetanide alternatives, in the false belief that bumetanide has troubling side effects that cannot be managed/mitigated.

The study is by some clever guys in Cambridge Massachusetts.

Another group of clever guys from MIT burned through $40 million dollars a few years ago trying to develop R-Baclofen for Fragile-X and autism.  After that Roche-funded clinical trial failed, R-Baclofen has now been resurrected and a new trial is planned, with different end points (measures of success).

Today we see why many people should indeed respond positively to R-Baclofen, but the mode of action is entirely different to the one originally targeted by the clever guys from MIT.

Tucked away in the supplementary material of today’s paper we see that R-Baclofen increases the expression of the transporter (KCC2) that takes chloride out of neurons. So, R-Baclofen is doing the same thing as Bumetanide, just to a lesser extent and in a different way.  Both lower intracellular chloride.

That means that people responsive to bumetanide should get a further boost from R baclofen, but you might need a lot of it.

Clever they may be, but these researchers do not know how to communicate their findings.  I had to dig through the supplementary tables to extract the good stuff, which is a list of what substances increase KCC2 in regular brains (Table S1) and specifically in Rett Syndrome brains (Table S2).

This blog does rather bang on about blocking/inhibiting NKCC1 that lets chloride into neurons, you can of course alternatively open up KCC2 to let the chloride flood out. This latter strategy is proposed by the MIT researchers.

What really matters is the ratio KCC2/NKCC1.  In people with bumetanide-responsive autism, which pretty clearly will include girls with Rett Syndrome, you want to increase KCC2/NKCC1. So, block/down-regulate NKCC1 and/or up-regulate KCC2.

·        NKCC1↓

·        KCC2↑

The researchers identified 14 compounds.  To be useful as drugs these compounds have to be able to cross the blood brain barrier to be of much use, many do not.

In the paper they call KCC2 expression-enhancing compounds KEECs.

We have five approved drugs to add to the list that are functionally the same to primary hit compounds. 

·        Sunitinib

·        Crenolanib

·        Indirubin Monoxiome

·        Cabozantinib

·        TWS-119


The researchers went on to test just two compounds in Rett syndrome mice; they picked piperine (from black pepper) and KW 2449 (a leukemia drug)

Even R-baclofen pops up, with a “B score” of 6.65 (needs to be >3 to increase KCC2 expression).

Pharmacological enhancement of KCC2 gene expression exerts therapeutic effects on human Rett syndrome neurons and Mecp2 mutant mice.

Abstract

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. There are currently no approved treatments for RTT. The expression of K⁺/Cl^- cotransporter 2 (KCC2), a neuron-specific protein, has been found to be reduced in human RTT neurons and in RTT mouse models, suggesting that KCC2 might play a role in the pathophysiology of RTT. To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the KCC2 gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of KCC2 gene expression in genome-edited human reporter neurons. From an unbiased screen of more than 900 small-molecule chemicals, we have identified a group of compounds that enhance KCC2 expression termed KCC2 expression-enhancing compounds (KEECs). The identified KEECs include U.S. Food and Drug Administration-approved drugs that are inhibitors of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3β (GSK3β) pathways and activators of the sirtuin 1 (SIRT1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment with hit compounds increased KCC2 expression in human wild-type (WT) and isogenic MECP2 mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in Mecp2 mutant mice ameliorated disease-associated respiratory and locomotion phenotypes. The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders involving dysregulation of KCC2.

Table S1. KEECs identified from screening with WT human KCC2 reporter neurons.

Table S2. KEECs identified from screening with RTT human KCC2 reporter neurons

Note Baclofen, Quercetin, Luteolin etc

Fig. 3. Identification of KEECs that increase KCC2 expression in human RTT neurons

B score >3 indicates compounds potentially increasing KCC2 expression

In cultured RTT neurons, treatment with KEECs KW-2449 and BIO restored the impaired KCC2 expression and rescued deficits in both GABAergic and glutamatergic neurotransmissions, as well as abnormal neuronal morphology. Previous data suggested that disrupted Cl− homeostasis in the brainstem causes abnormalities in breathing pattern (64), consistent with breathing abnormalities seen in mice carrying a conditional Mecp2 deletion in GABAergic neurons (67). The reduction in locomotion activity observed in the Mecp2 mutant mice has also been attributed to abnormalities in the GABAergic system (65). Therefore, treatment with the KEEC KW-2449 or piperine may ameliorate disease phenotypes in MeCP2 mutant mice through restoration of the impaired KCC2 expression and GABAergic inhibition.

Most KEECs that enhanced KCC2 expression in WT neurons, including KW-2449, BIO, and resveratrol, also induced a robust increase of KCC2 reporter activity in RTT neurons (Fig. 3, A and B; a complete list of hit compounds is provided in table S2). The increase in KCC2 signal induced by KEECs was higher in RTT neurons than in WT neurons,

1. 
   

Our results establish a causal relationship between reduced FLT3 or GSK3 signaling activity and increased KCC2 expression.

Two hit compounds, resveratrol and piperine, act on different pathways than the kinase inhibitors, activating the SIRT1 signaling pathway (50) and the TRPV1 (51), respectively

Thus, our data demonstrate that activation of the SIRT1 pathway or the TRPV1 channel enhances KCC2 expression in RTT human neurons.

The group of KEECs reported here may help to elucidate the molecular mechanisms that regulate KCC2 gene expression in neurons. A previous study conducted with a glioma cell line showed that resveratrol activates the SIRT1 pathway and reduces the expression of NRSF/REST (50), a transcription factor that suppresses KCC2 expression (52). Our results demonstrate that resveratrol increases KCC2 expression by a similar mechanism, which could contribute to the therapeutic benefit of resveratrol on a number of brain disease conditions (68, 69). We also identified a group of GSK3 pathway inhibitors as KEECs. Overactivation of the GSK3 pathway has been reported in a number of brain diseases (70). Thus, our results suggest that GSK3 pathway inhibitors could exert beneficial effects on brain function through stimulating KCC2 expression. Another major KEEC target pathway, the FLT3 kinase signaling, has been investigated as a cancer therapy target (71, 72). Although FLT3 is expressed in the brain (73), drugs that target FLT3 pathway have not been extensively studied as potential treatments for brain diseases. Our results provide the first evidence that FLT3 signaling in the brain is critical for the regulation of key neuronal genes such as KCC2. Therefore, this work lays the foundation for further research to repurpose a number of clinically approved FLT3 inhibitors as novel brain disease therapies

Our results are valuable for the development of novel therapeutic strategies to treat neurodevelopmental diseases through rectification of dysfunctional neuronal chloride homeostasis. Because of the lack of pharmaceutical reagents that enhance KCC2 expression, bumetanide, a blocker of the inward chloride transporter NKCC1 that counteracts KCC2, has been used as an alternative (74). Bumetanide treatment has shown benefits in treating symptoms in mouse models of fragile X syndrome (75) and Down’s syndrome (76) and was shown to confer symptomatic benefit to human patients with autism or fragile X syndrome (77, 78). These findings strongly suggest that pharmacological restoration of disrupted chloride homeostasis may provide symptomatic treatment for various neurodevelopmental and neuropsychiatric disorders. However, NKCC1 lacks the neuron- restricted expression pattern of KCC2 and is also expressed in nonbrain tissue including kidney and inner ears (79), consistent with knockout of Nkcc1 in mouse model leading to deafness and imbalance (30). Therefore, bumetanide treatment may trigger undesirable side effects, thus severely limiting its therapeutic application. In contrast, the expression of KCC2 is restricted to neurons, and a number of the KEECs identified in this study that enhance KCC2 expression in neurons are Food and Drug Administration–approved and have not elicited any severe adverse effects in clinical trials (80–83). The promising efficacy of KEECs demonstrated in this study and the known safety of the KCC2 target warrant further preclinical and clinical studies to investigate these drugs and their derivatives as potential therapies for neurodevelopmental diseases.

In summary, in this work, we investigated the efficacy of KEECs to rescue a number of well-documented cellular and behavior phenotypes of RTT, including impaired GABA functional switch, reductions in excitatory synapse number and strength, immature neuronal morphology (53, 54), as well as an increase in breathing pauses and a decrease in locomotion (84). It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases (17, 85) including epilepsy (86–88), schizophrenia (19, 20, 89), brain and spinal cord injury (21, 90), stroke and ammonia toxicity conditions (91–93), as well as the impairments in learning and memory observed in the senile brain (23). Thus, a phenotypically diverse array of brain diseases may benefit from enhancing the expression of KCC2. The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. There are currently no approved treatments for RTT. The expression of K+/Cl− cotransporter 2 (KCC2), a neuron-specific protein, has been found to be reduced in human RTT neurons and in RTT mouse models, suggesting that KCC2 might play a role in the pathophysiology of RTT. To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the KCC2 gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of KCC2 gene expression in genome-edited human reporter neurons. From an unbiased screen of more than 900 small-molecule chemicals, we have identified a group of compounds that enhance KCC2 expression termed KCC2 expression– enhancing compounds (KEECs). The identified KEECs include U.S. Food and Drug Administration–approved drugs that are inhibitors of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3 (GSK3) pathways and activators of the sirtuin 1 (SIRT1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment with hit compounds increased KCC2 expression in human wild-type (WT) and isogenic MECP2 mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in Mecp2 mutant mice ameliorated disease-associated respiratory and locomotion phenotypes. The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders involving dysregulation of KCC2.

By screening these KCC2 reporter human neurons, we identified a number of hits KCC2 expression–enhancing compounds (KEECs) from ~900 small-molecule compounds. Identified KEECs were validated by Western blot and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments on cultured human wild-type (WT) and isogenic RTT neurons, as well as on organotypic mouse brain slices. Pharmacological and molecular biology experiments showed that identified KEECs act through inhibition of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3b (GSK3b) kinases, or activation of the sirtuin 1 (SIRT1) or transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment of RTT neurons with KEECs rescued disease-related deficits in GABA functional switch, excitatory synapses, and neuronal morphological development. Last, injection of the identified KEEC KW-2449 or piperine into a Mecp2 mutant mice ameliorated behavioral phenotypes including breathing pauses and reduced locomotion, which represent important preclinical data, suggesting that the KEECs identified in this study may be effective in restoring impaired E/I balance in the RTT brain and provide symptomatic treatment for patients with RTT.

Fig. 2. KEEC treatment–induced enhancement of KCC2 protein and mRNA expression in cultured organotypic mouse brain slices and a hyperpolarizing EGABA shift in cultured immature neurons.

(E to G) KCC2 and NKCC1 mRNA expression induced by FLT3 inhibitors including sunitinib (n = 4), XL-184 (n = 6), crenolanib (n = 4), or a structural analog of BIO termed indirubin monoxime (n = 6). The calculated ratios of KCC2/NKCC1 mRNA expression are shown in (G). A.U., arbitrary units

Our results are valuable for the development of novel therapeutic strategies to treat neurodevelopmental diseases through rectification of dysfunctional neuronal chloride homeostasis. Because of the lack of pharmaceutical reagents that enhance KCC2 expression, bumetanide, a blocker of the inward chloride transporter NKCC1 that counteracts KCC2, has been used as an alternative (74). Bumetanide treatment has shown benefits in treating symptoms in mouse models of fragile X syndrome (75) and Down’s syndrome (76) and was shown to confer symptomatic benefit to human patients with autism or fragile X syndrome (77, 78). These findings strongly suggest that pharmacological restoration of disrupted chloride homeostasis may provide symptomatic treatment for various neurodevelopmental and neuropsychiatric disorders. However, NKCC1 lacks the neuron restricted expression pattern of KCC2 and is also expressed in nonbrain tissue including kidney and inner ears (79), consistent with knockout of Nkcc1 in mouse model leading to deafness and imbalance (30). Therefore, bumetanide treatment may trigger undesirable side effects, thus severely limiting its therapeutic application. In contrast, the expression of KCC2 is restricted to neurons, and a number of the KEECs identified in this study that enhance KCC2 expression in neurons are Food and Drug Administration–approved and have not elicited any severe adverse effects in clinical trials (80–83). The promising efficacy of KEECs demonstrated in this study and the known safety of the KCC2 target warrant further preclinical and clinical studies to investigate these drugs and their derivatives as potential therapies for neurodevelopmental diseases.

In summary, in this work, we investigated the efficacy of KEECs to rescue a number of well-documented cellular and behavior phenotypes of RTT, including impaired GABA functional switch, reductions in excitatory synapse number and strength, immature neuronal morphology (53, 54), as well as an increase in breathing pauses and a decrease in locomotion (84). It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases (17, 85) including epilepsy (86–88), schizophrenia (19, 20, 89), brain and spinal cord injury (21, 90), stroke and ammonia toxicity conditions (91–93), as well as the impairments in learning and memory observed in the senile brain (23). Thus, a phenotypically diverse array of brain diseases may benefit from enhancing the expression of KCC2. The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders.

The science-light version:-

Drug screen reveals potential treatments for Rett syndrome

An experimental leukemia drug and a chemical in black pepper ease breathing and movement problems in a mouse model of Rett syndrome, according to a new study.

Rett syndrome is a rare brain condition related to autism, caused by mutations in the MECP2 gene. Because the gene is located on the X chromosome, the syndrome occurs almost exclusively in girls. No drugs are available to treat Rett.

The team screened 929 compounds from three large drug libraries, including one focused on Rett therapies. They found 30 compounds that boost KCC2’s expression in the MECP2 neurons; 14 of these also increased the protein’s expression in control neurons.

The team tested two of the identified compounds in mice with mutations in MECP2: KW-2449, which is a small molecule in clinical trials for leukemia, and piperine, an herbal supplement and component of black pepper. These mice have several traits reminiscent of Rett. They are prone to seizures, breathing problems, movement difficulties and disrupted social behavior.

Injecting the mice with either drug daily for two weeks improved the animals’ mobility relative to untreated mice. The drugs also eased the mice’s breathing problems, decreasing the frequency of pauses in breathing (apnea). The findings appeared in July in Science Translational Medicine.

 

Piperine, Resveratrol and analogs thereof

Piperine and Resveratrol are commercially available supplements.

Resveratrol has been mentioned many times in this blog.  It has numerous beneficial properties, to which we can now add increasing KCC2 expression, but it is held back by its poor ability to cross the blood barrier.

The other natural substance highlighted in the study is piperine. Piperine is the substance that gets added to curcumin to increases its bioavailability and hopefully get its health benefits.

Piperine has been recently been found to be a positive allosteric modulator of GABA_A receptors.

It may be that piperine has 2 different effects on GABA, or maybe it is just the same one?

The result is that people are trying to develop modified versions of piperine that could be patentable commercial drugs.

Piperine also activated TRPV1 receptors.

You might wonder what is the effect in humans of plain old piperine in bumetanide-responsive autism.

Invitro blood–brain-barrier permeability predictions for GABAA receptor modulating piperine analogs

The alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABA_A) receptors. In order to reach their target sites of action, these compounds need to enter the brain by crossing the blood–brain barrier (BBB). We here evaluated piperine and five selected analogs (SCT-66, SCT-64, SCT-29, LAU397, and LAU399) regarding their BBB permeability. Data were obtained in three in vitro BBB models, namely a recently established human model with immortalized hBMEC cells, a human brain-like endothelial cells (BLEC) model, and a primary animal (bovine endothelial/rat astrocytes co-culture) model. For each compound, quantitative UHPLC-MS/MS methods in the range of 5.00–500 ng/mL in the corresponding matrix were developed, and permeability coefficients in the three BBB models were determined. In vitro predictions from the two human BBB models were in good agreement, while permeability data from the animal model differed to some extent, possibly due to protein binding of the screened compounds. In all three BBB models, piperine and SCT-64 displayed the highest BBB permeation potential. This was corroborated by data from in silico prediction. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human BBB models, and moderate to high in the animal BBB model. Efflux ratios (ER) calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux transporters.

The alkaloid piperine, the major pungent component of black pepper (Piper nigrum L.), was recently identified as a positive allosteric γ-aminobutyric acid type A (GABAA) receptor modulator. The compound showed anxiolytic-like activity in behavioral mouse models, and was found to interact with the GABAA receptors at a binding site that was independent of the benzodiazepine binding site [1,2]. Given that the compound complied with Lipinski’s “rule of five” [1], it represented a new scaffold for the development of novel GABAA receptor modulators [1–3]. Given that piperine also activates the transient receptor potential vanilloid 1 (TRPV1) receptors [4] which are involved in pain signaling and regulation of the body temperature [5,6], structural modification of the parent compound was required to dissect GABAA and TRPV1 activating properties

For drugs acting on the central nervous system (CNS), brain penetration is required. This process is controlled by the blood-brain barrier (BBB), a tight layer of endothelial cells lining the brain capillaries that limits the passage of molecules from the blood circulation into the brain [10]. Since low BBB permeability can reduce CNS exposure [11], lead compounds should be evaluated at an early stage of the drug development process for their ability to permeate the BBB [12].

Conclusions

Piperine and five selected piperine analogs with positive GABAA receptor modulatory activity were screened in three in vitro cell-based human and animal BBB models for their ability to cross the BBB. Data from the three models differed to some extent, possibly due to protein binding of the piperine analogs. In all three models, piperine and SCT-64 displayed the highest BBB permeation potential, which could be corroborated by in silico prediction data. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human models, and moderate to high in the animal model. ER calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux. In addition to the early in vitro BBB permeability assessment of the compounds, further studies (such as PK and drug metabolism studies) are currently in progress in our laboratory. Taken together, these data will serve for selecting the most promising candidate molecule for the next cycle of medicinal chemistry optimization

Conclusion

My conclusions are a little different to the MIT researchers

“The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders.”

This assumes that you cannot safely use bumetanide/azosemide, which you can.  Open your eyes and look at France, where several hundred children with autism are safely taking bumetanide.

”It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases”

We have copious evidence that elevated chloride is a feature of many conditions, not just Rett’s and an effective cheap therapy has been sitting in the pharmacy for decades.

In the clinical trial of R-Baclofen that failed, there were some positive effects on some subjects.  Were the positive effects just caused by the effect of Baclofen in increasing KCC2 expression?

Should R-Baclofen become a cheap generic, it might indeed become a useful add-on for those with bumetanide-responsive. Regular Baclofen (Lioresal) is an approved drug, but it does have some side effects, so most likely R-baclofen will have side effects in some.

Baclofen itself in modest doses has little effect on bumetanide-responsive autism.

A cheap side-effect free KCC2 enhancer would be a good drug for autism, although cheap, safe NKCC1 blockers already exist. 

I have no idea if piperine benefits bumetanide-responsive autism.  Piperine has long been used in traditional medicine.

The TRPV1 receptor also affected by piperine plays a role in pain and anxiety.

We saw in the post below that TRPV1 controls cortical microglia activation and that GABARAP modulates TRPV1 expression.

So, TRPV1 and GABA_A receptors are deeply intertwined.

  

GABAa receptor trafficking, Migraine, Pain, Light Sensitivity, Autophagy, Jacobsen Syndrome,Angelman Syndrome, GABARAP, TRPV1, PX-RICS, CaMKII and CGRP ... Oh and the"fever effect"

Is Piperine going to make autism better, or worse?

GABAa receptor trafficking, Migraine, Pain, Light Sensitivity, Autophagy, Jacobsen Syndrome, Angelman Syndrome, GABARAP, TRPV1, PX-RICS, CaMKII and CGRP ... Oh and the "fever effect"

The mechanism controlling transporting just the “right” number of GABA_A receptors

Today’s post is not for the faint-hearted.  It is another one that could just keep on rolling.  Ling will like it.

It again shows that GABA_A receptors are at the centre of much autism, whether single gene or idiopathic. Today we highlight what can go wrong as these receptors are “transported”.

Today’s post also draws on several quite recent papers. It seeks to tie together some previous things mentioned in this blog like the symptoms of pain, particularly felt in the head, sensory sensitivity with dysfunction processes like autophagy and linking it all back to the GABA_A receptor.  There is even a link at the end to the "fever effect", which occurs when a high temperature in some people causes a marked improvement in their autism symptoms.

We will come across some expensive drugs like Erenumab, the medical food PEA (Palmitoylethanolamide) and indeed Natasa’s favourite, CBD (Cannabidiol) and a newcomer CBDV (Cannabidivarin).   

We come across a protein called GABARAP (GABA_A receptor associated protein) for the first time in this blog.  There is a vast amount in this blog about the GABA_A receptor, how and why to modulate it. 

CaMKII makes an appearance, this is a protein kinase that is miss-regulated in much neurological disease. It changes the effect of many other proteins, acting just like a switch, by chemically adding phosphate groups to them. We have previously seen how important the protein kinases PKA, PKB and PKC are to autism.  Today add CaMKII to the list.

We come across another distinctive “face” of autism, this time it is Jacobsen syndrome, which I think is easily spotted by the trained eye, or some facial recognition software.  Jacobsen syndrome is a rare chromosomal disorder resulting from deletion of genes from chromosome 11 that includes band 11q24. This may include the gene that encodes the protein PX-RICS and, if so, it will lead to “autism”. Loss of that gene should be treatable with a GABA agonist.     

We also come back to that happy puppet syndrome (Angelman syndrome) which usually involves loss of the gene UBE3A, from chromosome 15. What I found interesting was that both Jacobsen syndrome and Angelman syndrome should share impaired GABA_A receptor trafficking as a feature. They each have a different impediment that should reduce the number of functioning GABA_A receptors. In the case of Angelman the impediment is CaMKII inhibition, in Jacobsen it is lack of the protein PX-RICS. Angelman syndrome may well respond to the same therapy as Jacobsen syndrome – a GABA agonist, of just a PAM (positive allosteric modulator, to “turn up the volume”).

Back to GABARAP

GABARAP has multiple functions:

1.     Transport of freshly minted GABA_A receptors

In order for newly minted GABA_A receptors to get to their final destination it requires four “helpers”: GABARAP, PX-RICS, 14-3-3 and Dynactin.  In addition, you need a dose of CaMKII. If you lack any one of these four, you will end up with reduced expression of GABA_A receptors. If CaMKII is overactivated you get too many GABA_A receptors.

In Jacobsen Syndrome there is reduced GABA_A receptor trafficking/transport, leading to reduced surface expression. (in effect not enough functioning GABA_A receptors in situ).  In some people with this syndrome the part of their DNA which encodes PX-RICS is missing.  This lack of PX-RICS produces autism.  The autism-like behavioural abnormalities in PX-RICS-deficient mice are ameliorated by enhancing inhibitory synaptic transmission with a GABA_AR agonist.

2.     GABARAP modulates TRPV1 expression

GABARAP also does something totally different, it modulates TRPV1 ion channels, that we have previously touched on in this blog.  This then triggers a cascade of effects relating to pain, neuralgia, migraine headaches, microglial activation, epilepsy and indeed longevity.

The simple function of TRPV1 is detection and regulation of body temperature. In addition, TRPV1 provides a sensation of scalding heat and pain. TRPV1 is also known as the capsaicin receptor.  Capsaicin is the active component of chilli peppers.

TRPV1 not only plays a role in pain, but is suggested to play a role in migraine. In migraine TRPV1 plays a role along with calcitonin gene-related peptide receptor (CGRPR). TRPV1 determines how much of the CGRPR protein is produced. CGRPR affects your metabolism broadly and as such plays a key role in longevity.  Ablation of select pain sensory receptors (TRPV1) or the inhibition of CGRP are associated with increased metabolic health and longevity.

Erenumab/Aimovig is a medication which targets CGRPR for the prevention of migraine. It was the first of the group of CGRPR antagonists to be FDA approved in 2018. It is a form of monoclonal antibody therapy in which antibodies are used to block the receptors for the protein CGRP, thought to play a major role in starting migraines.

Recent evidence suggests that TRPV1 may contribute to the onset and progression of some forms of epilepsy;  Cannabidivarin  (CBDV) and cannabidiol (CBD), activate and desensitize TRPV1.

TRPV1 also plays a crucial role in the activation of microglia. As the researchers put it “TRPV1 channels are critical brain inflammation detectors … microglia shifted toward an anti-inflammatory phenotype when TRPV1 is lacking. “

So, if we jump a few steps forward we can see that desensitizing TRPV1 might be helpful for people with: -

·        Some epilepsy

·        Some neuralgia

·        Perhaps some with chronic migraine

·        People with activated microglia, which is most autism

We also can see that a dysfunction in GABARAP may itself contribute to worsening the above conditions via its effect on TRPV1.

Non-psychotropic plant cannabinoids, cannabidivarin (CBDV) and cannabidiol (CBD), activate and desensitize transient receptor potential vanilloid 1 (TRPV1) channels in vitro: potential for the treatment of neuronal hyperexcitability

Epilepsy is the most common neurological disorder, with over 50 million people worldwide affected. Recent evidence suggests that the transient receptor potential cation channel subfamily member 1 (TRPV1) may contribute to the onset and progression of some forms of epilepsy. V Since the two nonpsychotropic cannabinoids cannabidivarin (CBDV) and cannabidiol (CBD) exert anticonvulsant activity in vivo and produce TRPV1-mediated intracellular calcium elevation in vitro, we evaluated the effects of these two compounds on TRPV1 channel activation and desensitization and in an in vitro model of epileptiform activity. Patch clamp analysis in transfected HEK293 cells demonstrated that CBD and CBDV dose-dependently activate and rapidly desensitize TRPV1, as well as TRP channels of subfamily V type 2 (TRPV2) and subfamily A type 1 (TRPA1). TRPV1 and TRPV2 transcripts were shown to be expressed in rat hippocampal tissue. When tested on epileptiform neuronal spike activity in hippocampal brain slices exposed to a Mg2+-free solution using multielectrode arrays (MEAs), CBDV reduced both epileptiform burst amplitude and duration. The prototypical TRPV1 agonist, capsaicin, produced similar, although not identical effects. Capsaicin, but not CBDV, effects on burst amplitude were reversed by IRTX, a selective TRPV1 antagonist. These data suggest that CBDV antiepileptiform effects in the Mg2+-free model are not uniquely mediated via activation of TRPV1. However, TRPV1 was strongly phosphorylated (and hence likely sensitized) in Mg2+-free solution-treated hippocampal tissue, and both capsaicin and CBDV caused TRPV1 dephosphorylation, consistent with TRPV1 desensitization. We propose that CBDV effects on TRP channels should be studied further in different in vitro and in vivo models of epilepsy.

TRPV1 channels are critical brain inflammation detectors and neuropathic pain biomarkers in mice

The capsaicin receptor TRPV1 has been widely characterized in the sensory system as a key component of pain and inflammation. A large amount of evidence shows that TRPV1 is also functional in the brain although its role is still debated. Here we report that TRPV1 is highly expressed in microglial cells rather than neurons of the anterior cingulate cortex and other brain areas. We found that stimulation of microglial TRPV1 controls cortical microglia activation per se and indirectly enhances glutamatergic transmission in neurons by promoting extracellular microglial microvesicles shedding. Conversely, in the cortex of mice suffering from neuropathic pain, TRPV1 is also present in neurons affecting their intrinsic electrical properties and synaptic strength. Altogether, these findings identify brain TRPV1 as potential detector of harmful stimuli and a key player of microglia to neuron communication.

TRPV1 controls cortical microglia activation

In the healthy mature brain, microglial cells play a role in immune surveillance and ensure the maintenance of brain homeostasis. Upon injuries these cells shift to an activated state characterized by drastic changes in the cellular shape, functional behavior and by the release of different proinflammatory and immunoregulatory factors^58,59. Conforming to the capsaicin-mediated induction of microglial chemotaxis²⁹, we investigated whether TRPV1 stimulation regulates the morphology of microglial cells…. Thus, stimulation of TRPV1 induced a pro-inflammatory phenotype of microglia from WTs. Conversely, microglia shifted toward an anti-inflammatory phenotype when TRPV1 is lacking.

Angelman syndrome

Angelman syndrome (Happy puppet syndrome) is a genetic disorder that mainly affects the nervous system. Symptoms include a small head and a specific facial appearance, severe intellectual disability, developmental disability, speaking problems, balance and movement problems, seizures, and sleep problems. Children usually have a happy personality and have a particular interest in water. The symptoms generally become noticeable by one year of age.  Angelman syndrome is typically due to a new mutation rather than one inherited from a person's parents. Angelman syndrome is due to a lack of function of part of chromosome 15 inherited from a person's mother. Most of the time, it is due to a deletion or mutation of the UBE3A gene.

CaMKII inhibition underlies Angelman Syndrome

CaMKII

CaMKII is a serine/threonine-specific protein kinase that is regulated by the Ca²⁺/calmodulin complex. CaMKII is involved in many signaling cascades and is thought to be an important mediator of learning and memory. CaMKII is also necessary for Ca²⁺ homeostasis and reuptake in cardiomyocytes, chloride transport in epithelia, positive T-cell selection, and CD8 T-cell activation.

Misregulation of CaMKII is linked to Alzheimer’s disease, Angelman syndrome, and heart arrhythmia.

Emerging role of CaMKII in neuropsychiatric disease

Recent evidence for CaMKII dysregulation in psychiatric diseases is reviewed.

Changes in postsynaptic structure and function appear to be central to multiple diseases.

Altered regulation of the CaMKIIα gene promoter may be a common mechanism among diseases.

CaMKII dysregulation in diverse brain regions may account for myriad disorders.

Although it has been known for decades that hippocampal calcium/calmodulin (CaM)-dependent protein kinase II (CaMKII) plays an essential role in learning and memory consolidation, the roles of CaMKII in other brain regions are only recently being explored in depth. A series of recent studies suggest that CaMKII dysfunction throughout the brain may underlie myriad neuropsychiatric disorders, including drug addiction, schizophrenia, depression, epilepsy, and multiple neurodevelopmental disorders, perhaps through maladaptations in glutamate signaling and neuroplasticity. I review here the structure, function, subcellular localization, and expression patterns of CaMKII isoforms, as well as recent advances demonstrating that disturbances in these properties may contribute to psychiatric disorders.

A Novel Human CAMK2A Mutation Disrupts Dendritic Morphology and Synaptic Transmission, and Causes ASD-Related Behaviors

Characterizing the functional impact of novel mutations linked to autism spectrum disorder (ASD) provides a deeper mechanistic understanding of the underlying pathophysiological mechanisms. Here we show that a de novo Glu183 to Val (E183V) mutation in the CaMKIIα catalytic domain, identified in a proband diagnosed with ASD, decreases both CaMKIIα substrate phosphorylation and regulatory autophosphorylation, and that the mutated kinase acts in a dominant-negative manner to reduce CaMKIIα-WT autophosphorylation. The E183V mutation also reduces CaMKIIα binding to established ASD-linked proteins, such as Shank3 and subunits of l-type calcium channels and NMDA receptors, and increases CaMKIIα turnover in intact cells. In cultured neurons, the E183V mutation reduces CaMKIIα targeting to dendritic spines. Moreover, neuronal expression of CaMKIIα-E183V increases dendritic arborization and decreases both dendritic spine density and excitatory synaptic transmission. Mice with a knock-in CaMKIIα-E183V mutation have lower total forebrain CaMKIIα levels, with reduced targeting to synaptic subcellular fractions. The CaMKIIα-E183V mice also display aberrant behavioral phenotypes, including hyperactivity, social interaction deficits, and increased repetitive behaviors. Together, these data suggest that CaMKIIα plays a previously unappreciated role in ASD-related synaptic and behavioral phenotypes.

SIGNIFICANCE STATEMENT Many autism spectrum disorder (ASD)-linked mutations disrupt the function of synaptic proteins, but no single gene accounts for >1% of total ASD cases. The molecular networks and mechanisms that couple the primary deficits caused by these individual mutations to core behavioral symptoms of ASD remain poorly understood. Here, we provide the first characterization of a mutation in the gene encoding CaMKIIα linked to a specific neuropsychiatric disorder. Our findings demonstrate that this ASD-linked de novo CAMK2A mutation disrupts multiple CaMKII functions, induces synaptic deficits, and causes ASD-related behavioral alterations, providing novel insights into the synaptic mechanisms contributing to ASD.

Jacobsen Sydrome

The signs and symptoms of Jacobsen syndrome can vary. Most affected people have delayed development of motor skills and speech; cognitive impairment; and learning difficulties. Behavioral features have been reported and may include compulsive behavior; a short attention span; and distractibility. Many people with the condition are diagnosed with attention deficit-hyperactivity disorder (ADHD). The vast majority of people with Jacobsen syndrome also have a bleeding disorder called Paris-Trousseau syndrome, which causes abnormal bleeding and easy bruising. 

People with Jacobsen syndrome typically have distinctive facial features, which include small and low-set ears; wide-set eyes (hypertelorism) with droopy eyelids (ptosis); skin folds covering the inner corner of the eyes; a broad nasal bridge; down-turned corners of the mouth; a thin upper lip; and a small lower jaw (micrognathia). Affected people often have a large head (macrocephaly) and a skull abnormality called trigonocephaly, giving the forehead a pointed appearance.

The Autism-Related Protein PX-RICS Mediates GABAergic Synaptic Plasticity in Hippocampal Neurons and Emotional Learning in Mice

GABAergic dysfunction underlies many neurodevelopmental and psychiatric disorders. GABAergic synapses exhibit several forms of plasticity at both pre- and postsynaptic levels. NMDA receptor (NMDAR)–dependent inhibitory long-term potentiation (iLTP) at GABAergic postsynapses requires an increase in surface GABA_ARs through promoted exocytosis; however, the regulatory mechanisms and the neuropathological significance remain unclear. Here we report that the autism-related protein PX-RICS is involved in GABA_AR transport driven during NMDAR–dependent GABAergic iLTP. Chemically induced iLTP elicited a rapid increase in surface GABA_ARs in wild-type mouse hippocampal neurons, but not in PX-RICS/RICS–deficient neurons. This increase in surface GABA_ARs required the PX-RICS/GABARAP/14–3-3 complex, as revealed by gene knockdown and rescue studies. iLTP induced CaMKII–dependent phosphorylation of PX-RICS to promote PX-RICS–14-3-3 assembly. Notably, PX-RICS/RICS–deficient mice showed impaired amygdala–dependent fear learning, which was ameliorated by potentiating GABAergic activity with clonazepam. Our results suggest that PX-RICS–mediated GABA_AR trafficking is a key target for GABAergic plasticity and its dysfunction leads to atypical emotional processing underlying autism.

There is a growing consensus that autism arises from the atypical regulation of the excitation/inhibition balance within specific neural microcircuitry. In terms of neural inhibition, autism is closely related to dysfunctional inhibitory signaling mediated by the γ-aminobutyric acid (GABA) type A receptors (GABA_ARs). Impaired presynaptic release of GABA and postsynaptic trafficking of GABA_ARs lead to autistic-like social behavior in mouse models of autism. There is a significant reduction in the number of GABA_ARs and GABAergic activity in certain brain areas of autistic individuals. Genetic association studies have revealed that several GABA_AR subunits are linked to an increased risk for autism. GABA_AR–mediated signaling is thus essential for the proper regulation of the excitation/inhibition balance associated with socio-emotional cognition.

PX-RICS, GABARAP and 14-3-3ζ/θ are localized in the specific dendritic compartments that are immunopositive for organelle markers for the endoplasmic reticulum (ER), ER exit sites and the trans-Golgi network. This structure, termed the dendritic satellite secretory pathway, is comprised of the dendritic ER and the Golgi outposts and is involved in the local synthesis, processing and transport of membrane-integral or secretory proteins in dendrites. The rapid increase in surface-expressed GABA_ARs after NMDA stimulation could be explained by the localization of the PX-RICS–dependent trafficking machinery in the dendritic secretory compartments.

Several lines of evidence suggest that the dysregulation of GABA signaling underlies atypical social behavior in autism However, there has been no report describing deficits in GABAergic plasticity that contribute to autistic features. The present study has shown that PX-RICS is essential for GABAergic iLTP and that loss of the PX-RICS function in mice leads to impaired cued fear learning. Cued fear learning is closely associated with GABA_AR–mediated activity and plasticity in the amygdala and is inversely correlated with the severity of autistic symptoms. Considering all of these findings, we thus reason that PX-RICS–dependent GABA_AR transport may play critical roles in emotional learning in the amygdala through the control of GABAergic synaptic plasticity and that the impairment of this transport mechanism may lead to improper socio-emotional processing, resulting in autistic-like atypical social behavior (Supplementary Fig. 7). Further elucidation of the functional link between GABAergic plasticity and socio-emotional learning could lead to a better understanding of autism pathogenesis and treatment. 

We have previously identified and characterized two splicing isoforms of GTPase-activating proteins specific for Cdc42 predominantly expressed in neurons of the cerebral cortex, amygdala and hippocampus: RICS (ARHGAP32 isoform 2) and PX-RICS (ARHGAP32 isoform 1) . RICS regulates NMDAR–mediated signaling at the postsynaptic density and axonal elongation at the growth cone. In contrast, PX-RICS forms an adaptor complex with GABARAP and 14-3-3ζ/θ to facilitate steady-state trafficking of the N-cadherin/β-catenin complex and GABA_ARs. PX-RICS is also responsible for autistic-like features observed in more than half of the patients with Jacobsen syndrome (JBS) [3]. Mice lacking PX-RICS/RICS show marked decreases in surface-expressed GABA_ARs and GABA_AR–mediated inhibitory synaptic transmission, resulting in various autistic-like behaviors and autism-related comorbidities. Rare single-nucleotide variations in PX-RICS are also linked to non-syndromic autism, schizophrenia and alexithymia. These findings strongly suggest that dysfunction of PX-RICS–mediated GABA_AR trafficking has severe effects on socio-emotional processing of the brain.

Our previous study described above showed that PX-RICS and other components of the GABA_AR trafficking complex are required for constitutive transport of the receptor. In this study, we have focused on the role of PX-RICS in the activity–induced promotion of GABA_AR trafficking during iLTP. Here we show that PX-RICS–mediated GABA_AR trafficking is also involved in NMDAR activity–dependent trafficking of GABA_ARs and that PX-RICS is a key target of CaMKII for regulating GABAergic synaptic plasticity. Furthermore, we show that PX-RICS dysfunction in mice leads to impaired amygdala–dependent emotional learning, which manifests as autistic-like social behavior [3].

Supplementary Fig. 7. PX-RICS–mediated GABAAR trafficking underlies NMDAR–dependent GABAergic iLTP PX-RICS, GABARAP and 14-3-3s are assembled to form an adaptor complex that interconnects γ2-containing GABAARs (cargo) and dynein/dynactin (motor). Interaction

of PX-RICS with 14-3-3s depends on the phosphorylation activity of CaMKII, and this interaction is a critical regulatory point for GABAAR trafficking. When CaMKII activity is at a basal level, the PX-RICS–mediated trafficking complex has a role in steady-state transport of GABAARs to maintain the number of surface GABAARs as needed for proper synaptic inhibition.3 Neural activity that evokes moderate Ca2+ influx through NMDAR preferentially increases the activated form of CaMKII and elicits its translocation to inhibitory synapses, where it phosphorylates target proteins such as gephyrin and the GABAAR β3 subunit. Phosphorylated gephyrin and the GABAAR β3 subunit regulate the surface dynamics of GABAARs such as lateral diffusion and synaptic confinement. The present study has revealed that PXRICS

is a downstream CaMKII target associated with anterograde transport of

GABAARs. Enhanced PX-RICS phosphorylation increases the PX-RICS–14-3-3 complex and thereby drives de novo GABAAR surface expression, resulting in GABAergic iLTP. Dysfunction of this trafficking mechanism in the amygdala causes impaired GABAergic synaptic plasticity, which may contribute to deficits in socioemotional behavior as observed in PX-RICS/RICS–deficient mice and JBS patients with autism.

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

Jacobsen syndrome (JBS) is a rare congenital disorder caused by a terminal deletion of the long arm of chromosome 11. A subset of patients exhibit social behavioural problems that meet the diagnostic criteria for autism spectrum disorder (ASD); however, the underlying molecular pathogenesis remains poorly understood. PX-RICS is located in the chromosomal region commonly deleted in JBS patients with autistic-like behaviour. Here we report that PX-RICS-deficient mice exhibit ASD-like social behaviours and ASD-related comorbidities. PX-RICS-deficient neurons show reduced surface γ-aminobutyric acid type A receptor (GABAAR) levels and impaired GABAAR-mediated synaptic transmission. PX-RICS, GABARAP and 14-3-3ζ/θ form an adaptor complex that interconnects GABAAR and dynein/dynactin, thereby facilitating GABAAR surface expression. ASD-like behavioural abnormalities in PX-RICS-deficient mice are ameliorated by enhancing inhibitory synaptic transmission with a GABAAR agonist. Our findings demonstrate a critical role of PX-RICS in cognition and suggest a causal link between PX-RICS deletion and ASD-like behaviour in JBS patients.

TRPV1

We now come back to TRPV1, which we saw is modulated by GABARAP.

GABAA receptor associated protein (GABARAP) modulates TRPV1 expression and channel function and desensitization

Transient receptor potential vanilloid (TRPV1) transduces noxious chemical and physical stimuli in high-threshold nociceptors. The pivotal role of TRPV1 in the physiopathology of pain transduction has thrust the identification and characterization of interacting partners that modulate its cellular function. Here, we report that TRPV1 associates with γ-amino butyric acid A-type (GABA_A) receptor associated protein (GABARAP) in HEK293 cells and in neurons from dorsal root ganglia coexpressing both proteins. At variance with controls, GABARAP augmented TRPV1 expression in cotransfected cells and stimulated surface receptor clustering. Functionally, GABARAP expression attenuated voltage and capsaicin sensitivity of TRPV1 in the presence of extracellular calcium. Furthermore, the presence of the anchor protein GABARAP notably lengthened the kinetics of vanilloid-induced tachyphylaxia. Notably, the presence of GABARAP selectively increased the interaction of tubulin with the C-terminal domain of TRPV1. Disruption of tubulin cytoskeleton with nocodazole reduced capsaicin-evoked currents in cells expressing TRPV1 and GABARAP, without affecting the kinetics of vanilloid-induced desensitization. Taken together, these findings indicate that GABARAP is an important component of the TRPV1 signaling complex that contributes to increase the channel expression, to traffic and cluster it on the plasma membrane, and to modulate its functional activity at the level of channel gating and desensitization.

‘Entourage’ effectsof N‐palmitoylethanolamide and N‐oleoylethanolamide on vasorelaxation to anandamide occur through TRPV1 receptors

Age-Dependent Anti-seizure and Neuroprotective Effect of Cannabidivarin in Neonatal Rats

Neonatal seizures and seizures of infancy represent a significant cause of morbidity. 30–40% of infants and children with seizures will fail to achieve seizure remission with current anti-epileptic drug (AED) treatment. Moreover, pharmacotherapy during critical periods of brain development can adversely affect nervous system function. We, and others, have shown that early life exposure to AEDs including phenobarbital, phenytoin, and valproate are associated with induction of enhanced neuronal apoptosis during a confined period of postnatal development in rats. Thus, identification of new therapies for neonatal/infantile epilepsy syndromes that provide seizure control without neuronal toxicity is a high priority.

Current clinical trials report that modulation of the cannabinoid system with the phytocannabinoid cannabidiol exerts anti-seizure effects in children with epilepsy. While cannabidiol and the propyl analog cannabidivarin (CBDV) display anti-seizure efficacy in adult animal models of seizures/epilepsy, they remained unexplored in neonatal models. Therefore, we investigated the therapeutic potential of CBDV in multiple neonatal rodent seizure models. To evaluate the therapeutic potential of CBDV, we tested its anti-seizure efficacy in five models of neonatal seizures: pentylenetetrazole (PTZ), DMCM, hypoxia, kainate and NMDA-evoked spasms, each representing a different clinical seizure phenotype. We also evaluated the preclinical safety profile in the developing brain.

Postnatal day (P) 10 or P20 male, Sprague-Dawley rat pups were pretreated with CBDV or vehicle prior to chemically or hypoxia induced seizures. CBDV only displayed anticonvulsant effects in the P20 rat pups in the PTZ and DMCM models, with no effect on seizure severity or latency in the P10 animals. Therefore, we next measured the relative expression of known targets for CBDV (TRPV1, TRPA1) to determine a mechanism for which CBDV is anticonvulsant in P20, but not P10 animals. The P20 animals show increased expression of TRPV1 in key brain regions implicated in epileptogenic activity.

Together, these results indicate that modulation of the cannabinoid system in a receptor independent manner can provide seizure control in developing animals, but in an age specific manner. Further, during a developmentally sensitive neonatal period, drugs targeting the cannabinoid system do not induce neuronal apoptosis characteristic of many other AEDs. These results provide some of the first systemic, preclinical data evaluating CBDV in pediatric models of epilepsy.

Cannabidivarin (CBDV) vs. Placebo in Children With Autism Spectrum Disorder (ASD)

Weight-based dosing of 10 mg/kg/day of CBDV for 12 weeks

Primary Outcome Measures  :

1.     Aberrant Behavior Checklist-Irritability Subscale (ABC-I) [ Time Frame: Change in ABC-I from Baseline to Week 12 (Change over 12 weeks) ]

Change in ABC-I from Baseline to Endpoint

Safety and Tolerability of Cannabidivarin (CBDV) in Children and Young Adults With Autism Spectrum Disorder

  

Lack of Autophagy will reduce the number of GABA_A receptors, by blocking GABARAP function

Regular readers will recall that one feature of autism and many other neurological diseases is a reduction in autophagy, which I likened to an intra-cellular garbage collection service. 

The very recent paper below shows that lack of autophagy blocks GABARAP from its job to transport freshly minted GABA_A receptors.

If correct, this actually has very wide implications.

Autophagy links MTOR and GABAsignaling in the brain

The disruption of MTOR-regulated macroautophagy/autophagy was previously shown to cause autistic-like abnormalities; however, the underlying molecular defects remained largely unresolved. In a recent study, we demonstrated that autophagy deficiency induced by conditional Atg7 deletion in either forebrain GABAergic inhibitory or excitatory neurons leads to a similar set of autistic-like behavioral abnormalities even when induced following the peak period of synaptic pruning during postnatal neurodevelopment. Our proteomic analysis and molecular dissection further revealed a mechanism in which the GABA_A receptor trafficking function of GABARAP (gamma-aminobutyric acid receptor associated protein) family proteins was compromised as they became sequestered by SQSTM1/p62-positive aggregates formed due to autophagy deficiency. Our discovery of autophagy as a link between MTOR and GABA signaling may have implications not limited to neurodevelopmental and neuropsychiatric disorders, but could potentially be involved in other human pathologies such as cancer and diabetes in which both pathways are implicated.

Conclusion

You may have skipped to the conclusion to avoid all the science.

The conclusion is simple, you need to keep your GABA_A receptors in tip top form if you want to avoid the symptoms of autism.

o   You need the right number of them

o   You need the right balance among the five constituent subunits

o   You need the correct level of chloride inside neurons so the receptors are not “working backwards”

All of the genes that encode proteins involved in the above are individually “autism genes”, because any one of them can disrupt the process.

Whether it is Dravet syndrome (GABA_A receptor α₂ subunit), Angelman syndrome, Jacobsen syndrome, Down syndrome or numerous other autism syndromes, not to mention idiopathic autism, check the above 3 bullet points.

Tune up/down your GABA_A receptors!

Desensitizing TRPV1 looks interesting and not just for epilepsy.  TRPV1 appears to be essential for microglia in the in brain to be activated.  We know that in autism microglia in the brain are permanently activated, as if there was a threat.

I do think there is cross-talk (feedback loops etc) going on here, for example you can treat the severe epilepsy in Dravet syndrome by any of the following:-

·        KBr, to lower intracellular chloride

·        Low dose clonazepam to affect α subunits of GABA_A receptors

·        CBD or CBDV to modify TRPV1

Note that Dravet syndrome is caused by a mutation in the gene that encodes the sodium ion channel Nav1.1, the dysfunction of GABA_A receptors is a secondary effect. Also of interest is that the seizures that occur in Dravet syndrome are often triggered by hot temperatures or fever, so you can see how TRPV1 is indeed likely involved.  More generally in idiopathic autism, we have the "fever effect" when high temperatures trigger a reduction in autistic behaviors, making it the opposite of Dravet syndrome. 

On the one hand the biology behind the various problems may look horribly complicated and interwoven, the solutions appear to be much simpler and you have multiple options.

I await the results of the autism clinical trial of CBDV (Cannabidivarin) with interest.

Just impaired autophagy may lead to a reduction in GABA_A receptors and the appearance of autistic features in an otherwise “normal” brain. This reminds us again of why autism is not a medical diagnosis, it is just a vague/subjective observation, which, in severe cases, should then trigger a thorough medical investigation.