Wednesday, 23 October 2019

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 GABAA 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 GABAA 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 GABAA 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 (GABAA receptor associated protein) for the first time in this blog.  There is a vast amount in this blog about the GABAA 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 GABAA receptor trafficking as a feature. They each have a different impediment that should reduce the number of functioning GABAA 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”).


GABARAP has multiple functions:

1.     Transport of freshly minted GABAA receptors

In order for newly minted GABAA 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 GABAA receptors. If CaMKII is overactivated you get too many GABAA receptors.

In Jacobsen Syndrome there is reduced GABAA receptor trafficking/transport, leading to reduced surface expression. (in effect not enough functioning GABAA 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 GABAAR 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 detectorsmicroglia 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.

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 factors58,59. 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 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.

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 GABAARs 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 GABAAR transport driven during NMDAR–dependent GABAergic iLTP. Chemically induced iLTP elicited a rapid increase in surface GABAARs in wild-type mouse hippocampal neurons, but not in PX-RICS/RICS–deficient neurons. This increase in surface GABAARs 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 GABAAR 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 (GABAARs). Impaired presynaptic release of GABA and postsynaptic trafficking of GABAARs lead to autistic-like social behavior in mouse models of autism. There is a significant reduction in the number of GABAARs and GABAergic activity in certain brain areas of autistic individuals. Genetic association studies have revealed that several GABAAR subunits are linked to an increased risk for autism. GABAAR–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 GABAARs 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 GABAAR–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 GABAAR 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 GABAARs. 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 GABAARs and GABAAR–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 GABAAR 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 GABAAR 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 GABAAR trafficking during iLTP. Here we show that PX-RICS–mediated GABAAR trafficking is also involved in NMDAR activity–dependent trafficking of GABAARs 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 GABAA 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.


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 (GABAA) 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.

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


Lack of Autophagy will reduce the number of GABAA 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 GABAA receptors.
If correct, this actually has very wide implications.

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 GABAA 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.


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

The conclusion is simple, you need to keep your GABAA 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 (GABAA 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 GABAA 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 GABAA 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 GABAA 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 GABAA 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.


  1. Confirmed: I like this post!
    It elegantly puts together several known diseases and pathways and joins them in the broad but treatable category of GABAar dysfunction. The greatest takeaway for me is possibly the connection from CamkII, but I'm not sure because there are several conclusions here that you've saved for us readers to draw.

    Capsaicin receptors and "spray fire in my head"?


    1. Ling, there are indeed lots of possible conclusions.

      Feedbucket continues not to work, so I have added a previous 20 comments function on the lower right side of the blog. If viewed on a smart phone you cannot see it. You need a tablet or PC.

    2. Yes, I noticed.
      In mobile phone view you can scroll down to the bottom of a page and click 'View web version' to see the right panel content.

      I'll have to look into Angelman more, the phenotype is similar to my daughter. If CamkII is down, this must blunt NMDAr signalling as well(?)


    3. Ling, it gets rather complicated. To read all about current knowledge, take a look at this:-

      The CaMKII/NMDA receptor complex controls hippocampal synaptic transmission by kinase-dependent and independent mechanisms

    4. That one looks like a gem. Thank you!


    5. What your paper above says that caught my interest is that it is not only the Ca2+ current through the NMDAr that activates CaMKII, maybe more important for LTP is that CaMKII is activated by the binding to a specific subunit of the NMDAr.
      This can't be seen in the graphics of this post, and neither Wikipedia seems knowledgeable of it.
      Details perhaps, but it also means that just enhancing Ca2+ flow through NMDArs might not be enough to get more LTP/better memory, the available number of NMDA receptors that contain the subunit is also important.
      Incidentally magnesium, used as a mild memory enhancer, raises the expression of this NMDAr subunit while otherwise being a NMDAr blocker.


  2. Fever effect : But I am daughter always worsens during fever.....different subgroup? carla marta

    1. Carla Marta, I was surprised by that article. Prof Hollander a while back replicated the fever effect by placing people in hot tubs, to raise their temperature, so no connection to IL-17, it was just raising their body temperature.

      I think the fever effect is only genuinely present in a minority of cases.

      Modifying cytokine expression is actually a very good way to treat autism, but entirely unrelated to the fever effect.

      Most people feel worse when they are sick, regardless of whether they have autism.

      As with Dravet syndrome, there will be people whose symptoms get worse as their temperature goes up (regardless of them being ill).

      It is important not to draw false conclusions.

    2. I don't know if it has ever been mentioned by other readers, but there is a company called avacen medical that is selling a device which raises body temperature by inserting the hand in a heated vacuum chamber. It is FDA cleared for the treatment of arthritis and body pains, but there is also an enrollment for a pilot study for autism called “The Fever Effect: Do kids with autism do better when they use a device to warm their blood? Effects of a six-week AVACEN Treatment Method on Autism Spectrum Disorder”.

    3. Thanks Michalis, this has not been mentioned. A soak in a hot bath might be a simpler method.

  3. Do you believe Resveratrol can effectively help with Autophagy. Or is it ever-elusive? I will search your wonderful blog but was curious thoughts. Has anyone seen any results from Resveratrol in asd? Thanks, CA

    1. Just was checking your blog -- sounds like "Pterostilbene is like a super potent version of resveratrol." Have many of you tried this and found this?

    2. CA, some readers do find a benefit from Resveratrol and Pterostilbene. If you want a supplement to trial, I would opt for Pterostilbene.

      In general I think you need drugs to have a major impact on autism.

  4. Is this relevant to this article and Peter can you give your sense with CamKII: Antineuronal Antibodies in a Heterogeneous Group of Youth and Young Adults with Tics and Obsessive-Compulsive Disorder
    Conclusion: Our study suggested a significant correlation of streptococcal-associated tics and OCD with elevated anti-D1R and antilysoganglioside antineuronal antibodies in serum concomitant with higher activation of CaMKII in human neuronal cells. Youth and young adults with chronic tics and OCD may have underlying infectious/immunologic etiology. Thanks, PE

    1. PE, "patients with tics and OCD had significantly increased activation of CaMKII activity compared with patients with only tics or only OCD"

      Activation/inhibition of CaMKII will have many effects and as stated in the main blog post it is implicated in many neurological disorders.

      Your paper is relevant to my article. Inhibiting CaMKII might be helpful as a therapeutic intervention. But in conditions like Angelman Syndrome you want the opposite therapy, you want to activate CaMKII.

  5. I just came across some very interesting research concerning the relationship between salt intake and accumulation of the tau protein (Alzheimer's).

    Press Release:


    Now what is very interesting with respect to this research is not so much the accumulation of tau via salt intake, but the mechanism of action for this effect to occur. What the researchers essentially found is a link that suggests a high-salt diet leads to the small intestine inducing an inflammatory response which releases copious amounts of Interleukin 17 (IL-17) which then travels to the brain where it reduces blood flow via a reduction in nitric oxide. In other words, you can think of excess salt as being the antithesis of drinking beet juice. This reduction in blood flow is what allows the tau buildup which causes cognitive impairment.

    Children with autism tend to have elevated levels of IL-17 which could primarily be from any number of other reasons many research papers have already explored in the autism space, but this research linking salt to tau suggests restricting salt in the diet of those with autism who may be sensitive to it (this is just pure speculation on my part), could reduce levels of IL-17 and improve autism symptoms.

    The issue seems to happen in the gut, so diuretics which waste sodium from the blood likely won't do anything from my reading of all of this as it is an immune response from excess salt that seems to be the problem. It may also be the case that a low-salt or no salt diet could help while the constituents of salt (sodium and chloride) are supplemented back into the diet in forms which do not aggravate this IL-17 immune response.

    1. Incidentally, sodium bicarbonate (baking soda) is a possible non-salt form of sodium. Also, you coud replace the the chloride in NaCl with KCl.

    2. Very interesting Tyler, I think that if you understand autism you will end up understanding many other disorders from Alzheimer's to MS.

      It would seem that Agmatine and/or beet juice is wise for autism to raise eNOS and NO. Both are also a good idea to avoid Alzheimer's, at least in research models.

      Down syndrome features early onset Alzheimer's.

      Anti-IL17 treatment ameliorates Down syndrome phenotypes in mice

    3. Well I just came across another bit of research posted in the last couple of days concerning IL-17A which is a bit of a head scratcher:

      From my look at this, what the researchers are arguing is that maternal immune activation during pregnancy causes a reduction in IL-17A receptors in the somatosensory cortex due to receptor desensitization (and likely other areas of the brain) causing these neurons to be hyperactive after birth as they have a low number of IL-17A receptors. When there is an immune challenge, as opposed to just an artificially induced fever, IL-17A is produced in abundance and this attenuates hyperactive neurons in the somatosensory cortex by strongly and persistently activating the IL-17A receptors. Of course, eventually the receptors will autoregulate back to a preprogrammed state of IL-17A equilibrium (i.e. a tolerance) and so the "fever effect" won't last.

      I could be reading this wrong as I have yet to read the full paper on the subject as I just came across it, so don't start adding massive amounts of salt to your child's diet to quiet their somatosensory cortex and increase their socialability just yet (-:

  6. So CAMKIIa is the kinase that connects NMDA receptors, L-type calcium channels and GABAa receptors. Very very cool. Not to forget that CamKII also (up)regulates AMPA receptors and thereby increase synaptic strength.

    Ok, now I have a number of questions! :-D

    I had not seen the term "GABAergic iLTP" before. Is it a fancy way of saying "noise reduction" or is there some real link to enhanced LTP/memory that I haven't grasped yet?

    I would really like to read more on the mechanisms of CaMKII - being so central it deserves a post of its own.
    Given that CACNA1C is implicated in the susceptibility of all the great neurological diseases it would be nice to get that connection clarified too. If hypertension runs in a family - what does that mean for CaMKII? What happens theoretically if you activate CaMKII more than normal? Is Calmodulin also implicated in autism?

    Other questions regarding this post:
    Will bumetanide work if neuronal chloride levels are in range (and the dysfunction is instead in the number of surfacing GABAars?)
    -Maybe this can be answered by looking at what happens in NT people using Bumetanide, do they get sleepy etc.
    Is TRPV1 expression modulated by GABARAP independently from GABARAPs function as a GABAar transporter?
    -That is, is TRPV1 only interesting if your GABARAP for some reason is out of balance, or is it interesting for anyone with activated microglia?
    Didn't those GABAar also had to be clustered to work perfectly?I vaguely remember an older post on the topic.


    1. Ling, I am not an expert in this field.

      Here is another paper that again includes the role of clustering of GABAa receptors. It also explains the role and interplay of excitatory and inhibitory neurons.

      Role of GABAAR trafficking in the plasticity of inhibitory synapses

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  8. Hi, I've been visiting this site recently as my diagnosis is ASD and OCD.. I would like to ask for now if there is there a way to improve my emotional awareness? I mean I almost don't know when I'm not feeling nothing ( Most of my day I don't feel body sensation so I think I can say that I don't feel nothing ) and I don't know if I've inability to feel pleasure.. because I don't even know how pleasure feels like.. what can I do? I'm totally lost... I want to be able to say what I'm feeling on every second of my days.

    1. The best person to answer this would be one of our Aspie readers.

      One OTC product that may be of benefit is the probiotic Biogaia Protectis usually given to children with stomach problems. It has a secondary effect of increasing the amount of the hormone Oxytocin, which affects emotions. It certainly does this in my son.

      Oxytocin nasal spray has been used in several autism clinical trials, but the effect does not last long. The bacteria in the probiotic (L. reuteri DSM 17938) continuously produces oxytocin and so could well be better than the nasal spray.

    2. Then should I buy the probiotic biogaia protectis and take it daily?

    3. You will only need one pill to know if it helps. The effect is that fast. It is worth a try.

    4. okok will buy it then.

  9. Thought you (and likely only you) might be interested in this research on the first three-dimensional structure of KCC1:

    Press Release:


    Of course it is not KCC2, but I thought you might be interested nevertheless.

    1. Tyler, thanks for this.

      The pharmaceutical scientists hoping to develop a new drug for NKCC1, told me that very little is known about the detail of KCC2/NKCC1 and all the isoforms. Any new drug will not be available for very many years, so best to make best use of what we have today.


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