Showing posts with label TRPV. Show all posts
Showing posts with label TRPV. Show all posts

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.

Thursday, 4 July 2019

Home/Clinic based Photobiomodulation/Laser Therapy in Autism - acting on Light Sensitive Ion Channels, Mitochondria, Lymph Nodes and more

Photobiomodulation underlying mechanisms at the cellular and molecular levels. Light at 600–850 nm is absorbed by the mitochondrial electron transfer chain and leads to upregulation of the neuronal respiratory capacity. The near-infrared light at range of 900– 1100 nm is absorbed by structured water clusters formed in or on a heat/light-gated ion channels. An increase in vibrational energy of water cluster leads to perturb the protein structure and opening the channel which ultimately allows modulation of intracellular Ca2+ levels. The absorption of green light by neuronal opsin photoreceptors (OPN2-5) activates transient receptor potential channels which causes nonselective permeabilization to Ca2+ , Na+ , and Mg2+ . The cryptochromes (a class of flavoprotein blue-light signaling receptors) absorb blue light and seems to activate the transducing cellular signals via part of the optic nerve to the suprachiasmatic nucleus in the brain, which is important in regulation of the circadian clock

Today we return to the idea of using low power lasers to treat autism.  This follows on from the original post that reviewed a credible clinical trial that compared laser therapy with a sham red light therapy.  My conclusion was either the researchers cheated, or it really did work.   It is a pity, but experience shows us that cheating does occur in published research. I also pondered whether a cheaper LED device could give the same benefit of an expensive laser.

Low Level Laser Therapy (LLLT) for Autism – seems to work in Havana

Our reader RD has been busy at home applying the research, first using LEDs to no avail, before moving on to an expensive laser device, which does provide a benefit.  Today we dig a little deeper about what might be going on inside the brains of people treated with such devices. Click below to read RD's extensive comments and interesting links.

Some autism therapies involving the use of expensive gadgets do set alarm bells ringing, but the more you look into Photobio-modulation, which is the new name of Low Level Laser Therapy (LLLT), the more credible it becomes.  There has been a great deal of recent research regarding other neurological conditions, autism only rarely gets a mention. The same therapy has been used on different parts of the body for several decades in Russia and some other countries. Where we live physiotherapists use Photobiomodulation/LLLT to treat numerous types of ache and pain.

It is still early days for Photobiomodulation and the brain. A lot depends on which parts of the brain you want to target; there are even plans for using the mouth, nose and ears as entry points to reach different parts of the brain.

Heat/light sensitive ion channels

Many human diseases are associated with ion channel dysfunctions (channelopathies).  Many people with autism have either genetic or acquired channelopathies of one kind or another.

Today our focus on light introduces us to a class of ion channels activated by heat and/or light.

We should immediately recall the so called “fever effect” in autism where in some people a rise in body temperature improved their autism, sometimes dramatically. The fever effect was replicated by one US researcher having people sit in a hot tub.


 Five control subjects without a history of fever completed the hyperthermia condition at 102 °F, and demonstrated the safety and feasibility of the study. Ten subjects with ASD and a history of fever response were enrolled and completed the hyperthermia condition (102 °F) and control condition (98 °F) at the aquatic therapy pool. Improvement in social cognition and repetitive/restrictive behaviors were observed at the hyperthermia condition (102 °F) on parent (SRS, RBS-R) and rater (CGI-I) assessments. Pupillometry biomarker and gene expression can be correlated with clinical improvement. Side effects were minimal, and were the same as those observed in a hot tub/sauna (redness, nausea).


We demonstrated improvement of socialization and repetitive and restricted behaviors at the hyperthermia condition (102 °F), and that we could reliably and safely increase children’s temperatures into the fever range (mean max temperature of 101.7 °F). This temperature increase was observed to cause significant and convergent improvement on clinician ratings (CGI-I) and parent ratings (SRS, RBS-R), both of which were kept blinded to the temperature of the pool. Interestingly, each child’s fever response history was correlated with the improvements observed at the elevated temperature. Those with a history of marked fever response had the most observable behavior changes. Behavior changes observed for each child were similar to those observed by parents during febrile episodes, including increased cooperation, communication and social reciprocity and decreased hyperactivity and inappropriate vocalizations. Although multiple rationales have been posited, this is the first study looking at the direct effect of temperature on ASD symptomatology.

TRPV1 and Autism

There has been a link suggested between TRPV1 and autism.  SHANK3 is a single gene type of autism, often used to study autism.

In control mice, SHANK3 tethers a protein called TRPV1 to the surface of sensory neurons, where it detects heat and chemical signals. Those signals activate TRPV1, causing calcium ions to flood into the cell, leading to a painful sensation.
Neurons from control mice show a robust influx of calcium ions in response to capsaicin, the chemical that gives chili peppers their heat. But the chemical triggers significantly less calcium flow into neurons from SHANK3 mice.
The study stokes curiosity about the connection between autism and TRPV1. This protein aids heart and lung function, and has been linked to addiction, anxiety and depression, says Camilla Bellone, assistant professor of neuroscience at the University of Geneva in Switzerland, who was not involved in the study. “It would be really interesting to see if TRPV1 dysfunction could explain other [features] associated with autism,” she says.

Pain, Rett Syndrome, MECP2 and TRPV1

It appears to be not just SHANK3 autism that has a TRPV1 connection, so does the all-female Rett Sydrome. Here the connection relates to unusual pain sensitivity in Rett Sydrome. Many people with autism have an unusual relationship with pain.

Although TRPV1 was expressed in MeCP2-positive TG neurons innervating the tongue in both wild-type and Mecp2+/- mice, a significantly smaller number of TRPV1-positive neurons were observed in the tongues of heterozygotes compared to wild-types. Together, these data suggest that the hypoalgesia observed in this mouse model is induced by the inhibition of TRPV1 expression, and this expression is dependent in part on MeCP2 signaling.
These findings suggest that tongue heat sensitivity and inflammatory hyperalgesia are dependent on TRPV1 expression in TG neurons that innervate the tongue and that this expression is regulated by MeCP2 signaling, supporting a role for MeCP2 in pain modulation. Hypoalgesia is a potentially dangerous condition that may result in more severe tissue damage from burns or other physical trauma due to a blunted pain withdrawal reflex.  Understanding how MeCP2 modulates pain might lead to therapies that improve the pain sensitivity in Rett syndrome patients, as well as treatments that might help to reduce neuropathic pain associated with other genetic or acquired conditions.

TRPV Channels in Mast Cells as a Target for Low-Level-Laser Therapy

Low-level laser irradiation in the visible as well as infrared range is applied to skin for treatment of various diseases. Here we summarize and discuss effects of laser irradiation on mast cells that leads to degranulation of the cells. This process may contribute to initial steps in the final medical effects. We suggest that activation of TRPV channels in the mast cells forms a basis for the underlying mechanisms and that released ATP and histamine may be putative mediators for therapeutic effects.

Modulation of TRPV channel gating by light-switched ligand. Putative modulation of an azo-chromophore between cis- and trans-form by light leading to activation of TRPV channel opening. As an example TRPV activation by the cis-form is cartooned.

We have shown in this review that laser irradiation in the visible and IR as well as UV range can modulate the function and expression of TRPV ion channels, and in particular TRPV1, TRPV2, and TRPV4. This may form the basis for effect of LLLT. As Ca2+-permeable ion channels, their activation may contribute to the laser-induced increase in intracellular Ca2+ that triggers degranulation and endocytotic release of ATP. Such light-induced mechanism may contribute to the basis of the medical effects of LLLT. This hypothesis still needs confirmation in animal tests and clinical trials.

Proposed Mechanisms of Photobiomodulation or Low-Level Light Therapy

Photobiomodulation (PBM) also known as low-level laser (or light) therapy (LLLT), has been known for almost 50 years but still has not gained widespread acceptance, largely due to uncertainty about the molecular, cellular, and tissular mechanisms of action. However, in recent years, much knowledge has been gained in this area, which will be summarized in this review. One of the most important chromophores is cytochrome c oxidase (unit IV in the mitochondrial respiratory chain), which contains both heme and copper centers and absorbs light into the near-infra-red region. The leading hypothesis is that the photons dissociate inhibitory nitric oxide from the enzyme, leading to an increase in electron transport, mitochondrial membrane potential and ATP production. Another hypothesis concerns light-sensitive ion channels that can be activated allowing calcium to enter the cell. After the initial photon absorption events, numerous signaling pathways are activated via reactive oxygen species, cyclic AMP, NO and Ca2+, leading to activation of transcription factors. These transcription factors can lead to increased expression of genes related to protein synthesis, cell migration and proliferation, anti-inflammatory signaling, anti-apoptotic proteins, antioxidant enzymes. Stem cells and progenitor cells appear to be particularly susceptible to LLLT.


Light sensitive ion channels

The most well-known ion channels that can be directly gated by light are the channelrhodopsins (ChRs), which are seven-transmembrane-domain proteins that can be naturally found in algae providing them with light perception. Once activated by light, these cation channels open and depolarize the membrane. They are currently being applied in neuroscientific research in the new discipline of optogenetics [35].
However, members of another broad group of ion-channels are now known to be light sensitive [36]. These channels are called "transient receptor potential" (TRP) channels as they were first discovered in a Drosophila mutant [36] and are responsible for vision in insects. There are now at least 50 different known TRP isoforms distributed amongst seven subfamilies [37], namely the TRPC (‘Canonical’) subfamily, the TRPV (‘Vanilloid’), the TRPM (‘Melastatin’), the TRPP (‘Polycystin’), the TRPML (‘Mucolipin’), the TRPA (‘Ankyrin’) and the TRPN (‘NOMPC’) subfamilies (see Figure 2). A wide range of stimuli modulate the activity of different TRP such as light, heat, cold, sound, noxious chemicals, mechanical forces, hormones, neurotransmitters, spices, and voltage. TRP are calcium channels modulated by phosphoinositides [38].


Low levels of red/NIR light can interact with cells, leading to changes at the molecular, cellular and tissue levels. Each tissue, however, can respond to this light-interaction differently, although it is well known that the photons, especially in the red or NIR, are predominantly absorbed in the mitochondria [132]. Therefore, it is likely that even the diverse results observed with PBM share the basic mechanism of action. What happens after the photon absorption is yet to be fully described, since many signaling pathways seem to be activated. It seems that the effects of PBM are due to an increase in the oxidative metabolism in the mitochondria [133]. Different outcomes can occur depending on the cell type, i.e. cancer cells that tend to proliferate when PBM is delivered [88]. In this review we have not discussed the response of cells and tissues to wavelengths longer than NIR, namely far IR radiation (FIR) (3 µm to 50 µm). At these wavelengths water molecules are the only credible chromophores, and the concept of structured water layers that build up on biological lipid bilayer membranes has been introduced to explain the selective absorption [134]. Nevertheless FIR therapy has significant medical benefits that are somewhat similar to those of PBM [135], and it is possible that activation of light/heat sensitive ion channels could be the missing connection between the two approaches.
As we have shown, PBM can regulate many biological processes, such as cell viability, cell proliferation and apoptosis, and these processes are dependent on molecules like protein kinase c (PKC), protein kinase B (Akt/PKB), Src tyrosine kinases and interleukin-8/1a (IL-8/1a). The effects of light on cell proliferation can be stimulatory at low fluences (which is useful in wound healing, for instance), but could be inhibitory at higher light doses (which could be useful in certain types of scar formation such as hypertrophic scars and keloids) [131].
The applications of PBM are broad. Four clinical targets, however, are the most common: shining light on injured sites to promote healing, remodeling and/or to reduce inflammation; on nerves to induce analgesia; on lymph nodes in order to reduce edema and inflammation; and on trigger points (a single one of as many as 15 points) to promote muscle relaxation and to reduce tenderness. Since it is non invasive, PBM is very useful for patients who are needle phobic or for those who cannot tolerate therapies with non-steroidal anti-inflammatory drugs [83].
The positive outcomes depend on the parameters used on the treatment. The anti-inflammatory effect of light in low intensity was reported on patients with arthritis, acrodermatitis continua, sensitive and erythematous skin, for instance [136]. With the same basic mechanism of action, which is the light absorption by mitochondrial chromophores, mainly Cox, the consequences of PBM are various, depending on the parameters used, on the signaling pathways that are activated and on the treated tissue. In order to apply PBM in clinical procedures, the clinicians should be aware of the correct parameters and the consequences for each tissue to be treated. More studies have to be performed in order to fill the gaps that still linger in the basic mechanisms underlying LLLT and PBM.

Photobiomodulation improves the frontal cognitive function of older adults.


The frontal lobe hypothesis of age-related cognitive decline suggests that the deterioration of the prefrontal cortical regions that occurs with aging leads to executive function deficits. Photobiomodulation (PBM) is a newly developed, noninvasive technique for enhancing brain function, which has shown promising effects on cognitive function in both animals and humans. This randomized, sham-controlled study sought to examine the effects of PBM on the frontal brain function of older adults.


Thirty older adults without a neuropsychiatric history performed cognitive tests of frontal function (ie, the Eriksen flanker and category fluency tests) before and after a single 7.5-minute session of real or sham PBM. The PBM device consisted of three separate light-emitting diode cluster heads (633 and 870 nm), which were applied to both sides of the forehead and posterior midline, and delivered a total energy of 1349 J.


Significant group (experimental, control) × time (pre-PBM, post-PBM) interactions were found for the flanker and category fluency test scores. Specifically, only the older adults who received real PBM exhibited significant improvements in their action selection, inhibition ability, and mental flexibility after vs before PBM.


Our findings support that PBM may enhance the frontal brain functions of older adults in a safe and cost-effective manner.

Brain Photobiomodulation Therapy: a Narrative Review.

Brain photobiomodulation (PBM) therapy using red to near-infrared (NIR) light is an innovative treatment for a wide range of neurological and psychological conditions. Red/NIR light is able to stimulate complex IV of the mitochondrial respiratory chain (cytochrome c oxidase) and increase ATP synthesis. Moreover, light absorption by ion channels results in release of Ca2+ and leads to activation of transcription factors and gene expression. Brain PBM therapy enhances the metabolic capacity of neurons and stimulates anti-inflammatory, anti-apoptotic, and antioxidant responses, as well as neurogenesis and synaptogenesis. Its therapeutic role in disorders such as dementia and Parkinson's disease, as well as to treat stroke, brain trauma, and depression has gained increasing interest. In the transcranial PBM approach, delivering a sufficient dose to achieve optimal stimulation is challenging due to exponential attenuation of light penetration in tissue. Alternative approaches such as intracranial and intranasal light delivery methods have been suggested to overcome this limitation. This article reviews the state-of-the-art preclinical and clinical evidence regarding the efficacy of brain PBM therapy.

Because neural tissues contain large amounts of mitochondrial CCO, application of red to NIR lights (600–850) for brain PBM therapy is highly attractive. The main problem so far has been getting enough light into the brain to accomplish the beneficial effects. In recent years, irradiation in the wavelength range between 980 and 1100 nm has been growing rapidly, and its different mechanisms of action including stimulation of ion channels and water molecules suggest it might even be combined with red/NIR. Improving cerebral metabolic function, stimulating neurogenesis and synaptogenesis, regulating neurotransmitters, and providing neuroprotection via anti-inflammatory and antioxidant biological signaling are the most important effects of brain PBM therapy (Fig. 4). The overall results from extensive preclinical and clinical studies in the brain PBM field suggest that modest levels of red and NIR light show biostimulatory effects without any thermal damage, and could improve neurobehavioral deficits associated with many brain disorders. Nevertheless, it is still not completely clear whether chronic repetition of brain PBM will be necessary for sustained clinical benefit, especially in psychological and neurodegenerative disorders. Owing to the beneficial impacts of brain PBM therapy in depression and anxiety, new trials for other psychiatric disorders such as schizophrenia autism, , bipolar, attention-deficit hyperactivity, and obsessive–compulsive disorders might well emerge in the future. Development of new techniques for effective light delivery to deeper structures of the brain is crucial, because of involvement of the limbic system and midbrain abnormalities seen in some brain disorders. In this respect, intracranial and intranasal irradiation methods, as well as the oral cavity route, even via the ear canal could be options. Although therapeutic influences of intracranial PBM therapy has been focused on PD researches, it is postulated that developing this technique also potentially effective for those conditions that are associated with limbic system dysfunctions such as anhedonia, anxiety, as well as impaired emotional processing. Preliminary evidence of benefit has been obtained in autism spectrum disorders. There is an epidemic of AD that is expected to hit the Western world as the overall population ages, and there has been a noticeable lack of any effective pharmacological therapies that have been approved for AD. Although the evidence for the effectiveness of PBM in the treatment of AD is still very preliminary, it is possible that PBM will play an even larger role in society in years to come if clinical trials now being conducted are successful. The authors conclude that clinic or home-based PBM therapy using laser or LED devices will become one of the most promising strategies for neurorehabilitation in upcoming years


Our reader RD is well ahead of the curve with his PBM/LLLT investigation. I do not see this kind of therapy being adopted by mainstream Western medicine, even if it did work.  It has been used in other countries for many decades by medical doctors, for all kinds of conditions, but that fact does not cut it with most Western doctors.  There are  practitioners of PBM/LLLT in Western countries, but they tend to be on the fringes of medicine, which puts PBM/LLLT clearly in the crank therapy category for most qualified Western doctors.

On the basis that we should keep an open mind about all kinds of therapies, we should consider PBM further. It is apparently safe at the power levels used. It may look a little strange, but it is non-invasive and the therapy does not take long. A single device could easily be used to treat many people, so the high price should not remain a barrier.

I was very surprised to hear that a local speech therapy company is now offering “neurofeedback therapy” using an expensive machine they have bought. I was very suspicious of a recent study carried out in Florida that was put forward to support this therapy using a commercial device, since of the 42 children in the group that had the actual therapy only 17 completed the 12 week trial and came back for the evaluation.  The trial included a similar sized group who had a sham therapy.  The likelihood of completing the trial was the same in both groups, which also looks odd.

Of the 83 subjects that completed the evaluation at the enrollment time, 34 returned for the POST evaluation after the 12 weeks of home based therapy.

If the results were so good, why did the majority of parents walk away during the trial? I was going to suggest to the speech therapist that perhaps those few thousands of euros/dollars might have been better spent on a laser, or perhaps the lottery.

For me, one big question about the laser is about how the device is used. Depending on what you believe the mode of action to be, you would have to use it in completely different ways.

If the benefit relates to improved mitochondrial function, you should really be able to measure this benefit using a PET scan that measures glucose uptake to each part of the brain. This was the method proposed by Polish researchers to show how some people benefit from a ketogenic diet to improve power/ATP output from different parts of the brain.

You would hope other researchers would try and replicate the benefit in autism, but the first group have already patented the laser idea.

Hopefully our reader RD will perfect this therapy and we await his feedback.

I did recently write about the recently discovered lymphatic system within the brain. One proposed benefit of PBM/LLLT is improved drainage of lymph. I thought that was interesting; if it was actually true then this therapy could potentially be used to prevent the onset of Alzheimer’s. We saw in that post that faulty lymph drainage may allow the accumulation of waste products (plaques etc) in aging brains and then Alzheimer's develops. Targeting the relevant lymph node with PBM/LLLT might be an alternative to the drug therapy currently being developed.

I am told that lymphatic drainage is currently "the big thing" in autism in the US, alongside anything to do with CBD (cannabis). Hopefully in the fb world of autism they have noted that in MS the problem with the brain's lymphatic system was not drainage, but the ingress of inflammatory messengers from the body into the brain, suggesting the opposite therapy.