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.

https://epiphanyasd.blogspot.com/2018/12/low-level-laser-therapy-lllt-for-autism.html?showComment=1560875374458#c8257042607661710259

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.

HYPERTHERMIA AND THE IMPROVEMENT OF ASD SYMPTOMS

 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).

Discussion

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.

Autism gene may double as pain processor

  

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.

SHANK3 deficiency impairsheat hyperalgesia and TRPV1 signaling in primary sensory neurons

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.

REduced MeCP2 signaling leads to hypoalgesia in a mouse model of Rett syndrome

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.

TRP ion channels in thermosensation, thermoregulation and metabolism

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.

MOLECULAR MECHANISMS OF PBM

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].

Conclusions

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.

OBJECTIVES:

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.

METHODS/DESIGNS:

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.

RESULTS:

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.

CONCLUSIONS:

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

Conclusion

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.

https://www.frontiersin.org/articles/10.3389/fneur.2018.00537/full

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.

Learning from GABAa Dysfunction in Huntington’s Disease – useful ideas for Autism therapies?

Today’s post is really for the regular readers of this blog who are interested in the GABA switch and Bumetanide. It is not light reading.  We see how advanced some Taiwanese researchers are in their understanding of GABA_A dysfunctions in Huntington’s Disease.

Taipei 101, briefly the world’s tallest building

It is an excellent paper and much of it is applicable to autism. There are some omissions, but you will struggle to find a more complete paper.

They even go into the detail of altered the sub-unit expression of GABA_A receptors that occurs as the disease progresses. I think that correcting sub-unit miss-expression has great potential in treating some autism.

Huntington’s is an inherited brain disorder that first manifests itself around the age of 40 and then progresses for the next 15 to 20 years.

Much autism is present prior to birth but there is a progression that occurs as the brain develops in early childhood. Some people do seem to be entirely typical at birth and only around 2 years old develop symptoms. After 5 years old you cannot really develop “autism”, just the symptoms might not get noticed till later in life.

Schizophrenia only develops in early to mid-adulthood.

It is surprising to many people that such varied disorders share some similar aspects of biology.

In terms of practical interventions, in today’s paper these include:       

·        Inhibition of NKCC1 (bumetanide)

·        Activation of KCC2 (N-Ethylmaleimide)

·        Enhancer of CKB (creatine)

·        Inhibitor of WNK/SPAK

·        Activation of extra-synaptic GABAa receptors (taurine, progesterone)

·        Activation of synaptic GABAa receptors (zolpidem, alprazolam)

·        Inhibition of GABA transport mechanism (Tiagabine)

One thing to note is that activating GABAa receptors may well have a negative effect in some people.

Sub-unit specific therapies, like very low dose clonazepam targeting α3, are not mentioned in this paper, nor is the role of GABAb on NKCC1/KCC2 expression.

We are familiar with Bumetanide as an NKCC1 blocker intervention in autism, but looking at the list there are other common autism therapies (creatine and taurine) and the female hormone progesterone. We come upon the beneficial effect of female hormones on a regular basis in this blog (estradiol, pregnenalone, progesterone …).  We even saw how a sub-SSRI dose of Prozac increases the amount of the neurosteroid 3α-hydroxy-5α-pregnan-20-one (Allo) that potently, positively, and allosterically modulates GABA action at GABA_A receptors. Progesterone is converted to Allo in the body.

 

Here is the excellent paper on Huntington’s:-

Insights into GABAAergic system alteration in Huntington’s disease

                                                                                                               

An overview of the g-aminobutyric acid (GABA) signalling system. (a) GABA homeostasis is regulated by neurons and astrocytes. GABA is synthesized by GAD65/67 from glutamate in neurons, while astrocytic GABA is synthesized through MAOB. The release of GABA is mediated by membrane depolarization in neurons and Best1 in astrocytes. The reuptake of GABA is mediated through GAT1 in neurons and GAT3 in astrocytes. The metabolism of GABA is mediated by GABA-T in neurons and astrocytes. The reuptake of GABA in astrocytes is further transformed into glutamine via the TCA cycle and glutamine synthetase (GS). The glutamine is then transported to neurons and converted to glutamate for regeneration of GABA.

(b) GABAA receptors are heteropentameric complexes assembled from 19 different subunits. The compositions of different subunits determines the subcellular distributions and functional properties of the receptors. Phasic inhibition is mediated via the activation of synaptic GABAA receptors following brief exposure to a high concentration of extracellular GABA. Tonic inhibition is mediated via the activation of extrasynaptic GABAA receptors by a low concentration of ambient GABA.

c) The excitatory inhibitory response of GABA is driven by the chloride gradient across cell membranes, which can be determined via two cation–chloride cotransporters (NKCC1 and KCC2). The high expression of NKCC1 during the developmental stage maintains higher intracellular [Cl2] via chloride influx to the cell. The activation of GABAA receptors at an early developmental stage results in an outward flow of chloride and an excitatory GABAergic response. As neurons mature, the high expression of KCC2 maintains lower intracellular [Cl2] via chloride efflux out of the cell. The activation of GABAA receptors on mature neurons results in the inward flow of chloride and an inhibitory GABAergic response.

An excerpt showing data on sub-unit misexpression in different parts of the brain at different stages of the disease

5.2. Modulation of chloride homeostasis via cation – chloride cotransporters

Emerging evidence suggests that chloride homeostasis is a therapeutic target for HD. Pharmacological agents that target cation–chloride cotransporters (i.e. NKCC1 or KCC2) therefore might be used to treat HD (figure 3b). Of note, dysregulation of cation–chloride cotransporters and GABA polarity was associated with several neuropsychiatric disorders [70,134–139] (reviewed in [27,140]). Such abnormal excitatory GABAA receptor neurotransmission can be rescued by bumetanide, an NKCC1 inhibitor that decreases intracellular chloride concentration. Bumetanide is an FDA-approved diuretic agent that has been used in the clinic. It attenuates many neurological and psychiatric disorders in preclinical studies and some clinical trials for traumatic brain injury, seizure, chronic pain, cerebral infarction, Down syndrome, schizophrenia, fragile X syndrome and autism (reviewed in [141]). Daily intraperitoneal injections of bumetanide also restored the impaired motor function of HD mice. The effect of bumetanide is likely to be mediated by NKCC1 because genetic ablation of NKCC1 in the striatum also rescued the motor deficits in R6/2 mice. This study uncovered a previously unrecognized depolarizing or excitatory action of GABA in the aberrant motor control in HD. In addition, chronic treatment with bumetanide also improved the impaired memory in R6/2 mice [69], supporting the importance of NKCC1 in HD pathogenesis. Owing to the poor ability of bumetanide to pass through the blood–brain barrier, further optimization of bumetanide and other NKCC1 inhibitors is warranted [142,143]. Disruption of KCC2 function is detrimental to inhibitory transmission and agents to activate KCC2 function would be beneficial in HD. However, no agonist of KCC2 has been described until very recently [144,145]. A new KCC2 agonist (CLP290) has been shown to facilitate functional recovery after spinal cord injury [145]. It would be of great interest to evaluate the effect of KCC2 agonists on HD progression. Another KCC2 activator, CLP257, was found to increase the cell surface expression of KCC2 in a rat model of neuropathic pain [146]. Post-translational modification of KCC2 by kinases may modulate the function of KCC2. The WNK/ SPAK kinase complex, composed of WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase), is known to phosphorylate and stimulate NKCC1 or inhibit KCC2 [147]. Thus, compounds that inhibit WNK/SPAK kinases will result in KCC2 activation and NKCC1 inhibition. Some compounds have been noted as potential inhibitors of WNK/SPAK kinases and need to be further tested for their effects on cation –chloride cotransporters [148–150]. An alternative mechanism to activate KCC2 is manipulation of its interacting proteins (e.g. CKB [65,66]). Because CKB could activate the function of KCC2 [65,66], CKB enhancers may increase the function of KCC2. In HD, reduced expression and activity of CKB is associated with motor deficits and hearing impairment [68,88]. Enhancing CKB activity by creatine supplements ameliorated the motor deficits and hearing impairment of HD mice. It is worthwhile to further investigate the interaction of KCC2 and CKB in GABAergic neurotransmission and motor deficits in HD. The depolarizing GABA action with altered expression levels of NKCC1 or KCC2 is associated with neuroinflammation in HD brains [32,69]. Blockade of TNF-a using Xpro1595 (a dominant negative inhibitor of soluble TNF-a) [151] in vivo led to significant beneficial effects on disease progression in HD mice [152] and reduced the expression of NKCC1. It would be of great interest to test the effect of other anti-inflammatory agents [153] on the function and expression of NKCC1 and GABAergic inhibition. Neuroinflammation is implicated in most neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease [154,155], and the interaction of cation–chloride cotransporters and neuroinflammation in GABAergic neurotransmission may also play a critical role in other neurodegenerative diseases.

Figure 2. Molecular mechanism(s) underlying the abnormal GABAAergic system in HD. (a) In the normal condition, adult neurons express high KCC2 and few NKCC1 to maintain the lower intracellular chloride concentration, which results in an inward flow of chloride when GABAA receptors are activated. Astrocytes function normally for the homeostasis of glutamate, potassium and glutamate/GABA-glutamine cycle. (b) In Huntington’s disease, reduced GABAA receptor-mediated neuronal inhibition is associated with enhanced NKCC1 expression and a decreased expression in KCC2 and membrane localized GABAA receptors. The dysregulated GABAAergic system might be caused by mutant HTT, excitotoxicity, neuroinflammation or other factors. Mutant HTT in neurons alters the transcription of genes (GABAAR and KCC2) through interactions with transcriptional activators (SP1) and repressors (REST/NRSF). Mutant HTT in neurons also disrupts the intracellular trafficking of GABAARs to the cellular membrane. HD astrocytes have impaired homeostasis of extracellular potassium/glutamate (due to deficits of astrocytic Kir4.1 channel and glutamate transporters, Glt-1) and cause neuronal excitability, which might be related to the changes of KCC2, NKCC1 and GABAAR. The activity of KCC2 could be affected through its interacting proteins, such as CKB and mHTT. Neuroinflammation, which is evoked by the interaction of HD astrocyte and microglia, enhances NKCC1 expression in neurons at the transcriptional level through an NF-kB-dependent pathway. HD astrocytes also have compromised astrocytic metabolism of glutamate/GABA–glutamine cycle that contributes to lower GABA synthesis.

Notably, neuroinflammation and the GABA neurotransmitter system are reciprocally regulated in the brain (reviewed in [104,105]). Specifically, neuroinflammation induces changes in the GABA neurotransmitter system, such as reduced GABAA receptor subunit expression, while activation

of GABAA receptors likely antagonizes inflammation.

TNF-a, a proinflammatory cytokine, induces a downregulation of the surface expression of GABAARs containing a1, a2, b2/3 and g2 subunits and a decrease in inhibitory synaptic strength in a cellular model of hippocampal neuron culture [106]. The same group further demonstrated that protein phosphatase 1-dependent trafficking of GABAARs was involved in the TNF-a evoked downregulation of GABAergic neurotransmission [107]. Upregulation of TNF-a also negatively impacts the expression of GABAAR a2 subunit mRNA and thus decreases the presynaptic inhibition in the dorsal root ganglion in a rat experimental neuropathic painmodel [108]. Conversely, blockade of central GABAARs in mice by aGABAAR antagonist increased both the basal and restraint stress-induced plasma IL-6 levels [109]. Inhibition of GABAAR activation by picrotoxin increased the nuclear translocation of NF-kB in acute hippocampal slice preparations [110]. Collectively, neuroinflammation

weakens the inhibitory synaptic strength in neurons, at least partly, through the reduction of GABAARs.

The reduced expression and function of GABAARs may further increase inflammatory responses. It remains elusive whether the same mechanism occurs in the inflammatory environment in HD brains.

hyperexcitability resulting from deficiency of astrocytic Kir4.1 might have also contributed to neuronal NKCC1 upregulation and altered GABAergic signalling in HD brains.

Figure 3. Strategy to target (a) GABAAR and (b) cation–chloride cotransporters as potential therapeutic avenues. (a) The GABAergic system is influenced directly by agents that (1) target synaptic GABAAR, (2) increase tonic GABA current or interfere with synaptic GABA concentrations via a reduction of GABA reuptake (3), and (4) block GABA metabolism.

5.1. Modulating the GABAA receptor as a therapeutic target

In view of the presently discovered HD-related deficit in the GABA system, the question arises whether HD patients can benefit from drugs that stimulate the GABA system (figure 3a). HD patients suffer from motor abnormalities and

non-motor symptoms, including cognitive deficits, psychiatric symptoms, sleep disturbance, irritability, anxiety, depression and an increased incidence of seizures [74,77,116,117].

Seizures are a well-established part of juvenile HD but no more prevalent in adult-onset HD than in the general population [73,74,118]. Several pharmacological compounds can enhance inhibitory GABAergic neurotransmission by targeting GABAAR and thereby producing sedative, anxiolytic, anticonvulsant and muscle-relaxant effects. A recent study demonstrated that zolpidem, a GABAAR modulator that enhances GABA inhibition mainly via the a1-containing GABAA receptors, corrected sleep disturbance and electroencephalographic abnormalities in symptomatic HD mice (R6/2) [119]. Alprazolam, a benzodiazepine-activating GABA receptor, reversed the dysregulated circadian rhythms and improved cognitive performance of HD mice (R6/2) [120].

In addition, progesterone, a positive modulator of GABAAR, significantly reversed the behavioural impairment in a 3-nitropropionic acid (3-NP)-induced HD rat model [121]. Apart from modulating the activity of the GABAergic system by interfering directly with the receptor, pharmacological agents can also interfere with synaptic GABA concentrations. Tiagabine, a drug that specifically blocks the GABA transporter (GAT1) to increase synaptic GABA level,was found to improve motor performance and extend survival inN171-82Q and R6/2 mice [122]. It is also worth evaluating whether vigabatrin, a GABA-T inhibitor that blocksGABAcatabolismin neurons and astrocytes [123], plays a role in the compromised astrocytic glutamate–GABA–glutamine cycling [56]. Interestingly, taurine exerted GABAA agonistic and antioxidant activities in a 3-NP HD model and improved locomotor deficits and increased GABA levels [124]. However, several early studies failed to provide the expected benefits of GABA analogues in slowing disease progression in HD patients [125–127]. For example, gaboxadol, an agonist for the extrasynaptic d-containing GABAA receptor, failed to improve the decline in cognitive and motor functions of five HD patients during a short two-week trial, but it caused side effects at the maximal dose [125]. Interestingly, although treatment with muscimol (a potent agonist of GABA receptors) did not improve motor or cognitive deficits in 10HDpatients, it did ameliorate chorea in the most severely hyperkinetic patient [126]. The therapeutic failure of GABA stimulation in early clinical trials does not argue against the importance of GABAergic deficits in HD pathogenesis. The alteration of GABAergic circuits plays a primary role or is a compensatory response to excitotoxicity, and it may contribute to HD by disrupting the balance between the excitation and inhibition systems and the overall functions of neuronal circuits. Because the subunits of the GABAA receptor are brain region- or neuron subtypespecific, the choice of drugs may have distinct effects on the brain region or neuronal population targeted [128–130]. For example, the expression of GABAAR subunits is differentially altered in MSNs and other striatal interneurons in HD 54,60]. The early involvement of D2-expressing MSNs can cause chorea [131], while dysfunctional PV-expressing interneurons can cause dystonia in HD patients [132]. Specific alteration in neuronal populations and receptor subtypes during HD progression needs to be taken into consideration when treating the dysfunction of GABAergic circuitry.

Notably, striatal tonic inhibition mediated by the dcontaining GABAARs may have neuroprotective effects against excitotoxicity in the adult striatum [63]. Because the reductions in d-containing GABAARs and tonic GABA currents in D2-expressing MSNs have been observed in early HD [32,39,40,54,61], it would be of great interest to evaluate the effects of several available compounds, such as alphaxalone and ganaxolone [133], that target d-containing GABAARs, in animal models of HD.

(b) GABAAR-mediated signalling in HD neurons is depolarizing due to the high intracellular chloride concentration caused by high NKCC1 expression and low KCC2 expression. Rescuing the function of cation–chloride cotransporters can occur via (1) inhibition of NKCC1 activity using bumetanide, (2, 3) increase in KCC2 function using a KCC2 activator or CKB enhancer, and (4) inhibitors of WNK/SPAK kinases.

5.2. Modulation of chloride homeostasis via cation–chloride cotransporters

Emerging evidence suggests that chloride homeostasis is a therapeutic target for HD. Pharmacological agents that target cation–chloride cotransporters (i.e.NKCC1 orKCC2) therefore might be used to treat HD (figure 3b). Of note, dysregulation of cation–chloride cotransporters and GABA polarity was associated with several neuropsychiatric disorders [70,134–139] (reviewed in [27,140]). Such abnormal   receptor neurotransmission can be rescued by bumetanide, an NKCC1 inhibitor that decreases intracellular chloride concentration. Bumetanide is an FDA-approved diuretic agent that has been used in the clinic. It attenuates many neurological and psychiatric disorders in preclinical studies and some clinical trials for traumatic brain injury, seizure, chronic pain, cerebral infarction, Down syndrome, schizophrenia, fragile X syndrome and autism (reviewed in [141]). Daily intraperitoneal injections of bumetanide also restored the impaired motor function ofHDmice (R6/2, Y-T Hsu,Y-GChang, Y-CLi, K-YWang, H-MChen, D-J Lee, C-HTsai, C-C Lien,YChern 2018, personal communication). The effect of bumetanide is likely to be mediated by NKCC1 because genetic ablation of NKCC1 in the striatum also rescued the motor deficits in R6/2 mice (Y-T Hsu, Y-G Chang, Y-C Li, K-Y Wang, H-M Chen, D-J Lee, C-H Tsai, C-C Lien, Y Chern 2018, personal communication). This study uncovered a previously unrecognized depolarizing or excitatory action of GABA in the aberrant motor control in HD. In addition, chronic treatment with bumetanide also improved the impaired memory in R6/2 mice [69], supporting the importance of NKCC1 in HD pathogenesis. Owing to the poor ability of bumetanide to pass through the blood–brain barrier, further optimization of bumetanide and other NKCC1 inhibitors is warranted [142,143].

Disruption of KCC2 function is detrimental to inhibitory transmission and agents to activate KCC2 function would be beneficial in HD. However, no agonist of KCC2 has been described until very recently [144,145]. A new KCC2 agonist (CLP290) has been shown to facilitate functional recovery after spinal cord injury [145]. It would be of great interest to evaluate the effect of KCC2 agonists on HD progression. Another KCC2 activator, CLP257, was found to increase the cell surface expression of KCC2 in a rat model of neuropathic pain [146]. Post-translational modification of KCC2 by kinases may modulate the function of KCC2. The WNK/SPAK kinase complex, composed of WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase), is known to phosphorylate and stimulate NKCC1 or inhibit KCC2 [147]. Thus, compounds that inhibit WNK/SPAK kinases will result in KCC2 activation and NKCC1 inhibition.

Some compounds have been noted as potential inhibitors of WNK/SPAK kinases and need to be further tested for their effects on cation–chloride cotransporters [148–150]. An alternative mechanism to activate KCC2 is manipulation of its interacting proteins (e.g. CKB [65,66]). Because CKB could activate the function of KCC2 [65,66], CKB enhancers may increase the function of KCC2. In HD, reduced expression and activity of CKB is associated with motor deficits and hearing impairment [68,88]. Enhancing CKB activity by creatine supplements ameliorated the motor deficits and hearing impairment of HD mice. It is worthwhile to further investigate the interaction of KCC2 and CKB in GABAergic neurotransmission and motor deficits in HD. The depolarizing GABA action with altered expression levels of NKCC1 or KCC2 is associated with neuroinflammation in HD brains [32,69]. Blockade of TNF-a using Xpro1595 (a dominant negative inhibitor of soluble TNF-a) [151] in vivo led to significant beneficial effects on disease progression in HD mice [152] and reduced the expression of NKCC1It would be of great interest to test the effect of other anti-inflammatory agents [153] on the function and expression of NKCC1 and GABAergic inhibition. Neuroinflammation is implicated in most neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease [154,155], and the interaction of cation–chloride cotransporters and neuroinflammation in GABAergic neurotransmission may also play a critical role in other neurodegenerative diseases.

Chloride extrusion enhancers as novel therapeutics for neurological diseases

Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

Upon activation by with-no-lysine kinases, STE20/SPS1-related proline–alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na⁺-Cl⁻ cotransporter (NCC) and Na⁺-K⁺-2Cl⁻ cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.

Chemical library screening for WNK signalling inhibitors using fluorescence correlation spectroscopy.

WNKs (with-no-lysine kinases) are the causative genes of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), and form a signal cascade with OSR1 (oxidative stress-responsive 1)/SPAK (STE20/SPS1-related proline/alanine-rich protein kinase) and Slc12a (solute carrier family 12) transporters. We have shown that this signal cascade regulates blood pressure by controlling vascular tone as well as renal NaCl excretion. Therefore agents that inhibit this signal cascade could be a new class of antihypertensive drugs. Since the binding of WNK to OSR1/SPAK kinases was postulated to be important for signal transduction, we sought to discover inhibitors of WNK/SPAK binding by screening chemical compounds that disrupt the binding. For this purpose, we developed a high-throughput screening method using fluorescent correlation spectroscopy. As a result of screening 17000 compounds, we discovered two novel compounds that reproducibly disrupted the binding of WNK to SPAK. Both compounds mediated dose-dependent inhibition of hypotonicity-induced activation of WNK, namely the phosphorylation of SPAK and its downstream transporters NKCC1 (Na/K/Cl cotransporter 1) and NCC (NaCl cotransporter) in cultured cell lines. The two compounds could be the promising seeds of new types of antihypertensive drugs, and the method that we developed could be applied as a general screening method to identify compounds that disrupt the binding of two molecules.

N-Ethylmaleimide increases KCC2 cotransporter activity by modulating transporter phosphorylation

K⁺/Cl⁻ cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl⁻ levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABA_A) receptors, which are Cl⁻-permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.

                                                                  

Tiagabine (trade name Gabitril) is n anticonvulsant medication produced by Cephalon that is used in the treatment of epilepsy. The drug is also used off-label in the treatment of anxiety disorders and panic disorder.

Tiagabine is approved by U.S. Food and Drug Administration (FDA) as an adjunctive treatment for partial seizures in individuals of age 12 and up. It may also be prescribed off-label by physicians to treat anxiety disorders and panic disorder as well as neuropathic pain (including fibromyalgia). For anxiety and neuropathic pain, tiagabine is used primarily to augment other treatments. Tiagabine may be used alongside selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, or benzodiazepines for anxiety, or antidepressants, gabapentin, other anticonvulsants, or opioids for neuropathic pain.^[4]

Tiagabine increases the level of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, by blocking the GABA transporter 1 (GAT-1), and hence is classified as a GABA reuptake inhibitor (GRI).

Conclusion

Today’s post shows how you need to read well beyond the autism research, not to miss something useful.

Some of today’s suggested therapies for Huntington’s are likely to help some types of autism, but some will certainly have a negative effect in some people.  For example, increasing the amount of GABA in the CNS would do my son no good at all.

The emerging field of drugs that enhance KCC2 should be very beneficial to all those with autism who are bumetanide responders.

Enhancing CKB with creatine is interesting. Creatine is a muscle building supplement used by body builders and some DAN doctors. It does have interactions at high doses.