Modulating Neuronal Chloride via WNK

Today’s post is a little complicated, but should be relevant to parents already using bumetanide to reduce the severity of autism.

Tuning neurons via Cl-sensitive WNK

The science behind today’s post only started to evolve twenty years ago when it became understood how chloride enters and exits the neurons in your brain. Nonetheless there is now a vast amount of research and there are parts that have not yet been covered in this blog. 

A moving target

The first thing to realize is that trying to reduce the elevated level of chloride found in much autism is very much an ongoing battle. Chloride is flowing in too fast via NKCC1 and exiting too slowing via KCC2.

If you want to reduce the entry via NKCC1, or increase the exit via KCC2, either of these two strategies should lower the equilibrium level of chloride.  Most strategies in this blog target NKCC1, but in another disease (neuropathic pain) the target has been KCC2.

Whichever you target, the risk is that the body’s feedback loops come into play and undo some of your good work. This was highlighted recently in a paper by Kristopher Kahle at Yale, who looks likely to be joining this blog’s Dean’s List, which highlights the researchers who are really worth following. He is part of the new generation of higher quality researcherswho have an interest in autism.   

If all that was not complex, we have to realize that the number of these valves (cotransporters) that either let chloride enter or exit, is changing all the time.  Many factors relating to inflammation and pain affect the number of NKCC1 and KCC2 cotransporters, so in times of inflammation  you get a reduction KCC2 and/or an increase in NKCC1; hence a higher level of chloride in your neurons.

When people have a traumatic brain injury (TBI), they get an increase in NKCC1 and so an increase in neuronal chloride.  This makes the neurotransmitter GABA less inhibitory, this can lead to cognitive loss, behavioral changes and even a tendency to seizures.

In TBI not surprisingly you have elevated inflammatory signaling, such as via something called NF-κB. As pointed out by our reader AJ, when you take the supplement Astaxanthin, you reduce the expression of NKCC1 in TBI and this has been shown to be via NF-κB. So the potent antioxidant and broadly anti-inflammatory Astaxanthin is a good choice for people with elevated NF-κB.

Much is written in neuropathic pain research about KCC2 and drugs are being developed that could later be repurposed for autism (and indeed TBI). In neuropathic pain there is a lack of KCC2 expression and this is known to be linked to something called WNK1.  The WNK1 gene provides instructions for making multiple versions of the WNK1 protein. 

Mechanisms that control NKCC1 and KCC2

There are multiple mechanisms that affect the expression of NKCC1 and KCC2.  In some cases the two (NKCC1 and KCC2) are interrelated so either one is expressed or the other is expressed.  In the mature brain there should be KCC2, but little NKCC1.  

The current research by Kristopher Kahle is based on the recent discovery of a “rheostat” of chloride homeostasis, comprising the Cl^- sensitive WNK-SPAK kinases and the NKCC1/KCC2 cotransporters. This rheostat provides a way to reversibly tune the strength of inhibition in neurons.

In effect this means that inhibiting WNK should make GABA more inhibitory, which is the goal for all people who have elevated chloride in their neurons.   

Restoring GABA inhibition in a Rett syndrome mouse model by tuning a kinase-regulated Cl-rheostat

GABA_A receptors are ligand-gated Cl- channels. GABA_AR activation can elicit excitatory or inhibitory responses, depending on the intraneuronal Cl- concentration levels. Such levels are largely established by the Cl- co-transporters NKCC1 and KCC2. A progressive postnatal increase in KCC2 over NKCC1 activity drives the emergence of GABA_AR-mediated synaptic inhibition, and is critical for functional brain maturation. A delay in this NKCC1/KCC2 ‘switch’ contributes to the impairment of GABAergic inhibition observed in Rett syndrome, fragile X syndrome, and other neurodevelopmental conditions, such as epilepsy.

Kristopher Kahle and his colleagues aim to understand the mechanisms that govern these developmental changes in NKCC1/KCC2 activity. They hypothesize that an improved knowledge of these mechanisms will lead to the development of novel strategies for restoring GABAergic inhibition. The researchers propose to exploit their recent discovery of a ‘rheostat’ of Cl- homeostasis, comprising the Cl-sensitive WNK-SPAK kinases and the NKCC1/KCC2 cotransporters^1-3. This rheostat provides a phosphorylation-dependent way to reversibly tune the strength of synaptic inhibition in neurons.

The team will create genetic mouse models with inducible expression of phospho-mimetic or constitutively dephosphorylated WNK-SPAK-KCC2 pathway components. They will also develop novel WNK-SPAK kinase inhibitors that function as simultaneous NKCC1 inhibitors and KCC2 activators. These mouse models and compounds will be used to therapeutically restore GABA inhibition in the Rett syndrome MeCP2(R308/Y) mouse model. The researchers will use a combination of two-photon microscopy coupled with improved fluorescent optogenetic Cl- sensing, quantitative phosphoproteomics and patch-clamp electrophysiology to assess cellular and physiological changes in these mice.

Kinase-KCC2 coupling: Cl- rheostasis, disease susceptibility, therapeutic target.

The intracellular concentration of Cl⁻ ([Cl⁻]_i) in neurons is a highly regulated variable that is established and modulated by the finely tuned activity of the KCC2 cotransporter. Despite the importance of KCC2 for neurophysiology and its role in multiple neuropsychiatric diseases, our knowledge of the transporter's regulatory mechanisms is incomplete. Recent studies suggest that the phosphorylation state of KCC2 at specific residues in its cytoplasmic COOH terminus, such as Ser940 and Thr906/Thr1007, encodes discrete levels of transporter activity that elicit graded changes in neuronal Cl⁻ extrusion to modulate the strength of synaptic inhibition via Cl⁻-permeable GABA_A receptors. In this review, we propose that the functional and physical coupling of KCC2 to Cl⁻-sensitive kinase(s), such as the WNK1-SPAK kinase complex, constitutes a molecular “rheostat” that regulates [Cl⁻]_i and thereby influences the functional plasticity of GABA. The rapid reversibility of (de)phosphorylation facilitates regulatory precision, and multisite phosphorylation allows for the control of KCC2 activity by different inputs via distinct or partially overlapping upstream signaling cascades that may become more or less important depending on the physiological context. While this adaptation mechanism is highly suited to maintaining homeostasis, its adjustable set points may render it vulnerable to perturbation and dysregulation. Finally, we suggest that pharmacological modulation of this kinase-KCC2 rheostat might be a particularly efficacious strategy to enhance Cl⁻ extrusion and therapeutically restore GABA inhibition.

Dominant-negative mutation, genetic knockdown, or chemical inhibition of WNK1 in immature neurons (but not mature neurons) is sufficient to trigger a hyperpolarizing shift in GABA activity by enhancing KCC2-mediated Cl⁻ extrusion secondary to a reduction of Thr906/Thr1007 inhibitory phosphorylation (Friedel et al. 2015). These results extended previous work by Rinehart et al. (2009), who showed that KCC2 Thr906 phosphorylation inversely correlates with KCC2 activity in the developing mouse brain, and Inoue et al. (2012), who showed a phosphorylation-dependent inhibitory effect of taurine on KCC2 activity in immature neurons that was recapitulated by WNK1 overexpression in the absence of taurine. Together, these compelling data suggest that a postnatal decrease in WNK1-regulated inhibitory phosphorylation of KCC2 also contributes to increased KCC2 function (Fig. 5), and thus to the excitatory-to-inhibitory GABA shift that occurs during development. This also raises the possibility that dysfunctional phosphoregulation of these sites could be important in certain neurodevelopmental pathologies, like autism or neonatal seizures. An important issue of future investigation will be to determine how the increased levels of Cl⁻ in immature neurons affect WNK1 kinase activity. Could taurine, a factor known to activate WNK1 in immature neurons, achieve this by decreasing the sensitivity of WNK1 to Cl⁻?

Recently, a few groups have developed innovative high-throughput assays to screen for compounds that modulate KCC2 activity (Delpire et al. 2009, 2012; Gagnon et al. 2013), and one drug shows promise as a KCC2-dependent Cl⁻ extrusion enhancer with therapeutic effect in a model of neuropathic pain (Gagnon et al. 2013). These early but encouraging results require validation, but they establish the validity in vivo of the concept of GABA modulation via the pharmacological targeting of CCC-dependent Cl⁻ transport (Gagnon et al. 2013; Kahle et al. 2014a; Kaila et al. 2014). Could CCC phosphoregulatory mechanisms, normally employed to modulate transporter activity in response to perturbation or biological need, be harnessed to stimulate the KCCs (or inhibit NKCC1) for therapeutic benefit in disease states featuring an accumulation of intracellular Cl⁻?

Moreover, since the WNK kinases might also be the Cl⁻ sensors that detect changes in intracellular Cl⁻ (Piala et al. 2014), inhibiting these molecules might prevent feedback mechanisms that would counter the effects of targeting NKCC1 or KCC2 alone.

  

Chloride extrusion enhancers as novel therapeutics for neurological diseases

The K(+)-Cl(-) cotransporter KCC2 is responsible for maintaining low Cl(-) concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABA(A) and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl(-) transport. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl(-)]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl(-)]i. CLP257 restored impaired Cl(-) transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a drugable target for CNS diseases.  

Chloride sensing by WNK1 kinase involves inhibition of autophosphorylation

WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. Here, we found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation.

The WNK-SPAK/OSR1 pathway: Master regulator of cation-chloride cotransporters

The WNK-SPAK/OSR1 kinase complex is composed of the kinases WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase) or the SPAK homolog OSR1 (oxidative stress–responsive kinase 1). The WNK family senses changes in intracellular Cl⁻ concentration, extracellular osmolarity, and cell volume and transduces this information to sodium (Na⁺), potassium (K⁺), and chloride (Cl⁻) cotransporters [collectively referred to as CCCs (cation-chloride cotransporters)] and ion channels to maintain cellular and organismal homeostasis and affect cellular morphology and behavior. Several genes encoding proteins in this pathway are mutated in human disease, and the cotransporters are targets of commonly used drugs. WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate Cl⁻-importing, Na⁺-driven CCCs or inhibit the Cl⁻-extruding, K⁺-driven CCCs. These coordinated and reciprocal actions on the CCCs are triggered by an interaction between RFXV/I motifs within the WNKs and CCCs and a conserved carboxyl-terminal docking domain in SPAK and OSR1. This interaction site represents a potentially druggable node that could be more effective than targeting the cotransporters directly. In the kidney, WNK-SPAK/OSR1 inhibition decreases epithelial NaCl reabsorption and K⁺ secretion to lower blood pressure while maintaining serum K⁺. In neurons, WNK-SPAK/OSR1 inhibition could facilitate Cl⁻ extrusion and promote γ-aminobutyric acidergic (GABAergic) inhibition. Such drugs could have efficacy as K⁺-sparing blood pressure–lowering agents in essential hypertension, nonaddictive analgesics in neuropathic pain, and promoters of GABAergic inhibition in diseases associated with neuronal hyperactivity, such as epilepsy, spasticity, neuropathic pain, schizophrenia, and autism. 

Regulation of OSR1 and the sodium, potassium, two chloride cotransporter by convergent signals

The Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and the STE20/SPS1-related proline-, alanine-rich kinase directly regulate the solute carrier 12 family of cation-chloride cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. OSR1 andSTE20/SPS1-related proline-,alanine-rich kinase are activated by with no lysine [K] protein kinases that phosphorylate the essential activation loop regulatory site on these kinases. We found that inhibition of phosphoinositide 3-kinase (PI3K) reduced OSR1 activation by osmotic stress. Inhibition of the PI3K target pathway, the mammalian target of rapamycin complex 2 (mTORC2), by depletion of Sin1, one of its components, decreased activation of OSR1 by sorbitol and reduced activity of the OSR1 substrate, the sodium, potassium, two chloride cotransporter, in HeLa cells. OSR1 activity was also reduced with a pharmacological inhibitor of mTOR. mTORC2phosphorylated OSR1 on S339 in vitro, and mutation of this residue eliminated OSR1 phosphorylation by mTORC2. Thus, we identify a previously unrecognized connection ofthePI3K pathwaythroughmTORC2 to a Ste20 proteinkinase and ion homeostasis.

Significance

With no lysine [K] (WNK) protein kinases are sensitive to changes in osmotic stress. Through the downstream protein kinases oxidative stress-responsive 1 (OSR1) and STE20/SPS1related proline-, alanine-rich kinase, WNKs regulate a family of ion cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. We found that a major phosphoinositide 3-kinase target pathway, the mammalian target of rapamycin complex 2, also phosphorylates OSR1, coordinating with WNK1 to enhance OSR1 and ion cotransporter function.

Changes in tonicity regulate the WNK-OSR1/SPAK pathway to control ion cotransporters for volume and ion homeostasis. We find that mTORC2 also contributes to enhanced OSR1 activity. Inhibiting mTORC2 does not inhibit WNK1 activity, indicating PF1 and PF2regions.

We conclude that cell homeostasis requires the multi level integration of WNK osmosensing and PI3K survival pathways.

The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+–Cl−co-transporters

These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl− influx, we propose that the targeting of WNK–SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl− extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.

WNK Inhibitors

The first orally bioavailable pan-WNK-kinase inhibitor is WNK463.

“WNK463 is an orally bioavailable pan-WNK-kinase inhibitor. In vivo: WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.”\

WNK463 is available as a research drug.

It looks like WNK2 is also very relevant, perhaps more so than WNK1, because we are interested specifically in the brain, where there is a lot of WNK2. WNK3 also looks very relevant. There is also WNK4.

WNK2 Kinase Is a Novel Regulator of Essential Neuronal Cation-Chloride Cotransporters

Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl⁻-dependent ⁸⁶Rb⁺ uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl⁻ accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation.  

Inhibition of WNK3 Kinase Signaling Reduces Brain Damage and Accelerates Neurological Recovery After Stroke.

WNK3 KO mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared to WNK3 WT mice subjected to MCA occlusion. The neuroprotective phenotypes conferred by WNK3 KO were associated with a decrease in stimulatory hyper-phosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr²⁰³/Thr²⁰⁷/Thr²¹², as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or siRNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia.

CONCLUSION

These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury, and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection following ischemic stroke.

  

Conclusion

I think we can simplify all of this into:-

We already know that many people with autism benefit from making GABA more inhibitory.

There are currently two types of therapy:

1.     Reducing intracellular chloride

2.     Modifying GABA_A α3 subunit sensitivity (low dose clonazepam from Professor Catterall)

Reducing intracellular chloride

This can be achieved by:

·        Reducing the inflow via NKCC1 using bumetanide and in future years using drugs which better pass the blood brain barrier, e.g. the research drug BUM5. Consider improving the potency of the current drug bumetanide using an OAT3 inhibitor that will increase its concentration and half-life, apparently already possible with acetazolamide.

·        Increasing the outflow via KCC2, possible with the research drug CLP257  

·        Reducing the inflow via AE3, possible with Diamox/acetazolamide

·        Substituting Br^- for Cl^-, using potassium bromide

·        Changing the relative expression of NKCC2/KCC1

Changing the relative expression of NKCC1/KCC2

·        This can be done today by treating any underlying inflammation.  Inflammation shifts the NKCC2/KCC1 balance in a way that makes GABA more excitatory, which is bad. This might be achieved by targeting IL-6, NF-κB or just treating any GI problems and allergies.  Always treat the comorbidities of autism.  

·        Using WNK inhibitors it will hopefully be possible to manually tune the NKCC1/KCC2 balance, just like tuning a piano. One pan-WNK-kinase inhibitor is WNK463.

·        I continue to believe that RORα could be an effective way to increase KCC2 expression and this is something that is not so hard to test.

The Purkinje-RORa-Estradiol-Neuroligin-KCC2 axis in Autism

I will be keeping a look out for further papers by Dr Kahle and be interested in any WNK-SPAK/OSR1 inhibitors he proposes.  If I was him I would start with WNK463.

There is more to the story, because naturally I want to see how estradiol relates to WNK and finally wrap up this subject. Then we will know how to treat the immature neurons often found in autism. A case of forever young.

In a following post I intend to do that; here is a sneak, but complex, preview.

The Excitatory/Inhibitory Imbalance – GABAA stabilization via IP3R

This blog aims to synthesize the relevant parts of the research and make connections that point towards some potential therapeutic avenues.  Most researchers work in splendid isolation and concentrate on one extremely narrow area of interest.

The GABA_A reset, not functional in some autism

On the one hand things are very simple, if the GABA_A receptors function correctly and are inhibitory and the glutamate receptors (particularly NMDA and mGluRx) function correctly, there is harmony and a  perfect excitatory/inhibitory balance.

Unfortunately numerous different things can go wrong and you could write a book about each one.

As you dig deeper you see that the sub-unit make-up of GABA_A receptors is not only critical but changes.  The plus side is that you can influence this.

Today we see that the receptors themselves are physically movable and sometimes get stuck in the “wrong place”. When the receptors cluster close together they produce a strong inhibitory effect, but continual activation of NMDA receptors by the neurotransmitter glutamate - as occurs naturally during learning and memory, or in epilepsy - leads to an excess of incoming calcium, which ultimately causes the receptors to become more spread out, reducing how much the neuron can be inhibited by GABA. There needs to be a mechanism to move the GABA_A receptors back into their original clusters.

Very clever Japanese researchers have figured out the mechanism and to my surprise it involves one of those hubs, where strange things in autism seem to connect to, this time IP₃R.

Is dysregulated IP3R calcium signaling a nexus where genes altered in ASD converge to exert their deleterious effect?

I guess the Japanese answer to my question above is simple. YES,

A very recent science-light article by Gargus on IP3:-

Mighty element plays major part in autism

Now to the Japanese.

I wonder if Gargus has read the Japanese research, because both the cause and cure for the GABA_A receptors dispersing and clustering is an increase in calcium and both mediated by glutamate.  

The excitatory neurotransmitter glutamate binds to the mGluR receptor and activates IP₃ receptor-dependent calcium release and protein kinase C to promote clustering of GABA_A receptors at the postsynaptic membrane - the place on a neuron that receives incoming neurotransmitters from connecting neurons.

If Professor Gargus is correct, and IPR3 does not work properly in autism, the GABA_A receptors are likely not sitting there in nice neat clusters. As a result their inhibitory effect is reduced and neurons fire when they should not.

Gargus has found that in the types of autism he has investigated IP₃ receptor open as they should, but close too fast and so do not release enough calcium from the cell’s internal calcium store (the endoplasmic reticulum).

In particular the Japanese researchers found that:-

“Stabilization of GABA synapses by mGluR-dependent Ca²⁺ release from IP₃R via PKC”

If the IP₃ receptor does not stay open as long as it should, not enough Ca²⁺ will be released and GABA synapses will not be stabilized. Then GABA_A receptors will be diffused rather than being in neat clusters.

The science-light version of the Japanese study:-

How excitatory/inhibitory balance is maintained in the brain

Just as a thermostat is used to maintain a balanced temperature in a home, different biological processes maintain the balance of almost everything in our bodies, from temperature and oxygen to hormone and blood sugar levels. In our brains, maintaining the balance -- or homeostasis -- between excitation and inhibition within neural circuits is important throughout our lives, and now, researchers at the RIKEN Brain Science Institute and Nagoya University in Japan, and École Normale Supérieure in France have discovered how disturbed inhibitory connections are restored. Published in Cell Reports, the work shows how inhibitory synapses are stabilized when the neurotransmitter glutamate triggers stored calcium to be released from the endoplasmic reticulum in neurons.

"Imbalances in excitation and inhibition in the brain has been linked to several disorders," explains lead author Hiroko Bannai. "In particular, forms of epilepsy and even autism appear to be related to dysfunction in inhibitory connections."

One of the key molecules that regulates excitation/inhibition balance in the brain is the inhibitory neurotransmitter GABA. When GABA binds to GABAA receptors on the outside of a neuron, it prevents that neuron from sending signals to other neurons. The strength of the inhibition can change depending on how these receptors are spaced in the neuron's membrane.

While GABAA receptors are normally clustered together, continual neural activation of NMDA receptors by the neurotransmitter glutamate -- as occurs naturally during learning and memory, or in epilepsy -- leads to an excess of incoming calcium, which ultimately causes the receptors to become more spread out, reducing how much the neuron can be inhibited by GABA.

To combat this effect, the receptors are somehow continually re-clustered, which maintains the proper excitatory/inhibitory balance in the brain. To understand how this is accomplished, the team focused on another signaling pathway that also begins with glutamate, and is known to be important for brain development and the control of neuronal growth.

In this pathway glutamate binds to the mGluR receptor and leads to the release of calcium from internal storage into the neuron's internal environment. Using quantum dot-single particle tracking, the team was able to show that after release, this calcium interacts with protein kinase C to promote clustering of GABAA receptors at the postsynaptic membrane--the place on a neuron that receives incoming neurotransmitters from connecting neurons.

These findings show that glutamate activates distinct receptors and patterns of calcium signaling for opposing control of inhibitory GABA synapses.

Notes Bannai, "it was surprising that the same neurotransmitter that triggers GABAA receptor dispersion from the synapse, also plays a completely opposite role in stabilizing GABAA receptors, and that the processes use different calcium signaling pathways. This shows how complex our bodies are, achieving multiple functions by maximizing a limited number of biological molecules.

Pre-activation of the cluster-forming pathway completely prevented the dispersion of GABAA receptors that normally results from massive excitatory input, as occurs in status epilepticus -- a condition in which epileptic seizures follow one another without recover of consciousness. Bannai explains, "further study of the molecular mechanisms underlying the process we have uncovered could help develop treatments or preventative medication for pathological excitation-inhibition imbalances in the brain.

"The next step in understanding how balance is maintained in the brain is to investigate what controls which pathway is activated by glutamate. Most types of cells use calcium signals to achieve biological functions. On a more basic level, we believe that decoding these signals will help us understand a fundamental biological question: why and how are calcium signals involved in such a variety of biological phenomena?"

The full Japanese study:-

Bidirectional Control of Synaptic GABA_AR Clustering by Glutamate and Calcium

·        Bidirectional synaptic control system by glutamate and Ca²⁺ signaling

·        Stabilization of GABA synapses by mGluR-dependent Ca²⁺ release from IP₃R via PKC

·        Synaptic GABA_AR clusters stabilized through regulation of GABA_AR lateral diffusion

·        Competition with an NMDAR-dependent Ca²⁺ pathway driving synaptic destabilization

GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABA_A receptor (GABA_AR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP₃ receptor-dependent calcium release and protein kinase C to promote GABA_AR clustering and GABAergic transmission. This GABA_AR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses.

In this study, we demonstrate that the mGluR/IICR/PKC pathway stabilizes GABAergic synapses by constraining lateral diffusion and increasing clustering of GABA_ARs, without affecting the total number of GABA_AR on the cell surface. This pathway defines a unique form of homeostatic regulation of GABAergic transmission under conditions of basal synaptic activity and during recovery from E/I imbalances. The study also highlights the ability of neurons to convert a single neurotransmitter (glutamate) into an asymmetric control system for synaptic efficacy using different calcium-signaling pathways.

The most striking conceptual finding in this study is that two distinct intracellular signaling pathways, NMDAR-driven Ca²⁺ influx and mGluR-driven Ca²⁺ release from the ER, effectively driven by the same neurotransmitter, glutamate, have an opposing impact on the stability and function of GABAergic synapses. Sustained Ca²⁺ influx through ionotropic glutamate receptor-dependent calcium signaling increases GABA_AR lateral diffusion, thereby causing the dispersal of synaptic GABA_AR, while tonic mGluR-mediated IICR restrains the diffusion of GABA_AR, thus increasing its synaptic density. How can Ca²⁺ influx and IICR exert opposing effects on GABA synaptic structure? Our research indicates that each Ca²⁺ source activates a different Ca²⁺-dependent phosphatase/kinase: NMDAR-dependent Ca²⁺ influx activates calcineurin, while ER Ca²⁺ release activates PKC.

Taken together, these results lead us to propose the following model for bidirectional competitive regulation of GABAergic synapses by glutamate signaling. Phasic Ca²⁺ influx through NMDARs following sustained neuronal excitation or injury leads to the activation of calcineurin, overcoming PKC activity and relieving GABA_AR diffusion constraints. In contrast, during the maintenance of GABAergic synaptic structures or the recovery from GABA_AR dispersal, the ambient tonic mGluR/IICR pathway constrains GABA_AR diffusion by PKC activity, overcoming basal calcineurin activity. One possible mechanism of dual regulation of GABA_AR by Ca²⁺ is that each Ca²⁺-dependent enzyme has a unique sensitivity to the frequency and number of external glutamate release events and can act to decode neuronal inputs (Fujii et al., 2013xNonlinear decoding and asymmetric representation of neuronal input information by CaMKIIα and calcineurin. Fujii, H., Inoue, M., Okuno, H., Sano, Y., Takemoto-Kimura, S., Kitamura, K., Kano, M., and Bito, H. Cell Rep. 2013; 3: 978–987

Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References, Li et al., 2012xCalcium input frequency, duration and amplitude differentially modulate the relative activation of calcineurin and CaMKII. Li, L., Stefan, M.I., and Le Novère, N. PLoS ONE. 2012; 7: e43810

Crossref | PubMed | Scopus (29)See all References, Stefan et al., 2008xAn allosteric model of calmodulin explains differential activation of PP2B and CaMKII. Stefan, M.I., Edelstein, S.J., and Le Novère, N. Proc. Natl. Acad. Sci. USA. 2008; 105: 10768–10773

Crossref | PubMed | Scopus (44)See all References) in inhibitory synapses.

Tight control of E/I balance, the loss of which results in epilepsy and other brain and nervous system diseases/disorders, is dependent on GABAergic synaptic transmission (Mann and Paulsen, 2007xRole of GABAergic inhibition in hippocampal network oscillations. Mann, E.O. and Paulsen, O. Trends Neurosci. 2007; 30: 343–349

Abstract | Full Text | Full Text PDF | PubMed | Scopus (194)See all ReferencesMann and Paulsen, 2007). A recent study showed that the excitation-induced acceleration of GABA_AR diffusion and subsequent dispersal of GABA_ARs from synapses is the cause of generalized epilepsy febrile seizure plus (GEFS+) syndrome (Bouthour et al., 2012xA human mutation in Gabrg2 associated with generalized epilepsy alters the membrane dynamics of GABAA receptors. Bouthour, W., Leroy, F., Emmanuelli, C., Carnaud, M., Dahan, M., Poncer, J.C., and Lévi, S. Cereb. Cortex. 2012; 22: 1542–1553

Crossref | PubMed | Scopus (14)See all ReferencesBouthour et al., 2012). Our results indicate that pre-activation of the mGluR/IICR pathway by DHPG could completely prevent the dispersion of synaptic GABA_ARs induced by massive excitatory input similar to status epilepticus. Thus, further study of the molecular mechanisms underlying the mGluR/IICR-dependent stabilization of GABAergic synapses via regulation of GABA_AR lateral diffusion and synaptic transmission could be helpful in the prevention or treatment of pathological E/I imbalances, for example, in the recovery of GABAergic synapses from epileptic states

DHPG = group I mGluR agonist dihydroxyphenylglycine.

On a practical level you want to inhibit GABA_A  dispersion and promote GABA_A stabilization. How you might do this would depend on exactly what was the underlying problem.

If the problem is IP₃R not releasing enough calcium, you might activate PKC in a different way or just increase the signal from Group 1 mGluR. If the problem is too much calcium influx through NMDA receptors due to excess glutamate, you could increase the re-uptake of glutamate, via GLT-1, using Riluzole.  You could block the flow of Ca²⁺ through NMDA receptors using an antagonist.

The Japanese used dihydroxyphenylglycine (DHPG) as their Group 1 mGluR agonist.  DHPG is an agonist of mGluR1 and mGluR5.  We have come across mGluR5 many times before in this blog.  Mavoglurant is an experimental drug candidate for the treatment of fragile X syndrome.  It is an antagonist of mGluR5.

We have seen many times before that there is both hypo and hyper function possible and indeed that fragile X is not necessarily a good model for autism.

The selective mGluR5 agonist CHPG protects against traumatic brain injury, which would indeed make sense. Although, that research suggests an entirely different mechanism.

The selective mGluR5 agonist CHPG protects against traumatic brain injury in vitro and in vivo via ERK and Akt pathway

The calcium released by IP₃ works in complex way together with DAG (diacylglycerol ) to activate PKC (protein kinase C).

Ideally you would have enough calcium released from IP3, but you could also increase DAG. It depends which part of the process is rate-limiting.

Diacylglycerol (DAG) has been investigated extensively as a fat substitute due to its ability to suppress the accumulation of body fat.  Diglycerides, generally in a mix with monoglycerides are common food additives largely used as emulsifiers. In Europe, when used in food the mix is called E471.

Conclusion

On the one hand things are getting very complicated, but on the other we keep coming back to the same variables (IP3R, mGlur5, GABA_A etc.).

It is pretty clear that some very personalized therapy will be needed.  Is it an mGlur5 agonist or antagonist? Or quite possibly neither, because in different parts of the brain it will have a good/bad effect.

It does look like Riluzole should work well in some people.

A safe IP3R agonist looks a possibility. As shown in the diagram earlier in this post,IP3 is usually made in situ, but agonists exist.

In effect autism could be the opposite of Huntington’s disease. In Huntington’s,  type 1 IP₃ receptors are  more sensitive to IP₃, which leads to the release of too much Ca²⁺ from the ER. The release of Ca²⁺ from the ER causes an increase in concentrations of Ca²⁺inside cells and in mitochondria.

According to Gargus we should have reduced concentrations of Ca²⁺inside cells in autism.

I suspect it is much more complicated in reality, because it is not just the absolute  level of Ca²⁺ but rather the flow of Ca²⁺; so it matters where it is coming from. I think we likely have impaired calcium channel activity of multiple types in autism and the actual level of intracellular calcium will not tell you much at all.

As the Japanese commented, it is surprising that glutamate is the neurotransmitter that controls the clustering, or not, of GABA_A receptors.  This suggests that you cannot ignore glutamate and just “fix” GABA.

The Glutamate Side of Things

Some readers have suggested that since we have discovered so many ways to treat the GABA_A dysfunctions common in autism, it is time to look at the glutamate side of things. Glutamate is the main excitatory neurotransmitter and has to be in balance with the opposing influence of GABA.

The chart below is really a summary of what has already been covered in this blog.  To newcomers it will look complicated, to regular readers it is just bringing together everything we have already covered, even those tauopathies appear. Tau protein tangles appear in Alzheimer’s and some autism.

Glutamate excitoxicity is what happens when things go really wrong, for example in a severe autistic regression.  I doubt you could be in a permanent state like this.

I am beginning to wonder is my son’s summer time raging, though triggered by allergy, develops to a so-called glutamatergic storm.  It fades to nothing  by using a Cav1.2 channel blocker, which does indeed stop those allergy mast cells de-granulating, but it stops the calcium influx in the above chart.  Existing dysfunction in Cav1.2 and Cav1.4 puts you at risk of excitotoxicity.

The oxidative damage to mitochondria causes lipid peroxidation and in particular the 4-HNE produced will cause tau protein, from a recent post and Alzheimer’s, to produce tau tangles, a damaging feature of so-called tauopathies.

The nitrosative stress in particular damages the production of the Complex 1 enzyme leading to mitochondrial disease/dysfunction. The damaging peroxynitrates can be quenched using high doses of calcium folinate. Oxidative stress and the reduced level of GSH can be treated with antioxidants like NAC and ALA.  

Reduced reuptake of glutamate, known to be caused by elevated TNF-α and immune dysfunction, is treatable via upregulating the GLT-1 transporter (beta-lactam antibiotics, riluzole and bromocriptine).

Elevated BDNF is a biomarker of autism and unfortunately this increases the chances of glutamate excitotoxicity.

An inactivated GABA switch that leaves neurons immature, will result in GABA acting excitatory rather than inhibitory, this itself can trigger of glutamate excitotoxicity. Use bumetanide.

Some types of autism feature NMDA hyper-function, this is treatable.  A deviation of NMDA function in either direction (hypo or hyper) leads to autism, but you need to know which way it is, to treat it.

It is also possible to have over/under expression of NMDA receptors.