Connecting Estradiol with WNK, SPAK and OSR1; plus Taurine

Japan, home to today’s complicated research

Today’s post hopes to give a more complete picture of the various processes involved in shifting the immature neurons often found in autism towards the mature neurons, found in most people.  This stalled process is complex and may only apply to around half of all autism.

The post assumes prior knowledge from previous posts about the GABA switch and the KCC2 and NKCC1 chloride cotransporters.

The best graphic I found is below and includes almost everything. The paper itself is very thorough and I recommend the scientists among you read the paper rather than my post.

What we want to understand is why neurons did not switch from immature to mature, in the process I am calling the “GABA switch”.  We know a great deal about what happens before and after the switch and many processes that can be  involved, but the exact switch itself remains undefined.

In a previous post I highlighted that neuroligin 2 (NL2)/RORa may be the GABA switch, but there is no mention of neuroligins in the research reviewed today. 

Neuroligins, Estradiol and Male Autism

So when you read today’s mainly Japanese research, you should note that one key part is missing, the actual trigger mechanism.

The ideal way to make neurons transition from immature to mature is the way nature intended. That requires an understanding of the GABA switch mechanism.

Source and excellent paper:-

Development and regulation of chloride homeostasis in the central nervous system

 The important things you might not notice:

E is the female hormone estrogen/estradiol

T is testosterone. Testosterone can be converted to estradiol by aromatase.

DHT is another male hormone Dihydrotestosterone. DHT is synthesized from testosterone by the enzyme 5α-reductase. In males, approximately 5% of testosterone undergoes 5α-reduction into DHT. DHT cannot be converted into estrogen.

Relative to testosterone, DHT is considerably more potent as an agonist of the androgen receptor (AR). This may turn out to be very important.

T3 is the active thyroid hormone, triiodothyronine

In earlier posts we saw that in autism there can be a lack of aromatase and that there is reduced expression of estrogen receptor beta.

In the diagram below this leads to reduced estrogen and increased testosterone. If there is elevated DHT this will make the situation worse.  All this down-regulates ROR-alpha.

ROR-alpha affects numerous things and is another nexus which links biological processes that have gone awry in autism. By upregulating ROR-alpha multiple good effects may follow, these include increasing KCC2 and reducing NKCC1.

It is certainly possible that the GABA switch is mediated by RORa-estradiol-Neuoligin-2.  In which case the solution is to upregulate RORa which can be done in many ways (androgen receptor, estrogen receptors etc.)

The schematic illustrates a mechanism through which the observed reduction in RORA in autistic brain may lead to increased testosterone levels through downregulation of aromatase. Through AR, testosterone negatively modulates RORA, whereas estrogen upregulates RORA through ER.

androgen receptor = AR

estrogen receptor = ER

Going back to the complex first chart in this post, we want to increase KCC2 in the immature neuron and reduce NKCC1.

So we want lines with flat end going into NKCC1, for example from OXT (the Oxytocin surge during natural birth).

We want arrows going to KCC2, for example we want more PKC (Protein Kinase C) coming from those  mGluRs, that we have come across many times in this blog.

What we do not want is anything coming from WNK- SPAK- OSR1.

Reduced expression of the thyroid hormone T3 does affect the both KCC2 and NKCC1 expression the brain. One of my earlier posts did suggest central hypothyroidism in autism, this fitted in with the findings of the Polish researcher at Harvard, who I had some correspondence with.

Oxidative Stress, Central Hypothyroidism, Autism and You   

Another transcription factor that has been identified as a potent regulator of KCC2 expression is upstream stimulating factor 1 (USF1) as well as USF2. The USF1 gene has been linked to familial combined hyperlipidemia. 

It is thought that increasing the expression of USF1 with increase KCC2, but it will increase other things as well.

We also know that Egr4 may be an important component in the mechanism for trophic factor-mediated upregulation of KCC2 protein in developing neurons.

Early Growth Response 4 (EGR-4) is a transcription factor that activates numerous other processes.

It is known that the growth factor Neurturin upregulates EGR4, but it does not cross the blood brain barrier. It was considered as a possible therapy for Parkinson’s Disease. In the first chart in this post, NRTN is Neurturin.

Neurturin Evokes MAPK-Dependent Upregulation of Egr4 and KCC2 in Developing Neurons

It turns out that EGR4 is redox sensitive. In other words certain types of oxidative stress should upregulate EGR4.

Recent studies have demonstrated that zinc controls KCC2 activity via a postsynaptic metabotropic zinc receptor/G protein-linked receptor 39 mZnR/GPR39. The levels of both synaptic Zn²⁺ and KCC2 are developmentally upregulated. During the postnatal period, synaptic Zn²⁺ accumulation and KCC2 expression reach levels similar to those in adult brain.  The zinc transporter 1 (ZnT-1), which is present in areas rich in synaptic zinc, is expressed from the first postnatal week in cortex, hippocampus, olfactory bulb. In the cerebellum, the expression of ZnT-1 in purkinje cells is increased during the second postnatal week.

We have seen that in autism there are anomalies with zinc; in effect it is in the wrong place. Perhaps there is a problem with the zinc transporter in some autism. Decreased ZnT-1 is associated with mild cognitive impairment (MCI).

The male/female hormones play a key role in KCC2/NKCC1, but estradiol/estrogen has a very complex role.

Estradiol can have paradoxical effects.  Its effects can also vary depending on whether you are male or female.

“the effects of estradiol on chloride cotransporters or GABA_A signaling may depend upon the direction of GABA_A responses”

In effect this may mean if GABA is working normally we get one effect on KCC2/NKCC1, but if it is working in reverse (bumetanide responders) we may see the opposite effect.

In the above chart estrogen is shown as increasing KCC2 mRNA in males (a good thing) but inhibiting KCC2 mRNA in females. Messenger RNA (mRNA) is one step in the process of producing the protein (KCC2) from its gene. So the more mRNA the better, if you want more of that protein.

Estrogen also has an effect on OSR1. As shown in this Japanese paper, estrogen is having the opposite effect to what we want; it is inhibiting KCC2 and stimulating NKCC1.

There is research specifically focused on the effect of estrogen on NKCC1 and KCC2. It looks like in some circumstances the effect is good, while in others it will be bad.

From the perspective I have from my posts on RORa, I am expecting a positive effect. I expect in bumetanide responders, estrogen/estradiol will increase KCC2 and reduce NKCC1 and so lower the level of chloride in neurons.

You can also easily argue that estrogen should be bad. What is clear is that inhibiting WNK, SPAK and OSR1 should all be good.  That then brings us to taurine and the start of the WNK-SPAK- OSR1 cascade.

As we have seen in previous posts,  TrkB (tyrosine receptor kinase B) a receptor for various growth factors including  brain-derived neurotrophic factor (BDNF), plays a role. In much autism BDNF is found to be elevated.

ERK is also called MAPK.  The MAPK/ERK pathway is best known in relation to (RAS/RAF-dependent) cancers. This RAS/RAF/ERK1/2 pathway is also known to be upregulated in autism.  In today’s case, ERK is just causing an increase in Early Growth Response 4 (EGR4).

Activating PKC looks a good idea.  It also is the mechanism in some other Japanese research I covered in an old post.  You may recall that in autism sometimes the GABA_A receptors get physically dispersed and need to be brought back tightly together, otherwise they do not work properly.  This process required calcium to be released from the via IP3R to increase PKC.

The Excitatory/Inhibitory Imbalance – GABA_A stabilization via IP3R

Studies have indeed shown that PKC is reduced in some autism, which is what you might have expected. 

Finally, the other estradiol/estrogen papers:- 

Kinases SPAK andOSR1 are up regulated by estradiol and activate NKCC1 in the developing hypothalamus

In immature neurons the amino acid neurotransmitter, γ-aminobutyric acid (GABA) provides the dominant mode for neuronal excitation by inducing membrane depolarization due to Cl⁻ efflux through GABA_A receptors (GABA_ARs). The driving force for Cl⁻ is outward because the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC1) elevates the Cl⁻ concentration in these cells. GABA-induced membrane depolarization and the resulting activation of voltage-gated Ca²⁺ channels is fundamental to normal brain development, yet the mechanisms that regulate depolarizing GABA are not well understood. The neurosteroid estradiol potently augments depolarizing GABA action in the immature hypothalamus by enhancing the activity of the NKCC1 cotransporter. Understanding how estradiol controls NKCC1 activity will be essential for a complete understanding of brain development. We now report that estradiol treatment of newborn rat pups significantly increases protein levels of two kinases upstream of the NKCC1 cotransporter, SPAK and OSR1. The estradiol-induced increase is transcription dependent, and its time course parallels that of estradiol-enhanced phosphorylation of NKCC1. Antisense oligonucleotide-mediated knockdown of SPAK, and to a lesser degree of OSR1, precludes estradiol-mediated enhancement of NKCC1 phosphorylation. Functionally, knockdown of SPAK or OSR1 in embryonic hypothalamic cultures diminishes estradiol-enhanced Ca²⁺ influx induced by GABA_AR activation. Our data suggest that SPAK and OSR1 may be critical factors in the regulation of depolarizing GABA-mediated processes in the developing brain. It will be important to examine these kinases with respect to sex differences and developmental brain anomalies in future studies.

The ability of the brain to synthesize estradiol in discrete loci raises the specter of estrogens as widespread endogenous regulators of depolarizing GABA actions that broadly impact on brain development.

Disregulation in developmental excitatory GABAergic signaling has been shown to impair the development of neuronal circuits and may be a contributing factor in neurodevelopmental disorders such as epilepsy, autism spectrum disorders, and schizophrenia (Briggs and Galanopoulou, 2011; Pizzarelli and Cherubini, 2011; Hyde et al, 2011). Sex differences have been widely reported in all of these disorders, implicating a role for estradiol in their etiology. Targeting SPAK or OSR1 may allow for novel therapeutic options for these neural disorders.

  

Sexually dimorphic expression of KCC2 and GABA function 

GABA_A receptors have an age-adapted function in the brain. During early development, they mediate depolarizing effects, which result in activation of calcium-sensitive signaling processes that are important for the differentiation of the brain. In more mature stages of development and in adults, GABA_A receptors acquire their classical hyperpolarizing signaling. The switch from depolarizing to hyperpolarizing GABA_A-ergic signaling is triggered through the developmental shift in the balance of chloride cotransporters that either increase (ie NKCC1) or decrease (ie KCC2) intracellular chloride. The maturation of GABA_A signaling follows sex-specific patterns, which correlate with the developmental expression profiles of chloride cotransporters. This has first been demonstrated in the substantia nigra, where the switch occurs earlier in females than in males. As a result, there are sensitive periods during development when drugs or conditions that activate GABA_A receptors mediate different transcriptional effects in males and females. Furthermore, neurons with depolarizing or hyperpolarizing GABA_A-ergic signaling respond differently to neurotrophic factors like estrogens. Consequently, during sensitive developmental periods, GABA_A receptors may act as broadcasters of sexually differentiating signals, promoting gender-appropriate brain development. This has particular implications in epilepsy, where both the pathophysiology and treatment of epileptic seizures involve GABA_A receptor activation. It is important therefore to study separately the effects of these factors not only on the course of epilepsy but also design new treatments that may not necessarily disturb the gender-appropriate brain development.

1.3.2 GABA_A receptor signaling as sex-specific modifier of estradiol effects

To further understand the mechanisms underlying the higher expression of KCC2 in the female SNR, we examined the in vivo regulation of KCC2 mRNA by gonadal hormones. As previously stated, the perinatal surge of testosterone in male rats is required for the masculinization of most studied sexually brain structures. Unlike humans, in rats, this is usually through the estrogenic derivatives of testosterone, produced through aromatization, and less often through the androgenic metabolites, like dihydrotestosterone (DHT) (Cooke et al. 1998). To determine whether KCC2 is regulated by gonadal hormones, the effects of systemic administration of testosterone, 17β-estradiol or DHT on KCC2 mRNA expression in PN15 SNR were studied (Galanopoulou and Moshé 2003). Testosterone and DHT increased KCC2 mRNA expression in both male and female PN15 SNR neurons. In contrast, 17β-estradiol decreased KCC2 mRNA in males but not in females. These effects were seen both after short (4 hours) or long periods (52 hours) of exposure to the hormones. However, they occurred only in neurons in which active GABA_A-mediated depolarizations were operative (naïve male PN15 SNR neurons). Estradiol failed to downregulate KCC2 in neurons in which GABA_A receptors or L-type voltage sensitive calcium channels (L-VSCCs) were blocked (bicuculline or nifedipine pretreated PN15 male rat SNR), and in those that had already hyperpolarizing GABA_A signaling (female PN15 SNR neurons). This indicated that 17β-estradiol-mediated downregulation of certain calcium-regulated genes, like KCC2, shows a requirement for active GABA_A-mediated activation of L-VSCCs (Galanopoulou and Moshé 2003). In agreement with this model, in vivo administration of 17β-estradiol decreased pCREB-ir in male but not in female PN15 SNR neurons (Galanopoulou 2006). The idea that the effects of estradiol on chloride cotransporters or GABA_A signaling may depend upon the direction of GABA_A responses is also reverberated in other publications. In hippocampal pyramidal neurons of adult ovariectomized female rats, where GABA_A signaling is thought to be hyperpolarizing, 17β-estradiol had no effect on KCC2 expression (Nakamura et al. 2004). In contrast, in cultured neonatal hypothalamic neurons that still respond with muscimol-triggered calcium rises, thought to be due to the depolarizing effects of GABA_A receptors, 17β-estradiol delays the period with excitatory GABA_A signaling (Perrot-Sinal et al. 2001). However, a direct involvement of KCC2 in this process has not been demonstrated yet. Such findings indicate that GABA_A signaling can not only augment the existing sex differences through pathways directly regulated by its own receptors, but can also interact indirectly and modify the effects of important neurotrophic and morphogenetic factors, like estradiol, at least in some neuronal types (Galanopoulou 2005; Galanopoulou 2006). It is possible that perinatal exposure to higher levels of the estrogenic metabolites produced by the testosterone surge in male pups could be one factor that maintains KCC2 expression lower in males. In agreement, daily administration of 17β-estradiol in neonatal female rat pups, during the first 5 days of life, reduces KCC2 mRNA at postnatal day 15. This does not occur if 17β-estradiol is given only during the first 3 days of postnatal life (personal unpublished data).

Development and regulation of chloride homeostasis in the central nervous system

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the mature central nervous system (CNS). The developmental switch of GABAergic transmission from excitation to inhibition is induced by changes in Cl⁻ gradients, which are generated by cation-Cl⁻ co-transporters. An accumulation of Cl⁻ by the Na⁺-K⁺-2Cl⁻ co-transporter (NKCC1) increases the intracellular Cl⁻ concentration ([Cl⁻]_i) such that GABA depolarizes neuronal precursors and immature neurons. The subsequent ontogenetic switch, i.e., upregulation of the Cl⁻-extruder KCC2, which is a neuron-specific K⁺-Cl⁻ co-transporter, with or without downregulation of NKCC1, results in low [Cl⁻]_i levels and the hyperpolarizing action of GABA in mature neurons. Development of Cl⁻ homeostasis depends on developmental changes in NKCC1 and KCC2 expression. Generally, developmental shifts (decreases) in [Cl⁻]_i parallel the maturation of the nervous system, e.g., early in the spinal cord, hypothalamus and thalamus, followed by the limbic system, and last in the neocortex. There are several regulators of KCC2 and/or NKCC1 expression, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF), and cystic fibrosis transmembrane conductance regulator (CFTR). Therefore, regionally different expression of these regulators may also contribute to the regional developmental shifts of Cl⁻ homeostasis. KCC2 and NKCC1 functions are also regulated by phosphorylation by enzymes such as PKC, Src-family tyrosine kinases, and WNK1–4 and their downstream effectors STE20/SPS1-related proline/alanine-rich kinase (SPAK)-oxidative stress responsive kinase-1 (OSR1). In addition, activation of these kinases is modulated by humoral factors such as estrogen and taurine. Because these transporters use the electrochemical driving force of Na⁺ and K⁺ ions, topographical interaction with the Na⁺-K⁺ ATPase and its modulators such as creatine kinase (CK) should modulate functions of Cl⁻ transporters. Therefore, regional developmental regulation of these regulators and modulators of Cl⁻ transporters may also play a pivotal role in the development of Cl⁻ homeostasis.

PI3K/Akt Signaling Pathway Activates the WNK-OSR1/SPAK-NCC Phosphorylation Cascade in Hyperinsulinemic db/db Mice

The discovery that the dominant inhibitory neurotransmitter, GABA, is also the major source of excitation in the developing brain was so surprising and unorthodox it required years of converging evidence from multiple laboratories to gain general acceptance (Ben-Ari, 2002) and continues to draw challenges some 20 years after the initial reports (Rheims et al., 2009; Waddell et al., 2011). Fundamental developmental endpoints regulated by depolarizing GABA action include giant depolarizing potentials (Ben-Ari etal, 1989), leading to spontaneous activity patterns (Blankenship & Feller, 2010), activity dependent survival (Sauer and Bartos, 2010), neurite outgrowth (Sernagor et al., 2010), progenitor proliferation (Liu et al., 2005), and hebbian-based synaptic patterning (Wang & Kriegstein, 2008). We previously identified an endogenous regulator of depolarizing GABA action, the gonadal and neurosteroid estradiol, which both amplifies the magnitude and extends the developmental duration of excitatory GABA (Perrot-Sinal et al., 2001). Estradiol is a pervasive signaling molecule that varies in concentration between brain regions, across development and in males versus females, thereby contributing to variability in neuronal maturation. The present studies reveal that this steroid enhances depolarizing GABA effects by increasing levels of the signaling kinases SPAK and OSR1, which are upstream of the NKCC1 cotransporter. Estradiol mediated increases in NKCC1 phosphorylation are precluded by antisense oligonucleotide-mediated knockdown of SPAK, and to a lesser extent OSR1, exhibiting the necessity of these kinases for mediating estradiol’s effects. Furthermore, knockdown of either or both of these kinases significantly attenuated estradiol’s enhancement of intracellular Ca²⁺ influx in response to GABA_A activation.

Estradiol has widespread effects on cellular processes through both rapid, nongenomic actions on cell signaling, and slower more enduring effects by modulating transcriptional activity (McEwen, 1991). The combination of a long time course and a complete ablation of the effectiveness of estradiol by simultaneous administration of blockers of transcription or translation confirm that the cascade of events leading to estradiol enhancement of depolarizing GABA begins with increased gene expression. The ability of the brain to synthesize estradiol in discrete loci raises the specter of estrogens as widespread endogenous regulators of depolarizing GABA actions that broadly impact on brain development.

Disregulation in developmental excitatory GABAergic signaling has been shown to impair the development of neuronal circuits and may be a contributing factor in neurodevelopmental disorders such as epilepsy, autism spectrum disorders, and schizophrenia (Briggs and Galanopoulou, 2011; Pizzarelli and Cherubini, 2011; Hyde et al, 2011). Sex differences have been widely reported in all of these disorders, implicating a role for estradiol in their etiology. Targeting SPAK or OSR1 may allow for novel therapeutic options for these neural disorders.

The role of Taurine and TauT

The Japanese paper below suggests that what I have called in this blog, the “GABA switch” is in part mediated by intracellular taurine.

In immature neurons, taurine is taken up into cells through the TauT transporter and activates WNK-SPAK/OSR1 signaling.

TauT is the taurine transporter that lets taurine into cells.

So logically if you blocked the taurine transporter in people with permanently immature neurons, things might improve.

Taurine is present in the embryonic brain by transportation from maternal blood via placental TauT. In addition, fetuses ingest taurine-rich amniotic fluid. Although fetal taurine decreases postnatally, infants receive taurine via breast milk, which contains a high taurine concentration. 

Taurine Inhibits K⁺-Cl⁻ Cotransporter KCC2 to Regulate Embryonic Cl⁻ Homeostasis via With-no-lysine (WNK) Protein Kinase Signaling Pathway

Taurine Inhibits KCC2 Activity via Serine/Threonine Phosphorylation

Because KCC2 is known to be regulated by kinases (15, 17, 54,–,56), phosphorylation-related reagents were used to evaluate the effect on KCC2 activity. The tyrosine kinase inhibitor AG18 and tyrosine phosphatase inhibitor vanadate did not affect E_GABA (supplemental Table 1A). In contrast, the broad spectrum kinase inhibitor staurosporine (Staur) shifted E_GABA toward the negative in 15–20 min in the presence of taurine (control, −45.2 ± 0.3 mV; Staur, −47.6 ± 0.5 mV, n = 5, p = 0.002 (supplemental Fig. 3A and Table 1A). Considering that 1 h of taurine treatment did not have an effect on E_GABA (Fig. 2A), these results suggest that chronic but not acute taurine treatment inhibited KCC2 activity in a serine/threonine phosphorylation-dependent manner. Moreover, staurosporine also shifted KCC2-positive cell E_GABA significantly toward the negative in embryonic brain slices at E18.5 but was less effective in postnatal brain slices at P7 (control, −46.5 ± 0.8 mV; Staur, −51.0 ± 1.1 mV, n = 6, p = 0.007 at E18.5; control, −57.6 ± 1.7 mV; Staur, −59.1 ± 1.6 mV (n = 6, p = 0.06 at P7)) (supplemental Fig. 3B). In contrast, vanadate did not affect E_GABA at either age (supplemental Table 1B).

Hypothetical model of Cl⁻ homeostasis regulated by taurine and WNK-SPAK/OSR1 signaling during perinatal periods. To control the excitatory/inhibitory balance mediated by GABA, [Cl⁻]_i is regulated by activation of the WNK-SPAK/OSR1 signaling pathway via KCC2 inhibition and possibly NKCC1 activation (54, 58, 59). In immature neurons, taurine is taken up into cells through TauT and activates WNK-SPAK/OSR1 signaling (left). Red arrows and T-shaped bars indicate activation and inactivation, respectively. Later (possibly a while after birth), this activation pathway induced by taurine diminishes, resulting in release of KCC/NKCC activity (right), whereas SPAK/OSR1 signaling recovers somewhat upon adulthood. Interestingly, in contrast to kinase signaling leading to KCC2 inhibition, other kinases are also known to facilitate KCC2 activity (see “Discussion”). 

We observed that taurine is implicated in WNK activity. WNK signaling is activated by stimuli, such as osmotic stress; however, the precise pathway leading to activation is unknown (38, 59). Our results indicate that taurine uptake is crucial for WNK activation, and only intracellular taurine activates WNKs, which are also involved in osmoregulation (52). There are no significant osmolarity differences with or without 3 mm taurine (without taurine, 215 ± 2 mosm versus with taurine, 216 ± 4 mosm (n = 4–5, p = 0.41)). In addition, 3 mm GABA did not affect phosphorylation of SPAK/OSR1 (data not shown), which indicates a specific action of taurine. 

KCC2 gene up-regulation is essential for Cl⁻ homeostasis during development, and phosphorylation of KCC2 is another important factor (5, 12, 15, 18, 55, 56). Ser-940 phosphorylation regulates KCC2 function by modulating cell surface KCC2 expression (56). Tyr-1087 phosphorylation affects oligomerization, which plays a pivotal role in KCC2 activity without affecting cell surface expression (20, 55). Rinehart et al. (54) indicated that Thr-906 and Thr-1007 phosphorylation does not affect cell surface KCC2 expression. In our study, oligomerization and plasmalemmal localization were not affected by taurine (data not shown), suggesting that phosphorylation of these sites may provide another mechanism of KCC2 activity modulation. 

A number of neuron types are generated relatively early during embryonic development, such as Cajal-Retzius and subplate cells in the cerebral cortex, which play regulatory roles in migration. Several reports have shown that these early generated neurons in the marginal zone and subplate are activated by GABA and glycine (82,–,85). These early generated neurons can express KCC2 as early as the embryonic and neonatal stages (86). In addition, taurine is enriched in these brain areas (data not shown). Therefore, the present results suggest that KCC2 is not functional due to the distribution of taurine, which affects WNK-SPAK/OSR1 signaling and preserves GABAergic excitation. This signaling cascade may have broader important roles in brain development than previously reported.

Conclusion

I think we have pretty much got to the bottom of the current research on this subject.

There is plenty of ongoing Japanese involvement, which is good news.

You either find the GABA switch and, better late than never, finally activate it, or you modify the downstream processes as a therapy for immature neurons.  

Numerous things affect NKCC1/KCC2; so numerous therapies can potentially treat it.

The really clever solution would be to activate the GABA switch; that part I continue to think about.

Clearly, if you disrupt evolutionary processes like oxytocin and taurine passed from mother to baby there may be unexpected consequences.

Unusual levels of both male and female hormones and expression of estrogen/androgen receptors do play a role in the balance between NKCC1/KCC2 and so the level of chloride and hence how GABA behaves.

Inhibitors of WNK, SPAK and OSR1 are all promising potential therapies and I think these will emerge, since the big money of autism research is already backing this idea.

The TauT transporter is another possible target.

Hormone related options include a selective estrogen receptor beta agonist, an androgen receptor antagonist, and estradiol.  Unfortunately such therapy is quite likely to have unwanted side effects. So-called phytoestrogens like EGCG, from green tea, covered in a recent post are not very potent but if you had enough might show some effect.

For many reasons it looks like many people with autism could do with some more PKC (Protein Kinase C).

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.