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).

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.