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

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

Taipei 101, briefly the world’s tallest building

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

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

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

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

Schizophrenia only develops in early to mid-adulthood.

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

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

·        Inhibition of NKCC1 (bumetanide)

·        Activation of KCC2 (N-Ethylmaleimide)

·        Enhancer of CKB (creatine)

·        Inhibitor of WNK/SPAK

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

·        Activation of synaptic GABAa receptors (zolpidem, alprazolam)

·        Inhibition of GABA transport mechanism (Tiagabine)

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

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

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

 

Here is the excellent paper on Huntington’s:-

Insights into GABAAergic system alteration in Huntington’s disease

                                                                                                               

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

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

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

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

5.2. Modulation of chloride homeostasis via cation – chloride cotransporters

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

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

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

of GABAA receptors likely antagonizes inflammation.

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

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

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

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

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

5.1. Modulating the GABAA receptor as a therapeutic target

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

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

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

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

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

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

5.2. Modulation of chloride homeostasis via cation–chloride cotransporters

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

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

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

Chloride extrusion enhancers as novel therapeutics for neurological diseases

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

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

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

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

N-Ethylmaleimide increases KCC2 cotransporter activity by modulating transporter phosphorylation

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

                                                                  

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

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

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

Conclusion

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

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

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

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

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).