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.

Enhancing the effect of Bumetanide in Autism

Many readers of this blog, and some of those who leave comments, are using the Bumetanide therapy proposed by Ben-Ari and Lemonnier.

At some point it should become an approved autism drug and Ben Ari has already patented it for use in Down Syndrome, so I guess that will come later on.

I have been developing my own add-on therapies that might help people for whom a high level of intracellular chloride is part of their autism, or indeed Down Sydrome.  If Bumetanide has a profound impact on your autism, this is almost certainly you.

Monty, aged 13 with ASD

After 4 years of Bumetanide, it continues to be effective and if Monty stops taking it there is a gradual cognitive decline over a few days, presumably as chloride concentration gradually increases.

In spite of an odd temporary Tourette’s type verbal tic that developed after an infection before Christmas, I have been getting plenty of feedback that Monty has got cleverer in 2017.  So it looks like some bumetanide add-on does indeed work.

The Colosseum

Monty’s big brother continues to be a fan of Lego and indeed Nanoblocks from Japan.  Nanoblocks is like extremely small Lego.

Having completed the Colossuem, his latest Nanoblocks model, he asked Monty “where is it?”.

Back came the answer, unprompted, “Italy”.

This was a big surprise.

That was not the answer big brother expected, he expected no answer or a silly answer like “over there”.

Add-ons

The first is potassium bromide (KBr) which was the original epilepsy therapy 150 years ago.  One of its effects is that the bromide (Br^-) part competes with chloride (Cl-) to enter neurons and bromide is known to be faster.  As a result some of the chloride inside cells is replaced by bromide.  Bromide is extremely similar to chloride, but is not reactive; this is why it can be used with any anti-epileptic drug (AED) without fear of negative interactions.

KBr has an extremely long half-life, meaning that if you take it every day it will take 4-6 weeks to reach its stable level in your body.

KBr is used for pediatric epilepsy in Germany and Austria and for epilepsy in pet dogs all over the world.  

A dose of 8mg/kg is far below the dose used for epilepsy, but does have a bumetanide enhancing effect in one 50kg boy.

The even more recent add-on is based on the experience of our reader Petra’s son with Asperger’s, who found that taking his bumetanide with Greek coffee seemed to make it more effective.

It turns out that dopamine is known to increase the effect of diuretics on the chloride cotransport NKCC2 in your kidneys.  There is a myth that coffee is a diuretic, but it is clear where this myth has come from.  Coffee will increase diuresis and so does caffeine.

In the brain it is the chloride cotransporter NKCC1 that is also blocked by bumetanide.  So it would be plausible that dopamine/coffee/caffeine it might have the same effect on NKCC1 as it does on the very similar NKCC2.

The cheap and widely available 50mg caffeine tablets do seem to serve as a proxy for a steaming cup of Greek coffee.  Indeed 50mg of caffeine is more like a weak cup of instant coffee.

I did much earlier propose the use of Diamox/ Acetazolamide to reduce chloride.  It seems that in some neurons 2/3 of the chloride enters via NKCC1 and 1/3 via the exchanger AE3.  Diamox/ Acetazolamide works via AE3.

Diamox has some other ion channel effects, making it useful in some epilepsy.

Some readers of this blog use Diamox, but in Monty it seems to cause reflux.

Caffeine is a very simple add-on to try.

Neuroligins, Estradiol and Male Autism

Today’s post looks deeper into the biology of those people who respond to the drug bumetanide, which means a large sub-group of those with autism, likely those with Down Syndrome and likely some with schizophrenia.

It is a rather narrow area of science, but other than bumetanide treatment, there appears to be no research interest in further translating science into therapy.    So it looks like this blog is the only place to develop such ideas.

I did not expect this post would lead to a practical intervention, but perhaps it does. As you will discover, the goal would be to restore a hormone called estradiol to its natural higher level, perhaps by increasing an enzyme called aromatase, which appears to be commonly downregulated in autism.  This should increase expression of neuroligin 2, which should increase expression of the ion transporter KCC2; this will lower intracellular chloride and boost cognition.

It seems that those people using Atorvastatin may have already started this process, since this statin increases IGF-1 and insulin is one of the few things that increases the aromatise enzyme. 

This process is known as the testosterone-estradiol shunt.  In effect, by becoming slightly less male, you may be able to correct one of the key dysfunctions underlying autism. Options would include insulin, IGF-1, estradiol and a promoter of aromatase.




The testosterone – estradiol shunt

It would seem that this sub-group of autism is currently a little bit too male, which might be seen as early puberty and in other features. In this group the balance between testosterone and estradiol is affected not just in the brain, which is actually a good thing.  This should be measurable, if it is not visible.

  

NKCC1, KCC2 and AE3

As we have seen in earlier posts, some people with autism have too little of a transporter called KCC2 that takes chloride out of neurons and too much of NKCC1 that lets chloride in.  The result is an abnormally high level of chloride, which changes the way the GABA neurotransmitter functions.  This reduces cognitive function and increases the chance of seizures.

It is likely that a group may exist that has mis-expression of an ion exchanger called AE3. Potentially some have just an AE3 dysfunction and some may have AE3, KCC2 and NKCC1 mis-expression.  I will come back to this in a later post, but in case I forget, here is the link:

NKCC1 and AE3 Appear to Accumulate Chloride in Embryonic Motoneurons

“NKCC1 seems to be responsible for approximately two thirds of the steady-state chloride accumulation, whereas AE3 is responsible for the remaining third”

Genetic dysfunction of AE3 is not surprisingly associated with seizures and should respond to treatment with Diamox/Acetazolamide.

Block NKCC1 with Bumetanide and/or increase KCC2 expression

I was recently updating the Bumetanide researchers about my son’s near four years of therapy with their drug and my ideas to take things further.

My plan is to apply other methods to reduce intracellular chloride levels.  I think that over time, blocking NKCC1 with bumetanide may trigger a feedback loop that leads to a further increase in NKCC1 expression.  So bumetanide continues to work, but the effect is reduced. One way to further reduce intracellular chloride levels is to increase expression of KCC2, the transport that takes chloride out of neurons.

The best way to do this would be to understand why KCC2 is down regulated in the first place. I have touched on this in earlier posts, where I introduced neuroligin 2.

Today’s post will look at neuroligins in autism and how they are connected to the female hormone Estradiol.  We will also look at how estrogen receptor expression may help explain why more males have autism. Taken together this suggests that an  estrogen receptor agonist might be an effective autism therapy in this sub-group.

The difficulty with hormones is that, due to evolution, each one performs numerous different functions in different parts of the body and they react with each other.  So a little extra estradiol/estrogen might indeed increase neuroligin 2 expression and hence increase KCC2 expression in the brain, but it would have other effects elsewhere.  In female hormone replacement therapy care is usually taken to direct estradiol/estrogen to where it is needed, rather than sending it everywhere.

I suspect that in this subgroup of autism the lack of estradiol is body-wide, not just in the brain.  If not you would either need an estrogen receptor agonist that is cleverly developed to be brain specific, or take the much easier route of delivering an existing agonist direct to the brain, which may also be possible.

In the paper below NL2 and neuroligin-2 mean the same thing. 

An unexpected role of neuroligin-2 in regulating KCC2 and GABA functional switch

Background

GABA_A receptors are ligand-gated Cl^- channels, and the intracellular Cl^- concentration governs whether GABA function is excitatory or inhibitory. During early brain development, GABA undergoes functional switch from excitation to inhibition: GABA depolarizes immature neurons but hyperpolarizes mature neurons due to a developmental decrease of intracellular Cl^- concentration. This GABA functional switch is mainly mediated by the up-regulation of KCC2, a potassium-chloride cotransporter that pumps Cl^- outside neurons. However, the upstream factor that regulates KCC2 expression is unclear.

Results

We report here that KCC2 is unexpectedly regulated by neuroligin-2 (NL2), a cell adhesion molecule specifically localized at GABAergic synapses. The expression of NL2 precedes that of KCC2 in early postnatal development. Upon knockdown of NL2, the expression level of KCC2 is significantly decreased, and GABA functional switch is significantly delayed during early development. Overexpression of shRNA-proof NL2 rescues both KCC2 reduction and delayed GABA functional switch induced by NL2 shRNAs. Moreover, NL2 appears to be required to maintain GABA inhibitory function even in mature neurons, because knockdown NL2 reverses GABA action to excitatory. Gramicidin-perforated patch clamp recordings confirm that NL2 directly regulates the GABA equilibrium potential. We further demonstrate that knockdown of NL2 decreases dendritic spines through down-regulating KCC2.

Conclusions

Our data suggest that in addition to its conventional role as a cell adhesion molecule to regulate GABAergic synaptogenesis, NL2 also regulates KCC2 to modulate GABA functional switch and even glutamatergic synapses. Therefore, NL2 may serve as a master regulator in balancing excitation and inhibition in the brain.

  

Neuroligins and Neurexins

The following paper has an excellent explanation of neuroligins, neurexins and their role in autism.  It does get complicated.

Neuroligins and Neurexins Link Synaptic Function to Cognitive Disease

Neurexins (Nrxns) and neuroligins (Nlgns) are arguably the best characterized synaptic cell-adhesion molecules, and the only ones for which a specifically synaptic function was established^8,9. In the present review, we will describe the role of Nrxns and Nlgns as synaptic cell-adhesion molecules that act in an heretofore unanticipated fashion. We will show that they are required for synapse function, not synapse formation; that they affect trans-synaptic activation of synaptic transmission, but are not essential for synaptic cohesion of the pre- and postsynaptic specializations; and that their dysfunction impairs the properties of synapses and disrupts neural networks without completely abolishing synaptic transmission as^10–12. As cell-adhesion molecules, Nrxns and Nlgns probably function by binding to each other and by interacting with intracellular proteins, most prominently PDZ-domain proteins, but the precise mechanisms involved and their relation to synaptic transmission remain unclear. The importance of Nrxns and Nlgns for synaptic function is evident from the dramatic deficits in synaptic transmission in mice lacking Nrxns or Nlgns.

As we will describe, the role of Nrxns and Nlgns in synaptic function almost predestines them for a role in cognitive diseases, such as schizophrenia and autism spectrum disorders (ASDs), that have been resistant to our understanding. One reason for the difficulties in understanding cognitive diseaseas is that they may arise from subtle changes in a subset of synapses in a neural circuit, as opposed to a general impairment of all synapses in all circuits. As a result, the same molecular alteration may produce different circuit changes and neurological symptoms that are then classified as distinct cognitive diseases. Indeed, recent studies have identified mutations in the genes encoding Nrxns and Nlgns as a cause for ASDs, Tourette syndrome, mental retardation, and schizophrenia, sometimes in patients with the same mutation in the same family^13–27. Viewed as a whole, current results thus identify Nrxns and Nlgns as trans-synaptic cell-adhesion molecules that mediate essential signaling between pre- and postsynaptic specializations, signaling that performs a central role in the brain’s ability to process information and that is a key target in the pathogenesis of cognitive diseases.

Neuroligins and neurexins in autism

ASDs are common and enigmatic diseases. ASDs comprise classical idiopathic autism, Asperger’s syndrome, Rett syndrome, and pervasive developmental disorder not otherwise specified^73,74. Moreover, several other genetic disorders, such as Down syndrome, Fragile-X Mental Retardation, and tuberous sclerosis, are frequently associated with autism. Such syndromic forms of autism and Rett syndrome are usually more severe due to the nature of the underlying diseases. The key features of ASDs are difficulties in social interactions and communication, language impairments, a restricted pattern of interests, and/or stereotypic and repetitive behaviors. Mental retardation (~70% of cases) and epilepsy (~30% of cases) are frequently observed; in fact, the observation of epilepsy in patients with ASDs has fueled speculation that autism may be caused by an imbalance of excitatory vs. inhibitory synaptic transmission. In rare instances, idiopathic autism is associated with specialized abilities, for example in music, mathematics, or memory. The relation of ASDs to other cognitive diseases such as schizophrenia and Tourette’s syndrome is unclear. As we will see below with the phenotypes caused by mutations in Nlgns and Nrxns, the boundaries between the various disorders may not be as real as the clinical manifestations suggest.

A key feature of ASDs is that they typically develop before 2–3 years of age^73,74. ASDs thus affect brain development relatively late, during the time of human synapse formation and maturation. Consistent with this time course, few anatomical changes are associated with ASDs⁷⁵. An increase in brain size was repeatedly reported⁷⁶, but is not generally agreed upon⁷⁵. Thus, similar to other cognitive diseases, ASDs are not a disorder of brain structure but of brain function. Among cognitive diseases, ASDs are the most heritable (~ 80%), suggesting that they are largely determined by genes and not the environment. ASDs exhibit a male:female ratio of approximately 4:1, indicating that ASDs involve the X-chromosome directly, or that the penetrance of pathogenic genes is facilitated in males^73,74.

Mutations in many genes have been associated with familial ASDs. A consistent observation emerging from recent studies is the discovery of mutations in the genes encoding Nrxn1, Nlgn3, and Nlgn4. Specifically, seven point mutations, two distinct translocation events, and four different large-scale deletions in the Nrxn1 gene were detected in autistic patients^13–18. Ten different mutations in the Nlgn4 gene were observed (2 frameshifts, 5 missense mutations, and 3 internal deletions), and a single mutation in the Nlgn3 gene (the R451C substitution)^21–24. Besides these mutations, five different larger deletions of X-chromosomal DNA that includes the Nlgn4 locus (referred to as copy-number variations) were detected in autism patients^18,25–27.

In addition to the Nrxn/Nlgn complex, mutations in the gene encoding Shank3 – an intracellular scaffolding protein that binds indirectly to Nlgns via PSD-95 and GKAP (Fig. 1)⁶⁶ – may also be a relatively frequent occurrence in ASDs. An astounding 18 point mutations were detected in the Shank3 gene in autistic patients, in addition to several cases containing CNVs that cover the gene^18,77–82. Indeed, the so-called terminal 22q deletion syndrome is a relatively frequent occurrence that exhibits autistic features, which have been correlated with the absence of the Shank3 gene normally localized to this chromosome section. Shank3 is particularly interesting because it not only indirectly interacts with Nlgns, but also directly binds to CIRL/Latrophilins which in turn constitute α-latrotoxin receptors similar to Nrxns, suggesting a potential functional connection between Shank3 and Nrxns⁸³.

Overall, the description of the various mutations in the Nrxn/Nlgn/Shank3 complex appears to provide overwhelming evidence for a role of this complex in ASDs, given the fact that in total, these mutations account for a significant proportion of autism patients. It should be noted, however, that two issues give rise to skepticism to the role of this complex in ASDs.

First, at least for some of the mutations in this complex, non-symptomatic carriers were detected in the same families in which the patients with the mutations were found. Whereas the Nlgn3 and Nlgn4 mutations appear to be almost always penetrant in males, and even female carriers with these mutations often have a phenotype, the Shank3 point mutations in particular were often observed in non-symptomatic siblings^77,78. Thus, these mutations may only increase the chance of autism, but not actually cause autism.

Second, the same mutations can be associated with quite different phenotypes in different people. For example, a microdeletion in Nlgn4 was found to cause severe autism in one brother, but Tourette’s syndrome in the other²⁶. This raises the issue whether the ‘autism’ observed in patients with mutations in these genes is actually autism, an issue that could also be rephrased as the question of whether autism is qualitatively distinct from other cognitive diseases, as opposed to a continuum of cognitive disorders. In support of the latter idea, two different deletions of Nrxn1α have also been observed in families with schizophrenia^19,20, indicating that there is a continuum of disorders that involves dysfunctions in synaptic cell adhesion and manifests in different ways. Conversely, very different molecular changes may produce a similar syndrome, as exemplified by the quite different mutations that are associated with ASDs⁸⁴.

At present, the relation between the Nrxn/Nlgn synaptic cell-adhesion complex and ASDs is tenuous. On one hand, many of the mutations observed in familial ASD are clearly not polymorphisms but deleterious, as evidenced by the effect of these mutations on the structure or expression of the corresponding genes, and by the severe autism-like phenotypes observed in Nlgn3 and Nlgn4 mutant mice^85–87. On the other hand, the nonlinear genotype/phenotype relationship in humans, evident from the only 70–80% heritability and from the occasional presence of mutations in non-symptomatic individuals, requires explanation. Elucidating the underlying mechanisms for this incomplete genotype/phenotype relationship is a promising avenue to insight into the genesis of autism. Furthermore, in addition to the link of Nrxn1α mutations to schizophrenia^19,20, linkage studies have connected Nrxn3 to different types of addiction^88,89. It is possible that because of the nature of their function, mutations in genes encoding Nrxns and Nlgns constitute hotspots for human cognitive diseases.

  

As you will have seen from the above paper, whose author seems to be very well informed of the broader picture (a continuum of disorders that involves dysfunctions in synaptic cell adhesion, and even the link to addiction), neuroligins and neurexins are very relevant to autism and other cognitive disease.

Let’s get back on subject and focus on Neuroligin 2 

The very recent paper below mentions sensory processing defects and NLG2 alongside what we already have figured out so far.

Neuroligin 2 nonsense variant associated with anxiety, autism, intellectual disability, hyperphagia, and obesity

Abstract

Neuroligins are post-synaptic, cellular adhesion molecules implicated in synaptic formation and function. NLGN2 is strongly linked to inhibitory, GABAergic signaling and is crucial for maintaining the excitation-inhibition balance in the brain. Disruption of the excitation-inhibition balance is associated with neuropsychiatric disease. In animal models, altered NLGN2 expression causes anxiety, developmental delay, motor discoordination, social impairment, aggression, and sensory processing defects. In humans, mutations in NLGN3 and NLGN4 are linked to autism and schizophrenia; NLGN2 missense variants are implicated in schizophrenia. Copy number variants encompassing NLGN2 on 17p13.1 are associated with autism, intellectual disability, metabolic syndrome, diabetes, and dysmorphic features, but an isolated NLGN2 nonsense variant has not yet been described in humans. Here, we describe a 15-year-old male with severe anxiety, obsessive-compulsive behaviors, developmental delay, autism, obesity, macrocephaly, and some dysmorphic features. Exome sequencing identified a heterozygous, de novo, c.441C>A p.(Tyr147Ter) variant in NLGN2 that is predicted to cause loss of normal protein function. This is the first report of an NLGN2 nonsense variant in humans, adding to the accumulating evidence that links synaptic proteins with a spectrum of neurodevelopmental phenotypes

After some investigation I learned that both estradiol/estrogen and progesterone increase expression of neuroligin 2, at least in rats.

Increasing neuroligin 2/NLGN2/NL2 looks a promising strategy.

Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene

In addition, neuroligin 2 mRNA levels were increased by both 17beta-oestradiol (E(2)) and P(4), although P(4) administration upregulated gene expression to a greater extent than injection of E(2). These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E(2) and P(4), which are secreted periodically during the oestrous cycle.[1]

So a female steroid-regulated gene is down-regulated in male-dominated autism.  Another example of the protective nature of female hormones?  I think it is.

Estrogens Suppress a Behavioral Phenotype in Zebrafish Mutants of the Autism Risk Gene, CNTNAP2

Highlights

·         Zebrafish mutants of the autism risk gene cntnap2 have GABAergic neuron deficits

·         High-throughput behavioral profiling identifies nighttime hyperactivity in mutants

·         cntnap2 mutants exhibit altered responses to GABAergic and glutamatergic compounds

·         Estrogenic compounds suppress the cntnap2 mutant behavioral phenotype

Summary

Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.

Estrogen receptor expression may help explain why more males have autism

Estrogen is known to help protect premenopausal women from maladies such as stroke and impaired cognition. Exposure to high levels of the male hormone testosterone during early development has been linked to autism, which is five times more common in males than females.

The new findings of reduced expression of estrogen receptor beta as well as that of an enzyme that converts testosterone to estrogen could help explain the high testosterone levels in autistic individuals and higher autism rates in males, Pillai said.

It was the 5-to-1 male-to-female ratio along with the testosterone hypothesis that led Pillai and his colleagues to pursue whether estrogen might help explain the significant gender disparity and possibly point toward a new treatment.

"The testosterone hypothesis is already there, but nobody had investigated whether it had anything to do with the female hormone in the brain," Pillai said. "Estrogen is known to be neuroprotective, but nobody has looked at whether its function is impaired in the brain of individuals with autism. We found that the children with autism didn't have sufficient estrogen receptor beta expression to mediate the protective benefits of estrogen."

Comparing the brains of 13 children with and 13 children without autism spectrum disorder, the researchers found a 35 percent decrease in estrogen receptor beta expression as well as a 38 percent reduction in the amount of aromatase, the enzyme that converts testosterone to estrogen.

Levels of estrogen receptor beta proteins, the active molecules that result from gene expression and enable functions like brain protection, were similarly low. There was no discernable change in expression levels of estrogen receptor alpha, which mediates sexual behavior.

Estrogen receptor expression may help explain why more males have autism

The new findings of reduced expression of estrogen receptor beta as well as that of an enzyme that converts testosterone to estrogen could help explain the high testosterone levels in autistic individuals and higher autism rates in males

They also plan to give an estrogen receptor beta agonist -- which should increase receptor function -- to a mouse with generalized inflammation and signs of autism to see if it mitigates those signs. Inflammation is a factor in many diseases of the brain and body, and estrogen receptor beta agonists already are in clinical trials for schizophrenia.

The following trial was run by a psychiatrist; when I looked at why he thought estrogen might improve schizophrenia, there was no biological explanation.  He is trying to avoid the possible side effects by using of a selective estrogen receptor agonist.  I hope the trial successful.  The question is whether his subjects are starting out as extreme male or just male.

The Efficacy and Safety of a Selective Estrogen Receptor Beta Agonist (LY500307) for Negative Symptoms and Cognitive Impairment Associated With Schizophrenia (Beta)

http://grantome.com/grant/NIH/UH2-TR000955-01

Several lines of investigation have supported the potential therapeutic effects of estrogen for negative and cognitive symptoms in schizophrenia. However, estrogen has had limited therapeutic application for male and premenopausal patients with schizophrenia because of tolerability concerns including uterine cancer liability, and heart disease and feminization effects in men. Selective Estrogen Receptor Beta (ER beta) agonists are a new class of treatments that are relatively free of estrogen's primary side effects and yet have demonstrated estrogen-like effects in brain including improvement in cognitive performance and an association to extremes in social behavior. Thus, these agents may have a therapeutic role for cognitive and negative symptoms in schizophrenia. The primary objectives of this application are to determine if the selective ER beta agonist LY500307 significantly improves negative and cognitive symptoms in patients with schizophrenia. Secondary aims include assessing LY500307 effects on cerebral blood flow during working and episodic memory tasks with fMRI, and electrophysiological indices of auditory sensory processing and working memory. A single seamless phase 1b/2a adaptive design will be used to evaluate two LY500307 doses (25 mg/day and 75 mg/day) in the first stage of the trial (year 1 of the application) to determine which dose should be advanced to stage 2 (years 2and 3 of the application) or if the trial should be discontinued.

More generally:-

Learning and memory: Steroids and epigenetics

Highlights

Steroid hormones exert a considerable influence on several aspect of cognition.

Estrogens and androgens exert positive effects on cognitive functions.

Progesterone and allopregnanolone have variable effects on cognitive functions.

Glucocorticoids act to encode and store information of the emotional events.

Epigenetic modifications are a powerful mechanism of memory regulation.

Conclusion

More female hormones and less male hormones? Seems a good idea.

More of the aromatase enzyme ?  There are numerous drugs to reduce/inhibit aromatase but not specifically to increase it.

Insulin does increase aromatase, as does alcohol and being overweight.

The clever thing to do would be to just correct the reduced level of aromatase, or wait for a selective estrogen receptor beta agonist like LY500307 to come to the market.

In those who are extreme male, a little estradiol might be the simple solution, but not the amount that is currently taken by those that abuse it.  Yes people abuse estradiol – males who want to be females.

Antonio Hardan at Stanford did trial high dose pregnenolone, another hormone mainly found in females, that should increase progesterone.

Brief report: an open-label study of the neurosteroid pregnenolone in adults with autism spectrum disorder.

Overall, pregnenolone was modestly effective and well-tolerated in individuals with ASD.

This steroid should increase the level of progesterone and so might be expected to cause some side effects in males. You would expect it to have an effect on anxiety, but as we saw in an earlier post it should be quite dose specific.

Why Low Doses can work differently, or “Biphasic, U-shaped actions at the GABAa receptor”

So Hardan may have just picked the "wrong dose".

If he would like to trial 0.3mg of oral estradiol in adults with autism, I think he might find a positive response.