Treating Rett syndrome, some autism and some dementia via TrkA, TrkB, BDNF, IGF-1, NGF and NDPIH. And logically why Bumetanide really should work in Rett

Source: Rett Syndrome: Crossing the Threshold to Clinical Translation

 

Today’s post is on the one hand very specific to Rett syndrome, but much is applicable to broader autism and other single gene autisms.

Today’s post did start out with the research showing Bumetanide effective in the mouse model of Rett syndrome. This ended up with figuring out why this should have been obvious based on what we already know about growth factors that are disturbed in autism and very much so in Rett.

We even know from a published human case studies that Bumetanide can benefit those with Fragile X and indeed Down syndrome, but the world takes little notice.

If Bumetanide benefits human Rett syndrome would anyone take any notice?  They really should.

To readers of this blog who have a child with Rett, the results really are important.  You can even potentially link the problem symptoms found in Rett to the biology and see how you can potentially treat multiple symptoms with the same drug.

One feature of Rett is breathing disturbances, which typically consist of alternating periods of hyperventilation and hypoventilation.

Our reader Daniel sent me a link to paper that suggest an old OTC cough medicine could be used to treat the breathing issues.

The antitussive cloperastine improves breathing abnormalities in a Rett Syndrome mouse model by blocking presynaptic GIRK channels and enhancing GABA release

Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder caused mainly by mutations in the MECP2 gene. One of the major RTT features is breathing dysfunction characterized by periodic hypo- and hyperventilation. The breathing disorders are associated with increased brainstem neuronal excitability, which can be alleviated with antagonistic agents.

Since neuronal hypoexcitability occurs in the forebrain of RTT models, it is necessary to find pharmacological agents with a relative preference to brainstem neurons. Here we show evidence for the improvement of breathing disorders of Mecp2-null mice with the brainstem-acting drug cloperastine (CPS) and its likely neuronal targets. CPS is an over-the-counter cough medicine that has an inhibitory effect on brainstem neuronal networks. In Mecp2-null mice, CPS (30 mg/kg, i.p.) decreased the occurrence of apneas/h and breath frequency variation. GIRK currents expressed in HEK cells were inhibited by CPS with IC50 1 μM. Whole-cell patch clamp recordings in locus coeruleus (LC) and dorsal tegmental nucleus (DTN) neurons revealed an overall inhibitory effect of CPS (10 μM) on neuronal firing activity. Such an effect was reversed by the GABAA receptor antagonist bicuculline (20 μM). Voltage clamp studies showed that CPS increased GABAergic sIPSCs in LC cells, which was blocked by the GABAB receptor antagonist phaclofen. Functional GABAergic connections of DTN neurons with LC cells were shown.

These results suggest that CPS improves breathing dysfunction in Mecp2-null mice by blocking GIRK channels in synaptic terminals and enhancing GABA release.

  

Cloperastine (CPS) is a central-acting antitussive working on brainstem neuronal networks The drug has several characteristics. 1) It affects the brainstem integration of multiple sensory inputs via multiple sites including K+ channels, histamine and sigma receptors. 2) Its overall effect is inhibitory, suppressing cough and reactive airway signals. 3) With a large safety margin, it has been approved as an over-the-counter medicine in several Asian and European countries.  

With the evidence that DTN cells receive GABAergic recurrent inhibition, we tested whether the inhibitory effect of CPS was caused by enhanced GABAergic transmission. Thus, we recorded the evoked firing activity of DTN cells before and during bath application of CPS in the presence of 20 μM bicuculline. Under this condition, CPS failed to decrease the excitability of DTN neurons (F(1,9) = 0.41, P > 0.05; two‐way repeated measures ANOVA) (n=9) (Fig. 8), indicating that the inhibitory effect relies on GABAA synaptic input 

 

It appeared to me that the breathing issues might be considered as another consequence of the excitatory/inhibitory (E/I) imbalance that is a core feature of much severe autism.

In the case of Rett the lack of BDNF will make any E/I imbalance worse and that by treating the E/I imbalance we will produce the inhibitory effect from GABAa receptors that is needed to ensure correct breathing.  Note that in bumetanide responsive autism there is no inhibitory effect from GABAa receptors, the effect is excitatory.

I did wonder if arrhythmia (irregular heartbeat) is present in Rett, since the breathing problems in Rett are also seen as being caused by a dysfunction in the autonomic nervous system. Arrhythmia is actually a big problem for girls with Rett syndrome.  Regular readers of this blog might then ask about Propranolol, does that help?  It turns out to have been tried and it is not so helpful.  What is effective is another drug we have come across for autism, the sodium channel blocker Phenytoin.  Phenytoin is antiepileptic drug (AED) and it works by blocking voltage gated sodium channels.

Low dose phenytoin was proposed as an autism therapy and a case study was published from Australia. In a separate case study, phenytoin was used to treat self-injury that was triggered by frontal lobe seizures.

When you treat arrhythmia in Rett girls with Phenytoin does it have an impact on their breathing problems?

If you treat the girls with Phenytoin do they still go on to develop epilepsy?

What about if you add treatment with Bumetanide to reduce symptoms of autism? 

Lots of questions looking for answers.

 

What is Rett Syndrome?

Rett syndrome was first identified in the 1950s by Dr Andreas Rett as a disorder that develops in young girls.  Only as recently as 1999 was it determined that the syndrome is caused by a mutation in the MECP2 gene on the X chromosome.  The X chromosome is very important because girls have two copies, but boys have just one.  Rett was an Austrian like many other early researchers in autism like Kanner and Asperger. Even Freud was educated in Vienna. Eugen Bleuler lived pretty close by in Switzerland and he coined the terms schizophrenia, schizoid and autism. 

Rett syndrome is a rare genetic disorder that affects brain development, resulting in severe mental and physical disability.

It is estimated to affect about 1 in 12,000 girls born each year.

Rett is a rare condition, but among these rare conditions it is quite common and so there is a lot of research going on to find treatments.  The obvious one is gene therapy to get the brain to make the missing MeCP2 protein.

Rett syndrome is thankfully rare in absolute terms, but it is one of the best known development conditions that is associated with autism symptoms.

While Rett syndrome may not officially be an ASD in the DSM-5, the link to autism remains. Many children are diagnosed as autistic before the MECP2 mutation is identified and then the diagnosis is revised to RTT/Rett. 

Fragile X  syndrome (FXS), on the other hand, is the most common inherited cause of intellectual disability (ID), as well as the most frequent single gene type of autism.

In the meantime, the logical strategy is to treat the downstream consequences of the mutated gene. Much is known about these downstream effects and there overlaps with some broader autism and indeed dementia.

One area known to be disturbed in Rett, some other autisms and dementia is growth factors inside the brain. The best known growth factors are IGF-1 (Insulin-like Growth Factor 1), BDNF (brain-derived neurotrophic factor) and my favorite NGF (Nerve growth factor).

Without wanting to get too complicated we need to note that BDNF acts via a receptor called TrkB.  You can either increase BDNF or just find something else to activate TrkB, as pointed out to me by Daniel.

For readers whose children respond to Bumetanide they are benefiting from correcting elevated levels of chloride in neurons. Too much had been entering by the transporter NKCC1 and too little exiting via KCC2.

One of the effects of having too little BDNF and hence not enough activation of TrkB is that chloride becomes elevated in neurons.  If you do not activate TrkB you do not get enough KCC2, which is what allows chloride to exit neurons.

To what extent would TrkB activation be an alternative/complement to bumetanide in broader autism?

To what extent would TrkB activation be success in treating some types of chronic pain (where KCC2 is known to be down regulated)?

Low levels of BDNF are a feature of Rett and much dementia.

So you would want to:

·        Increase BDNF

·        Activate TRKB with something else

·        Block NKCC2 to compensate for the lack of KCC2

Note that BDNF is not reduced in all types of autism, just in a sub-group.

I note that there already is solid evidence in the research:-

Restoration of motor learning in a mouse model of Rett syndrome following long-term treatment with a novel small-molecule activator of TrkB

Reduced expression of brain-derived neurotrophic factor (BDNF) and impaired activation of the BDNF receptor, tropomyosin receptor kinase B (TrkB; also known as Ntrk2), are thought to contribute significantly to the pathophysiology of Rett syndrome (RTT), a severe neurodevelopmental disorder caused by loss-of-function mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Previous studies from this and other laboratories have shown that enhancing BDNF expression and/or TrkB activation in Mecp2-deficient mouse models of RTT can ameliorate or reverse abnormal neurological phenotypes that mimic human RTT symptoms. The present study reports on the preclinical efficacy of a novel, small-molecule, non-peptide TrkB partial agonist, PTX-BD4-3, in heterozygous female Mecp2 mutant mice, a well-established RTT model that recapitulates the genetic mosaicism of the human disease. PTX-BD4-3 exhibited specificity for TrkB in cell-based assays of neurotrophin receptor activation and neuronal cell survival and in in vitro receptor binding assays. PTX-BD4-3 also activated TrkB following systemic administration to wild-type and Mecp2 mutant mice and was rapidly cleared from the brain and plasma with a half-life of ∼2 h. Chronic intermittent treatment of Mecp2 mutants with a low dose of PTX-BD4-3 (5 mg/kg, intraperitoneally, once every 3 days for 8 weeks) reversed deficits in two core RTT symptom domains – respiration and motor control – and symptom rescue was maintained for at least 24 h after the last dose. Together, these data indicate that significant clinically relevant benefit can be achieved in a mouse model of RTT with a chronic intermittent, low-dose treatment paradigm targeting the neurotrophin receptor TrkB. 

Early alterations in a mouse model of Rett syndrome: the GABA developmental shift is abolished at birth

Genetic mutations of the Methyl-CpG-binding protein-2 (MECP2) gene underlie Rett syndrome (RTT). Developmental processes are often considered to be irrelevant in RTT pathogenesis but neuronal activity at birth has not been recorded. We report that the GABA developmental shift at birth is abolished in CA3 pyramidal neurons of Mecp2^−/y mice and the glutamatergic/GABAergic postsynaptic currents (PSCs) ratio is increased. Two weeks later, GABA exerts strong excitatory actions, the glutamatergic/GABAergic PSCs ratio is enhanced, hyper-synchronized activity is present and metabotropic long-term depression (LTD) is impacted. One day before delivery, maternal administration of the NKCC1 chloride importer antagonist bumetanide restored these parameters but not respiratory or weight deficits, nor the onset of mortality. Results suggest that birth is a critical period in RTT with important alterations that can be attenuated by bumetanide raising the possibility of early treatment of the disorder.

    

The GABA Polarity Shift and Bumetanide Treatment: Making Sense Requires Unbiased and Undogmatic Analysis

 

GABA depolarizes and often excites immature neurons in all animal species and brain structures investigated due to a developmentally regulated reduction in intracellular chloride concentration ([Cl⁻]_i) levels. The control of [Cl⁻]_i levels is mediated by the chloride cotransporters NKCC1 and KCC2, the former usually importing chloride and the latter exporting it. The GABA polarity shift has been extensively validated in several experimental conditions using often the NKCC1 chloride importer antagonist bumetanide. In spite of an intrinsic heterogeneity, this shift is abolished in many experimental conditions associated with developmental disorders including autism, Rett syndrome, fragile X syndrome, or maternal immune activation. Using bumetanide, an EMA- and FDA-approved agent, many clinical trials have shown promising results with the expected side effects. Kaila et al. have repeatedly challenged these experimental and clinical observations. Here, we reply to the recent reviews by Kaila et al. stressing that the GABA polarity shift is solidly accepted by the scientific community as a major discovery to understand brain development and that bumetanide has shown promising effects in clinical trials.

 

Back in 2013 a case study was published showing Bumetanide worked for a boy with Fragile X syndrome. A decade later and still nobody has looked to see if it works in all Fragile X. 

Treating Fragile X syndrome with the diuretic bumetanide: a case report

https://pubmed.ncbi.nlm.nih.gov/23647528/

We report that daily administration of the diuretic NKCC1 chloride co-transporter, bumetanide, reduces the severity of autism in a 10-year-old Fragile X boy using CARS, ADOS, ABC, RDEG and RRB before and after treatment. In keeping with extensive clinical use of this diuretic, the only side effect was a small hypokalaemia. A double-blind clinical trial is warranted to test the efficacy of bumetanide in FRX.

 

What do Rett syndrome and Fragile X have in common? 

In a healthy mature neuron the level of chloride needs to be low for it to function correctly (the neurotransmitter GABA to be inhibitory).

 

Rett and Fragile X are part of a large group of conditions that feature elevated levels of chloride in neurons.

 

Elevated chloride in neurons is treatable.

 

Is Bumetanide a cure for Rett syndrome, or Fragile X?

No it is not, but it is a step in that direction because it reverses a key defect present in at least some Rett and some Fragile X.

In the mouse model of Rett, bumetanide corrected some, but not all the problems caused by the loss of function of the MECP2 gene.

 

Moving on to IGF-1

IGF-1 is a growth hormone with multiple functions throughout aging. Production of IGF-1 is stimulated by GH (growth hormone).

The lowest levels occur in infancy and old age and highest levels occur around the growth spurt before puberty.

Girls with Turner syndrome, lack their second X chromosome and this causes a lack of growth hormones and female hormones. They end up with short stature and with features of autism. Treatment is possible with GH or indeed IGF-1.

In dementia one strategy is to increase IGF-1.  This same strategy is also being applied to single gene autisms like Rett and Pitt Hopkins.

Trofinetide and NNZ-2591 are improved synthetic analogues of peptides that occur naturally in the brain and are related to IGF-1. Trofinetide is being developed to treat Rett and Fragile X syndromes, NNZ-2591 is being developed to treat Angelman, Phelan-McDermid, Pitt Hopkins and Prader-Willi syndromes.

 

NGF (nerve growth factor)

Nerve growth factor does what it says (boosting nerve growth), plus much more. NGF plays a key role in the immune system, it is produced in mast cells, and it plays a role in how pain in perceived.

NGF acts via NGF receptors, not surprisingly, but also via TrkA receptors. We saw earlier in this post that BDNF acts via TrkB receptors.

Once NGF binds to the TrkA receptor it triggers a cascade of signalling via  the Ras/MAPK pathway and the PI3K/Akt pathway.  Both pathways relate to autism and Ras itself can play a role in intellectual disability. 

These are also cancer pathways and indeed NGF seems to play a role.  Beta cells in the pancreas produce insulin and these beta cells have TrkA receptors. In type 1 diabetes these beta cells die.  Beta cells need NGF to activate their TrkA receptors to survive.

Clearly for multiple reasons you need plenty of NGF.

Lack of NGF would be one cause of dementia and that is why Rita Levi-Montalcini choose to self-treat with NGF eye drops for 30 years. Rita won a Nobel prize for discovering NGF.

In Rett syndrome we know that the level of NGF is very low in the brain.

Logical therapies for Rett would seem to include:

·        NGF itself, perhaps taken as eye drops, but tricky to administer

·        A TrkA agonist, that would mimic the effect of NGF

·        The traditional medicinal mushroom  Lion’s Mane (Hericium erinaceus) 

We should note that effect of NGF acting via TrkA is mainly in the peripheral nervous system, not the brain.

It has long been known that Lions’ Mane (Hericium erinaceus) increases NGF but it was not clear why.  This has very recently been answered.

The active chemical has been identified to be N-de phenylethyl isohericerin (NDPIH).

The opens the door to synthesizing NDPIH as drug to treat a wide range of conditions from Alzheimer’s to Rett. 

Mushrooms Magnify Memory by Boosting Nerve Growth  

Active compounds in the edible Lion’s Mane mushroom can help promote neurogenesis and enhance memory, a new study reports. Preclinical trials report the compound had a significant impact on neural growth and improved memory formation. Researchers say the compound could have clinical applications in treating and preventing neurodegenerative disorders such as Alzheimer’s disease.

Professor Frederic Meunier from the Queensland Brain Institute said the team had identified new active compounds from the mushroom, Hericium erinaceus.

“Extracts from these so-called ‘lion’s mane’ mushrooms have been used in traditional medicine in Asian countries for centuries, but we wanted to scientifically determine their potential effect on brain cells,” Professor Meunier said.

“Pre-clinical testing found the lion’s mane mushroom had a significant impact on the growth of brain cells and improving memory.

“Laboratory tests measured the neurotrophic effects of compounds isolated from Hericium erinaceus on cultured brain cells, and surprisingly we found that the active compounds promote neuron projections, extending and connecting to other neurons.

“Using super-resolution microscopy, we found the mushroom extract and its active components largely increase the size of growth cones, which are particularly important for brain cells to sense their environment and establish new connections with other neurons in the brain.” 

 

Hericerin derivatives activates a pan‐neurotrophic pathway in central hippocampal neurons converging to ERK1/2 signaling enhancing spatial memory

The traditional medicinal mushroom Hericium erinaceus is known for enhancing peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. Here, we purified and identified biologically new active compounds from H. erinaceus, based on their ability to promote neurite outgrowth in hippocampal neurons. N-de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom, together with its hydrophobic derivative hericene A, were highly potent in promoting extensive axon outgrowth and neurite branching in cultured hippocampal neurons even in the absence of serum, demonstrating potent neurotrophic activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA-12 only partly prevented the NDPIH-induced neurotrophic activity, suggesting a potential link with BDNF signaling. However, we found that NDPIH activated ERK1/2 signaling in the absence of TrkB in HEK-293T cells, an effect that was not sensitive to ANA-12 in the presence of TrkB. Our results demonstrate that NDPIH acts via a complementary neurotrophic pathway independent of TrkB with converging downstream ERK1/2 activation. Mice fed with H. erinaceus crude extract and hericene A also exhibited increased neurotrophin expression and downstream signaling, resulting in significantly enhanced hippocampal memory. Hericene A therefore acts through a novel pan-neurotrophic signaling pathway, leading to improved cognitive performance.

 

Since the discovery of the first neurotrophin, NGF, more than 70 years ago, countless studies have demonstrated their ability to promote neurite regeneration, prevent or reverse neuronal degeneration and enhance synaptic plasticity. Neurotrophins have attracted the attention of the scientific community in the view to implement therapeutic strategies for the treatment of a number of neurological disorders. Unfortunately, their actual therapeutic applications have been limited and the potential use of their beneficial effects remain to be exploited. Neurotrophins, for example, have poor oral bioavailability, and very low stability in serum, with half-lives in the order of minutes  as well as minimal BBB permeability and restricted diffusion within brain parenchyma. In addition, their receptor signaling networks can confer undesired off-target effects such as pain, spasticity and even neurodegeneration. As a consequence, alternative strategies to increase neurotrophin levels, improve their pharmacokinetic limitations or target specific receptors have been developed. Identification of bioactive compounds derived from natural products with neurotrophic activities also provide new hope in the development of sustainable therapeutical interventions. Hericerin derivative are therefore attractive compounds for their ability to promote a pan-neurotrophic effect with converging ERK1/2 downstream signaling pathway and for their ability to promote the expression of neurotrophins. Further work will be needed to find the direct target of Hericerin capable of mediating such a potent pan-neurotrophic activity and establish whether this novel pathway can be harnessed to improve memory performance and for slowing down the cognitive decline associated with ageing and neurodegenerative diseases.

 

What this means is that there are 2 good reasons why Lion’s Mane should be helpful in Rett syndrome, both increasing BDNF and NGF.

  

Conclusion

Interestingly, one of the above papers is co-authored by a researcher from the European Brain Research Institute, founded by Rita Levi-Montalcini, the Nobel laureate who discovered NGF (Nerve growth factor). My top pick to test next in Rett syndrome would be NGF. Administration would have to follow Rita’s own example and be in the form of eye drops or follow the Lion’s Mane option, that has recently been further validated.

Rett syndrome is very well documented and many researchers are engaged in studying it.

As with broader autism, the problem is translating all the research into practical therapy today.

Clearly polytherapy will be required.

More than one type of neuronal hyperexcitability seems to be in play.

It looks like one E/I imbalance is the bumetanide responsive kind, that can be treated and will reduce autism symptoms and improve learning skills.  Then we have the hypoventilation/apnea for which Cloperastine looks a fair bet.  For the arrhythmia we have Phenytoin.  If there are still seizures after all that therapy it looks like sodium valproate is the standard treatment for Rett.

Sodium valproate is also an HDAC inhibitor and so has possibly beneficial epigenetic effects as a bonus.

I have always liked the idea of the Lion’s Mane mushrooms as a means to increase NGF (Nerve growth factor).  In today’s post we saw that it is the NDPIH from the mushrooms that acts to increase both BDNF and NGF.  You would struggle to buy NDPIH but you can buy these mushrooms. I did once buy the supplement version of these mushrooms and it was contaminated, so I think the best bet is the actual chemical or the actual mushroom.  One reader did write in once who is a big consumer of these mushrooms.

 

Lion's Mane Mushroom

Source: Igelstachelbart Nov 06

 

A Trk-B agonist that can penetrate the blood brain barrier would look a good idea.  There are some sold by the nootropic people.

7,8-dihydroxyflavone is such an agonist that showed a benefit in the mouse model.

 

7,8-dihydroxyflavone exhibits therapeutic efficacy in a mouse model of Rett syndrome

Following weaning, 7,8-DHF was administered in drinking water throughout life. Treated mutant mice lived significantly longer compared with untreated mutant littermates (80 ± 4 and 66 ± 2 days, respectively). 7,8-DHF delayed body weight loss, increased neuronal nuclei size and enhanced voluntary locomotor (running wheel) distance in Mecp2 mutant mice. In addition, administration of 7,8-DHF partially improved breathing pattern irregularities and returned tidal volumes to near wild-type levels. Thus although the specific mechanisms are not completely known, 7,8-DHF appears to reduce disease symptoms in Mecp2 mutant mice and may have potential as a therapeutic treatment for RTT patients.

Rett syndrome also features mitochondrial dysfunction and a variant of metabolic syndrome.  We have quite a resource available from broader autism, not much of it seems to have been applied in Rett.

You can see that in Rett less oxygen is available due to breathing issues and yet more oxygen is required due to “faulty” mitochondria. 

“Intensified mitochondrial O2 consumption, increased mitochondrial ROS generation and disturbed redox balance in mitochondria and cytosol may represent a causal chain, which provokes dysregulated proteins, oxidative tissue damage, and contributes to neuronal network dysfunction in RTT.”

https://www.frontiersin.org/articles/10.3389/fphys.2019.00479/full#:~:text=Rett%20syndrome%20(RTT)%2C%20an,inner%20membrane%20is%20leaking%20protons.

 

We have seen in this blog that 2 old drugs exist to increase oxygen levels in blood.  The Western world has Diamox (Acetazolamide) and the former soviet world has Mildronate/Meldonium. Mildronate also was suggested to have some wider potential benefit to mitochondria.

Rett is proposed as a neurological disorder with metabolic components, so based on what we have seen in this blog, you would think along the lines of Metformin, Pioglitazone and a lipophilic statin (Atorvastatin, Simvastatin or Lovastatin). 

The Anti-Diabetic Drug Metformin Rescues Aberrant Mitochondrial Activity and Restrains Oxidative Stress in a Female Mouse Model of Rett Syndrome

Statins improve symptoms of Rett syndrome in mice

The ultimate Rett cure will be one of the new gene therapies given to a baby before any significant progression of the disorder has occurred.

For everyone else, it looks like there is scope to develop a pretty potent individualized polytherapy, just by applying the very substantial knowledge that already exists in the research.

Good luck to Daniel and all the others seeking answers.

 

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).