Showing posts with label RORα. Show all posts
Showing posts with label RORα. Show all posts

Thursday, 3 October 2019

Elevated Prenatal Estradiol in Mothers/Babies – a protective reaction to stress that also predicts who will develop Autism? Time for Fetal Medicine?

There has been little mention in this blog about fetal medicine, but it is an area that does hold great promise.  At the Children’s Hospital of Philadelphia, they have been surgically treating babies with spina bifida prior to their birth for nearly twenty years. Early surgical intervention to the spine even allows for malformation of the brain to be self-repaired and this is visible on MRI scans. Such prenatal treatment can be 100% successful, resulting in there being no physical disability in adulthood. 

The more you read about neuroscience the more you realize how little we really know and so encouraging the brain to self-repair may indeed be the best strategy.  This is an avenue of research and not just with stem cells.  A similar approach is proving successful in treating skin cancer, you do not attack the cancer with drugs, you modify the immune system with a drug so it "wakes up" and does its job and kills the cancer cells.

        Skin cancer: Half of people surviving advanced melanoma

Hormones are an often-ignored area of autism research, but they are they on my Venn diagram simplification of autism.

We have seen how female hormones can be highly neuroprotective and that the estradiol/testosterone balance affects a key “switch” that controls gene expression RORalpha.

Today we see that researchers in Utah suggest that stress in the developing fetus with autism causes an increase in estradiol, as a protective mechanism, and this increase in estradiol can then be measured in the mother’s blood. They propose that this elevated estradiol is an advance warning of a baby with autism.

University of Utah researchers have discovered a link between increased levels of a type of estrogen in babies in their second trimester and risk for autism, according to a new study.

The findings could eventually help doctors identify babies at risk of autism early in their mothers’ pregnancies and monitor them more closely, as well as provide early interventions to ensure the children’s well-being, said Dr. Deborah A. Bilder, the study’s first author.

Both the control group and the group of mothers whose children had autism were selected so that 50% of each group had an exposure to a condition such as gestational diabetes, hypertension and preeclampsia. Previous studies have shown links between those conditions and autism risk.

The researchers looked at several different steroids in the blood samples. Bilder said she expected to find increased levels of steroids that were known to be associated with the conditions, like testosterone. She found those steroids, but they did not reach a statistical significance, according to Bilder.

Bilder also expected progesterone and testosterone in the kids who developed autism to be elevated.

“But that’s not what I found. Instead, what I found, is estradiol being elevated in the children who developed autism,” she said.

Estradiol is a type of estrogen. Lower levels of estradiol in a baby usually indicate a concern with the baby, and high estrogen levels are not currently associated with abnormal conditions.

But as the researchers looked at the steroid hormones that were measured, “what we realized is that the higher estrogen levels being produced by the placenta actually may be stimulating the baby’s development of his or her stress response.”
Usually, a baby’s stress response takes time so that when the pregnancy reaches full term, the baby has developed its own stress response. But elevated levels of estrogen cause the baby’s stress response to develop early, Bilder said.
That prepares babies, when there is an issue, to survive outside the mom. It causes early growth of the lungs, gut and skin so that if the baby doesn’t make it all the way through pregnancy, it’s more likely to survive, she said.

The findings indicated that in the babies with autism, something set off their stress response early.

Studies have shown that children with autism have an abnormal stress response, according to Bilder. She believes the mechanism that triggers the early stress response during pregnancy may still be affecting children with autism past delivery.

Bilder doesn’t think doctors should target the higher estradiol levels or try to lower them. Instead, because it signals a “protective mechanism, that baby is surviving,” doctors should target something that doesn’t jeopardize the baby’s survival.

“By being able to have a way of looking at the baby’s well-being in that regard, I think that opens up the door to considering how can you reduce the stress on that baby?” Bilder explained.
The full paper:-

Early Second Trimester Maternal Serum Steroid-Related Biomarkers Associated with Autism Spectrum Disorder

Epidemiologic studies link increased autism spectrum disorder (ASD) risk to obstetrical conditions associated with inflammation and steroid dysregulation, referred to as prenatal metabolic syndrome (PNMS). This pilot study measured steroid-related biomarkers in early second trimester maternal serum collected during the first and second trimester evaluation of risk study. ASD case and PNMS exposure status of index offspring were determined through linkage with autism registries and birth certificate records. ASD case (N = 53) and control (N = 19) groups were enriched for PNMS exposure. Higher estradiol and lower sex hormone binding globulin (SHBG) were significantly associated with increased ASD risk. Study findings provide preliminary evidence to link greater placental estradiol activity with ASD and support future investigations of the prenatal steroid environment in ASD.

Fig. 1 The placenta produces estradiol from DHEA of both maternal and fetal origin and shunts over 90% of estradiol into the maternal circulation. The volume of DHEA substrate determines placental estradiol production and subsequently maternal serum estradiol levels. DHEA exists primarily in its conjugated form DHEAS

This is interesting as are some other findings linking steroid hormones to future autism. Another paper highlights a mechanism where maternal stress only has damaging effects on the male fetus (Placental adaptation in response to PNMS is sex-dependent, leading to an increased risk of adverse neurodevelopmental effect in male compared to female).

This paper looks at the effect of maternal stress on serotonin and another group of hormones (Glucocorticoids).

Effects of prenatal maternal stress on serotonin and fetal development

Fetuses are exposed to many environmental perturbations that can influence their development. These factors can be easily identifiable such as drugs, chronic diseases or prenatal maternal stress. Recently, it has been demonstrated that the serotonin synthetized by the placenta was crucial for fetal brain development. Moreover, many studies show the involvement of serotonin system alteration in psychiatric disease during childhood and adulthood. This review summarizes existing studies showing that prenatal maternal stress, which induces alteration of serotonin systems (placenta and fetal brain) during a critical window of early development, could lead to alteration of fetal development and increase risks of psychiatric diseases later in life.


         Fig. 1. Proposed mechanism of fetal programming of psychiatric disorders involving placental serotonin system. Cortisol and placental serotonin are essential for fetal brain development. Prenatal maternal stress alters glucocorticoid (11b-HSD2, GR and CRH) and serotonin (SERT, 5-HT1A and 5-HT2A) systems as well as serotonin and glucocorticoid interaction in the placenta. These placental alterations lead to adverse neurodevelopment and programming leading to psychiatric disorders later in life. Placental adaptation in response to PNMS is sex-dependent, leading to an increased risk of adverse neurodevelopmental effect in male compared to female. 11b-HSD2: Type 2 11-beta hydroxysteroid dehydrogenase, GR: Glucocorticoid receptor, CRH: Corticotrophin releasing hormone, SERT: Serotonin transporter, 5-HT1A: Serotonin 1A receptor, 5-HT2A: Serotonin 2A receptor

It should be noted that estradiol is supposed to be elevated during pregnancy. Indeed, this elevation is suggested to explain why females with ADHD have far less symptoms during pregnancy (estradiol is good for ADHD). The study is highlighting a level of estradiol during pregnancy that is even higher than that normally expected.

Spina bifida is normally detected by ultrasound before 18 weeks of pregnancy. This is around the same time that in autism there appears to be elevated estradiol.  Hopefully other biomarkers will also be found.

Given this advance warning, there is potential for fetal medicine.

Only very recently was the first person in the UK treated for spina bifida using fetal surgery, almost two decades after the first operations in the US.

Fetal medicine for autism would not be surgical, rather pharmacological.  In mouse models it has already started.

Estradiol has many effects and I did write about DHED, an orally active, centrally selective estrogen and a biosynthetic prodrug of estradiol. DHED is estradiol just for the brain, without affecting the rest of the body.  I think many people would benefit from DHED, across the range from ADHD to TBI (Traumatic Brain Injury).

DHED, delivering Estradiol only to the Brain, also Lupron and Spironolactone

Estradiol may indeed prove to be a fetal biomarker for autism and DHED might be a useful drug for someone with autism (via ERβ and RORalpha).

Wednesday, 10 January 2018

A RORα Agonist for Autism?

Today’s post is again about RORα, which was suggested to be a nexus where different biological dysfunctions that lead to autism may converge. I think you can consider RORα like a dimmer switch on your lights, you need to adjust the brightness to give the effect you want.

Fine tuning RORα to tune autism gene expression

I recently came across some research where the scientist clearly has the same idea. He has been working on a synthetic RORα/γ agonist for some years and has investigated its use as both a cancer therapy and an autism therapy.
I have become rather interested in cancer therapies because there are so many overlaps between what can lead to cancer and what exists in autism. The big research money is of course in cancer research.
Tumor suppressor genes/proteins like PTEN and p53 have been shown to be disturbed in autism, as is Bcl-2. The Bcl-2 family of proteins regulate cell death (apoptosis); some members induce cell death and other inhibit it; the balance is important.
Generally it seems that most people with autism might benefit from more PTEN and Bcl-2. 

Autism is a developmental disorder of the nervous system associated with impaired social communication and interactions as well excessive repetitive behaviors. There are no drug therapies that directly target the pathology of this disease. The retinoic acid receptor-related orphan receptor α (RORα) is a nuclear receptor that has been demonstrated to have reduced expression in many individuals with autism spectrum disorder (ASD). Several genes that have been shown to be downregulated in individuals with ASD have also been identified as putative RORα target genes. Utilizing a synthetic RORα/γ agonist, SR1078, that we identified previously, we demonstrate that treatment of BTBR mice (a model of autism) with SR1078 results in reduced repetitive behavior. Furthermore, these mice display increased expression of ASD-associated RORα target genes in both the brains of the BTBR mice and in a human neuroblastoma cell line treated with SR1078. These data suggest that pharmacological activation of RORα may be a method for treatment of autism. 
The RORs have been linked to autism in human in several studies. In 2010, Nguyen and co-workers reported that RORα protein expression was significantly reduced in the brains of autistic patients and this decrease in expression was attributed to epigenetic alterations in the RORA gene. Additional work from this group demonstrated that multiple genes associated with autism spectrum disorder are direct RORα target genes and suggested that reduction of RORα expression results in reduced expression of these genes associated with the disorder leading to the disease. Independently, Devanna and Vernes demonstrated that miR-137, a microRNA implicated in neuropsychiatric disorders, targets a number of genes associated with autism spectrum disorder including RORA. There are also additional links between RORα and autism. Deficiency of Purkinje cells is one of the most consistently identified neuroanatomical abnormalities in brains from autistic individuals, and RORα is critical in development of the Purkinje cells. Significant circadian disruptions have also been recognized in autistic patients, and RORs play a critical role in regulation of the circadian rhythm., Additionally, the staggerer mouse displays behaviors that are associated with autism including abnormal spatial learning, reduced exploration, limited maze patrolling, and perseverative behavior relative to wt mice.

SR1078 is a relatively low potency compound with limited RORα efficacy (3–5 μM EC50Emax 40%), but the efficacy compares favorably to other classes of compounds that have been optimized such as a 38% decrease in the same model induced by the mGluR5 allosteric modulator GRN-529 and a 47% reduction by the mGluR5 antagonist MPEP. Both of these compounds have been optimized and display high potency (single digit nanomolar range at mGluR5) and strong efficacy., Thus, we believe that focused optimization of RORα ligands will provide compounds that will have improved efficacy in this model. It should also be noted that SR1078 has both RORα and RORγ agonist activity and a RORα selective agonist has not yet been developed. Thus, it is possible that the RORγ activity of this compound may also play a role in its efficacy in this model of autism. In summary, we have demonstrated that a synthetic RORα/γ agonist is able to increase the expression of key genes whose decrease in expression is associated with ASD both in cell culture and in vivo. Furthermore, the agonist decreases repetitive behavior in an animal model of autism suggesting that it is possible that ROR agonists may hold utility in treatment ASD. 

Activation of p53 function leading to cell-cycle arrest and/or apoptosis is a promising strategy for development of anti-cancer therapeutic agents. Here, we describe a novel mechanism for stabilization of p53 protein expression via activation of the orphan nuclear receptor, RORα. We demonstrate that treatment of cancer cells with a newly described synthetic ROR agonist, SR1078, leads to p53 stabilization and induction of apoptosis. These data suggest that synthetic ROR agonists may hold utility in the treatment of cancer.  

Results showed that levels of Bcl-2 decreased by 38% and 36% in autistic superior frontal and cerebellar cortices, respectively when compared to control tissues. By the same token, levels of P53 increased by 67.5% and 38% in the same brain areas in autistic subjects vs. controls respectively. Calculations of ratios of Bcl-2/P53 values also decreased by 75% and 43% in autistic frontal and cerebellar cortices vs. controls respectively. The autistic cerebellar values were significantly reduced (p < 0.08) vs. control only. There were no significant differences in levels of β-actin between the two groups. Additionally, there were no correlations between Bcl-2, P53, and β-actin concentrations vs. age or PMI in either group.
These results confirm and extend previous data that levels of Bcl-2 and P53 are altered in three important brain tissues, i.e. frontal, parietal, and cerebellar cortices of autistic subjects, alluding to deranged apoptotic mechanisms in autism.  

Increasing PTEN and Bcl-2 is already part of my Polypill, via the use of Atorvastatin.
There are of course many other genes miss-expressed in autism and we cannot give a drug for each one. We need to identify a handful of nexus, where multiple anomalies can be resolved with a single intervention.
It is good that Thomas Burris, the lead researcher, has been working on SR1078 for at least 6 years, let’s hope he continues to persevere.
I think it highly likely that some types of autism will need the opposite therapy, a RORα antagonist.
My method of attempting to modulate RORα will be different. I come back to my earlier gross simplification of autism :- 

As we have seen in earlier posts, the hormonal dysfunction, this time the balance between testosterone and estradiol, has a direct effect on RORα (and vice versa).

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

As you might know, many hormones are interrelated, so what are thought of as male/female sex hormones have much wider effects. They impact growth hormones and play a big role in calcium metabolism. They also affect serotonin.
We know that in most autism aromatase is reduced, estradiol is reduced and that there is reduced expression of estrogen receptor beta.
In the ideal world it might indeed be best to use an agonist or antagonist to fine tune RORα.
We have a chicken and the egg situation. Is RORα out of tune in autism because the hormones are disturbed, or vice versa?
We do know that hormones generally have feedback loops, but we also know that increasing a hormone like estradiol via obesity is not fully matched by a corresponding reduction in aromatase. So it looks highly plausible that you can tune RORα via estradiol, and that this could be a long term strategy, not just a short term strategy.
In the case of people with low T3 thyroid hormone centrally (in the brain), giving exogenous T3 may help initially, but in the long term it does not because feedback loops to the thyroid will reduce production of the pro-hormone T4. In the extreme you will make the thyroid gland shut down, this does happen to people using thyroid hormones for depression and even weight loss. 
T3 is quite commonly prescribed by alternative practitioners in the US for autism and also for depression in older people. In Europe this hormone is rarely even available. 
Many phytoestrogens are used as OTC autism therapies. These are dietary estrogens that are structurally similar to the human hormone estradiol and so produce estrogen-like effects. They include soy products, fenugreek, kudzu, EGCG etc.

Saturday, 16 December 2017

Turner Syndrome, Estradiol and Autism-lessons from the X Chromosome

This post is best read if you have reviewed the earlier ones regarding the estradiol/testosterone disturbances in autism and how they govern the RORα “switch” that then triggers a torrent of other dysfunctions. So the hormonal disturbance, if present, is a key point at which to make a potent intervention. 

Beauty is in the eye of the beholder

In the mass media it is now popular to dismiss the fact that autism is far more prevalent on boys than girls. In the scientific literature, fortunately, they stick to the facts and much is written about the sex differences in autism.
As we have seen in earlier posts, females have some natural defences against autism. They have two X chromosomes and of course they have those all-important neuroprotective female hormones (estrogen/estradiol, progesterone etc.). In effect, the more female you are, the more protection you have against idiopathic autism and any X-chromosome linked single gene autism. So a girl with Fragile-X syndrome is likely to be far less affected than her brother with same condition.
Recall that we all have 23 pairs of chromosomes and that the 23rd set contains two Xs in girls and in boys one X and one Y. The girls’ “spare” X chromosome is also what gives them their feminine features.  

It is interesting to look what happens to females who lack part of their second set of X- chromosomes. This diagnosis is called Turner Syndrome. As you might have guessed people with Turner Syndrome have much lower levels of female hormones and a higher incidence of autism, although some people find this controversial. The autism-like characteristics of TS include:-

·      Impairments in social functioning

·      Impairments in face and emotion processing

·      Spatial executive deficits

·      Poor social coping skills and increased immaturity

·      Hyperactivity and impulsivity

Turner syndrome occurs in 50 per 100,000 live-born females. Autism occurs about ten times for frequently, so about 500 per 100,000 live-born females.  Turner syndrome provides the extreme case of what happens when females have too little estrogen/estradiol.
I think you will find a large group of females with idiopathic autism (no identified genetic defects) have/had low levels of estradiol. I think this is the reason that facial recognition studies show that some females with idiopathic autism look different, (as do many boys, of course). We already know that most single gene types of autism produce tell-tale signs, often on the face (big ears, wide face, big/small head etc).

I am not suggesting that there is anything wrong with looking different; rather it may be a useful diagnostic tool and not an expensive or invasive one. Physical variation has long been used to identify genetic syndromes, before genetic testing became widely available.

Physical variation inside your head
We saw in an earlier post that MRI scans of the autistic brains actually do often show subtle differences, particularly when you use software to read them, rather than the naked eye. Traditionally doctors say that MRIs are “normal” in autism and cannot be used to diagnose it. Yet in a recent studies machine reading of MRIs was able to identify 70%-96% of autism cases.  Some of these are scans taken before birth.

This is interesting, because ultimately you might bypass the current very slow and subjective observational diagnosis process.

MRIs show a brain anomaly in nearly 70 percent of babies at high risk of developing the condition who go on to be diagnosed, laying the groundwork for a predictive aid for pediatricians and the search for a potential treatment

Predicting the future with brain imaging

In a new study, Emerson et al. show that brain function in infancy can be used to accurately predict which high-risk infants will later receive an autism diagnosis. Using machine learning techniques that identify patterns in the brain’s functional connections, Emerson and colleagues were able to predict with greater than 96% accuracy whether a 6-month-old infant would develop autism at 24 months of age. These findings must be replicated, but they represent an important step toward the early identification of individuals with autism before its characteristic symptoms develop.

MRI scanners are very widely used, but you do have to keep very still inside when they are in operation. The even harder part is the reading of the data. It is clear that some standardized machine reading (A/I artificial intelligence) process is required to notice every possible variation. You could have a centralized location where you just submit your MRI data, the center gets to keep the data and learn from it; and you get their insight as to what differences there might be.

Facial Differences vs MRI Brain Differences
I like to keep things simple and under my control.  In the short term we have to settle for facial differences, since any well-managed MRI process will be decades away.

Hormonal Variation in Autism
Hormonal differences were one of the key areas I identified years ago in this blog. Big/small heads result from disturbances in pro-growth signalling pathways. We should expect variations in bone-age, early/late onset of puberty and indeed big variations in height and weight.

In Turner Syndrome, the girls tend to be very short and they are often treated with growth hormones, as well as female/feminizing hormones.  
Great caution has to be taken when treating children with any hormones. When children are treated, it is for serious reasons like not achieving puberty, or having a serious growth delay (being very short).

Hormone Therapy During Pregnancy
In some countries hormones are given during pregnancy although I think this would be seen as odd/risky in some advanced countries.

We have already seen that couples who have difficulty producing a child often have a family history that includes autism. It was proposed by one serious fertility expert that what helps prevent miscarriage also helps prevent autism. This did sound odd when I first read about, but when you look in more depth there is a basis for this idea.
That expert has these two websites:-

Progesterone supplements have been recommended for more than 50 years for women struggling with infertility, but research now shows they can also help prevent miscarriage.

Tamoxifen, an estrogen receptor (ER) antagonist, is also used to treat infertility.
Estradiol is sometimes prescribed during pregnancy.
Testosterone is produced naturally during pregnancy.

All this is clearly beyond the scope of this blog, but perhaps altered female/male hormones during pregnancy might be a biomarker of some future autism and female hormones might be a protective therapy in the subgroup of pregnant mothers with low levels of these hormones and/or high levels of testosterone. Recall that human trials in the hospital ER have shown certain substances are highly neuroprotective (progesterone, atorvastatin etc) and when administered immediately after a traumatic brain injury markedly improve the outcome.                                         

Hormone Therapy for Autism
Hormone therapy in people with autism would be controversial, but we saw in an earlier post that via RORα the balance between testosterone and estradiol affects numerous biological relevant to autism.

Many pictures of girls/women with autism, that you can view online, suggest reduced levels of estradiol. Faces look more boy-like. Many males with autism are reported to have physical features of high testosterone and low estradiol. 
One example of many:-

Both faces in the above article show clear indications of autism. Since both young people do have autism, this should not surprise anyone.
My own conclusion is that if you have autism or Asperger’s, a little extra estradiol could therapeutic, particularly if you have physical features that reinforce this.
There are of course many males and females with autism who are physically indistinguishable from the rest of the world. The point of this post is to highlight that visible differences may help to define the sub-type of autism and indicate possibly effective therapies, that exist today.

Obesity and Estradiol
In an earlier post on estradiol, I pointed out that in males estradiol is made in your adipose (fat) tissue. In the US many people with autism are overweight, in part due to side effects from their likely un-needed psychiatric medications; this has the hidden benefit of increasing their estradiol levels, feminizing their behavior slightly and shifting RORalpha in the right direction.
This also means that losing weight should be helpful to obese females with estrogen receptor positive breast cancer.  Research does support this.

Asperger’s and too much Estradiol?
We saw in earlier posts that much autism is associated with reduced expression of estrogen receptor beta and low aromatase, so high testosterone and low estradiol.

We have seen on many occasions that when one extreme exists in autism, so usually does the other; so many big heads, but also some tiny ones, NMDAR hypofunction, but also hyperfunction.

There was a lot of talk a while back in the media about children undergoing therapy to change their gender, and it was highlighted that Asperger’s was much over-represented in this group. One expert got into trouble for suggesting that their autism was causing them to obsess about their identity and so mistakenly convince a boy that he would rather be a girl.  It seems that these days some clinicians are then all too willing to provide drug therapy and then operate on them, to make them female.  I do wonder if perhaps some of these boys with Asperger’s might have the other extreme of aromatise. That would give them too little testosterone and too much Estradiol.
I think measuring these hormones is quite a good idea, as I keep repeating, they go on to affect the critical “switch”  RORα, which then impacts a large number of biological processes implicated in autism.  In other words you can try to normalize a wide range of important autism variables, just be tweaking RORα, via estradiol/testosterone.

A boy with high testosterone, and so low estradiol, will likely exhibit physical signs of this, just like the girl with low estradiol. These are just pieces of the puzzle, in plain view, that can be used to understand each specific case of autism. And no machine reading of an MRI is required.

For those left wanting more:
A very thorough paper on Turner Syndrome:-

Turner syndrome (TS) is a neurogenetic disorder characterized by partial or complete monosomy-X. TS is associated with certain physical and medical features including estrogen deficiency, short stature and increased risk for several diseases with cardiac conditions being among the most serious. Girls with TS are typically treated with growth hormone and estrogen replacement therapies to address short stature and estrogen deficiency. The cognitive-behavioral phenotype associated with TS includes strengths in verbal domains with impairments in visual-spatial, executive function and emotion processing. Genetic analyses have identified the short stature homeobox (SHOX) gene as being a candidate gene for short stature and other skeletal abnormalities associated with TS but currently the gene or genes associated with cognitive impairments remain unknown. However, significant progress has been made in describing neurodevelopmental and neurobiologic factors underlying these impairments and potential interventions are on the horizon

We utilized an ultrasensitive assay to study estradiol levels in 34 girls with TS and 34 normal age-matched prepubertal girls between the ages of 5 and 12 years. The average estradiol level in the girls with TS (6.4 +/- 4.9 pmol/l estradiol equivalents) was significantly lower than in the normal prepubertal girls (12.7 +/- 10.8 pmol/l estradiol equivalents; p < 0.01). Girls with TS were significantly shorter, and weighed less than the normal prepubertal girls, as expected. The estradiol level was not significantly correlated with height, bone age, 


Saturday, 3 June 2017

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. 

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:-

 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.

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 GABAA signaling may depend upon the direction of GABAA 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 GABAA 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.

Studies have indeed shown that PKC is reduced in some autism, which is what you might have expected. 
Finally, the other estradiol/estrogen papers:- 

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 GABAA receptors (GABAARs). 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 GABAAR 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.


GABAA 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, GABAA receptors acquire their classical hyperpolarizing signaling. The switch from depolarizing to hyperpolarizing GABAA-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 GABAA 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 GABAA receptors mediate different transcriptional effects in males and females. Furthermore, neurons with depolarizing or hyperpolarizing GABAA-ergic signaling respond differently to neurotrophic factors like estrogens. Consequently, during sensitive developmental periods, GABAA 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 GABAA 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 GABAA 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 GABAA-mediated depolarizations were operative (naïve male PN15 SNR neurons). Estradiol failed to downregulate KCC2 in neurons in which GABAA 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 GABAA signaling (female PN15 SNR neurons). This indicated that 17β-estradiol-mediated downregulation of certain calcium-regulated genes, like KCC2, shows a requirement for active GABAA-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 GABAA signaling may depend upon the direction of GABAA responses is also reverberated in other publications. In hippocampal pyramidal neurons of adult ovariectomized female rats, where GABAA 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 GABAA receptors, 17β-estradiol delays the period with excitatory GABAA signaling (Perrot-Sinal et al. 2001). However, a direct involvement of KCC2 in this process has not been demonstrated yet. Such findings indicate that GABAA 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).

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.

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 GABAA 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 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 EGABA (supplemental Table 1A). In contrast, the broad spectrum kinase inhibitor staurosporine (Staur) shifted EGABA 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 EGABA (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 EGABA 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 EGABA 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.

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).