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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.


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).












28 comments:

  1. Hi Peter,

    Thank you for another fantastic post! Again, I have learned a great deal from this post, and really appreciate how you make connections between various papers and lines of research that are very relevant to us.

    When I read this post something struck me and I wonder if you could provide your thoughts on it. Many months ago, I tried Homotaurine as an option (if memory serves me right, I was trying to find a natural mimic for something, may have been Acamprosate) and my daughter felt off after having it, and then fell asleep soon after (which was unusual for her at that time of day. So I stopped it right away but it stuck with me as that has been the only item I've ever tried that to me had a negative effect. Could it be that she already had too much Taurine and the supplement just overloaded her Taurine levels (hence the not feeling well and zonking)? If so, does that tell me anything (i.e. she's more likely to benefit from Bumetanide)?

    Thanks very much as always Peter, for a tremendous post and your insights!

    AJ

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    1. AJ, one known marker for bumetanide responders is a paradoxical reaction to Valium, instead of being calming it makes them agitated. This is because GABA is working "in reverse" in those people. I do not know if this applies to 100% of bumetanide responders.

      Maybe there is also an unusual response to taurine, I do not know.

      One reader just commented that in their child it took one month with bumetanide 1 mg twice a day in a 5 year old girl to see the response. At a lower dose there was no response.

      So when you make your trial, it is best to keep going, to be absolutely sure if your daughter is a responder or not.

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    2. Peter and AJ, for those wit sulfa allergies, shouldn't they avoid bumetanide? And it is recommended to tey spironolactone instead? Maybe AJ for your daughter the reaction to taurine, since it is a sulfur containing amino acid, she is sensitive ? Have you tried molybdenum to see if it helps her tolerate sulfury things more? Does she react negatively to sulfury foods? I am living vicariously through everyone's bumetanide experiements and not trying to seem like I have it all figured out - ha! My doctor was reluctant to rx it when I first asked. But considering persuading her again until I heard about sulfa allergy issues. My son has sulfa med allergies.

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    3. Tanya, if your son is allergic to a class of medications called sulfonamides, he might also be allergic to bumetanide. This is something to ask your doctor to look into. She might suggest taking a very small single dose to check if your son has an allergic response.

      Spironolactone is a good diuretic for people allergic to sulfonamides, but it will not have the "autism effect" of bumetanide.

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    4. Are there any other known markers for bumetanide responders?
      I could probably get valium easier than bumetanide, and the following reaction would be a good hint to whether I should pursue my hunt for this elusive drug, or put my efforts on finding other interventions.
      /Ling

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    5. Yes Peter, it was a reaction to Bactrim which is a sulfonamide,when he was young - he had a mild Stevens-Johnson reaction, peeling skin on his eyelids up to the eyebrows..I now wonder if that was the trigger for his mast cell activation....

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    6. I think any benzodiazepine, like valium, may have a reverse effect(i.e. make anxiety). This was the observation that lead to the idea of bumetanide being therapeutic in autism. I do not know if this applies to 100% of responders.

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    7. Hi Peter, Tanya, and Community,

      Peter - thank you for your response! I actually tried to order Bumetanide some time ago from an online Mexican pharmacy (that's a sentence I never thought I would say a year ago). But my Visa provider doesn't allow any payments to online Mexican pharmacies and the Mexican pharmacy wouldn't ship to Canada (probably because it would get stopped at the border), so I'm going back to plan A and seeing another physician soon who I hope will be prescribe Bumetanide. Once I finally do get it, I will definitely heed your advice - I do want to ensure that it does / does not work so will likely start with 0.5mgs X 2, and then titrate up to 1mg X 2 per day if nothing happens in the first month.

      Out of interest Peter, here is an interesting article and paper about Histamine and Tourette's:

      https://medicalxpress.com/news/2017-06-tourette-like-tics-mice-histamine.html

      http://www.pnas.org/content/early/2017/05/31/1704547114

      And here's an interesting paper on plasma amino acid levels in ASD boys versus controls:

      http://psychiatriapolska.pl/uploads/images/PP_2_2017/ENGver359Bugajska_PsychiatrPol2017v51i2.pdf

      And finally, maybe the most interesting paper of the 3 - "Rescue of impaired sociability and anxiety-like behavior in adult cacna1c-deficient mice by pharmacologically targeting eIF2α":

      http://www.nature.com/mp/journal/vaop/ncurrent/full/mp2017124a.html

      "Remarkably, systemic treatment with ISRIB, a small molecule inhibitor that suppresses the effects of phosphorylated eIF2α on mRNA translation, was sufficient to reverse the social deficit and elevated anxiety-like behavior in adult cacna1c fbKO mice. ISRIB additionally normalized the lower protein synthesis and higher E/I ratio in the PFC"

      Have a great night Peter!

      AJ

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    8. Hi Tanya,

      Hope all is well!

      Peter is far more knowledgeable about this than I am, and his advice sounds reasonable to me (i.e. testing a very small dose to see if any issues).

      As far as sulfur containing foods, my daughter hasn't shown any issues, and one of the supplements I'm currently giving her is aged garlic and I believe that has sulfur in it, with no negative effects, so I assume she's OK with sulfur.

      As I noted in my post a few minutes ago, I've had a real adventure in trying to access Bumetanide, with no success yet. Seeing a new doc in a couple of months, and hoping that the new doc is open to this. I'm going to make a strong case using all the trial results and other info I can of parents using this off-label and can only hope.

      As I had mentioned a while ago, in my research, I had found that Astaxanthin may be an option against NKCC1 as per the following:

      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007682/

      I can't say it's done wonders, but I still use it, and the issue may be dosing, as I don't know what the equivalent would be to 1mg X 2 of Bumetanide, if that's even reasonable with Astaxanthin. It hasn't hurt so I keep using it.

      I did just post the following and wonder if we can find a natural method to address the finding:

      http://www.nature.com/mp/journal/vaop/ncurrent/full/mp2017124a.html

      My other potential option is Claritin. Peter has shown that it may be helpful, so that's on my radar as a next potential option.

      Have a wonderful night Tanya!

      AJ

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    9. AJ, the third paper is particularly interesting.

      In mice harboring a loss of cacna1c (the gene for the calcium channel Cav1.2) they found reduced activity of mTORC1 and its downstream mRNA translation initiation factors eIF4B and 4EBP1, as well as elevated phosphorylation of eIF2α.

      ISRIB is one two experimental drugs that inhibits phosphorylation of eIF2α.

      ISRIB improved anxiety and sociability in your study and cognition in another study.

      I think in autism there often is a problem with Cav1.2, but it may be the opposite problem, i.e. too much.

      In the mouse study they wanted in shift the E/I balance towards excitatory, in autism we usually want to shift it towards inhibition.

      As with Gargus and his IP3R theory, we seem to keep coming back to aberrant calcium channels, but they can be dysfunctional in both directions.

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    10. Thanks AJ - astaxanthin is an appealing option to trial for this theory. For my son, I don't think my dr would ever rx bumetanide. And with sulfa allergies in his history, I don't think I could ever be cowboy enough to try even the tiniest dose - much less having to get it from an on-line pharmacy. I guess when you've witnessed stevens johnson reaction and also have another son with an epi pen and had to witness anaphylaxis in your child, it puts the fear in you. ha! I am just interested to learn from others - when there may be things in common with my son's symptoms/reactions - learning frm when things go wrong, even slightly. Helps figure out sub types better and that may work in my kid. Very UN scientific, I know, but none of the scientists are making much head way in figuring it out. Except Naviaux seems to be the closest - with his selection process for kids in his study. Thanks for sharing your research - I look forward to reading these papers tonight.

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    11. AJ I am in Canada and was able to get my paediatrician to prescribe butenamide, I provided the study info and he gave me a standing order to test electrolytes. It should not to be a problem. If you need help email me at nieszka_m@hotmail.com

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    12. Nieszka, it is great to hear that your paediatrician was open to consider relevant clinical trials. This is exactly the way it should be. I hear of more and more similar cases. Hopefully he will see the positive effect and prescribe it to his other patients with autism and tell his peers. Treatment by mainstream clinicians is the best outcome.

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    13. Hi Nieszka,

      That is wonderful, and thank you so much for kindly offering your assistance! I will email you later today when I get home from work.

      AJ


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  2. For anyone looking to try a long-shot "herbal" remedy that would be easy to trial, there is some new research suggesting the existence of an endocannabinoid transporter which if inhibited would increase endocannabinoid signaling in the brain, leading to anti-inflammatory effects (probably good for autism):

    Press Release:

    https://www.sciencedaily.com/releases/2017/06/170606090806.htm

    Paper:

    http://www.pnas.org/content/early/2017/05/30/1704065114

    Now in this research, they created a drug that acted as a powerful endcannabinoid reuptake inhibitor. Further research on this drug will likely lead to some people trying to get a higher high with marijuana, but for therapeutic purposes this drug may be relevant to autism.

    The researchers were originally inspired by the effects of Echinacea which has been an herbal remedy for helping to prevent colds for probably thousands of years. They searched for similar compounds that they thought would upregulate the endocannabinoid system and found one that worked well.

    Now as best I know, Echinacea is safe and I have never ever heard of an Echinacea overdose, so it is possible it might help some autisms (just to be clear this is just an idea of mine, with no proof behind it). Echinacea is also pretty cheap and here in North America you can just make some tea yourself by picking some purple coneflowers growing off a local highway if you want to (though buying it in tablet form probably makes a lot more sense).

    Speaking of cannabinoids, there was also some other very interesting research on THC that suggests low-doses (not enough to get you high) reduce anxiety while greater than low doses increase anxiety:

    Press Release:

    https://www.sciencedaily.com/releases/2017/06/170602155252.htm

    Paper:

    http://www.drugandalcoholdependence.com/article/S0376-8716(17)30220-X/fulltext

    So maybe a little Echinacea could help with anxiety problems and perhaps general brain inflammation issues related to autism since it would increase cannabinoid signaling just enough to have benefits, but not so much that you would get the negative effects people experience from marijuana use. This intervention is also so cheap and simple that I will likely trial this once I do a little more research to see if anyone else has noticed spontaneously improving symptoms from Echinacea. And if Echinacea does improve symptoms, even a little bit, then perhaps these researchers studying their endocannabinoid reuptake inhibitor could be pointed in autism's direction somehow.

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    1. So I did a little more research on this and low and behold there are several discussions out there about people trying to use Echinacea in combo with Cannabis to get a higher high including this one:

      https://www.reddit.com/r/DrugNerds/comments/1dkjqn/can_echinacea_extract_be_used_as_a_marijuana/

      I also looked deeper into any prior research on hypothesized mechanisms of action and came across some very interesting stuff with Echinacea and its potential relevance to autism, starting with this paper:

      https://www.researchgate.net/publication/7232261_Alkylamides_from_Echinacea_are_a_new_class_of_cannabinomimetics_-_Cannabinoid_type_2_receptor-dependent_and_-independent_immunomodulatory_effects

      which shows IL-6 upregulation but downregulation of other various cytokines including TNF-a.

      On the contrary, this paper shows an upregulation in TNF-a mRNA transcription but not an increase in TNF-a protein:

      http://www.sciencedirect.com/science/article/pii/S0014579304013183

      Peter had an old blog post I remembered (he has several discussing IL-6 and TNF-a) but here is one from several years ago that seems very interesting here:

      https://epiphanyasd.blogspot.com/2014/03/single-dose-of-il-6-antibodies-or-tnf.html

      So in light of all of this, it seems like Echinacea would make things worse for autism via its immunostimulant properties, however, I found multiple anecdotal reports of parents claiming Echinacea helped with behaviors (they seemed to think Echinacea would clear their child's body of toxins and viruses somehow).

      But of course when dealing with receptor based issues, you want things to be in equilibrium so perhaps Echinacea might cause cannabinoid receptor downregulation which in the long-run would decrease overall cannabinoid signaling sensitivity.

      I suppose in this situation, there is only one way to find out.

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  3. Hi Ling, instead of using valium, why don't you use Ponstan for a couple of days? Maybe you did that already? I remember Knut saying that both Ponstan and Bumetanide could lower chloride levels and the responders are those who have the paradoxical respond to valium.
    I have never used valium for my son, but when I use even very low dose benzodiazepine he gets extremely agitated.
    There are research papers supporting the benzodiazepine parodoxical reaction for Bumetanide responders.
    Not knowing that my son was on the spectrum when he was a child, I used Ponstan syrup each time he didn't feel well and possibly this prevented him from regression.

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  4. Hi AJ, very interesting papers, indeed.
    My son was found marginally low in protein synthesis. Doctor says no clinical significance.
    He also has Tourette's, not officially diagnosed, but it's obvious, he blinks his left eye and shows "self grooming behaviours" which is very distressing.
    Fot targeting both histamine and elf2a, do you see any practical interventions?

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  5. Peter,

    Would you or anyone else have thoughts on why DMG, which I gave to my son only for four days caused such a big explosion...rage, trauma, SIB. And though things seem to be stabilizing, some remnants are still there...some hand biting.

    I read a parents comment on another forum, whose child developed serious head banging on DMG and it took many years for the issue to resolve. This has really left me cold.

    Could this kind of reaction to DMG be an indicator about other possible stuff to steer clear off? He was alright on mb12.

    I feel if parents share more about trials that go seriously wrong, it would be as helpful, if not more, as reporting the things that worked...for two weeks (sarcasm). Though it does make you look and feel like an ass or even outright criminal.

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    1. Kritika, DMG is an agonist of the glycine binding site of NMDA receptors. These receptors play a key role in glutamate excitotoxicity, which I think you have just triggered in your son. I suspect this was the cause of the rage and SIB.

      Many different things can lead to glutamate excitotoxicity.

      I expect things will gradually get back to normal.

      This probably tells you that son is more likely to have NMDAR hyper-function than hypo-function.

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    2. Dear Peter,

      Thank you so much. Actually I was suspecting role of glutamate receptors and its relation with glycine here...oxalate issues are often also triggered with glycine in some and I was supplementing vit c too which could have triggered a crisis. Well, I have some information about my son now.

      Yesterday, I gave him 600 mg NAC and it did seem to have a beneficial effect. Today, morning, he floored our neighbour and a cop by smiling and waving at them spontaneously. Do not know if it was NAC (you said it has a very short half life) or him just feeling good. Unfortunately, I cant do NAC too regularly as it leads to gi discomfort for him. But last time I tried NAC, it seemed to have some carry over effects so will just test if dosing with a gap works.

      Thank you once again and kind regards

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    3. I probably was the parent who reported head banging from DMG, which lasted for years. It is important to note while DMG caused SIB in my son, it also made him communicate with us by taking my hand and bringing me to the object of his interest. It is hard to say why DMG caused SIB because of its multiple actions. I personally dont think SIB was caused by extra Glycine or NMDA activation. In fact, I think those were positives because when we gave TMG, we didnt see any problems. DMG also blocks BHMT, which converts homocesteine into methinione. And perhaps that is where the problem is. Without BHMT and proper transulfuration function (e.g. CBS mutation or lack of Serine), you get an overload of homocesteine. My son also becomes irritable on sulfur containing supplements like Curcumin. So, for him, the theory that SIB was caused by overload of homocesteine makes sense. My son doesnt have CBS mutation. But he has DAOA mutation, which reduces Serine (and NMDA activity) and makes him 3x more prone to become schizophrenic. And how is schizophrenia treated? By giving Glycine! Because Glycine, just like Serine, is a co-agonist of NMDA receptors. In fact, there are clinical trials of Sarcosine, which blocks Glycine conversion and increases its concentration, as an effective treatment of scizophtenia.

      In short, TMG helps BHMT to remove homocesteine by donating its methyl group. By doing so, it is converted to DMG, whech is then converted to Glycine and Sarcosine. But, DMG also blocks BHMT. So, giving it as a supplement results in overload of homocesteine.
      V

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    4. A couple of things to add to my previous post. Besides the DAOA mutation, which limits Sarcosine used for homocesteine conversion and NMDA activation, my son also has homozygous MTHFR A1298C mutation that limits folate, which is also used to convert homocysteine into methione. There are only two paths to convert homocesteine into methione: through folate and through BHMT. So, if you blick both (one by MTHFR mutation and the other by DMG supplementatio), you will create too much homocysteie. Interestingly, one could suggest giving B6 and zinc to help with homocysteine conversion into glutathione. But in my son, B6 and zinc cause irritability, and it is quite possibly caused by B6 an zinc helping to create dopamine or some other action. It is all complicated.
      V

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    5. Kritika, almost all the DAN/MAPS-type interventions can have negative effects in some people. The same is true with the interventions I use.

      You can take the view, like Tanya, that you can learn a lot from which interventions have a negative reaction, but it is often hard to be sure why there was a negative reaction.

      It is prudent to test interventions on yourself first, since your child’s genes are Mum’s and Dad’s (with mitochondrial genes all from Mum). If you have a negative reaction best not to give it to your son.

      Even when some drugs are finally approved for autism, there will still be those who show negative effects.

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    6. Peter,

      Maternal inheritance....well, testing reactions to drug/supplements is not as simple as you make it out to be. My son has an aberrant biochemistry..neurotypicals don't go flying into rage on supplements. They do when they are very sick sometimes. I have taken all the sups that he took. Though, I agree testing on yourself is better than no testing at all.

      Things are not black and white..I think every parent is treading a grey line here, thinking out the pros and cons. Anfd
      And every parent has a child who is different and every child has a parent who is different too. I have experienced SIB first hand and I worship parents who try to work around this and going all out with a urgency into trying out every intervention out there which might help with this. But when every thing you try only makes things worse, you did something which by mischance turned your happy child into a tragic monster, you naturally want to be very very careful. Probably it would be prudent to work things up slowly, strengthen the foundation, try and get a better understanding. So, no parent can speak on behalf of or against another.

      We all are living stories which are so similar but are also so different.

      Respectful regards

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    7. Kritika, you could try paternal inheritance and try your therapies out on the Dad. I found verapamil has a beneficial effect on me.

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    8. Peter,

      Very creative suggestion....unfortunately made with the presumption that all contributors to paternal inheritance...dads..would be as sweet, cooperative and willing as you.

      Different kids, different parents, different trajectories.

      Sorry, not getting into a,spat with you, just expressing ground realities.

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    9. Dear V,

      First, I am an admirer. The kind of information you have put out here and on the other forum is so helpful.

      My son did not show any positives on DMG unlike your son. And his SIB was not harmless, funny head banging. He was terrified, traumatised and aggressive at the same time, wanting us to help him get rid of what hurt his body and soul so, but we did not know how to help him. He was screaming in pain, tears rolling down his face and his head, hands, feet and penis hurt. He bit his hand, hit his head and penis with his tab, tried to wrench off the offending organ and when I tried to help he pulled my hair. The most tragic part that he was aware of what had come over him and he gestured me to stay away. Finally, he went off to sleep, curled up, distant and lonely, an ancient response to deep suffering, as I watched from a distance. In other societies, this would be like being possessed. I am sharing all this to express the emotional toll it takes on a parent when they watch suffering unfold in front of their eyes which somehow they caused unwittingly.

      My son does not respond favourably to most drugs/supplements and its not a question of to which ones but when the intolerance strikes and in what magnitude...it could be days, it could be weeks. I was supplementing him with low dose of methyl folate and folinic acid before I added DMG. We have not done many tests, just the routine ones which all come out perfect. I think it would be reasonable to dig a little deeper with the lab works.

      You have given some indicators and the exhaustive list of drugs/sups with possible effects or side effects which you have provided on the other site is so useful.

      Thank you so much

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