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Showing posts with label Pregnenolone. Show all posts
Showing posts with label Pregnenolone. Show all posts

Friday 23 December 2016

Neuroligins, Estradiol and Male Autism


Today’s post looks deeper into the biology of those people who respond to the drug bumetanide, which means a large sub-group of those with autism, likely those with Down Syndrome and likely some with schizophrenia.
It is a rather narrow area of science, but other than bumetanide treatment, there appears to be no research interest in further translating science into therapy.    So it looks like this blog is the only place to develop such ideas.
I did not expect this post would lead to a practical intervention, but perhaps it does. As you will discover, the goal would be to restore a hormone called estradiol to its natural higher level, perhaps by increasing an enzyme called aromatase, which appears to be commonly downregulated in autism.  This should increase expression of neuroligin 2, which should increase expression of the ion transporter KCC2; this will lower intracellular chloride and boost cognition.
It seems that those people using Atorvastatin may have already started this process, since this statin increases IGF-1 and insulin is one of the few things that increases the aromatise enzyme. 

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




The testosterone – estradiol shunt



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

  

NKCC1, KCC2 and AE3

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

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


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

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

Block NKCC1 with Bumetanide and/or increase KCC2 expression

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

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

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

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

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

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

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


Background

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

Results

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

Conclusions

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

  
Neuroligins and Neurexins

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





Neurexins (Nrxns) and neuroligins (Nlgns) are arguably the best characterized synaptic cell-adhesion molecules, and the only ones for which a specifically synaptic function was established8,9. In the present review, we will describe the role of Nrxns and Nlgns as synaptic cell-adhesion molecules that act in an heretofore unanticipated fashion. We will show that they are required for synapse function, not synapse formation; that they affect trans-synaptic activation of synaptic transmission, but are not essential for synaptic cohesion of the pre- and postsynaptic specializations; and that their dysfunction impairs the properties of synapses and disrupts neural networks without completely abolishing synaptic transmission as1012. As cell-adhesion molecules, Nrxns and Nlgns probably function by binding to each other and by interacting with intracellular proteins, most prominently PDZ-domain proteins, but the precise mechanisms involved and their relation to synaptic transmission remain unclear. The importance of Nrxns and Nlgns for synaptic function is evident from the dramatic deficits in synaptic transmission in mice lacking Nrxns or Nlgns.

As we will describe, the role of Nrxns and Nlgns in synaptic function almost predestines them for a role in cognitive diseases, such as schizophrenia and autism spectrum disorders (ASDs), that have been resistant to our understanding. One reason for the difficulties in understanding cognitive diseaseas is that they may arise from subtle changes in a subset of synapses in a neural circuit, as opposed to a general impairment of all synapses in all circuits. As a result, the same molecular alteration may produce different circuit changes and neurological symptoms that are then classified as distinct cognitive diseases. Indeed, recent studies have identified mutations in the genes encoding Nrxns and Nlgns as a cause for ASDs, Tourette syndrome, mental retardation, and schizophrenia, sometimes in patients with the same mutation in the same family1327. Viewed as a whole, current results thus identify Nrxns and Nlgns as trans-synaptic cell-adhesion molecules that mediate essential signaling between pre- and postsynaptic specializations, signaling that performs a central role in the brain’s ability to process information and that is a key target in the pathogenesis of cognitive diseases.

Neuroligins and neurexins in autism


ASDs are common and enigmatic diseases. ASDs comprise classical idiopathic autism, Asperger’s syndrome, Rett syndrome, and pervasive developmental disorder not otherwise specified73,74. Moreover, several other genetic disorders, such as Down syndrome, Fragile-X Mental Retardation, and tuberous sclerosis, are frequently associated with autism. Such syndromic forms of autism and Rett syndrome are usually more severe due to the nature of the underlying diseases. The key features of ASDs are difficulties in social interactions and communication, language impairments, a restricted pattern of interests, and/or stereotypic and repetitive behaviors. Mental retardation (~70% of cases) and epilepsy (~30% of cases) are frequently observed; in fact, the observation of epilepsy in patients with ASDs has fueled speculation that autism may be caused by an imbalance of excitatory vs. inhibitory synaptic transmission. In rare instances, idiopathic autism is associated with specialized abilities, for example in music, mathematics, or memory. The relation of ASDs to other cognitive diseases such as schizophrenia and Tourette’s syndrome is unclear. As we will see below with the phenotypes caused by mutations in Nlgns and Nrxns, the boundaries between the various disorders may not be as real as the clinical manifestations suggest.

A key feature of ASDs is that they typically develop before 2–3 years of age73,74. ASDs thus affect brain development relatively late, during the time of human synapse formation and maturation. Consistent with this time course, few anatomical changes are associated with ASDs75. An increase in brain size was repeatedly reported76, but is not generally agreed upon75. Thus, similar to other cognitive diseases, ASDs are not a disorder of brain structure but of brain function. Among cognitive diseases, ASDs are the most heritable (~ 80%), suggesting that they are largely determined by genes and not the environment. ASDs exhibit a male:female ratio of approximately 4:1, indicating that ASDs involve the X-chromosome directly, or that the penetrance of pathogenic genes is facilitated in males73,74.

Mutations in many genes have been associated with familial ASDs. A consistent observation emerging from recent studies is the discovery of mutations in the genes encoding Nrxn1, Nlgn3, and Nlgn4. Specifically, seven point mutations, two distinct translocation events, and four different large-scale deletions in the Nrxn1 gene were detected in autistic patients1318. Ten different mutations in the Nlgn4 gene were observed (2 frameshifts, 5 missense mutations, and 3 internal deletions), and a single mutation in the Nlgn3 gene (the R451C substitution)2124. Besides these mutations, five different larger deletions of X-chromosomal DNA that includes the Nlgn4 locus (referred to as copy-number variations) were detected in autism patients18,2527.

In addition to the Nrxn/Nlgn complex, mutations in the gene encoding Shank3 – an intracellular scaffolding protein that binds indirectly to Nlgns via PSD-95 and GKAP (Fig. 1)66 – may also be a relatively frequent occurrence in ASDs. An astounding 18 point mutations were detected in the Shank3 gene in autistic patients, in addition to several cases containing CNVs that cover the gene18,7782. Indeed, the so-called terminal 22q deletion syndrome is a relatively frequent occurrence that exhibits autistic features, which have been correlated with the absence of the Shank3 gene normally localized to this chromosome section. Shank3 is particularly interesting because it not only indirectly interacts with Nlgns, but also directly binds to CIRL/Latrophilins which in turn constitute α-latrotoxin receptors similar to Nrxns, suggesting a potential functional connection between Shank3 and Nrxns83.

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

First, at least for some of the mutations in this complex, non-symptomatic carriers were detected in the same families in which the patients with the mutations were found. Whereas the Nlgn3 and Nlgn4 mutations appear to be almost always penetrant in males, and even female carriers with these mutations often have a phenotype, the Shank3 point mutations in particular were often observed in non-symptomatic siblings77,78. Thus, these mutations may only increase the chance of autism, but not actually cause autism.

Second, the same mutations can be associated with quite different phenotypes in different people. For example, a microdeletion in Nlgn4 was found to cause severe autism in one brother, but Tourette’s syndrome in the other26. This raises the issue whether the ‘autism’ observed in patients with mutations in these genes is actually autism, an issue that could also be rephrased as the question of whether autism is qualitatively distinct from other cognitive diseases, as opposed to a continuum of cognitive disorders. In support of the latter idea, two different deletions of Nrxn1α have also been observed in families with schizophrenia19,20, indicating that there is a continuum of disorders that involves dysfunctions in synaptic cell adhesion and manifests in different ways. Conversely, very different molecular changes may produce a similar syndrome, as exemplified by the quite different mutations that are associated with ASDs84.

At present, the relation between the Nrxn/Nlgn synaptic cell-adhesion complex and ASDs is tenuous. On one hand, many of the mutations observed in familial ASD are clearly not polymorphisms but deleterious, as evidenced by the effect of these mutations on the structure or expression of the corresponding genes, and by the severe autism-like phenotypes observed in Nlgn3 and Nlgn4 mutant mice8587. On the other hand, the nonlinear genotype/phenotype relationship in humans, evident from the only 70–80% heritability and from the occasional presence of mutations in non-symptomatic individuals, requires explanation. Elucidating the underlying mechanisms for this incomplete genotype/phenotype relationship is a promising avenue to insight into the genesis of autism. Furthermore, in addition to the link of Nrxn1α mutations to schizophrenia19,20, linkage studies have connected Nrxn3 to different types of addiction88,89. It is possible that because of the nature of their function, mutations in genes encoding Nrxns and Nlgns constitute hotspots for human cognitive diseases.

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

Let’s get back on subject and focus on Neuroligin 2 
The very recent paper below mentions sensory processing defects and NLG2 alongside what we already have figured out so far.

Abstract


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

After some investigation I learned that both estradiol/estrogen and progesterone increase expression of neuroligin 2, at least in rats.
Increasing neuroligin 2/NLGN2/NL2 looks a promising strategy.


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

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

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


Highlights


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

·         High-throughput behavioral profiling identifies nighttime hyperactivity in mutants

·         cntnap2 mutants exhibit altered responses to GABAergic and glutamatergic compounds

·         Estrogenic compounds suppress the cntnap2 mutant behavioral phenotype

Summary


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


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

The new findings of reduced expression of estrogen receptor beta as well as that of an enzyme that converts testosterone to estrogen could help explain the high testosterone levels in autistic individuals and higher autism rates in males, Pillai said.
It was the 5-to-1 male-to-female ratio along with the testosterone hypothesis that led Pillai and his colleagues to pursue whether estrogen might help explain the significant gender disparity and possibly point toward a new treatment.

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

Comparing the brains of 13 children with and 13 children without autism spectrum disorder, the researchers found a 35 percent decrease in estrogen receptor beta expression as well as a 38 percent reduction in the amount of aromatase, the enzyme that converts testosterone to estrogen.
Levels of estrogen receptor beta proteins, the active molecules that result from gene expression and enable functions like brain protection, were similarly low. There was no discernable change in expression levels of estrogen receptor alpha, which mediates sexual behavior.



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

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

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



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

More generally:-


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

Estrogens and androgens exert positive effects on cognitive functions.

Progesterone and allopregnanolone have variable effects on cognitive functions.

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

Epigenetic modifications are a powerful mechanism of memory regulation.


Conclusion

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

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

Insulin does increase aromatase, as does alcohol and being overweight.
The clever thing to do would be to just correct the reduced level of aromatase, or wait for a selective estrogen receptor beta agonist like LY500307 to come to the market.

In those who are extreme male, a little estradiol might be the simple solution, but not the amount that is currently taken by those that abuse it.  Yes people abuse estradiol – males who want to be females.
Antonio Hardan at Stanford did trial high dose pregnenolone, another hormone mainly found in females, that should increase progesterone.


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

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


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




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

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

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



 
  


Thursday 21 January 2016

2016 To-do List

I expect many readers of this blog have a list of things to trial in 2016; I certainly do.

Monty’s older brother, codenamed Ted, did say to me recently, “I thought you said you’d be all finished with this, in a couple of years”; that was indeed the intention.  


A medicine cabinet to be proud of, but not mine


It has now been three years.  I never really intended to go so deeply into the science, and I never expected there to be so many “obvious” things un/under-investigated by researchers.

Most people diagnosed these days with “autism” are fortunate to be relatively mildly affected.  Parents of those kids likely find this blog rather shocking; how can so many pills be needed and still you want more?

Some other people also diagnosed with autism, face really big challenges, not limited to:-
  
     ·        Unable to talk
·        Unable to walk
·        Unable to eat (must use G tube)
·        Unable to be toilet trained
·        Unable to read
·        Unable to write
·        Have seizures 

So when asked by a teacher at school, if Monty, now aged 12, has severe autism I responded in the negative.  He does not tick any of the above boxes.

If you have more than “mild autism” it seems that there are likely many dysfunctions and the more you treat, the better the result.  A quest without an end.


School

Ted hates his relatives discussing his school grades and I agree with him that they are entirely his business.  We all know that typical kids vary in how smart they are and how motivated they are.  NT kids tend to get the grades they deserve.

I do break these rules with Monty, but that is because I really want to show that when a person has numerous neurological dysfunctions, as those found in classic autism, if you treat them with science (not with bleach and other nonsense), you can end up in a different, better place. 99.99999% of the world do not know this; perhaps 500 people do know.

Improving IQ will improve the person’s ability to understand and compensate for the dysfunctions that have not been treated.  

Grading academic performance at school is something we all understand and along with its limitations.  We have all been there, so let's use it.

Kids with classic autism do not get the grades they potentially deserve.  Most can be made smarter and it is easy to measure.

Before coming to my to-do list, I did receive another question about what exactly is the effect of bumetanide. 

When I collected Monty from school the other day, his assistant was proudly holding up the latest “quick fire” math test, where speed is seemingly even more important than the right answer.

So Monty, the only one with autism, came first and by a long way. 3 minutes and 35 seconds, with the runner up taking 3:56.  He got 90% correct, but that is enough to keep first place.   The previous test before Christmas he got 100%, but finished 7th out of 16 on speed.  It must be the turkey.

The questions are very simple, since you have to be very fast; but until the age of 9, and the introduction of Bumetanide, the class teacher would never have dreamt of having Monty compete at all.  Coming a distant last in everything would be disheartening, for the teacher. Monty would not have even noticed, let alone cared.

People with Classic Autism, or what Knut termed SDA (strict definition autism), are usually hopeless academically; but with Bumetanide, it does not have to be that way. 

Many people with classic autism leave school 18 years old, still at the level of single digit addition and subtraction, or perhaps up to 20.

If you reach the academic level of Grade 2 (Year 3 in the UK system), that of a typical 7 or 8 year old, by the time you “graduate” high school, you are doing above average.









So Ted is not alone in being able to get good grades.  The PolyPill is indeed worth all the bother.



To-do list


I did have to go through by supply cupboard to see what I had not got round to testing and that I still think has some potential merit.  Some things did get thrown out.

Some old ideas are worth revisiting.

·        Biotin (high dose)
This did seem to have a marginal positive effect and is both cheap and harmless. 

·        Pregnenolone (very low dose)
This also appeared to have some positive effect and should affect GABA subunit expression. High doses have been used in a Stanford clinical trial. We saw in earlier posts that allopregnanolone possesses biphasic, U-shaped actions at the GABAA receptor, meaning that a tiny dose can have the same effect as a large dose.
 I like low doses.  

Old ideas worth developing:-

·        Miyairi 588 bacteria, but at higher doses

This is the bacteria used as a probiotic in Japan for humans, since the 1940s.  It is also added to animal feed to avoid inflammatory disease and so produce healthier animals.

The science showed that it should be helpful to raise Butyrate levels.  It can be achieved directly via supplementation, with sodium butyrate, and indirectly by adding a butyrate-producing bacteria, such as Clostridium Butyricum or Miyari 588.

I have been using a tiny dose of Miyari 588 for months.  It achieves what it is sold for in Japan, in that it reduces gas, which is the only obvious negative side effect of Monty’s Polypill, other than diuresis.

The positive side effect of the Polypill is near perfect asthma control.  Asthma is an auto-immune/inflammatory disease, highly comorbid with autism. 

The effect of Miyari 588 is reversible because this bacteria cannot survive long in the intestines, which is why you have to take it every day.  It crowds out some of the other bacteria in the intestines, but they will soon grow back.


New ideas already in this blog:-

·        Diamox

I did suggest on several occasions that it might be possible to get a “Bumetanide plus” effect by adding Diamox.

Diamox (Acetazolamide) is another diuretic and it is a carbonic anhydrase inhibitor


Acetazolamide is a carbonic anhydrase inhibitor, hence causing the accumulation of carbonic acid Carbonic anhydrase is an enzyme found in red blood cells that catalyses the following reaction:



hence lowering blood pH, by means of the following reaction that carbonic acid undergoes:


The mechanism of diuresis involves the proximal tubule of the kidney. The enzyme carbonic anhydrase is found here, allowing the reabsorption of bicarbonate, sodium, and chloride. By inhibiting this enzyme, these ions are excreted, along with excess water, lowering blood pressure, intracranial pressure, and intraocular pressure. By excreting bicarbonate, the blood becomes acidic, causing compensatory hyperventilation, increasing levels of oxygen and decreasing levels of carbon dioxide in the blood

This change in bicarbonate will also affect the AE3 and NDAE exchangers.

As you will see in the figure below the regulation of bicarbonate HCO3- and pH is directly connected to chloride Cl- homeostasis.  This means that via AE3 and NDAE you can affect intracellular chloride levels by change the level of HCO3-

In turns this means that Diamox (Acetazolamide) really should have an effect on the level of intracellular chloride.

This in turn suggested to me that Diamox could augment the effect that bumetanide has on NKCC1.

 In the case that Bumetanide can lower intracellular chloride, but not to the optimal level to correct the GABA dysfunction, Diamox might be able to lower chloride levels a little further so further shifting GABA to inhibitory.










http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317631/

Neuronal activity results in significant pH shifts in neurons, glia, and interstitial space. Several transport mechanisms are involved in the fine-tuning and regulation of extra- and intracellular pH. The sodium-independent electroneutral anion exchangers (AEs) exchange intracellular bicarbonate for extracellular chloride and thereby lower the intracellular pH. Recently, a significant association was found with the variant Ala867Asp of the anion exchanger AE3, which is predominantly expressed in brain and heart, in a large cohort of patients with idiopathic generalized epilepsy. To analyze a possible involvement of AE3 dysfunction in the pathogenesis of seizures, we generated an AE3-knockout mouse model by targeted disruption of Slc4a3. AE3-knockout mice were apparently healthy, and neither displayed gross histological and behavioral abnormalities nor spontaneous seizures or spike wave complexes in electrocorticograms. 



After only a couple of days of Diamox, it is pretty clear that there is indeed a “bumetanide plus” effect.  So the same changes that were noted when starting bumetanide appear again.

A promising start to 2016.



·        Ponstan

This is the NSAID that is also suggested to be useful to affect the ion channels expressed by the genes ANO 2/4/7 & KCNMA1.  We saw in this post

http://epiphanyasd.blogspot.com/2015/12/autism-treatments-proposed-by-clinical.html

where Knut highlighted that Fenamates act as CaCC inhibitors and also stimulate BKCa channel activity.  Ponstan is a Fenamate.



·        Vitamin A

This was Maja’s discovery, that in some people vitamin A will stimulate oxytocin, via upregulation of CD38.


·        Zinc

Zinc should affect GABA, particularly in immature neurons.  Zinc homeostasis is disturbed in some autism and perhaps, in some people, a small dose of zinc may actually have a positive effect.  Simple to check.

Clioquinol, the drug that shifts zinc to the “right” place, is not without risks.


·        Picamilon

Once the GABA switch has been repaired, it may be time for a little extra GABA.  GABA should not be able to cross the blood brain barrier (BBB), but in the form of Picamilion, it does cross the BBB.


·        Inositol

This it naturally produced in the body from glucose and used to be known as vitamin B8.  In some people Inositol reduces OCD and stereotypy.  Simple to check.


·        Montelukast

This is an asthma drug, considered very safe in children, that Dr Kelley (formerly of Johns Hopkins and likely the cleverest autism clinician)  uses in children with AMD, as a short term therapy, when they are sick and, very interestingly, before immunizations.  This is to avoid further mitochondrial damage.  Montelukast is a leukotriene receptor antagonist (LTRA) used for the maintenance treatment of asthma and to relieve symptoms of seasonal allergies.

Dr Kelley also uses Ibuprofen as a short term therapy to counter the effects of increased cytokine production.  Montelukast is more potent and has different side effects, meaning it might be a better choice than ibuprofen for some people.

Ibuprofen may be OTC, but, more than very occasional use, can cause side effects in many people.  These side effects are caused by NSAIDs also being COX-2 inhibitors, which leads to stomach and intestinal adverse reactions.

Since I have determined that in the case of autism I deal with, the surge in cytokines like IL6 causes behavioral regression, Montelukast might be a good alternative to Ibuprofen to treat some types of autism flare.  

So a new addition to the autism flare-up toolkit, I hope.

  

Ideas not yet in this blog:-

·        Curcumin

Curcumin, and particularly some of the substances within it, have been shown to have very interesting autism-relevant effects, particularly in vitro (in test tubes).  Whether taking curcumin orally, in reasonable doses, produces any of these effects in humans is a big question.  Many such substances like luteolin and resveratrol fail to meet expectations in humans, due to poor bioavailability.

There are various ways to improve the bioavailability of curcumin, so it seems worth investigating.



·        5-loxin

Frankincense has been used for 5,000 years.  More recently, two thousand years ago, three wise men did bring gifts of gold, frankincense, and myrrh.

Frankincense is an aromatic resin obtained from trees of the genus Boswellia.  Boswellia is used for inflammatory conditions like arthritis in a similar way to curcumin.

There are six boswellic acids, one is most active. This fraction is called AKBA. 5-Loxin is a boswellia supplement claiming to deliver a high standardized level of AKBA.

5-Loxin does seem to help some people with arthritis, but does it have any benefit for the pro-inflammatory aspects found in some autism?  I am not expecting much, but you never know.

  
Ideas suggested to me by others, that look interesting:-


·        Mint/Menthol

This is Natasa’s discovery and there is evidence to show that Menthol does indeed affect GABAA receptors.



These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA-mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA-related pharmacotherapies.

  
·        NIAGEN / Nicotinamide Riboside

This was highlighted by Tyler and is another potential therapy for oxidative stress.  Not as cheap as peppermint, but definitely interesting, perhaps particularly for those with autism and mitochondrial dysfunction.

Also note that there are odd recurring links between some autism and obesity. This is not the first anti-obesity therapy that potentially has some benefit for autism.



Summary
As NAD+ is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38—both NAD+ consumers—increases NAD+ bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD+ precursor with the ability to increase NAD+ levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD+ levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.
  



Low-grade chronic inflammation (metaflammation) is a major contributing factor for the onset and development of metabolic diseases, such as type 2 diabetes, obesity, and cardiovascular disease. Nicotinamide riboside (NR), which is present in milk and beer, is a functional vitamin B3 having advantageous effects on metabolic regulation. However, the anti-inflammatory capacity of NR is unknown. This study evaluated whether NR modulates hepatic nucleotide binding and oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Male, 8-week-old KK/HlJ mice were allocated to the control or NR group. NR (100 mg/kg/day) or vehicle (phosphate-buffered saline) was administrated by an osmotic pump for 7 days. Glucose control, lipid profiles, NLRP3 inflammasome, and inflammation markers were analyzed, and structural and histological analyses were conducted. NR treatment did not affect body weight gain, food intake, and liver function. Glucose control based on the oral glucose tolerance test and levels of serum insulin and adiponectin was improved by NR treatment. Among tested lipid profiles, NR lowered the total cholesterol concentration in the liver. Histological and structural analysis by hematoxylin and eosin staining and transmission electron microscopy, respectively, showed that NR rescued the disrupted cellular integrity of the mitochondria and nucleus in the livers of obese and diabetic KK mice. In addition, NR treatment significantly improved hepatic proinflammatory markers, including tumor necrosis factor-alpha, interleukin (IL)-6, and IL-1. These ameliorations were accompanied by significant shifts of NLRP3 inflammasome components (NLRP3, ASC, and caspase1). These results demonstrate that NR attenuates hepatic metaflammation by modulating the NLRP3 inflammasome

  

  

  

An apparently crazy idea of my own, but actually serious:-


·        Propolis tincture, without the propolis

The BIO 30 Propolis from New Zealand is a (mild) PAK1 inhibitor.  One reader is convinced of its cognitive enhancing effects in autism .  I also think it had an effect, but in our case not as potent as that reader.  Now I am wondering what was it that produced this effect. 

Most propolis is made as a tincture with ethanol.  Propolis is not soluble in water.  They typically use 70% ethanol to make propolis tincture.  “Non-alcoholic” tinctures use glycol.

In the last post we saw ethanol has pronounced effects on several GABAA receptor subunits, mainly delta but also alpha, including possibly down regulating alpha 5.

So was it the propolis, or the ethanol that has the effect?

Propolis tincture is either made with ethanol (grain alcohol) or if it is “alcohol free” they use propylene glycolPropylene glycol actually is a food ingredient but it is also used to de-ice aircraft in winter.  Ethylene glycol is the antifreeze in your car and you would not want to drink that.

Compared to ethanol, glycol can dissolve less propolis, 

A quick check of school chemistry reminds us that if it is an –ol , it’s an alcohol.

·        Alcohols have at least one hydroxyl group
·        Diols have two hydroxyl groups

Propylene glycol is  C3H8Oand as you can see below it has two hydroxyl groups (the – OH), so it is both a diol and an alcohol. 






So your Propolis tincture can be ethanol-free, but it cannot be alcohol-free.  Someone might point that out to the supplement makers.

It also should be noted that propylene glycol has known effects on GABA very similar to ethanol.


  
This suggests that the users of ethanol-free BIO30 may also be seeing responses unrelated to propolis.

Propylene glycol even has an E-number, it is E1520.  It is cheap and they even sell it on Amazon.

Food grade ethanol is normally not sold to the public.

In lay terms, ethanol and alcohol are interchangeable, so one corner of the supermarket contains food grade ethanol, with some impurities.

Japanese research suggests that these impurities are much more potent than ethanol in modulating GABA receptors.  It is the fragrant compounds that accumulate over the years on wooden barrels that cause this effect.

The twenty drops of propolis suggested to me by the Japanese PAK1 researcher/doctor contained about 1ml of ethanol.  It seems that to get an effect on GABA similar to this amount of ethanol would require a much smaller amount to well-aged Japanese whiskey.

So if someone over 18 responds well to twenty drops of BIO 30 propolis, it would helpful if they could compare the effect with 1ml of Propylene glycol (E1520), 1ml of ethanol, if they find it, and with a few drops of well-aged whiskey.