The Purkinje-RORa-Estradiol-Neuroligin-KCC2 axis in Autism











Add testosterone/estradiol to those dysfunctional hormones


This blog is about noticing connections and making things a little simpler to understand.  Today�s post is going to be a good example; all those odd sounding things like Purkinje cells and neuroligins all fitting nicely together.

Today we see how a central hormonal dysfunction (testosterone/estradiol) can lead to an ion channel dysfunction (NKCC1/KCC2) at one end of the chain and at the other explains the absence of many Purkinje cells in the autistic cerebellum, which leads to some of the observed features of autism.

I am calling it the Purkinje-RORa-Estradiol-Neuroligin-KCC2 axis, or Purkinje-KCC2 axis for short.

We also get to see how melatonin fits in here and see why disturbed sleeping patterns should be expected in someone affected by the Purkinje- KCC2 axis.

I should point out that not everyone with autism is likely affected by the Purkinje-NKCC1 axis, but I think it will apply to a majority of those with non-regressive, multigenic, strictly defined autism (SDA).

We saw in a recent post how the enzyme aromatase acts in the so-called  testosterone � estradiol shunt.





I suggested that lack of aromatase was leading to too little estradiol which then affected neuroligin 2 (NL2) which then caused down-regulation of the KCC2 cotransporter that takes chloride out of neurons. This then caused neurons to remain in a permanent immature state.

Digging a little deeper we find recent research that shows how the control loops that balance aromatase act through RORA/RORa, RORa  (retinoic acid-related orphan receptor alpha.















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



RORa (retinoic acid-related orphan receptor alpha.)


RORa certainly has a long full name. Retinoic acid is a metabolite of vitamin A (retinol).

RORa does some clever things.

RORa is necessary for normal circadian rhythms

ROR-alpha is expressed in a variety of cell types and is involved in regulating several aspects of development, inflammatory responses, and lymphocyte development

RORa is involved in processes that regulate metabolism, development, immunity, and circadian rhythm and so shows potential as drug targets. Synthetic ligands have a variety of potential therapeutic uses, and can be used to treat diseases such as diabetes, atherosclerosis, autoimmunity, and cancer. T0901317 and SR1001, two synthetic ligands, have been found to be RORa and ROR? inverse agonists that suppress reporter activity and have been shown to delay onset and clinical severity of multiple sclerosis and other Th17 cell-mediated autoimmune diseases. SR1078 has been discovered as a RORa and ROR? agonist that increases the expression of G6PC and FGF21, yielding the therapeutic potential to treat obesity and diabetes as well as cancer of the breast, ovaries, and prostate. SR3335 has also been discovered as a RORa inverse agonist.

RORs are also called nuclear melatonin receptors. Many people with autism take melatonin to balance circadian rhythms and fall asleep.

The reduced estrogen levels in women during menopause likely caused them not to sleep due to the effect on RORa.

So it would appear that some of what is good for menopausal women may actually be helpful for some people with autism.



Many Genes affected by RORa



Most exciting, the researchers say, is that 426 of RORA�s gene targets are listed in AutismKB, a database of autism candidates maintained by scientists at Peking University in Beijing, and 49 in SFARI Gene.



Therapeutic Effect of a Synthetic RORa/? Agonist in an Animal Model of Autism



Autism is a developmental disorder of the nervous system associated with impaired social communication and interactions as well excessive repetitive behaviors. There are no drug therapies that directly target the pathology of this disease. The retinoic acid receptor-related orphan receptor a (RORa) is a nuclear receptor that has been demonstrated to have reduced expression in many individuals with autism spectrum disorder (ASD). Several genes that have been shown to be downregulated in individuals with ASD have also been identified as putative RORa target genes. Utilizing a synthetic RORa/? agonist, SR1078, that we identified previously, we demonstrate that treatment of BTBR mice (a model of autism) with SR1078 results in reduced repetitive behavior. Furthermore, these mice display increased expression of ASD-associated RORa target genes in both the brains of the BTBR mice and in a human neuroblastoma cell line treated with SR1078. These data suggest that pharmacological activation of RORa may be a method for treatment of autism.



For those who like natural substances, some research from Japan.

            Abstract

The retinoic acid receptor-related orphan receptors a and ? (RORa and ROR?), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. In this study, we investigated the effects of isoflavones on RORa/? activity and the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In doxycycline-inducible CHO stable cell lines, we found that four isoflavones, biochanin A (BA), genistein, formononetin, and daidzein, enhanced RORa- or ROR?-mediated transcriptional activity in a dose-dependent manner. In an activation assay of the Il17a promoter using Jurkat cells, these compounds enhanced the RORa- or ROR?-mediated activation of the Il17a promoter at concentrations of 1 � 10(-6)M to 1 � 10(-5)M. In mammalian two-hybrid assays, the four isoflavones enhanced the interaction between the RORa- or ROR?-ligand binding domain and the co-activator LXXLL peptide in a dose-dependent manner. In addition, these isoflavones potently enhanced Il17a mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin, but showed slight enhancement of Il17a gene expression in RORa/?-knockdown EL4 cells. Immunoprecipitation and immunoblotting assays also revealed that BA enhanced the interaction between ROR?t and SRC-1, which is a co-activator for nuclear receptors. Taken together, these results suggest that the isoflavones have the ability to enhance IL-17 gene expression by stabilizing the interactions between RORa/? and co-activators. This also provides the first evidence that dietary chemicals can enhance IL-17 gene expression in immune cells.



Genistein is a common supplement.  It is a pytoestrogen and unfortunately these substances lack potency in real life.  In test tubes they have interesting properties, but they are poorly absorbed when taken orally and so unless they are modified they are likely to have no effect in the usual tiny doses used in supplements.

This is true with very many products sold as supplements.

Sometimes care is taken to improve bioavailability as with some expensive curcumin supplements, like Longvida.

Trehalose, a supplement referred to recently in comments on this blog, is another interesting natural substance that lacks bioavailablity.  Analogs of this natural substance have been produced that are much better absorbed and are now potential drugs.




Purkinje Cells







Back in 2013 I wrote a post about Purkinje cells.

          Pep up those Purkinje cells


Loss of Purkinje cells is one of the few non-disputed abnormalities in autism. 

These cells are some of the largest neurons in the human with an intricately elaborate dendritic arbor, characterized by a large number of dendritic spines. Purkinje cells are found within the Purkinje layer in the cerebellum. Purkinje cells are aligned like dominos stacked one in front of the other. Their large dendritic arbors form nearly two-dimensional layers through which parallel fibers from the deeper-layers pass. These parallel fibers make relatively weaker excitatory (glutamatergic) synapses to spines in the Purkinje cell dendrite, whereas climbing fibers originating from the inferior olivary nucleus in the medulla provide very powerful excitatory input to the proximal dendrites and cell soma. Parallel fibers pass orthogonally through the Purkinje neuron's dendritic arbor, with up to 200,000 parallel fibers[2] forming a Granule-cell-Purkinje-cell synapse with a single Purkinje cell. Each Purkinje cell receives ca 500 climbing fiber synapses, all originating from a single climbing fiber.[3] Both basket and stellate cells (found in the cerebellar molecular layer) provide inhibitory (GABAergic) input to the Purkinje cell, with basket cells synapsing on the Purkinje cell axon initial segment and stellate cells onto the dendrites.

Purkinje cells send inhibitory projections to the deep cerebellar nuclei, and constitute the sole output of all motor coordination in the cerebellar cortex.

In humans, Purkinje cells can be harmed by a variety causes: toxic exposure, e.g. to alcohol or lithium; autoimmune diseases; genetic mutations causing spinocerebellar ataxias, Unverricht-Lundborg disease, or autism; and neurodegenerative diseases that are not known to have a genetic basis, such as the cerebellar type of multiple system atrophy or sporadic ataxias.

Purkinje cells are some of the largest neurons in the human brain and the most important.

Neuronal maturation during development is a multistep process regulated by transcription factors. The transcription factor RORa (retinoic acid-related orphan receptor a) is necessary for early Purkinje cell maturation but is also expressed throughout adulthood.

The active form (T3) of thyroid hormone  controls critical aspects of cerebellar development, such as migration of postmitotic neurons and terminal dendritic differentiation of Purkinje cells. T3 action on the early Purkinje cell dendritic differentiation process is mediated by RORa.

In autism we have seen that oxidative stress may lead to low levels of T3 in the autistic brain.  We now see that low levels of RORa are also likely in autsim.

The combined effect would help explain the loss of Purkinje cells in autism.







Neuropathological studies, using a variety of techniques, have reported a decrease in Purkinje cell (PC) density in the cerebellum in autism. We have used a systematic sampling technique that significantly reduces experimenter bias and variance to estimate PC densities in the postmortem brains of eight clinically well-documented individuals with autism, and eight age- and gender-matched controls. Four cerebellar regions were analyzed: a sensorimotor area comprised of hemispheric lobules IV�VI, crus I & II of the posterior lobe, and lobule X of the flocculonodular lobe. Overall PC density was thus estimated using data from all three cerebellar lobes and was found to be lower in the cases with autism as compared to controls. These findings support the hypothesis that abnormal PC density may contribute to selected clinical features of the autism phenotype.



Estradiol � Neuroligin 2 to KCC2

We saw in a recent post how reduced levels of estradiol could lead to KCC2 underexpression via the action of neuroligin 2.





Conclusion

So in my grossly oversimplified world of autism, I think I have a plausible case for the Purkinje-KCC2 axis.  I think that in addressing this axis numerous other issues would also be solved ranging from sleep issues to those hundreds of other genes whose regulation is at least partly governed by RORa.

The KCC2 end of the axis can be treated by bumetanide, diamox/acetazolamide, potassium bromide and possibly by intranasal IGF-1/insulin.  


How to address the rest of the Purkinje-KCC2 axis?


        More RORa, or just a RORa agonist.

        More aromatase

        Genistein may help, but you would need it by the bucket load, due to bioavailability issues

        Estrogen receptor agonists

        Exogenous estradiol

The simplest is the last one and really should be trialed on adult males with autism.  The dose would need to be much lower than the feminizing dose, so 0.2mg would seem a good starting dose for such a study.

Due to the feedback loops somethings may work short term, but not long term. 


















The Clever Ketogenic Diet for some Autism


I have covered the Ketogenic Diet (KD) in earlier posts. 

There are more and more studies being published that apply the KD to mouse models of autism.

Calling the KD a diet does rather under sell it.  The classic therapeutic ketogenic diet was developed for treatment of pediatric epilepsy in the 1920s and was widely used into the next decade, but its popularity waned with the introduction of effective epilepsy drugs.

There are various exclusion diets put forward to treat different medical conditions; some are medically accepted but most are not, but that does not mean they do not benefit at least some people.

When it comes to the ketogenic diet (KD) the situation is completely different, this diet is supposed to be started in hospital and maintained under occasional medical guidance. The KD was developed as a medical therapy to treat pediatric epilepsy.  It is very restrictive which is why it is used mainly in children, since they usually will (eventually) eat what is put in front of them.

The KD was pioneered as a medical therapy by researchers at Johns Hopkins in the 1920s, over the years they have shown that most of the benefit of the KD can be achieved by the much less restrictive Modified Atkins Diet (MAD).  The first autism mouse study below suggests something similar �Additional experiments in female mice showed that a less strict, more clinically-relevant diet formula was equally effective in improving sociability and reducing repetitive behavior�.


What about the KD in Autism?

Most people with autism, but without epilepsy, will struggle to get medical help to initiate the KD.  Much research in animal models points to the potential benefit of the KD.




        Drug treatments are poorly effective against core symptoms of autism.


        Ketogenic diets were tested in EL mice, a model of comorbid autism and epilepsy.


        Sociability was improved and repetitive behaviors were reduced in female mice.


        In males behavioral improvements were more limited.


        Metabolic therapy may be especially beneficial in comorbid autism and epilepsy.


The core symptoms of autism spectrum disorder are poorly treated with current medications. Symptoms of autism spectrum disorder are frequently comorbid with a diagnosis of epilepsy and vice versa. Medically-supervised ketogenic diets are remarkably effective nonpharmacological treatments for epilepsy, even in drug-refractory cases. There is accumulating evidence that supports the efficacy of ketogenic diets in treating the core symptoms of autism spectrum disorders in animal models as well as limited reports of benefits in patients. This study tests the behavioral effects of ketogenic diet feeding in the EL mouse, a model with behavioral characteristics of autism spectrum disorder and comorbid epilepsy. Male and female EL mice were fed control diet or one of two ketogenic diet formulas ad libitum starting at 5 weeks of age. Beginning at 8 weeks of age, diet protocols continued and performance of each group on tests of sociability and repetitive behavior was assessed. A ketogenic diet improved behavioral characteristics of autism spectrum disorder in a sex- and test-specific manner; ketogenic diet never worsened relevant behaviors. Ketogenic diet feeding improved multiple measures of sociability and reduced repetitive behavior in female mice, with limited effects in males. Additional experiments in female mice showed that a less strict, more clinically-relevant diet formula was equally effective in improving sociability and reducing repetitive behavior. Taken together these results add to the growing number of studies suggesting that ketogenic and related diets may provide significant relief from the core symptoms of autism spectrum disorder, and suggest that in some cases there may be increased efficacy in females.






        The BTBR mouse has lower movement thresholds and larger motor maps relative to control mice.


        The high-fat low-carbohydrate ketogenic diet raised movement thresholds and reduced motor map size in BTBR mice.


        The ketogenic diet normalizes movement thresholds and motor map size to control levels.


Autism spectrum disorder (ASD) is an increasingly prevalent neurodevelopmental disorder characterized by deficits in sociability and communication, and restricted and/or repetitive motor behaviors. Amongst the diverse hypotheses regarding the pathophysiology of ASD, one possibility is that there is increased neuronal excitation, leading to alterations in sensory processing, functional integration and behavior. Meanwhile, the high-fat, low-carbohydrate ketogenic diet (KD), traditionally used in the treatment of medically intractable epilepsy, has already been shown to reduce autistic behaviors in both humans and in rodent models of ASD. While the mechanisms underlying these effects remain unclear, we hypothesized that this dietary approach might shift the balance of excitation and inhibition towards more normal levels of inhibition. Using high-resolution intracortical microstimulation, we investigated basal sensorimotor excitation/inhibition in the BTBR T + Itprtf/J (BTBR) mouse model of ASD and tested whether the KD restores the balance of excitation/inhibition. We found that BTBR mice had lower movement thresholds and larger motor maps indicative of higher excitation/inhibition compared to C57BL/6J (B6) controls, and that the KD reversed both these abnormalities. Collectively, our results afford a greater understanding of cortical excitation/inhibition balance in ASD and may help expedite the development of therapeutic approaches aimed at improving functional outcomes in this disorder.





Background

Gastrointestinal dysfunction and gut microbial composition disturbances have been widely reported in autism spectrum disorder (ASD). This study examines whether gut microbiome disturbances are present in the BTBRT?+?tf/j (BTBR) mouse model of ASD and if the ketogenic diet, a diet previously shown to elicit therapeutic benefit in this mouse model, is capable of altering the profile.

Findings

Juvenile male C57BL/6 (B6) and BTBR mice were fed a standard chow (CH, 13 % kcal fat) or ketogenic diet (KD, 75 % kcal fat) for 10�14 days. Following diets, fecal and cecal samples were collected for analysis. Main findings are as follows: (1) gut microbiota compositions of cecal and fecal samples were altered in BTBR compared to control mice, indicating that this model may be of utility in understanding gut-brain interactions in ASD; (2) KD consumption caused an anti-microbial-like effect by significantly decreasing total host bacterial abundance in cecal and fecal matter; (3) specific to BTBR animals, the KD counteracted the common ASD phenotype of a low Firmicutes to Bacteroidetes ratio in both sample types; and (4) the KD reversed elevated Akkermansia muciniphila content in the cecal and fecal matter of BTBR animals.

Conclusions

Results indicate that consumption of a KD likely triggers reductions in total gut microbial counts and compositional remodeling in the BTBR mouse. These findings may explain, in part, the ability of a KD to mitigate some of the neurological symptoms associated with ASD in an animal model.





        We evaluated, throughout a systematic review, the studies with a relationship between autism and ketogenic diet.


        Studies points to effects of KD on behavioral symptoms in ASD through the improve score in Childhood Autism Rating Scale (CARS).


        Reviewed studies suggest effects of KD especially in moderate and mild cases of autism.


        KD in prenatal VPA exposed rodents, as well in BTBR and Mecp2 mice strains, caused attenuation of some autistic-like features.



Autism spectrum disorder (ASD) is primarily characterized by impaired social interaction and communication, as well as restricted repetitive behaviours and interests. The utilization of the ketogenic diet (KD) in different neurological disorders has become a valid approach over time, and recently, it has also been advocated as a potential therapeutic for ASD. A MEDLINE, Scopus and Cochrane search was performed by two independent reviewers to investigate the relationship between ASD and the KD in humans and experimental studies. Of the eighty-one potentially relevant articles, eight articles met the inclusion criteria: three studies with animals and five studies with humans. The consistency between reviewers was ? = 0.817. In humans, the studies mainly focused on the behavioural outcomes provided by this diet and reported ameliorated behavioural symptoms via an improved score in the Childhood Autism Rating Scale (CARS). The KD in prenatal valproic acid (VPA)-exposed rodents, as well as in BTBR and Mecp2 mice strains, resulted in an attenuation of some autistic-like features. The limited number of reports of improvements after treatment with the KD is insufficient to attest to the practicability of the KD as a treatment for ASD, but it is still a good indicator that this diet is a promising therapeutic option for this disorder.



Conclusion

Since very many parents do not want to use drugs to treat autism, it is surprising more people do not try the ketogenic diet (KD) or at least the KD-lite, which is the Modified Atkins Diet (MAD).
I think you have to be pretty rigid about the MAD, if you go MAD-lite you will likely achieve little; rather like thinking you have a Mediterranean diet because you buy the occasional bottle of olive oil.
Many children with epilepsy who started out on the KD continue in adulthood with the Modified Atkins Diet (MAD).
There is anecdotal evidence that people with mitochondrial disease benefit from the KD.
All in all, it is hard to argue that the KD/MAD should not be the first choice for those choosing to treat autism by diet. It really does have science and clinical study to support it.

In some people with autism it appears that when you eat is as important as what you eat.  There can be strange behaviors just after eating, presumably caused by a spike in blood sugar, or for others before breakfast. 

In regressive autism (AMD) Dr Kelley, from Johns Hopkins, wrote that:- 


Another important clinical observation is that many children with mitochondrial diseases are more symptomatic (irritability, weakness, abnormal lethargy) in the morning until they have had breakfast, although this phenomenon is not as common in AMD as it is in other mitochondrial diseases.  In some children, early morning symptoms can be a consequence of compromised mitochondrial function, whereas, in others, a normal rise in epinephrine consequent to a falling blood glucose level in the early morning hours can elicit agitation, ataxia, tremors, or difficulty waking.  In children who normally sleep more than 10 hours at night, significant mitochondrial destabilization can occur by the morning and be evident in biochemical tests, although this is less common in AMD than in other mitochondrial disorders.  When early morning signs of disease are observed or suspected, giving uncooked cornstarch (1 g/kg; 1 tbsp = 10g) at bedtime effectively shortens the overnight fasting period.  Uncooked cornstarch, usually given in cold water, juice (other than orange juice), yogurt, or pudding, provides a slowly digested source of carbohydrate that, in effect, shortens overnight fasting by 4 to 5 hours.



I still find it rather odd that none of Dr Kelley's work on treating regressive autism has been published in any scientific or medical journal.  After all, he was a leading staff member at one of the world's leading hospitals.  He is no quack.  It is extremely wasteful of knowledge and clinical insights that could help improve the lives of something greater than 0.2% of the world's young children.  That is a lot of people.












Tideglusib, Repairing Dental Cavities, Wnt signaling, GSK-3 and Autism


Kings College in London seem to be more effective in dentistry than autism; they have just published research showing how they effectively regrew a tooth to repair a cavity.  That is rather clever.

Perhaps soon to be a thing of the past?


Using biodegradable collagen sponges to deliver the treatment, the team applied low doses of small molecule glycogen synthase kinase (GSK-3) inhibitors to the tooth. They found that the sponge degraded over time and that new dentine replaced it, leading to a complete, natural repair.




The full paper is here:- 




All very well, but what has this got to do with Autism?

As regular readers will be aware, autism turns out to be multigenic (it involves lots of different genes) and no single gene seems to account for more than one or two percent of cases.  A small number of any of hundreds of possible genes can be disturbed and then affect so-called signaling pathways  that control our bodies.  These pathways have evolved over millions of years and can seem quite unnecessarily complex.  The pathways overlap with each other and at certain critical points it seems like different genetic dysfunctions can lead to the same dysfunctional point, or nexus.  
We previously saw one such nexus, IPR3, suggested by Gargus:-




 but another one may be the Synaptic Wnt/GSK3� signaling hub. 


We came across Wnt signaling in earlier posts.  Among other things, it relates to those RASopathies that often lead to cognitive dysfunction; but RAS dysfunction can also lead to common cancers, so called RAS-dependent cancers.

Wnt signaling is also involved in hair growth and hair greying, as one of our more adventurous readers experienced.  So using a PAK1 inhibitor to modulate the Wnt pathway may make your hair go grey.

BCL-2 is another autism gene that affects hair growth/loss.

It has been suggested by some of the very clever researchers (Chauhan and Chauhan) that the BDNF-Akt-Bcl2 anti-apoptotic signaling pathway is compromised in the brain of autistic subjects.

So while the gene Bcl-2 might be the dysfunction in one per cent of people, in more cases it is the pathway along which Bcl-2 lies, that is the problem.

There is also so called cross-talk between pathways connecting Bcl-2 to RAS.

Then you will see that some drugs affect both Bcl-2 and RAS.  So on the one hand things get much more complicated than just 20,000 different genes, but on the other hand the really good interventions will likely solve multiple dysfunctions. This is why we have talk about a nexus, or hub, where different dysfunctions lead to common points.

It makes sense to focus on identifying the limited number of these hubs, rather than getting lost in thousands of possibly dysfunctional genes. 


GSK-3 (Glycogen synthase kinase 3)

This area is very complex and really only a few people, mainly cancer researchers, and at least one dentist, understand it.

In essence, among other effects, GSK-3 inhibitors activate Wnt signaling. 

Glycogen synthase kinase 3 is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. First discovered in 1980 as a regulatory kinase for its namesake, Glycogen synthase ]GSK-3 has since been identified as a kinase for over forty different proteins in a variety of different pathways.  In mammals GSK-3 is encoded by two known genes, GSK-3 alpha (GSK3A) and GSK-3 beta (GSK3B). GSK-3 has recently been the subject of much research because it has been implicated in a number of diseases, including Type II diabetes (Diabetes mellitus type 2), Alzheimer's Disease, inflammation, cancer, and bipolar disorder. 


Glycogen synthase kinase-3 (GSK3) may be the busiest kinase in most cells, with over 100 known substrates to deal with. How does GSK3 maintain control to selectively phosphorylate each substrate, and why was it evolutionarily favorable for GSK3 to assume such a large responsibility? GSK3 must be particularly adaptable for incorporating new substrates into its repertoire, and we discuss the distinct properties of GSK3 that may contribute to its capacity to fulfill its roles in multiple signaling pathways. The mechanisms regulating GSK3 (predominantly post-translational modifications, substrate priming, cellular trafficking, protein complexes) have been reviewed previously, so here we focus on newly identified complexities in these mechanisms, how each of these regulatory mechanism contributes to the ability of GSK3 to select which substrates to phosphorylate, and how these mechanisms may have contributed to its adaptability as new substrates evolved. The current understanding of the mechanisms regulating GSK3 is reviewed, as are emerging topics in the actions of GSK3, particularly its interactions with receptors and receptor-coupled signal transduction events, and differential actions and regulation of the two GSK3 isoforms, GSK3a and GSK3�. Another remarkable characteristic of GSK3 is its involvement in many prevalent disorders, including psychiatric and neurological diseases, inflammatory diseases, cancer, and others. We address the feasibility of targeting GSK3 therapeutically, and provide an update of its involvement in the etiology and treatment of several disorders.



GSK-3 and Autism

The good news is that the Alzheimer�s researchers have already developed a GSK-3 inhibitor, the current favourite is called Tideglusib.  This is also the one the clever dentists at King�s College used.

Researchers in Santiago, Chile, have proposed the role of GSK-3 in the onset and development of ASDs through direct modulation of Wnt/�-catenin signaling.





 Figure 1: Wnt/�-catenin signaling in ASDs. Wnt binding to FZD-LRP5/6 complex receptor at the membrane recruits the destruction complex and inhibits GSK3� activity thus stabilizing �-catenin in the cytoplasm and nucleus. Activation of the Wnt/�-catenin pathway facilitates synaptic plasticity through the activation of voltage gated ion channels that allows activation of CAMK and CREB mediated transcription. Mutations in TSC associated with ASD prevent �-catenin degradation which results in a gain of function of the Wnt pathway. In the presynaptic terminal cadherin mediated cell adhesion between synapses is weakened by phosphorylation of �-catenin and synaptic vesicle clustering is enhanced through DVL1. Clustering is also dependent on NLGN/NRXN cell adhesion complexes. Both lithium (LiCl) and VPA activate Wnt/�-catenin signaling through inhibition of GSK3� activity. Conversely, in the absence of a Wnt ligand, activated GSK3� targets �-catenin for proteosome-mediated degradation. Mutations associated with DISC1 fail to inhibit GSK3� and thus activate Wnt/�-catenin pathway. In the presynaptic side Wnt signaling buffering of synaptic vesicles is inhibited and adherens junctions mediated by cadherins are strengthened.

This becomes more interesting because a clinical trial has already been put in motion to trial Tideglusib in autism.  I am not sure if the Canadian researchers are just trying an Alzheimer�s drug on the off-chance it might help autism, or whether they are really up to speed with their Wnt signaling pathway.  I suspect the former, but it does not really matter.



This might be of interest to our reader Alli in Switzerland.


Conclusion 

It pays to read the science reports that appear to have nothing to do with autism.










Enhancing the effect of Bumetanide in Autism


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

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

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

Monty, aged 13 with ASD

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

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


The Colosseum

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

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

Back came the answer, unprompted, �Italy�.

This was a big surprise.

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



Add-ons

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

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

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

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

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

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

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

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

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

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

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

Caffeine is a very simple add-on to try.





Ketones and Autism Part 4 � Inflammation, Activated Microglia, CtBP, the NLRP3 Inflammasome and IL-1�

This series of posts on ketones and the ketogenic diet (KD) is nearly finished and I am glad that I made favourable comments about the KD ea...