Thursday, 20 October 2016

Clinical Trial of Mega-dose Folinic Acid in Autism

The common form of Leucovorin Calcium is for injection, but it exists in 
tablet form. Maybe another opportunity for intra-nasal delivery?

As pointed out by Tyler, Richard Frye has published his trial on the effect of mega-dose folinic acid in children with autism and language impairment.

FRAA (folate receptor-αautoantibody) status was predictive of response to treatment.  This means that people who are FRAA positive are likely to really benefit from folinic acid treatment.

There are different types of folinic acid.  Dr Frye uses Calcium Leucovorin (Calcium Folinate), which is used in chemotherapy.  It is given by intramuscular injection or orally.

Dr Frye uses the oral form.

Folinic acid should be distinguished from folic acid (vitamin B9). However, folinic acid is a vitamer for folic acid, and has the full vitamin activity of this vitamin.

The dose is huge by normal standards of vitamin B9.  It was 2mg/kg per day (maximum 50mg per day) in two equally divided doses with half of the target dose given during the first 2 weeks. 

Two folate-related biomarkers were investigated. FRAA titers, both blocking and binding, were analyzed. Plasma free reduced-to-oxidized glutathione redox ratio was determined. Folate-related vitamins and minerals were measured. Serum total folate and vitamin B12 were measured 

Of 93 children with ASD, 60% and 44% were positive for blocking and binding FRAAs, respectively.

We sought to determine whether high-dose folinic acid improves verbal communication in children with non-syndromic autism spectrum disorder (ASD) and language impairment in a double-blind placebo control setting. Forty-eight children (mean age 7 years 4 months; 82% male) with ASD and language impairment were randomized to receive 12 weeks of high-dose folinic acid (2mgkg−1 per day, maximum 50mg per day; n=23) or placebo (n=25). Children were subtyped by glutathione and folate receptor-αautoantibody (FRAA) status. Improvement in verbal communication, as measured by a ability-appropriate standardized instrument, was significantly greater in participants receiving folinic acid as compared with those receiving placebo, resulting in an effect of 5.7 (1.0,10.4) standardized points with a medium-to-large effect size (Cohen’s d=0.70). FRAA status was predictive of response to treatment. For FRAA-positive participants, improvement in verbal communication was significantly greater in those receiving folinic acid as compared with those receiving placebo, resulting in an effect of 7.3 (1.4,13.2) standardized points with a large effect size (Cohen’s d=0.91), indicating that folinic acid treatment may be more efficacious in children with ASD who are FRAA positive. Improvements in subscales of the Vineland Adaptive Behavior Scale, the Aberrant Behavior Checklist, the Autism Symptom Questionnaire and the Behavioral Assessment System for Children were significantly greater in the folinic acid group as compared with the placebo group. There was no significant difference in adverse effects between treatment groups. Thus, in this small trial of children with non-syndromic ASD and language impairment, treatment with high-dose folinic acid for 12 weeks resulted in improvement in verbal communication as compared with placebo, particularly in those participants who were positive for FRAAs.

Separate analyses were conducted for each biomarker of folate metabolism (Table 2A). In general, improvement in verbal communication was significantly greater in participants on folinic acid as compared with those on placebo for participants with abnormal folate metabolism (i.e., FRAA positive, low glutathione redox ratio). For participants with biomarkers indicating more normal folate metabolism (i.e., FRAA negative, high glutathione redox ratio) improvement in verbal communication was not significantly different between groups.

This study suggests that FRAAs predict response to high-dose folinic acid treatment. This is consistent with the notion that children with ASD and FRAAs may represent a distinct subgroup.61 Other factors such as genetic polymorphisms in folate-related genes or mitochondrial dysfunction may be important in determining treatment response but were not examined in this study. When methylcobalamin was combined with folinic acid, improvement in communication as well as glutathione redox status was found.48 Indeed, future studies will be needed to define factors that predict response to treatment, investigate optimal dosing and help understand whether other compounds could work synergistically with folinic acid.


This study, and previous ones, suggest that > 50% of people tested have what Frye is calling positive Folate Receptor Antibody Status.  This combined with oxidative stress, as measured by low glutathione redox ratio, looks a like a good predictor of who will benefit from Calcium Folinate.

Clearly using tablets, as opposed to the usual injections, means that less of the folinic acid actually reaches the brain.  As was discussed in an earlier post, there are other forms of folate, like Metafolin, that are OTC.

Can Metafolin perform the same function as  Calcium Leucovorin?

It would be useful to know how much Metafolin = 2mg/kg of Calcium Leucovorin.  

The only way to find out would be to ask someone taking Calcium Leucovorin.

Metafolin® is a proprietary ingredient directly usable by the human organism, involved in lowering homocysteine blood levels, and the only form of folate able to cross the blood-brain barrier. In addition, Metafolin® does not mask a vitamin B12-deficiency and presents no risk of an accumulation of unmodified folic acid in the body.”

I suppose readers will now want to measure Folate Receptor Antibody (FRA) status and look for Calcium Leucovorin.  Our regular reader Roger may want to give his insights; perhaps he wants to see if Metafolin can do the job of Calcium Leucovorin?

Any side effects, Roger, after long term use of Calcium Leucovorin?

Thursday, 13 October 2016

Multigenerational Epigenetic Change Stimulating Inflammatory Disease

Multigenerational transmission of nicotine-induced effects. The diagram illustrates the experimental design and findings of Rehan et al. [4]. Pregnant dams (F0 generation) are injected with nicotine or nicotine + rosiglitazone. The lungs and gonads of both male and female offspring (F1 generation) of nicotine-treated dams exhibit epigenetic changes, and the lungs show an asthma-like functional phenotype (blue nicotine-induced changes). These nicotine effects are not seen in the offspring of animals treated with nicotine + rosiglitazone. Offspring of F1 mated pairs (F2 generation) exhibit the same nicotine-induced changes to lung function as their parents, even though they were not exposed to drug.

Today’s post is again filling in some gaps in this blog to date.

A big question in autism is whether the incidence is increasing or not.  According to the now best-selling autism author Silberman, incidence is not increasing at all; it is just that diagnosis is much better than it was half a century ago.  So it is not an “autism epidemic”, rather a “diagnosis epidemic”.

I did not buy Siberman’s book and while I would like to believe he has accurately assessed the facts, in this case he really has not.

Psychiatrists have done none of us any favours by constantly changing the definition of autism and clinicians have never adequately collated data on those who match those criteria.

It does actually matter whether or not incidence of autism is increasing, because this would then stimulate research as to why.  In time this better understanding would lead to therapeutic avenues.

Being neither a professional researcher, nor a best-selling author, my level of evidence can be a little lower.  In earlier posts we saw incidence of ASD (autism, Asperger’s and PDD-NOS) is around one percent of both the child and adult population.  Many adults with Asperger’s and milder dysfunctions were never diagnosed as children, because they did not have speech delay or great cognitive difficulties.

The autism figures are always of low quality, but there is an opinion that underlying them is a real increase in severe autism, as well as the increased diagnosis of milder autism due to lowering of the diagnostic threshold.

The data I would like to see is the incidence of severe autism over the last few decades, but it does not exist.  All we have is anecdotes.

I remember asking my retired doctor mother how many patients had autism in her medical practice of about 10,000, where she saw all the children.  They did not have any and apparently until the Wakefield autism-MMR business nobody even talked about autism.

Hidden away in a group of 10,000 there “should be” about 100 with some degree of autism.  About 30 might have quite severe autism, many with MR/ID and epilepsy. 30 sounds a lot, but it is only one or two births a year.  People with severe autism live half as long as typical people, so you would not see many past middle age. I suppose it was easy to just diagnose mental retardation and then put the child into “care” when the parents could not cope.  

When a friend of mine from graduate school asked our alumni group of 200 how many had a child with autism there were six responses.  None were Asperger’s, all were strictly defined autism (SDA).

Some disease surprisingly does correlate with educational level.  I recently read that IBS/IBD is much more common among more educated people.

So my take is that hidden in all those poor quality statistics is a rise in the incidence of strictly defined autism (SDA).  Just as it is known that there has been a rise in inflammatory disease like asthma.

Asthma and COPD are really well researched and we know at least some of the reason why they have become more common.  I think the same general mechanism is behind the increase in SDA.

By understanding this mechanism you can then try and reverse it.  This is already being done in COPD research and some of the single gene autisms like Pitt Hopkins.

The mechanism is epigenetics, where you can modify when genes turn on, or turn off.  COPD is a severe disease because an environmental factor (normally smoking) has caused the body's oxidative stress response genes to be turned off.  Pitt Hopkins is caused by an insufficient expression of the TCF4 gene.  This was unlikely to have been caused by epigenetic changes, but could potentially be treated by using epigenetics to turn on the TCF4 gene.

Today’s post highlights pretty convincing research that shows how an environmental factor, smoking in this case, can cause heritable epigenetic changes.  It shows how a Grandparent smoking increases asthma incidence in the grandchildren.

Other than sending the message that smoking can affect the health of your future grandchildren, it becomes clear that many other environmental insults could also be heritable.  The accumulation of these insults over generations affects the incidence of certain diseases, particularly those complex ones often caused by multiple hits (cancer, autism etc.).
This makes me recall how it is theorized that epilepsy can develop as an acquired channelopathy.  We saw how the threshold for a person’s first seizure is quite high, but after the first seizure the threshold falls.  The proposed mechanism is called an acquired channelopathy.  This means that one of the many ion channels whose dysfunction is known to lead to epilepsy has been permanently disturbed.  The ion channel can now behave aberrantly with little provocation,

Ion channel diseases are classified as ‘acquired’ or ‘genetic’. Genetic ion channel disorders of the brain generally manifest as epilepsy, migraine, paroxysmal dyskinesia or episodic ataxia.

Acquired channelopathies can be caused by antibodies which target specific ion channels or by toxins which block voltage-gated ion channels. Altered transcription of ion channels may contribute to many acquired neurological ion channel disorders.

Mutations in genes which encode subunits of CNS sodium, potassium, calcium channels, GABAA and nicotinic receptors have been reported in association with various epilepsy syndromes.

While genetic (inherited) ion channel disorders may be the cause of most people’s epilepsy, it is suggested that acquired channelopathies are also involved.  Perhaps both are present?

 the “acquired channelopathy” hypothesis suggests that proepileptic channel characteristics develop during epilepsy.

In summary, cell type-specific information on epilepsy-related ion channel modifications can explain and support AED strategies. Precisely those inhibitory ion channels which appear to be effective AED targets in preclinical tests are the ones upregulated in DG GCs during TLE. These data indicate that cell-endogenous ion channel homeostasis mechanisms could be used as “channelacoid” archetypes in the search of antiepileptic strategies. In particular, the enhancement of static shunt via combined K/Cl/cation leak channel support appears to be a promising strategy.

The science, though complex, is still in its infancy.  You do wonder if acquired channelopathy cannot be caused by epigenetic changes to the genes encoding the ion channel.

Nicotine, your genes and those of your heirs

Finally, the subject of today’s post, the research showing the epigenetic effects of nicotine. In place of nicotine you could likely substitute other environment damage such as intense air pollution in cities like Beijing.  Another example below is lead pollution. 

 First the easier to read article:-

"Our results therefore indicate that the increased disease risk associated with smoking is partly caused by epigenetic changes. A better understanding of the molecular mechanism behind diseases and reduced body function might lead to improved drugs and therapies in the future," 

Now the more interesting study that shows how the effect of nicotine is passed down the generations to non-smokers.

Multigenerational transmission of nicotine-induced effects. The diagram illustrates the experimental design and findings of Rehan et al. [4]. Pregnant dams (F0 generation) are injected with nicotine or nicotine + rosiglitazone. The lungs and gonads of both male and female offspring (F1 generation) of nicotine-treated dams exhibit epigenetic changes, and the lungs show an asthma-like functional phenotype (blue nicotine-induced changes). These nicotine effects are not seen in the offspring of animals treated with nicotine + rosiglitazone. Offspring of F1 mated pairs (F2 generation) exhibit the same nicotine-induced changes to lung function as their parents, even though they were not exposed to drug.

A recent preclinical study has shown that not only maternal smoking but also grandmaternal smoking is associated with elevated pediatric asthma risk. Using a well-established rat model of in utero nicotine exposure, Rehan et al. have now demonstrated multigenerational effects of nicotine that could explain this 'grandmother effect'. F1 offspring of nicotine-treated pregnant rats exhibited asthma-like changes to lung function and associated epigenetic changes to DNA and histones in both lungs and gonads. These alterations were blocked by co-administration of the peroxisome proliferator-activated receptor-γ agonist, rosiglitazone, implicating downregulation of this receptor in the nicotine effects. F2 offspring of F1 mated animals exhibited similar changes in lung function to that of their parents, even though they had never been exposed to nicotine. Thus epigenetic mechanisms appear to underlie the multigenerational transmission of a nicotine-induced asthma-like phenotype. These findings emphasize the need for more effective smoking cessation strategies during pregnancy, and cast further doubt on the safety of using nicotine replacement therapy to reduce tobacco use in pregnant women.

More on epigenetic changes related to heart disease.

Finally the effect down the generations of lead, a known neurotoxin.

We report that the DNA methylation profile of a child’s neonatal whole blood can be significantly influenced by his or her mother’s neonatal blood lead levels (BLL). We recruited 35 mother-infant pairs in Detroit and measured the whole blood lead (Pb) levels and DNA methylation levels at over 450,000 loci from current blood and neonatal blood from both the mother and the child. We found that mothers with high neonatal BLL correlate with altered DNA methylation at 564 loci in their children’s neonatal blood. Our results suggest that Pb exposure during pregnancy affects the DNA methylation status of the fetal germ cells, which leads to altered DNA methylation in grandchildren’s neonatal dried blood spots. This is the first demonstration that an environmental exposure in pregnant mothers can have an epigenetic effect on the DNA methylation pattern in the grandchildren.


As regards autism, heritable epigenetic changes could well explain the increase in strictly defined autism (SDA) that cannot be explained away in terms of widening diagnostic criteria and awareness.

With respect to many diseases it is hardly surprising that they are becoming more prevalent if we accumulate the environmental insults experienced by our ancestors, via heritable epigenetic changes.  Where this will lead in future generations?

There are further studies looking at the role of PPAR gamma agonists (the rosiglitazone given to protect the mouse from epigenetic change) and HDAC inhibitors, which together can do very clever things regarding epigenetics.

You may recall the broccoli sprout extract being given by John Hopkins researchers to protect Beijing residents from the effects of severe air pollution.  The sulforaphane produced is an HDAC inhibitor.  

The mouse studies showed how to protect a mouse from epigenetic change occurring, what would be more interesting would be studies looking at reversing that change, once it has already occurred.

The only bad thing in the Mediterranean diet/lifestyle is smoking; just imagine how healthy the Greeks would be without smoking 2,000 cigarettes per adult per year, compared to 1,000 in the US.

Thursday, 29 September 2016

Probiotics – Science and Pseudoscience

Once anyone starts to make claims that some autism is treatable, people respond in different ways.  Those applying what has always been taught in medical school, that autism is untreatable,  will either think you are making it all up, or worse, you are some evil person taking advantage of parents in emotional distress.

The very few people who read the research about things like metabolic errors and intracellular signaling may well take a different view. Also the oncology/cancer researchers who themselves think about sub-types of disease that are induced by specific signaling pathways (like RAS-induced cancers for example), may well see the sense in experimentation like that in this blog.

Medicine does indeed say that autism, Down Syndrome and ID/MR are untreatable; however current science does not support this.  Your local doctor applies medicine; he is likely totally out of his depth when it comes to where science is in 2016.

My posts are just my take on the science, I am well aware that some clever neurologists have looked at this blog and think it is all fantasy.  The doctors who have a child with autism and read this blog tend to look from a different perspective and with a much more open mind.  Once you find one therapy that is truly effective, bumetanide in our case, then there can be no turning back.

There are all kinds of diets, supplements and therapies promoted by various people, I wish them all well.

The problem any future science-based autism clinicians will have is that they inevitably get mixed up with other types.  In the US they already go to the same autism conferences, which surprises me. People then think, "Oh well if Professor X is here from Ivy League college Y, then everyone must be legit".  Big mistake. You need to be on really top form to separate out all the pseudoscience, and on occasion you may get it wrong. 


I used to be a skeptic of probiotic bacteria, that is until I was prescribed some little glass vials about a dozen years ago.  I had some side effect from an antibiotic prescribed for an ear infection.  I still recall the ENT doctor calling out (not in English) and asking what to prescribe for the GI side effects.  When I took his prescription to the pharmacy I received a pack of glass vials and a small saw blade.  You used the saw to cut the neck of the vial then you added water to the white fungus growing in the vial and poured into a glass of water, which you then drank.

It most definitely worked.

Even today when I tell my doctor relatives in the UK that probiotics work wonders for diarrhea, all I get is strange looks.

So I am already sold on the fact that probiotic bacteria can do great things for stomach problems.

I spoke to a friend in Denmark this week who has been ill much of the year and finally his problems have been diagnosed as stemming from Ulcerative Colitis.  His first symptom was actually a blood clot.  It turns out that inflammatory bowel diseases (IBD), like ulcerative colitis, increase your risk of blood clots.

So I told my friend to read up on VSL#3 and Viviomixx, which do seem to help IBD, and also to read up on melatonin in the IBD research.

Probiotics and Inflammatory Disease

Looking at immune health more generally we saw how the probiotic Miyairi 588 is used to produce butyric acid which can improve immune health.  This is why cost conscious farmers put it in their animal feed to produce healthier, faster growing animals.

We saw that an alternative is just to add sodium butyrate to the food.  This is done is both livestock and some humans.

Butyrate is an HDAC inhibitor and so is thought to have epigenetic effects.

Probiotics and the Brain

You might be able to convince your doctor that a probiotic bacterium can be good for your stomach, but would you convince him that it could be good for the brain?

I must admit I also would like to see some scientific evidence, beyond anecdotes - even my own anecdotes.

So finally today’s featured scientific study:-

 There is increasing, but largely indirect, evidence pointing to an effect of commensal gut microbiota on the central nervous system (CNS). However, it is unknown whether lactic acid bacteria such as Lactobacillus rhamnosus could have a direct effect on neurotransmitter receptors in the CNS in normal, healthy animals. GABA is the main CNS inhibitory neurotransmitter and is significantly involved in regulating many physiological and psychological processes. Alterations in central GABA receptor expression are implicated in the pathogenesis of anxiety and depression, which are highly comorbid with functional bowel disorders. In this work, we show that chronic treatment with L. rhamnosus (JB-1) induced region-dependent alterations in GABAB1b mRNA in the brain with increases in cortical regions (cingulate and prelimbic) and concomitant reductions in expression in the hippocampus, amygdala, and locus coeruleus, in comparison with control-fed mice. In addition, L. rhamnosus (JB-1) reduced GABAAα2 mRNA expression in the prefrontal cortex and amygdala, but increased GABAAα2 in the hippocampus. Importantly, L. rhamnosus (JB-1) reduced stress-induced corticosterone and anxiety- and depression-related behavior. Moreover, the neurochemical and behavioral effects were not found in vagotomized mice, identifying the vagus as a major modulatory constitutive communication pathway between the bacteria exposed to the gut and the brain. Together, these findings highlight the important role of bacteria in the bidirectional communication of the gut–brain axis and suggest that certain organisms may prove to be useful therapeutic adjuncts in stress-related disorders such as anxiety and depression.

The study is interesting because it shows that a bacterium can modify GABA subunit expression in the brain, but when the vagus nerve is removed the effect is lost.  So it is pretty likely that in humans the vagus nerve is the conduit to the brain, as has many times been suggested, but here we have some pretty conclusive supporting evidence.

For a less science heavy explanation of the study:-

Belly bacteria boss the brain

Gutmicrobes can change neurochemistry and influence behavior

I did a post about the vagus nerve a while back and there is an easy to read article here:-

Viva vagus: Wandering nerve could lead to range of therapies

My old posts:-

The Vagus Nerve and Autism

Cytokine Theory of Disease & the Vagus Nerve


Individual GI bacteria have very specific effects.  In people with neurological dysfunctions the possibility genuinely exists to delivery therapies to brain via the gut.  This might have been seen as pseudoscience a decade ago, but now it is part of science, but not yet medicine.

Many other clever things going on in your gut.  The long awaited CM-AT pancreatic enzyme therapy, from a company called Curemark, is now entering its phase 3 trial (thanks Natasa). Click below. 

Blüm is the study of CM-AT, a biologic, for the treatment of Autism.

The Curemark lady, Joan Fallon, has collected numerous patents regarding various mixtures of pancreatic enzymes and even secretin.  Secretin was an autism therapy that was written off many years ago, but is still used by some DAN type doctors.

Some comments on this blog from parents of kids in the early CM-AT trials are supportive of its effect.

Pancreatic enzymes (e.g. Creon) are already used as a therapy for people who lack pancreatic enzymes and many people with autism have taken them.

Curemark have never published any of their trial data which annoys at least one of our medical researcher readers.  If you have so many patents, why not share your knowledge?

Sunday, 25 September 2016

Excitotoxicity triggered by GABAa dysfunction

This blog, as you will have noticed, does rather meander through science of autism.  As a result there are some gaps and unanswered questions.

The blog talks a lot about the neurotransmitter GABA and the excitatory/inhibitory imbalance.  We have ended up with some therapies based on this that do seem to help many people.

The opposing (excitatory) neurotransmitter is glutamate which affects the NMDA, AMP and mGlu receptors.

It appears that in autism there is an unusually high level of glutamate, but another issue looks likely to be at specific receptors, for example mGluR5

This does get very complicated and lacks any immediate therapies. 

One very interesting insight was that you can repurpose the existing cheap generic GABAB drug Baclofen to treat NMDAR-hypofunction. 

This seems to work really well at low doses with many people with Asperger’s.  People with more severe autism do not seem to respond to low doses, however some do to higher doses.  The more potent version R Baclofen is a research drug.

GABAb-mediated rescue of altered excitatory–inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR-hypofunction

Reduced N-methyl-D-aspartate-receptor (NMDAR) signaling has been associated with schizophrenia, autism and intellectual disability. NMDAR-hypofunction is thought to contribute to social, cognitive and gamma (30–80 Hz) oscillatory abnormalities, phenotypes common to these disorders.

Constitutive NMDAR-hypofunction caused a loss of E/I balance, with an increase in intrinsic pyramidal cell excitability and a selective disruption of parvalbumin-expressing interneurons. Disrupted E/I coupling was associated with deficits in auditory-evoked gamma signal-to-noise ratio (SNR). Gamma-band abnormalities predicted deficits in spatial working memory and social preference, linking cellular changes in E/I signaling to target behaviors. The GABAB-receptor agonist baclofen improved E/I balance, gamma-SNR and broadly reversed behavioral deficits.


We have touched on this subject on a few occasions but today, excitotoxicity is the focus of this post.
Excitotoxicity looks likely to be present in much autism and helps to connect all the various dysfunctions that we can read about in the literature.

It is a little scary because you cannot know to what extent this process is reversible.  It looks like in milder cases it should be treatable, whereas in extreme cases damage will be irreversible.

Excitotoxicity is the pathological process by which nerve cells are damaged or killed by excessive stimulation by neurotransmitters, particularly glutamate. This occurs when receptors for the excitatory neurotransmitter glutamate (glutamate receptors) such as the NMDA receptor and AMPA receptor are overactivated by glutamatergic storm. 

Unfortunately you can trigger glutamate excitotoxity via a dysfunction in GABAA receptors.

For example if you severely inhibit GABAA receptors you kill brain cells, but it was the reaction in glutamate signaling that did the damage.  GABA is supposed to be inhibitory; in some autism it is not and then Glutamate gets out of balance.  This does lead to excess firing of neurons, which seems to degrade cognition, but it will tend towards glutamate excitotoxity.

When you see the cascade of events triggered by glutamate excitotoxity you will see how this really helps to explain biological finding in autism, even mitochondrial dysfunctions.

You can then trace this all back to the faulty GABA switch caused by too little KCC2 and too much NKCC1.

Then you can look at other neurological conditions that feature glutamate excitotoxity, like traumatic brain injury and neuropathic pain, and you see that the research shows low expression of KCC2.

This then suggests that much of autism would have been prevented if you could increase KCC2.  You would not just fix the E/I imbalance but you would avoid all the damage done by excitotoxity.

Just how early you would have to correct KCC2 expression is not clear.  For sure it is a case of better late than never, but how much damage caused by excitotoxicity is reversible?

Good News

The good news is that because KCC2 underexpression is a feature of many conditions there is plenty of research money being spent looking for answers.  When they find a solution for increasing KCC2 to treat neuropathic pain, or spinal cord injury (SCI), the drug can be simply re-purposed for autism.

The French government is funding research into increasing KCC2 to treat SCI.  They are starting with serotin  5-HT2A receptor agonists.  Regular readers without any memory loss may recall that back in the 1960 Lovaas was giving LSD to people with autism at UCLA.  LSD is a potent 5-HT2A receptor agonist.  The French are also looking at BDNF to upregulate KCC2 and then they plan to have a blind test where they try all the chemicals they have in their library.  The French are of course doing their trials in test tubes.

When I looked at this subject a while back, I looked for existing therapies that are known to be safe and should be effective.

Treating KCC2 Down-Regulation in Autism, Rett/Down Syndromes, Epilepsy and Neuronal Trauma ?

My conclusion then was that intranasal insulin was the best choice.

Excitoxicity in Autism

Autism is a debilitating neurodevelopment disorder characterized by stereotyped interests and behaviours, and abnormalities in verbal and non-verbal communication. It is a multifactorial disorder resulting from interactions between genetic, environmental and immunological factors. Excitotoxicity and oxidative stress are potential mechanisms, which are likely to serve as a converging point to these risk factors. Substantial evidence suggests that excitotoxicity, oxidative stress and impaired mitochondrial function are the leading cause of neuronal dysfunction in autistic patients. Glutamate is the primary excitatory neurotransmitter produced in the CNS, and overactivity of glutamate and its receptors leads to excitotoxicity. The over excitatory action of glutamate, and the glutamatergic receptors NMDA and AMPA, leads to activation of enzymes that damage cellular structure, membrane permeability and electrochemical gradients. The role of excitotoxicity and the mechanism behind its action in autistic subjects is delineated in this review

The influx of intracellular calcium triggers the induction of inducible nitric oxide (iNOS) and phosphorylation of protein kinase C. Increased iNOS enhances nitric oxide (NO•) production in excess, whereas protein kinase C activates phospholipase A2 which in turn results in the generation of pro-inflammatory molecules The subsequent generation of free radicals can inhibit oxidative phosphorylation and damage mitochondrial enzymes involved in the electron transport chain, which mitigate energy production .

Reactive intermediates such as peroxynitrates and other peroxidation products hamper the normal function of mitochondrial enzymes by impairing oxidative phosphorylation and inhibiting complex II of the electron transport chain. Moreover, lipid peroxidation products, such as 4-hydroxynonenal (4-HNE) can interact with synaptic protein and impair transport of glucose and glutamate, thereby decreasing energy production and increasing excitotoxic sensitivity

Overstimulation of the glutamate receptors, NMDA and AMPA, leads to the release of other excitotoxins resulting in the accumulation of glutamate. Indeed, excess glutamate concentrations results in an increase in calcium levels in the cytosol. This effect is attributed to the fact that excessive glutamate allows calcium channel to open for longer periods of time, leading to increased influx of calcium into cells. Calcium triggers inducible nitric oxide and protein kinase C that produce free radicals, ROS and arachidonic aid. Generation of these oxidants results in mitochondrial dysfunction and accumulation of pro-inflammatory molecules and finally cell death. Free radicals interact with the mitochondrial and cellular membrane to form lipid peroxidation. 4-HNE is a major destructive product of this process. Lipid peroxidation prevents the dephosphorylation of excessively phosphorylated tau protein, significantly interfering with microtubule function. It has also been shown to inhibit glutathione reductase needed to convert oxidised glutathione to its functional reduced form

The mechanism responsible for excitotoxicity and neuronal cell death is diverse. Experimental studies have shown that the apoptotic and/or necrotic cell death may be due to the severity of NMDA damage or can be dependent on receptor subunit composition of neurons (Bonfoco et al. 1995; Portera-Cailliau et al. 1997). Pathological events related to this mode of action can be loss of cellular homoeostasis with acute mitochondrial dysfunction leading to hindrance in ATP production. Moreover, glutamatergic insults can cause cell death by the action of one or more molecular pathways which involves the action of signaling molecules such as cysteine proteases, mitochondrial endonucleases, peroxynitrite, PARP-1 and GAPDH in the excitotoxic neurodegeneration pathway.

Intracellular calcium levels also rely on voltage-dependent calcium channels and Na exchangers . The Na?/Ca2? exchanger is a bi-directional membrane ion transporter, which during membrane depolarisation or the opening of the gated sodium channels, transports sodium out of the cell and calcium into the cell. AMPA-type glutamate receptors are highly permeable to calcium and its over expression can lead to excitotoxicity. The Ca2? permeability capability of AMPA-type glutamate receptors relies on the presence or the absence of the GluR2 subunit in the receptor complex. Reduced GluR2 expression permits the construction of AMPA receptors with high Ca2? permeability and contributes to neuronal defect and excitotoxicity. Another mechanism is the release of calcium from internal stores such as the endoplasmic reticulum and mitochondria. It results in mitochondrial dysfunction, reduction in ATP synthesis and ROS generation.

Voltage gated channels found in dendrites and cell bodies of neurons modulate neuronal excitability and calcium-regulated signaling cascades (Dolmetsch et al. 2001; Catterall et al. 2005). Point mutations in the gene encoding the L-type voltage-gated channels Ca v1.2 (CACNA1C) and Ca v1.4. (CACNA1F) prevent voltage-dependent inactivation of these genes. This causes the channel to open for longer time, leading to excessive influx of calcium.


Autism is a multifactorial disorder characterized by neurobehavioral and neurological dysfunction. Excitotoxicity is the major neurobiological mechanism that modulates diverse risk factors associated with autism. It is triggered by potential mutation in ion channels and signalling pathways, viral and bacterial pathogens, toxic metals and free radical generation. Over expression of glutamate receptors and increased glutamate levels leads to increased calcium influx and oxidative stress and progressive cellular degeneration and cell death. Genetic defect, such as mutation in voltage gated or ligand channels that regulate neuronal excitability leads to defect in synaptic transmission and excitotoxic condition in autism. Mutation in BKCa and Ca v1.2 channels also results in excess calcium influx Sodium, potassium and chloride channels also play important roles in maintaining homoeostasis of neuronal cells, and decreased channel activity leads to destabilization of membrane potential and excitotoxicity. Moreover, over expression of BDNF results in hyperexcitability. Excessive BDNF and NMDA receptor activity increases the neurotransmitter release and excitotoxic vulnerability. Given that autism is a multifaceted disorder with multiple risk factors, more precise studies are needed to explore the signalling pathways that influence emergence of excitotoxicity in ASDs.

Some relevant reading for those interested:-

GABAergic/glutamatergic imbalance relative to excessive neuroinflammation in autism spectrum disorders



Autism spectrum disorder (ASD) is characterized by three core behavioral domains: social deficits, impaired communication, and repetitive behaviors. Glutamatergic/GABAergic imbalance has been found in various preclinical models of ASD. Additionally, autoimmunity immune dysfunction, and neuroinflammation are also considered as etiological mechanisms of this disorder. This study aimed to elucidate the relationship between glutamatergic/ GABAergic imbalance and neuroinflammation as two recently-discovered autism-related etiological mechanisms.


Twenty autistic patients aged 3 to 15 years and 19 age- and gender-matched healthy controls were included in this study. The plasma levels of glutamate, GABA and glutamate/GABA ratio as markers of excitotoxicity together with TNF-α, IL-6, IFN-γ and IFI16 as markers of neuroinflammation were determined in both groups.


Autistic patients exhibited glutamate excitotoxicity based on a much higher glutamate concentration in the autistic patients than in the control subjects. Unexpectedly higher GABA and lower glutamate/GABA levels were recorded in autistic patients compared to control subjects. TNF-α and IL-6 were significantly lower, whereas IFN-γ and IFI16 were remarkably higher in the autistic patients than in the control subjects.


Multiple regression analysis revealed associations between reduced GABA level, neuroinflammation and glutamate excitotoxicity. This study indicates that autism is a developmental synaptic disorder showing imbalance in GABAergic and glutamatergic synapses as a consequence of neuroinflammation.
Keywords: Autism, Glutamate excitotoxicity, Gamma aminobutyric acid (GABA), Glutamate/GABA, Tumor necrosis factor-α, Interleukin-6, Interferon-gamma, Interferon-gamma-inducible protein 16

Postmortem brain abnormalities of the glutamate neurotransmitter system in autism.


Subjects with autism may have specific abnormalities in the AMPA-type glutamate receptors and glutamate transporters in the cerebellum. These abnormalities may be directly involved in the pathogenesis of the disorder.

Pathophysiologyof traumatic brain injury

General pathophysiology of traumatic brain injury
The first stages of cerebral injury after TBI are characterized by direct tissue damage and impaired regulation of CBF and metabolism. This ‘ischaemia-like’ pattern leads to accumulation of lactic acid due to anaerobic glycolysis, increased membrane permeability, and consecutive oedema formation. Since the anaerobic metabolism is inadequate to maintain cellular energy states, the ATP-stores deplete and failure of energy-dependent membrane ion pumps occurs. The second stage of the pathophysiological cascade is characterized by terminal membrane depolarization along with excessive release of excitatory neurotransmitters (i.e. glutamate, aspartate), activation of N-methyl-d-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolpropionate, and voltage-dependent Ca2+- and Na+-channels. The consecutive Ca2+- and Na+-influx leads to self-digesting (catabolic) intracellular processes. Ca2+ activates lipid peroxidases, proteases, and phospholipases which in turn increase the intracellular concentration of free fatty acids and free radicals. Additionally, activation of caspases (ICE-like proteins), translocases, and endonucleases initiates progressive structural changes of biological membranes and the nucleosomal DNA (DNA fragmentation and inhibition of DNA repair). Together, these events lead to membrane degradation of vascular and cellular structures and ultimately necrotic or programmed cell death (apoptosis).

Excitotoxicity and oxidative stress
TBI is primarily and secondarily associated with a massive release of excitatory amino acid neurotransmitters, particularly glutamate.854 This excess in extracellular glutamate availability affects neurons and astrocytes and results in over-stimulation of ionotropic and metabotropic glutamate receptors with consecutive Ca2+, Na+, and K+-fluxes.2273 Although these events trigger catabolic processes including blood–brain barrier breakdown, the cellular attempt to compensate for ionic gradients increases Na+/K+-ATPase activity and in turn metabolic demand, creating a vicious circle of flow–metabolism uncoupling to the cell.1650
Oxidative stress relates to the generation of reactive oxygen species (oxygen free radicals and associated entities including superoxides, hydrogen peroxide, nitric oxide, and peroxinitrite) in response to TBI. The excessive production of reactive oxygen species due to excitotoxicity and exhaustion of the endogenous antioxidant system (e.g. superoxide dismutase, glutathione peroxidase, and catalase) induces peroxidation of cellular and vascular structures, protein oxidation, cleavage of DNA, and inhibition of the mitochondrial electron transport chain.31160 Although these mechanisms are adequate to contribute to immediate cell death, inflammatory processes and early or late apoptotic programmes are induced by oxidative stress.11

Knocking down of the KCC2 in rat hippocampal neurons increases intracellular chloride concentration and compromises neuronal survival

Non-technical summary

‘To be, or not to be’– thousands of neurons are facing this Shakespearean question in the brains of patients suffering from epilepsy or the consequences of a brain traumatism or stroke. The destiny of neurons in damaged brain depends on tiny equilibrium between pro-survival and pro-death signalling. Numerous studies have shown that the activity of the neuronal potassium chloride co-transporter KCC2 strongly decreases during a pathology. However, it remained unclear whether the change of the KCC2 function protects neurons or contributes to neuronal death. Here, using cultures of hippocampal neurons, we show that experimental silencing of endogenous KCC2 using an RNA interference approach or a dominant negative mutant reduces neuronal resistance to toxic insults. In contrast, the artificial gain of KCC2 function in the same neurons protects them from death. This finding highlights KCC2 as a molecule that plays a critical role in the destiny of neurons under toxic conditions and opens new avenues for the development of neuroprotective therapy.

New understanding of brainchemistry could prevent brain damage after injury

Sciences de la vie, de la santé et des écosystèmes : Neurosciences (Blanc SVSE 4) 2010

The potassium-chloride transporter KCC2 : a new target for the treatment of neurological diseases

A decrease in synaptic inhibition –disinhibition- appears to be an important substrate in several neuronal disorders, such as spinal cord injury (SCI), neuropathic pain... Glycine and GABA are the major inhibitory transmitters in the spinal cord. An important emerging mechanism by which the strength of inhibitory synaptic transmission can be controlled is via modification of the intracellular concentration of chloride ions ([Cl-]i) to which receptors to GABA/glycine are permeable. Briefly, a low [Cl-]i is a pre-requisite for inhibition to occur and is maintained in healthy neurons by cation-chloride co-transporters (KCC2) in the plasma membrane, which extrude Cl-. We showed recently (Nature Medicine, accepted for publication) that these transporters are down-regulated after SCI, thereby switching the action of GABA and glycine from inhibition to excitation; this can account for both SCI-induced spasticity and chronic pain. KCC2 transporters therefore appear as a new target to restore inhibition within neuronal networks in pathological conditions. The present project aims at reducing spasticity and chronic pain after SCI by up-regulating KCC2. 
An important part will consist in identifying new compounds that increase the cell surface expression and/or the functionality of KCC2. Two strategies are considered. 1) Serotonin and BDNF will be tested on the basis of preliminary experiments and/or previous reports in other areas of the central nervous system indicating that these two compounds may affect the expression of KCC2. 2)Testing a large amount of compounds available in a library (“blind test”) to sort out KCC2-modulating molecules. This task can only be done in vitro on an assay that enables to easily visualize and quantify cell surface expression of KCC2, in response to these molecules (HEK293 cells). The few compounds isolated at the end of this task will then be tested on cultures of motoneurons (both mouse motoneurons and human motoneurons derived from induced pluripotent cells) and characterized further (potential toxicity, ability to cross the Brain Blood Barrier and effect on internalization and endocytosis of KCC2). 
The selected candidate compounds will enter into the in vivo validation phase aimed at increasing the expression of KCC2 following spinal cord injury (SCI; both contusion and complete spinal cord transection). The selected hits will be applied by intrathecal injections in SCI rats and their effects on KCC2 expression in the plasma membrane of motoneurons will be tested by means of western blots and immunohistochemistry. Their efficacy in increasing the cell-surface expression of KCC2 will also be tested electrophysiologically in vitro (i.e. their ability to hyperpolarize ECl). Functionally, their efficacy in reducing both SCI-induced spasticity and chronic pain will be assessed. 
Genetic tools will be used to increase the expression of KCC2 in some spinal neurons. This task will be done in collaboration with teams in the USA. Lentiviral vectors aimed at increasing KCC2 in the host cells, after parenchymal injection, have been developed in San Diego. A transgenic mouse model with a conditional tamoxifen-induced overexpression of KCC2 has been developed in Pittsburgh. The rationale for this part of the project is to use these genetic tools in the chronic phase of SCI to reduce spasticity and chronic pain. 
The last part of the project will focus on more fundamental issues regarding the relationship between the SCI-induced downregulation of KCC2 and the development of spasticity and chronic pain. 
The significance of the expected results goes far beyond the scope of SCI, since altered chloride homeostasis resulting from mutation or dysfunction of cation-chloride cotransporters has been implicated in various neurological disorders such as, for instance, ischemic seizures neonatal seizures and temporal lobe epilepsy. 

KCC2 escape from neuropathic pain

Activationof 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2.

 In healthy adults, activation of γ-aminobutyric acid (GABA)(A) and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl(-)](i)), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), E(IPSP), in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT(2A) receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT(2A)R agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).


Very little is certain in autism, in great part because only about 200 brains have ever been examined post mortem.  There are many theories, but very many more sub-types of autism.

GABAA dysfunction due to the faulty GABA switch never increasing KCC2 expression in the first weeks of life, triggering glutamate excitotoxicity and all that follows would go a long way to explaining my son’s type of autism. It might well explain 30+% of all autism.

Clearly other causes of excess glutamate would lead to a similar result.