Pioglitazone for Autism and Specifically Summertime Raging and Verapamil-responsive Autism?

 

Adult-sized people with autism can cause property damage and much worse.

I am told that summertime raging is a common problem encountered by neurologists, but it remains poorly understood and usually remains untreated.

The most common worry for parents of toddlers diagnosed with severe autism is their lack of speech.

By the time these children reach adulthood, the biggest worry for parents is often aggression and self-injury. Often it is the mother who faces the worst episodes of aggression, which is a really cruel turn of events.

Aggression is usually not present in young children with autism, in some people it never develops, but in others it later becomes established as a learned behavior and then you are stuck with how to deal with it.

One of my own therapy targets has long been to improve cognitive function; this can indeed be achieved and then you can improve important daily living skills (adaptive function). Some steps that you can take to improve cognition, and indeed speech, have a downside in that they increase anxiety, which may lead to aggression. Calcium Folinate (Leucovorin) does cause aggression in a significant minority of people.  I think that low dose Roflumilast (60mcg) is cognitive enhancing, as proposed by the researchers at 100mcg, but it does seem to increase edginess/anxiety. DMF (Dimethyl fumarate) increases alertness, which is a good thing, but too much alertness will make you anxious.

When dealing with a full sized adult, which is more important, increased cognition/speech or avoiding explosive aggression?

Clearly there is a need for a compromise.

In adults with severe autism, living at home, entirely extinguishing aggressive behavior looks like the number one treatment goal.

For children in mainstream school, following the regular curriculum, cognitive function has to be a top priority.  Fortunately, this is our case, but only after starting Bumetanide therapy in 2012.

It looks like you can potentially have the best of both worlds - increased IQ and adaptive function, but without aggressive behavior. That is my own experience, but it was not simple.

Pioglitazone has been covered quite extensively in this blog and it is again featuring in the research. Pioglitazone is an interesting old drug used to treat people with type 2 diabetes; the phase 2 trial for autism has been completed.  I doubt there will be a phase 3 trial due to the high costs. Pioglitazone is broadly anti-inflammatory; it reduces the pro-inflammatory cytokine IL-6 and increases the anti-inflammatory cytokine IL-10.

We have seen in early posts how important is IL-6 and that it plays a key role in both allergy and even how milk teeth roots “dissolve” and then permanent teeth erupt. This transition to permanent teeth is another common cause of raging in autism, in our case it was mostly wintertime raging. 

IL-6, either directly or indirectly, seems to negatively affect behavior.

 

PPAR gamma

In earlier posts there was a lot about the various PPARs. These are used in medicine as targets to treat conditions like high cholesterol and type 2 diabetes.

Resveratrol and Pterostilbene are the OTC supplements that some readers are using. Sytrinol is another such supplement, but its cognitive benefit unfortunately just lasts a few days.

Here is a relatively recent paper on the subject, for those seeking the details. 

 

Nuclear Peroxisome Proliferator-Activated Receptors (PPARs) as Therapeutic Targets of Resveratrol for Autism Spectrum Disorder

 

Or just look up the old posts in this blog:- 

https://epiphanyasd.blogspot.com/search/label/PPAR%CE%B3

PPARs are rather complicated, but do seem to be very relevant.  For example, the master regulator of mitochondrial biogenesis, something called PGC-1 alpha, is activated by PPAR gamma. If you have mitochondrial dysfunction that included a reduced number of mitochondria, you might want to make more mitochondria. A PPAR gamma agonist might be beneficial.

Dysregulation of PGC-1 alpha is associated with neurodegenerative and metabolic disorders including Parkinson's, Alzheimer's and Huntington's.

Outside this blog, there is some interest in PGC-1 alpha and autism, particularly in connection with oxidative stress and mitochondrial dysfunction.

 

“In conclusion, we demonstrated mitochondrial oxidative stress may affect a significant subgroup of ASD children and that the SIRT1/PGC-1α signaling pathway may be a promising medical treatment for ASD.”

Source: Role of SIRT1/PGC-1α in mitochondrial oxidative stress in autistic spectrum disorder

It does look like PPARs can be targeted and provide a benefit for at least some types of autism. My choice is Pioglitazone.

 

Dumber in the Summer

In parallel with summertime raging comes the phenomenon I called “Dumber in the Summer”, where cognitive function regresses.

Monty’s assistant told me recently there is no “Dumber in the Summer” this year, and I opened my medicine cupboard and explained why this is indeed the case.

At least in our case, when you resolve summertime raging, you also protect against cognitive regression. That therapy involves Verapamil, Pioglitazone and allergy therapies, Dymista spray (azelastine + fluticasone) plus Ceterizine and Clemastine. Clemastine also has the pro-myelination effect and stabilizes microglia.

 

Pioglitazone Side effects

In the stage 2 trials for autism doses of 0.25 mg/kg, 0.5 mg/kg and 0.75 mg/kg were all found to be safe and well tolerated.

As a summertime add-on therapy it appears very well tolerated.

In adults with type 2 diabetes, who will tend to be overweight and not so healthy, there are common side effects.  At one point, it was thought that there was an association between this drug and bladder cancer. Now this is thought not to be the case.

For adults with severe untreated autism, who are aggressive and self-injure, these behaviors very much limit where they can live and what they can do during the day. Life expectancy is also severely reduced. If Pioglitazone can help control these behaviors, some side effects are likely a price worth paying. 

 

Conclusion

Pioglitazone, by the standards of autism drugs, has plenty of evidence in the literature, regarding both mouse models and humans, to support an n=1 trial.  It addresses neuro-inflammation, one key feature of autism and it has beneficial effects on mitochondria.

Pioglitazone abolishes autistic-like behaviors via the IL-6 pathway

In a small cohort of autistic children, daily treatment with pioglitazone eased some autistic behaviors, such as irritability, lethargy, stereotypy, and hyperactivity, without significant side effects

 pioglitazone treatment inhibits the secretion of proinflammatory factors, such as nitric oxide and IL-6, and enhances the levels of the secretion of anti-inflammatory factors IL-4 and IL-10. Therefore, considering the results of Qiu and Li and our present findings, pioglitazone acted to benefit autistic-like behaviors possibly via the inhibition of IL-6 secretion in astrocytes stimulated by LPS, which inhibited the neuroinflammatory response.

 

I think for people whose child with autism has a behavioural or cognitive regression in summer, there is good reason to expect a benefit.  They very likely have allergies or other autoimmune conditions.

For people who deal with aggression and self-injury in a person who responds partially, but not 100%, to Verapamil, they may find that Pioglitazone helps to complete their anti-aggression therapy.

Our doctor reader Agnieszka did her best to collect case studies of people with autism responsive to Verapamil, but not enough parents wanted to participate.

Based on the comments section in this blog, it would look like our reader George in Romania has a son whose son’s aggression is reduced by Verapamil.  If some aggression persists in summer, I think there is a very good chance that Pioglitazone will help reduce it.  George did recently share with us the the anti-inflammatory Probiotic Lactobacillus Plantarum 299v, from the previous post and widely used for irritable bowel syndrome (IBS), improved his son's speech.  

Note that the research clearly shows that most autism has an "inflammatory" element, but the exact nature varies (for details read the work of Paul Ashwood at the MIND Institute).  There are very many different anti-inflammatory therapies that are reported to benefit specific people, but there are no unifying therapies that work for all. Some will inevitably make non-responders worse and potentially dramatically so, like L.reuteri ATCC PTA 6475, found in Biogaia Gastrus. Trial and error seems unavoidable if you want to find an effective therapy.

The research proposes Pioglitazone as a year round therapy for idiopathic autism.  In the phase 2 trial almost half of the children were deemed to be responders to the treatment; not a bad result. I think it also has potential as just a summertime add-on therapy. We used it last summer and now again this summer.

People with a diagnosis of mitochondrial disease, who also present with lethargy, might be another target group because of PGC-1 alpha.

Eubiotics for GI Dysfunction and some Autism

  

Today’s post is about some drugs/supplements that have already been discussed in earlier posts.  Rifaximin, used in cycles, is an effective part of our reader Maja’s therapy, while Sodium Butyrate was highlighted long ago by our reader in Switzerland, Alli.

I had a consultation with a gastroenterologist last week and came away with a prescription for Rifaximin, microencapsulated Sodium Butyrate and Lactobacillus Plantarum 299v. Where we live, these are all inexpensive. Rifaximin is an antibiotic with extra benefits and costs about 7 euros (9 dollars). 

A course of Rifaximin can cost $2,000 in the United States.

I was pleased to read that the private equity owners of a pharmaceutical company that raised the price of a common thyroid drug by 6000% have just been fined $140 million in the UK.

Advanz Pharma and former private equity owners were fined £100m by markets watchdog

Perhaps some of our US readers should query the crazy price of drugs in the US with their congressman? Very many cheap old drugs are ultra expensive in the US, even insulin is over-priced. Not a good model of a market economy. 

 

Eubiotics – a big business

You may very well never have come across the term eubiotic before, but it is already a multi-billion dollar business.  A eubiotic is something that changes the gut microbiome to improve health. The big business to date are additives to animal feed, rather than products for human health.

Eubiotics work for humans as well. Rifaximin is an antibiotic but it also has the additional properties of a eubiotic. 

“These include: modulation of the microflora of the gastrointestinal tract by promoting the growth of Lactobacilli and Bifidobacteria strains (the so-called “eubiotic” effect) as well as modulation of bacterial metabolism, including inhibition of the hydrocarbon-derived pathways.  This drug is also capable of reducing the virulence of enteropathogenic Escherichia coli strains by inhibiting the expression of enterotoxins or adhesive factors. Interestingly, Rifaximin is distinguished by several anti-inflammatory activities mainly exerted by the pregnane X receptor (PXR), expressed primarily in the gastrointestinal tract, the small intestine, and the colon. Due to the activity described above, Rifaximin is called a eubiotic, not an antibiotic.”

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497137/

Rifaximin, like vancomycin, is usually thought of as a GI antibiotic; it stays in your gut and almost none ends up in your blood.  Both drugs are used to kill off bacteria in your gut. This is all vancomycin does, so it is not classed as a eubiotic. Rifaximin, however, goes on to perform further functions as a eubiotic, so it models your gut flora in a beneficial way.

Rifaximin is almost a wonder drug for IBS-D (irritable bowel syndrome with diarrhea).  It is also a common therapy for SIBO (small intestinal bacterial overgrowth), but while it works well for some, it actually makes things worse for some others.

Rifaximin is used both as a therapy for an acute GI problem and preventatively. It can be used in cycles, like a few days every month.

Maja is in a good position, because where she lives Rifaximin costs a few euros/dollars.

People with IBS-D in the United States often cannot afford monthly cycles of Rifaximin.

Other kinds of eubiotics include prebiotics, other probiotics, all kinds of clever fiber, inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS) etc.  I did cover psychobiotics in an earlier post, these are probiotic bacteria that are used to reduce anxiety, ADHD and other psychiatric symptoms.

Psychobiotics (PS128) for Autism, Stereotypy and Sometimes Effective Therapies for what might be SIBO (Rifaximin and Herbal)

  

Sodium Butyrate

Sodium buyrate produces butyric acid when you swallow it.  Butyric acid is what gives rancid butter its smell.  Butyric acid is one of the big eubiotics used in the animal feed industry. I did cover the very old Japanese probiotic MIYAIRI 588 (full name is Clostridium butyricum MIYAIRI 588) a long time ago in this blog.  This probiotic, in use since the Second World War, produces butyric acid in your gut by fermentation.  In Japan this probiotic is used in humans and more recently as an additive to animal feed, to produce healthier, bigger, chickens and pigs. 

Our reader MG in Hong Kong recently reported that MIYAIRI 588 was beneficial in his case. 

My gastroenterologist prescribed me Microencapsulated Sodium Butyrate, which is covered in the research and has encouraging results. When you see the word microencapsulated, you might start feeling some pain developing in your wallet, rather than in your gut, but again, this product called Integra and made in Poland,  was not so pricey - about EUR 10 ($12) for 60 capsules. One capsule contains 150 mg of sodium butyrate in tiny particles covered in triglycerides.  I have no idea if it is going to do me any good, but the research suggests it is beneficial for certain types of GI dysfunction and will strengthen the intestinal gut barrier (the equivalent of the blood brain barrier). 

Butyric acid has several different modes of action, one is as an HDAC inhibitor, which was covered in earlier posts. HDAC inhibitors can change gene transcription, which is potentially very useful, including in the prevention and treatment of some cancers. The potent HDAC inhibitors from cancer therapy show effect in some types of single gene autism.

Autism-Like Social Deficits Reversed by Epigenetic Drug 

There are different classes of HDAC inhibitor and you would need to match the type of autism with the appropriate type of HDAC inhibitor.  Valproic acid is another common HDAC inhibitor sitting on the shelf of many people with autism plus epilepsy. 

Lactobacillus Plantarum 299v 

Lactobacillus plantarum 299v has been shown to improve symptoms of IBS (Irritable Bowel Syndrome).  It prevents Clostridium difficile-associated diarrhea among patients receiving antibiotic treatment.  It is also known to be immunomodulatory, shifting the balance away from pro-inflammatory cytokines.

The role of Lactobacillus plantarum 299v in supporting treatment of selected diseases 

Alterations in composition of human gut microbiome can lead to its dysbiosis. It is associated with gastrointestinal side effects during anti-cancer treatment, antibiotics administration, or infectious agents. There are studies confirming positive effect of consuming Lactobacillus plantarum 299v on intestinal microflora. This review summarizes the current knowledge about the role of L. plantarum 299v in supporting treatment of selected diseases, such as cancer, irritable bowel syndrome (IBS), and Clostridium difficile infection. The immunomodulating properties of L. plantarum 299v include an increase in the level of anti-inflammatory cytokines, which reduce the risk of cancer and improve the efficacy of regimens. The intake of L. plantarum 299v provides benefits for IBS patients, mainly due to normalization of stool and relief of abdominal pain, which significantly improves the quality of life of IBS patients. In addition, the intake of L. plantarum 299v prevents C. difficile-associated diarrhea among patients receiving antibiotic treatment. Due to the limited possibilities of treating these diseases and numerous complications of cancer treatment, there is a need for new therapeutic strategies. The administration of L. plantarum 299v seems to be useful in these cases. 

 

Bacteria could aid autistics

Might a daily dose of friendly bacteria help treat autism? UK researchers hope probiotics will soothe the gut problems linked to autism and may even ease psychological symptoms. They are planning a clinical trial to test the idea.

The proposed health benefits of probiotic bacteria are well known. The beneficial bugs are thought to out-compete other gut bacteria that can cause diarrhoea and ill health.

Children with autism are known to have higher levels of one group of 'bad' bacteria, Clostridia, in their guts, explains Glenn Gibson from the University of Reading. So he hopes probiotic food supplements that lower levels of Clostridia will allay some symptoms of autism.

He is not suggesting that the bad bacteria cause autism: genetic and environmental factors are both likely to contribute to the complex disorder, the cause of which is unknown. But toxic by-products of the bacteria may be absorbed into the blood and travel to the brain, where they may play a role in ill health.

At present, the researchers are honing their choice of bacteria. There are many different types of good bacteria, so it is important to choose one that can compete effectively against Clostridia.

One candidate, called Lactobacillus plantarum 299v, looks especially promising. The bacterium binds to the gut lining and stimulates its growth. As well as out-competing other bacteria, it also lowers gut pH, which helps the digestive tract to fight infection. It stays in the gut for days and has never been associated with any health problems.

 

Conclusion

I am always surprised how many common drugs that you come across have potential to be repurposed to benefit  some people with autism.

It really shows how effective therapy, for at least some people with autism, is already in the medicine cabinet at home, or more likely over at the grandparents’ house.

(statins, calcium channel blockers, asthma/COPD drugs, other blood pressure drugs, diuretics, type 2 diabetes drugs)

I thought my gastroenterologist’s therapy was quite enlightened. I hope his diagnosis is accurate; I am not entirely convinced, but time will tell.  The diagnosis from doctor number one was kidney stones and now I am on doctor number three. An accurate diagnosis is not always a simple matter, as autism parents know only too well.

I did meet Dr Federico Balzola a while back. He is an Italian gastroenterologist with a keen interest in autism. He is an associate of Dr Arthur Krigsman, a US gastroenterologist heavily involved with autistic patients. In some countries the connection between GI problems and autism is still a taboo subject, seemingly because Dr Andrew Wakefield was a gastroenterologist.  

 

I am always surprised how many young Aspies have symptoms of IBS or IBD. I would actually like to know if this is mainly a problem in childhood and adolescence, which I suspect is the case. 

One of my most popular posts was another one about gastroenterology, which really surprised me.

Eosinophilic Esophagitis – another Granulocyte Disorder Associated with Autism

 

Google is discontinuing subscribe by email feature

 

Many readers have been receiving new posts automatically by email.

This was a free service provided by Feedburner, part of Google.  This service is about to be discontinued by Google.

I have added a new service provided by a company called Follow.it, which should provide the follow by email service.

In theory, the people who subscribed to the Google service should be migrated over to the new service.  

If it does not work, you can just subscribe again in the top right corner of the Web version of the blog. 

The smart phone version of the blog may not display this feature, because of the small screen size.

 

 

Leaky ATP from either Mitochondria or Neurons in Fragile X and Autism

 

 

For leaky ATP, Popeye might want to try Dexpramipexole and

Suramin, or even the already approved Mirapex

If you are old enough to be a parent, you will have encountered problems with some kind of leak.  A leaky roof, a leaky pipe, a leaky washing machine, an air-conditioning unit... The list goes on, the older you get.

I have been preoccupied by fixing a leak recently.  We have a large roof terrace and, in the winter, water started leaking from the ceiling in the floor below.  I improvised a system to catch all the water, but still I had to find the source of the leak.

I did finally find the source of the problem and most importantly without digging up 95% of the terrace.  Now I have to put the 5% back together again.

Leaks are often extremely difficult to locate, because water always finds the easiest path and the dripping you see might have originated from a leak far away.  Nobody wants to fix leaks, because it can be a pretty thankless task and you can cause plenty of damage in the process, without solving the problem.  So, as with fixing autism, I ended up doing much of the fixing myself.  The damage had actually been there since the house was built, hidden under ceramic tiles.

I recently read about leaky ATP in Fragile-X, where ATP leaks from the mitochondria into the cell.

This fits neatly into Professor Naviaux’s belief that ATP is leaking from the cell into the extracellular space, as the basis for his concept of the cell danger response, as a unifying and treatable feature of most autism.

Sounds complicated?

Just think of it as bunch of leaks you need to fix.

 

 What is ATP? 

ATP has many functions:- 

·        It is the fuel your cells need to function.

·        It is a signalling molecule within a cell and importantly between different cells.

·        It is used to make your DNA

  

Mitochondria

Each cell in your brain contains many mitochondria and these are where ATP is produced. Mitochondria die and are replaced, whereas if the host brain cell dies, it is lost forever. Cell death in the brain is bad news.

The ATP – ADP Cycle 

You can think of ATP as a fully charged battery.  Once the energy has been used up the flat battery is called ADP and it goes back for recharging in the mitochondria.  It is a continuous cycle.

ADP is powered back to ATP through the process of releasing the chemical energy available in food; this is constantly performed via aerobic respiration in the mitochondria. This process is also called OXPHOS and has been covered in previous posts.  In most mitochondrial disease the problem is that one of the four mitochondrial enzyme complexes is insufficient; this means that the ATP-ADP cycle is restricted.  There is then insufficient energy to power the brain in times of peak energy requirement.  This can cause loss of myelination and ultimately cell death.

 

  

ATP in Fragile X

It looks like in Fragile X the mitochondria in the brain do not work properly. ATP is leaking from the mitochondria and this stops synapses from maturing. 

A synapse is just the junction between one neuron and its neighbour.

The immature synapse manifests as autistic behavior.  When you plug the leak with Dexpramipexole, a drug trialed for ALS and now asthma, dendritic spines mature and autistic behavior is reduced.

To what extent this leakage occurs in idiopathic autism is unknown, but we know that impaired dendritic spine formation/morphology is a key feature of most autism and that it can be modified, although the sooner you start the better the result will be.

It looks to me that some people diagnosed with mitochondrial disease based on blood tests may actually have leaking ATP which then affects metabolic pathways and shows up with odd blood test results, that is then misdiagnosed as mitochondrial disease.  Note that many people diagnosed with mitochondrial disease show no response to therapy.

In Professor Naviaux’s theory, the ATP leak is from the cell membrane, like the outer wall of the cell.  He thinks that ATP is leaking and this then sends a false danger signal to the rest of your brain.  This is his Cell Danger Response (CDR).  Because the brain thinks it is under attack it is set in a permanent pro-inflammatory state, this gets in the way of basic functions the developing brain needs to complete.  This might explain why the microglia (the brain’s immune cells) are found to be permanently activated in autism; this then means that they do not carry out their regular brain housekeeping activities very well, like pruning synapses.

Naviaux wants to plug the leaks in the cell wall using Suramin, which is an old anti-parasite drug made by Bayer, the giant German company.

The link between the Fragile X research from Yale and Naviaux’s work at UCSD is that ATP needs to be kept in the right place for the brain to function correctly.

Leaky ATP will cause you big problems.

 

 

Now for the supporting research

 

Leaky ATP in Fragile X

 

Fragile X syndrome traits may stem from leaky mitochondria

The persistent leak influences which metabolic pathway the cell uses to generate energy, the team discovered by using a technique called mass spectrometry. For example, fragile X neurons produce more enzymes associated with glycolysis — a pathway commonly used by immature cells — than do typical neurons. Previous studies have shown altered mitochondrial metabolism in people with other forms of autism2.

Adding dexpramipexole to the cells of fragile X mice decreased production of lactate dehydrogenase and other enzymes linked to glycolysis, suggesting that closing the leak causes the neurons to start to use different, more mature metabolic pathways.

Giving injections of dexpramipexole to fragile X model mice lessened their hyperactivity, repetitive behaviors and excessive grooming — traits that are reminiscent of those seen in people with autism and in those with fragile X syndrome. Mice that received the dexpramipexole injections also had neurons with more mature dendritic spines and decreased levels of protein synthesis.

Dexpramipexole has been tested in people with the neurological disease amyotrophic lateral sclerosis and found safe, but it is unclear how it would affect young people if taken over sustained periods of time.

 

ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome

Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the Fmr1^-/y mouse have a mitochondrial inner membrane leak contributing to a "leak metabolism." In human Fragile X syndrome (FXS) fibroblasts and in Fmr1^-/y mouse neurons, closure of the ATP synthase leak channel by mild depletion of its c-subunit or pharmacological inhibition normalizes stimulus-induced and constitutive mRNA translation rate, decreases lactate and key glycolytic and tricarboxylic acid (TCA) cycle enzyme levels, and triggers synapse maturation. FMRP regulates leak closure in wild-type (WT), but not FX synapses, by stimulus-dependent ATP synthase β subunit translation; this increases the ratio of ATP synthase enzyme to its c-subunit, enhancing ATP production efficiency and synaptic growth. In contrast, in FXS, inability to close developmental c-subunit leak prevents stimulus-dependent synaptic maturation. Therefore, ATP synthase c-subunit leak closure encourages development and attenuates autistic behaviors.

 

Highlights 

·        ATP synthase c-subunit leak in Fragile X causes aberrant metabolism

·        Changes in ATP synthase component stoichiometry regulate protein synthesis rate

·        Inhibition of the leak normalizes synaptic spine morphology and Fragile X behavior

 

In Brief

Lack of FMRP in Fragile X neurons is associated with a leak in the ATP synthase, the blockade of which normalizes cellular and behavioral disease phenotypes.

 

Now they fix the leak using Dexpramipexole (Dex) and cyclosporine A (CsA)

 

 

We have found that the mitochondrial inner membrane leak of FX neurons and cells is caused by abnormal levels of ATP synthase c-subunit. The c-subunit leak causes persistence of a mitochondrial leak metabolic phenotype characterized by high glycolytic flux, high lactate levels, and increased levels of glycolytic and TCA enzymes. The leak also aberrantly elevates overall and specific protein synthesis; a decrease in c-subunit level or pharmacological inhibition of the ATP synthase leak reduces protein synthesis rates and decreases the levels of leak metabolism enzymes. In Fmr1/y synapses, stimulation-dependent protein synthesis is absent. This is correlated with a lack of stimulus induced EF2 phosphorylation and a lack of synthesis of the ATP synthase b-subunit. These abnormalities are readily reversed by ATP synthase leak inhibitors, suggesting that leak closure is required for the ATP-dependent phosphorylation of EF2 adjacent to mitochondria. EF2 phosphorylation may regulate the change in subsets of proteins synthesized and may be correlated with- the overabundant synthesis of enzymes supporting a high flux glycolytic/TCA cycle ‘‘leak’’ metabolism indicative of metabolic immaturity. Consistent with the hypothesis that the c-subunit leak is also a major cause of synapse immaturity, we find that inhibition of the ATP synthase leak allows the maturation of synapses and normalizes autistic behaviors.

 

 

 

Closing Leaky Mitochondria Halts Behavioral Problems in Fragile X, Study Suggests

“In Fragile X neurons, the synapses fail to mature during development. The synapses remain in an immature state and this seems to be related to their immature metabolism,” she said.

The investigators tested whether closing the leak to boost the efficiency of ATP production would lessen behavioral abnormalities.

They first saw that nerve cells treated with an ATP synthase inhibitor named dexpramipexole (Dex) — a form of the common Parkinson’s therapy Mirapex ER (pramipexole) and previously tested as a treatment for amyotrophic lateral sclerosis — increased the levels of ATP.

Two-day treatment with Dex also reversed autistic-like behaviors, namely excessive time spent grooming and compulsive shredding of the animals’ nests. The treatment also reduced hyperactivate behaviors.

“We find that inhibition of the ATP synthase leak allows for the maturation of synapses and normalizes autistic behaviors in a mouse model of [fragile X],” the team wrote.

Jonas and her team now intend to further test the effectiveness of this and other leak-closing therapies for improving learning.

The lab is conducting a study assessing the role of leaky membranes in memory formation. Findings could pave the way for novel therapeutics for fragile X and autism, as well as for Alzheimer’s disease.

 

 

 

Dr Naviaux and Suramin for Autism

 

I have covered Suramin in previous posts.  There is a presentation below by Prof Naviaux that is for lay people, it is good to hear directly from the man himself.

 

Autism Treatment, the cell danger response and the SAT1 trial

https://youtu.be/pqd_BoCeRUw

In essence he says that when cells are stressed, they leak ATP and this creates the cell danger response.  If you have suramin in your bloodstream, it plugs the ATP channels and stops it leaking out of the cell and so blocks the cell danger response.

It is the cell danger response that is causing the symptoms we see as autism.

  

Conclusion

Who to call to fix an ATP leak?

If it is a case of Fragile X, there looks to be potential solution, but you will definitely not find it at your local doctor’s office.

For a mouse with Fragile X, you might choose Dexpramipexole.  Dexpramipexole was developed as a therapy for ALS (motor neuron disease), but failed in phase 3 trials and is now being developed for asthma.

For a human, the logical place to start would be the already approved Mirapex, which is currently used to treat Parkinson's disease and restless legs syndrome.

Mirapex - a miracle for Fragile X?

Clearly somebody should make a clinical trial of the existing drug.

I expect what will happen is that the Yale researchers will come up will a new drug that can be patented as a novel therapy for Fragile X.  This way they get to make some money, but a decade is wasted.

Is leaky ATP from mitochondria an issue in broader autism, beyond Fragile X? That is still unknown, but the Yale researchers seem to think their work has potential application in both autism and Alzheimer’s.

In the case of broader autism, Dr Naviaux and his partner Kuzani have some competition from Paxmedica.  Both groups seek to monetize Dr Naviaux’s published research.

It looks like the German giant Bayer does not want to help either group.  Instead of just tapping into Bayer’s existing production of Suramin, Kuzani and Paxmedica will have to figure out how to produce Suramin.

This all helps us to understand why there still are no approved therapies for core Autism or indeed Fragile X and yet there is a mountain of research.  Too many barriers and interests to overcome.

If you want to fix leaky ATP any time soon, you will be doing it mainly by yourself.  This has been my experience with most other kinds of leak!

 

 

Betaine (TMG) and Gene Therapy as potential alternatives to Bumetanide Treatment in Autism?

Betaine (also known as TMG, or trimethylglycine) is a methyl derivative of glycine, first isolated from sugar beet and hence its name.

Today’s post was prompted by our reader, and Covid home-school instructor, AJ.  He raised the question of whether betaine can be used like Bumetanide to normalize chloride levels in neurons.

I am combing this idea with news from Genoa in Italy, where they have developed gene therapy as an alternative to Bumetanide and in their words :-

“This sets the stage for the development of a gene therapy approach to overcome the shortcomings of bumetanide treatment.”

The interesting thing is that neither of these ideas come from autism research.  The idea to use Betaine was stumbled upon and was then written up in a Norwegian case study about Creatine transporter deficiency.  The Italians are trying to improve cognition in brain disorders and their model of choice was Down syndrome. 

As we have seen time and again, elevated chloride within neurons is a common feature of many types of brain disorders from some idiopathic autism, to Down syndrome, to adult conditions such as Parkinson’s disease.  Today we learn that it is may well be a feature of Creatine Transporter Deficiency.

I have been rather wary of writing about any kind of gene therapy, because it seemed either too far ahead of its time, or just absurdly expensive.  There are some new $1+ million treatments.

This may be about to change given that the Biontech (AKA Pfizer vaccine), Moderna, Janssen (Johnson & Johnson) and Oxford AstraZeneca vaccines for Covid 19 are all based on gene therapy.

The Biontech people are really clever and were already trying to treat various kinds of cancer and other condition using gene therapy, before they developed their highly successful Covid vaccine.

The Italians in Genoa used an adeno-associated virus (AAV)-mediated RNA interference (RNAi) to target and reduce neuronal NKCC1 expression, rescue neuronal Cl-  homeostasis, GABAergic transmission, and cognitive deficits.   The benefit was still there 6 months after the injection.

Don’t worry if the above paragraph makes little sense. Just read on.

The same type of adeno-associated virus (AAV) vector is the platform for gene therapy delivery used in the Astra Zeneca, Janssen and the Russian Sputnik covid vaccines.

The virus is just the delivery system (vector) to get some genetic code into cells.

The Oxford-AstraZeneca COVID-19 vaccine uses a chimpanzee adenoviral vector. It delivers the gene that encodes the SARS-CoV-2 spike protein, to our cells.  Our cells then transcribe this gene into messenger RNA, or mRNA, which in turn prompts our cellular machine to make the spike protein in the main body of the cell. The mRNA molecule behaves essentially like a recipe.  Then our cells present the spike protein on the cell surface, prompting our immune system to make antibodies and mount T cell responses.

Biontech and Moderna are pioneers of mRNA vaccines, which bypass one step in the above process. They do not require our cells to make the messenger RNA, or mRNA.  They have already made it for you.

 

Gene therapy for autism?

Single gene autisms are all potential candidates for gene therapy.

The problem is that most autism and all Down syndrome is polygenic, there can be hundreds of miss-expressed genes.

But the researchers in Italy show us that even polygenic autism and Down syndrome can benefit from therapy targeting a single gene.  You just have to select the right one.

The problem is the price. Covid vaccines are made in huge quantities and are cheap.

Customized gene therapy is ultra expensive, in part because each therapy has to be approved individually.

 

An NKCC1 Gene Therapy?

The Italians have already made the NKCC1 Gene Therapy.  The question is will it ever going be available to humans with Down Syndrome, Autism or even Parkinson’s disease?

Restoring neuronal chloride homeostasis with anti-NKCC1 gene therapy rescues cognitive deficits in a mouse model of Down syndrome

A common feature of diverse brain disorders, is the alteration of GABA-mediated inhibition due to aberrant intracellular chloride homeostasis induced by changes in the expression and/or function of chloride transporters. Notably, pharmacological inhibition of the chloride importer NKCC1 is able to rescue brain-related core deficits in animal models of these pathologies and some human clinical studies. Here, we show that reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down syndrome (DS) restores intracellular chloride concentration, efficacy of GABA-mediated inhibition and neuronal network dynamics in vitro and ex vivo. Importantly, AAV-mediated neuron-specific NKCC1 knockdown in vivo rescues cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a mechanistic link between NKCC1 expression and behavioral abnormalities in DS mice, and establish a molecular target for new therapeutic approaches, including gene therapy, to treat brain disorders characterized by neuronal chloride imbalance.

 

This sets the stage for the development of a gene therapy approach to overcome the shortcomings of bumetanide treatment.

This highlights a causative role of NKCC1 upregulation in learning and memory deficits in adult Ts65Dn mice, thus also validating brain NKCC1 as a target for ameliorating cognitive disabilities in DS. Furthermore, our neuro-specific knockdown approach points to neurons as major players in the NKCC1- dependent cognitive impairment in DS mice. Nevertheless, we cannot exclude that other cell types which also express NKCC1 (e.g. glial cells) could still play a role in the overall cognitive impairment that characterizes DS.

Despite the very large and fast-increasing literature both on animal models and patients indicating positive outcomes upon bumetanide treatment, there is not yet a strong demonstrated direct link between NKCC1 inhibition, restoration of Cl- homeostasis and full GABAergic inhibitory signaling, and rescue of brain deficits.  Moreover, bumetanide has strong diuretic activity, triggering ionic imbalance, and potential ototoxicity 25,26.  This hampers its use for clinical applications in lifelong treatments4,27 and may strongly jeopardize treatment compliance along years of treatment.  Moreover, bumetanide was given systemically in most studies, and the suboptimal brain pharmacokinetic profile of the drug28 raises questions on its mechanism of action29.  Here, we demonstrate that adeno-associated virus (AAV)-mediated RNA interference (RNAi) to target (and reduce) neuronal NKCC1 expression rescues neuronal Cl- homeostasis, GABAergic transmission, and cognitive deficits in the Ts65Dn mouse model of Down syndrome. This sets the stage for the development of a gene therapy approach to overcome the shortcomings of bumetanide treatment.

 

“Thus, our results indicate the efficacy of long-term AAV9-mediated neuro-specific NKCC1 knockdown in rescuing cognitive deficits in Ts65Dn mice.”

 

“Besides establishing a causal link between NKCC1 upregulation and cognitive impairment in DS, our data also provide a proof-of-concept for a neuro-specific RNAi gene therapy approach to restore hippocampus-dependent cognitive behaviors in adult animals specifically in the brain, and without affecting peripheral organs (e.g., the kidney). This is particularly relevant in the context of the current clinical trials repurposing the strong diuretic bumetanide to treat brain disorders with impaired chloride homeostasis3.  Importantly, we achieved a comparable degree of long-term cognitive rescue with two different amiR sequences against NKCC1, underlining the specificity of our approach.”

  

Gone Fishing

If a trip to Italy for gene therapy is not realistic, this takes us back to AJ’s idea, which is to use Betaine.  The correct version is TMG or glycine betaine, and confusingly not Betaine HCl.

Fish love the taste of betaine.

Betaine was first isolated from sugar beet.

I recall from my time at the sugar factory, when I was 18, that once you have sliced up the sugar beet and extracted as much sugar as possible you are left with the pulp.  This pulp is dried, molasses is added back and then it is made into pellets.  The pellets are fed to cattle and horses.  They taste pretty bad in my opinion.

To humans it tastes bad because of the beet molasses by-product.

The molasses by-product from sugar cane tastes great to humans.  That is why they make rum in the Caribbean, and not in England or Canada.

Brown sugar from a sugar beet factory is made by adding sugar cane molasses to white sugar from beet.  It is a cheat really.

Cows love sugar beet by-products.

It turns out that fish love betaine HCl.

Betaine HCl is an excellent natural attractor that stimulates a strong, prolonged feeding response from carp and many other coarse fish.

Betaine HCl is now used to induce feeding in the fish farming industry

As our reader Tyler has highlighted, Betaine HCl, that fish like and is available is a cheap supplement is not the same as the Betaine used in the medical case study. Confusingly, the original Betaine (TMG, or called glycine betaine) gave way to a class of compounds all called betaines. One of these betaines is betaine HCL.

In most cases, in the medical literature when they refer to Betaine, they mean glycine betaine, also known as TMG.

Betaine HCl is used to increase acidity in your stomach. The effect of betaine compounds other than glycine betaine/TMG on NKCC1 is unknown.

Glycine Betaine (TMG) and NKCC1

It seems that betaine reduces your level of NKCC1 RNA. 

In your DNA are the instructions to make the NKCC1 transporter. To go from these instructions to actually making the transporters you need RNA.

In some autism there are too many NKCC1 transporters, so put simply there was too much NKCC1 RNA. So, if you can find a substance that reduces NKCC1 RNA, you might well solve the problem.

The caveat is that the substance must not also increase KCC2 RNA.  This appears to be what taurine does.

Here, finally, is AJ’s paper:

Treatment experience in two adults with creatine transporter deficiency

Background

Creatine transporter deficiency (CTD) is an X-linked form of intellectual disability (ID) caused by SCL6A8 mutations. Limited information exists on the adult course of CTD, and there are no treatment studies in adults.

Methods

We report two half-brothers with CTD, 36 and 31 years at intervention start. Their clinical phenotypes were consistent with CTD, and intervention was indicated because of progressive disease course, with increased difficulties speaking, walking and eating, resulting in fatigue, and malnutrition. We therefore performed treatment trials with arginine, glycine and a proprietary product containing creatine and betaine, and then a trial supplementing with betaine alone. Results In the older patient, glycine and arginine were accompanied by adverse effects, while betaine containing proprietary product gave improved balance, speech and feeding. When supplementation stopped, his condition deteriorated, and improved again after starting betaine supplement. Betaine supplementation was also beneficial in the younger patient, reducing his exhaustion, feeding difficulties and weight loss, making him able to resume his protected work.

Discussion & conclusion

We report for the first time that betaine supplement was well tolerated and efficient in adults with CTD, while arginine and/or glycine were accompanied by side effects. Thus, betaine is potentially a new useful treatment for CTD patients. We discuss possible underlying treatment mechanisms. Betaine has been reported to have antagonistic effect on NKCC1 channels, a mechanism shared with bumetanide, a medication with promising results in both in autism and epilepsy. Further studies of betaine's effects in well-designed studies are warranted.

 

The mechanism of betaine’s assumed favorable effect is unknown. We do not know whether betaine influences the cell creatine content in itself or its effects are more aspesific. However, we would like to present some hypotheses. First, betaine may have effect in CTD by modulating GABA-transmission. Betaine has been reported to have an antagonistic effect on NKCC1 channels, which also influences GABAergic neurotransmission. Inhibiting NKCC1 is a mechanism shared with bumetanide, a well-known diuretic medication that in recent years has been found to influence GABAergic transmission, and thereby it has been found promising in treatment of several brain conditions, including autism, and epilepsy. NKCC1 inhibition by bumetanide has also been tried with success in other rare neurodevelopmental disorders fragile X syndrome and tuberous sclerosis. Second, betaine’s properties as an osmolyte may be of importance, as betaine has similarities with creatine in being an osmolyte. Osmotic properties are thought to be one of the central mechanism behind bumetanide’s efficacy in treating brain disorders. Thus, it could be speculated that the lack of intracellular creatine in CTD may result in inefficient osmolyte regulation, and that betaine supplementation replaces the lacking creatine and thereby improves the neuronal adaption to salinity changes, edema or cellular dehydration. Betaine has osmolyte properties that even makes it act as a “chemical chaperone” increasing the stability of cell and membrane proteins. Fourth, it is possible that betaine has some effect through modifying methylation. Methylation of GAA by GAMT to form creatine is a rate-limiting step in the creatine synthesis by neurons. Betaine could stimulate this by donating methyl groups to SAMe, which donates a methyl group to GAA to form creatine. This might reduce the burden when body demands more methyl groups for creatine synthesis. Similar mechanisms may be responsible for a beneficial effect of both betaine and s-adenosyl methionine (SAMe). However, as creatine and GAA share the same transporter, one would not expect GAA to enter the GAMTexpressing cells in patients suffering from CTD. Still, it cannot be excluded that there is some rest function in the creatine transporter, and that increased endogenous synthesis improves the condition slightly. Furthermore, it is possible that CTD increases the need for methylation agents in general, as creatine supplementation has been found to reduce the need for other methylation agents [34]. Thus, it is likely that betaine may have a positive effect in CTD by improving methylation capacity for other reactions than those directly involved in creatine production. Betaine’s effect on muscle may be also of importance, as animal studies have shown that muscles growth improves with betaine [35], which potentially could have had a positive impact on our patients fatigue and weight loss. To summarize, betaine has several properties that make it likely that it will have a beneficial effect in CTD, especially the properties as an osmolyte, a down regulator of the NKCC1 channel and an influencer of GABAergic transmission. These properties are similar to the properties of bumetanide, a promising new medication for treatment of autism and epilepsy, which are common symptoms of CTD. Further research is needed, however, to elucidate the role of betaine in CTD.

If you read the detail of the old paper that is referred to in the above paper, you see that betaine is not blocking the NKCC1 channels as suggested, but it seems to be reducing the number of them.  The net effect may be the same, but the process is very different.

 

Expression and regulation of the Na+/K+/2Cl− cotransporter NKCC1 in rat liver and human HuH-7 hepatoma cells

The expression of sodium potassium chloride cotransporter 1 (NKCC1) was studied in different liver cell types. NKCC1 was found in rat liver parenchymal and sinusoidal endothelial cells and in human HuH-7 hepatoma cells. NKCC1 expression in rat hepatic stellate cells increased during culture-induced transformation in the myofibroblast-like phenotype. NKCC1 inhibition by bumetanide increased α₁-smooth muscle actin expression in 2-day-cultured hepatic stellate cells but was without effect on basal and platelet-derived-growth-factor-induced proliferation of the 14-day-old cells. In perfused rat liver the NKCC1 made a major contribution to volume-regulatory K⁺ uptake induced by hyperosmolarity. Long-term hyperosmotic treatment of HuH-7 cells by elevation of extracellular NaCl or raffinose concentration but not hyperosmotic urea or mannitol profoundly induced NKCC1 mRNA and protein expression. This was antagonized by the compatible organic osmolytes betaine or taurine. The data suggest a role of NKCC1 in stellate cell transformation, hepatic volume regulation, and long-term adaption to dehydrating conditions.

 

Aha!  Glycine Betaine and Taurine – not so fast 

You have to check the effect on both NKCC1 and KCC2.  One lets chloride into neurons and the lets it out.  You want to block NKCC1 and not KCC2, otherwise you undo all the good you have done.

Both glycine betaine (TMG) and taurine are already used as autism supplements at low doses.  The paper below suggest that Taurine is not a good idea for people with high levels of chloride within neurons.

 

Taurine inhibits K+-Cl- cotransporter KCC2 to regulate embryonic Cl- homeostasis via with-no-lysine (WNK) protein kinase signaling pathway

GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.

 

So, it is likely only Glycine Betaine (TMG) may be of potential benefit, in the case of lowering chloride.

 

Glycine Betaine in the broader research

 

Betaine in Inflammation: Mechanistic Aspects and Applications

Betaine is known as trimethylglycine and is widely distributed in animals, plants, and microorganisms. Betaine is known to function physiologically as an important osmoprotectant and methyl group donor. Accumulating evidence has shown that betaine has anti-inflammatory functions in numerous diseases. Mechanistically, betaine ameliorates sulfur amino acid metabolism against oxidative stress, inhibits nuclear factor-κB activity and NLRP3 inflammasome activation, regulates energy metabolism, and mitigates endoplasmic reticulum stress and apoptosis. Consequently, betaine has beneficial actions in several human diseases, such as obesity, diabetes, cancer, and Alzheimer’s disease.

 

Betaine is a stable and nontoxic natural substance. Because it looks like a glycine with three extra methyl groups, betaine is also called trimethylglycine . In addition, betaine has a zwitterionic quaternary ammonium form [(CH3)3N+ CH2COO−] (Figure 1). In the nineteenth century, betaine was first identified in the plant Beta vulgaris. It was then found at high concentrations in several other organisms, including wheat bran, wheat germ, spinach, beets, microorganisms, and aquatic invertebrates. Dietary betaine intake plays a decisive role in the betaine content of the body. Betaine is safe at a daily intake of 9–15 g for human and distributes primarily to the kidneys, liver, and brain. The accurate amount of betaine intake generally relies on its various sources and cooking methods. Besides dietary intake, betaine can be synthesized from choline in the body. Studies report that high concentrations of betaine in human and animal neonates indicate the effectiveness of this synthetic mechanism.

  

Boosting amino acid derivative may be a treatment for schizophrenia

Many psychiatric drugs act on the receptors or transporters of certain neurotransmitters in the brain. However, there is a great need for alternatives, and research is looking at other targets along the brain's metabolic pathways. Lack of glycine betaine contributes to brain pathology in schizophrenia, and new research shows that betaine supplementation can counteract psychiatric symptoms in mice.

 

 

Supplement treats schizophrenia in mice, restores healthy “dance” and structure of neurons Repurposed drug works by building cells’ skeleton and transportation network

 

 

Conclusion

Early on in the Covid saga, I saw interviews with both the Moderna researchers and the Oxford (AstraZeneca) researchers. Both claimed that they designed their vaccines over a weekend.  This was made possible by the Chinese releasing the DNA code of the virus.

When you think about gene therapy for autism and Down syndrome, the same likely applies; much could be achieved over a weekend.

The expensive and time-consuming part is the testing and approval process.

In the Covid pandemic the approval process was modified to allow for emergency use.  Perhaps this should also be the case for all gene therapies?

What use is a $2 million therapy for autism or Down syndrome?

In theory, if you gave your gene therapy prior to birth or shortly thereafter, it might be fully curative.  Realistically, by the time you get the therapy it is just going to be beneficial and you will still need other ongoing therapies.

Note that gene therapy normally applies to just one gene.  In Down syndrome people have a third copy of all, or just part, of Chromosome 21.  This results directly in the miss-expression of hundreds of genes from that chromosome.

The gene that encodes NKCC1 is on Chromosome 5, which has nothing directly to do with Down syndrome.

The NKCC1 transporter is over-expressed in Down syndrome as a down stream consequence of the disorder. It is caused by the “faulty GABA switch”, referred to in earlier posts.

The Italian gene therapy to lower chloride in neurons and so raise cognition, has numerous applications, in people currently of all ages, so there is a big potential market.

Why not gene therapy for all single gene autisms?  It could be a highly productive use of the researcher’s weekends, for a year or two.

The issue is who would pay for the $20 to $30 million approval process, for each gene?

Maybe some of the billions in profit from clever Covid vaccines could be used for pro bono gene therapy?  Highly unlikely.

Biontech, who are the brains behind the Pfizer vaccine, do have plans to develop gene therapy for other medical conditions.  I think these will be ultra expensive,

That brings me back to Glycine Betaine (TMG), is 10g a day of this supplement really going to reduce the expression of NKCC1 transporters in neurons and so lower chloride within neurons?  It seems to work in creatine transporter deficiency, is all we can say.  

Glycine betaine, at much lower doses, has been used by DAN and now MAPS doctors for decades. They use it as a “methyl-donor”.  There is a combination of real science and hocus-pocus surrounding DNA methylation. 

 DNA Methylation and Susceptibility to Autism Spectrum Disorder