Modulating Wnt Signaling in Autism and Cancer

Source: LDL receptor-related proteins 5 and 6 in Wnt/β-catenin signaling: Arrows point the way

In earlier posts I have covered various signaling pathways such as Wnt, mTOR and the unusually sounding Hedgehog.

You can go into huge detail if you want to understand these pathways, or just take a more superficial view. In most cases, things only start to go wrong if you are hypo/hyper (too little/too much) in these pathways.

We saw with mTOR that most people with autism are likely to have too much activity and so might benefit from mTOR inhibition, but a minority will have the opposite status and stand to benefit from more mTOR activity.

When it comes to Wnt signaling the research suggests the same situation. Wnt signaling is likely to be aberrant, but both extremes exist.

Wnt signaling networks in autism spectrum disorder and intellectual disability

Given the large volume of genetic data, analyzing each gene on its own is not a feasible approach and will take years to complete, let alone attempt to use the information to develop novel therapeutics. To make sense of independent genomic data, one approach is to determine whether multiple risk genes function in common signaling pathways that identify signaling “hubs” where risk genes converge. This approach has led to multiple pathways being implicated, such as synaptic signaling, chromatin remodeling, alternative splicing, and protein translation, among many others. In this review, we analyze recent and historical evidence indicating that multiple risk genes, including genes denoted as high-confidence and likely causal, are part of the Wingless (Wnt signaling) pathway. In the brain, Wnt signaling is an evolutionarily conserved pathway that plays an instrumental role in developing neural circuits and adult brain function.

While the human genetic data is an important supporting factor, it is not the only one. There are a number of mouse genetic knockout (KO) models targeting Wnt signaling molecules, describing molecular, cellular, electrophysiological, and behavioral deficits that are consistent with ASD and ID. Furthermore, the genes involved in Wnt signaling are of significant clinical interest because there are a variety of approved drugs that either inhibit or stimulate this pathway.

There are many drugs developed and tested as modulators of Wnt signaling in the cancer field that could potentially be repurposed for developmental cognitive disorders. In cases where a reduction in Wnt signaling is thought to underlie the pathology of the disorder, usage of compounds that elevated canonical Wnt signaling could be applied. An example of this is GSK-3β inhibitors that have failed in cancer trials but may be effective for ASDs and ID (e.g., Tideglusig, ClinicalTrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT02586935","term_id":"NCT02586935"}}NCT02586935). In cases where elevated Wnt signaling is thought to contribute to disease pathology, there are many potential options to inhibit canonical Wnt signaling using chemicals (Fig. 1) that inhibit the interaction between β-catenin and its targets (e.g., inhibiting β-catenin interaction with the TCF factors), disheveled inhibitors (through targeting of the PDZ domain which generally inhibit the Frizzled–PDZ interaction), and tankyrase inhibitors (e.g., XAV939, which induces the stabilization of axin by inhibiting the poly (ADP)-ribosylating enzymes tankyrase 1 and tankyrase 2)

As autism candidates emerge, cancer pathway rises to top

In recent years, strong autism ties have cropped up for one group of genes in particular: those that make up a well-known signaling pathway called WNT, which also has strong links to cancer. This pathway is especially compelling because some people with autism carry mutations in various members of it, including one of its central players: beta-catenin¹. What’s more, studies from the past year indicate that several of the strongest autism candidate genes, including CHD8 and PTEN, interact with this pathway.

“There might be a particular subgroup of genes associated with autism that could all be feeding into or be regulating this pathway,” says Albert Basson, reader in developmental and stem cell biology at King’s College London, who studies CHD8 and WNT. “That clearly has emerged as a relatively major theme over the last few years.”

The connection between cancer and some autism is over-activated pro-growth signaling pathways. Many signaling pathways have growth at one extreme and cell death at the other. In cancer you actually want cell death to suppress tumor growth; in much autism there is also too much growth.  

Many cancers are associated with elevated signaling of mTOR, Wnt and indeed Hedgehog.  These are targets for cancer drug therapy and so there is already a great deal known.

A complication is that in a developmental neurological condition, like autism, it also matters when these signaling pathways were/are disturbed. For example Wnt signaling is known to play a role in dendritic spines and synaptic pruning, some of this is an ongoing process but other parts are competed at an early age, so it would matter when you intervene to modulate these pathways.

Historically cancer therapies involve potent drugs, often with potent side effects, however in recent years there has been growing awareness that some safe existing drugs can have equally potent anti-cancer effects. Many of these drugs are anti-parasite drugs, but even the very widely used diabetes drug Metformin has been shown to have significant anti-cancer effects, not to forget Simvastatin.

Many autism pathways/genes play a role in cancer (RAS, PTEN) and the upstream targets considered in cancer research are also autism targets.  For example many human cancers are RAS dependent and in theory could be treated by a RAS inhibitor, but after decades of looking nobody has found one. So instead scientists go upstream to find another target that will indirectly reduce RAS. This led to the development of PAK1 inhibitors that will reduce RAS.

RAS plays a role in some types of intellectual disability and indeed autism. The collective term is RASopathy.  Logically, drugs that modulate RAS to treat cancer might be helpful in modulating RAS for some autism.

Most types of cancers are complex and so there are multiple potential targets to attack them, but also the same target can have multiple possible approaches. RAS dependent cancers can be targeted via Wnt and even Hedgehog signaling.

This may sound all very complicated but does it have any relevance to autism?

It apparently does because almost all these pathways are known to be disturbed hypo/hyper in autism.  This means that clever insights developed for cancer can be repurposed for autism.

Anti-parasite drugs and Cancer

It is indeed remarkable how many anti-parasite drugs have an anticancer effect and indeed there is a much maligned theory to justify this.

Clinical Evaluation of a New Form of Cancer Therapy Based on the Principles of Atavistic Metamorphosis

Quite possibly it is just a coincidence.

There are many ways to kill parasites, one of which involves starving them of ATP. ATP is the fuel that is produced in your mitochondria.

Cancer cells and many parasites use a very inefficient way to produce ATP that does not require oxygen. In normal human cells the process followed is known as OXPHOS, by which glucose and oxygen from the blood is converted into ATP (energy) is very efficient. Only when you run low on oxygen, like a marathon runner at the end of the race, can you run into trouble because there is not enough oxygen for OXPHOS.  What happens next is anaerobic respiration, when a different process takes over to make ATP. It is much less efficient and causes lactic acidosis which makes marathon runners' muscles hurt.

A cheap anti-parasite drug Pyrvinium targets anaerobic respiration and starves the parasite of ATP and thus kills it. Another common children’s anti-parasite drug albendazole also works by starving the parasite of ATP.

Other anti-parasite drugs work in different ways.

We already know from the autism trials of Suramin, another anti-parasite drug,  that it works via P2X and P2Y purinergic channels.

Ivermectin  binds to glutamate-gated chloride channels (GluCls) in the membranes of invertebrate nerve and muscle cells, causing increased permeability to chloride ions, resulting in cellular hyper-polarization, followed by paralysis and death.  Fortunately in mammals ivermectin does not cross the BBB.

Ivermectin is also a PAK1 inhibitor and a positive allosteric modulator of P2X7.

Both PAK1 and P2X7 are relevant to many cancers and so not surprisingly research shows that Ivermectin has an anti-cancer effect.

Ivermectin appears to have a positive effect in some autism, but strangely it does not cross the BBB.

Mebendazole is another extremely cheap children’s anti-parasite drug which has remarkable potential anti-cancer properties. It inhibits hedgehog signaling and, via the inhibition of TNIK, it is a Wnt inhibitor.

Unfortunately in the US the private sector has also noticed the anticancer effects of Mebendazole and albendazole and they have recently become astronomically expensive. Mebendazole (MBZ), which costs almost nothing in many countries, now costs hundreds of dollar per dose in the US under the name Emverm. Outside of the US, Mebendazole is OTC in many developed countries. In poor countries it is donated free by big pharma.

In the cancer research they consider taking advantage of the fact that cimetidine (a cheap H2 antihistamine) interacts with Mebendazole to increase its bioavailability. Cimetidine is by chance another generic drug also being considered to be repurposed for cancer.

While some anti-parasite drugs like Suramin have side effects or cannot be taken regularly like Ivermectin, others are seen as safe for continued use even at high doses (e.g. Mebendazole and albendazole).  

Anti-parasite drugs and Autism

Just as many anti-parasite drugs seem to have a positive effect on some cancers it looks likely that the same may be true for autism.  This does not mean that parasites cause either cancer or autism.

We know from Professor Naviaux that some people respond to Suramin.

Two people who comment on this blog have found their child responds to PAK1 inhibitors, one of which is the drug Ivermectin.

There are groups of people on the internet who think parasites cause autism and you will find some of them if you google “autism mebendazole”, but there are some very valid reasons why some people’s autism may respond to mebendazole, but nothing to do with little worms.

Potency of Anticancer drugs

Failed anticancer drugs are already considered as possible drugs to treat neurological conditions.

The same pathways do seem to be involved in some cancer and some neurological conditions, but the severity by which that pathway is affected may be very different, so a new drug may lack potency to treat a type of cancer but be potent enough to benefit others.

In the case of the anti-parasite drugs Ivermectin and indeed mebendazole the dosage being used in current cancer studies are very much higher than normally used.

Very little mebendazole makes its way out of your intestines and so researchers counter this by using a dose 15 times higher and even taking advantage of the interaction with the H2 antagonist cimetidine to boost bioavailability.

The standard human dose of Ivermectin is 3mg, but in the cancer trials (IVINCA trial - IVermectin IN CAncer) in Switzerland and Spain the trial dose is 12, 30 and 60 mg.

So when it comes to autism and the possible repurposing of these drugs, the cancer studies will give valuable safety information, but the likely dose required to fine-tune these signaling pathways will likely be a tiny fraction of the cancer dose.

The newly developed cancer drugs that fail in clinical trials, may have potential in autism but it is unlikely that anyone will develop them, test them and bring them to the market.

The clever thing for autism seems to be to keep an eye on the existing generic drugs considered to benefit the overlapping cancer pathways.

Conclusion

Aberrant Wnt signaling has been identified by researchers as playing a key role in autism; the Simons Foundation is among those now funding further research.

In practical terms you can be either hypo or hyper, but hyper seems more likely. It may be a case of shutting the stable door after the horse has bolted, because the ideal time to modulate Wnt signaling is probably as a baby, or before. Nonetheless some older people may indeed benefit from modulating Wnt; the Simons Foundation must also believe so.

In the case of people with hyperactive Wnt signaling, there is a case to make for the potential use of the cheap anti-parasite drug Mebendazole.

The drug Mebendazole (MBZ) can found in three states/polymorphs called Polymorph A, B or C. This is relevant because they do not cross the blood brain barrier to the same extent.

Brain Penetration and Efficacy of Different Mebendazole Polymorphs in a Mouse Brain Tumor Model  

To treat brain tumors, or indeed potentially some autism, you need MBZ-B or MBZ-C, it looks like MBZ-A does not cross the blood brain barrier.

Fortunately, MBZ-C is  the polymorph found most commonly in generic mebendazole tablets.  

Ivermectin is known not to cross the blood brain barrier but yet has been shown to show anti-tumor activity in brain cancer. The anti-cancer effect is thought to be as a PAK1 inhibitor, but this effect must be occurring outside the brain. Some people do use Ivermectin for autism.

The people using Ivermectin for autism are told they cannot use it continuously. Perhaps as the high dose cancer trials evolve the safety advice may change.

PAK inhibitors and potentially treating some Autism using Grandpa’s Medicine Cabinet

I wrote several posts about why PAK1 inhibitors should be beneficial in some autism and indeed some schizophrenia.

We also saw that PAK1-blocking drugs could be potentially useful for the treatment of neurofibromatosis type 2, in addition to RAS-induced cancers and neurofibromatosis type 1.

One problem with drugs developed for cancer is that, even if they finally get approved, they tend to be ultra-expensive.  Production volumes are low because even if they “work” they do not prolong life for so long and cancer has numerous sub-types.

Cheap drugs are ones used to treat common chronic conditions like high blood pressure, high cholesterol and indeed treatment of male lower urinary tract symptoms (LUTS), like benign prostatic hyperplasia (BPH).

A small number of readers of this blog have confirmed the beneficial effect of PAK inhibitors in their specific sub-types of autism.  The problem is that there are no potent PAK1 inhibitors suitable for long term use that are readily available.

The anti-parasite drug Ivermectin is an extremely cheap PAK1 inhibitor, but cannot be used long term, due to its other effects.

Propolis containing CAPE (Caffeic Acid Phenethyl Ester) is a natural PAK1 inhibitor, but may not be sufficiently potent as is reported by people with neurofibromatosis.

You would think somebody would just synthesize CAPE (Caffeic Acid Phenethyl Ester) artificially and then higher doses could be achieved.

PAK Inhibitors and Treatment of Prostate Enlargement

I was rather surprised that research has recently been published suggesting that PAK inhibitors could be used to treat the prostate enlargement, common in most older men. 

P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate

Abstract

Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients’ adherence is particularly low to combination therapies of 5α-reductase inhibitors and α₁-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1–10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions.

It looks like a PAK inhibitor could potentially solve both the key problems in BPH and so replace the current therapies.

Existing Drugs for LUTS/BPH

Undoubtedly someone is going to wonder whether existing drugs for LUTS/BPH might improve autism.  This is actually possible, but totally unrelated to PAK1 inhibition and RASopathies.

Existing drugs are in two classes, 5α-reductase inhibitors and α₁-adrenoceptor antagonists.

α-adrenoceptor antagonists

Alpha blockers relax certain muscles and help small blood vessels remain open. They work by keeping the hormone norepinephrine (noradrenaline) from tightening the muscles in the walls of smaller arteries and veins, which causes the vessels to remain open and relaxed. This improves blood flow and lowers blood pressure.

Because alpha blockers also relax other muscles throughout the body, these medications can help improve urine flow in older men with prostate problems.

Selective α₁-adrenergic receptor antagonists are often used in BPH because it is the α₁-adrenergic receptor that is present in the prostate.

 α ₂-adrenergic receptors are present elsewhere in the body

Alpha-2 blockers are used to treat anxiety and post-traumatic stress disorder (PTSD). They decrease sympathetic outflow from the central nervous system. Post-traumatic stress disorder is an anxiety disorder that is theorized to be related to a hyperactive sympathetic nervous system.

Alpha-2 receptor agonists for the treatment of post-traumatic stress disorder

So a nonselective alpha blocker, like one given to an older man with high blood pressure and BPH, might well have an effect on some kinds of anxiety.

You would think that a selective alpha 2 blocker might be interesting, how about Idazoxan?

Idazoxan is a drug which is used in research. It acts as both a selective α2 adrenergic receptor antagonist, and an antagonist for the imidazoline receptor. Idazoxan has been under investigation as an antidepressant, but it did not reach the market as such. More recently, it is under investigation as an adjunctive treatment in schizophrenia. Due to its alpha-2 receptor antagonism it is capable of enhancing therapeutic effects of antipsychotics, possibly by enhancing dopamine neurotransmission in the prefrontal cortex of the brain, a brain area thought to be involved in the pathogenesis of schizophrenia.

Mirtazapine is a cheap generic drug used at high doses for depression.  It happens to be a selective alpha 2 blocker, but it has numerous other effects as well.  One reader of this blog does respond very well to Mirtazapine.

So realistically in Grandpa’s medicine cabinet there might a selective alpha 1 agonist or a non-selective alpha agonist, it is the latter type that might have an effect on some kinds of autism.

5α-reductase inhibitors

The pharmacology of 5α-reductase inhibition involves the binding of NADPH to the enzyme followed by the substrate. Specific substrates include testosterone, progesterone, androstenedione, epitestosterone, cortisol, aldosterone, and deoxycorticosterone.

Beyond being a catalyst in testosterone reduction, 5α-reductase isoforms I and II reduce progesterone to 5α-dihydroprogesterone (5α-DHP) and deoxycorticosterone to dihydrodeoxycorticosterone (DHDOC).

In vitro and animal models suggest subsequent 3α-reduction of DHT, 5α-DHP and DHDOC lead to neurosteroid metabolites with effect on cerebral function.

These neurosteroids, which include allopregnanolone, tetrahydrodeoxycorticosterone (THDOC), and 5α-androstanediol, act as potent positive allosteric modulators of GABA_A receptors, and have anticonvulsant, antidepressant, anxiolytic, prosexual, and anticonvulsant effects.

Inhibition of 5α-reductase results in decreased conversion of testosterone to DHT.

This, in turn, results in slight elevations in testosterone and estradiol levels. 

In BPH, DHT acts as a potent cellular androgen and promotes prostate growth; therefore, it inhibits and alleviates symptoms of BPH. In alopecia, male and female-pattern baldness is an effect of androgenic receptor activation, so reducing levels of DHT also reduces hair loss.

A new look at the 5alpha-reductase inhibitor finasteride

Finasteride is the first 5alpha-reductase inhibitor that received clinical approval for the treatment of human benign prostatic hyperplasia (BPH) and androgenetic alopecia (male pattern hair loss). These clinical applications are based on the ability of finasteride to inhibit the Type II isoform of the 5alpha-reductase enzyme, which is the predominant form in human prostate and hair follicles, and the concomitant reduction of testosterone to dihydrotestosterone (DHT). In addition to catalyzing the rate-limiting step in the reduction of testosterone, both isoforms of the 5alpha-reductase enzyme are responsible for the reduction of progesterone and deoxycorticosterone to dihydroprogesterone (DHP) and dihydrodeoxycorticosterone (DHDOC), respectively. Recent preclinical data indicate that the subsequent 3alpha-reduction of DHT, DHP and DHDOC produces steroid metabolites with rapid non-genomic effects on brain function and behavior, primarily via an enhancement of gamma-aminobutyric acid (GABA)ergic inhibitory neurotransmission. Consistent with their ability to enhance the action of GABA at GABA(A) receptors, these steroid derivatives (termed neuroactive steroids) possess anticonvulsant, antidepressant and anxiolytic effects in addition to altering aspects of sexual- and alcohol-related behaviors. Thus, finasteride, which inhibits both isoforms of 5alpha-reductase in rodents, has been used as a tool to manipulate neuroactive steroid levels and determine the impact on behavior. Results of some preclinical studies and clinical observations with finasteride are described in this review article. The data suggest that endogenous neuroactive steroid levels may be inversely related to symptoms of premenstrual and postpartum dysphoric disorder, catamenial epilepsy, depression, and alcohol withdrawal.

This would suggest that a 5α-reductase inhibitor, like finasteride, that might be among Grandpa’s tablets might very well have an effect on someone with GABAa dysfunction, this includes very many people with autism, schizophrenia and Down Syndrome.

Whether the effect will be good or bad is hard to say, and may well depend on whether other drugs that target GABA or NMDA receptors are being used. Due to their other effects, 5α-reductase inhibitors are usually only used in adults.

Merck developed a lower dose form of finasteride, called Prospecia to treat baldness, usually in men.  It is 20% the normal potency used for BPH.

Side effects

The current BPH drugs cause side effects in some people.  PAK1 inhibitors may also have some side effects.

Conclusion

Going back in the days of living with your extended family might make treating many people’s autism much simpler.  It looks like many older people’s drugs can be repurposed for some types of autism (ion channel modifying diuretics, calcium channel blockers, statins, even potentially intranasal insulin in some).  Because older people’s drugs are so widely used they are well understood and inexpensive.  

Clearly the research on PAK inhibitors for LUTS/BPH is at an early stage, but there is a huge potential market.   A widely available PAK1 inhibitor might be a big help to some people with autism, neurofibromatosis, other RASopathies, not just Grandpa’s prostate.

In addition to FRAX486 and IPA3, why doesn’t someone try synthetic CAPE, i.e. without the bees, as a PAK inhibitor?

Bioactivity and chemical synthesis of caffeic acid phenethyl ester and its derivatives.

There is far more chance of a PAK1 inhibitor coming to market for LUTS/BPH, or certain cancers than for autism.  That is a fact of life.

As for 5α-reductase inhibitors, like finasteride, we know from Hardan’s study on Pregnenolone at Stanford that this hormone can have a positive effect and we know that various natural steroid metabolites will modulate GABA subunits.  So it is quite likely that finasteride is going have a behavioral effect.  Perhaps Hardan would like to trial finasteride 5mg and 1mg (Prospecia) in some adults with autism. I suspect it will make some people “worse” and others somewhat “better”; so please do not report the “average” response, highlight the nature of the positive responders.

Intranasal Insulin for Some Autism vs IGF-1 and NNZ-2566

 

Very often the simplest solutions are the best and very often, when fault finding a problem, people overlook the obvious.  

I seem to be forever having to mend things and I find this all the time.

Back in 2013, when I knew much less about autism, I wrote about the experimental use of insulin like growth factor 1 (IGF-1) in autism.  

It’s a Small World – IGF-1 and NNZ-2566 in Autism

It turned out that in autism the many different growth factors can be disturbed (too much, or too little) and this variation does indeed define some specific types of autism.  For example in Rett Syndrome there are very low levels of Nerve Growth Factor (NGF); low levels of NGF in some older people is the cause of their dementia.  In more common types of autism NGF is actually elevated.

IGF-1 is very well studied.

 

IGF-1 is a primary mediator of the effects of growth hormone (GH). 

Growth hormone is made in the anterior pituitary gland, is released into the blood stream, and then stimulates the liver to produce IGF-1. IGF-1 then stimulates systemic body growth, and has growth-promoting effects on almost every cell in the body, especially skeletal muscle, cartilage, bone, liver,kidney, nerves, skin, hematopoietic cell, and lungs. This would explain why adults abusing GH may end up needing hip and knee replacements.

Before getting into the science, IGF-1 has long been available as a drug to treat children with growth delays.  In the US this drug is being used on children with a type of autism called Phelan-McDermid Syndrome.

Now, regular readers will recall from my last post on intranasal insulin that it was in this very syndrome that there was a successful intranasal insulin.

So most likely without delving into the science at all it looks like IGF-1 and intranasal insulin are both options to treat the same dysfunction.

Using IGF-1

Clinical Trial in 22q13 Deletion Syndrome (Phelan-McDermid Syndrome)

Using Intranasal Insulin

Intranasal insulin to improve developmental delay in children with 22q13 deletion syndrome: an exploratory clinical trial.

NNZ-2566

This is an Australian drug that is a modified version of IGF-1 (a so called analog).  They modified it so that it can be taken orally rather than by injection.  The developer has a very thorough presentation showing why they think it should be effective in autism.  

  

NNZ-2566 - Rationale for use in Autistic Spectrum Disorders

The Science

The first thing to note is that insulin and IGF-1 act as messengers.  Disruption in growth factor signaling can have serious consequences.

Insulin and IGF-1 both activate the same insulin receptor (IR).

Most people think that insulin is a just a hormone produced in their pancreas that regulates the amount of glucose (sugar) in their blood.  It does of course do that, but it actually does much more.

  

Insulin receptors are expressed all over the body including the brain.

Here is a relatively simple presentation explaining the role of insulin signaling in the brain:-

The role of insulin receptor signaling in the brain

Now for the diehard scientists among you that have been reading about all those signaling pathways that lie behind autism, cancer and many other hard to treat conditions, look at the graphic below.

We know the importance of RAS.  Impaired RAS signaling underlies the RASopathies, one feature of which is cognitive loss (MR/ID), another is autism.

We also know the importance of Akt (PKB/protein kinase B) in some types of autism.  PTEN appears again.

So irrespective of an undoubtedly important effect on glucose and insulin resistance, we should expect activation of insulin receptors in the brain, in some types of autism, to have a further positive effect.

It would seem to be a potential therapy for RASopathies.

As is often the case, there are extreme dysfunctions of RAS and I suggest there are more mild dysfunctions.

I suggest that some people with autism and some cognitive dysfunction have a partial RASopathy.

Since autism contains both extremes of many dysfunctions, there will undoubtedly be types of autism that respond negatively, or not at all, to activation of insulin receptors in the brain.

Practicalities

Nobody likes injections and that is necessary to give IGF-1.

NNZ-2566 is an experimental autism drug and on past performance that means it will take decades to reach the market, if ever.

That leaves insulin which was sitting all along in your local pharmacy.

Intranasal insulin was once investigated for use in diabetics, but it did not work.  It is not absorbed into the blood stream.

This is of course the huge advantage for people with autism, since we only want to activate the insulin receptors in the brain.  If you are not diabetic why would you want to have any effects in the rest of the body?

Indeed there are known major side effects of injecting IGF-1 or GH (growth hormone) into adults.  All kinds of things start growing and this can lead to terrible results.

The fact that all the studies show that intranasal insulin does not enter the blood stream and so lower blood glucose levels, makes it a much better drug for autism than IGF-1 or indeed NNZ-2566.

Insulin

There are various types of insulin and the main difference is that some are modified to be longer acting.

The basic insulin is soluble or clear insulin, and nowadays is synthetic rather than derived from pigs.  Examples include Humulin Regular/R/S by Lilly.

The standard concentration is 100 IU/ml.

The trials in Alzheimer’s and other conditions varied in dosage but generally used about 20 to 40 IU per day.

This is not a trivial dose.  If injected, rather than inhaled, that dose would have a significant effect on lowering blood sugar and would be dangerous.

My antihistamine nasal spray gives a metered dose of 0.14 ml.

So without any dilution, if filled with off the shelf insulin it would dispense 14 IU per spray.

So no special high tech drugs, dilutants/diluents or dispensers appear to be necessary. Some trials do use fancy inhalers, like the one in the video at the end of this post.

To be prudent it might be wise to dilute the insulin so as to gradually increase the dose.  Maybe in some people the nasal membrane is more permeable than in others.  Some of the trials did this, but most did not.

A fridge is required, because insulin needs to be kept chilled.

I do wonder why nobody seems to be researching this in autism.  Silly point, as one insulin researcher commented on the earlier post; there is no big money to be made, hence no interest.

Insulin & Alzheimer’s

The reasons that intranasal insulin improves Alzheimer’s, and likely will Down Syndrome, may differ to those help in (some) autism.

Beta amyloid is key to Alzheimer’s (and early onset Alzheimer’s in Down Syndrome) but is not a known issue in autism.  Central insulin resistance is an issue in Alzheimer’s and might well be in autism.  

Perhaps people with mitochondrial dysfunction (an energy conversion dysfunction) might particularly benefit from increased glucose uptake in the brain.  It appears that mitochondrial dysfunction plays a role in insulin resistance. 

Role of Mitochondrial Dysfunction in Insulin Resistance

The activation of the RAS pathway might be highly beneficial to some people with autism.  

Here is a good film, which refers to the studies from previous posts and shows the effect on one man with Alzheimer's. 

Insulin and Alzheimer's (approx. 22 min.)

 You also see their fancy inhaler device.

mTOR – Indirect inhibition, the Holy Grail for Life Extension and Perhaps Some Autism

 Not cheap at about $1,000 for just 140mg

Life extension may come as a surprise, but it is interesting because it is well studied and, in mice at least, easy to measure.  Most research into mTOR relates to cancer, but this is a very complex condition. With various feedback loops it means that sometimes the actual effect is the opposite of what was predicted.  For example, a substance that can help prevent cancer can actually become harmful later and promote its growth.

Direct inhibition of mTOR with Everolimus and similar drugs (variants/analogs of Rapamycin, all called Rapalogs) has not been as successful as hoped in cancer research.  Trials of direct inhibition of mTOR will shortly start in one rare single gene type of autism (TSC).  The drugs are so expensive that many providers do not want to pay for them.

As you will see mTOR is just one process in a cloud of interrelated processes.  Almost everything has a role/effect:- growth factors, cytokines, amino acids, mitochondria, dendritic spines, PPAR gamma, hormones, oxidative stress, autophagy ….

While it would be nice to think that a single protein complex like mTORC1 or mTORC2 is the root of all evil in autism, I rather doubt it can be so simple.

The knowledge that one factor controlling mTORC1 and mTORC2 is oxidative stress, does raise the possibility that, yet again, the root problem could be oxidative stress.  

Nonetheless, we will see in today’s post that too much mTOR activity is clearly not good and that it is associated with lots of bad things:-

·        Epilepsy

·        Autistic behaviours

·        Food allergies

·        Mitochondrial dysfunction

·        Cognitive impairment

as well as aging, cancer ….

Indirect reduction in mTOR activity

Rather than the very expensive first and second generation mTOR inhibiting drugs developed for cancer,  I think the safe way forward for autism (and aging) may be indirect reduction in mTOR activity, and there is already a wide choice of methods.

Ketogenic Diet, (or just reduction in carbohydrate intake)

This diet has been used for a hundred years to control epilepsy, which it now seems can be triggered by elevated mTOR.  Research has shown that the ketogenic diet reduces mTOR. 

Low glycemic index diet

This is a low carbohydrate, no sugar diet, typical of someone with diabetes.  It avoids rapid change in blood sugar.  This will lower mTOR and has recently been shown in a mouse model to improve autistic behaviors.

Growth factors

The blood levels of growth factors such as insulin and IGF-1 reflect the fed status of the organism. When food is plentiful, levels of these growth factors are sustained and promote anabolic cell processes such as translation, lipid biosynthesis, and nutrient storage via mTORC1.  So, dietary restriction, which lowers IGF-1, will reduce mTOR; but it will also reduce growth.

Note that one autism therapy under trial does just the opposite, it is to increase IGF-1 levels via injections of IGF-1.

Increase amino acids, particularly leucine

Ask any body builder about BCAA (Branch Chained Amino Acids)

  

Reduce oxidative stress

We know how to do that

NMDA agonists

NMDA receptor activation decreases mTOR signaling activity. 

This might be achieved using D-Cycloserine, Aminocyclopropanecarboxylic acid, cis-2,3-Piperidinedicarboxylic acid, Aspartic acid, Glutamic acid, Quinolinate, Homocysteic acid, D-Serine, L-Serine, D-Alanine, L-Alanine, ACPL

Note that D-Cycloserine is used in autism and D-Serine is used in schizophrenia

Increase PTEN, for example with a Statin drug

Reduce RAS signaling, for example with a Statin drug

I am not the first person to realize this.  Here is a very highly cited paper:-

Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress

  

Since the body is controlled via feedback loops, there might exist a clever way to “trick” the body into lowing mTOR.  For example PPAR gamma, which we have come across in earlier posts, is controlled via mTOR.  If you stimulate PPAR gamma externally this might well have an effect back stream on mTOR activity, via these feedback loops.  Just like if you supplement Melatonin, you will likely affect the behaviour back stream of the pineal gland.

mTOR and Aging

A surprising number of emerging autism therapies are actually also put forward by the life extension people.  In case you did not know, there is a small industry of pills and potions dedicated to making you live longer.  Some serious institutions like MIT and Harvard are involved, as in the paper below.

Rapalogs and mTOR inhibitors as anti-aging therapeutics

We earlier saw that PAK-1 is probably there to make sure you do eventually die, reducing mTOR signaling can probably extend your lifetime and, more importantly, your healthy lifetime.

Ketogenic Diet

We did see a case report a while back from Martha Herbert, from Harvard, who has a good result with the ketogenic diet. 

Autism and Dietary Therapy: Case Report and Review of the Literature

  

  

The Science of mTOR

In the following section there are numerous scientific papers explaining mTOR, so you can choose just how deep you want to go into the details.

You may notice on the complex diagram below various substances that we have already encountered in this blog as relevant to autism.

·        PTEN ( increased by Statins) reduced in some autism

·        Growth factors (disturbed in autism and therapeutic to some)

·        Ras / Rasopathy (increased by statins, linked to some autism and MR/ID )

·        Wnt (affects morphology of those dendritic spines, malformed in autism)

·        Lipid metabolism/synthesis (disturbed in autism)

·        TSC1  (tuberous sclerosis autism variant)

·        PPAR alpha and gamma affecting inflammation

·        Mitochondrial metabolism, dysfunctional in autism

·        Autophagy was explained in recent post and, if impaired, will degrade cellular health and function, particularly in mitochondria

·        Note Stress/Hypoxia, we have mentioned Hypoxia before.  REDD1 inhibits mTOR.  REDD1 was first identified as a gene induced by hypoxia and DNA damage, other environmental stresses such as energy stress, glucocorticoid treatment and reactive oxygen species have also been reported to induce REDD1 transcription  

Pathway Description: The mechanistic target of Rapamycin (mTOR) is an atypical serine/threonine kinase that is present in two distinct complexes.

The first, mTOR complex 1 (mTORC1), is composed of mTOR, Raptor, GβL, and DEPTOR and is inhibited by Rapamycin. It is a master growth regulator that senses and integrates diverse nutritional and environmental cues, including growth factors, energy levels, cellular stress, and amino acids. It couples these signals to the promotion of cellular growth by phosphorylating substrates that potentiate anabolic processes such as mRNA translation and lipid synthesis, or limit catabolic processes such as autophagy. The small GTPase Rheb, in its GTP-bound state, is a necessary and potent stimulator of mTORC1 kinase activity, which is negatively regulated by its GAP, the tuberous sclerosis heterodimer TSC1/2. Most upstream inputs are funneled through Akt and TSC1/2 to regulate the nucleotide-loading state of Rheb. In contrast, amino acids signal to mTORC1 independently of the PI3K/Akt axis to promote the translocation of mTORC1 to the lysosomal surface where it can become activated upon contact with Rheb. This process is mediated by the coordinated actions of multiple complexes, notably the v-ATPase, Ragulator, the Rag GTPases, and GATOR1/2.

The second complex, mTOR complex 2 (mTORC2), is composed of mTOR, Rictor, GβL, Sin1, PRR5/Protor-1, and DEPTOR. mTORC2 promotes cellular survival by activating Akt, regulates cytoskeletal dynamics by activating PKCα, and controls ion transport and growth via SGK1 phosphorylation.

Aberrant mTOR signaling is involved in many disease states including cancer, cardiovascular disease, and diabetes.

 Source: http://www.cellsignal.com/common/content/content.jsp?id=pathways-mtor-signaling#sthash.RhBXxjat.dpuf

mTOR: from growth signal integration to cancer, diabetes and ageing

Growth factors regulate mTORC1

Energy and stress regulate mTORC1

mTOR regulates metabolism in mammals

mTOR in fasting and starvation

mTOR, over-feeding, and insulin sensitivity

One of the most efficient forms of energy storage are triglycerides, because they provide a high energetic yield per unit of mass. mTORC1 mediates lipid accumulation in fat cells

mTORC1 may impact on PPAR-γ activity by increasing its translation¹¹⁸ and by activating the transcription factor SREBP-1c . Active SREBP-1c enhances PPAR-γ activity and transactivates a set of genes directly involved in lipid synthesis. At present, the molecular links between mTORC1, SREBP-1c and PPAR-γ activity remain to be clarified.

Thus, mTORC1 coordinates food intake with energy storage at multiple levels, from central control of food seeking to energy storage and expenditure in peripheral tissues. This multi-level regulation explains the profound consequences that dysregulated mTOR signaling exerts on human metabolism.

Aging

Due to its role at the interface of growth and starvation, mTOR is a prime target in the genetic control of ageing, and evidence from genetic studies supports the view that mTOR may be a master determinant of lifespan and ageing in yeast, C. elegans, flies and mice.

The only ‘natural’ method available to counter ageing is dietary restriction (DR), where the caloric intake is decreased anywhere from 10% to 50%. DR appears to act mainly through the inhibition of mTORC1, and genetic inactivation of mTORC1 pathway components provides no additional benefit over DR. In mice, DR causes lifespan extension and changes in gene expression profile similar to those resulting from loss of S6K1 further supporting the view that DR acts through inhibition of mTORC1

Finally, it remains to be seen whether limiting mTOR activity in adult humans would really enable a longer lifespan, or it would only bring an increase in the quality of life and the way we age, without necessarily affecting how long we live.

mTOR in food allergy

mTOR plays an important role in cow's milk allergy-associated behavioral and immunological deficits

Highlights

mTOR pathway is implicated in gut–brain axis of food allergy-induced ASD-like behavior.

Food allergy is associated with enhanced mTOR signaling in the brain and gut.

mTORC1 inhibitor Rapamycin improved the behavioral deficits of allergic mice.

Rapamycin reduced mTORC1 activity in the brain and gut of allergic mice.

Rapamycin inhibited food allergy and increased the number of Treg cells in the ileum.

5. Conclusions

In conclusion, the current studies provide strong and first evidence

that the enhanced mTOR signaling pathway in the brain as well as in the intestines plays a pivotal role in the behavioral and immunological changes in CMA mice. mTOR might be the linking pin involved in gut-immune-brain axis in ASD and the intestinal tract could be a potential target in the treatment of patients with ASD and comorbid intestinal symptoms. It is a compelling hypothesis that an enhanced mTOR activity throughout the body may account for both the behavioral as well as the gastrointestinal dysfunctions in patients with ASD. Whether inhibition of mTOR is able to treat both allergic and behavioral deficits of ASD patients remains to be further investigated. Importantly, increased gastrointestinal deficits and in particular behavioral abnormalities are commonly reported in other neurodevelopmental diseases such as attention deficit hyperactivity disorder (ADHD), multiple sclerosis , schizophrenia, Parkinson's disease , however the role of mTOR needs to be investigated. Our findings on the gut-immune-brain connection in a murine model of CMA indicate that targeting mTOR signaling pathway might be applicable to various neurological disorders. Future studies focusing on the mTOR signaling pathway should shed more light on the effective treatment of ASD and other neurodevelopmental disorders.

  

mTOR and Autism

Autism-related deficits via dysregulatedeIF4E-dependent translational control

Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of Rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signaling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)—an eIF4E repressor downstream of mTOR—or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.

 Reversing autism by targeting downstream mTOR signaling
 Autism spectrum disorders (ASDs) are a group of clinically and genetically heterogeneous neurodevelopmental disorders characterized by impaired social interactions, repetitive behaviors and restricted interests. The genetic defects in ASDs may interfere with synaptic protein synthesis. Synaptic dysfunction caused by aberrant protein synthesis is a key pathogenic mechanism for ASDs Understanding the details about aberrant synaptic protein synthesis is important to formulate potential treatment for ASDs. The mammalian target of the Rapamycin (mTOR) pathway plays central roles in synaptic protein. Recently, Gkogkas and colleagues published exciting data on the role of downstream mTOR pathway in autism

Previous studies have indicated that upstream mTOR signaling is linked to ASDs. Mutations in tuberous sclerosis complex (TSC) 1/TSC2, neurofibromatosis 1 (NF1), and Phosphatase and tensin homolog (PTEN) lead to syndromic ASD with tuberous sclerosis, neurofibromatosis, or macrocephaly, respectively. TSC1/TSC2, NF1, and PTEN act as negative regulators of mTOR complex 1 (mTORC1), which is activated by phosphoinositide-3 kinase (PI3K) pathway. Activation of cap-dependent translation is a principal downstream mechanism of mTORC1. The eIF4E recognizes the 5′ mRNA cap, recruits eIF4G and the small ribosomal subunit. The eIF4E-binding proteins (4E-BPs) bind to eIF4E and inhibit translation initiation. Phosphorylation of 4E-BPs by mTORC1 promotes eIF4E release and initiates cap-dependent translation. A hyperactivated mTORC1–eIF4E pathway is linked to impaired synaptic plasticity in fragile X syndrome, an autistic disorder caused by lack of fragile X mental retardation protein (FMRP) due to mutation of the FMR1 gene, suggesting that downstream mTOR signaling might be causally linked to ASDs. Notably, one pioneering study has identified a mutation in the EIF4E promoter in autism families, implying that deregulation of downstream mTOR signaling (eIF4E) could be a novel mechanism for ASDs.As an eIF4E repressor downstream of mTOR, 4E-BP2 has important roles in synaptic plasticity, learning and memory. Writing in their Nature article, Gkogkas and colleagues reported that deletion of the gene encoding 4E-BP2 (Eif4ebp2) leads to autistic-like behaviors in mice. Pharmacological inhibition of eIF4E rectifies social behavior deficits in Eif4ebp2 knockout mice. Their study in mouse models has provided direct evidence for the causal link between dysregulated eIF4E and the development of ASDs.Are these ASD-like phenotypes of the Eif4ebp2 knockout mice caused by altered translation of a subset mRNAs due to the release of eIF4E? To test this, Gkogkas et al. measured translation initiation rates and protein levels of candidate genes known to be associated with ASDs in hippocampi from Eif4ebp2 knockout and eIF4E-overexpressing mice. They found that the translation of neuroligin (NLGN) mRNAs is enhanced in both lines of transgenic mice. Removal of 4E-BP2 or overexpression of eIF4E increases protein amounts of NLGNs in the hippocampus, whereas mRNA levels are not affected, thus excluding transcriptional effect. In contrast, the authors did not observe any changes in the translation of mRNAs coding for other synaptic scaffolding proteins. Interestingly, treatment of Eif4ebp2 knockout mice with selective eIF4E inhibitor reduces NLGN protein levels to wild-type levels. These data thus indicate that relief of translational suppression by loss of 4E-BP2 or by the overexpression of eIF4E selectively enhances the NLGN synthesis. However, it cannot be ruled out that other proteins (synaptic or non-synaptic) may be affected and contribute to animal autistic phenotypes.Aberrant information processing due to altered ratio of synaptic excitation to inhibition (E/I) may contribute to ASDs. The increased or decreased E/I ratio has been observed in ASD animal models  In relation to these E/I shifts, Gkogkas et al then examined the synaptic transmission in hippocampal slices of Eif4ebp2 knockout mice. They found that 4E-BP2 de-repression results in an increased E/I ratio, which can be explained by the increase of vesicular glutamate transporter and spine density in hippocampal pyramidal neurons. As expected, application of eIF4E inhibitor restores the E/I balanceFinally, in view of the facts that genetic manipulation of NLGNs results in ASD-like phenotypes with altered E/I balance in mouse models  and NLGN mRNA translation is enhanced concomitant with increased E/I ratio in Eif4ebp2 knockout mice, Gkogkas et al. tested the effect of NLGN knockdown on synaptic plasticity and behaviour in these mice . NLGN1 is predominantly postsynaptic at excitatory synapses and promotes excitatory synaptic transmission. The authors found that NLGN1 knockdown reverses changes at excitatory synapses and partially rescues the social interaction deficits in Eif4ebp2 knockout mice. These findings thus established a strong link between eIF4E-dependent translational control of NLGNs, E/I balance and the development of ASD-like animal behaviors (Figure 1).
In summary, Gkogkas et al. have provided a model for mTORC1/eIF4E-dependent autism-like phenotypes due to dysregulated translational control (Gkogkas et al., 2013). This novel regulatory mechanism will prompt investigation of downstream mTOR signaling in ASDs, as well as expand our knowledge of how mTOR functions in human learning and cognition. It may narrow down therapeutic targets for autism since targeting downstream mTOR signaling reverses autism. Pharmacological manipulation of downstream effectors of mTOR (eIF4E, 4E-BP2, and NLGNs) may eventually provide therapeutic benefits for patients with ASDs.

  

mTOR Inhibition: From Aging to Autism and Beyond

3.3. Autism

As with epilepsy, the link between aberrant mTOR activation and autism is strongest in tuberous sclerosis complex; between 20 and 60% of tuberous sclerosis patients are diagnosed with autism [219, 237], which may account for 1–4% of all autism cases [238]. In addition to tuberous sclerosis, however, there is growing evidence that dysregulated mTOR activity may contribute to a wider variety of autism spectrum disorders. As with epilepsy, mutations in PTEN that lead to aberrant activation of mTOR are associated with autism [239]. In addition, mutations in the downstream mTOR target eukaryotic translation initiation factor 4E (eIF4E) have also been associated with autism [240]. There is also evidence for a strong association between macrocephaly (large head size) early in life and autism spectrum disorders, as well as genetic diseases linked to autism and mTOR hyperactivation, including tuberous sclerosis complex, neurofibromatosis type I, Lhermitte-Duclos syndrome, and Fragile X syndrome [241]. Taken together these data suggest that disinhibited mTOR may cause, or at least contribute to, many cases of autism spectrum disorder. Clinical trials are ongoing to assess whether Everolimus can reduce autistic symptoms in tuberous sclerosis patients.

5. Conclusion

Given the breadth of pathological conditions where mTOR has already been implicated, it seems likely that additional therapeutic uses for mTOR inhibitors will be discovered in the near future. While potential negative effects of mTOR inhibition need to be addressed, they appear generally manageable and, as new mTOR inhibitors continue to be developed, it may be possible to maximize the beneficial effects of targeted mTOR inhibition while reducing adverse effects.

mTOR signaling at a glance

This paper is very comprehensive and this graphic has everything you could ever need to know.  You can use it to figure out your own therapy.

 Source: http://jcs.biologists.org/content/122/20/3589/F1.poster.jpg

mTOR and seizures

Role of the mTOR signaling pathway in epilepsy

Epilepsy, a common neurological disorder and cause of significant morbidity and mortality, places an enormous burden on the individual and society. Presently, most drugs for epilepsy primarily suppress seizures as symptomatic therapies but do not possess actual antiepileptogenic or disease-modifying properties. The mTOR (mammalian target of Rapamycin) signaling pathway is involved in major multiple cellular functions, including protein synthesis, cell growth and proliferation and synaptic plasticity, which may influence neuronal excitability and be responsible for epileptogenesis. Intriguing findings of the frequent hyperactivation of mTOR signaling in epilepsy make it a potential mechanism in the pathogenesis as well as an attractive target for the therapeutic intervention, and have driven the significant ongoing efforts to pharmacologically target this pathway. This review explores the relevance of the mTOR pathway to epileptogenesis and its potential as a therapeutic target in epilepsy treatment by presenting the current results on mTOR inhibitors, in particular, Rapamycin, in animal models of diverse types of epilepsy. Limited clinical studies in human epilepsy, some paradoxical experimental data and outstanding questions have also been discussed.

The ketogenic diet inhibits the mammalian target of Rapamycin (mTOR) pathway

  

The ketogenic diet (KD) is an effective treatment for epilepsy, but its mechanisms of action are poorly understood. We investigated the hypothesis that KD inhibits mammalian target of Rapamycin (mTOR) pathway signaling. The expression of pS6 and pAkt, markers of mTOR pathway activation, was reduced in hippocampus and liver of rats fed KD. In the kainate model of epilepsy, KD blocked the hippocampal pS6 elevation that occurs after status epilepticus. As mTOR signaling has been implicated in epileptogenesis, these results suggest that the KD may have anticonvulsant or antiepileptogenic actions via mTOR pathway inhibition.

Everolimus: A Novel Treatment for Seizures?

Seizure-dependent mTOR activation in 5-HT neurons promotes autism-like behaviors in mice

Highlights

•

Tsc1 deletion in neurons causes epilepsy and autism-like behaviors in mice.

•

Epileptiform activity spreads to the brainstem.

•

mTOR becomes hyperactivated in 5-HT neurons following seizure onset.

•

mTOR hyperactivity in 5-HT neurons causes autism behaviors.

•

Autism-like behaviors can be reversed following treatment with Rapamycin.

---

Abstract

Epilepsy and autism spectrum disorder (ASD) are common comorbidities of one another. Despite the prevalent correlation between the two disorders, few studies have been able to elucidate a mechanistic link. We demonstrate that forebrain specific Tsc1 deletion in mice causes epilepsy and autism-like behaviors, concomitant with disruption of 5-HT neurotransmission. We find that epileptiform activity propagates to the raphe nuclei, resulting in seizure-dependent hyperactivation of mTOR in 5-HT neurons. To dissect whether mTOR hyperactivity in 5-HT neurons alone was sufficient to recapitulate an autism-like phenotype we utilized Tsc1flox^/flox;Slc6a4-cre mice, in which mTOR is restrictively hyperactivated in 5-HT neurons. Tsc1flox^/flox;Slc6a4-cre mice displayed alterations of the 5-HT system and autism-like behaviors, without causing epilepsy. Rapamycin treatment in these mice was sufficient to rescue the phenotype. We conclude that the spread of seizure activity to the brainstem is capable of promoting hyperactivation of mTOR in the raphe nuclei, which in turn promotes autism-like behaviors. Thus our study provides a novel mechanism describing how epilepsy can contribute to the development of autism-like behaviors, suggesting new therapeutic strategies for autism.

mTOR inhibition via carbohydrate restriction

Is there a role for carbohydrate restriction in the treatment and prevention of cancer?

  

Low glycemic index diet reduces symptoms of autism in mice

  

Dietary glycemic index modulates the behavioral and biochemical abnormalities associated with autism spectrum disorder

Amino acids and mTOR

Amino acids activatemTOR complex 2 via PI3K/Akt signaling

The activity of mammalian target of Rapamycin (mTOR) complexes regulates essential cellular processes, such as growth, proliferation or survival. Nutrients such as amino acids are important regulators of mTOR Complex 1 (mTORC1) activation, thus affecting cell growth, protein synthesis and autophagy.

Here, we show that amino acids may also activate mTOR Complex 2 (mTORC2). This activation is mediated by the activity of class I PI3K and of Akt. Amino acids induced a rapid phosphorylation of Akt at Thr308 and Ser473. Whereas both phosphorylations were dependent on the presence of mTOR, only Akt phosphorylation at Ser473 was dependent on the presence of rictor, a specific component of mTORC2. Kinase assays confirmed mTORC2 activation by amino acids. This signaling was functional, as demonstrated by the phosphorylation of Akt substrate FOXO3a. Interestingly, using different starvation conditions, amino acids can selectively activate mTORC1 or mTORC2. These findings identify a new signaling pathway used by amino acids underscoring the crucial importance of these nutrients in cell metabolism and offering new mechanistic insights.

Finally, this report shows the crucial importance of dietary restriction/starvation conditions for understanding the amino acid signaling. Several studies show the effects of amino acid intake in obesity [23,27,28], and of dietary restriction in human cancers [79,80]. Although more physiological studies are needed to link these effects to mTOR complex regulation, it is noteworthy that a study in human muscle shows activation of both mTORC1 and mTORC2 by ingestion of

a leucine-enriched amino acid-carbohydrate mixture [86]. It has been recently described that branched-chain amino acid dietary supplementation increased the average life span in mice and cardiac and skeletal muscle improvement [87] validating the physiological relevance of amino acid supplementation. In this context, we now report that cell supplementation with amino acids can activate both mTOR complexes (Figures 10 and 11). In summary, this manuscript shows for the first time that amino acids can activate mTORC1 and mTORC2 complexes, thus underscoring the crucial importance of these nutrients in cell metabolism and offering new mechanistic insights with potential therapeutic applications in cancer, obesity and aging.

  

Branched-chain amino acid supplementation promotes survival and supports cardiac and skeletal muscle mitochondrial biogenesis in middle-aged mice.

Recent evidence points to a strong relationship between increased mitochondrial biogenesis and increased survival in eukaryotes. Branched-chain amino acids (BCAAs) have been shown to extend chronological life span in yeast. However, the role of these amino acids in mitochondrial biogenesis and longevity in mammals is unknown. Here, we show that a BCAA-enriched mixture (BCAAem) increased the average life span of mice. BCAAem supplementation increased mitochondrial biogenesis and sirtuin 1 expression in primary cardiac and skeletal myocytes and in cardiac and skeletal muscle, but not in adipose tissue and liver of middle-aged mice, and this was accompanied by enhanced physical endurance. Moreover, the reactive oxygen species (ROS) defense system genes were upregulated, and ROS production was reduced by BCAAem supplementation. All of the BCAAem-mediated effects were strongly attenuated in endothelial nitric oxide synthase null mutant mice. These data reveal an important antiaging role of BCAAs mediated by mitochondrial biogenesis in mammals.

  

Amino acid deficiency causing Autism

Amino-acid deficiency underlies rare form of autism

A rare, hereditary form of autism has been found — and it may be treatable with protein supplements.

Genome sequencing of six children with autism has revealed mutations in a gene that stops several essential amino acids being depleted. Mice lacking this gene developed neurological problems related to autism that were reversed by dietary changes, a paper published today in Science shows¹.

Some children with autism have low blood levels of amino acids that can't be made in the body.

“This might represent the first treatable form of autism,” says Joseph Gleeson, a child neurologist at the University of California, San Diego, who led the study. “That is both heartening to families with autism, and also I think revealing of the underlying mechanisms of autism.”

He emphasizes, however, that the mutations are likely to account for only a very small proportion of autism cases. “We don’t anticipate this is going to have implications for patients in general with autism,” says Gleeson. And there is as yet no proof that dietary supplements will help the six children, whose mutations the researchers identified by sequencing the exome — the part of the genome that codes for proteins.

In mice, at least, the chemical imbalance can be treated. The mutant mice had neurological problems typical of mouse versions of autism, including tremors and epileptic seizures. But those symptoms disappeared in less than a week after the mice were put on diets enriched in branched-chain amino acids.

Gleeson’s team has tried supplementing the diets of the children with this form autism, using muscle-building supplements that contain branched-chain amino acids. The researchers found that the supplements restore the children's blood levels of amino acids to normal. As for their autism symptoms, Gleeson says, the “patients did not get any worse and their parents say they got better, but it’s anecdotal”.

  

Restoration of Normal Cerebral Oxygen Consumption with Rapamycin Treatment in a Rat Model of Autism–Tuberous Sclerosis

  

This paper is very recent and suggests, at least in one mouse model, that oxygen consumption in the brain is dysfunction and that this was rescued using the mTOR inhibitor Rapamycin.

  

Tuberous sclerosis (TSC) is associated with autism spectrum disorders and has been linked to metabolic dysfunction and unrestrained signaling of the mammalian target of Rapamycin (mTOR). Inhibition of mTOR by Rapamycin can mitigate some of the phenotypic abnormalities associated with TSC and autism, but whether this is due to the mTOR-related function in energy metabolism remains to be elucidated. In young Eker rats, an animal model of TSC and autism, which harbors a germ line heterozygous Tsc2 mutation, we previously reported that cerebral oxygen consumption was pronouncedly elevated. Young (4 weeks) male control Long–Evans and Eker rats were divided into control and Rapamycin-treated (20 mg/kg once daily for 2 days) animals. Cerebral regional blood flow (¹⁴C-iodoantipyrine) and O₂ consumption (cryomicrospectrophotometry) were determined in isoflurane-anesthetized rats. We found significantly increased basal O₂ consumption in the cortex (8.7 ± 1.5 ml O₂/min/100 g Eker vs. 2.7 ± 0.2 control), hippocampus, pons and cerebellum. Regional cerebral blood flow and cerebral O₂ extractions were also elevated in all brain regions. Rapamycin had no significant effect on O₂ consumption in any brain region of the control rats, but significantly reduced consumption in the cortex (4.1 ± 0.3) and all other examined regions of the Eker rats. Phosphorylation of mTOR and S6K1 was similar in the two groups and equally reduced by Rapamycin. Thus, a Rapamycin-sensitive, mTOR-dependent but S6K1-independent, signal led to enhanced oxidative metabolism in the Eker brain. We found decreased Akt phosphorylation in Eker but not Long–Evans rat brains, suggesting that this may be related to the increased cerebral O₂ consumption in the Eker rat. Our findings suggest that Rapamycin targeting of Akt to restore normal cerebral metabolism could have therapeutic potential in tuberous sclerosis and autism.

Mitochondrial Dysfunction  and mTOR

  

Mitochondrial Dysfunction and Its Relationship with mTOR Signaling and Oxidative Damage in Autism Spectrum Disorders

  

Mitochondria are organelles that play a central role in processes related to cellular viability, such as energy production, cell growth, cell death via apoptosis, and metabolism of reactive oxygen species (ROS). We can observe behavioral abnormalities relevant to autism spectrum disorders (ASDs) and their recovery mediated by the mTOR inhibitor Rapamycin in mouse models. In Tsc2+/- mice, the transcription of multiple genes involved in mTOR signaling is enhanced, suggesting a crucial role of dysregulated mTOR signaling in the ASD model. This review proposes that the mTOR inhibitor may be useful for the pharmacological treatment of ASD. This review offers novel insights into mitochondrial dysfunction and the related impaired glutathione synthesis and lower detoxification capacity. Firstly, children with ASD and concomitant mitochondrial dysfunction have been reported to manifest clinical symptoms similar to those of mitochondrial disorders, and it therefore shows that the clinical manifestations of ASD with a concomitant diagnosis of mitochondrial dysfunction are likely due to these mitochondrial disorders. Secondly, the adenosine triphosphate (ATP) production/oxygen consumption pathway may be a potential candidate for preventing mitochondrial dysfunction due to oxidative stress, and disruption of ATP synthesis alone may be related to impaired glutathione synthesis. Finally, a decrease in total antioxidant capacity may account for ASD children who show core social and behavioral impairments without neurological and somatic symptoms.

PTEN-type Autism and mTOR

Balancing Proliferation and Connectivity in PTEN -associated Autism Spectrum Disorder

Germline mutations in PTEN, which encodes a widely expressed phosphatase, was mapped to 10q23 and identified as the susceptibility gene for Cowden syndrome, characterized by macrocephaly and high risks of breast, thyroid, and other cancers. The phenotypic spectrum of PTEN mutations expanded to include autism with macrocephaly only 10 years ago. Neurological studies of patients with PTEN-associated autism spectrum disorder (ASD) show increases in cortical white matter and a distinctive cognitive profile, including delayed language development with poor working memory and processing speed. Once a germline PTEN mutation is found, and a diagnosis of phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome made, the clinical outlook broadens to include higher lifetime risks for multiple cancers, beginning in childhood with thyroid cancer. First described as a tumor suppressor, PTEN is a major negative regulator of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of Rapamycin (mTOR) signaling pathway—controlling growth, protein synthesis, and proliferation. This canonical function combines with less well-understood mechanisms to influence synaptic plasticity and neuronal cytoarchitecture. Several excellent mouse models of Pten loss or dysfunction link these neural functions to autism-like behavioral abnormalities, such as altered sociability, repetitive behaviors, and phenotypes like anxiety that are often associated with ASD in humans. These models also show the promise of mTOR inhibitors as therapeutic agents capable of reversing phenotypes ranging from overgrowth to low social behavior. Based on these findings, therapeutic options for patients with PTEN hamartoma tumor syndrome and ASD are coming into view, even as new discoveries in PTEN biology add complexity to our understanding of this master regulator

Intellectual Disability (MR) and mTOR

Collybistin binds and inhibits mTORC1 signaling: a potential novel mechanism contributing to intellectual disability and autism

Protein synthesis regulation via mammalian target of Rapamycin complex 1 (mTORC1) signaling pathway has key roles in neural development and function, and its dysregulation is involved in neurodevelopmental disorders associated with autism and intellectual disability. mTOR regulates assembly of the translation initiation machinery by interacting with the eukaryotic initiation factor eIF3 complex and by controlling phosphorylation of key translational regulators. Collybistin (CB), a neuron-specific Rho-GEF responsible for X-linked intellectual disability with epilepsy, also interacts with eIF3, and its binding partner gephyrin associates with mTOR. Therefore, we hypothesized that CB also binds mTOR and affects mTORC1 signaling activity in neuronal cells. Here, by using induced pluripotent stem cell-derived neural progenitor cells from a male patient with a deletion of entire CB gene and from control individuals, as well as a heterologous expression system, we describe that CB physically interacts with mTOR and inhibits mTORC1 signaling pathway and protein synthesis. These findings suggest that disinhibited mTORC1 signaling may also contribute to the pathological process in patients with loss-of-function variants in CB.

mTORC2 as opposed to mTORC1 as a target in Autism Research

The Role of the New mTOR Complex, MTORC2, in Autism Spectrum Disorders

The goal of my DOD-supported research is determine the role of the new mTOR complex (mTORC2) in Autism Spectrum Disorder (ASD). ASD individuals exhibit impaired social interactions, seizures and abnormal repetitive behavior. In addition, 70-80% of autistic individuals suffer from mental retardation. Autism is a heritable genetically heterogeneous disorder and mutations in negative regulators of the mammalian target of Rapamycin complex 1 (mTORC1) signaling pathway, such as PTEN were associated with ASD. Here, we show that in the hippocampus of Pten fb-KO mice – where Pten is conditionally deleted in the murine forebrain – the activity of both mTORC1 and mTORC2 is increased. In addition, Pten fb-KO mice exhibit seizures, learning and memory and social deficits. Our remarkable preliminary data show that genetic inhibition of mTORC2 activity in Pten-deficient mice significantly promotes survival. In addition, Pten-rictor fb- double KO (DKO) mice, in which mTORC2 activity is restored to normal levels, EEG seizures, learning and memory as well as social phenotypes, are all rescued. In the second year, we will study the molecular mechanism underlying this process. These insights hold the promise for new treatment of ASD.

1. Introduction:

Autism represents a heterogeneous group of disorders, which are defined as “autism spectrum disorders” (ASDs). ASD individuals exhibit common features such as impaired social interactions, language and communication, and abnormal repetitive behavior. In addition, 70-80% of autistic individuals suffer from mental retardation1-3. The major goal of this award is to determine the role of mTORC2 in two mouse models of ASD.

Recently, we have shown that mTORC2 plays a crucial role in long-term memory formation. Briefly, mice lacking mTORC2 showed impaired long-lasting changes in synaptic strength (L-LTP) as well as impaired long-term memory (LTM). In addition, we have found that by promoting mTORC2 activity, with a new agent A-443654, it facilitates L-LTP and enhances long-term memory formation in WT mice. Interestingly, mTORC2 activity is altered in both ASD patients and ASD mouse models harboring mutation in Tsc and Pten5,6. Hence, in this proposal we will test the hypothesis that the neurological dysfunction in several ASD mouse models is caused by dysregulation of mTORC2 rather than mTORC1 activity.

4. Key Research Accomplishment

- We developed a way to specifically block mTORC2 activity in Pten-deficient mice.

- Genetic deletion of mTORC2 prolongs the survival of Pten-deficient mice.

- Genetic deletion of mTORC2 dramatically attenuates seizures in Pten-deficient mice.

- Genetic deletion of mTORC2 improves cognitive and social phenotypes in Pten-deficient mice.

5. Conclusion

It has been proposed that the increased mTORC1 in Pten-deficient or Tsc-deficient mice causes the cellular and behavioral phenotypes associated with ASD. Our new data challenge this view and posit that the neurological dysfunction in ASD, at least in the Pten-ASD mouse model, is caused by dysregulation of mTORC2. Hence, these preliminary data are very important since they identified a new signaling pathway involved in ASD and seizure disorders that could be targeted and lead to the development of new treatments for ASD and seizure disorders.

E/I Imbalance in Schizophrenia and Autism

Common Mechanisms of Excitatory and Inhibitory Imbalance in Schizophrenia and Autism Spectrum Disorders

This paper looks really useful and does refer to mTOR, but is not open access

Autism Spectrum Disorders (ASD) and Schizophrenia (SCZ) are cognitive disorders with complex genetic architectures but overlapping behavioral phenotypes, which suggests common pathway perturbations. Multiple lines of evidence implicate imbalances in excitatory and inhibitory activity (E/I imbalance) as a shared pathophysiological mechanism. Thus, understanding the molecular underpinnings of E/I imbalance may provide essential insight into the etiology of these disorders and may uncover novel targets for future drug discovery. Here, we review key genetic, physiological, neuropathological, functional, and pathway studies that suggest alterations to excitatory/inhibitory circuits are keys to ASD and SCZ pathogenesis.

NMDA activation, Sociability and mTOR

NMDA receptor activation regulates sociability by its effect on mTOR signaling activity

Highlights

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Several syndromic forms of ASD are associated with disinhibited activity of mTORC1.

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Rapamycin, an inhibitor of mTORC1, improved sociability in mouse models of TSC.

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NMDA receptor-mediated neurotransmission regulates sociability in mice.

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NMDA receptor activation decreases mTOR signaling activity.

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D-Cycloserine improved sociability in the Balb/c and BTBR mouse models of ASD.

  

Abstract

Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORC1 in neurons (e.g., cerebellar Purkinje cells). mTORC1 is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that Rapamycin, an inhibitor of mTORC1, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORC1 overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORC1 activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are “drivers” of mTORC1 activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders.

Dendritic Spine Dysgenesis in Autism and mTOR

Dendritic spine dysgenesis in autism related disorders

The activity-dependent structural and functional plasticity of dendritic spines has led to the long-standing belief that these neuronal compartments are the subcellular sites of learning and memory. Of relevance to human health, central neurons in several neuropsychiatric illnesses, including autism related disorders, have atypical numbers and morphologies of dendritic spines. These so-called dendritic spine dysgeneses found in individuals with autism related disorders are consistently replicated in experimental mouse models. Dendritic spine dysgenesis reflects the underlying synaptopathology that drives clinically relevant behavioral deficits in experimental mouse models, providing a platform for testing new therapeutic approaches. By examining molecular signaling pathways, synaptic deficits, and spine dysgenesis in experimental mouse models of autism related disorders we find strong evidence for mTOR to be a critical point of convergence and promising therapeutic target.

3. Spine dysgenesis in autism related disorders Spine dysgenesis has been described in autopsy brains of several ARDs, their genetic causes ranging from hundreds of affected genes to one, with their pervasiveness relating to both severity and number of clinical symptoms. By examining common clinical phenotypes correlated to spine and synaptic abnormalities between the disorders, we can work to recognize causalities in dysgenesis and identify potential targets for therapeutic intervention.

4. mTOR: a convergence point of spine dysgenesis and synaptopathologies in ASD Dysgenesis of dendritic spines occurs in the majority of individuals afflicted with ARDs, as well as in most experimental mouse models of these syndromes. It would, therefore, follow that there must be a converging deregulated molecular pathway downstream of the affected genes and upstream of dendritic spine formation and maturation. Identifying this pathway will not only define a causal common denominator in autism-spectrum disorders, but also open new therapeutic opportunities for these devastating conditions. The Ras/ERK and PI3K/mTOR pathways, which regulate protein translation in dendrites near excitatory synapses, have received the most attention as such candidate convergence points

5. Conclusion Cajal once postulated, “the future will prove the great physiological role played by the dendritic spines” [229]. And indeed, it is now widely accepted that dendritic spines are the site of neuronal plasticity of excitatory synapses and the focal point for synaptopathophysiologies of ARDs. Individuals and mouse models of ARDs all display spine dysgenesis, with mTOR-regulated protein translation being a critical point of convergence. Deviations from optimal levels of protein synthesis correlate with the magnitude of dendritic spine pruning and LTD in ARDs. Alleviation of heightened mTOR activity rescues both synaptic and behavioral phenotypes in FXS and TS animals. Correcting mTOR signaling levels also reversed ARD phenotypes in adult fully symptomatic mice, challenging the traditional view that genetic defects caused irreversible developmental defects [230]. More excitingly, these observations demonstrate the potential of pharmacological therapies for neurodevelopmental disorders. The list of ARDs that have been reversed in adult symptomatic mice continues to grow, and also includes RTT [231], DS [232,233], and AS [92]. Together, these findings demonstrate the remarkable plastic nature of the brain and imply that if the causal denominator of ARDs could be found and therapeutically targeted, we may be able to allow the ARD brain to rewire itself and relieve clinical symptoms once believed to be irreversible. The analysis of correlative physiological and behavioral phenotypes and identification of the common mTOR pathway will hopefully provide such potential targets.

  

Clinical Trials

It will be interesting to see the results of the current trials on children with Tuberous Sclerosis Complex, a rare type of autism, that is the most likely to respond to mTOR inhibition.

Rapalogs for Autism Phenotype in TSC: A Feasibility Study (RAPT)

The purpose of this study is to assess the feasibility and safety of administering rapalogs sirolimus or everolimus, in participants with Tuberous Sclerosis Complex (TSC) and self-injury and to measure cognitive and behavioral changes, including reduction in autistic symptoms, self-injurious and aggressive behaviors, as well as improvements in cognition across multiple domains of cognitive function.

Efficacy of RAD001/Everolimus in Autismand NeuroPsychological Deficits in Children With Tuberous Sclerosis Complex (RAPIT)

Tuberous sclerosis complex (TSC) is a genetic disease that leads to mental retardation in over 50% of patients, and to learning problems, behavioral problems, autism and epilepsy in up to 90% of patients. The underlying deficit of TSC, loss of inhibition of the mammalian target of Rapamycin (mTOR) protein due to dysfunction of the tuberin/hamartin protein complex, can be rescued by everolimus. Everolimus has been registered as treatment for renal cell carcinoma and giant cell astrocytoma (SEGA). Evidence in human and animal studies suggests that mTOR inhibitors improve learning and development in patients with TSC.