Showing posts with label D-Serine. Show all posts
Showing posts with label D-Serine. Show all posts

Monday 3 April 2017

Different Types of Excitatory/Inhibitory Imbalance in Autism, Fragile-X & Schizophrenia

There is much written in the complex scientific literature about the Excitatory/Inhibitory (E/I) imbalance between neurotransmitters in autism. 

Many clinical trials have already been carried out, particularly in Fragile-X.  These trials were generally ruled as failures, in spite of a significant minority who responded quite well in some of these trials.

As we saw in the recent post on the stage II trial of bumetanide in severe autism, there is so much “background noise” in the results from these trials and it is easy to ignore a small group who are responders.  I think if you have less than 40%, or so, of positive responders they likely will get lost in the data. 

You inevitably get a significant minority who appear to respond to the placebo, because people with autism usually have good and bad days and testing is very subjective.

There are numerous positive anecdotes from people who participated in these “failed” trials.  If you have a child who only ever speaks single words, but while on the trial drug starts speaking full sentences and then reverts to single words after the trial, you do have to take note. I doubt this is a coincidence.

Here are some of the trialed drugs, just in Fragile-X, that were supposed to target the E/I imbalance:-

Metabotropic glutamate receptor 5 (mGluR5) antagonist

·        Mavoglurant

·        Lithium

mGluR5 negative allosteric modulator

·        Fenobam

N-methyl-D-aspartic acid (NMDA) antagonist

·        Memantine

Glutamate re-uptake promoter

·        Riluzole

Suggested to have effects on NMDA & mGluR5 & GABAA

·        Acamprosate

GABAB agonist

·        Arbaclofen

Positive allosteric modulator (PAM) of GABAA receptor

·        Ganaxolone

Best not to be too clever

Some things you might use to modify the E/I imbalance can appear to have the opposite effect, as was highlighted in the comments in the post below:-

So whilst it is always a good idea to try and figure things out, you may end up getting things the wrong way around, mixing up hypo and hyper.

The MIT people who work on Fragile-X are really clever and they have not figured it all out.

Fragile-X and Idiopathic Autism

Fragile-X gets a great deal of attention, because its biological basis is understood.  It results in a failure to express the fragile X mental retardation protein (FMRP), which is required for normal neural development.

We saw in the recent post about eIF4E, that this could lead to an E/I imbalance and then autism.

Our reader AJ started looking at elF4E and moved on to EIF4E- binding protein number 1.

In the green and orange boxes below you can find elF4E and elF4E-BP2.

This has likely sent some readers to sleep, but for those whose child has Fragile-X, I suggest they read on, because it is exactly here that the lack of fragile X mental retardation protein (FMRP) causes a big problem.  The interaction between FMRP on the binding proteins of elF4E, cause the problem with neuroligins (NLGNs), which causes the E/I imbalance.  Look at the red oval shape labeled FMRP and green egg-shaped NLGNs.

In which case, while AJ might naturally think Ribavirin is a bit risky for idiopathic autism, it might indeed be very effective in some Fragile-X.  You would hope some researcher would investigate this.

Can you have more than one type of E/I imbalance?

Readers whose child responds well to bumetanide probably wonder if they have solved their E/I imbalance.

I think they have most likely improved just one dysfunction that fits under the umbrella term E/I imbalance.  There are likely other dysfunctions that if treated could further improve cognition and behavior.

On the side of GABA, it looks like turning up the volume on α3 sub-unit and turning down the volume on α5 may help. We await the (expensive) Down syndrome drug Basmisanil for the latter, given that the cheap 80 year old drug Cardiazol is no longer widely available. Turning up the volume on α3 sub-unit can be achieved extremely cheaply, and safely, using a tiny dose of Clonazepam.

It does appear that targeting glutamate is going to be rewarding for at least some of those who respond to bumetanide.

One agonist of NMDA receptors is aspartic acid. Our reader Tyler is a fan of L-Aspartic Acid, that is sold as a supplement that may boost athletic performance.  

Others include D-Cycloserine, already used in autism trials; also D-Serine and L-Serine.

D-Serine is synthesized in the brain from L-serine, its enantiomer, it serves as a neuromodulator by co-activating NMDA receptors, making them able to open if they then also bind glutamate. D-serine is a potent agonist at the glycine site of NMDA receptors. For the receptor to open, glutamate and either glycine or D-serine must bind to it; in addition a pore blocker must not be bound (e.g. Mg2+ or Pb2+).

D-Serine is being studied as a potential treatment for schizophrenia and L-serine is in FDA-approved human clinical trials as a possible treatment for ALS/Motor neuron disease.  

You may be thinking, my kid has autism, what has this got to do with ALS/Motor neuron disease (from the ice bucket challenge)? Well one of the Fragile-X trial drugs at the beginning of this post is Riluzole, a drug developed for specially for ALS.  Although it does not help that much in ALS, it does something potentially very useful for some autism, ADHD and schizophrenia; it clears away excess glutamate.

Fragile-X is likely quite different to many other types of autism

I suspect that within Fragile-X there are many variations in the downstream biological dysfunctions and so that even within this definable group, there may be no universal therapies.  So for some people an mGluR5 antagonist may be appropriate, but not for others.

Even within this discrete group, we come back to the need for personalized medicine.

I do not think Fragile-X is a good model for broader autism.

Glutamate Therapies

There are not so many glutamate therapies, so while the guys at MIT might disapprove, it would not be hard to apply some thoughtful trial and error.

You have:


     ·        mGluR5 agonists (only research compounds)

·        mGluR5 positive allosteric modulators (only research compounds)

·        mGluR5 antagonists (Mavoglurant, Lithium)

·        mGluR5 negative allosteric modulators (Fenobam, Pu-erh tea decreases mGluR5 expression )

Today you can only really treat too much mGluR5 activity.  It there is too little activity, the required drugs are not yet available.  I wonder how many people with Fragile-X are drinking Pu-erh tea, it is widely available.

NMDA agonists

D-Cycloserine an antibiotic with similar structure to D-Alanine (D-Cycloserine was trialed in autism and schizophrenia)

ɑ-amino acids:

·         Aspartic acid (trialed and used  by Tyler, suggested for schizophrenia)

·         D-Serine (trialed in schizophrenia)

NMDA antagonists

·        Memantine (widely used off-label in autism, but failed in clinical trials)

·        Ketamine (trialed intra-nasal in autism)

Glutamate re-uptake promoters via GLT-1

·        Riluzole

·        Bromocriptine

·        Beta-lactam antibiotics

Thursday 11 June 2015

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. 

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:-

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.

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

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.

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.


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 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

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.


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.

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.

mTOR and seizures

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 (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.


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.

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



Amino acids and mTOR

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.


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

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”.


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
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

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

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 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

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

Several syndromic forms of ASD are associated with disinhibited activity of mTORC1.
Rapamycin, an inhibitor of mTORC1, improved sociability in mouse models of TSC.
NMDA receptor-mediated neurotransmission regulates sociability in mice.
NMDA receptor activation decreases mTOR signaling activity.
D-Cycloserine improved sociability in the Balb/c and BTBR mouse models of ASD.

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

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.

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.

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.