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

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

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Epileptiform activity spreads to the brainstem.

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

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mTOR hyperactivity in 5-HT neurons causes autism behaviors.

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

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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.

•

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.

  

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.

Autophagy, Mitophagy, Calpains and mTOR in Autism, but also in aging, cancer, diabetes, Alzheimer's, Parkinson's, and Huntington's etc.

 Source:  http://www.tanpaku.org/autophagy/

I am writing a science heavy post all about a protein called mTOR.  It is one of those "cancer proteins" that are now heavily researched, very complicated, but clearly very connected to autism.

In today’s lead-in post, that was not supposed to get complicated, I will introduce new terms, Autophagy, Mitophagy and Calpains. 

There are some very interesting implications from the research, not least that you can reduce mTOR levels just by eating (a lot) less.  Indeed, this “starvation” diet has now been shown by the University of Newcastle to be able to reverse the onset of type 2 diabetes.  It also may suggest another reason for those Somali Autism clusters in the US and Sweden, where refugees from Somalia have been settled.  Just as a starvation diet reduces mTOR, excessive eating increases mTOR.  Via several mechanisms we will see that autism associates with high levels of mTOR.  While the hygiene hypotheses can be used to explain these autism “hotspots” among Somali refugees, a completely different reason might be the switch from relative starvation to an overabundant diet; this would trigger an increase in mTOR and therefore the increase in autism (and later diabetes and cancer in the wider group).

In today’s post we will find out about Autophagy/Mitophagy and see how they are relevant to autism.

We will see how they are generally controlled by mTOR.  PINK1, which we encountered in a previous post will reappear, as will Verapamil, that L-type calcium channel blocker that seems to affect so many things.

Not only does verapamil appear protective towards developing type 2 diabetes, but also now Huntingdon’s Disease.

Autophagy

Autophagy is a very complex process.

Here is a relatively plain English explanation:

The word autophagy is derived from Greek words “auto” meaning self and “phagy” meaning eating. Autophagy is a normal physiological process in the body that deals with destruction of cells in the body.

It maintains homeostasis or normal functioning by protein degradation and turnover of the destroyed cell organelles for new cell formation.

During cellular stress the process of Autophagy is upscaled and increased. Cellular stress is caused when there is deprivation of nutrients and/or growth factors.

Thus Autophagy may provide an alternate source of intracellular building blocks and substrates that may generate energy to enable continuous cell survival.

Autophagy and cell death

Autophagy also kills the cells under certain conditions. These are form of programmed cell death (PCD) and are called autophagic cell death. Programmed cell death is commonly termed apoptosis.

Autophagy is termed a nonapoptotic programmed cell death with different pathways and mediators from apoptosis.

Autophagy mainly maintains a balance between manufacture of cellular components and break down of damaged or unnecessary organelles and other cellular constituents.

There are some major degradative pathways that include proteasome that involves breaking down of most short-lived proteins.

Autophagy and stress

Autophagy enables cells to survive stress from the external environment like nutrient deprivation and also allows them to withstand internal stresses like accumulation of damaged organelles and pathogen or infective organism invasion.

Autophagy is seen in all eukaryotic systems including fungi, plants, slime mold, nematodes, fruit flies and insects, rodents (laboratory mice and rats), humans.

Types of autophagy

There are several types of Autophagy. These are:-

·         microautophagy – in this process the cytosolic components are directly taken up by the lysosome itself through the lysosomal membrane.

·         macroautophagy – this involves delivery of cytoplasmic cargo to the lysosome through the intermediary of a double membrane-bound vesicle. This is called an autophagosome that fuses with the lysosome to form an autolysosome.

·         Chaperone-mediated autophagy – in this process the targeted proteins are translocated across the lysosomal membrane in a complex with chaperone proteins (such as Hsc-70).  

·         micro- and macropexophagy

·         piecemeal microautophagy of the nucleus

·         cytoplasm-to-vacuole targeting (Cvt) pathway

Autophagy & Autism

Loss of mTOR-dependent macroautophagy causes autistic-like synaptic pruning deficits

Developmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2 ± ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2 ± mice, but not in Atg7(CKO) neuronal autophagy-deficient mice or Tsc2 ± :Atg7(CKO) double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.

Verapamil, Autophagy and Calpains

Here we need to introduce another new term, the calpain.

Hyper activation of calpains is a feature of Alzheimer’s and Huntingdon’s disease.  This does lead to altered calcium homeostasis.

Nobody has really studied calpains and autism.  There is research into calpains and TBI (traumatic brain injury).

Since we know there is aberrant calcium channel activity in autism and even excessive physical calcium present in autistic brains, it seems possible that hyper activation of calpains may be occurring in autism.

We also know that calpains play a role in degrading PTEN, which then affects BDNF, in turn affecting mTOR activation.  So everything is highly interrelated.

Calpain may be released in the brain for up to a month after a head injury, and may be responsible for a shrinkage of the brain sometimes found after such injuries.

However, calpain may also be involved in a "resculpting" process that helps repair damage after injury.

Moreover, the hyperactivation of calpains is implicated in a number of pathologies associated with altered calcium homeostasis such as Alzheimer's disease

  

Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis

From: Biomarkers of Brain Injury

 Source:-  Stanford Huntingdon Disease (HD) Project

So if it was the case that in autism, as in HD, that there is excessive calpain activity, then it would be possible to increase autophagy simply by reducing the flow of calcium into the cells. 

So this might be yet another reason why Verapamil may be a good therapeutic choice for some people with autism.

Mitophagy & PINK1

Mitophagy is a necessary ongoing “spring cleaning” of damaged bits of mitochondria.

It appears that in some autism, this process goes awry and damaged mitochondria accumulate.

We saw in early posts that in brain samples from younger people with autism, abnormal mitochondria are typically found.

Mitochondrial abnormalities in temporal lobe of autistic brain

Children with autism have mitochondrial dysfunction, study finds

I should point out that there are various types of mitochondrial disease and dysfunction.

It appears that some people’s autism is solely the result of mitochondrial disease, but a much broader group have some mitochondrial dysfunction.

Mitophagy is the selective degradation of mitochondria by autophagy. It often occurs to defective mitochondria following damage or stress. This process was first mentioned by J.J. Lemasters in 2005, although lysosomes in the liver that contained mitochondrial fragments had been seen as early as 1962, “As part of almost every lysosome in these glucagon-treated cells it is possible to recognize a mitochondrion or a remnant of one. It was also mentioned in 1977 by scientists studying metamorphosis in silkworms, “...mitochondria develop functional alterations which would activate autophagy."  Mitophagy is key in keeping the cell healthy. It promotes turnover of mitochondria and prevents accumulation of dysfunctional mitochondria which can lead to cellular degeneration. It is mediated by Atg32 (in yeast) and NIP3-like protein X (NIX). Mitophagy is regulated by PINK1 and parkin protein. The occurrence of mitophagy is not limited to the damaged mitochondria but also involves undamaged ones.

Autophagy and mitophagy in cellular damage control

Mitochondrial dysfunction due to aberrant mTOR-regulated mitophagy in autism

This Mentored Research Scientist Development Award (K01) is designed to characterize the molecular mechanism underlying mitochondrial dysfunction in autism, with the eventual goal of identifying therapeutic interventions for mitochondrial defects. The applicant (Dr. Guomei Tang) is an Associate Research Scientist at Columbia University Medical Center (CUMC), where internationally renowned basic neuroscience research in psychiatry has been ongoing for many years. CUMC provides a rich environment that supports and encourages Dr. Tang's development and this K01 award will be instrumental for her successful transition to an independent research investigator. Dr. Tang has recruited an outstanding team of mentors, co-mentors, consultants and collaborators with extensive experience in mitochondrial biology and diseases, neuropathology, psychiatry neuropathology, neuroscience, molecular and cell biology, and mTOR-autophagy signaling. These experts will provide her with critical guidance and advice, and enhance her technical and scientific skills for the proposed research. The career development activities include tutorials, directed readings, course work, workshops for mitochondrial biology, skills in collaborating with clinicians and senior scientists, grant writing and presentations, and responsible conduct of research. Dr. Tang's long term research goal is to elucidate the molecular and cellular mechanisms underlying synaptic pathology in autism, and to provide insights into the pathogenesis and potential treatment for autism. To accomplish this, Dr. Tang will use a multidisciplinary approach combining biochemical, histological and imaging techniques to examine mitochondrial autophagy in postmortem autistic brain and mouse models. Her preliminary evidence indicates an association between mitochondrial defects and a dysregulation of mTOR-autophagy signaling in autistic brain. In mouse embryonic fibroblasts (MEFs) and neuronal cultures, mTOR hyperactivation inhibits autophagy, decreases mitochondrial membrane potential and causes an accumulation of damaged mitochondria. These results suggest that mitochondrial dysfunction in autism may result from aberrant mTOR- mediated mitophagy signaling. To address this hypothesis, Dr. Tang proposes 3 specific aims: 1) To determine whether mTOR hyper regulation inhibits neuronal mitophagy and causes mitochondrial dysfunction in ASD mouse models;2) To examine whether enhancing mitophagy rescues mitochondrial dysfunction in ASD mouse models; and 3) To confirm mitophagy defects in ASD postmortem brain and lymphoblasts. These data will be important for understanding the mechanism by which mTOR kinase regulates mitophagy, elucidating the mitochondrial pathophysiology that underlies ASD pathogenesis, and ultimately to design interventions effective in treatment. The knowledge and experience gained from this proposal will lead directly to a study of the effects of mitophagy defects and mitochondria dysfunction on synaptic pathology in autism, which will be proposed in an R01 grant application in 3-4 years of the award

Obesity & Autism

Briefly to return to obesity, since I just saw something interesting…

Since we know that over eating with increase mTOR and that hyper-activated mTOR in associated with several dysfunctions in autism, being obese and autistic is not a good idea.

In the US, where potent “psychiatric” drugs are widely prescribed for autism, almost a third of all adolescents with autism are obese, not just over-weight.  Weight gain is a known side effect of some of these drugs.

CDC Study Flags High Rate of Obesity among Teens with Autism

Conclusion

It would appear that hyperactivated mTOR in autism causes dysfunctions in autophagy/mitophagy.  This causes at least two subsequent dysfunctions:-

 ·        Synaptic pruning dysfunction.  There is a post all about this subject.

 Dendritic Spines in Autism – Why, and potentially how, to modify them

 ·        Mitochondrial dysfunction
 

If hyper activation of calpains is occurring in autism, this would explain some of the odd behaviour of Ca²⁺.  It would also again suggest Verapamil for a broader group of autism.

The numerous other connections between mTOR and autism, will be covered in upcoming post on mTOR, which will even include food intolerance. 

Primary and Secondary Dysfunctions in Autism - plus Candesartan

Sometimes the secondary event can completely overshadow the primary event.  
The above relates to dust explosions (in large silos containing grain, sugar etc.) rather than autism.

As we continue to investigate the science behind autism and associated possible therapies, it is becoming necessary to introduce some further segmentation.

I have referred to autism “flare-ups” many times, but even that term means very different things to different people.

We now have many examples of autism treatments (NAC, Bumetanide etc), once effective, suddenly stopping working in certain people.  This needs explaining.

We know from the research that in most cases, autism is caused by multiple “hits”, only when taken together do they lead to autism.

We also see the “double tap” variety of autism, when relatively mild autism later develops into something more serious, following some event, or trigger.  

Thanks to the internet, we know have numerous n=1 examples of certain drugs showing a positive effect in some people.  You do have to discount all those people trying to sell you something, or support the cause of others trying to sell you something.  We also have full access to all those people who have patented their clever ideas, although 99% never develop them.

Within all this information there are some very useful insights, which can help further our understanding of autism

Candesartan

A case in point is Candesartan, which one reader of this blog brought to my attention, in the comment below.  This drug is used to treat high blood pressure and is often combined with a diuretic.

A very recent study relating to neurodegenerative disease and Parkinson's especially:

http://www.sciencedaily.com/releases/2015/05/150512150022.htm

discusses the use of a new drug as well as another blood pressure drug sometimes used in conjunction with Bumetanide called Candesartan. Their goal in this study was to explore how to attenuate chronic microglial activation (a hallmark of autism) by targeting toll-like receptors TLR1 and TLR2 via these two drugs.

Candesartan also modulates NKCC2 activity:

http://www.ncbi.nlm.nih.gov/pubmed/18305093

which is interesting considering the original cited research above deals with attenuating microglial activation, rather than modulating the chloride levels within GABA inhibitory neurons as Bumetanide does.

Note that Bumetanide affects both NKCC1 and NKCC2 transporters.  NKCC1 is present in the brain at birth, but should not be present in the adult brain.  However, it appears to remain in a large sub-group of those with autism, causing GABA to remain excitatory.  NKCC2 is found specifically in the kidney, where it serves to extract sodium, potassium, and chloride from the urine so that they can be reabsorbed into the blood

This drug is, along with Minocycline, is one of the few that is known to have an effect on microglial activation.

In a clinical trial, Minocycline was shown to have no effect on autism.

I do feel this kind of assessment is too simplistic; so I was interested to see the actual effect of Candesartan in autism, albeit with n=1.

Conveniently somebody has filed a patent for the use of Candesartan in autism.  Within the document is the n=1 case report of its effect.

Example of treatment with Candersartan in autism

[00047] A 16 year old boy with autism was evaluated for behavioral management. He was frequently aggressive, primarily directed to himself but to others as well. These episodes were usually unprovoked but would also occur when his parents attempted to re direct him. The child was essentially non verbal except for echolalia. His comprehension to verbal re direction was limited, making non pharmacological interventions to his aggression limited.

[00048] His neurological exam was otherwise normal.

[00049] An MRI, EEG were normal. Routine studies, including examination for fragile x and other metabolic disorders were negative.

[00050] Prior medication trials included anti convulsants which were without benefit and atypical neuroleptics, which resulted in weight gain and unsatisfactory effects on behavior.

[00051] After obtaining consent from his parents, Candesartan was started. An initial dose of 8 mg resulted in significant attenuation of aggressive behavior. Blood pressure remained stable. After 2 weeks, the dose was raised to 16 mg. Further improvement in aggression was noted with no adverse lowering of blood pressure.

[00052] The patient has remained on Candesartan with beneficial anti aggression effects being maintained over one year.

[00053] A preferred dose found by the inventor to treat autism is approximately O.lmg/kg. In children, a liquid form may be used.

So we can conclude from this that in a non-verbal 16 year old boy with autism, with significant aggressive tendencies, this drug successfully reduced aggression.  Since he was on the drug for a year, there were no other major changes, such as language or cognitive function, otherwise they would surely be mentioned to support the patent.

I can of course look further into why Candesartan might have been effective.

Our blog reader suggested this research:-

Unraveling mystery of a-synuclein inneurodegenerative disease and reversing its course

"The real job of microglia is to keep the brain healthy by getting rid of pathogens as well as cellular debris," says Maguire-Zeiss, "However, in a diseased state microglia can become chronically activated, leading to a continuous onslaught of inflammation which is damaging to the brain."

In this study, the Maguire-Zeiss lab found that only a certain size structures of misfolded α-synuclein can activate microglial cells -- normal protein and even smaller forms of misfolded α-synuclein cannot. Then the researchers sought to discover precisely how microglia responded to misfolded α-synuclein; that is, which of its many "pattern recognition receptors" reacted to the toxic protein.

Microglia use many different pattern recognition proteins, called toll-like receptors (TLR), to recognize potential threats. The investigators found that misfolded α-synuclein caused TLR1 and TLR2 to come together into one complex (receptor), creating TLR1/2. They traced the entire molecular pathway from the protein's engagement of TLR1/2 at the cell surface to the production of inflammatory molecules.

Then Maguire-Zeiss and her team tested a drug, developed by researchers at the University of Colorado, which specifically targets TLR1/2. They also tested the hypertension drug candesartan, which can target TLR2. Both agents significantly reduced inflammation.

I found some other possible explanations:-

Angiotensin II AT1Receptor Blockade Ameliorates Brain Inflammation

Brain inflammation has a critical role in the pathophysiology of brain diseases of high prevalence and economic impact, such as major depression, schizophrenia, post-traumatic stress disorder, Parkinson's and Alzheimer's disease, and traumatic brain injury. Our results demonstrate that systemic administration of the centrally acting angiotensin II AT₁ receptor blocker (ARB) candesartan to normotensive rats decreases the acute brain inflammatory response to administration of the bacterial endotoxin lipopolysaccharide (LPS), a model of brain inflammation. The broad anti-inflammatory effects of candesartan were seen across the entire inflammatory cascade, including decreased production and release to the circulation of centrally acting proinflammatory cytokines, repression of nuclear transcription factors activation in the brain, reduction of gene expression of brain proinflammatory cytokines, cytokine and prostanoid receptors, adhesion molecules, proinflammatory inducible enzymes, and reduced microglia activation. These effects are widespread, occurring not only in well-known brain target areas for circulating proinflammatory factors and LPS, that is, hypothalamic paraventricular nucleus and the subfornical organ, but also in the prefrontal cortex, hippocampus, and amygdala. Candesartan reduced the associated anorexic effects, and ameliorated associated body weight loss and anxiety. Direct anti-inflammatory effects of candesartan were also documented in cultured rat microglia, cerebellar granule cells, and cerebral microvascular endothelial cells. ARBs are widely used in the treatment of hypertension and stroke, and their anti-inflammatory effects contribute to reduce renal and cardiac failure. Our results indicate that these compounds may offer a novel and safe therapeutic approach for the treatment of brain disorders.

However the underlying mechanism may indeed be (yet again) activating PPAR γ.

Involvement of PPAR-γ in the neuroprotective and anti-inflammatory effects of angiotensin type 1 receptor inhibition: effects of the receptor antagonist telmisartan and receptor deletion in a mouse MPTP modelof Parkinson's disease


This paper suggests that the effect of Candesartan on microglia is :-


"Several recent studies have shown that angiotensin type 1 receptor (AT1) antagonists such as candesartan inhibit the microglial inflammatory response and dopaminergic cell loss in animal models of Parkinson's disease. However, the mechanisms involved in the neuroprotective and anti-inflammatory effects of AT1 blockers in the brain have not been clarified. A number of studies have reported that AT1 blockers activate peroxisome proliferator-activated receptor gamma (PPAR γ). PPAR-γ activation inhibits inflammation, and may be responsible for neuroprotective effects, independently of AT1 blocking actions."

Primary Autism Dysfunctions

I define Primary Autism Dysfunctions as those core dysfunctions that are always present.

So in the case of Monty, aged 11 with ASD, the primary dysfunctions include:-

·        GABA_A dysfunction, due to over expression of NKCC1,  leading to excitatory imbalance

·        Oxidative stress

In some other people the primary dysfunctions are quite different:-

·        Mitochondrial disease

·        etc...

I think that most aggressive behavior resulting from these dysfunctions can be traced back to communication problems and frustration.  So if the person is non-verbal and cannot get what he/she wants, aggression may follow; or if the person has pain and cannot understand it or seek help he may lash out at his care giver.

Secondary Autism Dysfunctions

I define Secondary Autism Dysfunctions as additional dysfunctions that can appear and disappear over time, these are my "flare-ups".

These dysfunctions can be more disabling that the Primary Autism Dysfunctions and it appears these are the dysfunctions that may trigger un-prompted self-injury and other random aggression.

These secondary dysfunctions can be so strong that they completely outweigh the primary dysfunction, giving the effect that the treatment for the primary dysfunction has “stopped working”.

It appears that many  Secondary Autism Dysfunctions are linked to an “over activated immune system”.  It does appear that from the research that activated microglia is an expression of this immune state and we saw one researcher calling the microglia the brain's “immunostat”. 

So in the case of Monty, aged 11 with ASD, the secondary dysfunctions are:-

·        over activated immune system / activated microglia

·        mast cell degranulation as a trigger

·        Il-6 from dissolving milk teeth as a trigger

·        Emotional distress (aged 8, when his long-time assistant left) as trigger (Emotional distress is known to cause oxidative stress)

In other people the secondary dysfunctions may be similar or quite different, for example:-

·        over activated immune system / activated microglia

·        leaky gut with GI problems as a trigger

·        food intolerance as a trigger

·        bacterial infection, with remission while on antibiotics, as a trigger

·        etc …

So I think the trial of Minocycline may have failed because the subjects were only affected by Primary Autism Dysfunctions.

I think the 16 year old aggressive boy in the Candersartan patent most likely had big Secondary Autism Dysfunctions.  The drug reduced microglial activation and so damped the effect of whatever his particular triggers were.

So probably Minocycline should be trialed again, but only in people with autism and regular SIB and aggression.  Success would be measured as a reduction in violent events.

Drugs targeting Primary Autism Dysfunctions should show things like:-

·        Cognitive improvement

·        Increased speech

·        Improved social interactions

·        Reduction in stereotypy

·        Reduction in anxiety (in higher functioning cases)

So I could classify my own interventions as

Primary

·        Bumetanide

·        Low dose Clonazepam

·        NAC

·        Sulforaphane (broccoli)

·        Atorvastatin

·        Potassium

Secondary

·        Verapamil

·        Sytrinol/Tangeretin PPAR-γ agonist for microglia

·        Occasional use of Ibuprofen (anti IL-6 therapy)

·        Quercetin/Azelastine/ Fluticasone Propionate for mast cells

The over activated immune system/activated microglia needs a trigger

Just like a modern plastic explosive is completely harmless to touch and needs the combination of extreme heat and shock wave from a detonator, it appears that the activated microglia, commonly found in autism, is in itself harmless, like Play-Doh, without a trigger.

But with a trigger, you probably know what can happen next.

What about all those failed clinical trials? False Negatives?

So now you not only need to match the trial therapy with the correct sub-type of autism, but you also cannot reliably trial a drug for a Primary Dysfunction, if there is an "active" Secondary Dysfunction.

This is indeed the reason why I do not try new therapies during the summer pollen season.

Perhaps this partly explains why clinical trials in autism always seem to fail.