Pan-agonists of PPARs and PGC-1α in Mitochondrial Disease, Autism and Sport

Today’s post should be of interest to those concerned about mitochondrial disease and mTOR.

mTOR is a very important signaling cascade that often dysfunctional in autism. Many aspects of autism and its comorbidities can be traced back to mTOR.

The going is easier with a PPAR pan-agonist 

mTOR integrates the input from upstream pathways, including insulin, growth, and amino acids.   mTOR also senses cellular nutrient, oxygen, and energy levels. The mTOR pathway is a central regulator of metabolism and physiology, with important roles in the function of tissues including liver, muscle, adipose tissue, and the brain.  It is dysregulated in human diseases, such as diabetes, obesity, certain cancers and indeed autism.

One important process affected by mTOR is the creation of new mitochondria in your cells.  Each cell has many mitochondria, but in some people there are not enough and/or they may not work properly.  

Mitochondrial Disease and Autism

In the above post we saw that Oxidative phosphorylation (or OXPHOS in short) is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing energy.  This takes place inside mitochondria.

The five enzymes required have simplified names: complex I, complex II, complex III, complex IV, and complex V.

The most common problem in autism is a lack of complex 1, this leads to a lack in the production of energy (ATP) in cells.  In your muscles this will appear as a lack of exercise endurance and in your brain as a lack of cognitive function.

On that rather intimidating chart (below), all about mTOR, tucked away at the bottom right is PGC-1α.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is the master regulator of mitochondrial biogenesis.

PGC-1α may be also involved in controlling blood pressure, regulating cellular cholesterol homoeostasis, and the development of obesity.

PGC-1α is thought to be a master integrator of external signals. It is known to be activated by a many factors, including:-

·         Exercise  (gradual endurance training)

·         PPARδ , PPARγ and it was thought PPARα

·         AMPK (Metformin, or AICAR)

·         Sirt-1 (resveratrol and other polyphenolic ‎compounds)

Interestingly, massage therapy appears to increase the amount of PGC-1α which leads to the production of new mitochondria. Many autism parents believe in various massage therapies. 

Metformin is a very old drug to treat diabetes, it does activate AMPK but unfortunately it also inhibits the Complex 1 mitochondrial enzyme. This might explain why one reader of this blog found it had a negative effect in her son.  In some types of cancer metformin can be used to “starve” the cancer cells of energy and stop them proliferating.

Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis

AICAR was thought to have been used by cyclists in the 2009 Tour de France, it is a heart drug from the 1980s. It activates AMPK and increases nitric oxide production from endothelial nitric oxide synthase.

Here is the lower right part enlarged:-

  

The above chart, while complex does not give the complete picture regarding PPAR.

It appears that the type of PPAR that is needed to activate PGC-1α  is actually PPARδ  (PPAR delta). For a long time researchers thought it was PPAR α (PPAR alpha).

PPAR delta, but not PPAR alpha,activates PGC-l alpha gene transcription in muscle

PGC-1 alpha induces mitochondrial biogenesis in muscle and its activity has been related to insulin sensitization. Here, we report that fibrates induce PGC-1 alpha gene expression in muscle both in vivo and in vitro. However, only activation via PPAR delta but not PPAR alpha underlies this effect. PPAR delta induces PGC-1 alpha gene transcription through a PPAR-response element in the PGC-1 alpha promoter. Moreover, PGC-1 alpha coactivates the PPAR delta-responsiveness of its own gene. A further positive autoregulatory loop of control relies on the induction of PPAR6 expression by PGC-1 alpha. These data point to a distinct value of PPARdelta rather than PPAR alpha agonists in the improvement of oxidative metabolism in muscle.

Peroxisome proliferator-activated receptors (PPARs)

There was a post in this blog a long time ago about all the PPARs. There are three types (alpha, delta and gamma) just to confuse us, sometimes delta is called beta.

• α (alpha) - expressed in liver, kidney, heart, muscle, adipose tissue, and others
• β/δ (beta/delta) - expressed in many tissues but markedly in brain, adipose tissue, and skin
• γ (gamma) - although transcribed by the same gene, this PPAR through is expressed in three forms:
• γ1 - expressed in virtually all tissues, including heart, muscle, colon, kidney, pancreas, and spleen
• γ2 - expressed mainly in adipose tissue
• γ3 - expressed in macrophages, large intestine, white adipose tissue.

It does seem that activating alpha, gamma and delta has potential benefit.

The PPAR alpha agonist PEA is available as a supplement and as food for medical purposes In Italy and Spain.  It has been proposed for various inflammatory and pain syndromes. A large trial at a Skoda car factory in 1972 showed that PEA was protective against flu and the common cold.

Palmitoylethanolamide:A Natural Body-Own Anti-Inflammatory Agent, Effective and Safe against Influenza and Common Cold 

Fibrate drugs are PPAR alpha agonist drugs used to lower cholesterol. A key point here is that these drugs also activate other types of PPAR as well.

PPAR gamma agonists are widely used to treat diabetes.  They improve insulin sensitivity and decrease some inflammatory responses. They lower cholesterol.

PPAR delta has various antidiabetic effects and agonism of PPAR delta changes the body's fuel preference from glucose to lipids. Recently it was shown that PPAR delta can be activated to promote biogenesis of mitochondria.

It does appear likely that there is some interaction between the PPARs.

Using the mild PPAR gamma agonist, Sytrinol, which gives a long term cholesterol lowering effect, gives a short term cognitive and behavioral improvement in autism.

Pioglitazone is used to lower glucose levels in type 2 diabetes and is a PPAR gamma agonist.  It has been shown to have a positive effect in autism and more trials are in progress. It also binds to a lesser extent to PPAR alpha.

Our reader Maja is investigating whether Sytrinol will maintain its initial good effect when combined with a mild PPAR alpha agonist, like PEA. 

Pan-agonists of PPAR

Bezafibrate appears to be the best known “pan-agonist” of PPAR alpha, gamma and delta.

The PPARpan-agonist bezafibrate ameliorates cardiomyopathy in a mouse model of Barth syndrome 

   
Bezafibrate as treatment option in patients with mitochondrial complex I (CI) deficiency

These results support bezafibrate as a promising treatment option for specific subgroups of patients with CI deficiency.

Less well known is the natural substance Berberine. 

Berberine Is a Potent Agonist of Peroxisome Proliferator Activated Receptor Alpha

Berberine activates peroxisome proliferator-activated receptor gamma to increase atherosclerotic plaque stability in Apoe-/- mice with hyperhomocysteinemia. 

Effect of berberine on PPAR alpha/delta/gamma expression in type 2 diabetic rat retinae.

The multifaceted drug Telmisartan, from a recent post, is also a pan-agonist of PPARs. It is usually quoted as being a PPAR delta agonist. 

Telmisartan increases lipoprotein lipase expression via peroxisome proliferator-activated receptor-alpha in HepG2 cells.

Telmisartan is a promising cardiometabolic sartan due to its unique PPAR-gamma-inducing property

Angiotensin II receptor blockertelmisartan enhances running endurance of skeletal muscle through activation of the PPAR-δ/AMPK pathway.

AICAR

The drug AICAR is thought of as an AMPK activator rather than a PPAR agonist, but it does affect all three types of PPAR.

AICAR Protects against High Palmitate/High Insulin-Induced Intramyocellular Lipid Accumulation and Insulin Resistance in HL-1 Cardiac Cells by Inducing PPAR-Target Gene Expression

Treatment with AICAR induced gene expression of all three PPARs, but only the Ppara and Pparg regulation were dependent on AMPK.

Conclusion

It looks like some athletes, seeking an advantage, are already using the above strategies to improve their exercise endurance; having more mitochondria is of course a competitive advantage.  A list of all the substances banned in sport might be another good source of therapies not only for autism, but also dementia.

Since mitochondrial dysfunction is a feature of Parkinson’s, Huntington’s and Alzheimer’s there are some investigations ongoing. There is even a trial to perk up the mitochondria in people with Bipolar using Bezafibrate.

It is odd that Sytrinol has only a short term positive effect in most people with autism, although our reader RG’s daughter has a long term benefit. I suspect some people may need a pan-agonist, there may be some interaction/crosstalk/ feedback that we are not aware of.

It would be nice to have some data on the relative potency of Bezafibrate,  Telmisartan and Berberine across alpha, delta and gamma receptors, otherwise we are left with trial and error.

The advantage of Berberine is that it is an OTC supplement.

AICAR is also interesting.

Angiotensin II in the Brain & Therapeutic Considerations

In a previous post I suggested that another cheap generic drug (an ACE inhibitor) could potentially be repurposed to treat schizophrenia and some autism. The original idea was related more to modifying the immune/inflammatory response in the body, rather than the brain.  There is however plenty of research regarding Angiotensin within the brain and the numerous roles it plays.

Juggling - maximizing effects, while minimizing
drug interventions

You may recall in the earlier post that in both schizophrenia and autism there is elevated angiotensin II.

In the brain there are two types of angiotensin receptor, AT1 and AT2.  Their actions are opposing each other.

In many kinds of disease we would want to stimulate AT2, but inhibit AT1.

AT2 is thought to be important for cognitive function and is now a target for Alzheimer’s research.

Using an ACE inhibitor you reduce the amount of angiotensin II and so in effect inhibit both AT1 and AT2.

In theory angiotensin II should not cross the blood brain barrier (BBB), so we should be dealing with centrally produced (i.e. inside the brain) angiotensin II.  In practical terms it seems that people with high levels of angiotensin II may have a permeable BBB.

This is relevant because most ACE inhibitors do not cross the BBB, but the original ACE inhibitor called Captopril does cross the BBB.  So if a centrally acting ACE inhibitor were found to be required, it was discovered 40 years ago.

A therapy would ideally be targeted selectively at AT1 or AT2 receptors.  An AT1 blocker might treat for stress-induced disorders.  An experimental AT2 receptor agonist, called compound 21, is now available and is expected to reduce inflammation and oxidative stress.

Compound 21, a selective agonist of angiotensin AT2 receptors, prevents endothelial inflammation and leukocyte adhesion in vitro and in vivo.

Angiotensin II receptor AT1 antagonists are widely used drugs indicated for hypertension, diabetic nephropathy and congestive heart failure. They block effect of Angiotensin on AT1 and might be good in the brain.

We would like to increase the effect on AT2, we could do that with more Angiotensin II, but then we would make things worse with AT1.

                          Do nothing  ACE inhibitor    AT1 antagonist      AT2 agonist

Effect on AT1               none                            good                                     good                          none

Effect on AT2               none                            bad                                       none                          good

AT1 antagonists are widely available and seen as well tolerated.

AT1 antagonists appear to protect against Alzheimer’s.

The only AT2 agonist is an experimental drug called Compound 21.

The only ACE inhibitor that should affect AT2 in the brain is Captopril and so may be an unwise choice. It will reduce Angiotensin II in the brain and in the rest of the body.


Why were we interested in Angiotensin?

Targeting Angiotensin in Schizophrenia and Some Autism

In the original Angiotensin post in this blog we saw that in schizophrenia and some autism, that Angiotensin II is elevated.  We also saw that:-

·        Blocking angiotensin-converting enzyme (ACE) induces those potent regulatory T cells that are lacking in autism and modulates Th1 and Th17 mediated autoimmunity.  See my last post on Th1, Th2 and Th17. 

·        In addition, Angiotensin II affects the function of the NKCC1/2 chloride cotransporters that are dysfunctional in much autism and at least some schizophrenia.

·        It should also reduce any troubling high levels of leptin, which we saw in another post is an issue in most autism

So the idea was that many broadly anti-inflammatory effects of reducing Angiotensin II might be helpful in autism.

But what about inside the brain?

Angiotensin in the Brain

Here we do get to the science, but I will start with the conclusion. We actually want more effect from the Angiotensin AT2 receptor, which should give numerous benefits, but have no means of achieving this. What we can do is make sure we do not reduce AT2 activity, this means better to use and AT1 antagonist, rather than an ACE inhibitor.

The science supporting the use of an AT agonist follows:-

In the text you will see ARB and compound 21. Both are doing good things. The suggestion is that by doing all these good things there should be improved cognitive function; this has yet to be proved in human tests.

ARB = Angiotensin Receptor AT1 Blocker

Compound 21 = Angiotensin Receptor AT2 agonist

Roles of Brain Angiotensin II in Cognitive Function and Dementia

The brain renin-angiotensin system (RAS) has been highlighted as having a pathological role in stroke, dementia, and neurodegenerative disease. Particularly, in dementia, epidemiological studies indicate a preventive effect of RAS blockade on cognitive impairment in Alzheimer disease (AD). Moreover, basic experiments suggest a role of brain angiotensin II in neural injury, neuroinflammation, and cognitive function and that RAS blockade attenuates cognitive impairment in rodent dementia models of AD. Therefore, RAS regulation is expected to have therapeutic potential for AD. Here, we discuss the role of angiotensin II in cognitive impairment and AD. Angiotensin II binds to the type 2 receptor (AT₂) and works mainly by binding with the type 1 receptor (AT₁). AT₂ receptor signaling plays a role in protection against multiple-organ damage. A direct AT₂ receptor agonist is now available and is expected to reduce inflammation and oxidative stress and enhance cell differentiation. We and other groups reported that AT₂ receptor activation enhances neuronal differentiation and neurite outgrowth in the brain. Here, we also review the effect of the AT₂ receptor on cognitive function. RAS modulation may be a new therapeutic option for dementia including AD in the future.

Figure 1: Possible effect of angiotensin II on neurovascular unit. AT₂: angiotensin II type 2 receptor, AchR: acetylcholine receptor, BBB: blood brain barrier, and TGF-β: transforming growth factor β.

Figure 2: Effect of angiotensin II type 2 receptor signaling on cognitive function. AT₂: angiotensin II type 2 receptor, ATIP: AT₂ receptor-interacting protein, Id1: inhibitor of DNA binding protein 1, MMS2: methyl methanesulfonate-sensitive 2, NO: nitric oxide, SHP-1: Src homology 2 domain-containing protein-tyrosine phosphatase 1, and Ubc-13: ubiquitin conjugating enzyme 13.

Figure 3: Effect of angiotensin II on cognitive function. ACE: angiotensin converting enzyme inhibitor, AT₁: angiotensin II type 1 receptor, AT₂: angiotensin II type 2 receptor, and ARB: angiotensin II type 1 receptor blocker.

Continuous stimulation with angiotensin II may damage neurons via multiple cascades through AT₁ receptor stimulation. On the other hand, stimulation of the AT₂ receptor is expected to prevent neural damage and cognitive impairment (Figure 3). However, it is difficult to perform clinical intervention studies to confirm the results of animal studies because of the long-term progression of cognitive impairment. Moreover, in clinical practice, it is not possible to exclude the antihypertensive effect of RAS blockade on cognition in patients with hypertension. However, RAS modulation may be a new therapeutic option for dementia including AD in the future. Therefore, the hypothesis that RAS regulation affects future cognitive function should be confirmed with carefully designed clinical studies.

Which ARB (Angiotensin Receptor Blocker) for Autism?

Very many biological markers are disturbed in autism and many of them seem to be best ignored, you cannot “correct” them all.

However, there will be an underlying reason behind each one of them being disturbed.

As we saw in the recent post on metabolic syndrome, it is not uncommon to find a cascade of downstream problems that might seem to indicate a huge list of drugs.  A different approach is required, it is necessary to treat the underlying (upstream) problems and have a much shorter list of therapies.

We saw in the post on leptin that the elevated levels in autism are treatable, but is there any point?

We have a long list of other things that might be useful in autism and it would be nice to have a single therapy that might address many of them.

It appears that selecting the optimal ARB might give the opportunity to address numerous issues at once.

Telmisartan seems to have numerous potentially useful additional effects:

·        Acts as a PPAR gamma agonist, like the glitazone drugs shown effective in autism trials

·        Acts as a PPAR delta agonist, which should activate the impaired PPARδ  PGC-1α signaling pathway, and enhance mitochondrial biogenesis. This should help people with mitochondrial disease and should be evident by increased exercise endurance and, in theory, improved cognitive function.

·        Telmisartan regulates the Bcl-2 cancer gene, implicated in autism

BDNF-Akt-Bcl2 antiapoptotic signaling pathway is compromised in the brain of autistic subjects

While the effect in autism is complex, Telmisartan is already seen as a potent target for prevention and treatment in human prostate cancer

·        Telmisartan and other ARBs appear to give protection from Alzheimer’s Disease (suggested to be via its effect on PPAR gamma). Perhaps useful for young adults with Down Syndrome, where early onset Alzheimer’s is expected?

·       Telmisartan and other ARBs have a tendency to increase the level of potassium in blood. Up to 10% of people would experience mild hyperkalemia.  For people with autism taking bumetanide, this effect on potassium might actually be helpful. They would need to reduce their potassium supplementation, or might need none at all.

Telmisartan in clinical trials related to autism

As is repeatedly the case, schizophrenia research is again more advanced than autism research. A quick check showed this:-

Telmisartan Completed Phase 4 Trials for Schizoaffective Disorders / Schizophrenia Treatment

This is a 12-week, randomized, double-blinded, placebo-controlled trial of telmisartan 80 mg/day as an adjunctive to clozapine or olanzapine therapy, in 70 schizophrenia subjects to examine telmisartan's effect on glucose metabolism, weight, food intake, resting energy expenditure, and body composition. In addition, the study will examine insulin's effects on psychopathology and cognition.

Conclusion

We currently have no possibility of something like Compound 21, but Telmisartan looks very interesting and it would nice if those psychiatrists who have trialed it in schizophrenia would do the same in autism.  

It looks like the beneficial effects should come at a lower dose than that used to lower blood pressure. In the schizophrenia trial I think they used a higher dose (80mg) than necessary, I suppose they wanted to maximize their chance of success.  In order to minimize any possible negative effects, I would suggest the psychiatrists trial 20mg in youth with autism.

There will be a post on PPAR delta and mitochondrial disease, because there are at least two other ways to target mitochondrial disease in this way, if you do not like Telmisartan.  There is the cheap drug Bezafibrate and the supplement berberine.

Metabolic Syndrome & Autism

Today’s post is not just about autism.

Having written 370 posts in this autism blog, I sometimes feel that I am becoming a bit of an expert on diabetes (and COPD), which you might think has nothing to do with autism.

I was talking to a friend of mine who has type 2 diabetes; he was telling me about all the other things that are going wrong with him, because he actually has “metabolic syndrome”.

What exactly is metabolic syndrome?  It really is not a very good name. Sure you can have a metabolic system, but there are going to be many different ones.  It looks like in the world of medicine there is just one.

The common problem is that in late middle age many people get overweight around their waist, they also have increased blood pressure, high blood sugar and abnormal cholesterol, or triglyceride levels. This combination of symptoms is called metabolic syndrome and it increases your risk of heart disease, stroke, diabetes and much more. (see chart above, even high uric acid/gout is there)

The clever way to treat metabolic syndrome would be to treat the underlying molecular biology, rather than each symptom one by one.  This is not as hard as it may sound, just from reading about the biology of autism, I was telling my friend lots of things he could suggest to his doctor.

If you are going to take a drug to lower blood pressure, why not take the one that also protects your beta cells, the ones that produce insulin, from dying? If you are going to take an ACE inhibitor, why not take the one that will also improve your insulin sensitivity. Instead of taking a glitazone drug that is effective at lowering blood glucose, but has not been shown to reduce the long-term complications of diabetes (such as heart disease and stroke), why not take a single drug that does all three?

Metabolic Syndrome & Autism

It is not surprising to me that research shows that parents who develop metabolic syndrome have an increased likelihood of already having children with autism.

Nor is it a surprise that people with autism, or schizophrenia, have themselves a tendency to various kinds of metabolic syndrome; in fact I would suggest that autism is a metabolic syndrome, just not always the same kind.

It is not a surprise that the drugs produced to treat the classic metabolic syndrome seem to provide such a good hunting ground for autism drugs.

We know that glitazone drugs, being PPAR gamma agonists, should help some kinds of autism and also that PPAR delta agonists may help some with mitochondrial disease. The issue I have with glitazone drugs is their safety in long term use.  Another glitazone autism trial is underway in Canada. Glitazone drugs are used to improve insulin sensitivity in type 2 diabetics.

Bezafibrate is getting a well-deserved trial for mitochondrial disease. Through its action on PPAR, where it is a “pan-agonist”, it is thought that Bezafibrate should trigger biogenesis of mitochondria. Bezafibrate is an old drug to lower cholesterol.

One very interesting candidate drug for autism is Telmisartan which will be covered in a coming post on Angiotensin II in the brain.  Telmisartan is an Angiotensin AT1 agonist, which means it will lower blood pressure, but it does numerous other things. It happens also to be a PPAR gamma/delta agonist.  It improves insulin sensitivity and lower blood glucose levels.  It also modifies the immune system by reducing Il-17a, an important inflammatory cytokine found elevated in both autism and schizophrenia. It also reduces leptin release and prevents leptin resistance. Leptin levels are high in autism and leptin resistance is feature of obesity.

One of the drugs often prescribed to people with metabolic syndrome is Atorvastatin, which some readers of this blog have found improves the application of cognitive ability in their case of autism.

If I had metabolic syndrome, after losing weight, I would choose Atorvastatin, Verapamil and Telmisartan as my top three drugs; none of which are prescribed to that friend of mine. I would also add a glass of beetroot juice which is vasodilating; it is not a drug, but should do plenty of good. I would use an antioxidant like ALA (alpha-lipoic acid) and use sulforaphane to activate the body’s antioxidant genes via Nrf2; many side effects of metabolic syndrome are caused/aggravated by oxidative stress.

Zinc, Hedgehog Signaling, Shank2/3, NMDA/AMPA Inactivation and Autism

I am gradually tying up the loose ends in this blog. Today several issues are dealt with that are all connected by zinc. Some are extremely complicated and I will skip over the details.

Not that kind of hedgehog

1.     In those rather complicated graphics in the literature that explain signaling pathways, you may have noticed something called hedgehog signaling. This is a basic pathway present in all bilaterians - creatures with a head and tail/feet and a left and right. So flies, yes; but jelly fish, no.  In autism there is excessive hedgehog signaling.  Zinc deficiency is linked to activation of the hedgehog signaling pathway

2.     One of the commonly used models of autism is called Shank3; there is another one called Shank2.  Shank proteins are scaffold proteins that connect neurotransmitter receptors and ion channels to the actin cytoskeleton and G-protein-coupled signaling pathways.  Mutations in these genes are associated with autism. This gets very complicated.

3.     In trying to consider all types of excitatory–imbalance in autism we have yet to look into how low levels of zinc inactivate Shank2 (and so inactivate NMDA receptors) and also inactivate Shank3 reducing synaptic transmission via AMPA receptors as well.

4.     In earlier posts there have been references to zinc in autism and it was suggested that the Zn²⁺ ions are in the “wrong place”.

5.     In people with autism very often there appears to be high levels of copper, but low levels of zinc.

6.     There is a paradoxical relationship where high levels of zinc supplementation actually causes zinc deficiency in the hippocampus

  

While you might not read much about zinc and autism, it clearly is very relevant but only partially understood. 

Much of the early research regarding zinc and autism has been very simplistic and tells you little. Recently research has been far from trivial and is getting into the details; look for terms such as Shank2, Shank3, and even Shankopathies. 

If someone with autism is deficient in zinc, supplementation may indeed have a positive effect, but high doses of oral zinc will actually cause deficiency in the brain.  

In the brain, zinc is stored in specific synaptic vesicles by glutamatergic neurons and can modulate neuronal excitability. It plays a key role in synaptic plasticity and so in learning.   

Zinc can also be a neurotoxin, suggesting zinc homeostasis plays a critical role in the functional regulation of the central nervous system. Dysregulation of zinc homeostasis in the central nervous system that results in excessive synaptic zinc concentrations is believed to induce neurotoxicity through mitochondrial oxidative stress, the dysregulation of calcium homeostasis, glutamate excitotoxicity, and interference with intra-neuronal signal transduction. 

Zinc: indications in brain disorders

Zinc is the authoritative metal which is present in our body, and reactive zinc metal is crucial for neuronal signaling and is largely distributed within presynaptic vesicles. Zinc also plays an important role in synaptic function. At cellular level, zinc is a modulator of synaptic activity and neuronal plasticity in both development and adulthood. Different importers and transporters are involved in zinc homeostasis. ZnT-3 is a main transporter involved in zinc homeostasis in the brain. It has been found that alterations in brain zinc status have been implicated in a wide range of neurological disorders including impaired brain development and many neurodegenerative disorders such as Alzheimer's disease, and mood disorders including depression, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion disease. Furthermore, zinc has also been implicated in neuronal damage associated with traumatic brain injury, stroke, and seizure. Understanding the mechanisms that control brain zinc homeostasis is thus critical to the development of preventive and treatment strategies for these and other neurological disorders. 

For a full list of zinc transporters and disease associations click the link below

Zinc transporters, cellular location, tissue-specific expression, and disease associations. 

Most likely the problem in autism is caused by zinc transporters.  In schizophrenia it is suggested that the zinc transporter ZIP12/ SLC39A12 is over-expressed.

Increased cortical expression of the zinc transporter SLC39A12 suggests a breakdown in zinc cellular homeostasis as part of the pathophysiology of schizophrenia 

Hedgehog Signaling 

You may wonder what could be the connection between zinc, hedgehogs and autism, but today I am talking about a special kind of hedgehog, the evolutionarily conserved Hedgehog (Hh) pathway; there really is a connection. 

Sonic hedgehog is a protein that in humans is encoded by the SHH (sonic hedgehog) gene. Sonic hedgehog is one of three proteins in the mammalian signaling pathway family called hedgehog, the others being Desert hedgehog (DHH) and Indian hedgehog (IHH). SHH is the best studied of the hedgehog signaling pathway.  

Both sonic and Indian hedgehog are consistently found elevated in autism. Desert hedgehog gets much less attention, but was found to be reduced in one study from Saudi Arabia, no surprise they choose the desert variant. 

Sonic hedgehog is seen as the most important in development and is heavily implicated in some cancers. It plays a role in how your teeth grow, how your lungs grow, how your hair regenerates and very many other things. 

The next graphic is complicated and most people will skip it.

Pathway Description:

The evolutionarily conserved Hedgehog (Hh) pathway is essential for normal embryonic development and plays critical roles in adult tissue maintenance, renewal and regeneration. Secreted Hh proteins act in a concentration- and time-dependent manner to initiate a series of cellular responses that range from survival and proliferation to cell fate specification and differentiation.

Proper levels of Hh signaling require the regulated production, processing, secretion and trafficking of Hh ligands– in mammals this includes Sonic (Shh), Indian (Ihh) and Desert (Dhh). All Hh ligands are synthesized as precursor proteins that undergo autocatalytic cleavage and concomitant cholesterol modification at the carboxy terminus and palmitoylation at the amino terminus, resulting in a secreted, dually-lipidated protein. Hh ligands are released from the cell surface through the combined actions of Dispatched and Scube2, and subsequently trafficked over multiple cells through interactions with the cell surface proteins LRP2 and the Glypican family of heparan sulfate proteoglycans (GPC1-6).

Hh proteins initiate signaling through binding to the canonical receptor Patched (PTCH1) and to the co-receptors GAS1, CDON and BOC. Hh binding to PTCH1 results in derepression of the GPCR-like protein Smoothened (SMO) that results in SMO accumulation in cilia and phosphorylation of its cytoplasmic tail. SMO mediates downstream signal transduction that includes dissociation of GLI proteins (the transcriptional effectors of the Hh pathway) from kinesin-family protein, Kif7, and the key intracellular Hh pathway regulator SUFU.

GLI proteins also traffic through cilia and in the absence of Hh signaling are sequestered by SUFU and Kif7, allowing for GLI phosphorylation by PKA, GSK3β and CK1, and subsequent processing into transcriptional repressors (through cleavage of the carboxy-terminus) or targeting for degradation (mediated by the E3 ubiquitin ligase β-TrCP). In response to activation of Hh signaling, GLI proteins are differentially phopshorylated and processed into transcriptional activators that induce expression of Hh target genes, many of which are components of the pathway (e.g. PTCH1 and GLI1). Feedback mechanisms include the induction of Hh pathway antagonists (PTCH1, PTCH2 and Hhip1) that interfere with Hh ligand function, and GLI protein degradation mediated by the E3 ubiquitin ligase adaptor protein, SPOP.

In addition to vital roles during normal embryonic development and adult tissue homeostasis, aberrant Hh signaling is responsible for the initiation of a growing number of cancers including, classically, basal cell carcinoma, edulloblastoma, and rhabdomyosarcoma; more recently overactive Hh signaling has been implicated in pancreatic, lung, prostate, ovarian, and breast cancer. Thus, understanding the mechanisms that control Hh pathway activity will inform the development of novel therapeutics to treat a growing number of Hh-driven pathologies. 

Source:https://www.cellsignal.com/contents/science-pathway-research-stem-cell-markers/hedgehog-signaling-pathway/pathways-hedgehog#sthash.fZ0jSdvd.dpuf 

Sonic Hedgehog Protein correlates with severity of autism

The research does show that the more severe the autism, the higher is the level of sonic hedgehog protein.    

Relationship Between Sonic Hedgehog Protein, Brain-Derived Neurotrophic Factor and Oxidative Stress in Autism Spectrum Disorders 

 

Serum levels of Sonic hedgehog protein in control and autistic children.

Highly statistically significant Sonic hedgehog serum level in mild and severe autism 

Zinc deficiency activates hedgehog signaling 

Zinc Inhibits Hedgehog Autoprocessing

LINKING ZINC DEFICIENCY WITH HEDGEHOG ACTIVATION

Background: In many types of cancers zinc deficiency and overproduction of Hedgehog (Hh) ligand co-exist.
Results: Zinc binds to the active site of the Hedgehog-intein (Hint) domain and inhibits Hh ligand production both in vitro and in cell culture.
Conclusion: Zinc influences the Hh autoprocessing.

Significance: This study uncovers a novel mechanistic link between zinc and the Hh signaling pathway.  

DISCUSSIONZinc is an essential trace element, acting as a co-factor for >300 enzymes that regulate a variety of cellular processes and signaling pathways (38). Zinc is also a signaling molecule and can modulate synaptic activity (39). The imbalance of zinc homeostasis has been established in many pathological conditions (14–21), including many types of cancer and autism. However, the mechanistic role of zinc deficiency in these diseases remains poorly understood. 

ASD, with an astounding prevalence of ∼2% (43), is characterized by abnormal social interaction, communication, and stereotyped behaviors in affected children. The etiology of ASD is poorly understood, but both oxidative stress (44) and low zinc status have been reproducibly associated with ASD (16, 45). In astrocyte culture, Hh autoprocessing is promoted by H₂O₂ and low zinc level (Fig. 2A), offering a plausible mechanistic explanation for the recent observation of increased serum level of sonic Hh ligand in ASD (9). The resulting higher level of secreted Hh ligand may lead to the abnormal activation of Hh signaling pathway in both neurons and glial cells in the developing brain. A clinical feature of ASD, macrocephaly, also implicates Hh activation (46–48). Hh plays an important role in the early expansion of the developing brain and in regulating the cerebral cortical size (49, 50). In contrast, the opposite clinical feature, microcephaly, is observed in holoprosencephaly (51), which can be caused by mutations in the Hh autoprocessing domain (HhC) that reduce Hh ligand production (51–54). The abnormal activation of Hh pathway, even transiently by fluctuations in zinc level, may cause brain overgrowth, disrupting the proper development of neuronal network for language and social interactions. We, therefore, hypothesize that in ASD low zinc status promotes Hh autoprocessing and the generation of higher level of Hh ligand. Coupled with oxidative and/or genetic defects in other Hh signaling components, low zinc status may lead to abnormal activation of Hh signaling pathway during brain development, contributing to the complex etiology of ASD.

   

Zinc deficiency linked to activation of Hedgehog signaling pathway  

Indian Hedgehog Protein Levels in Autistic Children: Preliminary Results

The etiology of autism spectrum disorders (ASD) is not well known but recently we reported that the serum levels of sonic hedgehog (SHH) protein and brain-derived neurotrophic factor (BDNF) might be linked to oxidative stress in ASD. We hypothesized that Indian hedgehog (IHH) protein which belongs to SHH family may play a pathological role in the ASD. We studied recently diagnosed patients in early stages of ASD (n=54) and age-matched, cognitively normal, individuals (n=25), using serum levels of IHH protein. We found statistically significantly higher-levels of serum IHH protein in ASD subjects (p=0.001) compared to control subjects. Our findings are the first to report a role of IHH in ASD children, suggesting a possible pathological role-played by IHH in early-stage in ASD. Such measures might constitute an early biomarker for ASD and ultimately offer a target for novel biomarker-based therapeutic interventions.

   

Too much zinc causes Hippocampal Zinc Deficiency 

Before you rush to buy some zinc tablets, you should read the next study.  

High Dose Zinc Supplementation Induces Hippocampal Zinc Deficiency and Memory Impairment with Inhibition of BDNF Signaling

These results indicate that zinc plays an important role in hippocampus-dependent learning and memory and BDNF expression, high dose supplementation of zinc induces specific zinc deficiency in hippocampus, which further impair learning and memory due to decreased availability of synaptic zinc and BDNF deficit.

Consistent with previous reports, zinc supplementation in low dosage may increase the anxiety level [19], [32].The previous data regarding the low dose zinc supplementation on learning and memory was conflicting. Flinn JM et al. reported in a series of publications that enhanced zinc (10 ppm) consumption causes memory deficits in rats [19], [32] and potentiates memory impairment in transgenic disease mouse models [33], [34], while others observed improved performance of the animals in spatial memory tasks [35], [36]. In our experiments, we also observed improved performance of mice in contextual discrimination task. The underlying mechanism for the memory improvement by low dose zinc supplement needs further exploration. On the contrary, zinc supplementation in high dose resulted in impaired spatial memory. Interestingly, the memory deficit seemed to be highly hippocampus dependent, since high dose supplementation of zinc only impaired the performance of the mice in context discrimination but not in contextual conditioning 

The possible positive effect of zinc supplementation in Autism  

There was a Phase 1 clinical trial at Penn State (by Jeanette C. Ramer) looking at the level of copper and zinc in autism and then supplementing vitamin C and zinc.  The study was completed a few years ago but it looks like they never published the results.  We have to assume it was inconclusive, but it would nice if they published the results anyway. 

The study below was funded by the Autism Research Institute.  

Analysis of Copper and Zinc Plasma Concentration and the Efficacy of Zinc Therapy in Individuals with Asperger’s Syndrome, Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS)and Autism

Aim

To assess plasma zinc and copper concentration in individuals with Asperger’s Syndrome, Pervasive Developmental Disorder-Not Otherwise Specified (PDD-NOS) and autistic disorder, and to analyze the efficacy of zinc therapy on the normalization of zinc and copper levels and symptom severity in these disorders.

Subjects and methods

Plasma from 79 autistic individuals, 52 individuals with PDD-NOS, 21 individuals with Asperger’s Syndrome (all meeting DSM-IV diagnostic criteria), and 18 age and gender similar neurotypical controls, were tested for plasma zinc and copper using inductively-coupled plasma-mass spectrometry.

Results

Autistic and PDD-NOS individuals had significantly elevated plasma levels of copper. None of the groups (autism, Asperger’s or PDD-NOS) had significantly lower plasma zinc concentrations. Post zinc and B-6 therapy, individuals with autism and PDD-NOS had significantly lower levels of copper, but individuals with Asperger’s did not have significantly lower copper. Individuals with autism, PDD-NOS and Asperger’s all had significantly higher zinc levels. Severity of symptoms decreased in autistic individuals following zinc and B-6 therapy with respect to awareness, receptive language, focus and attention, hyperactivity, tip toeing, eye contact, sound sensitivity, tactile sensitivity and seizures. None of the measured symptoms worsened after therapy. None of the symptoms in the Asperger’s patients improved after therapy.

Discussion

These results suggest an association between copper and zinc plasma levels and individuals with autism, PDD-NOS and Asperger’s Syndrome. The data also indicates that copper levels normalize (decrease to levels of controls) in individuals with autism and PDD-NOS, but not in individuals with Asperger’s. These same Asperger’s patients do not improve with respect to symptoms after therapy, whereas many symptoms improved in the autism group. This may indicate an association between copper levels and symptom severity.  

  

Our study shows that autistic individuals have lower levels of zinc and significantly higher levels of copper when compared to neurotypical controls.

We do not know why copper doesn’t normalize after zinc therapy in Asperger’s patients but suggest that since symptom severity of these patients remains high, high copper levels are most likely associated with symptom severity.

Individuals in this study who presented to the Pfeiffer Treatment Center with depression (or anxiety) were tested for Zn, Cu and anti-oxidant levels. Based on deficiencies, they were then prescribed the appropriate dose of anti-oxidants. Pre-therapy patients represent those who were tested when they first presented and were not previously taking any Zn or anti-oxidants. Post-Therapy patients received anti-oxidant therapy (Vitamin C, E, B-6 as well as Magnesium, and Manganese if warranted), and Zn supplementation (as Zn picolinate), daily, for a minimum of 8 weeks. 

Trans-synaptic zinc mobilization 

I did write a post a while back about some very interesting findings from Taiwan.  

Altered Homeostasis in Autism: Cl-, K+, Ca2+, and quite possibly Zn2+ 

In their research they found that simply repositioning zinc improved social interaction in two models of autism and they proposed a trial in humans with a drug already licensed in Taiwan.  They also had to suggestions for people with autism.   

Clioquinol may help people with autism: study  

Hsueh recommends that people with autism who are diagnosed with zinc deficiency caused by the underexpression of the NMDAR receptor to increase their zinc intake by eating food high in zinc, such as oysters. She added that meat, which is rich in protein, helps boost zinc absorption.

Trans-synaptic zinc mobilization improves social interaction in two mouse models of autism through NMDAR activationgh NMDAR activation 

In the present study, we demonstrate that trans-synaptic Zn mobilization by clioquinol, a Zn chelator and ionophore (termed CQ hereafter), rescues the social interaction deficits in Shank2_/_ and Tbr1þ/_ mice. CQ mobilizes Zn from enriched presynaptic pools to postsynaptic sites, where it enhances NMDAR function through Src activation. These results indicate that postsynaptic Zn rescues social interaction deficits in distinct mouse models of ASDs, and suggest that reduced NMDAR function is associated with ASDs. 

In the present study, we found that trans-synaptic Zn mobilization improves social interaction in two distinct mouse models of ASD through postsynaptic Src and NMDAR activation. Our study suggests that CQ-dependent mobilization of Zn from pre- to postsynaptic sites—not Zn removal after chelation—might be useful in the treatment of ASDs. This unique transsynaptic Zn mobilization is supported by the following findings: 

(1) CQ failed to enhance NMDAR function in ZnT3_/_ mice, which lack the presynaptic Zn pool; and (2) Ca-EDTA, a membrane-impermeable Zn chelator that should chelate Zn in the synaptic cleft or extracellular sites, blocked CQ-dependent NMDAR activation. 

Finally, our study broadens the therapeutic potential of CQ. CQ has been used as a topical antiseptic or an oral intestinal amoebicide since 1930s, although the latter use has ceased for its controversial association with subacute myelo-optic neuropathy.

Recently, however, CQ-dependent chelation of Zn has been suggested for the treatment of neurological disorders including Alzheimer’s disease67, Parkinson’s disease68 and Huntington’s’ disease. Moreover, PBT2, a second-generation CQ-related compound under clinical trials, seems to be safe and improve cognitive deficits in patients with Alzheimer’s disease. 

Therefore, our study is the first to demonstrate the possibility of repositioning of the FDA-approved antibiotic, CQ, to ASDs based on a novel mechanism distinct from chelation. In addition, CQ-dependent trans-synaptic Zn mobilization might also be useful in other psychiatric disorders that are notable for being caused by a decrease in NMDAR function. 

In conclusion, our study suggests that trans-synaptic Zn mobilization rapidly improves social interaction in two independent mouse models of ASD through Src and NMDAR activation, and a new therapeutic potential of CQ in the treatment of ASDs.

MorM

Clioquinol (CQ) is an anti-fungal drug.

  

Shank3 and Autism/Schizophrenia

Shank3, which is found at synapses in the brain, is associated with neuro-developmental disorders such as autism and schizophrenia. 

The exact role of Shank3 is very complex and would take a long time fully understand. It particularly affects all the types of glutamate receptor, so the AMPA, NMDA and mGluRs in the diagram below. Note the green circle with zinc, Zn²⁺. 

Shank proteins particularly Shank2 and Shank3 are associated with autism and a Shank dysfunction is even called a “Shankopathy”. 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659167/figure/F2/

Schematic of the partial Shank protein interactome at the PSD with Shank3 as a model. A more complete list of Shank family interacting proteins is shown in Table 2. Protein domains in Shank family members are similar. Many interacting proteins interact with all three Shank family proteins (Shank1, Shank2, and Shank3) in in vitro assays. The proteins in red font are altered in Shank3 mutant mice. 

  

Here is a science-light article from New Zealand.

  Zinc found to reverse brain cell changes in autism

Cellular changes in the brain caused by genetic mutations that occur in autism can be reversed by zinc, according to research at the University of Auckland.

Medical scientists at the University’s Department of Physiology have researched aspects of how autism mutations change brain cell function for the past five years.

This latest work - a joint collaborative effort lead by neuroscientist collaborators in Auckland, America and Germany - was published today in the high impact journal, The Journal of Neuroscience.

The study was funded by the Marsden Fund and the Neurological Foundation.

Lead investigator at the University of Auckland, Associate Professor Johanna Montgomery from the University’s Department of Physiology and Centre for Brain Research, says “This most recent work, builds significantly from our earlier work showing that gene changes in autism decrease brain cell communication.”

”We are seeking ways to reverse these cellular deficits caused by autism-associated changes in brain cells," she says. “This study looks at how zinc can alter brain cell communication that is altered at the cellular level and we are now taking that forward to look at the function of zinc at the dietary and behaviour level."

“Autism is associated with genetic changes that result in behavioural changes,” says Dr Montgomery. “It begins within the cells, so what happens at a behavioural level indicates something that has gone wrong at the cellular level in the brain.”

International studies have found that normally there are high levels of zinc in the brain, and brain cells are regulated by zinc, but that zinc deficiency is prevalent in autistic children.

“Research using animal models has shown that when a mother is given a low zinc diet, the offspring will be more likely to display autistic associated behaviours,” she says.

“Our work is showing that even the cells that carry genetic changes associated with autism can respond to zinc.

“Our research has focused on the protein Shank3, which is localized at synapses in the brain and is associated with neuro-developmental disorders such as autism and schizophrenia,” she says.

“Human patients with genetic changes in Shank3 show profound communication and behavioural deficits. In this study, we show that Shank3 is a key component of a zinc-sensitive signalling system that regulates how brain cells communicate.”

“Intriguingly, autism-associated changes in the Shank3 gene impair brain cell communication,” says Dr Montgomery. ”These genetic changes in Shank3 do not alter its ability to respond to zinc”.

“As a result, we have shown that zinc can increase brain cell communication that was previously weakened by autism-associated changes in Shank3”.

“Disruption of how zinc is regulated in the body may not only impair how synapses work in the brain, but may lead to cognitive and behavioural abnormalities seen in patients with psychiatric disorders.”

“Together with our results, the data suggests that environmental/dietary factors such as changes in zinc levels could alter this protein’s signalling system and reduce its ability to regulate the nerve cell function in the brain,” she says.

This has applications to both autism and psychiatric disorders such as schizophrenia.

Dr Montgomery says the next stage of their research is to investigate the impact of dietary zinc supplements to see what impact it has on autistic behaviours.

“Too much zinc can be toxic, so it is important to determine the optimum level for preventing and treating autism and also whether zinc is beneficial for all or a subset of genetic changes that occur in Autism patients.”

Full paper

Shank3 Is Part of a Zinc-Sensitive Signaling System That Regulates Excitatory Synaptic Strength 

Shank3 is a multidomain scaffold protein localized to the postsynaptic density of excitatory synapses. Functional studies in vivo and in vitro support the concept that Shank3 is critical for synaptic plasticity and the trans-synaptic coupling between the reliability of presynaptic neurotransmitter release and postsynaptic responsiveness. However, how Shank3 regulates synaptic strength remains unclear. The C terminus of Shank3 contains a sterile alpha motif (SAM) domain that is essential for its postsynaptic localization and also binds zinc, thus raising the possibility that changing zinc levels modulate Shank3 function in dendritic spines. In support of this hypothesis, we find that zinc is a potent regulator of Shank3 activation and dynamics in rat hippocampal neurons. Moreover, we show that zinc modulation of synaptic transmission is Shank3 dependent. Interestingly, an autism spectrum disorder (ASD)-associated variant of Shank3

(Shank3R87C) retains its zinc sensitivity and supports zinc-dependent activation of AMPAR-mediated synaptic transmission. However, elevated zinc was unable to rescue defects in trans-synaptic signaling caused by the R87C mutation, implying that trans-synaptic increases in neurotransmitter release are not necessary for the postsynaptic effects of zinc. Together, these data suggest that Shank3 is a key component of a zinc-sensitive signaling system, regulating synaptic strength that may be impaired in ASD. 

Significance Statement 

Shank3 is a postsynaptic protein associated with neurodevelopmental disorders such as autism and schizophrenia. In this study, we show that Shank3 is a key component of a zinc-sensitive signaling system that regulates excitatory synaptic transmission. 

Intriguingly, an autism-associated mutation in Shank3 partially impairs this signaling system. Therefore, perturbation of zinc homeostasis may impair, not only synaptic functionality and plasticity, but also may lead to cognitive and behavioral abnormalities seen in patients with psychiatric disorders.

Figure 6. Model of zinc-dependent regulation of Shank3 dynamics and activation state. Our data suggest that zinc changes the conformation and association of Shank3 within dendritic spines, resulting in Shank3, which dynamically exchanges between three pools. In pool 1, Shank3 is in an active conformation in the presence of higher zinc (green squares). This conformation assembles into an active signaling complex that includes Homer, AMPARs, and Neuroligin, leading to enhanced synaptic transmission. When zinc levels are low, Shank3 is inactive and resides in two additional pools: one that is rapidly exchanging (red squares) and one that contains oligomerized Shank3 (bound red squares). Oligomerization is potentially mediated by its SAM domain. We propose that, during synaptic transmission, zinc released from vesicles or from intracellular stores could lead to real-time changes in synaptic strength through the recruitment of activated Shank3 into the PSD.

In summary, our studies reveal that Shank3 not only senses changes in postsynaptic zinc, but also is a key component of a zinc sensitive signaling pathway at excitatory synapses. Importantly, zinc homeostasis is disrupted in neuropsychiatric disorders including ASD (Curtis and Patel, 2008; Grabrucker et al., 2011a; Russo and Devito, 2011; Yasuda et al., 2011). Elevation of zinc has been shown to rescue normal social interaction via Src andNMDARactivation in Shank2 and Tbr1 ASD mouse models (Lee et al., 2015), whereas chronic zinc deficiency induces the loss of Shank2/3 and increases the incidence of ASD-related behaviors (Grabrucker et al., 2014). Together with our results, these data suggest that environmental/ dietary factors such as changes in zinc levels could alter the Shank3-signaling system and reduce the optimal performance of Shank3-dependent excitatory synaptic function. Therefore, strategies to activate this zinc-sensitive pathway could potentially restore the functionality of these synapses.

   

Zinc and Dopamine 

I know that some readers of this blog are interested in dopamine.  

Zn²⁺ reverses functional deficits in a de novo dopamine transporter variant associated with autism spectrum disorder

Conclusion

It is clear that zinc can play an important role in autism, but the research has a long way to go to really understand all of the issues. 

Impaired zinc homeostasis (equilibrium) is going to cause numerous effects. It will disturb all the glutamate receptors (AMPA, NMDA, mGluRs); in doing so it would disturb the brain’s excitatory-inhibitor balance.  

The research from Taiwan suggests that moving zinc from pre- to post-synaptic sites using an old drug called Clioquinol might be useful in the treatment of some autism. 

Some research suggests that correcting a low level of zinc, found in a blood test, using a supplement may have a beneficial effect. I suspect the impact is either small or highly variable, but simple to check. 

Low levels of zinc seem to be associated by high levels of copper. Supplementing zinc raises the level of zinc and also reduces the level of copper. 

Large amounts of supplemental zinc have a paradoxical effect of reducing the level of zinc in the hippocampus. 

The real issue is perhaps the transport of zinc within the brain, there are many zinc transporters and it is most likely that the problem in autism is caused by zinc transport rather than a lack of dietary zinc. Faulty zinc transporters are associated with numerous diseases, but only recently has autism research started to move from the simple idea of zinc deficiency to consider the role of specific zinc transporters, like ZIP2 and ZIP4.     

Supplementing zinc, along with scores of other things, has long been practiced by alternative therapists in autism. I could not find many reports of significant positive changes.

Hopefully, there will be a human trial of Clioquinol in Taiwan and, if there is, I hope they will check the expression Sonic Hedgehog and Indian Hedgehog.