Tuning Wnt Signaling for more/fewer hairs and to optimize Dendritic Spine Morphology in Autism

Today’s post is about another example of how evolution can play jokes on us.  It really is the case that a signaling pathway that controls hair growth is the same that determines the number and shape of dendritic spines in the brain.

This is good news not just for Homer Simpson but for people interesting in perking up behavior and cognitive function in autism.

The post also connects several subjects that we have previously encountered - dendritic spines which are abnormal in autism, Wnt signaling which is implicated in cancer (and autism), statins, Ivermectin, CAPE found in some propolis and verapamil.  There is plenty of research to back all these connections, but strangely nobody seems to be applying them to develop any practical therapies.

I introduced dendritic spines in an earlier post.  Each neuron in your brain has hundreds of protruding spines.

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

In that post I reported that PAK1, the gene NrCAM and the protein MTOR were all implicated in the dysfunction in both shape and number of these spines.

It now seems that there may be one even more critical pathway involved – Wnt. There are links between Wnt and PAK1, that appeared in several earlier posts.

You may recall that dendritic spines are constantly changing shape.  Their shape affects their function.  In many disorders, both the number and shape of the spines is dysfunctional.  It appears that the morphology (shape) can be modified, which implies you could affect behavior, memory, and cognitive function.

My follow up post of dendritic spines has yet to materialize, but here is a sneak preview, showing the progression of autism, schizophrenia and Alzheimer’s in terms of the number of dendritic spines.

Dendritic Spines and Wnt Signaling

Dendritic spines are constantly changing their shape and certain psychiatric disorders are characterized by different morphologies (shapes) of these spines.  It is not just the number of spines, but their shape which affects cognitive function, memory and behavior.

The Wnt signaling pathway also lies behind hair growth.

What is more, we know that Wnt signaling is dysfunctional in autism and we even now which the genes are that likely trigger of this dysfunction.

Wnt dysfunction is also involved in many types of cancer and therefore has been subject of much research.

The surprise came when I read that attempts are underway to “tune” Wnt signaling to control hair growth.  Why not autism?

This post is about tuning Wnt signaling to improve cognitive function and behavior.  This appears just as plausible as controlling hair growth.

The Wnt Signaling Pathways

Here is the Wikipedia explanation.

Wnt signaling pathway

The Wnt signaling pathways are a group of signal transduction pathways made of proteins that pass signals from outside of a cell through cell surface receptors to the inside of the cell. Three Wnt signaling pathways have been characterized: the canonical Wnt pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway. All three Wnt signaling pathways are activated by the binding of a Wnt-protein ligand to a Frizzled family receptor, which passes the biological signal to the protein Dishevelled inside the cell. The canonical Wnt pathway leads to regulation of gene transcription, the noncanonical planar cell polarity pathway regulates the cytoskeleton that is responsible for the shape of the cell, and the noncanonical Wnt/calcium pathway regulates calcium inside the cell. Wnt signaling pathways use either nearby cell-cell communication (paracrine) or same-cell communication (autocrine). They are highly evolutionarily conserved, which means they are similar across many species from fruit flies to humans.^[1][2]
Wnt signaling was first identified for its role in carcinogenesis, but has since been recognized for its function in embryonic development. The embryonic processes it controls include body axis patterning, cell fate specification, cell proliferation, and cell migration. These processes are necessary for proper formation of important tissues including bone, heart, and muscle. Its role in embryonic development was discovered when genetic mutations in proteins in the Wnt pathway produced abnormal fruit fly embryos. Later research found that the genes responsible for these abnormalities also influenced breast cancer development in mice.
The clinical importance of this pathway has been demonstrated by mutations that lead to a variety of diseases, including breast and prostate cancer, glioblastoma, type II diabetes, and others.^[3][4]

The Canonical Wnt pathway is dysfunctional in Autism

It is the canonical Wnt pathway that is dysfunction in autism and it is this same pathway plays a role in dendrite growth and suboptimal Wnt activity negatively affects the dendritic arbor.

A very thorough review of all the genetic evidence is provided in the following study:

A review of the evidence for the canonical Wnt pathway in autism spectrum disorders

Notably, the available genetic information indicates that not only canonical Wnt pathway activation, but also inhibition seems to increase autism risk. The canonical Wnt pathway plays a role in dendrite growth and suboptimal activity negatively affects the dendritic arbor. In principle, this provides a logical explanation as to why both hypo- and hyperactivity may generate a similar set of behavioral and cognitive symptoms.

The review highlights that, as we have seen before, some people with autism are hypo and some people are hyper; this means some people need Wnt signaling to be inhibited and other people need the opposite therapy.  The author points out that you really need some test to check which way you need your Wnt “tuned”.  

It sounds a bit like tuning the timing of the sparks inside your car engine, in the days before it was all electronic and self-tuning.  In theory you needed to measure the timing of the sparks with a special strobe light; but if you knew what you were doing you could just use your ears.  So in the same vein, you could make a small change to inhibit Wnt and see the result, if it made matters worse you just stop and go the other way.  As you will see later in this post, some of us are already tuning Wnt without even realizing it.

We have exactly the same issue with mGluR5, where you might need a positive/negative allosteric modulator to optimize brain performance.  Different variants of “autism” would be located either left or right of “top dead center”.

In that post we learnt that at MIT they are suggesting that errors in synaptic protein synthesis are behind several types of autism and that these errors can be corrected using either positive or negative stimulators of the receptor mGluR5.

Ketamine, Memantine, D-Cycloserin, Magnesium, Fenobam and yet more as Glutamatergic Modulators in Autism (and Fragile X)

For a more detailed understanding of Wnt signaling, see the paper below:-

Wnt signal transduction pathways

For Homer Simpson and others wanting more hair

Tuning Wnt signals for more or fewer hairs

Abnormal hair development and regeneration has been implicated in diseases of the skin (ie., hirsutism, alopecia, etc) or in open wounds when hair follicles are completely eliminated. To manage these clinical conditions, it is important to understand molecular pathways which regulate the number, size, growth and regeneration of hair follicles. Wnt signaling plays a fundamental role in this process. We need a deeper understanding so we can reliably adjust Wnt levels in existing follicles. This studies reviewed here have future translational value for skin regeneration following severe wound injuries or in the context of tissue engineering. Tuning the levels of Wnt ligands can directly modulate the number and growth of hairs. Using this new knowledge, we now know that Wnt activity can be modulated by adjusting the secretion of Wnt ligands, altering binding of ligands to receptors, inhibiting β-catenin translocation, or by regulating extra-follicular dermal Wnt and Wnt inhibitors.

How to tune Dendritic Spine Morphology

We have already encounter Brain-Derived Neurotropic Factor  (BDNF) in an earlier post.  You could think of BDNF as brain fertilizer.

Human Growth Factors, Autism and the Centenarian Nobel Laureate

“Older people and anyone with Retts Syndrome are likely to benefit from more NGF (Nerve Growth Factor).  In autism it appears possible that there was too much NGF and BDNF at a very early age, with levels then changing.  High levels of NGF and BDNF look a bad idea.  A lot more research is needed to understand what determines  NGF and BDNF levels.  It appears that BDNF may stay high in autism, but NGF levels.”

It has been shown that BDNF and Wnt signaling together regulate dendritic spine formation.

So, since in autism we have excess BDNF as the brain is developing, this might explain there are too many dendritic spines in autistic brains.  Too many spines and the wrong morphology (shape) would explain very many issues that have gone “wrong” in autistic brains.

Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation

Here, we show that Wnt signaling inhibition in cultured cortical neurons disrupts dendritic spine development, reduces dendritic arbor size and complexity, and blocks BDNF-induced dendritic spine formation and maturation. Additionally, we show that BDNF regulates expression of Wnt2, and that Wnt2 is sufficient to promote cortical dendrite growth and dendritic spine formation. Together, these data suggest that BDNF and Wnt signaling cooperatively regulate dendritic spine formation.

BDNF overexpression rapidly and robustly increases primary dendrite formation in cortical neurons (Horch et al., 1999; McAllister et al., 1997; Wirth et al., 2003). We reproduced this finding, and found that this increase was not blocked by overexpression of the Wnt inhibitors (Fig. S2), indicating that some aspects of BDNF modulation of dendrites remain intact in the presence of Wnt inhibitors. To further assess whether expression of the Wnt inhibitors impaired the signaling ability of BDNF, we analyzed autocrine induction of c-Fos expression by BDNF overexpression. c-Fos is an immediate early gene whose transcription is rapidly upregulated by BDNF (Calella et al., 2007; Gaiddon et al., 1996). We found that BDNF induced c-Fos expression was not reduced in neurons overexpressing any of the four Wnt inhibitors, suggesting that the ability of the inhibitors to interfere with BDNF-induced spine formation and spine head width expansion was not a result of decreased levels of BDNF signaling (Fig. S3).

Fig. 5

Wnt2 overexpression is sufficient to increase cortical dendrite length. (A) Representative cortical neurons expressing either EV or Wnt2. Quantification of the total dendrite length per neuron (B) and the number of dendritic endpoints per neuron (C) for ...

Fig. 6

Wnt2 overexpression increases dendritic protrusion density and influences spine shape on cortical neurons. (A) Representative dendritic segments of cortical neurons expressing either EV or Wnt2. (B) Quantification of dendritic protrusion density. (C) ...

Wnt inhibition and dendritic spine maturation

We found that a series of different Wnt signaling inhibitors were able to block BDNF-induced increases in dendritic spine density and dendritic spine head width

I think all this existing science really tells us a lot.

Back in the slow lane

In cancer research, decades have already been spent investigating Wnt signaling.

Wnt Signaling Inhibition: Will Decades of Effort Be Fruitful at Last?

Drugs that Enhance Wnt Signaling

Back in my world, with a little help from Google scholar, I rapidly find that drugs already exist that affect Wnt signaling.  Some very familiar names pop up.

Simvastatin Promotes Adult Hippocampal Neurogenesis by Enhancing Wnt/β-Catenin Signaling

SummaryStatins improve recovery from traumatic brain injury and show promise in preventing Alzheimer disease. However, the mechanisms by which statins may be therapeutic for neurological conditions are not fully understood. In this study, we present the initial evidence that oral administration of simvastatin in mice enhances Wnt signaling in vivo. Concomitantly, simvastatin enhances neurogenesis in cultured adult neural progenitor cells as well as in the dentate gyrus of adult mice. Finally, we find that statins enhance Wnt signaling through regulation of isoprenoid synthesis and not through cholesterol. These findings provide direct evidence that Wnt signaling is enhanced in vivo by simvastatin and that this elevation of Wnt signaling is required for the neurogenic effects of simvastatin. Collectively, these data add to the growing body of evidence that statins may have therapeutic value for treating certain neurological disorders.Simvastatin rescues cerebrovascular and memory-related deficits in mouse models of Alzheimer disease (AD) (Li et al., 2006; Tong et al., 2009, 2012), and recent meta-analysis of clinical studies concluded that statins provide a slight benefit in the prevention of AD and all-type dementia (Wong et al., 2013). While these effects have been attributed to reduction of inflammation, reduced oxidative stress, upregulated PI3K/AKT signaling, and enhanced neurogenesis, the mechanisms by which statins are beneficial in neurological disorders are not fully understood.Simva is under investigation for its potential therapeutic effects outside of hyperlipidemia treatment. While statins have been reported to enhance Wnt signaling in vitro, it was heretofore not known whether statins can enhance this pathway in vivo and in the context of neurogenesis. Here we provide evidence that oral simva treatment enhances Wnt signaling in the mammalian adult hippocampus. This is significant in that aside from lithium, no other clinically approved compound has been demonstrated to enhance Wnt signaling in the brain

You will find the element Lithium in your smart phone battery, but it is also a drug.

Lithium is useful in the treatment of bipolar disorder. Lithium salts may also be helpful for related diagnoses, such as schizoaffective disorder and cyclic major depression. The active part of these salts is the lithium ion Li⁺.

But, not surprisingly, Lithium has other effects, like activating Wnt signaling.

Lithium activates the Wnt and phosphatidylinositol 3-kinase Akt signaling pathways to promote cell survival in the absence of soluble survival factors.

Drugs that inhibit Wnt Signaling

There are drugs with the opposite effect, inhibiting Wnt signaling.

Verapamil Protects against Cartilage Degradation in Osteoarthritis by Inhibiting Wnt/β-Catenin Signaling

Abstract

In past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. FRZB, a soluble antagonist of Wnt signaling, has been studied in osteoarthritis (OA) animal models and OA patients as a modulator of Wnt signaling. We screened for FDA-approved drugs that induce FRZB expression and suppress Wnt/β-catenin signaling. We found that verapamil, a widely prescribed L-type calcium channel blocker, elevated FRZB expression and suppressed Wnt/β-catenin signaling in human OA chondrocytes. Expression and nuclear translocation of β-catenin was attenuated by verapamil in OA chondrocytes. Lack of the verapamil effects in LiCl-treated and FRZB-downregulated OA chondrocytes also suggested that verpamil suppressed Wnt signaling by inducing FRZB. Verapamil enhanced gene expressions of chondrogenic markers of ACAN encoding aggrecan, COL2A1 encoding collagen type II α1, and SOX9, and suppressed Wnt-responsive AXIN2 and MMP3 in human OA chondrocytes. Verapamil ameliorated Wnt3A-induced proteoglycan loss in chondrogenically differentiated ATDC5 cells. Verapamil inhibited hypertrophic differentiation of chondrocytes in the explant culture of mouse tibiae. Intraarticular injection of verapamil inhibited OA progression as well as nuclear localizations of β-catenin in a rat OA model. We propose that verapamil holds promise as a potent therapeutic agent for OA by upregulating FRZB and subsequently downregulating Wnt/β-catenin signaling.

Repositioning verapamil—for Wnt of an OA treatment

The river blindness drug Ivermectin and related macrocyclic lactones inhibit WNT-TCF pathway responses in human cancer

AbstractConstitutive activation of canonical WNT-TCF signaling is implicated in multiple diseases, including intestine and lung cancers, but there are no WNT-TCF antagonists in clinical use. We have performed a repositioning screen for WNT-TCF response blockers aiming to recapitulate the genetic blockade afforded by dominant-negative TCF. We report that Ivermectin inhibits the expression of WNT-TCF targets, mimicking dnTCF, and that its low concentration effects are rescued by direct activation by TCF^VP16. Ivermectin inhibits the proliferation and increases apoptosis of various human cancer types. It represses the levels of C-terminal β-CATENIN phosphoforms and of CYCLIN D1 in an okadaic acid-sensitive manner, indicating its action involves protein phosphatases. In vivo, Ivermectin selectively inhibits TCF-dependent, but not TCF-independent, xenograft growth without obvious side effects. Analysis of single semi-synthetic derivatives highlights Selamectin, urging its clinical testing and the exploration of the macrocyclic lactone chemical space. Given that Ivermectin is a safe anti-parasitic agent used by > 200 million people against river blindness, our results suggest its additional use as a therapeutic WNT-TCF pathway response blocker to treat WNT-TCF-dependent diseases including multiple cancers.

Effects of Caffeic Acid Phenethyl Ester in Wnt/β-Catenin Signaling Pathway on Human Neuroglioma

Previous studies have revealed that its anti-tumor function could be attributed to its ability to suppress the abnormal Wnt/β-catenin signaling pathway

What about hair loss/gain?

To quote from  the previous study on hair loss gain:-

“Using this new knowledge, we now know that Wnt activity can be modulated by adjusting the secretion of Wnt ligands, altering binding of ligands to receptors, inhibiting β-catenin translocation, or by regulating extra-follicular dermal Wnt and Wnt inhibitors.”

We have now learnt that the drug Verapamil is thought to be a Wnt inhibitor.  So it would be fair to assume that hair loss would be reported as a side effect of using Verapamil.  Indeed it is.

Dermatologic

Dermatologic side effects have included rash (up to 1.4%). Diaphoresis has been reported with intravenous verapamil. Arthralgia and rash, exanthema, hair loss, hyperkeratosis, macules, sweating, urticaria, Stevens-Johnson syndrome, and erythema multiforme have been reported during open trials/postmarketing experience.

What about Statins and hair?

So many millions of people take statins, of course somebody would claim it causes hair loss (alopecia).  I think it should cause hair gain.  As with Verapamil the effect on the hair growth would be much greater if it was applied to the skin and not taken orally.  Maybe older people would not go to the doctor to complain about hair gain?

Do statins cause alopecia?

Summary

·        As hair loss is a generally accepted male characteristic, drug-induced alopecia may be mistaken as part of a natural process and therefore under reported.

·        There have been reports of alopecia associated with the use of all UK licensed statins but there is insufficient data to confidently attribute hair loss to statin use.

·        Case studies suggest an association but as yet there is insufficient information to suggest a mechanism, make comparisons of the individual incidence of alopecia between the various statins or propose a class effect.

·        The greatest number of reports of alopecia is for simvastatin but this may be related to a greater market share or length of time on market.

It would seem that enough people lose hair from Verapamil for it to be a published side effect.  The same is not true for statins and I think hair loss may be coincidental.

But, maybe too much and too little Wnt signaling cause hair loss ?

Recall earlier in this post that Hans Otto Kalkman suggested that both too much and too little Wnt might cause similar behavioral and cognitive symptoms.  Perhaps the same is true with hair growth.

The canonical Wnt pathway plays a role in dendrite growth and suboptimal activity negatively affects the dendritic arbor. In principle, this provides a logical explanation as to why both hypo- and hyperactivity may generate a similar set of behavioral and cognitive symptoms.

For optimal hair growth perhaps there is an optimal amount of Wnt signaling? 

That might explain why a small number of people find Wnt inhibitors (Verapamil) and drugs that enhance Wnt (statins) cause hair loss.

That might mean that people with very full hair have optimal Wnt signaling?

So advise Homer Simpson to find out whether his Wnt signaling is hyper or hypo.  Then he might find either simvastatin or verapamil brings back his full head of hair.

Wnt signaling and Diabetes

Yet again we find another connection between Diabetes and autism.

In the pancreas  β-cells produce insulin. In diabetics these β-cells get destroyed.  It appears that Wnt signaling is involved in controlling these β-cells.  It has been proposed that they could be protected via this pathway.

Role of Wnt signaling in the development of type 2 diabetes.

 

Abstract

Type 2 diabetes is characterized by insulin resistance, insulin deficiency, and hyperglycemia. Susceptibility to type 2 diabetes has been linked to Wnt signaling, which plays an important role in intestinal tumorigenesis. Carriers of variants of the transcription factor 7-like 2 gene, an important component of the Wnt pathway, are at enhanced risk for developing type 2 diabetes. The modulation of proglucagon expression by Wnt activity may partially explain the link between Wnt signaling and diabetes, and one of the transcriptional and processing products of the proglucagon gene, the glucagon-like peptide-1 (GLP-1), exhibits a wide variety of antidiabetogenic activities. GLP-1 stimulates Wnt signaling in pancreatic beta cells, enhancing cell proliferation; thus, positive feedback between GLP-1 and Wnt signaling may result in increased proliferation, and suppressed apoptosis, of pancreatic cells. Since beta-cell protection is a potential treatment for type 2 diabetes, stimulation of Wnt activity may represent a valid therapeutic approach.

Wnt signaling: relevance to β-cell biology and diabetes

Here, we review emerging new evidence that Wnt signaling influences endocrine pancreas development and modulates mature β-cell functions including insulin secretion, survival and proliferation. Alterations in Wnt signaling might also impact other metabolic tissues involved in the pathogenesis of diabetes, with TCF7L2 proposed to modulate adipogenesis and regulate GLP-1 production. Together, these studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, highlighting the importance of further investigation of this pathway to develop new therapies for this disease.

Future Prospects of Wnt-Targeted Therapy for Treatment of Type 2 Diabetes

As with autism and cancer, the people with diabetes are also perhaps not benefiting from the latest science.

Preventing β-Cell Loss and Diabetes With Calcium Channel Blockers

Oral verapamil administration prevents β-cell apoptosis and STZ-induced diabetes.

The End.

Dr Dolittle, Autism and the Broccoli Sprouts

In the Dr Dolittle books and subsequent films, a man develops the power to communicate with animals.  It seems that one effect of broccoli sprout powder (and we assume Sulforaphane), in autism,  is an urge to talk, not only to humans, but also to animals.

Monty, aged 11 with ASD, took his first dose of 2.5ml of broccoli powder (Supersprouts brand from Australia) and after about half an hour developed euphoria.  The laughter later subsided and throughout the day he was very talkative.  This was relevant speech and not repeating things he had heard previously.  Other than the euphoria, which is the word chosen by elder brother Ted, a nice development was the desire to communicate with the animal world.

After a visit to his favourite ice cream shop, he looked up and saw the big railway bridge. “Bye bye railway station” commented Monty.  Walking up the hill we first passed a kitten, playing by the verge, “Hello baby kitten! Bye bye baby kitten!”  Then a big dog appeared “Hello big white dog and a woman! Bye bye big white dog and woman!”.  This was all rather unexpected.

The next day, another 2.5ml of broccoli powder and the same result.  Euphoria and lots of talking.

Then I decided to start experimenting with the dose.  I gave 1.25ml three times a day.

After the breakfast dose, no euphoria but still plenty of speech.  After lunch, the second dose and the return of mild euphoria.  After the evening dose, more euphoria.  The half-life of Sulforaphane in people is claimed to be about two hours.

Based on this limited experience, I think 2.5ml is about right.  There is no need for more.

  

Cost

I paid AU$ 110 (US$ 95 or GBP 60) for 300g of broccoli powder including shipping.

2.5ml of powder weighs 1.1g.  So using that daily dose of 2.5ml the cost would be 35 US cents (22 UK pence).

My earlier assumption was that a dose of about 18 g of fresh sprouts would produce the required level of Sulforaphane.  In theory, this would be 3 ml of broccoli powder, if it had 100% of the right enzymes in it and none of the bad stuff (called ESP, from the last post).  I was quite surprised at the effect of 2.5ml.  Johns Hopkins told me that most broccoli powders are no good; that is why I looked around before choosing the Australian product.

As a dosage comparison, this supplement is sold in Australia with a suggested daily dose of 5g, which equates to about 11 ml. 

So my “autism dose" looks quite conservative.  I think even half the suggested adult dose would make Monty completely hyper.

Note that the dose of the anti-oxidant NAC used in autism trials is 4X the usual adult dose of NAC and 2X the adult dose for adults with COPD (severe asthma).

The effect on an adult

I tried a scaled up dose myself, but sadly no euphoria followed.

  

Note

Monty is already taking a potent anti-oxidant called NAC, which has been investigated in an autism trial at Stanford.

The broccoli sprouts produce a substance called Sulforaphane (SFN).  This substance activates Nrf2 which upregulates “phase II enzymes”; they increase the body’s antioxidant response.  SFN is also an inhibitor of HDAC (Histone Deacetylase) and this may give SFN the ability to target aberrant epigenetic patterns.

SFN is therefore a secondary anti-oxidant.  It has been shown to improve the body’s response to cancer and environmental toxins.  The chemoprotective properties may result from SFN’s epigenetic properties or the anti-oxidant properties.

SFN was shown in a recent study at Johns Hopkins to improve autism in young adults.  It is not known definitively why it was effective.

Conclusion

My experiment indicates that, in classic autism, Sulforaphane (SFN) does provide a marked and immediate benefit over NAC alone, which is what I set out to determine.

Australian broccoli sprout powder appears to be a relatively cheap and effective way to make SFN at home. 

Sulforaphane, Epithiospecifier Proteins (ESP) or just Sulforadex for Autism

  

One reader of the last post on Sulforaphane raised the issue of whether she should cook her broccoli sprouts, to optimize her autism therapy.

This seemed a bit strange, since even the researchers at Johns Hopkins are eating their sprouts raw.  She does have a valid point.  It seems that while sprouts have large amounts of glucoraphanin and the required enzyme myrosinase, they also have something called Epithiospecifier Protein (ESP).  If there is much ESP present, instead of Sulforaphane you get a very similar compound called Sulforaphane Nitrile.  You can see that the “S” has been replaced by an “N”.

All is not lost, for those of you with sprouts growing in the kitchen.

Further research showed that the concentration of ESP in the sprouts peaks on the second day and that by day 5 has dropped dramatically.

It was also showed that raising the temperature of the sprouts to 60 degrees Celsius deactivated the ESP.  Heating Broccoli florets much beyond this then reduced the Sulforaphane produced, but not heating the sprouts.

Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli.

Abstract

Sulforaphane, an isothiocyanate from broccoli, is one of the most potent food-derived anticarcinogens. This compound is not present in the intact vegetable, rather it is formed from its glucosinolate precursor, glucoraphanin, by the action of myrosinase, a thioglucosidase enzyme, when broccoli tissue is crushed or chewed. However, a number of studies have demonstrated that sulforaphane yield from glucoraphanin is low, and that a non-bioactive nitrile analog, sulforaphane nitrile, is the primary hydrolysis product when plant tissue is crushed at room temperature. Recent evidence suggests that in Arabidopsis, nitrile formation from glucosinolates is controlled by a heat-sensitive protein, epithiospecifier protein (ESP), a non-catalytic cofactor of myrosinase. Our objectives were to examine the effects of heating broccoli florets and sprouts on sulforaphane and sulforaphane nitrile formation, to determine if broccoli contains ESP activity, then to correlate heat-dependent changes in ESP activity, sulforaphane content and bioactivity, as measured by induction of the phase II detoxification enzyme quinone reductase (QR) in cell culture. Heating fresh broccoli florets or broccoli sprouts to 60 degrees C prior to homogenization simultaneously increased sulforaphane formation and decreased sulforaphane nitrile formation. A significant loss of ESP activity paralleled the decrease in sulforaphane nitrile formation. Heating to 70 degrees C and above decreased the formation of both products in broccoli florets, but not in broccoli sprouts. The induction of QR in cultured mouse hepatoma Hepa lclc7 cells paralleled increases in sulforaphane formation.

So it would seem that if you want to eat the sprouts raw, you need to wait for five days before consuming them.  Not good to eat them when two days old.

If you cook them, you do risk affecting the myrosinase and then you might need to add back some more from another source, just as Nicole mentioned in her comment.  But some research implies the sprouts are heat stable.

This all starts to get rather complicated.

Personally I decided to buy freeze dried broccoli sprout powder from Australia.  They claim to measure for ESP, and there is very little.  Their myrosinase has not been deactivated in processing.

If true, their product is near ideal.  Is say near ideal, because one spoonful also has the taste of a plateful of broccoli.

Mine has now arrived and so I will serve one level teaspoonful a day.

Other research actually suggested that Daikon radish may be event better than broccoli.  Johns Hopkins chose to patent the broccoli.  In their research compound, they reacted broccoli sprouts with daikon radish sprouts to make a standardized Sulforaphane which is then freeze dried and kept frozen.

RADISH SPROUTS VERSUS BROCCOLI SPROUTS: A COMPARISON OF ANTI-CANCER POTENTIAL BASED ON GLUCOSINOLATE BREAKDOWN PRODUCTS

Comparison of bioactive phytochemical content and releaseof isothiocyanates in selected brassica sprouts

Daikon powder is readily available and is a potent source of heat stable myrosinase.

So I will seek to get the optimal output from my Australian sprout powder by adding a dash of Daikon powder.

A better way?  Sulforadex

This kitchen chemistry may all seem rather haphazard and indeed it is.

Rather than try and make 8 mg of Sulforaphane in your kitchen, would it not be better to buy 8 mg of standardized heat stable Sulforaphane in the pharmacy?

Sulforadex is potentially exactly that; it is an analog of Sulforaphane.  Trials have started in humans and at very much higher doses to check for toxicity and side effects.

Here is a link to the Phase 1 trial:-

http://www.richmondpharmacology.com/downloads/Publications/Evgen-BPS-poster-tiny-web.pdf

The only questions I have are:- is anyone 100% certain that Sulforaphane is the only beneficial compound produced by eating broccoli?  Is Sulforaphane the only compound present in Johns Hopkin’s frozen capsules?  When they react their broccoli sprouts with daikon sprouts in the lab, there are other compounds produced.

Monty, aged 11 with ASD, is by now remarkably accommodating when it comes to downing unappetizing potions.  NAC tastes pretty bad, unless you use the more expensive effervescent variety.  But this pales in comparison to what a spoonful of broccoli sprout powder tastes like (and looks like).

They also make this powder in capsule form, for those who can swallow them. 

The more appetising anti-oxidant would be a bar of high flavanol dark chocolate, as we discovered in the previous post.  As well as tasting better, it may quite possibly be just as effective.

Why not Cocoa Flavanols for Autism?

Why not Cocoa Flavanols for Autism?

  

Judging by my blog statistics, lots of people are interested in broccoli (Sulforaphane) to treat autism.  Thanks to the patents held by Johns Hopkins, you can expect to hear much more about Sulforaphane in the coming years.

Meanwhile, Columbia University and Mars, the chocolate people, have released a study showing that “flavanoids” in cocoa can do wonders for memory loss in older people.  In effect, they can restore memory in 60 years olds to where it was 20 or 30 years earlier.

If you take a step back and look at what is known by science about oxidative stress and antioxidants, all will become much clearer.

Oxidative Stress Pioneers

In an earlier post we met Paul Talalay, a German-American, who worked at Johns Hopkins.  He specializes in foods that protect you from cancer.  He is Mr Broccoli. 

It turns out that perhaps the real pioneer in this field is a 100% German, called Helmut Sies, who also studies foods that act as antioxidants and nutrients that provide protection from cancer.  We have his very detailed diagram below, that explains the relationship between many of the factors involved in oxidative stress.  I wish I had found it earlier.  I added the six outer boxes.

If you want to read clever studies about this subject, just include Helmut Sies in your search; for example “selenium Helmut Sies”.

Redox Pioneer: Professor Helmut Sies

On this graphic you will see GSH (Glutathione).  When you take NAC (N-acetylcysteine) you directly raise the level of GSH.  When eat broccoli you activate Nrf2, which is a Redox switch, just under the traffic light in the graphic.

When you eat certain flavonoids, like Cocoa, or carotenoids like lycopene (found in tomatoes), you again promote the anti-oxidative free radical scavenger effect.  Look in the blue boxes under diet.

Not on the diagram, we also have flavonolignans which are natural phenols composed of a part flavonoid and a part lignan. As pointed out in a comment in the last post by Seth Bittker, one interesting  flavonolignan is Silibinin, which has anti-oxidant and chemoprotective effects

Note the presence of (Coenzyme) Q10 in the yellow box.  This is part of the mitochondrial cocktail suggested by Dr Kelley from Johns Hopkins for regressive autism.  Q10 is depleted by statins.

Glutathione peroxidases, in the yellow box, are also very interesting.  These are selenium-containing enzymes.  GPx (x goes from 1 to 8)  catalyze the reduction of H₂O₂ and organic hydroperoxides to harmless products. This function helps to maintain membrane integrity and to reduce further oxidative damage to molecules such as lipids and lipoproteins with the associated increased risk of conditions such as atherosclerosis.  It appears GP₁ may be defective in autism and this is contributes to increased oxidative stress.  This area has been well studied due to its impact on heart disease.  You appear to be able to counter the lack of GPx with yeast-bound selenium, other forms of selenium do not work, due to a lack of bioavailability. A post will appear just on Selenium.

There are several other potent (exogenous) antioxidants that we have come across:-

• Alpha lipoic acid also known as ALA or Tioctic acid (found  in Dr Kelley’s cocktail)
•   L-Carnosine (studied by Dr Chez )
•  Vitamin C (suggested by many, including Dr Kelley)

Another day, another anti-oxidant

In human health, two well used anti-oxidant drugs are Alpha lipoic Acid (ALA,  also known as Tioctic acid) and N-acetyl cysteine (NAC).  They share many similar effects.

•       Potent antioxidant
•       Increase insulin sensitivity
•       Improve memory in those with mild cognitive          impairment
•       May lower blood pressure
•       Improve behavior in autism

NAC is widely used to treat Chronic obstructive pulmonary disease (COPD) and ALA is used to treat diabetic neuropathy. Perhaps they could be interchanged

·        NAC has a chemoprotective effect
·        ALA has been shown to induce cell cycle arrest in  human breast cancers      cells

Back to Cocoa Flavanols and Mars

This flurry of activity was driven by a well publicized study done at Columbia University Medical Center (CUMC), using a high cocoa flavanol concentration drink provided by Mars.

Dietary cocoa flavanols reverse age-related memory decline

   

In the CUMC study, 37 healthy volunteers, ages 50 to 69, were randomized to receive either a high-flavanol diet (900 mg of flavanols a day) or a low-flavanol diet (10 mg of flavanols a day) for three months. Brain imaging and memory tests were administered to each participant before and after the study. The brain imaging measured blood volume in the dentate gyrus, a measure of metabolism, and the memory test involved a 20-minute pattern-recognition exercise designed to evaluate a type of memory controlled by the dentate gyrus.
The high-flavanol group also performed significantly better on the memory test. “If a participant had the memory of a typical 60-year-old at the beginning of the study, after three months that person on average had the memory of a typical 30- or 40-year-old,” said Dr. Small. He cautioned, however, that the findings need to be replicated in a larger study—which he and his team plan to do.

This is very impressive.  But how do the other anti-oxidants compare?

Well, without funding from Mars, researchers only managed the money to test ALA and NAC on mice; but as you might expect, the result was similar.

The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice

Chronic administration of either LA or NAC improved cognition of 12-month-old SAMP8 mice in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific effects on motor activity, motivation to avoid shock, or body weight. These effects probably occurred directly within the brain, as NAC crossed the blood-brain barrier and accumulated in the brain. Furthermore, treatment of 12-month-old SAMP8 mice with LA reversed all three indexes of oxidative stress. These results support the hypothesis that oxidative stress can lead to cognitive dysfunction and provide evidence for a therapeutic role for antioxidants.

Cocoa Flavanols are good for your heart

This is also good news, but it does seem that antioxidants are generally very good for your heart.

First cocoa.

In this study blood pressure, glucose, insulin and cholesterol were all markedly affected for the better by the cocoa as was cognitive function.

This is great;  but it is what Helmut Sies has been telling the world for many years.

Benefits in Cognitive Function, Blood Pressure, and Insulin Resistance Through Cocoa Flavanol Consumption in Elderly Subjects With Mild Cognitive Impairment

The Cocoa, Cognition, and Aging (CoCoA) Study

Abstract—Flavanol consumption is favorably associated with cognitive function. We tested the hypothesis that dietary flavanols might improve cognitive function in subjects with mild cognitive impairment. We conducted a double-blind, parallel arm study in 90 elderly individuals with mild cognitive impairment randomized to consume once daily for 8 weeks a drink containing _990 mg (high flavanols), _520 mg (intermediate flavanols), or _45 mg (low flavanols) of cocoa flavanols per day. Cognitive function was assessed by Mini Mental State Examination, Trail Making Test A and B, and verbal fluency test. At the end of the follow-up period, Mini Mental State Examination was similar in the 3 treatment groups (P_0.13). The time required to complete Trail Making Test A and Trail Making Test B was significantly (P_0.05) lower in subjects assigned to high flavanols (38.10_10.94 and 104.10_28.73 seconds, respectively) and intermediate flavanols (40.20_11.35 and 115.97_28.35 seconds, respectively) in comparison with those assigned to low flavanols (52.60_17.97 and 139.23_43.02 seconds, respectively). Similarly, verbal fluency test score was significantly (P_0.05) better in subjects assigned to high flavanols in comparison with those assigned to low flavanols (27.50_6.75 versus 22.30_8.09 words per 60 seconds). Insulin resistance, blood pressure, and lipid peroxidation also decreased among subjects in the high-flavanol and intermediate-flavanol groups. Changes of insulin resistance explained _40% of composite z score variability through the study period (partial r2_0.4013; P_0.0001). To the best of our knowledge, this is the first dietary intervention study demonstrating that the regular consumption of cocoa flavanols might be effective in improving cognitive function in elderly subjects with mild cognitive impairment. This effect appears mediated in part by an improvement in insulin sensitivity.

There are more cocoa studies:-

Enhancing dentate gyrus function with dietary flavanols improves cognition in older adults  The effect of acute consumption of a flavonol-rich cocoa drink on cerebral vasomotor reactivity in African Americans

Mars Cocoa Science website

Cocoa Flavanols as a therapy for Autism

Based on the work of Helmut Sies and the trials funded by Mars, it is pretty obvious that 1,000mg of cocoa flavanols a day would very likely have a marked effect on someone with autism, assuming that is they were not already taking NAC, ALA, Carnosine, Broccoli, Sulforaphane or Selenium.  500 mg should also have an effect.

Choice of antioxidant

The question is what is the ultimate treatment for oxidative stress in autism?

I guess this will depend on exactly what type of autism you have (regressive or not), to what extent you have a mitochondrial dysfunction and whether you have any genetic dysfunction related to oxidative stress.

What works best in Billy, may be suboptimal in Charlie, but still much better than nothing at all.

It looks to me that NAC and ALA will likely be the most potent antioxidants.

If you live in the US, you can buy cocoa flavanols in standardized doses from Mars.  One capsule = 125mg of cocoa flavanols.   I have to add that I am far more inclined to believe Mars, than those supplement companies out there.  You can buy tablets saying they contain 50 mcg of Selenium, but what do they really contain? 

You can also buy “high flavanol” raw (non-alkalized) cocoa powder in big bags.  This lighter brown cocoa has lost far less of the flavonoids in the processing process.  In theory, a 5g teaspoon of the very best one will contain (on a good day) 415 mg of flavavols.

Mars are only supplying their CocoaVia products in North America, so if you want to try cocoa flavanols you have a few options:-

·        8.5 teaspoons of standard raw cocoa  (content will vary widely)

                or

·        1.2 teaspoons of “Chococru” upmarket raw cocoa

                or

·        4 capsules of CocoaVia from Mars  

Each of the above should give you 500mg of cocoa flavanols, which would look like a good starting point.  As with NAC, the studies show that the benefit increases the more you take, but the extra benefit drops off.

If somebody in the US tries CocoaVia, do let us know the result.

Not surprising, Mars tell us on the label that the product is not intended for children.  I do not suppose they ever thought of it being an autism therapy either.

I do like the idea of the redox switch, Nrf2, which Sulforaphane is known to activate.  I also like the idea of the enzyme GP₁ that acts as catalyst in the oxidation/reduction process.

The science is around 20 years old and nobody has yet figured it all out;  they probably will not conclusively do so in the next 20 years either.

Food for thought!