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Wednesday, 26 April 2017

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 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




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.





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. 



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.    



 



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 

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 (1421), 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 (4648). 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 (5154). 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.  



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.  


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.  


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.   


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.




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

  

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”. 





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.



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 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.  




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.













38 comments:

  1. Well the question here on Zinc that we have yet to resolve is what is the best form of supplemental zinc (for the brain) in a manner similar to how Magnesium Threonate is touted as a superior form of Magnesium for the brain since it is reputed to cross the blood brain barrier much better than any other form of magnesium. Some research I have seen has suggested the positive brain health effects are from the threonate and not the magnesium. Threonate is a metabolite of Vitamin C by the way and what is also interesting is that there is a compound called Zinc L-Threonate which has near perfect absorption which can easily make it toxic for this reason alone as too much zinc can cause a lot of problems. In fact, I don't even think you can get Zinc L-Threonate as a supplement (I looked around and only found research papers), but I could be wrong.

    ReplyDelete
  2. A very interesting paper on frontotemporal dementia just came out that might as well be written about autism for many reasons pertinent to several big topics on this blog:

    Press Release:

    https://www.sciencedaily.com/releases/2017/04/170424152530.htm

    Paper:

    http://www.pnas.org/content/early/2017/04/19/1700477114

    There is a lot of really interesting stuff here where you could literally replace the words "fronttemporal dementia" with autism and the paper would still make sense.

    The main takeaways here are that they claim to have proven (at least with frontotemporal dementia) that obsessive behaviors (excessive self-grooming in mice) are caused by microglial activation. Also, even though their model was produced by genetically mutating a particular type of protein called progranulin (which seems to induce frontotemporal dementia), they found that the obsessive compulsive behaviors were rescued by inactivating NF-KB in microglia which of course downregulated TNF-a expression from the microglia as well. This also caused medium spiny neurons (GABAergic cells which comprise 95% of the basal ganglia neurons) to reduce their hyperexcitable state as well and normalize OCD like behaviors.

    What is awesome about this is it suggests that reducing microglial activation or at least focusing on NF-KB and TNF-a signaling like a laser beam could yield very significant improvements in some core autism symptoms (well this paper does not actually claim that, but this is my strong opinion on the matter).

    Of course interventions/research on microglial hyperactivation in autism is nothing new, but I think this research in frontotemporal dementia may give added weight to taking a closer look at ways of modulating NF-KB and TNF-a signaling in the brain as to me at the moment this seems like some very low-hanging fruit worth picking.

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    Replies
    1. Tyler, it looks like the herb-rich Greek/Mediterranean diet, including the strong coffee, might be in order.

      Extract of Oregano, Coffee, Thyme, Clove, and Walnuts Inhibits NF-κB in Monocytes and in Transgenic Reporter Mice
      http://cancerpreventionresearch.aacrjournals.org/content/3/5/653.long

      This study shows that dietary plants may be potent modulators of NF-κB signaling both in vitro and in vivo, and thus support further investigation of consumption of these plant foods as part of a healthy diet or as a mode of chemoprevention

      Also note that oxidative stress activates NF-KB and so using NAC to reduce oxidative stress may control stereotypy in part by reducing NF-KB activation.

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    2. Hi Peter and Tyler,

      Any thoughts on PQQ as an option based on this paper:

      https://www.ncbi.nlm.nih.gov/pubmed/25314304

      Thanks in advance!

      AJ

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    3. AJ, here is list of the drugs that inhibit NF-KB. They screened 2,800 approved drugs and found just 19.

      Identification of Known Drugs that Act as Inhibitors of NF-κB Signaling and their Mechanism of Action
      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834878/

      One is a common asthma drug, Accolate.

      Delete
  3. so suplementing with Zn isn't useful?Is better to try with Clioquinol? But we don't know what dosis we have to give and how many times a day

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    Replies
    1. Yes, that just about summarizes where we are. We need to know if humans react to Clioquinol as positively as the mice.

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  4. Look forward to reading this tonight.
    Peter, what are your thoughts on this:
    https://www.ncbi.nlm.nih.gov/pubmed/15519738
    I just asked Valentina if she is using zinc in combination with histidine therapy because when I was considering histidine in the past, I jotted this down in my notes - that zinc is needed to activate histidine. (?) But I didn't look further in to it because I decided to back-burner the histidine trial..

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    Replies
    1. Tanya, some zinc supplements include histidine to improve bioavailability. Histidine is known to cause loss of zinc. So the two do seem to go together.

      Histidine is cheap if you buy as powder, since you do not need much. It looks like sugar crystals. I am interested in it primarily as yet another way to reduce histamine from allergy. I only just started with it, because I only now have an allergy to treat. 1g does have an effect; I now need to see how long it lasts to see whether the effect fades over time

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    2. Tanya, again about sodium bicarbonate, as I read, in addition to its natural antihistamine properties, it is also used as an antidote for calcium channel blockers toxicity.
      I know that your son is on Verapamil and I thought perhaps you find this relevant.
      Have you ever considered using it orally or use food/drink high in sodium bicarbonate?
      You've done lots of research on histamine and probably used some antihistamines. Do you have any experience with Benadryl?
      It is a sodium channel blocker.

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    3. Petra, I had to stop verapamil. I realized it was causing strange side effect of an issue involving his mouth: it seemed to be affecting muscle control. He also wasn't speaking as much while on it. So I stopped and went back to the other mast cell stabilizers he was on and continued with extra potassium. Yes, a bit of baking soda in water to drink has been a remedy we have used with him for years. But a couple of years ago I learned of a remedy combining baking soda and potassium bicarbonate for histamine and we use that now. It seems at this point whenever he has any edgy moment, we know it is related to gut and the baking soda remedy stops it. Yes we do use benadryl. It really helps. I don't give it to him everyday though. I had no idea it was a sodium channel blocker! thanks for sharing.

      Delete
  5. Tanya, I am doing histidine since december,must tell you that he is without tics since then. I have clear all my son´s kind of movements: dyskinesias, tics and stimming or hand clapping, as it is the only stim that he has. But tics have disappeared, dyskinesias are almost eliminated, they are stubborn and returns form time to time. But tics, never came back. I agree with Peter that 1 gr is a good dose, I wouldn´t give more than that and of course added zinc, no more than 40 mg.
    Valentina

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  6. Peter, thanks for the links about histamine and Tourette. TS is strongly related to histamine and dopamine neurotransmission. H3 receptor has been revealed as an important regulator of dopamine and sensory motor gating. Would you use an H3 agonist in my situation? I remember asking you about betahistine, but it is an H3 antagonist
    Valentina

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    Replies
    1. Valentina, if the tics have been absent for 4 months with histidine, you may have solved the problem. You might assume that the histidine has increased histamine in the brain and this is your H3 agonist. You also have H1 and H2 in the brain and they may also be affected. If 1g of histidine has a stable long term effect, that is great.

      Delete
    2. Perfect! that´s it, I didn´t notice them anymore, but as I was focused on dyskinesias,wich I have controlled thanks to Tyler, and my son has had many kind of movements, I was not fully aware ot this constant change about tics.
      Valentina

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  7. Hi everyone,

    First, kudos to Peter on another great post - I knew about SHH in general, but didn't know it's impact on autism, and certainly wasn't familiar with IHH.

    It looks like there are several natural products that can inhibit SHH:

    https://www.hindawi.com/journals/ecam/2013/748587/
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546096/

    Any thoughts on inhibiting HH would be appreciated.

    On another front, I found the following interesting article on A2A Receptors and autism:

    https://www.ncbi.nlm.nih.gov/pubmed/28438638

    I looked up A2A receptor agonists and it looks like Limonene is an easy to acquire option. Does anyone have any experience with Limonene, or have any thoughts on the paper / A2A receptors? I'm considering adding Limonene to my treatment protocol but wanted to see if anyone had any insights.

    Thanks everyone!

    AJ

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    Replies
    1. I posted some stuff on this subject a while ago on this blog concerning the use of A2A agonists for opening the blood brain barrier to allow larger more complex molecules not chelated to some biological molecule like a protein or sugar (such as drugs) to get through easier.

      I can't remember what drug they used off the top of my head (I am sure you can dig up the links I posted on the blog pretty easily yourself).

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  8. Hi everyone,

    Just in case it hadn't been mentioned and you weren't aware of Ovid Therapeutics recent initiation of a phase I trial for Fragile X and Angelman syndrome:

    http://www.ovidrx.com/ovid-therapeutics-announces-initiation-of-phase-1-clinical-trial-of-ov101-for-adolescents-with-angelman-syndrome-or-fragile-x-syndrome/

    And the creation of Vencerx can only help:

    http://www.biospace.com/News/former-pfizer-neuroscientist-to-helm-new-york/451295

    AJ

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  9. So apparently, maternal grandmother smoking has a dramatic risk factor for increasing an ASD diagnosis in her grandchildren (53%) according to a new study:

    Press Release:

    https://www.sciencedaily.com/releases/2017/04/170427091740.htm

    Paper:

    https://www.nature.com/articles/srep46179

    This is not quite comparable to the risk factors of obesity and gestational diabetes in increasing the odds of an autism diagnosis in progeny, but still a pretty big effect.

    Even though smoking rates have gone down quite a bit in the last several decades in the United States, in much of the rest of the world smoking continues to be enemy number one in the budgets of public health systems and now you can add autism to the list of increased existential costs of legalized tobacco.

    Of course what is most pertinent to discussion here is the "why" ASD diagnosis spikes so dramatically via either epigenetic mechanisms or perhaps direct genetic mechanisms from the DNA damage caused by smoking via alpha radiation and other carcinogens and mutagens in tobacco products, but especially cigarette smoke.

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  10. Peter Iam planning to give my son Diamox plus low dose valproate, 100 mg twice a day diamox and 125 mg a day of valproate,or perhaps even less valproate? what do you think?
    Valentina

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    Replies
    1. Valentina, a lot is written about treating Tardive Dyskinesia. It is thought to be associated with too much of the amino acid phenylalanine and your branch chained amino acids are thought to work by reducing phenylalanine.

      There have been other suggested therapies, like the vitamin B6 you use.

      Diamox was also shown effective in small trials.

      I thought you were trying to stop using Valproate, due to side effects.

      It is probably wise to avoid phenylalanine in diet. You do need some of it, but large amounts may not be wise if you have TD, so best avoid aspartame as in sugar-free drinks.

      Do you think you have TD and tics ? or are the tics just part of the TD.

      The mechanism behind Tourette's tic and TD may be similar, but I doubt it is identical.

      Did your son have tics before the developed TD from his drug therapy?

      Delete
    2. Peter, I believe that in the case of my son´s autism all leads to a permanent activated microglia, that react with mast cells to produce neuroinlammation. Even his electrical activity, I dare to say. Also,I have had concrete proof of immune responses, mainly during the last two years. Allergie, parasites, linfophenia, viral and bacterial infection that are products that goes to the brain. At that time, complex motor tics were in the highest point. It was the moment I disappeared from the blog, I was confused and looking for answears and treatments. To this, was added the prolonged use of risperidone for years, and dyskinesias,mainly tongue protrusion and orofacial movements within buccal cavity,started to mix up with complex tics that had a different origin. An when I returned to this blog and with both you and Tyler help I started to put out fires that came from diferent places. I was thinking of adding ALA.
      Valentina

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  11. Hello Peter, Tyler,

    After five days course of amoxicillin which has unceremoniously kicked my son down into a pit full of systemic adverse effects including loss of attention and focus, digestive issues, painful erections once again (just before falling asleep and focusing) and a still blocked nose. Folinic acid drove him into a state of almost euphoric mania for 18 hours. Searching on the net I did find irritability as a common side effect but there was no report of extended meaningless laughter. I am suspecting a major role of histamine here..I think fisetin was helping but have run out of stock. I do have the perilla oil but could you please tell me the dose for a five year old. Net search does not yield any information but does take you to Tyler's comment on your blog where he unfortunately does not mention the dose. Please help.

    Respectful regards

    ReplyDelete
  12. Hello Peter, Tyler,

    As mentioned earlier, day before yesterday, I gave my son less than one quarter of an 800 mcg folinic acid tablet (source naturals) along with oral mb12 and around ten hours later he was laughing like crazy..losing his senses would be an apt description as he could not even remember how to write simple numbers..irrerestible laughter. It was not a seizure as he was not zoned out and totally responsive, following basic instructions but the tasks demanding higher mental faculties were difficult..understandably as either you can laugh or you can focus. But today, around forty eight hours later, i was making him repeat some simple 'words'..Nana, nani, nini, nunu, mama,Dadi etc.and he responded perfectly. This clarity was lost for a long time for us...more than six months and i am not sure what the trigger was for that deterioration.

    Could this positive turnaround be a result of folinic acid in which case i could give him just a miniscule dose on alternate days.

    I had also given him l.johnsonii for the first time yesterday hoping to restore his digestion following antibiotics.

    Do share your insights or intuition. I am happy about the speech clarity and if I could just nail down the helpful intervention, unless it is a random event, it would be a giant step ahead for us.

    Regards

    ReplyDelete
    Replies
    1. Kritika, maybe your son benefits from the beta lactam effect on glutamate, but also has an allergy to this specific antibiotic. Beta lactam antibiotics affect GLT1 which reduces any excess glutamate, in some people this gives a big behavioral improvement. Next time ask your doctor for a beta lactam that is not based on penicilin.

      Delete
    2. Peter,

      I do know about beta lactam effect on some autisms and hence the behavioural improvements in some when on certain antibiotics, thanks to your blog. However, I was not reading too much into amoxicillins effects on my son (this is a standard prescription by our paed)other than negative behaviours arising out of gut dysbiosis. But the first one or two days on antibiotic are good after which the adverse effects start outweighing the positives.. So yes, your intuition may be correct as I was considering glutamate reduction behind the good things we saw with NAC despite of stomach discomfort.

      That said, the speech thing we saw was a good three four days following cessation of antibiotic. And can i infer something from his odd reaction to folinic acid? I did manage to find a few very cursory mentions of euphoria due to folate or folinic acid but nothing helpful.

      Nevertheless, thanks a lot
      Will now search your blog to once again read about glutamate and its antagonists. Reading your blog regularly has now educated me and I suppose many others enough that much of the scientific terms now do not seem like monkeytypes but propah English and most often ring a bell and not the church ones.

      Thanks for educating us

      Delete
  13. Peter, I started Diamox 125mg a day and half sprinkle 125 mg valproate.I have some questions. Don´t know if it is better this combination,or take out valproate completley?. Should I start with 250 mg diamox per day or only with 125 for a few days? with two bananas is enough?
    Valentina

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    Replies
    1. I would use just Diamox and start at 125mg, to see how it is tolerated. Bananas are a good idea, but potassium is less of an issue than with bumetanide.

      Delete
    2. Ok, thank you very much!

      Delete
  14. http://journal.frontiersin.org/article/10.3389/fimmu.2017.00442/full
    great article about autoimmune encephalits, hedgehog signaling, etc. My son had autoimmune encephalitis badly, and we are so much better now. Peter I love your blog, but it is harder for me to follow now as it seems things are so more complex than we I first read it (e.g. probably good likelihood that sulfuraphane would help your child :)

    ReplyDelete
    Replies
    1. It is more complex, but it now aims to cover everything, not just the more superficial parts of the subject. This is the only way to find all the possible effective therapies, of which there are many more. I did avoid the complex areas, but if you go step by step even the most complex subjects are within grasp.

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  15. Here is some research which may or may not pertain to blood flow issues in the brain and perhaps maybe a reason hyperbaric oxygen therapy produces short-term improvements in some people with autism:

    Press Release:

    https://www.sciencedaily.com/releases/2017/05/170501112612.htm

    Paper:

    https://www.nature.com/nm/journal/vaop/ncurrent/full/nm.4331.html

    Now before I say anything else, this research involves spinal cord injury and may have nothing to do with how things work in the brain with regards to oxygenation issues. Nevertheless, I thought it was interesting and might be a long-shot explanation worth thinking about.

    What the researchers found is that after a spinal cord injury (in rats), instead of an increase of oxygen to the injury site (for repair), they found that at the capillary level there was a decrease in oxygen instead which retarded the neuronal networks into malfunctioning (neurons need oxygen after all). They then dove deeper into why this was the case and they found an upregulation of the enzyme AADC which is the enzyme that both serotonin and dopamine share/compete in converting 5-HTP to serotonin and L-DOPA to dopamine. Furthermore, they found an upregulation of 5-HT1 (serotonin 1) receptors on a type of cell called a pericyte which is kind of an intermediary cell that does a lot of things with regards to blood vessel function. This suggests that too much serotonin signaling (and too much serotonin) is literally cutting off the oxygen to the spinal cord injury area.

    So the researchers then did two things: One, they inhibited the AADC enzyme from being expressed (reducing amine production such as serotonin or tryptamine) and oxygen levels increased; two, they increased oxygen levels through inhaled oxygen. Both interventions improved oxygenation to the capillaries and improved symptoms, although temporarily.

    Now, why is this interesting? Well hyperbaric oxygen therapy for autism in my opinion (and still in my opinion) is kind of a quack therapy with little to no scientific evidence behind it for improving autism symptoms. Nevertheless, to be a contrarian here what if HBOT did improve symptoms (temporarily) for some people due to a similar problem in the brain where too much AADC enzyme is being expressed and too many 5-HT1 receptors on pericytes exist, which has the effect of restricting oxygen flow to the capillaries in the brain which would dramatically impair neuronal function. When doing an actual study on HBOT, no long-term improvements would be found because it would only improve symptoms for a very short time (enough that anecdotal reports from parents would be favorable).

    In the maternal immune activation model of autism, one of the noticeable hallmarks is excess serotonin from the mother getting into the blood of the baby and perhaps dysregulating not just the neuronal aspects of serotonin, but also the many other functions of serotonin in the body and brain which of course includes regulating blood flow to the capillaries.

    Under the very big assumption (and only my assumption here) this research on rats and on spinal cord injuries might more broadly apply to the brain overall, other than BCAA therapy, I don't really know of any good way to limit AADC expression other than to limit the fuel itself (amines derived from tryptophan). There is of course the oxygen inhaling intervention, but elevated levels of inhaled oxygen is not something you want to do long-term (high levels of oxygen kill brain cells for one thing). Maybe there is a low-level of increased oxygen inhalation that would be safe, but I have no idea what that would actually be.

    Anyone have any other ideas with respect to AADC modulation other than BCAA therapy and/or diet restricting tryptophan, phenylalanine, and tyrosine?

    ReplyDelete
  16. Peter
    first - sorry for my English.
    We started with zinc a month ago (our Tobi is 5yo, unspecified pervasive disorder with autistic symptoms, fortunately verbal) and so far I can see some amelioration. However now I found some document about zinc supplements and going to be doubt - they
    claims the zinc blocks or suppress aromatase. Hence testosterone is elevated then.
    In your older posts you wrote aromatase rises estradiol which has neuro-protective efect. So do you thing is it true the zinc suppress aromatase?
    If yes, I should reconsider zinc supplementation.

    ReplyDelete
    Replies
    1. It looks like you need to take a large amount of zinc to reduce aromatase, so I would not worry, as long as you give a moderate dose of zinc. Too much zinc is not good for you for many reasons.

      Delete
  17. I have been reading quite a bit on this idea of elevated copper and low zinc. It's fascinating and I apprecaite you publishing and recognizing the research. I think in terms of dosage, there seems to be some confusion with elemental zinc. I read that a simple 50mg zinc gluconate, only contains 7mg of elemental zinc.

    http://www.aafp.org/afp/2009/0501/p768.html

    For my 9 year old on the spectrum, he would need to have at least 60-70mg of zinc gluconate, just to hit the RDA of 8mg.

    https://ods.od.nih.gov/factsheets/Zinc-HealthProfessional/

    Is this epidemic partially related to simple zinc deficiency and/or elevated copper (or other metals) levels, which could be from exposure from a host of different sources? I am simplifying this all of course, but after stumbling on to Pfeiffer's writings on schizophrenia back in the 1960's and 1970's, I am interested in seeing additional current research, if it exists.

    https://riordanclinic.org/wp-content/uploads/2014/12/The_Schizophrenias_Ours_to_Conquer-Riordan-Clinic-Books.pdf

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  18. Thank you for yet another brilliant post!

    Peter, you mention SHANK3 in this post, but also in earlier posts scattered on the blog. And it does get complicated.

    What would be your best suggestion on therapies to consider for a child with SHANK3/Phelan McDermid/22q13?

    /L

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    Replies
    1. Within the definition of Phelan-McDermid syndrome (PMS) there are wide variations, with between 30 and 190 genes possible affected, which may or may not include Shank3. There at least 12 other autism genes on the long arm of chromosome 22.

      If you have not already done, so I would try and have genetic testing to see exactly which genes are affected. Then you could figure out more precisely what might be therapeutic.

      If Shank3 is lost I would look at the rescue models of Shank3 autism in mice.

      https://gene.sfari.org/database/animal-models/genetic-animal-models/SHANK3#rescue-models-tab

      One intervention for example is IGF-1 (insulin-like growth factor 1)

      You can increase IGF-1 with a well known drug called Baclofen. Intranasal insulin should bind to IGF-1 receptors in the brain.

      In the above post the topic was zinc, and it looks like in Shank3 zinc is important. Zinc itself was suggested as a possible therapy and is easy to try.

      Delete
    2. Thank you very very much Peter. Your effort to help people seems endless. Where would we be without you (and Monty)?

      /L

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