Showing posts with label zinc. Show all posts
Showing posts with label zinc. Show all posts

Saturday 3 June 2017

Connecting Estradiol with WNK, SPAK and OSR1; plus Taurine

Japan, home to today’s complicated research

Today’s post hopes to give a more complete picture of the various processes involved in shifting the immature neurons often found in autism towards the mature neurons, found in most people.  This stalled process is complex and may only apply to around half of all autism.
The post assumes prior knowledge from previous posts about the GABA switch and the KCC2 and NKCC1 chloride cotransporters.
The best graphic I found is below and includes almost everything. The paper itself is very thorough and I recommend the scientists among you read the paper rather than my post.
What we want to understand is why neurons did not switch from immature to mature, in the process I am calling the “GABA switch”.  We know a great deal about what happens before and after the switch and many processes that can be  involved, but the exact switch itself remains undefined.
In a previous post I highlighted that neuroligin 2 (NL2)/RORa may be the GABA switch, but there is no mention of neuroligins in the research reviewed today. 

So when you read today’s mainly Japanese research, you should note that one key part is missing, the actual trigger mechanism.

The ideal way to make neurons transition from immature to mature is the way nature intended. That requires an understanding of the GABA switch mechanism.

Source and excellent paper:-

 The important things you might not notice:

E is the female hormone estrogen/estradiol

T is testosterone. Testosterone can be converted to estradiol by aromatase.

DHT is another male hormone Dihydrotestosterone. DHT is synthesized from testosterone by the enzyme 5α-reductase. In males, approximately 5% of testosterone undergoes 5α-reduction into DHT. DHT cannot be converted into estrogen.

Relative to testosterone, DHT is considerably more potent as an agonist of the androgen receptor (AR). This may turn out to be very important.

T3 is the active thyroid hormone, triiodothyronine

In earlier posts we saw that in autism there can be a lack of aromatase and that there is reduced expression of estrogen receptor beta.
In the diagram below this leads to reduced estrogen and increased testosterone. If there is elevated DHT this will make the situation worse.  All this down-regulates ROR-alpha.
ROR-alpha affects numerous things and is another nexus which links biological processes that have gone awry in autism. By upregulating ROR-alpha multiple good effects may follow, these include increasing KCC2 and reducing NKCC1.
It is certainly possible that the GABA switch is mediated by RORa-estradiol-Neuoligin-2.  In which case the solution is to upregulate RORa which can be done in many ways (androgen receptor, estrogen receptors etc.)

The schematic illustrates a mechanism through which the observed reduction in RORA in autistic brain may lead to increased testosterone levels through downregulation of aromatase. Through AR, testosterone negatively modulates RORA, whereas estrogen upregulates RORA through ER.

androgen receptor = AR

estrogen receptor = ER

Going back to the complex first chart in this post, we want to increase KCC2 in the immature neuron and reduce NKCC1.
So we want lines with flat end going into NKCC1, for example from OXT (the Oxytocin surge during natural birth).
We want arrows going to KCC2, for example we want more PKC (Protein Kinase C) coming from those  mGluRs, that we have come across many times in this blog.
What we do not want is anything coming from WNK- SPAK- OSR1.
Reduced expression of the thyroid hormone T3 does affect the both KCC2 and NKCC1 expression the brain. One of my earlier posts did suggest central hypothyroidism in autism, this fitted in with the findings of the Polish researcher at Harvard, who I had some correspondence with.

Oxidative Stress, Central Hypothyroidism, Autism and You   

Another transcription factor that has been identified as a potent regulator of KCC2 expression is upstream stimulating factor 1 (USF1) as well as USF2. The USF1 gene has been linked to familial combined hyperlipidemia. 
It is thought that increasing the expression of USF1 with increase KCC2, but it will increase other things as well.
We also know that Egr4 may be an important component in the mechanism for trophic factor-mediated upregulation of KCC2 protein in developing neurons.
Early Growth Response 4 (EGR-4) is a transcription factor that activates numerous other processes.
It is known that the growth factor Neurturin upregulates EGR4, but it does not cross the blood brain barrier. It was considered as a possible therapy for Parkinson’s Disease. In the first chart in this post, NRTN is Neurturin.

It turns out that EGR4 is redox sensitive. In other words certain types of oxidative stress should upregulate EGR4.
Recent studies have demonstrated that zinc controls KCC2 activity via a postsynaptic metabotropic zinc receptor/G protein-linked receptor 39 mZnR/GPR39. The levels of both synaptic Zn2+ and KCC2 are developmentally upregulated. During the postnatal period, synaptic Zn2+ accumulation and KCC2 expression reach levels similar to those in adult brain.  The zinc transporter 1 (ZnT-1), which is present in areas rich in synaptic zinc, is expressed from the first postnatal week in cortex, hippocampus, olfactory bulb. In the cerebellum, the expression of ZnT-1 in purkinje cells is increased during the second postnatal week.
We have seen that in autism there are anomalies with zinc; in effect it is in the wrong place. Perhaps there is a problem with the zinc transporter in some autism. Decreased ZnT-1 is associated with mild cognitive impairment (MCI).

The male/female hormones play a key role in KCC2/NKCC1, but estradiol/estrogen has a very complex role.
Estradiol can have paradoxical effects.  Its effects can also vary depending on whether you are male or female.

“the effects of estradiol on chloride cotransporters or GABAA signaling may depend upon the direction of GABAA responses”

In effect this may mean if GABA is working normally we get one effect on KCC2/NKCC1, but if it is working in reverse (bumetanide responders) we may see the opposite effect.
In the above chart estrogen is shown as increasing KCC2 mRNA in males (a good thing) but inhibiting KCC2 mRNA in females. Messenger RNA (mRNA) is one step in the process of producing the protein (KCC2) from its gene. So the more mRNA the better, if you want more of that protein.
Estrogen also has an effect on OSR1. As shown in this Japanese paper, estrogen is having the opposite effect to what we want; it is inhibiting KCC2 and stimulating NKCC1.
There is research specifically focused on the effect of estrogen on NKCC1 and KCC2. It looks like in some circumstances the effect is good, while in others it will be bad.
From the perspective I have from my posts on RORa, I am expecting a positive effect. I expect in bumetanide responders, estrogen/estradiol will increase KCC2 and reduce NKCC1 and so lower the level of chloride in neurons.
You can also easily argue that estrogen should be bad. What is clear is that inhibiting WNK, SPAK and OSR1 should all be good.  That then brings us to taurine and the start of the WNK-SPAK- OSR1 cascade.
As we have seen in previous posts,  TrkB (tyrosine receptor kinase B) a receptor for various growth factors including  brain-derived neurotrophic factor (BDNF), plays a role. In much autism BDNF is found to be elevated.
ERK is also called MAPK.  The MAPK/ERK pathway is best known in relation to (RAS/RAF-dependent) cancers. This RAS/RAF/ERK1/2 pathway is also known to be upregulated in autism.  In today’s case, ERK is just causing an increase in Early Growth Response 4 (EGR4).
Activating PKC looks a good idea.  It also is the mechanism in some other Japanese research I covered in an old post.  You may recall that in autism sometimes the GABAA receptors get physically dispersed and need to be brought back tightly together, otherwise they do not work properly.  This process required calcium to be released from the via IP3R to increase PKC.

Studies have indeed shown that PKC is reduced in some autism, which is what you might have expected. 
Finally, the other estradiol/estrogen papers:- 

In immature neurons the amino acid neurotransmitter, γ-aminobutyric acid (GABA) provides the dominant mode for neuronal excitation by inducing membrane depolarization due to Cl efflux through GABAA receptors (GABAARs). The driving force for Cl is outward because the Na+-K+-2Cl cotransporter (NKCC1) elevates the Cl concentration in these cells. GABA-induced membrane depolarization and the resulting activation of voltage-gated Ca2+ channels is fundamental to normal brain development, yet the mechanisms that regulate depolarizing GABA are not well understood. The neurosteroid estradiol potently augments depolarizing GABA action in the immature hypothalamus by enhancing the activity of the NKCC1 cotransporter. Understanding how estradiol controls NKCC1 activity will be essential for a complete understanding of brain development. We now report that estradiol treatment of newborn rat pups significantly increases protein levels of two kinases upstream of the NKCC1 cotransporter, SPAK and OSR1. The estradiol-induced increase is transcription dependent, and its time course parallels that of estradiol-enhanced phosphorylation of NKCC1. Antisense oligonucleotide-mediated knockdown of SPAK, and to a lesser degree of OSR1, precludes estradiol-mediated enhancement of NKCC1 phosphorylation. Functionally, knockdown of SPAK or OSR1 in embryonic hypothalamic cultures diminishes estradiol-enhanced Ca2+ influx induced by GABAAR activation. Our data suggest that SPAK and OSR1 may be critical factors in the regulation of depolarizing GABA-mediated processes in the developing brain. It will be important to examine these kinases with respect to sex differences and developmental brain anomalies in future studies.
The ability of the brain to synthesize estradiol in discrete loci raises the specter of estrogens as widespread endogenous regulators of depolarizing GABA actions that broadly impact on brain development.

Disregulation in developmental excitatory GABAergic signaling has been shown to impair the development of neuronal circuits and may be a contributing factor in neurodevelopmental disorders such as epilepsy, autism spectrum disorders, and schizophrenia (Briggs and Galanopoulou, 2011; Pizzarelli and Cherubini, 2011; Hyde et al, 2011). Sex differences have been widely reported in all of these disorders, implicating a role for estradiol in their etiology. Targeting SPAK or OSR1 may allow for novel therapeutic options for these neural disorders.


GABAA receptors have an age-adapted function in the brain. During early development, they mediate depolarizing effects, which result in activation of calcium-sensitive signaling processes that are important for the differentiation of the brain. In more mature stages of development and in adults, GABAA receptors acquire their classical hyperpolarizing signaling. The switch from depolarizing to hyperpolarizing GABAA-ergic signaling is triggered through the developmental shift in the balance of chloride cotransporters that either increase (ie NKCC1) or decrease (ie KCC2) intracellular chloride. The maturation of GABAA signaling follows sex-specific patterns, which correlate with the developmental expression profiles of chloride cotransporters. This has first been demonstrated in the substantia nigra, where the switch occurs earlier in females than in males. As a result, there are sensitive periods during development when drugs or conditions that activate GABAA receptors mediate different transcriptional effects in males and females. Furthermore, neurons with depolarizing or hyperpolarizing GABAA-ergic signaling respond differently to neurotrophic factors like estrogens. Consequently, during sensitive developmental periods, GABAA receptors may act as broadcasters of sexually differentiating signals, promoting gender-appropriate brain development. This has particular implications in epilepsy, where both the pathophysiology and treatment of epileptic seizures involve GABAA receptor activation. It is important therefore to study separately the effects of these factors not only on the course of epilepsy but also design new treatments that may not necessarily disturb the gender-appropriate brain development.

1.3.2 GABAA receptor signaling as sex-specific modifier of estradiol effects

To further understand the mechanisms underlying the higher expression of KCC2 in the female SNR, we examined the in vivo regulation of KCC2 mRNA by gonadal hormones. As previously stated, the perinatal surge of testosterone in male rats is required for the masculinization of most studied sexually brain structures. Unlike humans, in rats, this is usually through the estrogenic derivatives of testosterone, produced through aromatization, and less often through the androgenic metabolites, like dihydrotestosterone (DHT) (Cooke et al. 1998). To determine whether KCC2 is regulated by gonadal hormones, the effects of systemic administration of testosterone, 17β-estradiol or DHT on KCC2 mRNA expression in PN15 SNR were studied (Galanopoulou and Moshé 2003). Testosterone and DHT increased KCC2 mRNA expression in both male and female PN15 SNR neurons. In contrast, 17β-estradiol decreased KCC2 mRNA in males but not in females. These effects were seen both after short (4 hours) or long periods (52 hours) of exposure to the hormones. However, they occurred only in neurons in which active GABAA-mediated depolarizations were operative (naïve male PN15 SNR neurons). Estradiol failed to downregulate KCC2 in neurons in which GABAA receptors or L-type voltage sensitive calcium channels (L-VSCCs) were blocked (bicuculline or nifedipine pretreated PN15 male rat SNR), and in those that had already hyperpolarizing GABAA signaling (female PN15 SNR neurons). This indicated that 17β-estradiol-mediated downregulation of certain calcium-regulated genes, like KCC2, shows a requirement for active GABAA-mediated activation of L-VSCCs (Galanopoulou and Moshé 2003). In agreement with this model, in vivo administration of 17β-estradiol decreased pCREB-ir in male but not in female PN15 SNR neurons (Galanopoulou 2006). The idea that the effects of estradiol on chloride cotransporters or GABAA signaling may depend upon the direction of GABAA responses is also reverberated in other publications. In hippocampal pyramidal neurons of adult ovariectomized female rats, where GABAA signaling is thought to be hyperpolarizing, 17β-estradiol had no effect on KCC2 expression (Nakamura et al. 2004). In contrast, in cultured neonatal hypothalamic neurons that still respond with muscimol-triggered calcium rises, thought to be due to the depolarizing effects of GABAA receptors, 17β-estradiol delays the period with excitatory GABAA signaling (Perrot-Sinal et al. 2001). However, a direct involvement of KCC2 in this process has not been demonstrated yet. Such findings indicate that GABAA signaling can not only augment the existing sex differences through pathways directly regulated by its own receptors, but can also interact indirectly and modify the effects of important neurotrophic and morphogenetic factors, like estradiol, at least in some neuronal types (Galanopoulou 2005; Galanopoulou 2006). It is possible that perinatal exposure to higher levels of the estrogenic metabolites produced by the testosterone surge in male pups could be one factor that maintains KCC2 expression lower in males. In agreement, daily administration of 17β-estradiol in neonatal female rat pups, during the first 5 days of life, reduces KCC2 mRNA at postnatal day 15. This does not occur if 17β-estradiol is given only during the first 3 days of postnatal life (personal unpublished data).

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the mature central nervous system (CNS). The developmental switch of GABAergic transmission from excitation to inhibition is induced by changes in Cl gradients, which are generated by cation-Cl co-transporters. An accumulation of Cl by the Na+-K+-2Cl co-transporter (NKCC1) increases the intracellular Cl concentration ([Cl]i) such that GABA depolarizes neuronal precursors and immature neurons. The subsequent ontogenetic switch, i.e., upregulation of the Cl-extruder KCC2, which is a neuron-specific K+-Cl co-transporter, with or without downregulation of NKCC1, results in low [Cl]i levels and the hyperpolarizing action of GABA in mature neurons. Development of Cl homeostasis depends on developmental changes in NKCC1 and KCC2 expression. Generally, developmental shifts (decreases) in [Cl]i parallel the maturation of the nervous system, e.g., early in the spinal cord, hypothalamus and thalamus, followed by the limbic system, and last in the neocortex. There are several regulators of KCC2 and/or NKCC1 expression, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF), and cystic fibrosis transmembrane conductance regulator (CFTR). Therefore, regionally different expression of these regulators may also contribute to the regional developmental shifts of Cl homeostasis. KCC2 and NKCC1 functions are also regulated by phosphorylation by enzymes such as PKC, Src-family tyrosine kinases, and WNK1–4 and their downstream effectors STE20/SPS1-related proline/alanine-rich kinase (SPAK)-oxidative stress responsive kinase-1 (OSR1). In addition, activation of these kinases is modulated by humoral factors such as estrogen and taurine. Because these transporters use the electrochemical driving force of Na+ and K+ ions, topographical interaction with the Na+-K+ ATPase and its modulators such as creatine kinase (CK) should modulate functions of Cl transporters. Therefore, regional developmental regulation of these regulators and modulators of Cl transporters may also play a pivotal role in the development of Cl homeostasis.

The discovery that the dominant inhibitory neurotransmitter, GABA, is also the major source of excitation in the developing brain was so surprising and unorthodox it required years of converging evidence from multiple laboratories to gain general acceptance (Ben-Ari, 2002) and continues to draw challenges some 20 years after the initial reports (Rheims et al., 2009; Waddell et al., 2011). Fundamental developmental endpoints regulated by depolarizing GABA action include giant depolarizing potentials (Ben-Ari etal, 1989), leading to spontaneous activity patterns (Blankenship & Feller, 2010), activity dependent survival (Sauer and Bartos, 2010), neurite outgrowth (Sernagor et al., 2010), progenitor proliferation (Liu et al., 2005), and hebbian-based synaptic patterning (Wang & Kriegstein, 2008). We previously identified an endogenous regulator of depolarizing GABA action, the gonadal and neurosteroid estradiol, which both amplifies the magnitude and extends the developmental duration of excitatory GABA (Perrot-Sinal et al., 2001). Estradiol is a pervasive signaling molecule that varies in concentration between brain regions, across development and in males versus females, thereby contributing to variability in neuronal maturation. The present studies reveal that this steroid enhances depolarizing GABA effects by increasing levels of the signaling kinases SPAK and OSR1, which are upstream of the NKCC1 cotransporter. Estradiol mediated increases in NKCC1 phosphorylation are precluded by antisense oligonucleotide-mediated knockdown of SPAK, and to a lesser extent OSR1, exhibiting the necessity of these kinases for mediating estradiol’s effects. Furthermore, knockdown of either or both of these kinases significantly attenuated estradiol’s enhancement of intracellular Ca2+ influx in response to GABAA activation.

Estradiol has widespread effects on cellular processes through both rapid, nongenomic actions on cell signaling, and slower more enduring effects by modulating transcriptional activity (McEwen, 1991). The combination of a long time course and a complete ablation of the effectiveness of estradiol by simultaneous administration of blockers of transcription or translation confirm that the cascade of events leading to estradiol enhancement of depolarizing GABA begins with increased gene expression. The ability of the brain to synthesize estradiol in discrete loci raises the specter of estrogens as widespread endogenous regulators of depolarizing GABA actions that broadly impact on brain development.

Disregulation in developmental excitatory GABAergic signaling has been shown to impair the development of neuronal circuits and may be a contributing factor in neurodevelopmental disorders such as epilepsy, autism spectrum disorders, and schizophrenia (Briggs and Galanopoulou, 2011; Pizzarelli and Cherubini, 2011; Hyde et al, 2011). Sex differences have been widely reported in all of these disorders, implicating a role for estradiol in their etiology. Targeting SPAK or OSR1 may allow for novel therapeutic options for these neural disorders.

The role of Taurine and TauT
The Japanese paper below suggests that what I have called in this blog, the “GABA switch” is in part mediated by intracellular taurine.
In immature neurons, taurine is taken up into cells through the TauT transporter and activates WNK-SPAK/OSR1 signaling.
TauT is the taurine transporter that lets taurine into cells.

So logically if you blocked the taurine transporter in people with permanently immature neurons, things might improve.
Taurine is present in the embryonic brain by transportation from maternal blood via placental TauT. In addition, fetuses ingest taurine-rich amniotic fluid. Although fetal taurine decreases postnatally, infants receive taurine via breast milk, which contains a high taurine concentration. 

Taurine Inhibits KCC2 Activity via Serine/Threonine Phosphorylation
Because KCC2 is known to be regulated by kinases (15, 17, 54,,56), phosphorylation-related reagents were used to evaluate the effect on KCC2 activity. The tyrosine kinase inhibitor AG18 and tyrosine phosphatase inhibitor vanadate did not affect EGABA (supplemental Table 1A). In contrast, the broad spectrum kinase inhibitor staurosporine (Staur) shifted EGABA toward the negative in 15–20 min in the presence of taurine (control, −45.2 ± 0.3 mV; Staur, −47.6 ± 0.5 mV, n = 5, p = 0.002 (supplemental Fig. 3A and Table 1A). Considering that 1 h of taurine treatment did not have an effect on EGABA (Fig. 2A), these results suggest that chronic but not acute taurine treatment inhibited KCC2 activity in a serine/threonine phosphorylation-dependent manner. Moreover, staurosporine also shifted KCC2-positive cell EGABA significantly toward the negative in embryonic brain slices at E18.5 but was less effective in postnatal brain slices at P7 (control, −46.5 ± 0.8 mV; Staur, −51.0 ± 1.1 mV, n = 6, p = 0.007 at E18.5; control, −57.6 ± 1.7 mV; Staur, −59.1 ± 1.6 mV (n = 6, p = 0.06 at P7)) (supplemental Fig. 3B). In contrast, vanadate did not affect EGABA at either age (supplemental Table 1B).

Hypothetical model of Cl homeostasis regulated by taurine and WNK-SPAK/OSR1 signaling during perinatal periods. To control the excitatory/inhibitory balance mediated by GABA, [Cl]i is regulated by activation of the WNK-SPAK/OSR1 signaling pathway via KCC2 inhibition and possibly NKCC1 activation (54, 58, 59). In immature neurons, taurine is taken up into cells through TauT and activates WNK-SPAK/OSR1 signaling (left). Red arrows and T-shaped bars indicate activation and inactivation, respectively. Later (possibly a while after birth), this activation pathway induced by taurine diminishes, resulting in release of KCC/NKCC activity (right), whereas SPAK/OSR1 signaling recovers somewhat upon adulthood. Interestingly, in contrast to kinase signaling leading to KCC2 inhibition, other kinases are also known to facilitate KCC2 activity (see “Discussion”). 

We observed that taurine is implicated in WNK activity. WNK signaling is activated by stimuli, such as osmotic stress; however, the precise pathway leading to activation is unknown (38, 59). Our results indicate that taurine uptake is crucial for WNK activation, and only intracellular taurine activates WNKs, which are also involved in osmoregulation (52). There are no significant osmolarity differences with or without 3 mm taurine (without taurine, 215 ± 2 mosm versus with taurine, 216 ± 4 mosm (n = 4–5, p = 0.41)). In addition, 3 mm GABA did not affect phosphorylation of SPAK/OSR1 (data not shown), which indicates a specific action of taurine. 
KCC2 gene up-regulation is essential for Cl homeostasis during development, and phosphorylation of KCC2 is another important factor (5, 12, 15, 18, 55, 56). Ser-940 phosphorylation regulates KCC2 function by modulating cell surface KCC2 expression (56). Tyr-1087 phosphorylation affects oligomerization, which plays a pivotal role in KCC2 activity without affecting cell surface expression (20, 55). Rinehart et al. (54) indicated that Thr-906 and Thr-1007 phosphorylation does not affect cell surface KCC2 expression. In our study, oligomerization and plasmalemmal localization were not affected by taurine (data not shown), suggesting that phosphorylation of these sites may provide another mechanism of KCC2 activity modulation. 
A number of neuron types are generated relatively early during embryonic development, such as Cajal-Retzius and subplate cells in the cerebral cortex, which play regulatory roles in migration. Several reports have shown that these early generated neurons in the marginal zone and subplate are activated by GABA and glycine (82,,85). These early generated neurons can express KCC2 as early as the embryonic and neonatal stages (86). In addition, taurine is enriched in these brain areas (data not shown). Therefore, the present results suggest that KCC2 is not functional due to the distribution of taurine, which affects WNK-SPAK/OSR1 signaling and preserves GABAergic excitation. This signaling cascade may have broader important roles in brain development than previously reported.

I think we have pretty much got to the bottom of the current research on this subject.
There is plenty of ongoing Japanese involvement, which is good news.
You either find the GABA switch and, better late than never, finally activate it, or you modify the downstream processes as a therapy for immature neurons.  
Numerous things affect NKCC1/KCC2; so numerous therapies can potentially treat it.
The really clever solution would be to activate the GABA switch; that part I continue to think about.
Clearly, if you disrupt evolutionary processes like oxytocin and taurine passed from mother to baby there may be unexpected consequences.
Unusual levels of both male and female hormones and expression of estrogen/androgen receptors do play a role in the balance between NKCC1/KCC2 and so the level of chloride and hence how GABA behaves.
Inhibitors of WNK, SPAK and OSR1 are all promising potential therapies and I think these will emerge, since the big money of autism research is already backing this idea.
The TauT transporter is another possible target.
Hormone related options include a selective estrogen receptor beta agonist, an androgen receptor antagonist, and estradiol.  Unfortunately such therapy is quite likely to have unwanted side effects. So-called phytoestrogens like EGCG, from green tea, covered in a recent post are not very potent but if you had enough might show some effect.
For many reasons it looks like many people with autism could do with some more PKC (Protein Kinase C).

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.  


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.


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.


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.


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.  


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.