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, GSK3� and CK1, and subsequent processing into transcriptional repressors (through cleavage of the carboxy-terminus) or targeting for degradation (mediated by the E3 ubiquitin ligase �-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 Tbr1�/_ 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.













The Excitatory/Inhibitory Imbalance � GABAA stabilization via IP3R


This blog aims to synthesize the relevant parts of the research and make connections that point towards some potential therapeutic avenues.  Most researchers work in splendid isolation and concentrate on one extremely narrow area of interest.

The GABAA reset, not functional in some autism

On the one hand things are very simple, if the GABAAreceptors function correctly and are inhibitory and the glutamate receptors (particularly NMDA and mGluRx) function correctly, there is harmony and a  perfect excitatory/inhibitory balance.

Unfortunately numerous different things can go wrong and you could write a book about each one.

As you dig deeper you see that the sub-unit make-up of GABAAreceptors is not only critical but changes.  The plus side is that you can influence this.

Today we see that the receptors themselves are physically movable and sometimes get stuck in the �wrong place�. When the receptors cluster close together they produce a strong inhibitory effect, but continual activation of NMDA receptors by the neurotransmitter glutamate - as occurs naturally during learning and memory, or in epilepsy - leads to an excess of incoming calcium, which ultimately causes the receptors to become more spread out, reducing how much the neuron can be inhibited by GABA. There needs to be a mechanism to move the GABAA receptorsback into their original clusters.

Very clever Japanese researchers have figured out the mechanism and to my surprise it involves one of those hubs, where strange things in autism seem to connect to, this timeIP3R.





I guess the Japanese answer to my question above is simple. YES,


A very recent science-light article by Gargus on IP3:-






Now to the Japanese.






I wonder if Gargus has read the Japanese research, because both the cause and cure for the GABAAreceptors dispersing and clustering is an increase in calcium and both mediated by glutamate.  

The excitatory neurotransmitter glutamate binds to the mGluR receptor and activates IP3receptor-dependent calcium release and protein kinase C to promote clustering of GABAA receptors at the postsynaptic membrane - the place on a neuron that receives incoming neurotransmitters from connecting neurons.

If Professor Gargus is correct, and IPR3 does not work properly in autism, the GABAAreceptors are likely not sitting there in nice neat clusters. As a result their inhibitory effect is reduced and neurons fire when they should not.

Gargus has found that in the types of autism he has investigated IP3receptor open as they should, but close too fast and so do not release enough calcium from the cell�s internal calcium store (the endoplasmic reticulum).

In particular the Japanese researchers found that:-

�Stabilization of GABA synapses by mGluR-dependent Ca2+ release from IP3R via PKC�
If the IP3receptor does not stay open as long as it should, not enough Ca2+ will be released and GABA synapses will not be stabilized. Then GABAAreceptors will be diffused rather than being in neat clusters.

The science-light version of the Japanese study:-




Just as a thermostat is used to maintain a balanced temperature in a home, different biological processes maintain the balance of almost everything in our bodies, from temperature and oxygen to hormone and blood sugar levels. In our brains, maintaining the balance -- or homeostasis -- between excitation and inhibition within neural circuits is important throughout our lives, and now, researchers at the RIKEN Brain Science Institute and Nagoya University in Japan, and �cole Normale Sup�rieure in France have discovered how disturbed inhibitory connections are restored. Published in Cell Reports, the work shows how inhibitory synapses are stabilized when the neurotransmitter glutamate triggers stored calcium to be released from the endoplasmic reticulum in neurons.

"Imbalances in excitation and inhibition in the brain has been linked to several disorders," explains lead author Hiroko Bannai. "In particular, forms of epilepsy and even autism appear to be related to dysfunction in inhibitory connections."

One of the key molecules that regulates excitation/inhibition balance in the brain is the inhibitory neurotransmitter GABA. When GABA binds to GABAA receptors on the outside of a neuron, it prevents that neuron from sending signals to other neurons. The strength of the inhibition can change depending on how these receptors are spaced in the neuron's membrane.

While GABAA receptors are normally clustered together, continual neural activation of NMDA receptors by the neurotransmitter glutamate -- as occurs naturally during learning and memory, or in epilepsy -- leads to an excess of incoming calcium, which ultimately causes the receptors to become more spread out, reducing how much the neuron can be inhibited by GABA.

To combat this effect, the receptors are somehow continually re-clustered, which maintains the proper excitatory/inhibitory balance in the brain. To understand how this is accomplished, the team focused on another signaling pathway that also begins with glutamate, and is known to be important for brain development and the control of neuronal growth.

In this pathway glutamate binds to the mGluR receptor and leads to the release of calcium from internal storage into the neuron's internal environment. Using quantum dot-single particle tracking, the team was able to show that after release, this calcium interacts with protein kinase C to promote clustering of GABAA receptors at the postsynaptic membrane--the place on a neuron that receives incoming neurotransmitters from connecting neurons.

These findings show that glutamate activates distinct receptors and patterns of calcium signaling for opposing control of inhibitory GABA synapses.

Notes Bannai, "it was surprising that the same neurotransmitter that triggers GABAA receptor dispersion from the synapse, also plays a completely opposite role in stabilizing GABAA receptors, and that the processes use different calcium signaling pathways. This shows how complex our bodies are, achieving multiple functions by maximizing a limited number of biological molecules.

Pre-activation of the cluster-forming pathway completely prevented the dispersion of GABAA receptors that normally results from massive excitatory input, as occurs in status epilepticus -- a condition in which epileptic seizures follow one another without recover of consciousness. Bannai explains, "further study of the molecular mechanisms underlying the process we have uncovered could help develop treatments or preventative medication for pathological excitation-inhibition imbalances in the brain.

"The next step in understanding how balance is maintained in the brain is to investigate what controls which pathway is activated by glutamate. Most types of cells use calcium signals to achieve biological functions. On a more basic level, we believe that decoding these signals will help us understand a fundamental biological question: why and how are calcium signals involved in such a variety of biological phenomena?"


The full Japanese study:-





        Bidirectional synaptic control system by glutamate and Ca2+signaling

        Stabilization of GABA synapses by mGluR-dependent Ca2+release from IP3R via PKC

        Synaptic GABAAR clusters stabilized through regulation of GABAAR lateral diffusion

        Competition with an NMDAR-dependent Ca2+pathway driving synaptic destabilization

GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABAA receptor (GABAAR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3receptor-dependent calcium release and protein kinase C to promote GABAAR clustering and GABAergic transmission. This GABAAR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses.



In this study, we demonstrate that the mGluR/IICR/PKC pathway stabilizes GABAergic synapses by constraining lateral diffusion and increasing clustering of GABAARs, without affecting the total number of GABAAR on the cell surface. This pathway defines a unique form of homeostatic regulation of GABAergic transmission under conditions of basal synaptic activity and during recovery from E/I imbalances. The study also highlights the ability of neurons to convert a single neurotransmitter (glutamate) into an asymmetric control system for synaptic efficacy using different calcium-signaling pathways.

The most striking conceptual finding in this study is that two distinct intracellular signaling pathways, NMDAR-driven Ca2+ influx and mGluR-driven Ca2+release from the ER, effectively driven by the same neurotransmitter, glutamate, have an opposing impact on the stability and function of GABAergic synapses. Sustained Ca2+influx through ionotropic glutamate receptor-dependent calcium signaling increases GABAAR lateral diffusion, thereby causing the dispersal of synaptic GABAAR, while tonic mGluR-mediated IICR restrains the diffusion of GABAAR, thus increasing its synaptic density. How can Ca2+ influx and IICR exert opposing effects on GABA synaptic structure? Our research indicates that each Ca2+ source activates a different Ca2+-dependent phosphatase/kinase: NMDAR-dependent Ca2+influx activates calcineurin, while ER Ca2+ release activates PKC.


Taken together, these results lead us to propose the following model for bidirectional competitive regulation of GABAergic synapses by glutamate signaling. Phasic Ca2+ influx through NMDARs following sustained neuronal excitation or injury leads to the activation of calcineurin, overcoming PKC activity and relieving GABAAR diffusion constraints. In contrast, during the maintenance of GABAergic synaptic structures or the recovery from GABAAR dispersal, the ambient tonic mGluR/IICR pathway constrains GABAAR diffusion by PKC activity, overcoming basal calcineurin activity. One possible mechanism of dual regulation of GABAAR by Ca2+ is that each Ca2+-dependent enzyme has a unique sensitivity to the frequency and number of external glutamate release events and can act to decode neuronal inputs (Fujii et al., 2013xNonlinear decoding and asymmetric representation of neuronal input information by CaMKIIa and calcineurin. Fujii, H., Inoue, M., Okuno, H., Sano, Y., Takemoto-Kimura, S., Kitamura, K., Kano, M., and Bito, H. Cell Rep. 2013; 3: 978�987

Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References, Li et al., 2012xCalcium input frequency, duration and amplitude differentially modulate the relative activation of calcineurin and CaMKII. Li, L., Stefan, M.I., and Le Nov�re, N. PLoS ONE. 2012; 7: e43810

Crossref | PubMed | Scopus (29)See all References, Stefan et al., 2008xAn allosteric model of calmodulin explains differential activation of PP2B and CaMKII. Stefan, M.I., Edelstein, S.J., and Le Nov�re, N. Proc. Natl. Acad. Sci. USA. 2008; 105: 10768�10773

Crossref | PubMed | Scopus (44)See all References) in inhibitory synapses.

Tight control of E/I balance, the loss of which results in epilepsy and other brain and nervous system diseases/disorders, is dependent on GABAergic synaptic transmission (Mann and Paulsen, 2007xRole of GABAergic inhibition in hippocampal network oscillations. Mann, E.O. and Paulsen, O. Trends Neurosci. 2007; 30: 343�349

Abstract | Full Text | Full Text PDF | PubMed | Scopus (194)See all ReferencesMann and Paulsen, 2007). A recent study showed that the excitation-induced acceleration of GABAAR diffusion and subsequent dispersal of GABAARs from synapses is the cause of generalized epilepsy febrile seizure plus (GEFS+) syndrome (Bouthour et al., 2012xA human mutation in Gabrg2 associated with generalized epilepsy alters the membrane dynamics of GABAA receptors. Bouthour, W., Leroy, F., Emmanuelli, C., Carnaud, M., Dahan, M., Poncer, J.C., and L�vi, S. Cereb. Cortex. 2012; 22: 1542�1553

Crossref | PubMed | Scopus (14)See all ReferencesBouthour et al., 2012). Our results indicate that pre-activation of the mGluR/IICR pathway by DHPG could completely prevent the dispersion of synaptic GABAARs induced by massive excitatory input similar to status epilepticus. Thus, further study of the molecular mechanisms underlying the mGluR/IICR-dependent stabilization of GABAergic synapses via regulation of GABAAR lateral diffusion and synaptic transmission could be helpful in the prevention or treatment of pathological E/I imbalances, for example, in the recovery of GABAergic synapses from epileptic states


DHPG = group I mGluR agonist dihydroxyphenylglycine.

On a practical level you want to inhibit GABAA  dispersion and promote GABAA stabilization. How you might do this would depend on exactly what was the underlying problem.

If the problem is IP3R not releasing enough calcium, you might activate PKC in a different way or just increase the signal from Group 1 mGluR. If the problem is too much calcium influx through NMDA receptors due to excess glutamate, you could increase the re-uptake of glutamate, via GLT-1, using Riluzole.  You could block the flow of Ca2+ through NMDA receptors using an antagonist.

The Japanese used dihydroxyphenylglycine (DHPG) as their Group 1 mGluR agonist.  DHPG is an agonist of mGluR1 and mGluR5.  We have come across mGluR5 many times before in this blog.  Mavoglurantis an experimental drug candidate for the treatment of fragile X syndrome.  It is an antagonist of mGluR5.

We have seen many times before that there is both hypo and hyper function possible and indeed that fragile X is not necessarily a good model for autism.

The selective mGluR5 agonist CHPG protects against traumatic brain injury, which would indeed make sense. Although, that research suggests an entirely different mechanism.



The calcium released by IP3 works in complex way together with DAG (diacylglycerol ) to activate PKC (protein kinase C).





Ideally you would have enough calcium released from IP3, but you could also increase DAG. It depends which part of the process is rate-limiting.

Diacylglycerol(DAG) has been investigated extensively as a fat substitute due to its ability to suppress the accumulation of body fat.  Diglycerides, generally in a mix with monoglycerides are common food additives largely used as emulsifiers. In Europe, when used in food the mix is called E471.


Conclusion

On the one hand things are getting very complicated, but on the other we keep coming back to the same variables (IP3R, mGlur5, GABAAetc.).

It is pretty clear that some very personalized therapy will be needed.  Is it an mGlur5 agonist or antagonist? Or quite possibly neither, because in different parts of the brain it will have a good/bad effect.

It does look like Riluzole should work well in some people.

A safe IP3R agonist looks a possibility. As shown in the diagram earlier in this post,IP3 is usually made in situ, but agonists exist.

In effect autism could be the opposite of Huntington�s disease. In Huntington�s,  type 1 IP3 receptors are  more sensitive to IP3, which leads to the release of too much Ca2+ from the ER. The release of Ca2+ from the ER causes an increase in concentrations of Ca2+inside cells and in mitochondria.

According to Gargus we should have reduced concentrations of Ca2+inside cells in autism.

I suspect it is much more complicated in reality, because it is not just the absolute  level of Ca2+ but rather the flow of Ca2+; so it matters where it is coming from. I think we likely have impaired calcium channel activity of multiple types in autism and the actual level of intracellular calcium will not tell you much at all.

As the Japanese commented, it is surprising that glutamate is the neurotransmitter that controls the clustering, or not, of GABAAreceptors.  This suggests that you cannot ignore glutamate and just �fix� GABA.





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