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Showing posts with label Diacylglycerol. Show all posts
Showing posts with label Diacylglycerol. Show all posts

Friday 21 April 2017

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 GABAA receptors 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 GABAA receptors 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 receptors back 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 time IP3R.





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 GABAA receptors dispersing and clustering is an increase in calcium and both mediated by glutamate.  

The excitatory neurotransmitter glutamate binds to the mGluR receptor and activates IP3 receptor-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 GABAA receptors 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 IP3 receptor 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 IP3 receptor does not stay open as long as it should, not enough Ca2+ will be released and GABA synapses will not be stabilized. Then GABAA receptors 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 École Normale Supérieure 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 IP3 receptor-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 CaMKIIα 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 Novère, 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 Novère, 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 Lévi, 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.  Mavoglurant is 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, GABAA etc.).

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 GABAA receptors.  This suggests that you cannot ignore glutamate and just “fix” GABA.