Signal Transduction in the Brain: Transcript Part 4
Importance of DARPP-32 Phosphorylation, Spinophilin's Role
Importance of DARPP-32 Phosphorylation:
This is the structure of DARPP-32 in the rat. DARPP-32 is a very highly conserved protein in the brain--in the mammalian brain--and this is a rat sequence. It is a 205 amino acid protein and it is this threonine in here, in position 34, which gets phosphorylated by PKA and PKG and dephosphorylated by calcineurin. And when it gets phosphorylated it causes a dramatic change in the biological properties of this protein.
And here is what happens. Protein phosphatase-1 is one of the major serine ____protein phosphatases in the brain, and when DARPP-32 is phosphorylated on 3 and 34, it is a very potent inhibitor of protein phosphatase-1 with an IC50 and KI value of that 10 to the minus 9th, that is 1 nanomolar. The dephosphorylated form is completely ineffective in regulating and acting as a protein phosphatase inhibitor. So this is quite a significant effect because there is about 10 to the minus 5th molar DARPP-32 in these nerve cells. This means that a tiny bit of activity in the dopaminergic pathway in the brain, by causing a tiny bit of phosphorylation of DARPP-32, causes a substantial inhibition of protein phosphatase-1 activity. And this, in turn, is going to regulate the state of phosphorylation and physiological activity of a number of downstream effectors that I will get to in just a few minutes.
So we can now go back and modify the scheme to add the fact that the phosphorylated form of DARPP-32--but not the dephosphorylated form--is a very potent inhibitor of protein phosphatase-1.
The next question was, "What is the significance of this DARPP-32 /PP1 cascade?". And we used two approaches to answer this question. One was a series of studies done by Angus Naim in which he, in collaboration with a number of other individuals and other physiologists, was able to inject second messengers or protein kinases or protein phosphatases, or activators or inhibitors of these enzymes, and show that one could either mimic or antagonize these various neurotransmitters as predicted by the schemes that we were developing. The other approach used by Alan Fienberg, is to knock out the DARPP-32 gene and study the ensuing phenotype.
Studies on DARPP-32 knockout mice:
And what was found in those studies is summarized in this slide. All of the responses to dopamine to the psychostimulant drugs of abuse, to the antipsychotic drugs, and to steroids, were abolished in the DARPP-32 knockout mice. For example dopamine has a number of effects. It regulates ion pumps such as the sodium, potassium, ATPase, ion channels, such as all three classes of calcium channels. It controls the GABA release from these DARPP-32 containing nerve cells and it controls the state of phosphorylation and physiological inductance of the ___ subunit of AMPA receptors. All these effects are gone. Dopamine psychostimulants ___ expression of the early genes such as C-phos, this effect is knocked out.
Both in humans and in experimental animals, the psychostimulants like cocaine and amphetamine cause destruction of the dopaminergic neurons. And these effects you can measure in various ways. One of the ways we use is reactive gliosis.
These effects were also abolished in the DARPP-32 knockout mice. That was particularly significant because all these other things were occurring in the nerve cells that contained the DARPP-32. But this showed that when the cells didn't have DARPP-32 in them, then not only the nerve cells that contained the DARPP-32, but other nerve cells in all this circuitry were affected. [For example,] here the dopaminergic neurons were dramatically altered in their behavior by the DARPP-32, and the dopamine receptive neurons being present or absent. Anti-schizophrenic [drugs] have a variety of affects such as actions on locomotor activity, which were abolished. Dopamine and progesterone are required for sexual receptivity in females and one example is female responsiveness, an analogous story now with males.
So we will go back to this story of the master scheme and now we can add, based on these studies that Angus Naim did and the type of studies Allen Fienberg did--that there are a whole bunch of downstream effectors including, for example, these glutamate receptors, GABA receptors, various calcium channels, sodium channels, sodium pumps, transcription factors, which are under this control. So this is a rather interesting picture here. All these neurotransmitters converging on this integrating cell which then produces these coordinated responses. All these things have to work in synchrony, in a meaningful way. It can't be chaotic. And this is how it all happens, through this sort of hourglass if you will. All this coming in to this cascade and then controlling all this going out.
I mentioned that drugs of abuse work through this pathway. Now every known drug of abuse works through this pathway. I should say, every drug of abuse that my graduate students have told me about. I suspect they have got a couple of secret ones. Cocaine, amphetamine, opiates&I already told you about. Marijuana, nicotine, alcohol, LSD&If you have a vice, it should be on this list. Because it works through the DARPP-32 pathway.
Drugs of Abuse and DARPP-32-experimental support:
We have two types of experiments that support this. One, all these drugs of abuse regulate the state of phosphorylation of DARPP-32 and two, in the DARPP-32 knockout animals these drugs are no longer effective. So if you have kids of teenage high school years, you may want to inactivate their DARPP-32 for a few years and then they won't be into these things.
Back to the master plan. You will notice that these fast acting glutamate receptors--AMPA and NMDA--work through the DARPP-32/ PP1 cascade and that the DARPP-32/PP1 cascade, in fact, regulates these glutamate receptors. So from this you would predict that protein phosphatase-1 would be in the part of the cell where these receptors are, and where are these receptors located, the glutamate receptors?
This is a medium spiny neuron and you can see this is an elucidated drawing of a medium spiny neuron, a DARPP-32 containing medium spiny neuron. You can see where the name "medium spiny" neuron comes from. Medium in size and these are the dendrites and you can see that they are covered --unfortunately this is a computer rendition which doesn't look so good--but there are little processes here called spines, with spine heads and spine necks.
So what Angus Naim and ____prepared recombinant PP1 and then made antibodies to it and localized the PP1, and here you see immunoperoxase labeling in spine heads and spine necks, sparse labeling of the dendritic shaft, and sparse labeling the presynaptic terminals. These spine heads are exactly where these fast acting neurotransmitters are located. So this is very encouraging.
Spinophilin:
Patrick Alan then undertook the project of seeing whether he could find a chaperone, a trafficking protein, which would take PP1 to the spines and enrich them there. And he found a compound which he called spinophilin, the structure of which is shown here. It is an acting, binding domain, a PP1 binding domain, a PDZ domain. For those of you who are not familiar with PDZ domains, they are present in proteins that bind to the cytosolic domain of transmembrane proteins in the region of synapsis. And then there is a coiled coil domain&I don't have time to discuss these.
But it turns out that the spinophilin plays a very important role in the mechanisms by which dopamine regulates glutametergic transmission.
First, look at the localization. This is an immunoelectron micrograph of a brain, and here you can see this spine head intensely labeled for immunoperoxase labeling. It is exactly where the AMP and NMDA receptor were located. It is not present even in the spine neck and certainly not present in the dendritic shaft. Nor is it present in... you can see two nerve terminals containing their vesicles with the neurotransmitters in them innervating this spine, and there is no spinophilin there. So the spinophilin is localized exactly where you would like to see it
I am going to show you first a model, and then some experimental support for this. What the spinophilin appears to do is as follows. Here is an AMPA receptor, which is an ion channel, when glutamate activates it binds to the AMPA receptor, it causes a conformational change in this receptor and causes an increase in conductance. Chuck Stevens and I showed a number of years ago that if you phosphorylate these AMPA receptors they become more responsive to glutamate.
But in terms of this model, what we now know is that in the resting state it is kept dephosphorylating inactive and the reason for that is spinophilin tethers the PP1 in the vicinity of the AMPA receptor, keeps it in this dephosphorylating state. When a dopamine activates the D1 receptor, this D1 receptor then raises cyclic AMP, which activates PKA, which now does two things. Again synergistically it directly phosphorylates the receptor and it phosphorylates the DARPP-32 which inhibits PP1. So you have increased phosphorylation, decreased dephosphorylation. And this is a synergistic thing.
Experimental support for spinophilin's role:
I am going to show you one biochemical experiment and then three physiological experiments in support of this model. First the biochemical one. This is the study of Gretchen Snyder, and here in the left panel you see the dopamine regulates the phosphorylation of&this is a subunit of the AMPA receptor &and you can see that dopamine causes a several fold increase in phosphorylation which, we had shown, increases its responsivity to glutamate. In the right hand panel you see the same experiment done in a DARPP-32 knock out mouse. Here, the dopamine is ineffective in causing the phosphorylation of the AMPA receptors. So that means that the direct phosphorylation and the inhibition of dephosphorylation are required to get a measurable increase in phosphorylation.
I am going to show you three experiments now on the physiology of these cells. These were carried out by Zhen Yan and what Zhen found was that when she did a whole cell patch clamping of these cells, there was a rundown of the cell which is associated with dephosphorylation.
And here is one such experiment. Here she is working with AMPA current and this is what would have happened in the absence of intervention. But at this point, Zhen Yan put on the D1 agonist, caused an increase in phosphorylation, and restored the AMPA conductance, the responsivity to glutamate and then when she washed it out the rundown continued.
She could do the same thing with the DARPP-32 molecules if she took a phosphoDARPP-32 peptide which continued the active PP1 inhibitory domain, she prevented the rundown. But with the dephosphopeptide did not prevent the rundown.
And finally, using a specific low molecular weight inhibitor of PP1, she prevented the rundown, whereas a closely related analog (inaudible) did not do so.
So now we can go back to this scheme and add spinophilin here. Actually what Zhen Yan found was just like the studies I showed you for the AMPA receptor. She found the same thing for the NMDA receptor but not for the GABA receptor. So clearly spinophilin is associated with modulation of fast excitatory transmission, not fast inhibitory transmission. We know that the spinophilin is a phosphoprotein and we have preliminary evidence that it too is regulated by dopamine. So this provides ... and that phosphorylation by dopamine through the PK pathway controls the ability of the dopamine to other `pathways to regulate these fast excitatory synapses.
Now clearly the efficacy of these neurotransmitters in controlling all of these downstream effectors depends on the percentage of the DARPP-32 which is phosphorylated and therefore the degree of inhibition of PP1. And what that means is that it is a balance of the pressures between protein phosphorylation and protein dephosphorylation.
It turns out that everything I have told you so far is just a small part of the signaling. This is now regulated by a much bigger regulatory system which I will tell you a little bit about, which we found in the last year or two.
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