Question And Answer Session

SESSION CO-CHAIR COYLE: Thank you. This presentation is open for discussion.

DR. MADHAVARAO: You said that this transporter is found only is astrocytes, but the hydrolyzing enzyme is in oligodendrocytes, and also there is no mention of identification of NAA in astrocytes. So what is the significance of this being in astrocytes?

DR. GANAPATHY: This astrocyte culture that we do, as I have been listening to the talks, we are not characterizing these astrocytes in detail, like type I, type II, oligodendrocytes. All we did was to follow the published procedure for astrocyte cultures where the uptake of NAA had been demonstrated. And then we got these results from such cultures. Other investigators have already shown that there is a transport system for the N-acetylaspartate in this type of astrocyte cultures. So all I can say is that this is a crude culture of astrocytes, and they contain an N-acetylaspartate transport system. Therefore, in future studies, if you can do it in a differential way with the various types of astrocytes, i.e., oligodendrocytes, type I astrocytes, and type II astrocytes, we'll be able to resolve the issue of what is the role of the transport system in these different cell types.

PARTICIPANT: Do you think NAAG has an affinity for this transport system?

DR. GANAPATHY: Other investigators have shown that NAAG has a very low affinity for this transport system, as has been shown in the Journal of Neurochemistry paper.5

PARTICIPANT: Okay. Did you try NAAG?

DR. GANAPATHY: That is what we are planning to do now. NAAG is a tricarboxylate. NaCT is actually a tricarboxylate transporter and, therefore, it would be of interest to know whether this transporter can transport NAAG. We are doing those studies now, thinking that NaDC3 is the N-acetylaspartate transport system and NaCT is the NAAG transport system.

DR. ROSS: I have just one question in relation to succinate and citrate, both of which are transported. One thinks that these molecules on the whole are intramitochondrial and are never going anywhere. So what is the role of this transporter in the astrocyte?

DR. GANAPATHY: You will be surprised to know the plasma level of succinate and citrate. Succinate levels in plasma are about 30 micromolar. Citrate levels in the plasma are about 160 micromolar. These concentrations are quite significant.

In the brain, for example, if you take succinate with the 30 micromolar concentration with the N-acetylaspartate 100 micromolar, I would say that the primary function of this NaDC3 in the brain is really to transport the N-acetylaspartate, because you will see very little competition from succinate under in vivo conditions. However, NaDC3 is also expressed in other tissues, such as the placenta and the liver, where N-acetylaspartate may not be the physiological substrate. So, I am not suggesting that with NaDC3, the only function is the transport of N-acetylaspartate. Probably that is what it does in the brain, but it has other functions in the rest of the body, in other peripheral tissues.

PARTICIPANT: I guess this is the only transport talk on this. So could I ask a question about the other essential component of this? Is there any idea of how NAA may be transported out of the axon, whether that is an active process, whether it occurs along the entire axon or at specific locations along the axon?

DR. GANAPATHY: No. I have no idea. Here people were talking about the N-acetyl-aspartate synthesis in the mitochondria. So this transporter has nothing to do with the release of N-acetylaspartate from the mitochondria into the cytoplasm. This transporter is present on the plasma membrane of the cells.

Therefore, we are looking at the transport into cells, rather than the release from the cells. Therefore, the release of N-acetylaspartate from the neuron should be using different mechanisms. It will have nothing to do with NaDC3.

DR. NAMBOODIRI: One question. Your in situ studies indicate that the transport is present more in oligodendrocytes and astrocytes. Now, is it possible that with respect to oligodendrocytes, it is transporting into cells, and with respect to astrocytes, it is transporting out of the cells? Do you have any reason to think that this may be what is happening?

DR. GANAPATHY: I don't know. I know very little about the biological functions of N-acetylaspartate because I came to this area because of my interest in transport.

So you all have to help me in trying to understand why this N-acetylaspartate transport system is present in astrocytes as well as in the oligodendrocytes and why the hydrolyzing enzyme is present only in the oligodendrocytes. And, what could be the other roles of N-acetylaspartate in astrocytes?

PARTICIPANT: Your talk I think raises a question of whether the transport mechanism in astrocytes is a means by which NAA actually reaches the circulation. The endfeet of the astrocyte connect with the endothelial cells in the brain capillaries. And, therefore, one needs to transport NAA on the node to the astrocyte.

There are patients who are now receiving succinate who have Canavan's disease. I am wondering now whether succinate is really helpful in that it is inhibiting the transport of NAA out of the nervous system.

PARTICIPANT: Could I just add to Ed's question, too? I'm wondering if you looked at the transporter distribution on other cell types, like ependymal cells and endothelial cells.

DR. GANAPATHY: No. I am not telling you that the transport system is responsible for the release of N-acetylaspartate. It is actually for the uptake into the cells. I say this mainly because it is sodium-coupled, with three sodium ions coupled to the transport of one NAA. It is electrogenic.

It would take a very high concentration of N-acetylaspartate in the cytoplasm in order to reverse this transport function in the opposite direction. Thermodynamically, we can predict that N-acetylaspartate transport via NaDC3 has the ability to concentrate N-acetylaspartate inside the cell more than 1,000-fold, mainly because of the transmembrane sodium gradient and the membrane potential.

Therefore, if you have the N-acetylaspartate concentration inside the cell, say about 1,000 times much higher than what is outside, then you can change the direction of the transport system such that NaDC3 can function in the reverse direction. Only under these conditions, you can release the N-acetylaspartate from the cell to outside via NaDC3.

Now, with succinate therapy, you can argue that, actually, the succinate is taken up into the cell via NaDC3. And then succinate can feed into the citric acid cycle to make oxaloacetate and then aspartate. So, this pathway might feed into the synthesis of the N-acetylaspartate. And that might be the therapeutic beneficial use of the succinate therapy.

I don't know whether you all know that recently a G-protein-coupled receptor for succinate has been identified. When you are treating the patient with succinate, how do we know that the beneficial effect of succinate is actually due to just its transport into the cell? It may be acting on the G-protein-coupled receptor and doing something actually to the biology of the cell, because there is this specific G-protein-coupled receptor for succinate?

So I have been wondering whether the G-protein-coupled receptor for succinate can also interact with N-acetylaspartate. In the brain, for example, the G-protein-coupled receptor might use N-acetylaspartate as a high-affinity ligand. So this is something that the investigators interested in N-acetylaspartate should think about.

SESSION CO-CHAIR COYLE: I think we can open up this discussion to a general discussion for a few minutes.

DR. GANAPATHY: Sure.

SESSION CO-CHAIR COYLE: Do you have time for it?

DR. GANAPATHY: Yes.

DR. MOFFETT: Basically astrocytes make end-feet contacts with capillaries in the brain. Is it possible that extracellular NAA just has to be cleared from the system and astrocytes take it up and transport it to capillaries? Because it is known that NAA is excreted in the urine, in particular, in Canavan disease.

DR. GANAPATHY: In the kidney, the NaDC1 is expressed at a very high level. The NaDC1 has a low affinity for succinate as well as N-acetylaspartate. We lose very little succinate and citrate in the urine. NaDC3 in the kidney is present in the basolateral membrane where it plays a role in the entry of succinate and may be N-acetylaspartate into the tubular cells. NaDC1 in the kidney, on the other hand, is present in the brush border membrane and is responsible for reclamation of the succinate from the tubular filtrate.

Therefore, NaDC1 might be responsible for the reabsorption of N-acetylaspartate in the kidney. Once N-acetylaspartate that is present in the circulation is filtered at the glomerulus, it can be recaptured very effectively by NaDC1. So that would explain, actually, why we don't lose a lot of N-acetylaspartate in the urine. But then, we are talking about the release of the N-acetylaspartate from the astrocytes. I do not know which transport mechanism is responsible for this.

PARTICIPANT: Well, is the system you described reversible?

DR. GANAPATHY: It theoretically is. If N-acetylaspartate inside the cell is very high, for example, or the cells are depolarized thus reducing the driving force the function of the transport system as an entry mechanism, then you can reverse the direction of NaDC3, facilitating the release of N-acetylaspartate from the cells. This is possible because all these transport systems are bidirectional. And the direction is completely determined by the driving forces.

Therefore, if we can change the driving force, for example, for the glutamate transport systems such as GLT1, which is the sodium-coupled, you can change the direction of glutamate flux. Normally, glutamate is transported into the neurons or into the glial cells. But you can reverse it with appropriate changes in driving forces. There are certain pathological conditions in which glutamate transport occurs in the reverse mode, i.e., glutamate is released from the neurons, thus increasing excitotoxicity.

Therefore, NaDC3 is not a unidirectional transport system. It is bidirectional, and the direction is purely determined by the driving forces.

PARTICIPANT: Considering that the operative transporter in neurons is NaCT, what would be the effect of succinate on that?

DR. GANAPATHY: If you use the murine neuronal model system, I would expect the NaCT to interact with the citrate as well as the succinate, because rodent NaCT has as high an affinity for citrate as it has for succinate. In the case of human NaCT, the affinity for citrate is much higher than that for succinate. Therefore in human neuronal cultures, you would expect a much higher affinity for tricarboxylate compounds, like citrate, as the preferred substrate.

DR. NAMBOODIRI: I have one question. Is this transporter exclusively on the plasma membrane?

DR. GANAPATHY: Yes.

DR. NAMBOODIRI: Or is it also on other membranes?

DR. GANAPATHY: No. NaDC3 is present only in the plasma membrane because you don't find any sodium-coupled transport system anywhere else, for example, in the mitochondria. In the mitochondria, the inner mitochondrial membrane has a huge number of transport systems. All of them are driven by the proton gradient, which is ideal in the in vivo situation because you have a proton-motive force across the innermitochondrial membrane. But, you don't find any sodium-coupled transport system in the innermitochondrial membrane. So the moment you see a transport system which is sodium-coupled, you can think about only the plasma membrane as the most likely location of the transport system.

But, you can find a sodium-coupled transport system that is inside the cell but not functional. But these transport proteins are sitting there only for trafficking purposes because you can modulate that the transporter going into the intracellular store and then into the plasma membrane. Thus, you can modulate the density of the transporter in the plasma membrane acutely without the participation of de novo synthesis of new transporter protein. But you don't find any sodium-coupled transport system that is functional inside the cytoplasm or inside the cell.

DR. ROSS: To change the topic, this is a general discussion, right? When I read the title of your paper, I thought you were going to educate me on NAA transport. My understanding of that title was not the same as yours. NAA transport along the axon is something we have all been taught about. Is somebody going to illuminate me on that subject?

DR. GANAPATHY: You are talking about the transport of the N-acetylaspartate along the axon?

DR. GANAPATHY: Oh. I have no clue.

SESSION CO-CHAIR COYLE: On that note, I would like to thank the speakers for their very timely presentations and the audience for a vigorous discussion and my colleague Alessandro Burlina, who is the co-chair.

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