Question And Answer Session

DR. GONZALEZ: Questions?

DR. WEINBERGER: Obviously, I am obligated to ask a question. It's just a spectacular series of studies, and I congratulate you.

You know, I'm sitting here thinking, now where do we exactly disagree. I could get into little intimate details about whether you have put voxels in the same place that we have with our slices.

One of the things we found and other groups have found: If you measure NAA qua the chemical, as if the brain were a test tube, you don't find NAA differences in schizophrenia. If you measure very discrete regions of the brain, there are some discrete regions sampled somewhat exquisitely that tend to show differences.

Now I think where we have the most fundamental difference of the phase at which we approach this signal is that you have approached this as if the pathology is in NAA, the chemical. You know, we have no evidence of that, one way or the other.

We have approached NAA very much as a proxy of the biology of the neuronal system. I think what is fascinating about your data in terms of there being a pathology of the biosynthesis or processing of NAA and that that is the pathology of the disease, which I, by the way, as in all the histories of neurochemical explanation of schizophrenia, would be very dubious of -- but nevertheless, that is a reasonable hypothesis that has to be tested in other ways. You don't need to study brain to look at glutamate or NAA signals.

There are many other tissues that could be studied if these are fundamental causative mechanisms in the disease, because the genes are the same everyplace.

Let me just ask you this question about the glutamate cycling, which is very interesting. To the extent that there is a stoichiometric relationship of this glutamate cycle and glucose utilization, is this the cause of the problem or is this itself another passenger in the fact that the tissue is less active metabolically?

DR. ROSS: Good question. The Yale Magestretti hypothesis is that the two are tightly coupled and, despite that there are many pools of glutamate, really what we are looking at is turnover rate, which is neurotransmission.

Bicarbonate, the end product, which reflects global metabolism of glucose, is not different between our small set of patients and controls. So we would say, no, this is not a reflection of metabolic disturbance, broadly speaking.

I would be much more interested again in your view. Now that could be mitochondrial metabolism at another level. We are then again looking at a surrogate, and I think this is where Dr. Gonzalez' data is so fascinating.

Clearly, we now see in real life that you can reduce the rate of NAA synthesis. You could reduce -- there must be, we would hypothesize, reduced mitochondrial cycling. How we can only reduce two terms and not the third, we are still playing with that. But I would think in the long term it will turn out to be much more focused on what the brain is doing, what the astrocytes and neurons are doing in speaking to each other, than a genetic marker of glutamate activity outside the brain, for example.

So I would still like to keep it inside the brain and relate it to function.

DR. MATALON: I heard today measurement of NAA in the entire brain tissue. Is there value for this determination under specific circumstances? Let us say, when you do gene therapy or when you do something else, maybe that is a better yardstick than a Voxel.

DR. ROSS: You mean NAA total. That is Gonen's assay, which I have never attempted to reproduce, because it is tough. So you really have to ask him directly.

In our hands, I think we only need the NAA concentration for one reason, and that is you cannot calculate fraction enrichment of a molecule pool that you don't know the size of. So, actually, I would disagree with Michael Weiner's answer to you. The ratios are just as good. We don't really need all this fancy footwork.

You do need to correct for white and gray matter, but in terms of do we need whole brain NAA levels in this context? No. Of course, we wouldn't mind having it, but it is really not important. It matters in the region in which we are measuring, and ultimately, obviously, we are going to have to measure in the dorsolateral prefrontal cortex, which you point out is very important.

DR. WEINER: Brian, did I understand you correctly when you said that you weren't actually giving us the rate of NAA synthesis, but it was the rate relative to the rate of labeling of aspartate?

DR. ROSS: In the Canavan data we are really confident that we have a single point and a single step dynamic assay in which we have collected all the data, and we make a calculation, put our hands on our hearts, and that is a rate.

In the newer data, we are trying to find a cheap and cheerful way, and it is approximately correct to say that, if you enrich an aspartate and you enrich an NAA which is made from that aspartate, then measure the ratio, the difference or the ratio will reflect NAA synthesis. But you are absolutely right. It is not anywhere near the true rate.

Nevertheless, you would have to find an explanation, for example, in Alzheimer's Disease where we have low neuron numbers, how on earth can you get more NAA incorporation. Of course, I think, while we are even standing here, we can think of ways to explain that which wouldn't necessarily mean rate. But within those limitations, these are rates.

DR. NAMBOODIRI: The inhibition of the synthetic enzyme, the purified or partially purified enzyme. The inhibitor IC50; there is an inhibition constant that is 0.5 millimolar of NAA.

DR. ROSS: 0.5 millimolar? So we have much more than that.

DR. NAMBOODIRI: -- and the same activity at 0.5 millimolar NAA. So my question is: When you have a 20-plus fold decrease in NAA, let's say, from 10 millimolar to 0.5 millimolar, how can you expect an inhibition of the NAA synthesis? You would think that NAA synthesis is already inhibited.

DR. ROSS: Yes. I think the reason we all work in vivo is ultimately that we know, when you break the system up, regulatory mechanisms are no longer what they were. So we are trying to mimic the in vivo concentration.

So all we can do is we know roughly what the Ki for the enzyme is, and it is within the physiological range. So I think it is fair to say that there's end product inhibition of this enzyme in vivo, as predicted by the in vitro experiment, but not necessarily the same Ki.

DR. GONZALEZ: Okay, thank you very much. Thank you very much, Brian. That was really wonderful.

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