At A Glance:
- Professor, University of South Florida, Pharmacology and Gerontology, since 1998
- Associate Director: Institute on Aging, University of South Florida, since 1996
- Associate Professor, University of South Florida, Department of Pharmacology, 1992 to 1998
- Assistant Professor, USC School of Gerontology, 1988-1992
- Research Assistant Professor, USC, 1986-1988
- Post-Doctoral research, UCLA, Neurogerontology, 1981-1984
- Northwestern University, Ph.D, Neurobiology, 1981
- Northwestern University, BA, Philosophy/Psychology, 1974
A couple of years ago, Alzheimer’s researcher Dave Morgan wanted to know how similar his lab’s amyloid transgenic mice were to humans with Alzheimer’s disease — in other words, how reliable his mouse model was. He knew that Jeanne Loring, who was then at Incyte doing microarray experiments, had just done comprehensive gene-expression profiling of brain tissue from Alzheimer’s patients. So Morgan and his group at the University of South Florida Alzheimer’s Disease Research Laboratory sent their mouse RNA off to Loring at Incyte, for her to use in comparative cDNA microarray experiments.
“I think it was one of the really good uses for microarrays — to ask something really fundamental where you can get a lot of information, and ask a really simple question,” Loring told BioArray News.
The results, published June 15 in the Journal of Neuroscience, do more than confirm that the brains of Morgan’s mice are strikingly similar to those of human Alzheimer’s patients at the gene expression level: Morgan, Loring and colleagues have also identified a small group of genes that is reproducibly suppressed in the amyloid mice. These genes could provide drug researchers with a whole new class of targets, and a whole new approach to fighting the disease, according to Morgan.
BioArray News caught up with Morgan to discuss the process that led to these potentially pioneering results.
Given the plethora of microarray experiments showing differential expression in different tissues, what made your experiment so valuable?
It really identified for us that there was a very selective suppression of the expression of several genes linked to learning and memory processes in other systems. Given that these mice show a memory deficit and given that the suppression of these genes appeared specific to the brain regions that had amyloid, we thought that this [might] be one mechanism by which amyloid causes memory deficits in mice.
In microarray experiments people get so many upregulated, so many downregulated genes, and then they still have to fit this information into a pathway. What did you do next with this data?
We’ve gone and taken this set of genes and attempted to expand it by selecting more genes that we know are associated with learning and memory. We’ve got 11 genes we find are significantly downregulated in a middle-aged transgenic mouse brain hippocampus or cortex, the places that have amyloid. We’ve got about 30 that are not. We’ve been trying to survey using quantitative RT-PCR with sample sizes where we can do things like eight or 12 if we need to. That’s why we’re doing it that way. It permits us to do a larger sample size with specific genes. In any case, we’ve confirmed that these things are very strongly related to amyloid’s presence. For example, if we look [at gene expression] when the animals are younger and they don’t have any amyloid, the genes are not [suppressed]. If we look in brain regions that have less amyloid, they’re [suppressed] less. We’ve basically done most of the experiments that we can to convince ourselves that this is truly due to the presence of the amyloid material and not the transgene itself.
We’ve also done another experiment: Some of these genes are what we call immediate early genes. They are actually induced by novelty. If an animal is just sitting around watching TV in his home cage, these genes are normally at a low level. But if you take him out of his cage and you show him some new environment, all of a sudden these genes get induced. The idea is that he is learning new information about that environment. One of the things we didn’t know from the results that we had was whether this was due to a reduced basal expression of these genes or whether there was a problem at induction. So we actually did an experiment where we took half the mice and we put them into a novel environment for five minutes and killed them half an hour later. And we took the other half of the mice and removed them from their home cages and killed them immediately. What we find is that the effects are really on the induced levels of the genes — that the basal levels of the genes are really unchanged in the transgenics, but the induced levels are about half of what they are normally. What this is also telling us is that when we go downstairs in the vivarium and grab some mice and put them into a cage and bring them up into our laboratory in order to sacrifice them, we’re inducing these genes.
So that could be a real caveat to anyone doing experiments with mouse neurogenomics, that you don’t induce them through your own experimental procedures.
Probably what I would argue is you want to induce them. But what it really tells you is that you need to do that in a controlled fashion. What we started doing once we found that these genes were modified is, we would have a runner who would go down into the vivarium every 10 minutes and grab another mouse, and bring them up, and we would let them sit upstairs for half an hour before we killed them, just to keep everything constant. But this is an interesting issue that I’ve never seen anyone doing microarray analysis worry about: Some of these genes have very short half-lives, and the manipulations immediately preceding the sacrifice of the mouse can have a profound impact on the overall expression of these genes.
Incyte doesn’t do microarrays anymore. Was this experiment done a while ago?
Yes. [Laughs]. It was done when they were still doing microarrays. We wanted to confirm the results. I’m a neurochemist by training. I’m very suspicious of the quantitation we get from microarray analysis. It’s fairly obvious that if you’ve got 10,000 spots on a [chip] and you end up with 40 that are different, the probability that that could occur by chance alone — it’s feasible. So, no matter how you do your statistics, it’s hard to overcome that issue, and the cost of doing the arrays, at least [at] that time, was [so much] that you couldn’t do the standard sample sizes of eight to 12 mice that you would do to convince yourself of other things. So, what we did is we got these results and sat on them for a while until we developed the quantitative RT-PCR procedure. We got a different set of mice with a larger sample size, and we basically confirmed many of the things that we saw on the microarray. I think there might have been one gene that we didn’t confirm. We didn’t test all of them. But, what we did test, we confirmed pretty much all along the line. At least 15 of the 40 changes that we saw along the microarray, we confirmed by RT-PCR. That’s what led us to go ahead and write this thing up and really try and push it forward.
So what are you doing now?
We’re trying to induce these genes in a cell system, just a neuron culture system, then see if we can get amyloid to, in fact, suppress that induction. If we can show that, then we can go in and start playing all the games with the intercellular signaling probes and dominant negatives and overexpression of permanently phosphorylated forms of all of the intercellular kinase-based regulators, and see if one or more of those pathways is somehow involved in this process. It can help us tremendously in understanding how amyloid may lead to memory dysfunction in a biological way that’s not involving destruction of neurons. So, we’re really hoping that this would create a whole new avenue of pharmacotherapy for the treatment of dementias. Right now, all the pharmacotherapies are focused on getting rid of the amyloid material — either blocking production with secretase inhibitors or enhancing clearance of amyloid with something like the vaccine that we’ve also played a role in, and also breaking up the fibrils [with] what we call plaque blusters, drugs that will disintegrate the fibular forms of the amyloid. If you look at other diseases — the one I would like to point to is heart disease — we’ve been remarkably successful in treating heart disease in the last half-century. People aren’t dying in their ‘50s from it; they’re dying in their ‘80s … And it hasn’t been because of just one finding, one drug category we’ve found. Instead it’s the combined action of things like anti-hypertension agents and cholesterol-lowering agents, and agents that block blood clotting, anticoagulant agents. … I think Alzheimer’s is going to be very much the same way.