ARF: Your work, although concentrating upon pathological states, seems applicable to both normal and disease-related biology. Is there a particular hypothesis which drives your work?
KD: When you look at an AD brain at post-mortem, the most obvious neuropathological features are the presence of plaques and tangles, but it is not obvious which of these lesions causes the damage, or whether what you are looking at is the toxic lesion or something that forms in response to it. In 1991, the discovery of pathogenic mutations in APP that affect APP processing and the generation of plaque-associated Aβ strongly linked Aβ elevation with disease etiology. We, and many other labs, therefore developed transgenic mice with APP transgenes to address the simple hypothesis that elevating Aβ levels through the overexpression of mutant or wild type human APP would lead to Alzheimer-type pathology. Early attempts along those lines failed to generate mice with robust amyloid pathology however, most likely because we now know that one has to achieve extremely high levels of APP/Aβ for a mouse to develop amyloid deposits. The idea that Aβ and pathology development were linked was further justified by the discovery of mutations in the presenilins, where the effect of pathogenic mutations is to elevate Aβ42 levels specifically. So far, all the genetic risk factors that have been identified can influence APP or Aβ so we still feel that modulating amyloid, or its precursor Aβ, is a valid approach to modeling AD.
ARF: So, the transgenic mouse in Alzheimer's research was an idea before its time, but its time has come?
KD: To some extent. Despite the initial problems in achieving sufficient levels of Aβ in the brain, the approach has held up and several mouse lines now have very robust amyloid pathology. Having thought that this would be sufficient to generate an animal model with full-blown AD though, we were surprised to find that the other prominent features of Alzheimer's disease— overt cell loss and the development of tangles—did not follow. So it would seem that we've achieved some of the pathology of AD but not all, although many of the missing features may be present, but more subtle, in the mouse than in humans.
ARF: How much do you think that those observations detract from the "Amyloid Hypothesis"?
KD: Initially I was disturbed that mice with so much amyloid did not show more features of the human disease, but the more closely we look at our transgenic models, the more we see features that do resemble Alzheimer's. For example, a subtle feature in amyloid depositing transgenic mice that is similar to AD is that increasing amyloid load is associated with disruption of the cholinergic system (Wong et al., 1999). In addition, the evidence that amyloid or Aβ accumulation is associated with cognitive impairment as proposed early on by Karen Hsiao and Paul Chapman, is also gaining more credibility as several of the amyloid models now report behavioral changes or electrophysiological disturbances. Unfortunately the field is still confounded to some extent by the lack of standardized test paradigms and different genetic backgrounds, so there is still contradictory data, but overall, many of the models are showing some form of impairment linked to increasing amyloid/Aβ load. The absence of severe cell loss in mice with large amounts of amyloid is still puzzling, but there are several possible explanations ranging from differences in mouse neurobiology to problems in quantifying cell or tissue loss, shrinkage or dysfunction. To address this, my colleagues Joe Helpern and Craig Branch at NKI have been developing protocols for functional and structural MRI which have been informative in identifying how the transgenic mouse brain responds to amyloid load. The great advantage of MRI is that live mice can be studied longitudinally, and their disease progression can be mapped, which we hope will have utility in establishing correlates between events that can be seen by immunohistochemistry or behavioral analysis for example, and those that can be seen by imaging.
ARF: Your paper (Duff et al., 1996) provided important data linking presenilin mutation and beta-amyloid peptide increases in vivo. Do you think that the beta-amyloid peptide is the lynch pin molecule in the progression of the disease? Are you open to other explanations?
KD: Although I still believe that Aβ or amyloid accumulation is critical for the initiation of AD because of the genetic data, I think that several other factors are likely to be important in the progression of the disease. In the human AD brain, it is very likely that pathogenic tau contributes significantly to the degenerative phenotype, and the identification of mutations in tau that are linked to other neurodegenerative diseases greatly strengthens that speculation. The role of the inflammatory system is also likely to be very important, including the potential role of microglia in the generation or clearance of amyloid from the brain. Genetic analysis may yet help us to identify susceptibility loci in genes involved in these types of pathways, which may not be directly linked to Aβ or amyloid accumulation, but to the brain's ability to deal with it.
ARF: You mentioned some of the alternative hypotheses concerning Alzheimer's disease. It seems you have been involved in many of the areas of Alzheimer's research, including ApoE, beta-APP, and presenilins. And more recently you've done work in tau proteins. Could you talk about that?
KD: Because the amyloid mice are now, albeit in their own way, showing so many of the features of human AD, I think the next big challenge is to generate tangles. Because there are differences in the forms of tau present in the mouse and human brain, we have humanized the tau environment of the mouse by generating transgenics that express all the forms of human tau. This type of mouse is currently being crossed to mice that make amyloid deposits and we are hoping that the resulting offspring will have both amyloid and tau pathology so we can examine the relationship between these two important features of the disease. This may help answer the age-old question of what comes first, plaques or tangles and help us address how each contributes to the AD phenotype.
ARF: Can you describe the differences between your tau transgenic mouse and the Goedert (Gotz et al., 1995) one?
KD: The transgenic mice that we have created carry a genomic transgene that includes the human tau promoter and all the coding and regulatory elements of the human tau gene within a PAC vector. When introduced into the mouse by microinjection, the human tau protein is represented in the brain with the correct spatial and temporal distribution. So we've set the scene for replicating the conditions expected in the human AD brain. The difference between the genomic tau mouse and others is that most only express a single isoform of human tau, which is not under the control of the tau promoter. So they don't get the exact same distribution you'd expect in the human brain, nor the ability for several human isoforms to aggregate as they do in AD tangles.
ARF: Do you have any preliminary data?
KD: Not yet for the crosses, but in collaboration with Peter Davies we have characterized the tau mice and have shown that they are making all the expected isoforms of human tau, and that the protein is expressed at high levels and with the correct distribution. There is four or five times more human tau in the mouse brain than mouse tau, and the levels are comparable to what one finds in the human brain. In addition, the human tau is able to be phosphorylated at sites that are thought to be significant in AD.
ARF: Wow. Well, on the topic of "patterns," how do you explain the neuroanatomical specificity of the progression of Alzheimer's disease?
KD: This can be a difficult question for people working with transgenics to address as transgene derived proteins under the control of a heterologous promoter are often not distributed at the same levels or in the same pattern as the endogenous protein. Interpreting regional differences in, for example, plaque development can therefore be misleading. Having said that, several of the APP transgenic mice do seem to demonstrate some of the neuroanatomical correlates that one sees in human Alzheimer's cases. For example, in both the transgenic mouse and the AD brain, deposits develop early in the entorhinal cortex, but are rarely seen in the cerebellum, even though Aβ is found in both regions. There seem to be some areas of the brain that are particularly vulnerable to Aβ accumulation that is difficult to explain given our current knowledge. It may be informative to see what local factors are associated with those areas and in this respect, the regional presence of proteases, inhibitors or chaperones may be significant, or it could be that more general mechanisms such as blood flow or clearance are less well able to prevent Aβ or amyloid from accumulating in sensitive brain regions.
ARF: Regarding genetics, you have done important work both in transgenics and also in hard-core basic genetics. How do you think the road ahead shapes up for genetics in Alzheimer's research?
KD:There's still a lot of genetic analysis being done but it's work with a diminishing return as it takes more work to find fewer and fewer genes, and the associations are becoming less clear-cut as the genetics becomes more complicated. As in the past, it is likely that most of the new genetic findings will identify proteins that can be linked to the amyloid/Aβ pathway, albeit by six degrees of separation in some cases, but it is likely that other susceptible pathways will be identified as amyloid load and degree of dementia do not always correlate. A convincing genetic association between tau and AD is still missing however, which weakens the argument that tau itself can initiate, or modulate pathology directly.
ARF: Dr. Selkoe and others have touted the gamma-secretase as a possible therapy target. Do you think there'll be a good therapy option soon in the field?
KD: If, as the genetic data suggests, Aβ elevation or accumulation truly is the cause of AD, and amyloid modulation is the appropriate therapeutic goal, then we should be close to achieving that goal.
ARF: Yes! It seems as though transgenic mice, including those which you developed, provide an extraordinary asset. Have you had much such goings-on?
KD: The mice that I have developed are very useful in that respect because they have such a robust and predictable amyloid phenotype. This makes them very useful for looking at the effects of amyloid modulation not only because the results can be obtained quickly, but because relatively few mice are required for statistical significance, and subtle changes in Aβ can be seen more easily. Now we are at the stage where excellent models have been created, and drugs have been developed, but often we cannot get the two things together because of commercial and legal restrictions imposed both by academic institutions and industry.
ARF: Do you see roadblocks to the development of therapies?
KD: Yes, they have already significantly slowed down progress in the AD field. Academia has changed a lot even in the past five years and in several areas, it is tied up by legal considerations. Especially in the case of transgenic mice, you can't transfer animal resources freely between labs without a material transfer agreement, and sometimes the terms of the agreement, especially reach-through rights, make it impossible for two institutions, even academic institutions, to interact. This has been particularly evident with the Mayo-owned APP mice. Unfortunately, once one institution acts bullishly, other institutions often follow suit. It negatively impacts science and wastes money as resources have to be re-created.
ARF: Can you give an example?
KD: Yes, I get endless requests from academics for the doubly transgenic PS/APP mice, especially for pilot studies for grants. Unfortunately, the mice have to be obtained separately from two institutions, accompanied by legal paperwork and often a lengthy delay, then they must be crossed, genotyped and aged at the requestee's institute before they can be used for their intended proof of concept experiment. Often it is not worth the time and resources and potentially important studies do not get done, all because I cannot send out a couple of mice to other academics. It's a real shame.
ARF: The unfortunate thing is that the closer we get to findings of therapeutic value, the slower the progress will be and that's the time when the most speed seems merited.
KD: For drug companies, there are huge financial, legal and downstream royalty considerations to deal with when licensing the mice and it often takes years before they can get their drugs into the model, even for pilot studies. Putting a drug candidate into a transgenic model is often the last step between drug development and clinical trials and for some, it's a decisive step in determining whether the drug will be clinically relevant and should be taken further. Although it doesn't affect the process of taking the drug to market--I don't think that FDA approval depends on using a transgenic model-- it's a great bonus if it shows utility in a model. Certainly, companies pay a lot of money to have mouse resources for preliminary studies, and when there is money involved it affects the way people behave, even in 'not-for-profit' foundations.
ARF: It seems as though it also undermines some important foundations of academia--interaction and collaboration.
KD: Yes, it does, I have experienced it first-hand, both in my career and my research, and I have seen radical changes in the field in the past five years.
ARF: What a conflict. It seems complicated, and I hope it gets addressed.
KD: It is beginning to be addressed. I recently participated in an NIH panel on transgenic models and we made several suggestions as to how the models should be maintained and distributed in the future. Although it is very complicated because many academics have industrial links, or wish to use proprietary treatments in an academic setting, guidelines for improved access to NIH funded animals and less restrictive legal documents were proposed. Unfortunately though, even the question of who owns a transgenic line can be very complicated, especially if the animal carries a mutation as patents filed to cover a mutation will try to also cover mice created using that mutation.
ARF: ...So it's gone beyond transgenics...
KD: Yes, The genetics field has always been particularly prone to commercial and legal influences. If you discover a mutation then a whole battery of lawyers will get involved. The mutation may be used for diagnostic purposes, the development of transgenic mice and potential drug discovery...the whole painful legal process usually starts with the identification of a genetic association.
ARF: So it used to be, the first thing you'd do upon discovering a mutation is write a paper, and now the first thing you'd do is call a lawyer.
KD: I wouldn't be surprised.
ARF: Maybe it is an outcome that is related to success in the field; growing pains, or something.
KD: Yes, it is a predictable outcome of high-impact science, and one that we cannot turn back from, but as a field, we need to impose some rules on ourselves so that the most aggressive and self-serving individuals don't determine the future outcome of our efforts.
ARF: Perhaps this is a good segue, then. Do you have any advice to younger investigators in this shifting field?
KD: Hm, yes: remain interactive. Try to be open-minded, generous and as open and honest as possible and beware of conflict of interest. Conflict of interest is very destructive, but what constitutes it is often a personal judgment that most people won't agree on.
ARF: Okay, and is there anything special to younger female researchers?
KD: Getting started with a female-friendly mentor in a healthy lab environment can be very important. If you're going for an interview, its usually not that difficult to spot a good lab - there should be a reasonable number of women in the lab in general but more significantly, a couple at the higher levels. In general, I think women in science need to be more confident, more bold in discussions as junior women scientists still tend to be shy in academic forums, and, often through their own reticence, do not gain credit for their own work and ideas. Overall, I have not had any discrimination problems that I couldn't overcome, and although having a social life is almost impossible, I enjoy being a scientist.
ARF: Well, I think we've taken enough of your time. Thanks very much, Dr. Duff, for being interviewed for our web site. We truly appreciate your time, in addition to the important insights and refreshing candor.
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