It’s safe to assume that for most Alzheimer researchers, the concept of a “supermodel” does not involve long legs and a pretty face (at least not at work). Rather, the term would conjure up the image of a furry, pointy-nosed creature encumbered with the big three brain pathologies—amyloid plaques, neurofibrillary tangles, neuronal loss—that characterize the human disease. In the past 2 years, Carol Colton, Michael Vitek, and their colleagues at Duke University Medical Center in Durham, North Carolina, have produced not one but two such models. They did it not by adding more genes or mutations to the mice, but by taking something away.
That something is nitric oxide synthase 2 (NOS2, or iNOS), an inducible enzyme that pumps out NO during acute immune responses. The group’s latest work, which appears in the February 13 issue of the Journal of Neuroscience, indicates that knocking out NOS2 in the Tg-SwDI mouse strain, a model of cerebral amyloid angiopathy with associated cognitive dysfunction, causes a progression to tau pathology, significant neuronal loss in the hippocampus, and further behavioral impairments.
The work reinforces an earlier report from Hana Dawson, Colton, and Vitek showing NOS2 knockout in the Tg2576 APP-expressing mouse also produced tangles and neuronal loss (see ARF related news story). Besides offering a more complete model of AD, the mice strongly indicate a protective role for NO against amyloid pathology.
In the new study, first author Donna Wilcock’s interest in vascular amyloid led her to the Tg-SwDI mice. The strain, originally created by coauthor William Van Nostrand of Stony Brook University, New York, carries a human APP gene with the Swedish, Dutch, and Iowa mutations, which causes massive accumulation of amyloid mainly in association with blood vessels. Crossing these mice with NOS2-/- mice resulted in offspring with abundant hyperphosphorylation and aggregation of endogenous tau, which appeared mostly in proximity to the vascular amyloid. The mice also display neuronal loss, with up to a 35 percent decrease in cell counts in some regions of the hippocampus.
Interestingly, in the new mice pathology progresses while Aβ levels stay flat. That contrasts with the Tg2576 mice, where NOS2 knockout resulted in elevated amyloid deposition and changes in the Aβ40/42 ratio. The finding strengthens the case that NOS2 knockout is the main reason for the accelerated pathology, the authors believe. “We don’t know the reason, but somehow, NOS2 deletion seems to just lift the brakes on the progression of the pathology,” Wilcock told ARF. That progression was accompanied by a decline compared to the Tg-SwDI parental strain in spatial memory function, as tested in the radial arm water maze and the Barnes maze.
Because these mice accumulate mainly vascular amyloid, the researchers were particularly interested in the fate of a subset of neurons that are associated with blood vessels, i.e., the NPY neurons. These neurons have been previously reported to be preferentially vulnerable in AD (Kowall and Beal,1988), and the same effect was observed in the Tg-SwDI/NOS2-/- mice. In the hippocampus, the overall number of NPY neurons dropped to half, and up to 65 percent went missing in the CA3 region. The scientists have not looked at other neuronal subsets yet.
Why does the absence of NOS2 accelerate pathology? It’s not clear, but the investigators think that NOS knockouts may emulate a more “human” NO environment, Wilcock says. “Mice microglia tend to make much more NO in response to a given stimulus than do human microglia. One hypothesis could be that high levels of NO production in APP transgenic mice are neuroprotective, and therefore pathology does not progress beyond amyloid deposition. By removing iNOS, the nitric oxide balance is now more like in the human brain, and therefore amyloid deposition can stimulate the other AD pathologies.” (For more on this topic, see Q&A with Carol Colton, below).
The mice offer a new opportunity to study the links between amyloid deposition and tau in a model where tau pathology is solely dependent on Aβ accumulation, not on tau overexpression or mutation. “The NOS2 knockout alone doesn’t show tau pathology, so obviously the amyloid is the stimulus,” Wilcock said. “We know how to affect amyloid by vaccination or by secretase inhibitors. Now we have a model where we can see what happens to endogenous tau. When we remove amyloid, do we remove the tau pathology? Or once tau gets started, does it stay? There are so many questions we can answer with this model.”
“It’s a mouse, so there are always going to be limitations, but I think it’s a more complete model than we have had before,” Wilcock added. “In addition, we feel that reproducing the pathology of the Tg2576/NOS2-/- in a different mutant APP mouse solidifies the approach of NOS2 deletion as a way to advance pathology in transgenic mice.”—Pat McCaffrey
Q&A with Carol Colton. Questions by Pat McCaffrey.
Q: How do you explain the effect of NOS2 knockouts in the AD mice?
A: First, NO has gotten a bad rap for many years. Part of the reason is because NO has multiple effects, some of which are damaging to surrounding tissue. So when you think about NOS2 in the immune response, the first thing everybody thinks about is NOS2 generating NO, which combines with superoxide to make peroxynitrite, resulting in severe oxidative stress and bystander tissue injury.
Even though NO can cause bystander tissue injury, the effect of NO is very concentration dependent. We’ve been working for the past few years to define what, in fact, are levels of NO in the brain. We would like to know what is realistic for us to expect and what’s not realistic, and whether we can associate a cell biological function with various levels of NO. Doug Thomas and David Wink have shown this happens in other cell types (Thomas et al., 2004).
NO has various ranges of effects that depend on the integrated level of NO. At low levels, NO activates cGMP and cGMP signaling pathways. At higher levels NO has a protective mode; for example, it inhibits caspase 3, or it can activate the Akt pathway or HIF. At the very highest levels, NO can react with various oxygen species, not just superoxide, to initiate oxidation, nitrosation, and nitration reactions, which can inactivate or damage proteins.
However, based on our new mouse models, we find that NO is not always a killer. Thus, although NO levels can reach damaging levels in some acute immune responses, and particularly in vitro, it is very difficult to achieve that highest level of NO in vivo. This was shown to be true in an interesting study using activated microglia by Duport and Garthwaite (Duport and Garthwaite, 2005)
Q: You mentioned a human/mouse difference, too.
A: One of the things I had spent a long time working on previously is differences in NO production by human compared to rodent microglia. Human cells do not make the same level of NO in vitro. We and others (Weinberg, 1998) tried for a number of years to figure out why. If you take a mouse culture of microglia or macrophages, and you take a human culture of microglia or macrophages, and you stimulate them the same way, the NO levels in the human culture are so low they are hardly detectible, while the mouse cells make abundant NO. This phenomenon has been studied for some years, and it is still not precisely clear why this difference occurs, although gene promoter differences may be a factor. It is important to stress, however, that NOS2 and NO participate in the human immune response, and under the complex stimulation condition found in vivo, measurable levels of NO can be made during disease in the human body.
The point is when you take a mouse that has relatively high NO levels and you pull down the NO levels with the NOS knockout, we believe we are making it more “human-like,” unmasking the protective effects of NO. We think we are now seeing the loss of NO-mediated protective mechanisms that are normally there in the mouse when the brain is chronically exposed to Aβ peptides and amyloid deposits.
Q: So it boils down again to the idea that the level of NO is the critical factor. How does loss of NO increase pathology in the APP NOS2 knockout mice?
A: We have evidence to show that caspase activation is no longer suppressed in the bigenic mice. In addition, we have preliminary evidence (unpublished) that suggests that NO regulates matrix metalloprotease 9, thus affecting Aβ degradation. Also, Akt is probably activated differently when we take away NOS2 and iNOS generated NO.
In general, removal of NO when Aβ and amyloid deposits are present is likely to remove that wonderful broad middle range, where NO has neuroprotective actions in the brain. The idea that NO is neuroprotective has recently gained momentum.
Q: You are saying that in the mouse model, you remove NO and see pathology because you have removed a layer of protection. What do you think is going on in people?
A: The integrated level of NO depends on its production and on its removal. The various NOS isoforms produce different levels of NO, with iNOS being the largest producer. However, there are many scavengers of NO including metals, superoxide anion or other oxidants, nitration reactions, etc. In addition, NOS activity and the production of NO may vary depending on endogenous inhibitors, the level of arginase 1, or arginine levels itself. (Arginine is the sole substrate for NOS.) We are currently very interested in finding out where the NO goes; is it consumed and if so, by what reaction? It may be that it is never actually made. Valina Dawson many years ago showed that superoxide anion can be made by NOS when arginine levels are low. So even if NOS is expressed, its activity may not represent NO production.
An interesting study shows that, in AD, NOS2 expression increases in cortical neurons that do not ordinarily express iNOS (Fernández-Vizarra et al., 2004). On the face of it, these data seem contradictory to our idea that decreased NO levels are critical to the pathology of AD. The immunocytochemistry in their study is quite good, so then my question is, Why is that happening?
My first impulse is to say it’s a protective response, an attempt by the cell to return NO to appropriate levels. Again, if the level of NO is the critical factor, it really doesn’t matter where the NO is generated or by what isoform as long as the crucial level is achieved in that specific region of the brain. I have no proof that my hypothesis is correct right now, but we are trying to directly discover if NO is lost in AD. I don’t have the definitive answer, and it is very important to realize that direct measurement of chronic tissue levels of NO is very difficult. Most commonly, surrogate markers such as nitrotyrosine or other products of NO’s reactions are used as a gauge of NO levels. Using genes that are significantly regulated by NO level can also be helpful to “measure” NO in the brain. For example, heme oxygenase I and thrombospondin are inducible genes whose mRNA expression levels are inversely related to NOS and to NO levels. Both of these genes increase in AD.
The other area we are currently exploring is to discover if superoxide rather than NO may be made by the NOS isoforms in AD. This action of NOS would clearly add to the oxidative imbalance already observed in AD. All of these ideas present intriguing possibilities. We do not have a definitive handle on them yet, but we have certainly opened a lot of doors with interesting and perhaps useful new directions to explore. We do have indications that the NO is probably changing in the direction that we think in human AD, but we clearly have a lot more work to do.
Q: Are there changes in iNOS or NO with aging?
A: There are some reports that iNOS goes up and some reports that it goes down. It’s a contentious question with disparate data, typical of the NO field. And again, you have to bear in mind that even if you do see an increase in NOS expression, it does not mean that NOS is making NO.
Polymorphisms in the NOS2 promoter region have been observed in the human population and appear to play an important role in the prevention of and/or recovery from cerebral malaria. Interestingly, this is not related to the parasite burden but is related to other factors which may involve protective actions of NO.
- Kowall NW, Beal MF. Cortical somatostatin, neuropeptide Y, and NADPH diaphorase neurons: normal anatomy and alterations in Alzheimer's disease. Ann Neurol. 1988 Feb;23(2):105-14. PubMed.
- Thomas DD, Espey MG, Ridnour LA, Hofseth LJ, Mancardi D, Harris CC, Wink DA. Hypoxic inducible factor 1alpha, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide. Proc Natl Acad Sci U S A. 2004 Jun 15;101(24):8894-9. PubMed.
- Duport S, Garthwaite J. Pathological consequences of inducible nitric oxide synthase expression in hippocampal slice cultures. Neuroscience. 2005;135(4):1155-66. PubMed.
- Weinberg JB. Nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: a review. Mol Med. 1998 Sep;4(9):557-91. PubMed.
- Fernández-Vizarra P, Fernández AP, Castro-Blanco S, Encinas JM, Serrano J, Bentura ML, Muñoz P, Martínez-Murillo R, Rodrigo J. Expression of nitric oxide system in clinically evaluated cases of Alzheimer's disease. Neurobiol Dis. 2004 Mar;15(2):287-305. PubMed.
- Wilcock DM, Lewis MR, Van Nostrand WE, Davis J, Previti ML, Gharkholonarehe N, Vitek MP, Colton CA. Progression of amyloid pathology to Alzheimer's disease pathology in an amyloid precursor protein transgenic mouse model by removal of nitric oxide synthase 2. J Neurosci. 2008 Feb 13;28(7):1537-45. PubMed.