PD Guide
Pre-Clinical Research Models
Research models for PD provide essential systems in which PD pathology can be studied and potential therapeutics can be tested. Models of PD can represent a single symptom, pathological process, or genetic abnormality, and can also embody more complex representations that come closer to the presentation of the disease in humans. There are two main methods for generating research models: genetic manipulation and pharmacological approaches.
- Endnote
- Chenjian Li, Ph.D, Assistant Professor, Weill Medical College of Cornell University
- Kirsten Carlson, PhD, Associate Director, Research Programs, Michael J. Fox Foundation for Parkinson's Research (MJFF)
A discussion of the new BAC Lrrk2 mouse model developed by Dr. Chenjian Li and colleagues, published today in Nature Neuroscience. Dr. Kirsten Carlson of The Michael J. Fox Foundation, which partially funded this work, asks Dr. Li about the model's features and limitations and his hopes for its widespread use by the PD research community. More information on this strain may be found in the Jax Mice Database.
KC: What is unique about your model and what need does it fulfill within the PD research community?
CL: It’s the first reported mouse model for LRRK2 mutations, and it recapitulates some key PD-like phenotypes. An interesting feature of this mouse is its age-dependent and progressive movement deficit which is L-dopa responsive. Additionally, the construct makes use of a bacterial artificial chromosome (BAC) which covers the whole human LRRK2 gene.
The PD research community always needs a variety of models. Take alpha-synuclein for example: although there have been many very interesting models, each useful in its own way, researchers are still generating new ones (fragment models, specific promoter-driven ones, etc). Our LRRK2 model compliments this list of PD models, and it offers some novel features. It is worth mentioning that our mice and BAC clones are not the end of PD model building efforts, instead, they are the beginning of further work. Together with our collaborators, we have already started using these validated BACs to engineer other human disease related mutations, as well as other manipulations for testing specific hypotheses genetically. For example, we are currently engineering GTPase dead and kinase dead mutations on top of the R1441G mutation to address whether the GTPase or kinase domains of LRRK2 are required for LRRK2-mediated pathogenesis.
KC: How does your model mimic human PD?
CL: It recapitulates the progressive reduction of movement at the behavioral level, dopamine release deficit at the circuitry level, and DA neuron atrophy and neurite degeneration at the histopathological level. Hyperphosphorylation of tau is another feature we observed. Although tauopathy is only a minor aspect of idiopathic PD, it is worth pointing out that some LRRK2 patients did have tauopathy in the absence of Lewy body pathology.
KC: Are there pathological features present in the model that are not related to PD?
CL: So far we haven’t found any. All of the phenotypes we observed to date are related to human PD. However, we have only had time to look at the “cardinal” PD phenotypes, and therefore naturally that’s what we see. It is possible that later, something new will come up. However, if that happens, we shouldn’t just discard those observations as irrelevant; instead, we should go back to human patients and ask if those pathological features are present but overlooked before.
KC: What features of human PD are missing from your model?
CL: Overt and robust DA neuronal death. Although there is statistically significant DA cell body atrophy and a striking DA neurite degeneration, cell death is not yet significant at the age of 10 months. It will be important to examine for this at a later time point.
Another missing feature is robust Lewy body pathology. This might be due to the fact that there is no human alpha-synuclein in these mice. Mice have endogenous alpha-synuclein which is not prone to aggregation. We are currently examining whether there are micro-aggregates, which has been suggested as a possible toxic species.
KC: How would your model be best used to study PD?
CL: Probably the best usage is simply to let people use it in whichever experiment they feel compelled to pursue. I believe that fellow scientists in academia or industry are very intelligent and creative, and each has some great ideas to test---be it mechanistic studies or drug testing. Of course, we also have directions that we are passionate to pursue, such as investigating LRRK2 kinase substrates and signaling pathways, axonal and dendritic degeneration, and some drug testing including small molecules or RNAi approaches, DA neurons induced from stem cells for replacement therapy, etc.
KC: Replication and verification of these features will be important - is the model publicly available?
CL: Absolutely. Not only for replication and verification, but most importantly to test new hypotheses.
We made these mice for us and for fellow scientists in the field. Weill Cornell Medical College has standard MTAs for academic labs and contracts for industry; the model is currently being re-derived at Jackson Laboratories and will be made available to the research community as soon as it is ready.
We hope that by providing this unique model to others, we can facilitate the development of new ideas. Additionally, we’re happy to combine efforts through collaboration for potentially maximum impact.
- Edward Weinstein, PhD, Director of Sigma Transgenic Research Center, Sigma-Aldrich
- Kirsten Carlson, PhD, Associate Director, Research Programs, Michael J. Fox Foundation for Parkinson's Research (MJFF)
Dr. Kirsten Carlson of MJFF interviews Dr. Edward Weinstein, Director of Sigma Transgenic Research Center for the latest on cutting edge work to create knockout rat models using Zinc Finger Nuclease technology. Don't miss the report in Science, published today!
KC: How might these findings impact how scientific research is conducted?
EW: I honestly believe that this is going the change the fundamentals behind choice of an animal model for researchers. Before the “knockout” was possible in the mouse, there was a lot more research conducted in rats, because rats have a lot of advantages over mice. Their size makes them easier to work with. They are better models for cardiovascular phenotypes and toxicology studies. In the fields of behavioral studies or addiction studies, rats are hands-down favorites over mice. But the mouse model was the only rodent that one could genetically manipulate; so many researchers began to focus on the mouse. Now everything has changed. People have been trying to make rat ES cells work for creating knockouts for about 25 years. With this new Zinc Finger Nuclease technology, we can make a knockout rat without having to work with ES cells. Now researchers can create their genetically modified rat that is tailor made for their studies. Simply put, researchers can now choose the model organism that makes sense for the biology that they are studying. Limitations have been removed.
KC: What is different about the technology used to generate these knockout rats as compared to what has previously been done in the field of rat genetics?
EW: Pronuclear injection technology has been available in the rat for quite some time, so researchers have had access to creation of transgenic rats, meaning that they can overexpress genes that have been randomly inserted into the rat genome. But the technology has never been available to make a knockout rat. The Zinc Finger Nuclease technology gets around the problem that we have never been able to successfully make rat knockouts in rat embryonic stem cells by allowing us to directly inject into the pronucleus of a one-cell rat embryo.
KC: How will differences in the technology affect the time required to produce knockout rat models?
EW: Typical knockout mice generally take 12 to 18 months to create. This is partially due to the multiple months spent working on the targeting vectors, the months spent manipulating and screening for low-frequency ES-cell mutation events, and partially due to the nature of the biology behind breeding chimera founders to establish colonies of mice. And a knockout rat has never been possible before. With the use of Zinc Finger Nucleases, we have created both knockout rats and knockout mice in just under 4 months, meaning we have gone from gene sequence to a founder Knockout animal. This advantage in time cannot be overstated. I believe this is going to fundamentally change how researchers view the usefulness of genetically modified animal models. In the past, one would have to expect to wait practically two years before getting meaningful phenotypic data on their model. This new ZFN technology completely shortens that timeline in a way that is almost difficult to comprehend.
KC: Are the genetic modifications passed on to offspring?
EW: The process described in the paper, ZFN microinjection into the pronucleus of a one-cell rat, allows one to change the genomic structure in a manner that is inherited. So once a rat model is created, the rat colony simply needs to be maintained through standard breeding and genotyping.
KC: Does the technology allow for disruption of multiple genes within the same organism?
EW: We know that the ZFN technology allows for disruption of multiple genes in vitro. This has been done a number of times in a few different cell lines. We should absolutely be able to do this in vivo as well. The simplest way is just by crossing two rat lines against each other, resulting in creation of a “double knockout” rat. But even if we are dealing with closely linked genes, the concept of knockout out multiple traits should be possible. My group is actually working on proving that as we speak.
KC: What do you anticipate may be the biggest limitations or challenges going forward?
EW: With this technology, we have made it possible to create a knockout rat, which is a huge accomplishment. But the field of knockout animals is actually very sophisticated. We want to be able to provide conditional knockout rats, humanized rats, and rats with targeting transgenic integration. These should all be possible through the use of Zinc Finger Nucleases and our R&D teams are working to optimize the processes.
KC: How would you suggest interested researchers find out more about Sigma-Aldrich’s capabilities in creating specific knockout rat models?
EW: Sigma-Aldrich has put a significant investment and commitment behind making Zinc Finger Nucleases available to the research community. We are the only licensed providers of ZFNs for research use and we have put a lot of support behind this product. Researchers should feel free to contact their local sales representative with any questions, or I would be happy to help direct them to the right person to talk to. They can always email us at SigmaTransgenics@sial.com.
Aron M. Geurts, Gregory J. Cost, Jeffrey C. Miller, Yevgeniy Freyvert, Bryan Zeitler, Vivian M. Choi, Shirin S. Jenkins, Adam Wood, Xiaoxia Cui, Xiangdong Meng, Anna Vincent, Stephen Lam, Russell C. DeKelver, Mieczyslaw Michalkiewicz, Rebecca Schilling, Jamie Foeckler, Shawn Kalloway, Hartmut Weiler, Séverine Ménoret, Ignacio Anegon, Gregory D. Davis, Lei Zhang, Edward J. Rebar, Philip D. Gregory, Fyodor D. Urnov, Howard J. Jacob, and Roland Buelow. Knockout Rats Produced via Embryo Microinjection of Designed Zinc Finger Nucleases. Science (July 24, 2009).
Geurts et al. have developed a truly significant piece of technology. Until now, genetically modified rats have been few and far between. This is a shame, since there are many areas of research – particularly in neuroscience – where rats are clearly preferable to mice. This new approach shows that it’s possible, with relative ease, to generate knock out rats in which one could study basic neurobiology. For many of us, this also raises the fascinating possibility of making new and more powerful models of human neurological disease. A major obstacle in the way of developing ways to treat diseases like Parkinson’s has been the lack of a really good rodent model, one that effectively mimics the disease state in humans. Without a good rodent model, it’s very hard to move forward with new therapeutics. Since there are autosomal recessive forms of Parkinsonism caused by mutations in one of at least three genes (Parkin, DJ1 and Pink1), it seems very likely that specifically deleting one or more of these genes in a rat could lead to new and exciting models of the disease. This has the potential to revolutionize the PD field, and move us further, and faster, along the road to developing more effective treatments.
As outlined in the Q+A and in Paul Murphy’s response, this really is an intriguing step forward in the attempt to model neurodegeneration in laboratory animals. I think it’s been interesting that frank cell loss, one of the real hallmarks of neurodegeneration in humans, has been relatively difficult to model using genetic tools related to either Alzheimer’s or Parkinson’s, at least within the lifespan of a mouse. The question here is whether moving to a rat will push this any further forward. The answer right now is that we don’t know and until someone makes a good rat model we won’t know, but I think there are a couple of reasons to suspect that a rat is at least worth trying.
Of the things that are different between mice and men, perhaps relevant to PD are that humans are relatively large bipeds. Maintaining an erect posture presumably requires dopaminergic neurons in the substantia nigra to burst fire relatively often, constantly initiating and terminating small movements. One guess for why nigral neurons are relatively sensitive in parkinsonism is that they have to work hard and over relatively long distances, having long complex projections. So, moving to a larger species that might spend more time in an upright posture may get us closer to the human situation. It may also be helpful that rats live longer, if age is an important variable in PD, perhaps giving cells a longer timeframe to degenerate. As some empirical support for trying rats over mice, it is interesting that many of the viral models of alpha-synuclein expression seem to have a little more robust nigral cell loss than mice, although it is difficult to make absolute comparisons across different studies.
Balanced against these advantages are some obvious disadvantages of moving to rat models. The wealth of molecular resources developed for mouse models including many transgenic lines and a fully sequenced genome, will take some time to replicate in any other species. And, of course, with larger animals comes a requirement for more housing space for longer periods and their associated costs. Taking up a rat model will depend, I think, on whether it has a clear phenotype that have face validity for the human disease to which it is related. Finding age-related, progressive and relatively selective neuronal loss would be the gold standard, but a robust biochemical or physiological phenotype would at least be something to work with.
But if rats work, what next? The logical extension would be to move to non-human primates where both the promise and the investment would be much greater. There have been a small number of transgenic monkeys made now and the possibilities of performing knockout/knockin is really quite intriguing.