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Sheep in HD Research

Animal models are a crucial tool for HD research. Clinical trials invariably involve animal testing of some kind. From developing our knowledge of the immune system, to helping in the fight against cancer, animals have contributed an immeasurable wealth of knowledge to medicine. Although mouse models are currently the primary candidate for experimentation in HD research, many new animal models, such as sheep and pig, are being developed for clinical research. Though this article will explore the role of sheep in HD, it is helpful to first understand why mouse models are used in HD research, and why different models are needed for continued research.1

Mouse Model Basics

Mouse models have been the primary mainstay of HD research for decades, and they possess several qualities that make them suitable for many types of medical experimentation. Mice are relatively cheap, easy to raise, and have short life cycles, making them an affordable and easy-to-study substitute for human test subjects. Specifically, important to HD research, mice possess a homolog, or gene similar in function, to the HTT gene, which is responsible for the disease in humans. Though no pathogenic form of the gene has been found in mice, several mouse models of HD have been created. Some of these models: 1) disrupt the native mouse Htt gene, 2) replace it entirely with the mutant human HTT gene, 3) introduce a small fragment of the HTT gene containing the CAG mutation, or 4) introduce the CAG expansion in the mouse Htt gene.1 For more on specific mouse HD models see our article linked at the bottom of the page.
The Problems with Mouse Models

While the use of mouse models to study HD has provided much insight, these models have several drawbacks. Mice have drastically shorter lifespans than humans, and while this can be helpful in decreasing study time, it can also provide a significant barrier to research. For example, many mouse models show little evidence of the disease, possibly because the afflicted mice die of natural causes long before the effects of the mutant version of the HTT gene present. Furthermore, those models that do show effects of the disease (often models that overexpress a fragment of the HTT gene containing the mutation) also show too rapid of a decline. In short, it is hard to study the pre-symptomatic effects of HD in mouse models.1

Compounding these issues, mice also have a relatively smooth cerebral cortex, the region of the brain responsible for higher brain functions. This smooth cortex anatomy, known as lissencephalic, differs significantly from the cortex anatomy of humans, which is known as glyrencephalic. In a glyrencephalic brain the cerebral cortex is full of folds, known as gyri, that increase the total surface area, complexity, and function of the cortex. Conditions that lead to a lack of gyri in humans cause intellectual disabilities, so it is likely that the lack of gyri in mouse cortexes drastically changes the effects of the mutant Htt gene in mice.2,3,4

Furthermore, the sheer size difference between human and mouse brains presents a challenge in applying mouse models to HD research. This poses barriers to testing methods of surgical delivery of therapies, and limits the number of imaging techniques that can be used to study the mouse brain.1

As a solution to the limitations of the mouse model, other animal models are currently being established. For the rest of the article we will focus on sheep, and alternative animal model that has seen much development.
Why Sheep?
Like mice, sheep possess a homolog to the human HTT gene. Sheep also have lifespans ranging between 10 and 20 years, glyrencephalic cerebral cortexes, and relatively large brains, all direct improvements over the mouse model.1

Furthermore, similarities between the human and sheep brain extend far beyond folding of the cerebral cortex. In addition to being glyrencephalic, sheep cerebral cortexes contain many of the same subcortical structures as those of humans. Sheep and humans also share a development in the dorsal striatum that is not found in mice. This section of the brain, which is influential in the brain’s reward system, is one uniform section in mice, but in humans and sheep it is made up of two separate sections; the putamen and the caudate nucleus. Given that the dorsal striatum is a key area of degradation in HD, this similarity is a key advantage of using sheep in HD research.2,4

Aside from these factors, sheep also have traits that make them ideal to study. Sheep are herd animals, meaning they exhibit complex social activities not seen in mouse models. For example, sheep can differentiate the faces of other members of their herds, and are capable of spatial reasoning tasks far beyond the ability of mice.5

Sheep are also open-air grazing animals, and thus require far less expensive housing and feed than other large domesticated animals. Furthermore, centuries of agricultural research have developed powerful and cost-effective reproductive technologies for sheep, such as advanced artificial insemination and cloning techniques, that do not exist for other species. In fact, the reproductive science of sheep may be better understood than that of humans.1,6

Though high cost, myths that sheep are completely unintelligent, and long-life span have traditionally limited researchers from developing a comprehensive understanding of sheep neural anatomy and cognitive testing regimes, recent work in the field, some of which will be covered further in this article, has built much of the necessary foundation for testing HD sheep models.

How are Sheep Models Created?

Though the idea to create a transgenic sheep model for HD first originated in 1996, it wasn’t until late 2005 that the first such model was created by researchers primarily from the University of Auckland, Harvard Medical School, University of Adelaide, the South Australian Research and Development Institute, Swansea University, and the University of Cardiff. The researchers created six separate transgenic sheep with a full copy of the mutant, human HTT gene. They first constructed a transgene, or transplantable section of DNA, and then injected it into multiple single-celled sheep zygotes in a process known as transgenisis. The original transgene contained the full HTT gene (including its promoter, a section of DNA necessary for gene expression) with 69 CAG repeats which was derived from a human HD patient. The unusually high number of repeats was selected to ensure that the resulting animal would be at least partially symptomatic for the disease. The zygotes were then implanted into a surrogate and allowed to develop.6,7

Each of the resulting six transgenic sheep showed various levels of mutant huntingtin. This is because simple transgenisis via microinjection (as occurred in this case) leads to random integration of the transgene into the sheep genome. The desired gene can be located in an area of DNA where it will be expressed often, or it may not be expressed at all. The entire gene may not even end up intact, which can further alter the output of the gene, and the levels of mutant huntingtin produced.

The most promising of the transgenic sheep (affectionately dubbed “Kiwi”) was bred for further research using a technique called IVF, in which a female sheep (or ewe) is induced to producing far more eggs than normal. Within a year, researchers had nearly 20 specimens of the model, and now hundreds of descendants of “Kiwi” have been born. The line is now referred to as “OVT73” (“OV” for ovine,”T” for transgenic, and “73” for the CAG repeat length which slightly increased after insertion), and at the time of this writing is the only large transgenic animal model for HD that contains the full mutant, human HTT gene.6

How are the effects of HD Measured in Sheep Models?

The OVT73 line is (so far) a pre-symptomatic model of HD. This means that the sheep that have been observed have not presented any of the symptoms of HD by 8 years of age, the age of the oldest OVT73 sheep. Though it is possible that one of these sheep might eventually develop HD symptoms, they may be completely asymptomatic. How, then, are they used to study the disease? Many different techniques can be used to examine subtle changes caused by the mutant HTT gene in the OVT73 model, and each of them can tell us more about the pathology of HD.

One of these methods is to compare the metabolites (substances involved in the maintenance and energy production of the body) found in control sheep to the metabolites found in OVT73 sheep. HD affects not only the nervous system of a patient, but their entire body. One of the earliest effects of the disease is a disruption of metabolism. By observing these pre-symptomatic sheep, and the changes in their metabolisms, researchers can observe how these changes affect the body in the long term, and thus gain further insights into the progression of HD.6,8

Another method used to study HD in OVT73 sheep is to examine how their circadian rhythms compare to those of normal sheep. Circadian rhythms are the patterns of activity, such as sleeping and eating, that an organism cycles through each day. A study led by Professor Jenny Morton of the University of Cambridge found that these patterns are interrupted in sheep populations consisting only of OVT73 sheep. This research not only strengthens the theory that circadian rhythm disruption is an early symptom of HD, but also hints that such a disruption may have an important role in the pathology of the disease.1,5

Though both findings had previously been seen in experiments with mice, follow up studies with OVT73 have confirmed these effects in a model more comparable to the HD patient.

These aren’t the only ways in which OVT73 is being studied. Research into the model has helped researchers learn an abundant amount about HD, and it is likely the model will contribute greatly to the further study of the disease.

Other HD Research in Sheep

Sheep are used generally in some forms of clinical testing, especially to test certain transplantationsedications, and surgery in general. Given the successful development of OVT73, sheep look to be a promising candidate for future preliminary HD pharmaceutical testing.9
Another transgenic sheep model also shows promise for HD research, though in an unlikely way. GM1 gangleosidosis is a progressive neurological genetic disorder caused by an overaccumulation of the GM1 fatty acid. In humans, the disease can cause rapid nervous system decline, leading to seizures, coma, and early death in juveniles as well as steady loss of motor skills in adults. A transgenic sheep model, similar to OTV73, was created to model the disease, though this model is symptomatic, and thus sheep of this variety show rapid accumulation of the GM1 fatty acid in their brain tissues.

One of the symptoms of HD appears to be the opposite of GM1 gangleosidosis. HD patients have greatly reduced levels of GM1 fatty acids in their brain tissue, and it is hypothesized that a lack of GM1 fatty acids in HD patients contributes to symptoms of the disease.9

Given that the process of extracting GM1 from GM1 gangleosidosis sheep for clinical purposes has already been shown safe for Parkinson’s Disease patients, clinical trials using extracted GM1 in HD patients may be on the horizon. The intervention is currently showing promising results in clinical trials with mice.10


Further Information

This HOPES article on transgenic mice explores how mice are used to research HD:

http://web.stanford.edu/group/hopes/cgi-bin/hopes_test/an-introduction-to-animal-models-of-huntingtons-disease/

 

This is a link to the original study where OVT73 was announced:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860888/

 

This a link to more information about the research regarding GM1 therapies, by HD Buzz, another fantastic resource for HD patients:
https://en.hdbuzz.net/199

 

Sources:

  1. Morton, J. A., & Howland, D. S. (2013). Large genetic animal models of Huntington’s Disease. Journal of Huntington’s Disease, 1(2), 3-19. 10.3233/JHD-130050
  2. Belleine, B. W., Delgado, M. R., Hikosaka, O. (2007). The role of the dorsal striatum in reward and decision-making. Journal of Neuroscience, 27(31). https://doi.org/10.1523/JNEUROSCI.1554-07.2007
  3. Lissencephaly. (2012). National Organization for Rare Disorders. Retrieved from https://rarediseases.org/rare-diseases/lissencephaly/
  4. Sun, T., & Henver, R. F. (2014). Growth and folding of the mammalian cerebral cortex: from molecules to malformations. Nature Reviews Neuroscience, 15(4), 217-232. 10.1038/nrn3707
  5. Morton, J. A., & Avanzo, L. (2011). Executive decision-making in the domestic sheep. Public Library of Science One, 6(1). journal.pone.0015752.
    6. Snell, R. G., personal communication, January 18, 2017
  6. Jacobsen, J. C., Bawden, C. S., Rudiger, S. R., McLaughlan, C. J., Reid, S. J., Waldvogel, H. J., MacDonald, M. E., Gusella, J. F., Walker, S. K., Kelly, J. K., Webb, G. C., Faull, R. L. M., Rees, M. I., & Snell, R. G. (2010) An ovine transgenic Huntington’s Disease model. Human Molecular Genetics, 19(10), 1873-1882. 10.1093/hmg/ddq063
  7. Handley, R. R., Reid, S. J., Patassini, S., Rudiger, S. R., Obolonkin, V., McLaughlan, C. J., Jacobsen, J. C., Gusella, J. F., Waldvogel, H. J., Bawden, C. S., Faull, R. L. M., Snell, R. G. (2016). Metabolic disruption identified in the Huntington’s Disease transgenic sheep model. Scientific Reports. 10.1038/srep20681
  8. Fox, L. (July 28, 2015). Would ewe believe it? GM1, sheep and Huntington’s Disease. HD Buzz. Retrieved from https://en.hdbuzz.net/199
  9. Pardo, Alba, et al. (2012). Ganglioside GM1 induces phosphorylation of mutant huntingtin and restores normal motor behavior in Huntington Disease mice. PNAS Neuroscience, 109(9), 3527-3533. https://www.ncbi.nlm.nih.gov/pubmed/22331905.