1Department of Biochemistry, University of Washington, 2Department of Neurology, University of Washington, 3Division of Genetics, Departments of Pediatrics and Cellular and Molecular Medicine, and the Institute for Genomic Medicine, University of California, San Diego - Rady Children’s Hospital
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Guyenet, S. J., Furrer, S. A., Damian, V. M., Baughan, T. D., La Spada, A. R., Garden, G. A. A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia. J. Vis. Exp. (39), e1787, doi:10.3791/1787 (2010).
We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.
To prevent bias, the experimenter performing the assessments should not have knowledge of the animal's genotype. Individual measures are scored on a scale of 0-3, with 0 representing an absence of the relevant phenotype and 3 representing the most severe manifestation. Each test is performed multiple times to ensure that the score is reproducible. Obesity will complicate the interpretation of all measures described. The investigator may wish to weigh mice following phenotype scoring to assess the possible role of adiposity in the results.
The ledge test is a direct measure of coordination, which is impaired in cerebellar ataxias and many other neurodegenerative disorders. This measure is the most directly comparable to human signs of cerebellar ataxia.
Hindlimb clasping is a marker of disease progression in a number of mouse models of neurodegeneration, including certain cerebellar ataxias .
Gait is a measure of coordination and muscle function.
Kyphosis is a characteristic dorsal curvature of the spine that is a common manifestation of neurodegenerative disease in mouse models . It is caused by a loss of muscle tone in the spinal muscles secondary to neurodegeneration.
With a sufficient number of animals, this procedure is capable of detecting phenotype differences between strains and within the same strain over time. For data analysis, calculate the score for each measure by taking the mean of the three measurements in each individual. Measures can be analyzed separately or combined into a composite phenotype score for each individual.
Chart the data and apply the appropriate statistical methods to determine significance. Figure 1 shows results from a composite phenotype assessment of a murine transgenic SCA7 model. In this floxed-SCA7-92Q transgenic model, the human ataxin-7 gene with 92 CAG repeats is flanked by loxP sites and expressed from a bacterial artificial chromosome. The composite phenotype score includes the clasping, ledge walking, gait and kyphosis assessments for a maximum possible score of 12. The progressive SCA7 phenotype in the floxed-SCA7-92Q transgenic mice is demonstrated by an increasing composite phenotype score, which is consistent with the progressive nature of the human disease.
Figure 1. Floxed-SCA7-92Q mice exhibit a progressive SCA7 phenotype that is significantly different from Non-Transgenic littermates beginning at 20 weeks (2-way ANOVA: Bonferroni post-hoc; ***P<0.001). Mice were assessed on a 0-3 scale each for ledge test, clasping, gait, and kyphosis. Average composite score for each genotype at each age was calculated. Bars represent SEM.
This protocol is designed to be a sensitive and easily performed evaluation of disease severity in mouse models of cerebellar ataxia. Individual components of the scoring system will be more or less effective in different mouse models of neurodegeneration.
Elements of this scoring system have been effectively used to assess a variety of mouse models of human neurodegenerative disease, including cerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy [1-3]. The ideal combination of tests will depend upon the disorder in question. This protocol was originally designed by the authors to evaluate transgenic mouse models of spinocerebellar ataxia type 7 (SCA7).
In the model of SAC7 employed to generate the data presented here, animals were sacrificed at the age of 40-43 weeks or sooner if the behavioral abnormalities progressed to a stage that an animal is no longer sufficiently mobile to independently maintain nutrition or hydration.
These studies were approved by the University of Washington Institutional Animal Care and Use Committee.
This work was supported by NIH grants NS052535 to G.A.G and EY014061 & EY014997 to A.R.L., as well as a UL1DE019583-02 consortium award.