Abstract
The R6/2 murine model of Huntington’s disease (HD) is extensively used in HD research. The current study replicates and extends previous work assessing the impact of housing R6/2 mice with healthy wild-type (WT) littermates on disease progression. The current study extends the previous finding by including male cohorts and the use of a standard diet and water regimen, as opposed to the enhanced diet used in the previous study. This study found that the inclusion of healthy wild-type (WT) littermates, alone, improved survivabilty in R6/2 mice, but did not have a significant impact on weight loss.
INTRODUCTION
Transgenic animal models of neurodegenerative diseases are critical tools in the analysis of etiological mechanisms underlying disease. Furthermore, the ability to control potentially confounding variables in an experiment increases the likelihood of success for potential treatments used in the disorders they are intended to model. The degree to which these animals recapitulate the pathology of a given disorder can greatly impact the utility of the model and its usefulness for assessing specific aspects of the disease it mimics.
Huntington’s disease (HD) is a fatal genetic disease characterized by progressive psychiatric and cognitive symptoms, motor impairment, and weight loss. HD is the result of an expanded CAG repeat in the gene encoding the huntingtin protein (HTT), which leads to an expanded polyglutamine stretch in HTT [1, 2]. The resultant mutant huntingtin protein (mHTT) is ubiquitously expressed and leads to cellular damage throughout the body, including specific brain regions, skeletal and cardiac muscle, pancreatic β-cells, adipocytes, and blood cells [3]. This progressive cellular damage results in the motor impairment, cognitive symptoms, weight loss, and ultimately death that characterizes HD.
The R6/2 mouse model of HD is one of the most widely used and well-characterized murine models of HD [4] and mimics many of the deficits observed in HD patients such as striatal atrophy, locomotor deficits, neuronal intranuclear inclusions, weight loss, and early death [5, 6]. One manipulation that has repeatedly been shown to ameliorate deficits and extend life in the R6/2 model is environmental enrichment [6–14]. Importantly, Morton and colleagues have shown that enriched environments, including the genotype of the cage-mate [7], can alter disease progression. In some cases the effects of enriched environments on disease progression can be sex-dependent [8].
Purpose
The purpose of the current study was to expand on the work from the Morton lab [7] by including a cohort of male mice, in the absence of altered feeding regimes. Carter and collegues [7] observed increased survivability in early phases of the disorder in female R6/2 mice when housed with wild-type (WT) cagemates and an enhanced diet. The current study was intended to examine the differences that may occur in both male and female mice in body weight and survivability when R6/2 mice are housed exclusively with hemizygous littermates as compared to R6/2 mice housed with WT littermates in non-enriched setting and in the absence of any dietary enhancements.
METHODS
R6/2 mice
All animal tests and procedures were performed in accordance with the Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at Central Michigan University under protocol #15-21.
For this study both male and female progeny were bred in house from ovarian transplanted R6/2 mice (B6CBA-R6/2 [CAG 160 +/- 5]; JAX stock 002810) from Jackson Lab (Bar Harbor, ME), crossed with C57BL/6 wild-type male mice. Sixteen female hemizygous R6/2 mice were used to assess weight loss and survivability (10 were housed on the mixed-genotype condition, which included at least one WT mouse and 6 were housed in the single-genotype condition, which included only R6/2 cage-mates). Eighteen male hemizygous R6/2 mice were used to assess weight loss and survivability (9 were housed with at least one WT and 9 were housed with only R6/2 cage-mates). All mice were weaned at three weeks of age and housed in groups of either single or mixed-genotype with 1 to 4 same-sex littermates in polypropylene tubs, which were lined with aspen chip bedding supplemented with a single paper towel for nesting. Mice had access to food and water ad libitum, in an environmentally controlled room on a 12:12 h reverse light cycle. Beginning at three weeks of age, mice were handled daily to habituate animals to handling by researchers.
Weight
Mice were weighed three times each week, beginning at seven weeks of age, until reaching ten weeks of age at which point they were weighed daily. After losing 20% of their baseline weight the mice were weighed twice daily and observed once in between weighing. Animal weights at the end of week 7 (pre-symptomatic) were used as the baseline weight for calculating percentage of baseline weight for analysis.
Survivability
Number of days at the age of death of R6/2 animals was recorded and used to assess statistical significance of survival duration between groups.
Statistics
All statistics were done using GraphPad Prism 8. A two way repeated measures ANOVA was used to compare weight over time. A product limit estimator, or the Kaplan-Meier estimator was used to analyze survival function between single genotype housed and mixed genotype housed R6/2 mice.
RESULTS
Weight
The critical time period of weight loss in R6/2 mice is between 8 and 12 weeks. Most mice continued to gain or maintain weight until 8 weeks of age, followed by severe weight loss progressing until death at approximately week 13. Two way repeated measures ANOVA with Tukey’s multiple comparisons test was done to compare weight differences between male single-genotype (n = 9), female single-genotype (n = 6), male mixed-genotype (n = 9), and female mixed-genotype (n = 7) housed animals between 8 and 12 weeks over the progression of the disease. There was no effect of housing condition on weight, F(3,27) = 0.32, p = 0.809. There was, as would be expected, a significant weight loss over time F(1.70, 45.82) = 94.16, p = <0.001. However, there was no interaction between housing condition and weight loss, F 12, 108) = 0.54, p = 0.888 (Fig. 1).
Weight. Two-way repeated-measures ANOVA showed no significant differences in percentage of weight loss between male single-gentotype, female single-genotype, male mixed-genotype, and female mixed-genotype housed animals over the course of the study (p = 0.888).
Survivability. R6/2 mice housed with WT littermates survived significantly longer than single genotype housed HD animals (p = 0.005).
Survivability
A Kaplan-Meier estimator was used to compare survival between male and female single-genotype housed animals and found no significant difference [log-rank (Mantel-Cox) chi square = 0.30, p = 0.581]. Kaplan-Meier estimator also compared survival between male and female mixed-genotype housed animals and found no significant difference [log-rank (Mantel-Cox) chi square = 0.35, p = 0.553]. As no sex differences were observed, groups were pooled and a Kaplan-Meier estimator was used to analyze survival function between single-genotype housed animals and mixed-genotype housed animals. Analysis revealed that R6/2 mice housed with WT littermates survived significantly longer than single-genotype housed R6/2 mice [log-rank (Mantel-Cox) chi square = 8.07, p = 0.005]. Single-genotype housed animals lived on average 92 days, while mixed-genotyped housed mice lived 101 days on average.
DISCUSSION
The purpose of the current study was to assess what impact the inclusion of WT littermates in the housing of both male and female R6/2 mice would have on their lifespan and progressive weight loss as a basic measure of disease progression and severity. It was hypothesized that even the limited enrichment that may be provided by a healthy compatriot would be enough to result in delayed weight loss and extended survival. Results of the current study partially supported this hypothesis, suggesting even subtle alterations of housing condition, namely the presence or absence of WT littermates, can have a significant impact on the lifespan of both male and female R6/2 mice, but even with WT compatriots, no alteration in weight loss was observed. Mixed-genotype housed mice lived on average 9 days longer than their single-genotype housed counterparts; 101 days compared to 92 days respectively. This accounts for nearly 10% of an R6/2 animals’ lifespan, and therefore, is an important experimental factor that should be taken into consideration in any R6/2 study assessing survivability as an outcome.
Unlike Carter and colleagues [7], we did not observe delayed weight loss with mixed-genotype housing of R6/2 mice. However, similar to their findings, we did show an increase in survivability with the inclusion of at least one WT littermate. Collectively, these findings suggest that WT littermates may have motivated or forced the otherwise sedentary R6/2 mice into activities that they may not have performed without more active cage mates (i.e., feeding, interaction, movement, etc.). It has further been suggested that the nesting behavior of the WT animals may additionally contribute to these differences [7], possibly by reducing disease-induced hypothermia from lack of physical activity or insufficient nesting. This difference in nesting behavior was observed in the current study, with single-genotype housed mice building no nests and frequently sitting or sleeping apart from one another. On the other hand, the mixed-genotype housed mice had fully constructed nests in which all animals slept, likely constructed by their WT cage-mates. The combination of increased activity, social interaction, and improved nesting conditions may offer some clues to the observed outcome; however, the precise underlying mechanisms responsible are unknown and beyond the scope of this short communication.
In conclusion, our findings show clear differences in lifespan between mixed and single-genotype housed R6/2 mice, supporting the findings of Carter and colleagues [7]. This represents the first study to examine these differences in both male and female mice with free access to food and water. The results of this study indicate that the genotype of cage-mates should be viewed as a critical variable when determining housing of R6/2 mice. Environmental enrichment has repeatedly been shown to have a significant impact on the lifespan of this mouse model and the current study suggests that even minor alterations in the environmental conditions of these animals can have a large impact. Housing condition is clearly an important factor to take into consideration when assessing past studies that used R6/2 mice, and is a critical factor that should be addressed when designing future studies. Further, as other studies of enrichment have suggested, these findings should be taken into consideration when comparing pre-clinical studies and when conducting clinical assessment and treatment of HD patients, given that our results suggest that even minor alterations in environmental conditions can play a significant role in the progression of HD.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
Footnotes
ACKNOWLEDGMENTS
The authors would like to thank Veronica Wilson for her assistance with animal care and monitoring throughout the course of this study. Funding for this project was provided by the Field Neurosciences Institute and the John G. Kulhavi Professorship in Neuroscience.