A Broad Phenotypic Screen Identifies Novel Phenotypes Driven by a Single Mutant Allele in Huntington’s Disease CAG Knock-In Mice

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing

Male <i>HdhQ111</i>/+ mice display a social recognition deficit.

<p>In the sample phase, an ovariectomized (OVX) female mouse was introduced into a cage with a male (A) or female (B) experimental <i>HdhQ111</i>/+ or Hdh+/+ mouse and time spent in olfactory investigation was recorded for 4 minutes after which time the OVX female was removed. After two hours, in the test phase, the experimental mouse was returned to the testing cage with both the familiar OVX mouse and a novel unfamiliar OVX female and time spent in olfactory investigation with both the familiar and unfamiliar mouse recorded for 4 minutes. Males: N=10 per genotype; females: N=10 per genotype. Bars show mean±SEM. * p<0.05; ** p<0.01 (2-tailed unpaired Student’s t-test comparing time spent investigating familiar and unfamiliar mouse). </p

Motor deficits in aged <i>HdhQ111</i>/+ mice.

<p><i>HdhQ111</i>/+ and Hdh+/+ mice at 46 weeks of age were tested on an automated CatWalk system (A-D) and for their ability to descend a vertical pole (E,F). <i>HdhQ111</i>/+ mice showed reduced Right by Left hindlimb stride length (A,B), a decreased cruciate step pattern frequency of pattern left front-right front-left hind-right hind (C,D) and an increased time to descend a vertical pole (total duration being the time taken to turn at the top of the pole and descend to the bottom). . For the CatWalk N=10 per sex and genotype; for the pole descent, males N=8 Hdh+/+, N=9 <i>HdhQ111</i>/+, females N=10 per genotype. Plotted are Tukey box-whisker graphs. All tests showed a significant genotype p value in a 2-way ANOVA. * Adjusted p value (p<0.05) following multiple testing correction.</p

Altered cardiovascular and lung parameters in <i>HdhQ111</i>/+ mice.

<p>A. Pulse was measured in 12 week mice (N=10 per group). A significant genotype p value (p<0.05) was obtained in a 2-way ANOVA. B. Tidal volume was measured in an independent cohort of 18 week mice (n=6 per group). Tidal volume and other lung function measures were analyzed independently in males and females by 2-tailed unpaired student’s t-test. * p<0.05. Plotted are Tukey box-whisker graphs. </p

Performance on the accelerating rotarod.

<p><i>HdhQ111</i>/+ and wild-type littermates were tested at 12 weeks (A, C, E) and 24 weeks (B, D, F) on an accelerating rotarod over three consecutive days and latency to fall (seconds, s) was recorded on each day (A-D). Improvement (E, F) was measured as the difference in latency to fall on day 3 compared to day 1, with positive values indicating improvement and negative values indicating a worsening performance. 12 weeks: N=20 <i>HdhQ111</i>/+ mice (10 males, 10 females and 21 Hdh+/+ mice (11 males, 10 females); 24 weeks: N= 20 <i>HdhQ111</i>/+ mice (10 males, 10 females) and 20 Hdh+/+ mice (10 males, 10 females). Bars show mean±SEM. * Adjusted p value (p<0.05) following multiple testing correction. </p

Free fed glucose levels in <i>HdhQ111</i>/+ and Hdh+/+ mice.

<div><p>Glucose levels were measured in free-fed mice at 16 weeks of age (A) (N=10 for each sex and genotype) and again at 19 weeks (B) in the same cohort of mice (N=10 males for each genotype, N=9 Hdh+/+ females and N=7 <i>HdhQ111</i>/+ females). </p> <p>* p<0.05 (2-tailed unpaired Student’s t-test). Plotted are Tukey box-whisker graphs. </p></div