Sex-dependent behavioral impairments in the HdhQ350/+ mouse line

Abstract

Huntington's Disease (HD) is an autosomal dominant neurodegenerative disease characterized by gradual deterioration of motor and cognitive functions and development of psychiatric deficits. Animal models provide powerful means to study the pathological processes, molecular dysfunctions and symptoms associated with HD. We performed a longitudinal behavioral study of the newly developed HdhQ350/+ mouse line, a knock-in model that expresses a repeat of 350 glutamines. We found remarkable sex-dependent differences on symptom onset and severity. While both sexes lose weight and grip strength, only HdhQ350/+ males have impaired motor coordination as measured by the rotarod and alterations in gait as measured by the catwalk assay. While HdhQ350/+ females do not exhibit impairment in motor coordination, we found a reduction in dark phase locomotor activity. Male and female HdhQ350/+ mice do not show anxiety as measured by the elevated plus maze or changes in exploration as measured by the open field test. To investigate these sex-dependent differences, we performed western blot analyses of striatal tissue. We measured equal mutant huntingtin protein expression in both sexes and found evidence of aggregation. We found the expected decrease of DARPP-32 expression only in female HdhQ350/+ mice. Remarkably, we found no evidence of reduction in synaptophysin or CB₁ receptors in HdhQ350/+ tissue of either sex. Our study indicates that male and female HdhQ350/+ mice differentially recapitulate select behavioral impairments commonly measured in other HD mouse models with limited sex-dependent changes in recognized histopathological markers. We conclude that expanded polyglutamine repeats influence HD pathogenesis in a sex-dependent manner.

Keywords: Huntington’s disease; Mouse model; Neurodegeneration; Polyglutamine disease; Protein aggregation; Sex-dependent.

Figure 1. General phenotype of HdhQ350/+ male and female mice compared to wild-type littermates

(A) Male and (B) female HdhQ350/+ mice showed reduced weight compared to their respective wild-type littermates. (C) When normalized to their respective wild-type littermates, both male and female HdhQ350/+ had similar reductions in weight. (D) Male and (E) female HdhQ350/+ mice showed reduced grip strength, starting at 30 weeks of age. (F) When normalized to their respective wild-type littermates, both sexes had similar reductions in grip strength. N=7–8 for all groups. Error bars represent S.E.M. and *p<0.05, **p<0.01, ***p<0.001 with two-way ANOVA with Bonferroni post-hoc test.

Figure 2. Spontaneous locomotor activity is affected in the dark, but not light, phase

(A) Male and (B) female HdhQ350/+ mice traveled less during the dark phase compared to their respective wild-type littermates. (C) Female HdhQ350/+ mice showed a sex-dependent decline in locomotor activity during the dark phase when normalized to their respective wild-type littermates. (D) Male and (E) female HdhQ350/+ mice traveled similarly to their respective wild-type littermates during the light phase. (F) There was no sex-dependent difference in locomotor activity during the light phase when normalized to their respective wild-type littermates. N=7–8 for all groups. Error bars represent S.E.M. and *p<0.05, ***p<0.001 with two-way ANOVA with Bonferroni post-hoc test.

Figure 3. Motor coordination is affected in male, but not female, HdhQ350/+ mice

Mice were tested over 3 days on an accelerating rotarod at various ages. (A) Male HdhQ350/+ mice showed reduced motor coordination on the second and third trial days at 60 weeks of age. (B) Male HdhQ350/+ mice had a lower average latency to fall at 60 weeks of age. (C) Female HdhQ350/+ mice had similar motor coordination on all trial days, compared to female wild-type littermates. (D) Female HdhQ350/+ mice had similar average latencies to fall at 40 and 60 weeks of age. N=7–8 for all groups. Error bars represent S.E.M. and *p<0.05, **p<0.01 with two-way ANOVA with Bonferroni post-hoc test.

Figure 4. Motor learning is affected in male, but not female, HdhQ350/+ mice

Fast motor learning was calculated by improved performance between the first and last trial on day 1. Slow motor learning was calculated by cumulative improved performance over all 3 days (See Materials and Methods). (A) HdhQ350/+ males showed significantly enhanced fast motor learning while (B) HdhQ350/+ females did not. (C) Comparison of male HdhQ350/+ mice to female HdhQ350/+ mice when normalized to their respective wild-type littermates in fast motor learning revealed no difference between sexes. (D) HdhQ350/+ males showed significantly enhanced slow motor learning while (E) HdhQ350/+ females did not differ from their respective wild-type littermates. (F) Comparison of male HdhQ350/+ mice to female HdhQ350/+ mice when normalized to their respective wild-type littermates revealed a significant difference in slow motor learning between sexes. N=8 for all groups. Error bars represent S.E.M. and *p<0.05, **p<0.01, with two-way ANOVA with Bonferroni post-hoc test.

Figure 5. HdhQ350/+ males and females show deficits in gait

(A) Male HdhQ350/+ mice do not have altered hind paw step cycle but (B) female HdhQ350/+ show increased hind paw step cycle at 40 and 60 weeks of age. (C) Male HdhQ350/+ mice have increased front paw stride length at 40 and 60 weeks of age but (D) female HdhQ350/+ mice do not. (E) Male HdhQ350/+ mice have reduced front paw base of support but (F) female HdhQ350/+ mice do not. N=6–8 for all groups. Error bars represent S.E.M. and *p<0.05, with two-way ANOVA with Bonferroni post-hoc test.

Figure 6. Psychiatric and cognitive functions are unaffected in HdhQ350/+ males and females

On the elevated plus maze, HdhQ350/+ (A) males and (B) females generally spent similar amounts of time in the closed arms than their respective wild-type littermates. HdhQ350/+ (C) males and (D) females spent similar amounts of time in the open arms than their respective wild-type littermates. Exploration was measured by the distance mice traveled within the first hour of being placed into the PhenoTyper chamber. (E) Male HdhQ350/+ mice and (F) female HdhQ350/+ mice covered similar distances than their respective wild-type littermates at 40 and 60 weeks of age. N=7–8 for all groups. Error bars represent S.E.M., with two-way ANOVA with Bonferroni post-hoc test.

Figure 7. mHtt expression and aggregation in striatal tissue of HdhQ350/+ males and females

Western blot of polyglutamine expansion at 40 and 60 weeks of age in (A) male and female HdhQ350/+ mice. Quantification of mHtt between male and female HdhQ350/+ mice showed no difference in protein expression levels. (B) SDDAGE blot comparing SDS solubility of striatal H350/+ tissue to yeast strains of 25 polyglutamines, 103 polyglutamines and prion Rnq1. Error bars represent S.E.M., with two-way ANOVA with Bonferroni post-hoc test.

Figure 8. HdhQ350/+ females, but not males, exhibit typical HD protein loss in the striatum

(A) Male HdhQ350/+ mice did not show changes in DARPP-32 protein expression at 40 or 60 weeks when compared to male wild-type littermates. (B) Female HdhQ350/+ mice showed significant reductions in DARPP-32 protein expression at 40 and 60 weeks of age. (C) Male HdhQ350/+ and (D) female HdhQ350/+ mice had no significant differences in average size of DARPP-32-positive cells compared to respective wild-type littermates. Representative images are cropped and captured at 40×. Scale bar denotes 10um. DAPI-positive cells quantified in the dorsolateral striatum showed no difference in (E) male HdhQ350/+ and (F) female HdhQ350/+ mice when compared to their respective wild-type littermates. N=3 for all groups. Error bars represent S.E.M. and **p<0.01, with two-way ANOVA with Bonferroni post-hoc test.