Mice lacking caspase-2 are protected from behavioral changes, but not pathology, in the YAC128 model of Huntington disease

Abstract

Background: Huntington Disease (HD) is a neurodegenerative disorder in which caspase activation and cleavage of substrates, including the huntingtin protein, has been invoked as a pathological mechanism. Specific changes in caspase-2 (casp2) activity have been suggested to contribute to the pathogenesis of HD, however unique casp2 cleavage substrates have remained elusive. We thus utilized mice completely lacking casp2 (casp2-/-) to examine the role played by casp2 in the progression of HD. This 'substrate agnostic' approach allows us to query the effect of casp2 on HD progression without pre-defining proteolytic substrates of interest.

Results: YAC128 HD model mice lacking casp2 show protection from well-validated motor and cognitive features of HD, including performance on rotarod, swimming T-maze, pre-pulse inhibition, spontaneous alternation and locomotor tasks. However, the specific pathological features of the YAC128 mice including striatal volume loss and testicular degeneration are unaltered in mice lacking casp2. The application of high-resolution magnetic resonance imaging (MRI) techniques validates specific neuropathology in the YAC128 mice that is not altered by ablation of casp2.

Conclusions: The rescue of behavioral phenotypes in the absence of pathological improvement suggests that different pathways may be operative in the dysfunction of neural circuitry in HD leading to behavioral changes compared to the processes leading to cell death and volume loss. Inhibition of caspase-2 activity may be associated with symptomatic improvement in HD.

Figure 1

Casp2-/- mice are protected from rotarod learning and accelerating rotorod deficits in the YAC128 mice. A-B) Naïve mice were trained at 4 months of age on a fixed speed (18 RPM) rotorod; 9 trials of 2 minutes each were conducted over three days. The time to first fall and the number of falls during each trial was recorded. Casp2-/- mice are unaffected on this task, compared to WT littermates as measured by time to first fall, while YAC128 mice show significant impairment that is rescued in casp2-/-;YAC128 mice (linear mixed effects model YAC128 F(1,109) = 7.74, p = 0.0064; casp2 F(1,109) = 1.43, p = 0.23; Interaction F(1,109) = 5.36, p = 0.023) or number of total falls (linear mixed effects model YAC128 F(1,109) = 9.45, p = 0.0026; casp2 F(1,109) = 1.09, p = 0.30; Interaction F(1,109) = 3.97, p = 0.049). Data represent mean +/- SEM. N = 33 WT mice, 28 YAC128 mice, 28 casp2-/- mice and 24 casp2-/-;YAC128. C) Mice were tested on an accelerating rotorod (5-40 RPM) at 4, 8 and 12 months. Each mouse performed 3 5-minute trials and the mean of three trials recorded. YAC128 mice perform worse than WT littermates on this task (i-two-way ANOVA, YAC128 F(1,51) = 23.07, p < 0.0001, age F(2,51) = 77.92, p < 0.0001; interaction F(2,51) = 1.61, p = 0.20), while casp2-/-;YAC128 mice do not perform significantly worse than casp2-/- littermates (ii-two-way ANOVA, YAC128 F(1,45) = 2.39, p = 0.13, age F(2,45) = 18.0, p < 0.0001; interaction F(2,45) = 0.11, p = 0.89). Data represent mean +/- SEM. N = 33 WT mice, 28 YAC128 mice, 28 casp2-/- mice and 24 casp2-/-;YAC128.

Figure 2

Casp2-/- mice are protected from cognitive symptoms of HD in the YAC128 mouse. A) Mice were trained in a swimming T-maze to find a submerged platform in one arm of the maze. Acquisition time of the platform location does not differ between genotypes during the first 12 trials (two-way repeated measures ANOVA genotype: F(3,638) = 0.56, p = 0.64). Before trial 1 of the reversal phase on day 5, the platform was switched to the opposite arm of the T-maze. Time to the platform in its new location differed by genotype across the 4 trials (two-way repeated measures ANOVA, genotype: F(3,147) = 2.90, p = 0.043). YAC128 mice take significantly longer to reach the platform on the first trial than Casp2-/-;YAC128 mice (19.5 seconds vs. 10.86 seconds, Bonferroni t = 3.205, p < 0.01). Data represent mean +/- SEM. N = 33 WT mice, 28 YAC128 mice, 28 casp2-/- mice and 24 Casp2-/-;YAC128 mice. B) Increased time to platform in the YAC128 mice was primarily due to increased perseveration during the first trial. YAC128 mice re-entered the previously correct arm more frequently than Casp2-/-;YAC128 mice (two-way repeated measures ANOVA Trial F(3,174) = 10.19, p < 0.0001; YAC128 F(3,174) = 2.04, p = 0.12; Interaction F(9,174) = 1.42, p = 0.18; Bonferroni post-hoc test significance indicated). N = 21 WT mice, 28 YAC128 mice, 17 casp2-/- mice and 24 Casp2-/-;YAC128 mice. C) YAC128;casp2-/- are rescued from deficits in a T-maze spontaneous alternation task. 7-month old Mice were exposed to a T-maze with a divider forcing them to choose one arm, which they were restrained to for 1 minute. After this familiarization trial, the mice re-entered the T-maze and their arm choice recorded. WT mice prefer the novel arm of the maze 79% of the time, while YAC128 mice only choose the novel arm 53% of the time. Casp2-/- and YAC128;casp2-/- choose the novel arm 77% and 73% of the time, similar to WT mice. N = 15 WT mice, 21 YAC128 mice, 17 casp2-/- mice and 15 Casp2-/-;YAC128 mice.

Figure 3

Casp2-/-;YAC128 mice are protected from pre-pulse inhibition deficits. Mice were tested for pre-pulse inhibition at 12 months of age. YAC128 mice show reduced PPI, compared to littermates, while casp2-/-;YAC128 mice do not (one-way ANOVA 2dB Genotype F(3,39) = 4.49, p = 0.0089; 4dB Genotype F(3,38) = 3.07, p = 0.04; 16dB Genotype F(3,37) = 2.25, p = 0.10; Newman-Keuls post-hoc test p-values indicated). Mean="+", horizontal bars = quartiles. N = 7 WT mice, 12 YAC128 mice, 9 casp2-/- mice and 12 Casp2-/-;YAC128 mice. Post-hoc Bonferroni genotype comparisons "*" indicates p < 0.05.

Figure 4

Casp2-/- mice are protected from locomotor symptoms of HD in the YAC128 mouse. At 7 months of age, YAC128 mice display decreased locomotor activity and increased anxiety, as measured by time spent in the center of the arena, during exploration of an open field as compared to WT mice. Casp2-/- and casp2-/-;YAC128 mice perform similarly to WT mice. Mean="+", horizontal bars = quartiles. N = 15 WT mice, 21 YAC128 mice, 17 casp2-/- mice and 15 Casp2-/-;YAC128 mice. Post-hoc Newman-Keuls genotype comparisons "*" indicates p < 0.05.

Figure 5

Casp2-/- mice are not protected from pathology. A-B) Fixed forebrain and testes weight in 12-month-old mice. YAC128 mice show forebrain atrophy that is not rescued by the absence of casp2 (summary statistics for two-way ANOVA indicated). Mean="+", horizontal bars = quartiles. N = 9 WT mice, 11 YAC128 mice, 9 casp2-/- mice, 14 Casp2-/-;YAC128 mice. C) Striatal volume, as determined by stereology, is reduced in YAC128 mice and not affected by caspase-2 expression. Mean="+", horizontal bars = quartiles, isolated dots = outliers. Factorial ANOVA F/p-values indicated. N = 19 WT mice, 25 YAC128 mice, 21 casp2-/- mice, 22 Casp2-/-;YAC128 mice.

Figure 6

Casp2-/- mice are not protected from brain pathology assayed with MRI. A) Volumes for brain structures were determined by automated segmentation of MRI images and expressed as Z-scores. YAC128 mice have smaller brains, with specific shrinkage in the basal ganglia and other sub-cortical gray matter structures. Casp2-/- mice are not protected from any of these losses (summary statistics for two-way ANOVA indicated). B) To control for overall brain atrophy, structure volumes were expressed as a percentage of brain volume. Relative striatal volume is decreased in the YAC128 mice, and not rescued in the casp2-/-;YAC128 mice (Post-hoc Bonferroni genotype comparisons "*" indicates p < 0.05). The cerebellum is relatively preserved in the YAC128 mice, due to selective forebrain degeneration. This phenotype is not ameliorated in the casp2-/-;YAC128 mice (Post-hoc Bonferroni genotype comparisons "*" indicates p < 0.05). Mean="+", horizontal bars = quartiles, isolated dots = outliers. N = 9 WT mice, 11 YAC128 mice, 9 casp2-/- mice, 14 Casp2-/-;YAC128 mice.