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Mice lacking caspase-2 are protected from behavioral changes, but not pathology, in the YAC128 model… Carroll, Jeffrey B; Southwell, Amber L; Graham, Rona K; Lerch, Jason P; Ehrnhoefer, Dagmar E; Cao, Li-Ping; Zhang, Wei-Ning; Deng, Yu; Bissada, Nagat; Mark Henkelman, R; Hayden, Michael R Aug 19, 2011

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Mice lacking caspase-2 are protected frombehavioral changes, but not pathology, in theYAC128 model of Huntington diseaseCarroll et al.Carroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59 (19 August 2011)RESEARCH ARTICLE Open AccessMice lacking caspase-2 are protected frombehavioral changes, but not pathology, in theYAC128 model of Huntington diseaseJeffrey B Carroll1, Amber L Southwell2, Rona K Graham2, Jason P Lerch3, Dagmar E Ehrnhoefer2, Li-Ping Cao2,Wei-Ning Zhang2, Yu Deng2, Nagat Bissada2, R Mark Henkelman3 and Michael R Hayden2*AbstractBackground: Huntington Disease (HD) is a neurodegenerative disorder in which caspase activation and cleavageof substrates, including the huntingtin protein, has been invoked as a pathological mechanism. Specific changes incaspase-2 (casp2) activity have been suggested to contribute to the pathogenesis of HD, however unique casp2cleavage substrates have remained elusive. We thus utilized mice completely lacking casp2 (casp2-/-) to examinethe role played by casp2 in the progression of HD. This ‘substrate agnostic’ approach allows us to query the effectof 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 featuresof HD, including performance on rotarod, swimming T-maze, pre-pulse inhibition, spontaneous alternation andlocomotor tasks. However, the specific pathological features of the YAC128 mice including striatal volume loss andtesticular degeneration are unaltered in mice lacking casp2. The application of high-resolution magnetic resonanceimaging (MRI) techniques validates specific neuropathology in the YAC128 mice that is not altered by ablation ofcasp2.Conclusions: The rescue of behavioral phenotypes in the absence of pathological improvement suggests thatdifferent pathways may be operative in the dysfunction of neural circuitry in HD leading to behavioral changescompared to the processes leading to cell death and volume loss. Inhibition of caspase-2 activity may beassociated with symptomatic improvement in HD.Keywords: Huntington’s Disease, neurodegeneration, caspase, magnetic resonance imagingBackgroundHuntington disease (HD) is a neurodegenerative disor-der characterized by progressive motor, cognitive andpsychiatric deficits [1] caused by an expanded poly-glu-tamine tract in the huntingtin (HTT) protein [2]. Neu-ropathologically, HD is characterized by early loss ofmedium spiny neurons (MSNs) in the striatum, accom-panied by gliosis and eventual progressive neuronal lossthroughout the brain [3].Caspases are a family of proteases initially described toplay critical roles in apoptotic cell death [4], whose non-apoptotic cellular functions are increasingly realized [5].HTT has been shown to be a substrate in vitro for cas-pases-1,2,3 and -6 [6-9]. A number of additional pro-teins whose mutation causes neurodegeneration are alsocaspase substrates, including the amyloid precursor pro-tein [10-12], tau [13], atrophin-1 [9], ataxin-3 [9],ataxin-7 [14] and the androgen receptor [9].The commonality of caspase cleavage of neurodegenera-tive disease proteins could reflect their degradative clear-ance during cell death. Alternatively, these cleavage eventscould mediate apoptotic signaling, as is the case for bid[15], XIAP [16] and the caspases themselves [17]. Support-ing the latter hypothesis, mutation of obligate aspartateresidues in the caspase recognition regions of HTT hasestablished that cleavage at amino acid 586, a caspase-6 site* Correspondence: mrh@cmmt.ubc.ca2Centre for Molecular Medicine and Therapeutics, Child and Family ResearchInstitute, Department of Medical Genetics, University of British Columbia,Vancouver, V5Z 4H4, CanadaFull list of author information is available at the end of the articleCarroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59© 2011 Carroll et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.in vitro, is crucial for the development of HD symptoms ina mouse model [18-20]. Analogous rescue is observed inmutant APP-transgenic mice resistant to caspase cleavageat aspartate-664 [21,22]. Further, prevention of caspase pro-cessing of both atrophin-1 [23] and ataxin-7 [14] reducestoxicity in vitro. These experiments suggest that caspase-mediated cleavage of neurodegenerative disease proteinsplays a role in the development of these conditions.Caspase-2 (casp2) has been implicated in both Alzhei-mer’s and Huntington disease. Dominant-negative casp2constructs rescue mutant HTT-induced toxicity inrodent neurons, and total casp2 levels are increased invulnerable neurons in human patient post-mortem tis-sue [6]. Furthermore, antisense to casp2 protects multi-ple neuronal cell lines from Aß1-42 induced toxicity[24-26]. Despite these disease associations, being thesecond mammalian caspase cloned [27,28] and its highdegree of evolutionary conservation [29] the role ofcasp2 in vivo has remained unresolved.While HTT itself is cleaved by casp2 at aspartate-552in vitro [6], this event is not crucial for development ofHD because mutating this site does not confer protec-tion from HD symptoms in a mouse model [18], andconstitutive cleavage at this site is observed in control,as well as diseased, brains [7]. However, experiments inrodent neurons with dominant-negative casp2 con-structs support the idea that activity of casp2 may con-tribute to mutant-HTT induced toxicity [6]. Whilecasp2 cleavage of HTT is thus unlikely to cause pathol-ogy in HD, other activities of casp2 may contribute tosigns and symptoms of HD.Very few casp2 cleavage substrates have beenreported: casp2 [30], golgin-160 [31], aII-spectrin [32],protein kinase C (delta) [33], and Bid [34]. Of theseevents, only golgin-160 cleavage is uniquely catalyzedby casp2 [31]. Furthermore, the reagents commonlyused as inhibitors and markers of casp2 activity basedon the binding between VDVAD pseudo-substrate andactive casp2 enzyme have been conclusively shown tobe nonspecific [35], limiting their use in complexsamples.To examine the role of casp2 in HD we thus under-took a ‘substrate agnostic’ approach. Rather than focus-ing on particular potential cleavage events, we studiedthe development of HD symptoms in the well-validatedYAC128 murine model of HD [36-38] when bred tocasp2 -/- mice [39] to determine whether developmentaland complete lack of casp2 may in some way modifythe phenotype of HD, without relying on proxy makersfor caspase activation.ResultsCasp2 mRNA and HTT protein levels in the brainCasp2 has been described as transcriptionally upregu-lated in the striatum of the YAC72 mouse model of HD[6] and total casp2 immunoreactivity is increased in thebrain of HD patients with significant striatal pathology[6]. To more directly examine caspase-2 transcription,we examined publically available microarray data [40]from early-stage (Vonsattel grade 0-2) human HD stria-tal tissue. This reveals no change in casp2 mRNA levelsin the striatum of HD patients (table 1). Levels of tran-scripts reduced in HD, such as HMG-CoA reductase[41], are reduced in HD patients in this population, sup-porting the robustness of the dataset (table 1).Consistent with this human data, quantitative real-time PCR (QRT-PCR) of striatal casp2 mRNA demon-strates that wild type (WT) and YAC128 mice haveequivalent levels throughout their life spans (table 1,two-way ANOVA, Genotype: F(1,27) = 0.073, p = 0.79;Age: F(3,27) = 2.48, p = 0.082; Interaction: F(3,27) =2.71, p = 0.065). Thus, in both a rigorously characterizedanimal model of HD, as well as patient material, we seeno evidence for transcriptional upregulation of casp2 inaffected tissues at a time when both the mice andhumans are displaying symptoms of HD.We then considered whether levels of full-lengthmutant HTT could be altered by expression of casp2-/-.Table 1 Casp2 expression in the striatum of YAC128 mice and human HD patientsSpecies Age (M) DiseaseStage*Gene Expression, HD/Control SEM t-value p-valueMouse 3 Casp2 .704 .063 1.78 > 0.05Mouse 6 Casp2 1.15 .11 0.56 > 0.05Mouse 9 Casp2 1.20 .12 0.71 > 0.05Mouse 12 Casp2 1.01 .12 0.08 > 0.05Human** 0-2 Casp2 0.996 -0.14 > 0.05Human** 0-2 HMGCR 0.739 -5.04 < 0.001Human striatal casp2 mRNA levels were extracted from publically available sources of microarray data (Affymetrix HG-U133A/B probe set 208050_s_at, HDBase.org-originally described in [66]). Mouse striatal casp2 levels were determined using QRT-PCR at 3, 6, 9 and 12 months of age. Casp2 is not upregulated in thestriatum of aging YAC128 mice or human HD patients.* Vonsattel neuropathological score at time of death [3]** Human data extracted from [40]Carroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59Page 2 of 12Using an antibody that recognizes long glutaminerepeats (1C2, Millipore MAB1574), we performed wes-tern blots with cortices of 12-month old YAC128 andcasp2-/-;YAC128 mice. There was no effect of genotypeon full-length mutant huntingtin levels (data not shown,t(4) = 0.12, p = 0.91). We have previously demonstratedthat the aa586 caspase-6 fragment of HTT is uniquelylinked to toxicity in the YAC128 model of HD [18-20].We determined the levels of the aa586 caspase-6 frag-ment in the cortex of 12 month old YAC128 andcasp2-/-;YAC128 mice, using the same western blots.These blots reveal no effect of genotype on aa586 frag-ment levels (data not shown, t(4) = 0.36, p = 0.74).Casp2 -/- mice are protected from motor and cognitivesymptoms of HDThe YAC128 murine model of HD robustly recapitu-lates many of the signs and symptoms of HD [37,38]. Inorder to examine the effect of casp2 on HD onset andprogression, we bred heterozygous YAC128 [38] mice toa casp2 -/- [39] and WT background.From two months of age YAC128 mice demonstrateperformance deficits when first exposed to a fixed-speed(18 RPM) 2-minute rotarod running task [37]. In thiscohort, at 4 months of age, no effect of casp2 ablation isobserved in WT mice on the time to first fall during 2-minute training sessions Figure 1A, gray and blacklines). As described [37], YAC128 mice perform signifi-cantly worse than WT mice, an effect which is rescuedin casp2-/-;YAC128 mice (Figure 1A, red and pink lines,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). The mean time to first fallacross all 9 trials is 68% of WT levels in the YAC128mice, but 94% of WT levels in the casp2-/-;YAC128mice, showing that casp2-/-;YAC128 mice are4 8 120100200300WTYAC128*******Age (Months)4 8 12Casp2-/-Casp2-/-;YAC128NSNSNSAge (Months)Mean Latency to Fall, Secondsi. ii.Casp2+/+ Casp2-/-0 2 4 6 8 10050100150Trial0 2 4 6 8 100246TrialTime to first fall, SecondsAverage Falls/2 MinutesWTYAC128Casp2-/-Casp2-/-;YAC128A. Time to first fall B. Total number of fallsC.Figure 1 Casp2-/- mice are protected from rotarod learning and accelerating rotorod deficits in the YAC128 mice. A-B) Naïve mice weretrained 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 falland the number of falls during each trial was recorded. Casp2-/- mice are unaffected on this task, compared to WT littermates as measured bytime 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 effectsmodel 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, 8and 12 months. Each mouse performed 3 5-minute trials and the mean of three trials recorded. YAC128 mice perform worse than WT littermateson 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), whilecasp2-/-;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-/- miceand 24 casp2-/-;YAC128.Carroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59Page 3 of 12performing at essentially WT levels on this motor task.Examination of the total number of falls during thetraining session validates the specific impairment of theYAC128 mice, which is again ameliorated in thecasp2-/-;YAC128 mice (Figure 1B, linear mixed effectsmodel 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).After training, mice were tested on a 5-minute accel-erating rotarod (5-40 RPM) task at 4, 8 and 12 monthsof age. As previously shown, YAC128 mice perform sig-nificantly worse than littermates on this task (Figure 1Ci, two-way ANOVA, YAC128 F(1,102) = 23.07, p <0.0001, Age F(2,102) = 77.92, p < 0.0001, Interaction F(2,102) = 1.61, p = 0.24). In mice lacking casp2, thiseffect is ameliorated (Figure 1C ii, two-way ANOVA,Genotype F(1,90) = 2.4, p = 0.13, Age F(2,90) = 18.0, p< 0.0001, Interaction F(2,90) = 0.11, p = 0.89). At 12months of age, YAC128 latency to fall is reduced by39% compared to WT mice (95.8 sec. vs. 157.6 sec,respectively). Casp2-/-;YAC128 mice remain on therotarod for an average of 154.6 seconds, 98% of WTlevels, suggesting complete rescue of this phenotype.In addition to motor impairment, YAC128 micedemonstrate clear cognitive and psychiatric deficits ana-logous to those seen in human HD patients [37,42,43].To probe perseverative behaviors, we used a previouslyvalidated swimming T-maze task [37]. At 12 months ofage there is no difference in swimming speed betweenany of the genotypes in the current study, and all fourgenotypes of mice learned to reach a submerged plat-form equally well during 12 training runs over 4 days(Figure 2, left side, two-way repeated measures ANOVA,genotype: F(3,58) = 0.56, p = 0.64). On day 5, the plat-form was switched to the opposite arm of the swimmingt-maze and YAC128 mice require significantly longer toreach the new platform location, while casp2-/-;YAC128mice perform similar to WT mice (Figure 2A, right side,two-way repeated measures ANOVA, genotype: F(3,58)= 2.90, p = 0.043, Bonferroni post-test trial 1, Casp2-/-;YAC128 vs. YAC128, t = 2.78, p < 0.05). This is primar-ily due to increased perseveration during the first rever-sal trial-the YAC128 mice re-enter the previouslycorrect arm of the maze more frequently than YAC128mice lacking casp2 (Figure 2B, repeated measures two-way ANOVA Bonferroni post-hoc tests indicated).These data suggest that YAC128 mice lacking casp2 areprotected from loss of cognitive flexibility-a cardinalearly feature of psychiatric disturbances in HD [44,45].To extend our understanding of the cognitive defectsunderlying this deficiency, we examined the spontaneousalternation of mice in a T-maze [46]. In this spatialmemory task mice are released at the base of the T-maze and allowed to choose an arm to explore. Whenre-exposed to the maze, normal mice recall the pre-viously visited arm of the T-maze and prefer to investi-gate the novel arm 79% of the time. YAC128 miceperform near chance in this task, visiting the new armof the maze only 53% of the time. Both casp2-/- andcasp2-/-;YAC128 mice perform similarly to WT micechoosing the new arm 77% and 73% of the time respec-tively (Figure 2C).HD patients show reduced pre-pulse inhibition (PPI)[47], as do YAC128 mice [37]. At 12 months of age star-tle amplitude in response to a 120dB noise is equivalentin all examined mice (one-way ANOVA, genotype: F(3,37) = 0.36, p = 0.78). In a PPI paradigm with a back-ground noise + 2dB, 4dB or 16dB warning tone theYAC128 mice have reduced PPI compared to WT mice(Figure 3B, 2dB Genotype F(3,39) = 4.49, p = 0.0089;4dB Genotype F(3,38) = 3.07, p = 0.04; 16dB GenotypeF(3,37) = 2.25, p = 0.10; Newman-Keuls post-hoc test p-values indicated). Lower intensity pre-pulses led to moreobvious deficits in the YAC128 mice, in agreement withpreviously published results [37]. This effect was amelio-rated in casp2-/-;YAC128 mice, who had normalized PPIcompared to YAC128 mice, suggesting that sensorimo-tor gating defects are corrected in YAC128 mice lackingcasp2.YAC128 mice are hypoactive during exploration of anopen field after about 6 months of age [38]. This loco-motor phenotype is rescued in the casp2-/-;YAC128mice at 7 months of age (Figure 4A, one way ANOVA F(3,67) = 4.30, p = 0.0079, Newman-Keuls post-hoc testsindicated). In addition to their hypoactive phenotype,the YAC128 mice show anxiety by spending less time inthe center of the open field arena, and entering the cen-ter zone less frequently [48]. This phenotype is alsoameliorated in the casp2-/-;YAC128 mice (Figure 4B,right, one way ANOVA F (3,67) = 5.73, p = 0.0015,Newman-Keuls post-hoc tests indicated).Casp2-/- mice are not protected from pathologicalfeatures of HDBrain weight is the simplest measure of neurodegenera-tion, and is decreased in the YAC128 mice [38]. In thepresent cohort, forebrain weight is reduced in theYAC128 mice, an effect which is not ameliorated by theabsence of casp2 (Figure 5A, linear mixed effects model,YAC128 F(1,39) = 4.5, p = 0.040; casp2 F(1,39) = 0.432,p = 0.52). Male YAC128 mice and human HD patientsalso have specific testicular degeneration [49], a pheno-type which is also not affected by the absence of casp2(Figure 5B, linear mixed effects model, YAC128: F(1,18)= 5.70, p = 0.028; Casp2: F(1,18) = 0.10, p = 0.75). LikeHD patients, YAC128 mice have progressive specificstriatal volume decreases [38,50]. Using stereologicaltechniques we observe reduced striatal volume in theCarroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59Page 4 of 12YAC128 mice that is not ameliorated in the casp2-/-;YAC128 mice (Figure 5C, two-way ANOVA YAC128: F(3,86) = 6.04, p = 0.016; casp2 F(3,86) = 0.17, p = 0.69;Interaction F(1,88) = 0.02, p = 0.88).To gain more detailed information on YAC128 neuro-pathology, we have developed and described magneticresonance imaging (MRI) techniques to generate volu-metric data for a number of CNS structures [50-53]. Thesetechniques allow the complete three-dimensional delinea-tion of structures, avoiding artificial truncation due totissue processing limitations. Measurements are conductedon brains in situ in the skull, minimizing processing andhandling artifacts. Also, using atlas based MRI techniquesit is possible to simultaneously determine volumes for alarge number of structures, rather than limiting analyses topre-determined regions of interest. Direct comparison ofMRI and stereological approaches to measuring striatalvolume in the YAC128 mice has shown that while thevolume loss detected by each technique is the same, MRI ismore accurate and therefore powerful [50,54].A.  Swimming T-maze with reversal phase, Time to Platform1 2 3 4 5 6 7 8 9 10 11 12510152025Trial1 2 3 4510152025Trial10152025Training Phase1015WTCasp2-/-YAC128Casp2-/-;YAC128Reversal PhaseTime to Platform (s)*C.  Spontaneous AlternationProportion AlternatingCasp2−/−;YAC128Casp2−/−YAC128WT0.0 0.2 0.4 0.6 0.8** ****Trial1 2Previously Targeted Arm Re-EntriesB.  T-maze PreseverationNot-alternatingAlternatingFigure 2 Casp2-/- mice are protected from cognitive symptoms of HD in the YAC128 mouse. A) Mice were trained in a swimming T-mazeto find a submerged platform in one arm of the maze. Acquisition time of the platform location does not differ between genotypes during thefirst 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 platformwas 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-wayrepeated measures ANOVA, genotype: F(3,147) = 2.90, p = 0.043). YAC128 mice take significantly longer to reach the platform on the first trialthan 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, 28YAC128 mice, 28 casp2-/- mice and 24 Casp2-/-;YAC128 mice. B) Increased time to platform in the YAC128 mice was primarily due to increasedperseveration during the first trial. YAC128 mice re-entered the previously correct arm more frequently than Casp2-/-;YAC128 mice (two-wayrepeated 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; Bonferronipost-hoc test significance indicated). N = 21 WT mice, 28 YAC128 mice, 17 casp2-/- mice and 24 Casp2-/-;YAC128 mice. C) YAC128;casp2-/- arerescued from deficits in a T-maze spontaneous alternation task. 7-month old Mice were exposed to a T-maze with a divider forcing them tochoose one arm, which they were restrained to for 1 minute. After this familiarization trial, the mice re-entered the T-maze and their arm choicerecorded. 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-/- miceand 15 Casp2-/-;YAC128 mice.Carroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59Page 5 of 12MRI data was acquired post-mortem, at twelvemonths of age-the same mice were used for both beha-vioral and MRI experiments. Total brain volume, asdetermined by MRI, was reduced in the YAC128 miceand not rescued in the casp2-/- mice, though reductionsobserved did not reach significance (Figure 6A, left,two-way ANOVA YAC128: F(1,39) = 3.10, p = 0.086;casp2: F(1,39) = 0.02, p = 0.88; Interaction F(1,39) =0.38, p = 0.54). MRI-detected striatal volume is specifi-cally reduced in the YAC128 mice, and not affected bycasp2 expression (Figure 6A, two-way ANOVAYAC128: F(1,39) = 13.14, p = 0.0008, casp2: F(1,39) =0.02, p = 0.85; Interaction: F(1,39) = 0.38, p = 0.54).Other brain structures, such as the thalamus, show atro-phy in the YAC128 mice [54,50], and human patients[55]. This atrophy was validated in the current cohortand was not affected by casp2 expression (Figure 6A,two-way ANOVA, YAC128: F(1,39) = 11.41, p = 0.0017;casp2: F(1,39) = 2.704, p = 0.11; Interaction: F(1,39) =0.09, p = 0.76).MRI-detected volumes are valuable measures of tissueatrophy because they can be normalized to total brainvolume. These “brain normalized” values highlight speci-fic regions of atrophy and preservation, by correcting forchanges in brain volume. We have shown that regionsof both atrophy (the striatum) and preservation (the cer-ebellum) can be observed with these measures in theYAC128 mice [50]. In agreement with the stereologicaldata, striatal volume, when considered as a fraction oftotal brain volume, is reduced in the YAC128 mice, andnot rescued in the casp2-/-;YAC128 mice (Figure 6B,Bonferroni post-hoc tests indicated). Analogously, thecerebellar cortex is increased in relative volume, due toits preservation in the face of HD-induced atrophy. Thiscerebellar preservation is not altered in the casp2-/-;YAC128 mice (Figure 6B, Bonferroni post-hoc testsindicated). These results validate the specific pathologi-cal tissue loss in the YA128 mice, the use of MRI toascertain these losses, and clearly demonstrate that theabsence of casp2 is not associated with amelioration ofthis loss.DiscussionThe primary finding of the current study is that casp2-/-mice are protected from behavioral and cognitive fea-tures of HD in the YAC128 model. It is remarkable thatevery behavioral and cognitive feature of HD examinedis completely rescued in the absence of caspase-2. How-ever, pathological phenotypes of HD including specificstriatal volume loss and testicular degeneration are notrescued in the casp-2-/- mice.Caspase-2 transcriptionThis work expands our understanding of the activationof casp2 in HD. An earlier report suggested that tran-scriptional up-regulation, secondary to BDNF loss, couldexplain increases in casp2 levels in HD [6]. The datapresented here do not support this hypothesis-humanmicroarray data indicates that striatal casp2 levels are***** **Total Distance Traveled Total Time in CenterCenter Duration (s)Distance Traveled (cm)A. B.Figure 4 Casp2-/- mice are protected from locomotorsymptoms of HD in the YAC128 mouse. At 7 months of age,YAC128 mice display decreased locomotor activity and increasedanxiety, 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 YAC128mice, 17 casp2-/- mice and 15 Casp2-/-;YAC128 mice. Post-hocNewman-Keuls genotype comparisons “*” indicates p < 0.05.Pre-pulse Intensity (+2dB, 4dB and 16dB)WTYAC128Casp2-/-Casp2-/-;YAC128020406080100 * **WTYAC128Casp2-/-Casp2-/-;YAC128*WTYAC128Casp2-/-Casp2-/-;YAC128Pre-Pulse Inhibition (%)Figure 3 Casp2-/-;YAC128 mice are protected from pre-pulse inhibition deficits. Mice were tested for pre-pulse inhibition at 12 months ofage. 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 Bonferronigenotype comparisons “*” indicates p < 0.05.Carroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59Page 6 of 12normal in early stage HD patients. Additionally, striatalcasp2 mRNA levels in YAC128 HD mice are equivalentto WT levels during the development of HD-like beha-vioral changes. Thus, increased casp2 transcription isunlikely to contribute to the development of HD.Caspase-2 -/- mice are protected from motor andcognitive features of HDCasp2-/- mice are protected from motor and cognitivedeficits seen in the YAC128 model of HD. Consistentwith previous reports, we show that the YAC128 micehave deficits during the learning phase of the rotarodtask at 4 months of age [37]. This learning deficit iscompletely reversed in the YAC128 mice lacking casp2,who are also protected from progressive motor impair-ment on the previously learned task. This demonstratesthat casp2-/-;YAC128 mice are protected from bothcognitive and motor aspects of this task.Data from the pre-pulse inhibition, swimming T-mazeand spontaneous alternation tasks presented here areconsistent with the cognitive flexibility deficits pre-viously observed in the YAC128 mice [37]. All thesetasks were performed significantly better by casp2-/-;YAC128 mice, relative to YAC128 mice. This suggeststhat YAC128 mice lacking casp2 are better able to mod-ify their behavior in response to environmental demandsthan normal YAC128 mice. Specific deficits in beha-vioral flexibility are a key feature of the cognitive defectsobserved in human HD patients [56,57], and improve-ment of these symptoms in casp2-/-;YAC128 mice sup-ports the idea that casp2 inhibition may have impact onneural function, leading to symptomatic benefit.Casp2-/- mice are not protected from pathologicalfeatures of HDDespite their protection from behavioral and cognitivesymptoms, the volume loss observed in the YAC128 micein structures such as the striatum and thalamus is not ame-liorated by the absence of casp2. In the periphery, patholo-gical atrophy of the testes is also not ameliorated incasp2-/-;YAC128 mice. The improved performance of thecasp2-/-;YAC128 mice on behavioral tasks in the face ofthis pathological tissue loss suggests that neuronal circuitsmediating disease relevant behavior are capable of augmen-tation leading to symptomatic benefit. These experimentsdemonstrate that loss of volume is not sufficient to causeall motor and cognitive features of HD in mice.Previous interventions in the YAC128 mice haveshown dissociation between neuropathological andbehavioral endpoints. Symptomatic treatment withcystamine results in neuroprotection without affectingbehavioral symptoms of HD [58]. Conversely, sympto-matic treatment with ethyl-eicosapentaenoic acid pro-vides relief from motor symptoms of HD, withoutA. Forebrain Weight at 12 months0.260.280.300.320.340.36Forebrain Weight (g)WTYAC128C2-/-C2-/-;YAC+YAC: F(1,39)=4.5 p=0.04C2: F(1,39)=0.43, p=0.52Interaction: F(1,39)= 0.10, p=0.750. Weight (g)WTYAC128C2-/-C2-/-;YAC+YAC: F(1,18)=5.69 p=0.023C2: F(1,18)=0.10, p=0.75Interaction: F(1,18)= 1.19, p=0.29B. Testes Weight at 12 monthsWT YAC128 Casp2-/-Casp2-/-;YAC128.75×101.0×101.3×101.5×10C. CNS Structure volumes, StereologyYAC: F(3,86)=6.04, p=0.016C2: F(3,86)=0.17, p=0.69Interaction: F(1,88)= 0.02, p=0.88Striatal Volume (μm3x1010)Figure 5 Casp2-/- mice are not protected from pathology. A-B)Fixed forebrain and testes weight in 12-month-old mice. YAC128mice show forebrain atrophy that is not rescued by the absence ofcasp2 (summary statistics for two-way ANOVA indicated). Mean="+”,horizontal bars = quartiles. N = 9 WT mice, 11 YAC128 mice, 9casp2-/- mice, 14 Casp2-/-;YAC128 mice. C) Striatal volume, asdetermined by stereology, is reduced in YAC128 mice and notaffected by caspase-2 expression. Mean="+”, horizontal bars =quartiles, isolated dots = outliers. Factorial ANOVA F/p-valuesindicated. N = 19 WT mice, 25 YAC128 mice, 21 casp2-/- mice, 22Casp2-/-;YAC128 mice.Carroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59Page 7 of 12altering neuropathological features [59]. These results, inconjunction with the current study, demonstrate thatsome aspects of pathological development are dissoci-able from tissue atrophy in HD. This dissociability ofpathological and behavioral symptoms highlights thepotential need for different approaches to treatment atdifferent stages of illness, and also for different signsand symptoms. The value of symptomatic therapy isclear; tetrabenazine, the only FDA-approved drug forHD, is used as an anti-chorea agent with no claim ofdisease modification [60].ConclusionsCasp2-/- mice are protected from a number of beha-vioral features of HD in the YAC128 mouse model. Thisprotection is seen in a number of paradigms, includingmotor learning, motor coordination, cognitive flexibility,spatial learning, sensorimotor gating and locomotorbehavior. Casp2-/- mice are not protected from patholo-gical signs of HD including testicular atrophy andregionally specific brain atrophy as assayed by stereologyand magnetic resonance imaging techniques. These find-ings lend weight to the concept that symptomaticimprovement is possible in HD even in the face of unal-tered pathology.A secondary goal of the current study was to objec-tively evaluate the usefulness of casp2 as a drug targetin HD. There are several caveats to careful interpreta-tion of these data. First, in these mice casp2 expressionis absent throughout development, rather than being20100-10-20-30Z-ScoreYAC: F(1,39)=13.143, p=0.0008C2: F(1,39)=1.117, p=0.30Interaction: F(1,39)= 1.21, p=0.28StriatumYAC: F(1,39)=3.09, p=0.086C2: F(1,39)=0.02, p=0.88Interaction: F(1,39)= 0.38, p=0.54Whole BrainYAC: F(1,39)=12.94, p=0.0009C2: F(1,39)=2.704, p=0.11Interaction: F(1,39)= 0.09, p=0.76ThalamusWTYAC128C2-/-C2-/-;YAC+WTYAC128C2-/-C2-/-;YAC+WTYAC128C2-/-C2-/-;YAC+**Striatal Volume, % Brain Volume1011121314** **Cerebellar Cortex Volume, % Brain VolumeA.B.Striatum Cerebellar CortexWTYAC128C2-/-C2-/-;YAC+WTYAC128C2-/-C2-/-;YAC+Figure 6 Casp2-/- mice are not protected from brain pathology assayed with MRI. A) Volumes for brain structures were determined byautomated segmentation of MRI images and expressed as Z-scores. YAC128 mice have smaller brains, with specific shrinkage in the basalganglia and other sub-cortical gray matter structures. Casp2-/- mice are not protected from any of these losses (summary statistics for two-wayANOVA indicated). B) To control for overall brain atrophy, structure volumes were expressed as a percentage of brain volume. Relative striatalvolume is decreased in the YAC128 mice, and not rescued in the casp2-/-;YAC128 mice (Post-hoc Bonferroni genotype comparisons “*” indicatesp < 0.05). The cerebellum is relatively preserved in the YAC128 mice, due to selective forebrain degeneration. This phenotype is not amelioratedin the casp2-/-;YAC128 mice (Post-hoc Bonferroni genotype comparisons “*” indicates p < 0.05). Mean="+”, horizontal bars = quartiles, isolateddots = outliers. N = 9 WT mice, 11 YAC128 mice, 9 casp2-/- mice, 14 Casp2-/-;YAC128 mice.Carroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59Page 8 of 12postnatally suppressed, as with a drug. Also, we havecompletely ablated, rather than reduced, the activities ofcasp2. Therefore it is difficult to extrapolate these find-ings with a genetic model to the effect of a drug, butthese beneficial effects clearly warrant additional investi-gation. The single mutation underlying HD has a largearray of effects on cells, and if the mutant HTT proteincannot be directly targeted for therapy, it may be neces-sary to target multiple aberrant processes to provideeffective therapy for HD. Based on findings here, casp2inhibition coupled with neuroprotective therapy may bean effective strategy to combat behavioral, cognitive andneuropathological features of HD.MethodsMice and breedingCaspase-2-/- mice [39] were obtained on the C57Bl/6strain, and backcrossed for at least 7 generations to theFVB/NJ strain before being used for experiments orbred to the YAC128 mouse. YAC128 (line 53) mice [38]were maintained on the FVB/NJ strain. Caspase-2+/-;YAC128+/- breeders were intercrossed and pups ofappropriate genotypes selected from the resultant pro-geny, to ensure subject mice were littermates. Theresulting mice (wild type, casp2-/-, YAC128 andcasp2-/-;YAC128) were genotyped for the rd allelewhich causes progressive retinal degeneration and blind-ness in the FVBN/J strain-all mice carried the FVBN/Jrd mutant allele. Mice were genotyped for the YAC128transgene and housed as previously described [38], andall animal experiments were conducted in accordancewith protocols approved by the University of BritishColumbia Committee on Animal Care.Quantitative Real-time PCR (QRT-PCR)Total RNA was extracted from dissected striata, frozenand stored at -80C using the RNeasy mini kit (Qiagen,74104). cDNA was prepared using 250 ng total RNAand the superscript-III first-strand synthesis kit witholigo-dT priming (Invitrogen, 11752-050). Primers usedincluded mouse casp2 forward: 5’-GAATGAACCTTATCGGGCATAACT-3’ and reverse: 5’-GATGACGGGT-GATAGTGTGAGACA-3’. Mouse actin forward: 5’-ACGGCCAGGTCATCACTATTG-3’ and reverse: 5’-CAAGAAGGAAGGCTGGAAAAGA-3’. QRTPCR wasconducted using SYBR Green PCR master mix (AppliedBiosystems, 4309155) in the ABI7500 instrument(Applied Biosystems) using the absolute quantificationstandard curve method.Behavioral assaysMice were single housed in microisolator cages with a12 h light/dark cycle. Mice were randomly coded andthe experimenter was blind to genotype. Motorcoordination and learning were examined using anaccelerating rotarod (UGO Basile, Comerio, Italy). Fortraining, naïve 4-month-old mice were given three trialsof 2 minutes on a fixed speed (18 RPM) task per day forthree days (9 trials total). The inter-trial interval was 2hours. Mice falling from the rod were returned, to amaximum of 10 falls/trial. The time to first fall and totalnumber of falls per trial were recorded. For longitudinalaccelerating rotarod assessment 4-, 8- or 12-month-oldmice were tested on a rod accelerating from 5 to 40RPM over 300 seconds. Latency to fall from the rod wasrecorded. 3 trials in 1 day were averaged to give meanperformance for each mouse at each age.Acoustic startle and prepulse inhibition (PPI) weremeasured in SR-LAB chambers (San Diego Instruments,San Diego, USA). Before use, the chambers were cali-brated using a vibrating standardization unit at 700 V(San Diego Instruments, San Diego, USA). After a 5-minute acclimatization period mice were exposed to 10050 ms startle stimuli with intensities ranging from back-ground level (70 dB) to 120 dB. Startle stimuli were pre-sented in pseudorandomized order in 10 blocks of 10trials, with a pseudorandomized 8-32 second inter-trialinterval.For the PPI task, the “pulse” was a 40 ms 120 dB sti-mulus. Eight blocks of trials were conducted-the firstand last of which were a series of 6 pulse only trials.The first block was used to determine the average startleintensity. The subsequent 6 blocks consisted of 6 trials:70 dB (background) alone ("no-pulse”), 120 dB for 40ms alone ("pulse”) and 4 pre-pulse trials with pre-pulseintensities of 72, 74, 78 and 86 dB (20 ms duration, pre-pulse interval of 100 ms). PPI was calculated as: [(First120 dB block response)-(PPI block response)]/(First 120dB block response). PPI stimuli were presented in pseu-dorandomized order with a 8-32 second inter-trialinterval.For the swimming T-maze + reversal task mice weretested in a white acrylic maze with arm dimensions 38× 14 cm [37]. The maze was filled with water and aplatform (10 × 14 cm) submerged below the water sur-face in one arm of the maze. Mice were released at thebase of the stem of the T and learned to swim to thesubmerged platform-the time to platform, total numberof arm entries and arm re-entries were recorded. Fortraining, mice received 4 trials per day for 3 days (12total trials) with a 45-minute inter-trial interval. On the5th day, the platform was switched to the opposite armof the maze and mice were required to change strategiesto find the platform in its new location. Mice received 4trials with a 45-minute inter-trial interval.For the spontaneous alternation task mice were testedin the maze described above for the swimming T-mazetask without water. A dividing wall was placed at theCarroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59Page 9 of 12intersection of the T extending into the stem of the T toencourage entry into a single goal arm. Mice wereplaced at the base of the T and allowed to choose a goalarm. A barrier was then lowered to prevent exit fromthe goal arm during a 1 minute exploration interval dur-ing which the central divider was removed. Mice werethen removed from the explored goal arm and immedi-ately placed at the base of the T and once again allowedto choose a goal arm. Mice who entered the previouslyexplored goal arm were given a score of 0 while micewho entered the novel arm were given a score of 1.For the open field exploration task mice were tested ina gray acrylic open topped box with dimensions 50 × 50× 20 cm under bright lighting. Mice were placed in thecorner of the box and allowed to explore for 10 min-utes. Trials were recorded using a ceiling mountedvideo camera and exploration tracks were analyzedusing EthoVision XT 7.0 software (Noldus).Pathology, including MRIMice were terminally anesthetized by intraperitonealinjection of 2.5% avertin, and transcardially perfusedwith 30 mL phosphate buffered saline, followed by 30mL 4% ice-cold paraformaldehyde in PBS. Testes weredissected out and weighed after fixation. Heads wereremoved and skin, lower jaws ears and cartligenous nosetip dissected away. Skulls were post-fixed in 4% parafor-maldehyde at 4°C overnight. Skulls were soaked in PBS+ 0.01% NaAzide for 5 days at room temperature withrotation. Skulls were then enhanced in PBS + 0.02%NaAzide with 2 mM Prohance (Bracco Diagnostics, DIN02229056). A 7.0 Tesla MRI scanner (Varian Inc., PaloAlto, CA) with a 6 cm inner bore diameter insert gradi-ent set was used for all MRI scans. Parameters used foranatomical MRI scans were optimized for high efficiencyand gray/white matter contrast: T2-weighted, 3D fastspin echo, with a TR of 325 ms, and TEs of 10 ms perecho for 6 echoes, four averages, field-of-view of 14 ×14 × 25 mm3 and matrix size of 432 × 432 × 780 givingan image with 0.032 mm isotropic voxels. Total imagingtime for this MRI sequence is ~12 hours [61]. Volumeswere then automatically segmented into 62 separateanatomical structures using automated image registra-tion techniques [62-64]. For graphing, volumes werestandardized and expressed as Z-scores. After imagingskulls were removed and brains weighed.StatisticsFor data with one independent variable an unpaired t-test or one-way ANOVA model was fitted and tested (asappropriate), followed by Newman-Keuls or Bonferronipost-hoc tests. Data with two or more independent vari-ables was analyzed by two-way ANOVA. To control forrepeated measurements and other sources of correlatederror data were analyzed using repeated measures two-way ANOVA or, when values were missing, by fitting alinear mixed effects (LME) model, followed by analysisof variance testing. In LME models, each mouse wasassigned a random intercept. T-tests, one- and two-wayANOVA’s were performed using Prism 4.0 software,and linear mixed effects models were done using the Rlanguage and environment including the “nlme” linearmixed effects model package [65].FundingThis work was supported by the Michael Smith Founda-tion for Health Research [ST-SGS-00835(06-1)BM,00495(06-1)BM], Canadian Institutes of Health Research[CGD-85375], Huntington Society of Canada [LandmarkGraduate Award, 2005] and CHDI Inc. [TREAT-HD].List of AbbreviationsANOVA: analysis of variance; LME: linear mixed effects model; Casp2:caspase-2; HD: Huntington’s Disease; Htt: Huntingtin (gene, PROTEIN);YAC128: Yeast artificial chromosome, 128 CAG repeats; PPI: Pre-pulseinhibition; MRI: Magnetic resonance imaging.AcknowledgementsThe authors would like to thank Drs. David Vaux, Sharad Kumar and Lien Hofor providing the caspase-2-/- mice used in this study. The authors wouldlike to thank the animal staff at the Centre for Molecular Medicine andTherapeutics, in particular Huijun (Mark) Wang and Qingwen Xia, for theirexcellent technical support. M.R.H. is a Killam University Professor and holdsa Canada Research Chair in Human Genetics and Molecular Medicine. R.M.H.holds a Canada Research Chair in Imaging. MRH is a Killam UniversityProfessor and holds a Canada Research Chair in Human Genetics andMolecular Medicine. RMH holds a Canada research chair in imagingtechnologies in human disease and preclinical models.Author details1Centre for Molecular Medicine and Therapeutics, Child and Family ResearchInstitute, Program in Neuroscience, University of British Columbia, Vancouver,V5Z 4H4, Canada. 2Centre for Molecular Medicine and Therapeutics, Childand Family Research Institute, Department of Medical Genetics, University ofBritish Columbia, Vancouver, V5Z 4H4, Canada. 3The Mouse Imaging Centre,The Hospital for Sick Children, Toronto, M5T 3H7, Canada.Authors’ contributionsJBC conceived and designed the study, performed behavioral,immunohistochemical and statistical analyses and drafted the manuscript.ALS performed behavioral experiments, statistical analyses and aided indrafting the manuscript. WNZ performed behavioral analyses. RKG, DEEparticipated in the conception and design of the study, and contributed tothe draft of the manucript. YD performed molecular experiments confirminggenotypes of mice. NB perfused the mice used in the study and managedthe animal colony. JPL and RMH conducted MRI analyses and generatedvolumetric data from brain regions, using a novel suite of tools. MRHconceived and designed the study and drafted the manuscript. All authorshave read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 10 May 2011 Accepted: 19 August 2011Published: 19 August 2011References1. Walker F: Huntington’s disease. Lancet 2007, 369(9557):218-228.Carroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59Page 10 of 122. The Huntington’s Disease Collaborative Research Group: A novel genecontaining a trinucleotide repeat that is expanded and unstable onHuntington’s disease chromosomes. Cell 1993, 72(6):971-983.3. Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, Richardson EP:Neuropathological classification of Huntington’s disease. 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Hodges A, Strand AD, Aragaki AK, Kuhn A, Sengstag T, Hughes G,Elliston LA, Hartog C, Goldstein DR, Thu D, et al: Regional and cellulargene expression changes in human Huntington’s disease brain. Hum MolGenet 2006, 15(6):965-977.doi:10.1186/1750-1326-6-59Cite this article as: Carroll et al.: Mice lacking caspase-2 are protectedfrom behavioral changes, but not pathology, in the YAC128 model ofHuntington disease. Molecular Neurodegeneration 2011 6:59.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitCarroll et al. Molecular Neurodegeneration 2011, 6:59http://www.molecularneurodegeneration.com/content/6/1/59Page 12 of 12


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