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Wild-type huntingtin ameliorates striatal neuronal atrophy but does not prevent other abnormalities in… Van Raamsdonk, Jeremy M; Pearson, Jacqueline; Murphy, Zoe; Hayden, Michael R; Leavitt, Blair R Dec 5, 2006

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ralssBioMed CentBMC NeuroscienceOpen AcceResearch articleWild-type huntingtin ameliorates striatal neuronal atrophy but does not prevent other abnormalities in the YAC128 mouse model of Huntington diseaseJeremy M Van Raamsdonk, Jacqueline Pearson, Zoe Murphy, Michael R Hayden* and Blair R LeavittAddress: Department of Medical Genetics and Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia, Vancouver, V5Z 4H4, BC, CanadaEmail: Jeremy M Van Raamsdonk - jeremy@cmmt.ubc.ca; Jacqueline Pearson - jacqui@cmmt.ubc.ca; Zoe Murphy - zmurphy@cmmt.ubc.ca; Michael R Hayden* - mrh@cmmt.ubc.ca; Blair R Leavitt - bleavitt@cmmt.ubc.ca* Corresponding author    AbstractBackground: Huntington disease (HD) is an adult onset neurodegenerative disorder caused by apolyglutamine expansion in the huntingtin (htt) protein. Htt function is essential for embryonicsurvival as well as normal function during the postnatal period. In addition to having roles intranscription and transport, recent evidence demonstrates that wild-type htt is neuroprotective invivo. To determine whether treatment with wild-type htt would be beneficial in HD, we crossedthe YAC128 mouse model of HD with mice that over-express wild-type htt (YAC18 mice) togenerate YAC128 mice that over-express wild-type htt (YAC18/128 mice).Results: YAC18/128 mice were found to express mutant htt at the same level as YAC128 miceand wild-type htt at the same level as YAC18 mice. YAC18/128 mice show no significantbehavioural improvement compared to YAC128 mice in the rotarod test of motor coordinationor in an automated open field test. In the brain, YAC18/128 mice show no significant improvementin striatal volume, striatal neuronal numbers or striatal DARPP-32 expression compared toYAC128 mice. In contrast, striatal neuronal cross-sectional area showed significant improvementin YAC18/128 mice compared to YAC128 mice.Conclusion: While the over-expression of wild-type htt results in a mild improvement in striatalneuropathology in YAC128 mice, our findings suggest that treatment with wild-type htt may notbe sufficient to ameliorate the symptoms of HD in this model.BackgroundHuntington disease (HD) is an autosomal dominant dis-order resulting from a trinucleotide CAG expansion in theHD gene. While the expression of mutant htt is sufficientlevels of wild-type htt in HD patients may also contributesignificantly to the pathogenesis of HD [4]. In support ofthis, we have recently demonstrated that the loss of wild-type htt in YAC128 mice significantly worsens motor per-Published: 05 December 2006BMC Neuroscience 2006, 7:80 doi:10.1186/1471-2202-7-80Received: 09 August 2006Accepted: 05 December 2006This article is available from: http://www.biomedcentral.com/1471-2202/7/80© 2006 Van Raamsdonk et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 9(page number not for citation purposes)to cause HD-like symptoms with normal expression levelsof wild-type htt [1-3], recent data suggests that decreasedformance, survival and striatal neuronal size [5].BMC Neuroscience 2006, 7:80 http://www.biomedcentral.com/1471-2202/7/80Htt function is essential for development as micehomozygous for the targeted inactivation of the mouseHD gene are embryonic lethal [6-8]. Furthermore,decreasing htt levels by 50% or more from birth results inneurological abnormalities [7,9,10]. The expression ofwild-type htt is also essential postnatally as mice express-ing decreased levels of wild-type htt in the forebrainbeginning at postnatal day 5 were shown to have a pro-gressive neurological phenotype [11]. Clearly, decreasedwild-type htt levels alone can lead to phenotypic abnor-malities independent of mutant htt.As the functions of wild-type htt become more clear, itseems that one of the most critical functions for htt maybe in promoting neuronal survival. In vitro studies havedemonstrated that the over-expression of wild-type httprotects cells against various insults including 3-nitropro-prionic acid, a toxin which damages the striatum and hasbeen used to model HD [12]. It has also been shown thatover-expression of wild-type htt can specifically protectagainst polyglutamine toxicity in vitro [13]. This findinghas been extended in vivo where increased expression ofwild-type htt eliminated apoptotic degeneration in thetestis caused by the expression of mutant htt [5,14]. Themechanism by which htt protects neurons may be directby sequestration of the pro-apoptotic protein HIP-1 [15]or indirectly mediated through htt's effect on expressionand transport within the cell as htt has been shown to beinvolved in both the transcription and movement of thebrain derived neurotrophic factor [16-18]. In contrast,fragments of mutant htt have been shown to also protectagainst some forms of injury (excitotoxicity) through adifferent mechanism, likely the induction of a stressresponse [19].In HD, there is evidence that striatal neurons die throughexcitotoxic mechanisms [20,21]. As such, we have previ-ously examined the ability of htt to protect neurons in vivoagainst two different excitotoxic neurotoxins. For theseexperiments, we used YAC18 mice that over-express httfrom a yeast artificial chromosome containing the entirehuman HD gene with 18 CAG repeats [22]. After intra-peritoneal injection of kainic acid, YAC18 mice showeddramatically less neuronal loss in the hippocampus com-pared to WT controls [42]. Similarly, YAC18 mice showedsignificant, htt dose-dependent protection against lesionscaused by intrastriatal injection of quinolinic acid [23].Based on the clear demonstration of in vivo protectionagainst excitotoxic cell death in YAC18 mice and the gen-eral protective effect of wild-type htt that has been demon-strated in vitro, we designed this experiment to determineif the over-expression of wild-type htt would be beneficialthe presence of at least one copy of the mutant HD gene),it is plausible that this decrease in htt's neuroprotectivefunction will make neurons more susceptible to the toxic-ity of mutant htt. A decrease in wild-type htt levels mayalso affect htt's role in transcription and transport withinthe cell [16-18,24]. In fact many of htt's assayable func-tions have been shown to be disrupted by polyglutamineexpansion [12,16,17,23] and polyglutamine expansionalso alters htt's ability to interact with its interacting pro-teins [24]. Thus, treatment of HD with wild-type htt maybe beneficial by compensating for the loss of wild-type httfunction and or through htt's general neuroprotectiveeffect.For this experiment we used the YAC128 mouse model ofHD which recapitulates many aspects of the human dis-ease [3,25]. These mice exhibit progressive motor dysfunc-tion, cognitive impairment and selectiveneurodegeneration. YAC128 mice were crossed to YAC18mice (which over-express full-length wild-type htt) to gen-erate YAC128 mice that over-express wild-type htt(YAC18/128 mice). We show that over-expression ofwild-type htt in YAC128 mice results in a mild improve-ment in striatal neuropathology but does not improvemotor dysfunction.ResultsGeneration of YAC128 mice that over-express wild-type huntingtinTo determine whether wild-type htt could protect againstmutant htt toxicity in the brain, we crossed YAC128 micewith YAC18 mice to generate YAC128 mice that over-express wild-type htt (YAC18/128 mice). In order to max-imize the amount of neuroprotection imparted by over-expression of wild-type htt, we used the highest expressingYAC18 line (line 212) available. We have previouslyshown that YAC18 line 212 mice express wild-type htt at2–3 times endogenous levels [22,23,26] and exhibit thegreatest degree of neuroprotection against quinolinic acidtoxicity of all the YAC18 lines [23]. To confirm the highexpression of htt in line 212 mice, we examined totalwild-type huntingtin in line 212 and WT mice by Westernblotting with polyclonal bkp1 antibody [27]. As previ-ously reported, we found that line 212 mice express wild-type htt at levels that are more than two times the level ofwild-type htt expression in WT mice (Fig. 1A, (WT: 0.386± 0.014 arbitrary units, YAC18: 0.942 ± 0.056 arbitraryunits, p = 0.02).After breeding YAC18 and YAC128 mice together, YAC18/128 mice were generated in equal proportions to WT,YAC18 and YAC128 mice indicating normal embryonicsurvival. Since the YAC transgenes used to generate YAC18Page 2 of 9(page number not for citation purposes)in treating HD. Since HD patients have at least a 50%genetic reduction in wild-type htt levels (resulting fromand YAC128 mice express human htt, we examined trans-genic htt expression with the human specific htt antibody,BMC Neuroscience 2006, 7:80 http://www.biomedcentral.com/1471-2202/7/80HD650 (Fig. 1B; [3]). We also examined total htt expres-sion using MAB2166 which detects both mouse andhuman htt (Fig. 1B). As expected, WT mice (non-trans-genic FVB/N) express wild-type mouse htt and no humanhtt. YAC18 mice express increased levels of wild-type httwhich are accounted for by increased human htt expres-sion. YAC128 mice express wild-type htt at the same levelas WT mice and human mutant htt. Finally, YAC18/128mice express increased levels of wild-type htt and mutanthuman htt. Importantly, the level of mutant htt expres-sion was equal between the YAC128 and YAC18/128 miceindicating that the over-expression of wild-type htt didnot down-regulate the expression of mutant htt (Fig. 1C;YAC128: 3389 ± 197 arbitrary units, YAC18/128: 3303 ±316 arbitrary units, p = 0.8; N = 3).Over-expression of wild-type huntingtin does not improve motor function in YAC128 miceTo examine the effect of wild-type htt on the motor dys-function present in the YAC128 mice, we monitoredmotor coordination on the rotarod from 2 to 12 monthsof age. While ANOVA revealed an overall effect of geno-type on rotarod performance (genotype: F(3,36) = 7.9, p <0.001, N = 8 WT, 9 YAC128, 16 YAC18, 8 YAC18/128),there were no significant differences between the YAC128mice and YAC18/128 at any time point (YAC128: 147 ±13 seconds, YAC18/128: 134 ± 14 seconds, pYAC128vsYAC18/128 = 0.5, YAC18: 164 ± 19 seconds). Both groups per-formed significantly worse than WT mice (WT: 227 ± 15seconds, p < 0.001).We have previously reported early hyperactivity and latehypoactivity in YAC128 mice compared to WT mice [3,5].As such, we compared the activity of YAC18/128 mice andYAC128 mice at 2 and 12 months of age to determine ifwild-type htt expression could ameliorate the abnormalactivity pattern present in YAC128 mice. We observed nosignificant improvement in activity in YAC18/128 micecompared to YAC128 mice (2 months – YAC128: 334 ±13 beam breaks, YAC18/128: 320 ± 12 beam breaks,pYAC128vsYAC18/128 = 0.5, YAC18: 324 ± 11 beam breaks; 12months – YAC128: 274 ± 19 beam breaks, YAC18/128:289 ± 15 beam breaks, pYAC128vsYAC18/128 = 0.5, YAC18:277 ± 11; N = 8 WT, 9 YAC128, 16 YAC18, 8 YAC18/128).Overall, increasing wild-type htt expression did not pro-vide a significant behavioural benefit to YAC128 mice.While this study was not powered to demonstrate signifi-cant differences in survival, the number of YAC18/128mice surviving to 12 months was similar to what we nor-mally observe in YAC128 mice [Table 1; 12 month sur-vival – WT males: 91%, YAC128 males: 73%, YAC18/128males: 73% (8 of 11 mice), WT females: 90%, YAC128Huntingtin expression in YAC18/128 miceFigure 1Huntingtin expression in YAC18/128 mice. YAC18 and YAC128 mice were crossed to generate YAC18/128 mice. A. To confirm high levels of htt over-expression in YAC18, line 212 mice we performed Western blots on whole brain lysates. We found that line 212 mice have 2.4 times the levels of wild-type htt as WT mice (WT: 0.386 ± 0.014 arbitrary units, YAC18: 0.942 ± 0.056 arbitrary units, p = 0.02). B. Total htt and human htt levels were assessed by Western blotting with MAB2166 and HD650 antibody respectively. The human specific HD650 antibody was used to detect htt expressed from the YAC transgenes in YAC18, YAC128 and YAC18/128 mice. Western blots performed with HD650 antibody indicate that YAC18/128 mice express both wild-type htt and mutant htt from YAC transgenes. As expected WT mice express no human htt, YAC18 mice express only wild-type human htt and YAC128 mice express only mutant human htt. Examination of total htt levels with MAB2166 antibody reveals that all mice express similar levels endog-enous wild-type htt. C. Quantification of protein expression reveals that YAC18/128 mice express mutant htt at the same level as YAC128 mice (YAC128: 3389 ± 197 arbitrary units, YAC18/128: 3303 ± 316 arbitrary units, p = 0.8). N = 3 mice per group. Error bars indicate standard error of the mean. A.U. = arbitrary units.WTYAC18YAC18/128YAC128Mutant httWT httActinBCWild-type htt Mutant httHuman Huntingtin Expression (A.U.)YAC18YAC18YAC18/128YAC18/128YAC128YAC128Mutant httWT htt (human)Human HttHD650Total HttMAB2166WT htt (mouse)AWTWTYAC18YAC18WTWTYAC18YAC18050010001500200025003000350040004500WT httActinPage 3 of 9(page number not for citation purposes)females: 93%, YAC18/128 females: 100% (4 of 4 mice)].BMC Neuroscience 2006, 7:80 http://www.biomedcentral.com/1471-2202/7/80Mice surviving to 12 months of age were sacrificed and weexamined brain and testicular weight as these have beenshown to be decreased in YAC128 mice [3,5]. In bothcases, there was a significant difference between YAC128and WT mice which was not improved by the over-expres-sion of wild-type htt (Brain weight – WT: 403 ± 7 mg,YAC18: 402 ± 4 mg, YAC128: 384 ± 4 mg, YAC18/128:382 ± 3 mg, pYAC128vsYAC18/128 = 0.6, N = 17 WT, 17YAC128, 14 YAC18, 17 YAC18/128; Testis weight – WT:164 ± 5 mg, YAC18: 152 ± 7 mg, YAC128: 142 ± 5 mg,YAC18/128: 123 ± 4 mg, pYAC128vsYAC18/128 = 0.01, N = 7WT, 8 YAC128, 10 YAC18, 10 YAC18/128). Unexpect-edly, the testicular weight in YAC18/128 mice was signif-icantly less than in YAC128 mice and there was a trendtowards decreased testicular weight in YAC18 mice com-pared to WT. This suggests the possibility that high levelsof htt expression may be detrimental in the testis.Over-expression of wild-type huntingtin results in mild improvement in striatal neuropathology in YAC128 miceYAC128 mice demonstrate clear striatal neuropathologyat 12 months of age with decreased striatal volume, stri-atal neuronal loss, striatal neuronal atrophy anddecreased striatal DARPP-32 expression [3,28]. To deter-mine whether the expression of wild-type htt could amel-iorate these striatal phenotypes we examined the striata ofYAC18/128 mice. Comparing YAC18/128 mice andYAC128 mice revealed no significant improvement in stri-atal volume (Fig. 2A; YAC128: 11.3 ± 0.2 mm3, YAC18/128: 11.6 ± 0.2 mm3, pYAC128vsYAC18/128 = 0.3, YAC18 =12.5 ± 0.3 mm3; N = 17 WT, 17 YAC128, 14 YAC18, 17YAC18/128), striatal neuronal numbers (Fig. 2B; YAC128:1.56 ± 0.03 million neurons, YAC18/128: 1.59 ± 0.03 mil-lion neurons, p = 0.4, YAC18: 1.64 ± 0.03 million neu-rons; N = 17 WT, 17 YAC128, 14 YAC18, 17 YAC18/128)or striatal DARPP-32 expression (Fig. 2C; YAC128: 828 ±28 arbitrary units, YAC18/128: 868 ± 22 arbitrary units,pYAC128vsYAC18/128 = 0.3, YAC18: 1012 ± 19 arbitrary units;mice showed significant abnormalities compared to WTmice. In contrast, increasing wild-type htt expression inYAC128 mice resulted in a significant reduction in striatalneuronal atrophy (Fig. 2D; YAC128: 96.2 ± 1.6 um2,YAC18/128: 108 ± 1.9 um2, pYAC128vsYAC18/128 < 0.001,YAC18: 110 ± 1.7 um2; N = 17 WT, 17 YAC128, 14YAC18, 17 YAC18/128) with the striatal neuronal cross-sectional area in YAC18/128 mice being almost restoredto wild-type (WT: 110 ± 3 um2, p = 0.6).DiscussionBased on recent work demonstrating a neuroprotectivefunction of wild-type htt and suggestions that loss of wild-type htt function contributes to HD pathogenesis, weinvestigated the therapeutic potential of wild-type htt inthe YAC128 mouse model of HD. We found that over-expression of wild-type htt in YAC128 mice resulted in amild improvement in striatal neuropathology with no sig-nificant improvement in behavioural phenotypes.The effect of over-expression of wild-type htt in YAC128mice is summarized in table 2. A robust finding of thisstudy was that over-expression of wild-type htt in YAC128mice restored striatal neuronal size. Similarly, we havefound that decreasing wild-type htt levels in YAC128 miceresults in decreased neuronal size [5]. Combined, theseresults suggest that wild-type htt levels influence neuronalsize and suggest that loss of wild-type htt may contributeto the striatal neuronal atrophy observed in HD. An alter-nate possibility is that striatal neuronal size is moreresponsive to mildly beneficial effects of treatments as thismeasure has been shown to exhibit the most dramaticimprovements in therapeutic trials in mouse models ofHD [29-31]. The effect of wild-type htt on neuronal sizemay be related to htt's ability to increase BDNF transcrip-tion [17] and transport [16] since BDNF promotes the sur-vival and differentiation of striatal neurons.Table 1: Effect of modulating wild-type huntingtin levels on survival in YAC128 mice.Sex Genotype Deaths Total Mice Percent SurvivingMales WT 4 46 91%YAC18/128 3 11 73%YAC128 12 48 75%YAC128-/- 11 15 27%Females WT 5 42 88%YAC18/128 0 4 100%YAC128 5 66 92%YAC128-/- 3 19 84%YAC128-/- mice express mutant htt and no wild-type htt. YAC128 mice express mutant htt and 2 copies of wild-type htt. YAC18/128 express mutant htt and increased levels of wild-type htt.Page 4 of 9(page number not for citation purposes)N = 10 WT, 12 YAC128, 10 YAC18, 8 YAC18/128) withincreased wild-type htt expression. In each case, YAC128In contrast to the significant improvement in striatal neu-ronal size, there was no significant effect of wild-type httBMC Neuroscience 2006, 7:80 http://www.biomedcentral.com/1471-2202/7/80over-expression on open field activity, striatal volume,striatal neuronal counts or striatal DARPP-32 levels inYAC128 mice, despite a trend towards improvement. Inparallel with these experiments we examined the effect ofeliminating wild-type htt expression in YAC128 mice andfound that there was a trend towards decreased striatalvolume, striatal neuronal counts and striatal DARPP-32expression which did not reach significance [5] (see Addi-tional file 1 for summary). It is possible that the 12 monthtime point chosen to assess neuropathology in theseexperiments was too late in the disease process and thatdifferences in severity were masked by a ceiling effect orIn our previous study we observed a significant increase ofboth rotarod performance and survival when wild-typehtt levels were increased in YAC128 -/- mice to wild-typelevels [5]. In this experiment, we did not observe any fur-ther improvement in either rotarod performance or sur-vival with the over-expression of wild-type htt suggestingthat there may be ceiling effect for the amount that wild-type htt can improve these outcome measures. While thisstudy did not have enough power to demonstrate a signif-icant improvement in survival, the fact that the percentageof mice surviving to 12 months was similar to YAC128mice suggests that the over-expression of wild-type hunt-Over-expression of wild-type htt results in mild improvements in striatal neuropathology in YAC128 miceFigur  2Over-expression of wild-type htt results in mild improvements in striatal neuropathology in YAC128 mice. Comparison of striatal phenotypes between YAC128 and YAC18/128 mice revealed that over-expression of wild-type htt resulted in no significant change in striatal volume (panel A: YAC128: 11.3 ± 0.2 mm2, YAC18/128: 11.6 ± 0.2 mm2, p = 0.3), striatal neuronal counts (panel B: YAC128: 1.56 ± 0.03 million neurons, YAC18/128: 1.59 ± 0.03 million neurons, p = 0.4) or striatal DARPP-32 expression (panel C: YAC128: 828 ± 28 arbitrary units, YAC18/128: 868 ± 22 arbitrary units, p = 0.3). In contrast, over-expression of wild-type htt resulted in a significant improvement in striatal neuronal cross-sectional area (panel D: YAC128: 96.2 ± 1.6 um2, YAC18/128: 108 ± 1.9 um2, p < 0.001). For each outcome measure, YAC128 mice show a signifi-cant deficit compared to WT mice. N = 17 WT, 17 YAC128, 14 YAC18, 17 YAC18/128 except for striatal DARPP-32 expres-sion where N = 10 WT, 12 YAC128, 10 YAC18, 8 YAC18/128. Error bars show standard error of the mean. ** p < 0.01. *** p < 0.001.WTYAC18YAC128YAC18/128**************ADCB1.05E+101.10E+101.15E+101.20E+101.25E+101.30E+10Striatal Volume(um^3)14000001450000150000015500001600000165000017000001750000Striatal Neuronal Counts60%70%80%90%100%110%DARPP-32 Staining Intensity(Percent of WT)80859095100105110115120Cross-Sectional Area(um^2)Page 5 of 9(page number not for citation purposes)that we have missed differences in the onset of striatalneuropathology.ingtin does not have a dramatic effect on survival inYAC128 mice. The survival deficit in the YAC128 andBMC Neuroscience 2006, 7:80 http://www.biomedcentral.com/1471-2202/7/80YAC18/128 mice were only observed in male mice thusconfirming our previous observations [5]. Unexpectedly,we found that increasing wild-type huntingtin expressionin YAC128 mice resulted in further decreases in testicularmass. Combined with a trend towards decreased testicularmass in YAC18 mice, this suggests the possibility thatexpression of htt beyond a certain threshold may result intesticular atrophy which is exacerbated by the presence ofmutant htt. It is also possible that the human origin of theover-expressed htt contributes to the testicular phenotype.Unfortunately, we are not aware of a mouse model thatover-expresses wild-type mouse htt at high levels thatwould permit testing of this hypothesis.In this study, we used the YAC128 mouse model of HDwhich transgenically expresses mutant huntingtin atapproximately 75% of endogenous levels [3]. These micehave two intact copies of the wild-type HD gene and wehave previously shown that they express wild-type htt atthe same level as WT mice [5]. As such, YAC128 miceexpress higher levels of wild-type htt protein than patientswith HD. The fact that the levels of wild-type htt arealready increased in YAC128 mice may diminish the ther-apeutic benefit we observe in YAC18/128 mice by furtherover-expressing wild-type htt. To more directly assess thetherapeutic benefit of wild-type htt in HD, one couldover-express mouse wild-type htt in a knockin mousemodel of HD to assess the effect of wild-type htt on earlydisease phenotypes in these mice.The results of this study are congruent with comparisonsof homozygous and heterozygous HD patients and HDmouse models which suggest that mutant htt has a greaterinfluence on the disease phenotype than wild-type htt.Examination of disease severity in patients homozygousand heterozygous for mutations in the HD gene havereported either no difference or that homozygous HDpatients are more severely affected [32-37]. Two inde-pendent studies have also examined the phenotype ofHD knock-in mice exhibited a more severe phenotypethan heterozygous HD knock-in mice, but the differenceswere mild [38,39]. These studies suggest that mutant httmaintains many of the critical functions of wild-type httas replacement of wild-type htt with mutant htt has onlya mild effect on phenotype. However, it has been shownthat polyglutamine expansion disrupts htt's neuroprotec-tive function [12,23], at least part of htt's role in transcrip-tion [17] and transport [16], and also affects theinteraction of htt with its interaction partners [24]. Sinceincreasing mutant htt expression alone is known to resultin a more severe phenotype in mice [40], it suggests thatthe increase in phenotypic severity between heterozygousand homozygous patients and animal models is mainlycaused by the increase in mutant htt levels which is in linewith our findings that increasing levels of wild-type htthas only a small impact on the disease phenotype.These findings are surprising given the importance of httfunction and its demonstrated neuroprotective abilities.Htt is essential for embryonic development and decreasesin htt expression alone lead to abnormal phenotypes [6-8,10,11,41]. Further, htt has been shown to protect cellsfrom death both in vitro and in the testis and in the brain[12-14,23]. While htt has been shown to specifically pro-tect against polyglutamine toxicity both in vitro [13] andin the testis [5,14], our findings here indicate a milderprotective effect against mutant htt toxicity in the brain.Given that wild-type htt exhibits protection against excito-toxic neurotoxins [23], our finding that wild-type httmildly improves striatal neuropathology in YAC128 miceis not inconsistent with excitotoxicity contributing to thepathogenesis of HD.ConclusionOverall, our results demonstrate that the over-expressionof wild-type htt in YAC128 mice results in a mildimprovement in striatal neuropathology. Based on theclear effect of htt over-expression on striatal neuronal size,Table 2: Effect of over-expression of wild-type huntingtin on HD-like phenotypes in the YAC128 mouse model of HD.Phenotype Percent Difference YAC128 Compared to WTPercent Difference YAC18/128 Compared to WTPercent Rescue with WT htt ExpressionSignificanceRotarod -36% -41% None p = 0.5Activity – 2 Months +6% +2% 74% P = 0.5Activity – 12 Months -10% -6% 47% P = 0.5Striatal Volume -9% -7% 21% p = 0.3Striatal Neuronal Counts -6% -3% 38% p = 0.4Striatal DARPP-32 Expression-18% -14% 22% p = 0.3Striatal Neuronal Size -12% -2% 87% p < 0.001Page 6 of 9(page number not for citation purposes)mice that are homozygous for a targeted expansion of theHD gene (HD knock-in mice). In both cases, homozygousit appears that htt function may be important in maintain-ing neuronal health. Despite the protective function ofBMC Neuroscience 2006, 7:80 http://www.biomedcentral.com/1471-2202/7/80wild-type htt, our results suggest that mutant htt toxicity isprimarily responsible for the pathognomic striatal neu-ropathology in HD and that treatment of HD with wild-type htt may not be sufficient to ameliorate the symptomsof the disease.MethodsMiceYAC18 and YAC128 mice that express human wild-type ormutant htt from a yeast artificial chromosome and wild-type littermates were used for these experiments [3,22].For YAC18 mice, we used the high-expressing line 212which expresses wild-type htt at 2–3 times endogenouslevels [22]. Mice were maintained on the FVB/N (CharlesRiver, Wilmington, MA) background strain. Mice weregroup housed with a normal light-dark cycle (lights on at6:00 AM, lights off at 8:00 PM) in a clean facility and givenfree access to food and water. All experiments were carriedout in accordance with protocols approved by the UBCCommittee on Animal Care and the Canadian Council onAnimal Care. The results shown are the combined resultsfrom male and female mice. We examined the data forboth sexes separately and found no differences from thecombined results.Behavioural analysisMotor coordination was assessed on an acceleratingrotarod (UGO Basile, Comerio, Italy) as previouslydescribed [5]. After training at 2 months, mice were testedbimonthly from 2 to 12 months of age with a maximumscore for each trial of 300 seconds. Open field activity wasassessed in an automated open field apparatus (San DiegoInstruments, San Diego, California). Activity was meas-ured at 2 months and at 12 months during a ten minuteopen field trial. Activity was measured as the number ofbeam crosses in the trial. Clean cages were used for eachtrial. For behavioural assessment we used 8 WT (5 F, 3 M),9 YAC128 (5 F, 4 M), 16 YAC18 (9 F, 7 M) and 8 YAC18/128 (3 F, 5 M) mice.Western blottingProtein levels were measured from homogenized wholebrain lysates from a total of 3 mice per genotype. A low-bis acrylamide gel was run with 100 μg of total protein persample for a total of 600 volt-hours. Proteins were trans-ferred to a membrane at 24 volts for 1.5 hours. Blots werethen probed with antibodies for either total htt(MAB2166, Chemicon, Temecula, California) or specifi-cally human htt [HD650, (Slow et al., 2003a)] followedby an anti-mouse, peroxidase-conjugated secondary anti-body before enhanced chemiluminescent detection. Pro-tein levels were quantified using Quantity One software(Biorad, Hercules, CA).NeuropathologyNeuropathology was carried out on 17 WT (11 F, 6 M), 17YAC128 (10 F, 7 M), 14 YAC18 (8 F, 6 M) and 17 YAC18/128 (8 F, 9 M) mice. Mice were perfused with 3% parafor-maldehyde in phosphate buffered saline. Brains and testiswere post-fixed in 3% paraformaldehyde for 24 hours andthen equilibrated with PBS prior to weighing. Subse-quently, brains were infiltrated with sucrose (25% inPBS), frozen and sectioned on a cryostat (Microm HM 500M, Richard-Allan Scientific, Calamazoo, Michigan).A series of 25 μm coronal sections spaced 200 μm apartwere stained with NeuN primary antibody (1:100 dilutionin 5% NGS, 0.1% T-X-100, PBS; Chemicon) overnight atroom temperature, biotinylated anti-mouse secondaryantibody (1:200 dilution in 1% NGS, 0.1% T-X-100, PBS)for 2 hours at room temperature and incubated in ABCreagent (ABC Elite kit, Vector) for 2 hours at room temper-ature before detection with metal-enhanced DAB solution(Pierce, Rockford, Illinois).Striatal volume was determined using Stereoinvestigatorsoftware (Microbrightfield, Williston, Virginia). Briefly,the perimeter of the striatum was traced using a 2.5Xobjective in each section of the coronal series and the soft-ware calculated the volume of the entire structure. Subse-quently, neuronal profiles in a 25 μm × 25 μm countingframe were counted with a 550 μm by 550 μm grid for allgrids the fell within the outlined areas. The counts werethen extrapolated to estimate the total number of neuronsin the striatum. To determine neuronal cross-sectionalareas, a single matched section from each animal wasstained with an Alexa488-conjugated NeuN antibody(Chemicon). Mounted sections were analyzed using Ster-eoinvestigator to outline the perimeter of all clearlydefined neurons within a 550 μm × 550 μm grid of 25 μm× 25 μm counting frames with the 100X objective. Onaverage 32 neurons per mouse were assessed for a total ofmore than 450 neurons per genotype.For measurement of DARPP-32 expression, sections werestained with rabbit anti-DARPP-32 antibody (ChemiconAB 1656, 1:1000). After 3 washes with PBS, sections wereincubated in Cy3-conjugated goat anti-rabbit antibody(1:500; Jackson ImmunoResearch Inc., West Grove, Penn-sylvania). Pictures of mounted sections were taken usingMetaMorph Imaging System (Molecular Devices, Down-ingtown, Pennsylvania) and the intensity of the fluores-cent stain within the striatum was measured. 10 WT, 12YAC128, 10 YAC18 and 8 YAC18/128 were used for thisanalysis.Immunohistochemistry for markers of neuronal healthPage 7 of 9(page number not for citation purposes)was performed as described above using the followingantibodies: synaptophysin (1:100; BD Transduction Lab-BMC Neuroscience 2006, 7:80 http://www.biomedcentral.com/1471-2202/7/80oratories, Mississauga, Ontario), calbindin (1:2000; pro-vided by Ken Bainbridge, University of British Columbia,Canada), EM48 (1:500; provided by Xiao-Jiang Li, EmoryUniversity, U.S.A) and 8-hydroxy-2-deoxyguanosine(1:500; Japan Institute for the Control of Aging, FukuroiCity, Japan).Statistical analysisOverall effects of genotype were determined by one wayANOVA. Repeated measures ANOVA analysis was used foranalysis of differences in rotarod performance. The signif-icance of differences between YAC128 and YAC18/128mice was determined by either the Tukey post-hoc test fol-lowing ANOVA or a student's t-test.Authors' contributionsJVR conceived the study, designed the experiment, com-pleted the neuropathological assessment of the miceexcept for the striatal DARPP-32 levels, carried out theWestern blot for human htt, prepared the figures for themanuscript and wrote the manuscript. JP carried out thebehavioural analysis including rotarod and open fieldtesting. ZM completed the western blot for total htt. MRHand BRL contributed to the conception and design of theexperiment and were also involved in editing the manu-script. All of the authors have read and approved the finalmanuscript.Additional materialAcknowledgementsWe would like to thank Daniel Rogers, Nagat Bissada, Kuljeet Vaid and Ge Lu for their technical assistance. This work was supported by grants from the Huntington's Disease Society of America, the High Q Foundation and the Huntington Society of Canada. JVR has been supported by the Canadian Institutes of Health Research, the Michael Smith Foundation for Health Research and the Huntington Society of Canada. BRL and MRH are sup-ported by the Canadian Institutes of Health Research, the Huntington Soci-ety of Canada, the Hereditary Disease Foundation and the Canadian Genetic Diseases Network. MRH is a Killam University Professor and holds a Canada Research Chair in Human Genetics.References1. Hodgson JG, Agopyan N, Gutekunst CA, Leavitt BR, LePiane F, Singa-raja R, et al.: A YAC mouse model for Huntington's disease2. Reddy PH, Williams M, Charles V, Garrett L, Pike-Buchanan L, Whet-sell WO Jr, et al.: Behavioural abnormalities and selective neu-ronal loss in HD transgenic mice expressing mutated full-length HD cDNA.  Nat Genet 1998, 20:198-202.3. 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