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Human Molecular Genetics Advance Access originally published online on November 8, 2005
Human Molecular Genetics 2005 14(24):3823-3835; doi:10.1093/hmg/ddi407
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Selective degeneration and nuclear localization of mutant huntingtin in the YAC128 mouse model of Huntington disease

Jeremy M. Van Raamsdonk1,2, Zoe Murphy1,2, Elizabeth J. Slow1,2, Blair R. Leavitt1,2 and Michael R. Hayden1,2,*

1Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada V6T 1Z3 and 2Centre for Molecular Medicine and Therapeutics, British Columbia Research Institute for Children's and Women's Health, Vancouver, BC, Canada V5Z 4H4

* To whom correspondence should be addressed at: Centre for Molecular Medicine and Therapeutics, British Columbia Research Institute for Children's and Women's Health, 980 West 28th Avenue, Vancouver, BC, Canada, V5Z 4H4. Email: mrh{at}cmmt.ubc.ca

Received August 13, 2005; Accepted October 21, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Huntington disease (HD) is an adult onset neurodegenerative disorder that predominantly affects the striatum and cortex despite ubiquitous expression of mutant huntingtin (htt). Here we demonstrate that this pattern of selective degeneration is present in the YAC128 mouse model of HD. At 12 months, YAC128 mice show significant atrophy in the striatum, globus pallidus and cortex with relative sparing of the hippocampus and cerebellum (striatum: –10.4%, P<0.001; globus pallidus: –10.8%, P=0.04; cortex: –8.6%, P=0.001; hippocampus: +0.3%, P=0.9; cerebellum: +2.9%, P=0.6). Similarly, neuronal loss at this age is present in the striatum (–9.1%, P<0.001) and cortex of YAC128 mice (–8.3%, P=0.02) but is not detected in the hippocampus (+1.5%, P=0.72). Mutant htt expression levels are similar throughout the brain and fail to explain the selective neuronal degeneration. In contrast, nuclear detection of mutant htt occurs earliest and to the greatest extent in the striatum—the region most affected in HD. The appearance of EM48-reactive mutant htt in the nucleus in the striatum at 2 months coincides with the onset of behavioral abnormalities in YAC128 mice. In contrast to YAC128 mice, the R6/1 mouse model of HD, which expresses exon 1 of mutant htt, exhibits non-selective, widespread atrophy along with non-selective nuclear detection of mutant htt at 10 months of age. Our findings suggest that selective nuclear localization of mutant htt may contribute to the selective degeneration in HD and that appropriately regulated expression of full-length mutant htt in YAC128 mice results in a pattern of degeneration remarkably similar to human HD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Huntington disease (HD) is an adult onset neurodegenerative disorder that is characterized by motor dysfunction, cognitive impairment and psychiatric disturbances. HD is caused by a trinucleotide CAG repeat expansion in the HD gene which is translated into an expanded polyglutamine stretch in the huntingtin (htt) protein. HD is part of a family of at least nine disorders caused by a CAG expansion in the affected gene (1Go). In each of these disorders, only a specific population of neurons degenerate despite widespread expression of the mutant protein. Furthermore, in each polyglutamine disorder, different populations of neurons are affected. Although it is believed that the protein context of the polyglutamine stretch determines which neurons are affected, the mechanism of selective damage in these diseases is poorly understood.

The neuropathology of HD is characterized by atrophy and neuronal loss in the striatum. Striatal volume loss in early stages of HD has been measured as 53% compared with controls using magnetic resonance imaging (2Go,3Go). Although the magnitude of atrophy was less than that in the striatum, significant volumes losses of 41 and 23% were reported in the globus pallidus and cortex of HD brains, respectively (2Go,4Go). In contrast, the hippocampus and the cerebellum are relatively spared. HD patients show a 9% decrease in hippocampal volume and no change at all in cerebellar volume compared with unaffected control subjects (2Go). Neuronal loss in HD patients is also selective approaching 90% loss in the striatum and 40% loss in the cortex (5Go). In the striatum, GABAergic medium-sized spiny neurons are most affected (6Go,7Go), whereas large neurons in layers III, V and VI are lost in the cortex (60Go,61Go).

Examination of htt expression in the brain has revealed no correlation between htt expression and disease pathology. Both in situ hybridization and western blotting has revealed that htt expression is highest in the cerebellum, a region that is relatively unaffected in HD (8Go–12Go). Similarly, htt expression is high in the hippocampus where neuropathology is slight (8Go,9Go,12Go). In contrast, htt expression in the cortex is similar to hippocampal htt expression but the cortex shows significant atrophy and neuronal loss (8Go,12Go). Finally, htt expression in the striatum is less than that in the cerebellum, hippocampus and cortex, but this is the region of greatest degeneration (8Go,11Go,12Go). Thus, regional differences in htt expression are observed within the brain but do not account for the selective degeneration in HD.

We previously generated the YAC128 mouse model to study the pathogenesis of HD (13Go). These mice express mutant htt under its endogenous regulatory elements from a yeast artificial chromosome (YAC) transgene. YAC128 mice exhibit progressive motor dysfunction, cognitive impairment, impaired lifespan and striatal and cortical degeneration thereby recapitulating many aspects of the human disease (13Go–15Go). However, the selectivity of atrophy and cell loss has not been examined in these mice, nor has the regional expression of mutant htt.

This work was designed to assess the regional specificity of cerebral damage in the YAC128 mouse model of HD. To assess the mechanism behind selective degeneration in HD, we examined the relationship between mutant htt toxicity and expression within the brain, where atrophy was used as a measure of toxicity. We also assessed the potential contribution of nuclear localization of mutant htt to the region-specific pathology in YAC128 brain. Importantly, the use of the YAC128 mouse model permitted us to compare the time course of nuclear detection of mutant htt with the development of behavioral abnormalities and neuropathology.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Selective degeneration in the brain of YAC128 mice
HD is characterized by selective degeneration in the basal ganglia and cortex with relative sparing of other regions including the hippocampus and cerebellum. We have previously demonstrated striatal and cortical atrophy in the YAC128 mouse model of HD (13Go). Here we sought to determine whether the atrophy in YAC128 mice was limited to regions affected in HD. To this end, we examined the volume of the striatum, cortex, globus pallidus, hippocampus and cerebellum in a cohort of 12-month-old YAC128 mice and WT littermates.

Overall brain weight was decreased 4% in YAC128 mice compared with wild-type mice (Fig. 1A; WT: 411±6 mg, YAC128: 394±4 mg, P=0.05). The volume of the striatum in YAC128 mice was 10.4% less than that in WT mice, suggesting that the small global changes in brain weight may be explained by larger changes in select regions of the brain (Fig. 1B; ANOVA (genotype): F(2, 68)=4.6, P=0.01; striatum—WT: 12.1±0.1 mm3, YAC128: 10.8±0.2 mm3, P<0.001). We found a similar 10.8% decrease in the volume of the globus pallidus in YAC128 mice compared with WT mice (Fig. 1B; WT: 1.60±0.06 mm3, YAC128: 1.43±0.04 mm3, P=0.04). Cortical volume was also decreased in YAC128 mice compared with WT mice but to a lesser extent than the striatum or globus pallidus (Fig. 1B; –8.6%, WT: 16.5±0.02 mm3, YAC128: 15.1±0.03 mm3, P=0.001). In contrast, the volumes of the hippocampus and cerebellum were unaffected in YAC128 mice (Fig. 1B; hippocampus—WT: 0.0355±0.001 mm3, YAC128: 0.0356±0.001 mm3, P=0.9; cerebellum—WT: 44.3±0.2 mm3, YAC128: 45.6±0.1 mm3, P=0.6). Furthermore, examination of cerebellar volume in 18-month-old YAC128 mice and WT controls revealed that even at this late age there is no cerebellar atrophy in YAC128 mice (WT: 53.1±0.02 mm3, YAC128: 55.0±0.1 mm3, P=0.5). Thus, the overall decrease in brain weight in YAC128 mice is caused by degeneration in select regions of the brain and the regional specificity in YAC128 mice is similar to that seen in patients with HD.



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  		Figure 1. YAC128 mouse model of HD exhibits selective neurodegeneration which is not explained by differences in expression of mutant huntingtin. At 12 months of age, YAC128 mice (N=8) and wild-type littermate controls (N=8) were perfused. Brain weight was measured prior to stereological assessment of regional volumes within the brain. (A) Brain weight was significantly decreased in YAC128 mice compared with WT controls (WT: 411±6 mg, YAC128: 394±4 mg, P=0.05). (B) The overall decrease in brain weight resulted from atrophy of specific regions of the brain. The striatum, globus pallidus and cortex all showed significant atrophy in YAC128 mice compared with WT mice (striatum—WT: 12.1±0.1 mm3, YAC128: 10.8±0.2 mm3, P<0.001; globus pallidus—WT: 1.60±0.06 mm3, YAC128: 1.43±0.04 mm3, P=0.04; cortex—WT: 16.5±0.02 mm3, YAC128: 15.1±0.03 mm3, P=0.001). In contrast, the hippocampus and cerebellum were unaffected in YAC128 mice (hippocampus—WT: 0.0355±0.001 mm3, YAC128: 0.0356±0.001 mm3, P=0.9; cerebellum—WT: 44.3±0.2 mm3, YAC128: 45.6±0.1 mm3, P=0.6). (C) Examination of mutant htt expression throughout the brain revealed that mutant htt is more highly expressed in cerebellum than in other brain regions. (D) Quantification of htt expression levels indicates that, despite exhibiting the most severe neuropathology, the expression levels of mutant htt in the striatum are not greater than in other brain regions (cerebellum: 130±18 arbitrary units, cortex: 100±10 arbitrary units, striatum: 84±9 arbitrary units, hippocampus: 108±8 arbitrary units). Thus, the regional specificity of mutant htt toxicity is not a result of increased mutant htt expression in the affected regions of the brain. Abbreviations: Str, striatum; Ctx, cortex, Hip, hippocampus; Cer, cerebellum. Error bars indicate SEM. *P<0.05, **P<0.01.

 
To determine whether the pattern of neuronal loss in HD is also recapitulated in the YAC128 mouse model, we assessed neuronal numbers within the striatum, cortex and hippocampus by stereology. At 12 months of age, the number of striatal neurons was significantly decreased in YAC128 mice compared with WT mice (WT: 1.90±0.05 million neurons, YAC128: 1.73±0.03 million neurons, –9.1%, P=0.01). Counting DARPP-32-positive neurons within the striatum revealed a similar degree of cell loss suggesting that it is the medium-sized spiny neurons that are affected (J.M. Van Raamsdonk, unpublished). Examination of the region of cortex above the striatum revealed that there is also neuronal loss in the cortex of YAC128 mice (WT: 2.25±0.05 million neurons, YAC128: 2.06±0.05 million neurons, –8.3%, P=0.02). In contrast, YAC128 and WT mice showed no difference in the estimated number of neurons in the cellular layer of the hippocampus (WT: 1.42±0.05 million neurons, YAC128: 1.44±0.03 million neurons, +1.5%, P=0.72). Thus, as with regional volumes, neuronal loss in the YAC128 mouse model is selective and follows a similar pattern of degeneration as in HD patients.

Selective toxicity of mutant huntingtin in YAC128 mice is not due to higher levels of mutant huntingtin expression
In mouse models of HD, higher levels of mutant htt expression consistently result in greater toxicity as demonstrated by a more severe phenotype (16Go–18Go). However, in HD, the regions of the brain with the highest levels of htt expression are not the most severely affected in the disease (11Go,12Go). To determine whether the level of mutant and wild-type htt expression in each region of the brain could explain the observed selectivity in atrophy and neuronal loss in YAC128 mice, we examined htt expression in the striatum, cortex, hippocampus and cerebellum using the htt-specific MAB2166 antibody and controlling for protein loading with anti-ß-tubulin antibody. A two-way ANOVA revealed significant differences in htt expression between brain regions (F(3, 25)=9.2, P<0.001) but no difference between the pattern of expression between mutant and wild-type htt (F(1, 29)=0.6, P=0.4). HTT expression was highest in the cerebellum, approximately equal in the hippocampus and cortex and least in the striatum (Fig. 1C and D; mutant htt—cerebellum: 130±18 arbitrary units, cortex: 100±10 arbitrary units, striatum: 84±9 arbitrary units, hippocampus: 108±8 arbitrary units; wild-type htt—cerebellum: 124±12 arbitrary units, cortex: 100±7 arbitrary units, striatum: 80±6 arbitrary units, hippocampus: 99±5 arbitrary units). Thus, as in patients with HD, the regional specificity of toxicity in the brains of YAC128 mice is not explained by higher expression levels of mutant htt.

Selective nuclear localization of mutant huntingtin in YAC128 mice
Although wild-type huntingtin is primarily a cytoplasmic protein (19Go), polyglutamine expansion results in increased nuclear localization of htt and it has been suggested that mutant htt may be more toxic in the nucleus than the cytoplasm (20Go–22Go). To determine whether differences in the nuclear localization of mutant htt could account for the selective degeneration in YAC128 mice, we examined the localization of mutant htt in YAC128 mice by staining with EM48 antibody (23Go,24Go). This antibody was raised against the first 256 amino acids of htt and has a high affinity for fragments of mutant htt, in the nucleus especially those that have aggregated. Under the experimental conditions we used for immunohistochemistry, EM48 staining in WT mice is not observed at any age and we only detect mutant htt in the nucleus. Initially, we examined 3- and 12-month-old YAC128 mice to determine if nuclear localization of mutant htt by EM48 staining was greater in affected regions of the brain and whether nuclear EM48 staining increased with the progression of the disease.

At 3 months of age, the highest levels of mutant htt in the nucleus were detected in the striatum (Fig. 2A), with visibly darker EM48 staining in the lateral striatum compared with the medial striatum (Fig. 2E and F). Mutant htt was also detected in the nucleus in parts of the cortex (Fig. 2B and G), hippocampus (Fig. 2C, D and H) and cerebellum. In the cortex, layer IV shows the most nuclear staining of mutant htt at this age (Fig. 2B). In the hippocampus, nuclear EM48 staining was darkest in the dentate gyrus, detectable in the CA3 region and absent from the CA1 region (Fig. 2C, D and H). Quantification of the intensity of nuclear EM48 staining revealed that the nuclear localization of EM48-reactive mutant htt was significantly greater in the lateral striatum than in any other region (Fig. 2Q; region: F(5, 17)=23.8, P<0.001; lateral striatum versus any other region, P<0.001).



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  		Figure 2. Selective nuclear localization of mutant htt in the YAC128 mouse model of HD. A series of coronal sections from 3- and 12-month-old YAC128 mice were stained with EM48 for visualization of mutant htt localization within the cell. At 3 months of age, the striatum (A) shows more nuclear localization of mutant htt than the cortex (B) or hippocampus (C). Under high power, striatal neurons (E, F) show strong EM48 staining indicating high amounts of mutant htt in the nucleus which is greatest in the lateral striatum. Layer IV of the cortex (G), the CA3 regions of the hippocampus (H) and the dentate gyrus (D) show some nuclear staining of mutant htt at this age. At 12 months of age, the striatum (I, M), cortex (J, N) and hippocampus (K, L, O, P) all show more nuclear EM48 staining than at 3 months. The striatum is still more intensely stained than the cortex or hippocampus. Within the cortex, layers II and III show the greatest degree of nuclear detection of mutant htt. In the hippocampus, the dentate gyrus (L) and CA3 region (O) show greater EM48 staining than the CA1 region (P). Photographs A–C and I–K were taken using the 10x objective (scale bar=500 µm). Photographs D–H and L–P were taken using the 100x objective (scale bar=50 µm). (Q) Quantification of EM48 staining intensity as a measure of the nuclear localization of mutant htt confirmed that differences between regions and between 3 and 12 months were significant (3 months region: F(5, 17)=23.8, P<0.001; 12 months region: F(9,30)=15.5, P<0.001; age: F(1,35)=86.7, P<0.001). Abbreviations: Str, striatum; Ctx, cortex, Hip, hippocampus, DG, dentate gyrus; Cer, cerebellum; Lat, lateral; Med, medial. Error bars indicate SEM.

 
At 12 months of age, nuclear staining of mutant htt was increased in all regions of the brain (age: F(1, 35)=86.7, P<0.001). At 3 months of age, the striatum shows more nuclear staining of mutant htt than is present in other regions of the brain (Fig. 2I and M). In the cortex, layers II and III show the most intense nuclear EM48 staining (Fig. 2J and N). In the hippocampus, the dentate gyrus still shows the greatest nuclear staining of mutant htt, with more nuclear EM48 staining in the CA3 region than CA1 (Fig. 2K, L, O and P). Again, these differences were confirmed by quantification of staining intensity [Fig. 2Q; region: F(9, 30)=15.5, P<0.001; significant differences were found between lateral striatum and medial striatum (P=0.01), cortex layers IV–VI (P<0.001), the CA1 region of the hippocampus (P<0.001), the dentate gyrus (P=0.01) and the cerebellum (P<0.001)]. As has been previously reported (24Go), subcellular fractionation and western blotting confirmed the presence of both full-length and fragments of mutant htt in the nucleus in a pattern similar to WT mice at ages when EM48 staining was positive (Supplementary Material, Fig. S1).

At both time points, the highest levels of mutant htt in the nucleus are detected in the striatum despite the fact that mutant htt expression is lower in the striatum than that in other regions of the brain. Overall, the nuclear localization of EM48-reactive mutant htt occurs earlier and to a greater extent in the striatum than in other regions of the brain and is associated with this region being most affected in HD. Although nuclear detection of mutant htt increases as the disease progresses, even at 12 months of age we did not observe neuronal intranuclear inclusions (NIIs) in any region of the brain.

Non-selective atrophy and nuclear localization of mutant huntingtin in the R6/1 mouse model of HD
The R6 mouse models of HD express exon 1 of human mutant htt with approximately 130 glutamines under 1 kb of the proximal regulatory elements of the HD gene (16Go) Mutant htt expression in the brain of these mice follows a similar pattern to YAC128 mice with greatest htt expression in the cerebellum and lower levels of htt in the striatum (16Go). Cerebral atrophy and aggregation of mutant htt are uniform throughout the brain of R6/2 mice (16Go,25Go). R6/1 mice express lower levels of mutant htt than R6/2 mice with a smaller CAG expansion (116 CAG repeats) and accordingly have a more prolonged disease progression. To determine if there is a correlation between nuclear localization of mutant huntingtin and toxicity in R6/1 mice, we examined regional volumes and nuclear EM48 staining in the brains of 10-month R6/1 mice and WT controls.

At 10 months of age, brain weight was decreased 20% in R6/1 mice compared with WT controls (Fig. 3A; WT: 390±10 mg, R6/1: 320±15 mg, P=0.02). Examination of regional volumes revealed equivalent decreases in all of the regions analyzed including regions unaffected in HD (Fig. 3B). In R6/1 mice, striatal volume was decreased 27% compared with WT mice (Fig. 3B; ANOVA (genotype): F(1, 38)=66.6, P<0.001; WT: 14.2±0.1 mm3, R6/1: 10.3±0.1 mm3, P<0.001), the volume of the globus pallidus was decreased 23% compared with WT mice (WT: 1.21±0.02 mm3, R6/1: 0.93±0.02 µm3, P=0.09), cortical volume was decreased 25% compared with WT mice (Fig. 3B; WT: 23.2±0.2 mm3, R6/1: 17.2±0.2 mm3, P<0.001), the volume of the hippocampus was decreased 24% compared with WT mice (Fig. 3B; WT: 5.78±0.25 mm3, R6/1: 4.41±0.15 mm3, P=0.009) and the volume of the cerebellum was decreased 16% compared with WT mice (WT: 48.8±2.3 mm3, R6/1: 40.9±1.6 mm3, P=0.05). In contrast to the selective degeneration observed in the YAC128 mouse model, R6/1 mice exhibit diffuse and non-selective atrophy across the brain. We did not assess the selectivity of neuronal loss in R6/1 mice, as cell loss has never been reported in these animals (16Go).



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  		Figure 3. Widespread atrophy in the brain of R6/1 mice. Brain weight and regional atrophy within the brain was analyzed at 10 months of age. (A) Brain weight in R6/1 mice was decreased 20% compared with WT mice (WT: 390±10 mg, R6/1: 320±15 mg, P<0.05). (B) R6/1 mice showed uniform decreases in regional volumes across the different regions of the brain (striatum: –27%, WT: 14.2±0.1 mm3, R6/1: 10.3±0.1 mm3, P<0.001; globus pallidus: 23%, WT: 1.21±0.02 mm3, R6/1: 0.93±0.02 µm3, P=0.09; cortex: 25%, WT: 23.2±0.2 µm3, R6/1: 17.2±0.2 µm3, P<0.001; hippocampus: 24%, WT: 5.78±0.25 mm3, R6/1: 4.41±0.15 mm3, P=0.009; cerebellum: 16%, WT: 48.8±2.3 mm3, R6/1: 40.9±1.6 mm3, P=0.05). Thus, unlike the YAC128 mouse model, R6/1 mice show non-selective cerebral atrophy. Abbreviations: Str, striatum; GP, globus pallidus; Ctx, cortex, Hip, hippocampus; Cer, cerebellum. N=2 WT, 2 R6/1. Error bars indicate SEM. *P<0.05, **P<0.01, ***P<0.001.

 
Examination of EM48 staining in 10-month-old R6/1 mice revealed extensive nuclear localization of mutant htt throughout the brain. Nuclear staining of mutant htt was uniform throughout the striatum (Fig. 4A) and showed no layer specificity in the cortex (Fig. 4B) or region specificity in the hippocampus (Fig. 4C). Unlike YAC128 mice, there were also widespread inclusions in all regions in the brain, both nuclear and cytoplasmic (Fig. 4D and E–H). Quantification of EM48 staining intensity across the different regions of the brain showed little evidence of selectivity (Fig. 4I; region: F(9, 10)=0.7, P=0.7). In fact, the EM48 staining intensity of mutant htt in the nucleus was equal to or greater than that in the striatum in every other region in the brain. Thus, the nuclear localization of EM48-reactive mutant htt and atrophy in 10-month-old R6/1 mice are both non-selective.



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  		Figure 4. Widespread nuclear staining of mutant huntingtin and inclusions in the brain of R6/1 mice. Nuclear localization of mutant htt was assessed by immunohistochemistry using EM48 antibody. (A–C) Low-power images of EM48 staining taken using the 10x objective reveal extensive nuclear localization of mutant htt in the striatum, cortex and hippocampus (scale bar=500 µm). (D–H) High-power images taken using the 100x objective show clear nuclear localization of mutant htt and the occurrence of nuclear inclusions in R6/1 brains (scale bar=50 µm). (I) Quantification of EM48 staining intensity across the different regions of the brain showed no significant differences between the different regions of the brain (region: F(9, 10)=0.7, P=0.7). Thus, the nuclear localization of mutant htt in R6/1 brain appears to be non-selective. Abbreviations: Str, striatum; Ctx, cortex, Hip, hippocampus, DG, dentate gyrus; Cer, cerebellum; Lat, lateral; Med, medial. N=2 WT, 2 R6/1. Error bars indicate SEM.

 
Appearance of EM48-reactive mutant huntingtin in the striatum occurs at the onset of motor abnormalities
Next, we examined the time course of the nuclear detection of mutant htt in YAC128 mice to determine when it occurred relative to the development of motor and cognitive abnormalities. Cognitive deficits and hyperkinesia develop at 2 months of age in YAC128 mice (26Go). Accordingly, we stained coronal sections from 1-, 2- and 3-month-old YAC128 mice with EM48 to detect mutant htt in the nucleus. At 1 month of age, mutant htt was not detected in the nucleus in any region of the brain (Fig. 5). At 2 months of age, mutant htt was found in the nucleus in the striatum but not in any other brain region (Fig. 5), whereas at 3 months of age low levels of mutant htt are found in the nucleus in striatum, cortex and hippocampus (Fig. 5). Thus, the nuclear localization of EM48-reactive mutant htt in the striatum coincides with the onset of motor and cognitive abnormalities in YAC128 mice at 2 months of age.



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  		Figure 5. Time course of nuclear localization of EM48-reactive mutant huntingtin in YAC128 mice. At 1 month of age, mutant htt is not detected in the nucleus in any region of the brain. At 2 months of age, mutant htt is detected in the nucleus only in the striatum indicating selective nuclear localization. At 3 months of age, mutant htt is also detected in the nucleus in some subregions of the cortex and hippocampus but EM48 staining is much greater in the striatum. For comparison, nuclear localization of mutant htt was examined in shortstop mice that express an N-terminal fragment of mutant htt from the same YAC as YAC128 mice. At 2 months of age, shortstop mice show extensive nuclear staining of mutant htt in all regions of the brain. Photographs were taken with 10x objective (scale bar=500 µm).

 
Selective nuclear localization of mutant huntingtin is not mediated by HD gene promoter
To determine whether the selective detection of mutant htt in the nucleus is mediated by the HD gene promoter, we examined nuclear localization of mutant htt in mice expressing a fragment of mutant htt from the complete HD gene promoter. Shortstop mice express exons 1 and 2 of htt with approximately 120 polyglutamines from the same YAC as YAC128 mice (27Go). Accordingly, the mutant htt fragment in shortstop mice is expressed in the same pattern as full-length mutant htt in YAC128 mice. The level of mutant htt expression in shortstop mice is slightly greater than that in YAC128 mice and more than 3-fold greater than that in R6/1 mice.

Examination of coronal brain sections from 2-month-old shortstop mice reveals extensive EM48 staining in all regions of the brain (Fig. 5). Importantly, there are no significant differences between the intensity of EM48 staining in the different regions of the brain (region: F(7, 16)=1.4, P=0.3). Also, staining within each region was uniform and, at this age, no macroaggregates were detected. This is in contrast to YAC128 mice where mutant htt is only found in the nucleus at 2 months of age and suggests that the selective nuclear localization of mutant htt in YAC128 mice does not result from the presence of the complete HD gene promoter. The fact that shortstop mice do not exhibit striatal atrophy (27Go) despite the extensive nuclear localization of the mutant htt fragment suggests that not all fragments of mutant htt are toxic in the nucleus.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
In this paper, we demonstrate selective neurodegeneration in the YAC128 mouse model of HD which is similar to the pattern of degeneration observed in HD patients. As in the human disease, mutant htt toxicity does not correlate with mutant htt expression. Nuclear localization of EM48-reactive mutant htt occurs earlier and to a greater extent in the region most affected by the disease and may partially explain the regional specificity of mutant htt toxicity. In contrast, we show that both atrophy and nuclear staining of mutant htt are non-selective in R6/1 mice. Furthermore, the widespread nuclear detection of a mutant htt fragment in shortstop mice indicates that the nuclear localization of this fragment of mutant htt is not sufficient to cause toxicity and raises the question as to whether only specific fragments of mutant htt are toxic in the nucleus.

Selective cellular toxicity in YAC128 mice is similar to the characteristic pattern of neuronal damage in human HD
Examination of regional volumes within the brain of 12-month-old YAC128 mice revealed volume losses of 10.4, 10.8 and 8.6% in the striatum, globus pallidus and cortex, respectively, with no change in hippocampal or cerebellar volume. As brain weight was only decreased 4% in YAC128 mice compared with WT mice, it is apparent that degeneration in select regions is responsible for the overall brain atrophy. The pattern of selective degeneration in YAC128 mice is similar to human HD where volume losses of approximately 50, 40 and 25% are reported in the striatum (caudate and putamen), globus pallidus and cortex, respectively, with little or no volume changes in the hippocampus and cerebellum (2Go,4Go,28Go,29Go). In the human disease, brain weight in advanced cases can be reduced by up to 30% (30Go). This is less than the volume changes observed in the most affected regions of the brain again, indicating that the global brain atrophy is caused by degeneration of selective regions. We also show here that cell loss of 9.1 and 8.3% is present in the striatum and cortex, respectively, of YAC128 mice whereas neuronal numbers within the hippocampus are spared. Similarly, in human HD, the greatest cell loss has been reported in the striatum and motor cortex (5Go).

Although the pattern of selective degeneration is similar between YAC128 mice and human HD patients, the magnitude of change is much greater in humans. This may stem from marked differences in the age of onset (~39 years on average in humans versus 2–3 months in YAC128 mice) and or the duration of disease progression (~15 years in humans versus 9 months in mice at 12-month time point). Perhaps for this reason or because of other differences between species, animal models of HD have repeatedly demonstrated that mutant htt with CAG repeat sizes comparable to those seen in adult onset HD result in mild phenotypes, if any, in mice (31Go–34Go). In the YAC128 mouse model, we have increased the expression of mutant htt and used a CAG length that would produce a severe case of juvenile onset HD in humans in order to observe a clear phenotype within a limited time frame (13Go). It is also possible that if mice were permitted to live to the end stages of the disease that the magnitude of atrophy and cell loss would be more similar to that observed in human HD.

Selective degeneration in YAC128 mice does not result from differences in mutant htt expression but may be caused by selective nuclear localization of mutant htt
To determine if differences in mutant htt expression between regions of the brain could account for the selective degeneration observed in the YAC128 mice, we examined mutant htt expression within the brain. Mutant htt expression in YAC128 mice was found to be highest in cerebellum, moderate in the hippocampus and cortex and lowest in the striatum of the regions we examined. Thus it seems that the regions with highest htt expression are those with the greatest neuronal density (35Go) which follows from the fact that htt is more highly expressed in neurons than glia (9Go). Similarly, in studies of mutant htt RNA or protein levels in humans, it has been found that htt expression is higher in the cerebellum, hippocampus and cortex than in the striatum (8Go–12Go). Thus, the pattern of mutant htt expression is similar in the YAC128 mouse model and human HD and in neither case explains the selective degeneration that occurs.

In contrast to mutant htt expression, nuclear localization of mutant htt was greatest in the striatum—the region most affected in HD. Mutant htt was not detected in the nucleus at 1 month of age but was detected in the nucleus in the striatum at 2 months of age. Although detection of mutant htt in the nucleus at the time of onset of motor and cognitive abnormalities (13Go,36Go) does not demonstrate causality, it does suggest the possibility that accumulation of EM48-reactive mutant htt in the nucleus contributes to the neuronal dysfunction that manifests as behavioral abnormalities. In support of this, mutant htt is first detected in the nucleus in the R6/2 mouse model at 4.5 weeks of age (37Go), just prior to the onset of motor abnormalities at 5 weeks (38Go). A correlation between mutant htt in the nucleus and toxicity is also supported by the fact that nuclear detection of mutant htt increases as the disease progresses in YAC128 mice.

In addition to the regional specificity of the EM48 staining, we also observed selective nuclear localization of mutant htt within the striatum. The lateral striatum was found to have visually more intense EM48 staining than the medial striatum. Combined with our previous observation of increased neurodegeneration in the lateral striatum compared with the medial striatum in YAC models of HD (31Go), this supports the contribution of nuclear localization of mutant htt to the selective degeneration in HD. Interestingly, graded neuropathology is also observed in the striatum of HD patients where the medial striatum is more affected than the lateral striatum (39Go). This difference between the YAC128 mice and HD patients may stem from anatomical differences between the mouse and human brain.

Wild-type htt is primarily a cytoplasmic protein which is also detected in the nucleus (40Go,41Go). htt contains a nuclear export signal (NES) in its carboxy-terminus which can be separated from the amino-terminus of the protein when htt is cleaved (42Go). Thus, it is thought that wild-type htt functions in the cytoplasm and nucleus and can shuttle between them. Mutant htt is also found in both the cytoplasm and nucleus but shows increased nuclear localization compared with wild-type htt, especially N-terminal fragments of the mutant protein (20Go,40Go). This may result from decreased interaction of mutant htt with the nuclear pore translocated promoter region (Tpr) which is thought to transport htt out of the nucleus (43Go).

Many in vitro experiments have demonstrated that nuclear localization of mutant htt is important for its toxicity (20Go–22Go). Conversely, the demonstration that a mutant htt fragment linked to a nuclear localization signal causes the same phenotype as the fragment alone in mice suggests that mutant htt exerts its toxic effects in the nucleus (17Go). This is supported by the acceleration of behavioral onset in mice expressing HPRT with 150 CAG repeats when an nuclear localization signal (NLS) signal is added and a later onset when an nuclear export signal (NES) is added (44Go). In addition, preventing the nuclear localization of the mutant androgen receptor protein in spinal and bulbar muscular atrophy resulted in a complete amelioration of the disease symptoms, suggesting that nuclear localization is essential for the pathogenesis of this related polyglutamine disorder (45Go,46Go).

The adverse effect of mutant htt in the nucleus on cell function and survival may result from alterations in gene expression as mutant htt has been found to interact with a number of transcription factors within the nucleus (20Go,47Go,48Go). It has also been demonstrated that the presence of mutant htt in the nucleus increases expression of caspase 1 and induces activation of caspase 3 and release of cytochrome c thereby leading towards apoptosis (49Go). Irrespective of the mechanism of mutant htt toxicity within the nucleus, these findings provide a possible mechanism by which increased nuclear localization of mutant htt leads to increased toxicity as we observed in the striatum of YAC128 mice.

Early nuclear detection of mutant htt in the striatum has been previously reported in other mouse models of HD (24Go,50Go–55Go). In a knock-in model of HD with 72–80 CAG repeats, nuclear staining of mutant htt with EM48 was reported to be specific to the striatum with very little staining in the cortex, hippocampus or cerebellum at 24 months of age (56Go). This is similar to what we observed in the YAC128 mice at 2 months of age. In a knock-in model of HD with 140 CAG repeats, nuclear localization of EM48-reactive mutant htt appeared greatest in the striatum, less in the cortex and hippocampus and least in the cerebellum (51Go). In these mice, mutant htt is first observed in the nucleus in the striatum at 1 month, followed by the cortex and hippocampus at 2 months and the cerebellum at 4 months. This pattern is similar to what we observe in YAC128 mice. However, in these mice it was not possible to examine the relationship between nuclear localization of htt and cerebral damage because of the mild neuropathology in these mice.

Our work both supports and extends these previous studies. By using a full-length mouse model of HD which exhibits selective degeneration in the brain, we examined both the nuclear localization of mutant htt and mutant htt toxicity in the same mouse model. Accordingly, we are able to correlate early selective nuclear localization of mutant htt in the striatum with subsequent selective degeneration. Additionally, our work shows that the timing of nuclear localization of EM48-reactive mutant htt in the striatum coincides with the onset of behavioral abnormalities, preceding quantifiable striatal atrophy by 7 months.

Selective nuclear localization of mutant huntingtin may require expression of full-length protein and is not mediated by HD gene promoter
Staining with EM48 in mouse models expressing N-terminal fragments of mutant htt revealed the presence of nuclear inclusions throughout the brain (17Go,37Go). In these mice, aggregation and volume loss do not appear to be selective (16Go,17Go). Given the accumulating evidence that nuclear inclusions are not harmful (22Go,27Go,57Go,58Go), we examined the relationship between regional cerebral atrophy and nuclear localization of mutant htt in the R6/1 mouse model. In contrast to the YAC128 mouse model, we show that atrophy and nuclear staining of mutant htt are non-selective in the R6/1 mouse model of HD. The degree of volume loss was uniform in the striatum, cortex, globus pallidus, hippocampus and cerebellum. This is unlike the human disease where the hippocampus and cerebellum are relatively unaffected. However, the pattern of volume loss follows the pattern of nuclear localization of mutant htt which also occurs to a similar extent in all regions of the brain. The lack of selectivity in R6/1 mice may result from the absence of important regulatory elements controlling the expression of mutant htt or the expression of only an N-terminal fragment of htt.

To differentiate between these two possibilities, we utilized shortstop mice which express a mutant htt fragment under the same regulatory region as YAC128 mice. These mice showed extensive nuclear staining of mutant htt at 2 months of age which was not regionally selective. As both shortstop and YAC128 mice express htt from the same promoter, this suggests that the selective nuclear detection of mutant htt in YAC128 mice does not result from the presence of the complete HD gene promoter. It is plausible that the small fragments of mutant htt expressed in R6/1 mice and shortstop mice facilitate the entry of mutant htt into the nucleus in these models, but the full-length protein in YAC128 mice requires cleavage or an alternative transport mechanism to cross the nuclear envelope. We have previously reported cleavage of mutant htt in the cortex of YAC mice as early as 2 months of age (59Go), indicating that htt cleavage precedes detection of mutant htt in the nucleus. The absence of neuropathology in shortstop mice despite extensive nuclear localization of mutant htt indicates that, in addition to subcellular localization, other factors, such as the size of mutant htt fragment and mutant htt expression levels, are critical in determining toxicity.

In this paper, we confirm previous reports that both full-length and cleavage fragments of mutant htt are present in the nucleus (24Go). Although it is not certain which form of mutant htt is detected by EM48, recent data from our laboratory suggests that a specific fragment of mutant htt mediates toxicity in YAC128 mice (Graham et al., submitted for publication). This suggests that cleavage of mutant htt may be a critical step in the pathogenesis of HD and although full-length mutant htt is present in the nucleus, it is most likely a specific mutant htt fragment (or fragments) that mediates toxicity in the nucleus.

CONCLUSIONS
Overall, we show that the YAC128 mouse model of HD exhibits selective atrophy and neuronal loss in a pattern similar to that observed in human HD. Although the striatum does not show increased mutant htt expression, nuclear localization of EM48-reactive mutant htt occurs earlier and to a greater extent in the striatum, suggesting the possibility that selective nuclear localization of mutant htt may contribute to the selective neurodegeneration in HD. Furthermore, the appearance of EM48-reactive mutant htt in the nucleus coincides with the onset of behavioral abnormalities suggesting that this may contribute to neuronal dysfunction (see time course of symptoms in YAC128 mice in Supplemental Material, Fig. S2). A comparison to shortstop and R6/1 mice reveals non-selective nuclear detection of mutant htt in these mouse models and suggests that the expression of full-length mutant htt may be important in modeling the selective neuropathology in HD (Fig. 6). Finally, the absence of neuropathology in shortstop mice (27Go), despite extensive nuclear staining of mutant htt, indicates that nuclear localization of this specific fragment of mutant htt alone is not sufficient to cause pathology.



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  		Figure 6. Summary of findings in YAC128, shortstop and R6/1 mice. YAC128 mice show selective nuclear localization of mutant htt and selective atrophy in the brain. Shortstop mice show non-selective nuclear localization of mutant htt and exhibit no atrophy in any region of the brain. In contrast, R6/1 mice also show non-selective nuclear localization of mutant htt and non-selective atrophy in all regions of the brain. Combination of these results suggests that selective nuclear localization of mutant htt is not mediated by the HD gene promoter and may require the expression of full-length mutant htt. The fact that shortstop mice show extensive nuclear localization of mutant htt and no toxicity suggests that only specific fragments of mutant htt are toxic.

 

   MATERIALS AND METHODS
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mice
Transgenic HD mice expressing human htt with approximately 128 CAG repeats (YAC128) and their non-transgenic littermates (wild-type) were used for these experiments (13Go). Shortstop mice that express exons 1 and 2 of mutant htt from a YAC transgene were also examined (27Go). Mice were maintained on the FVB/N (Charles River, Wilmington, MA, USA) background strain. Mice were group housed with a normal light–dark cycle (lights on at 6:00 A.M. and lights off at 8:00 P.M.) in a clean facility and given free access to food and water. We also obtained brains from R6/1 mice that are transgenic for exon 1 of mutant htt (16Go). All experiments were carried out in accordance with the University of British Columbia animal care committee.

Detection of huntingtin expression
Western blots were performed on tissue samples frozen immediately after mice were asphyxiated with carbon dioxide. Protein lysates containing 80–100 µg of total protein were separated on either a 7.5% acrylamide gel or an 8% low-bis acrylamide gel for allelic separation of htt. Following transfer to a membrane at 24 V for 1.5 h, htt protein was detected using the htt-specific MAB2166 antibody (1:2000, Chemicon) followed by incubation with a peroxidase-conjugated, anti-mouse secondary antibody (1:5000) and enhanced chemiluminescent detection. Protein levels were quantified by measuring band density with Quantity One software (BioRad).

Regional volume loss and neuronal loss in brain
Mice were injected with heparin, terminally anesthetized by intraperitoneal injection of 2.5% avertin and perfused with 3% paraformaldehyde in phosphate-buffered saline (PBS). Brains were post-fixed in 3% paraformaldehyde for 24 h and then equilibrated with PBS. Subsequently, brains were infiltrated with sucrose (25% in PBS) and frozen on dry ice before mounting with Tissue-TEK O.C.T. compound (Sakura). Twenty-five-micrometer coronal sections were cut on a cryostat (Microm HM 500M) and collected in PBS.

A series of 25 µm coronal sections spaced 200 µm apart were stained with NeuN primary antibody (1:100 dilution in 5% NGS, 0.1% T-X-100, PBS, Ab; Chemicon) overnight at room temperature, biotinylated anti-mouse secondary antibody (1:200 dilution in 1% NGS, 0.1% T-X-100, PBS) for 2 h at room temperature and incubated in ABC reagent (ABC Elite kit, Vector) for 2 h at room temperature before detection with metal-enhanced DAB solution (Pierce).

Regional volumes were determined using Stereoinvestigator software (Microbrightfield). Briefly, the perimeter of each region was traced using a 2.5x objective in each section of the coronal series and the software calculated the volume of the entire structure. Striatal volume was measured from the start of the striatum to the start of the hippocampus. The volume of the entire globus pallidus was measured. Cortical volume was measured from the point where the corpus callosum crosses to the start of the hippocampus. Hippocampal volume was measured from the start of the hippocampus to the point where the CA3 region thickens and descends. The volume of the cerebellum was measured from a coronal series of unstained sections collected directly onto glass slides.

Striatal and cortical neuron counts were determined using Stereoinvestigator software. Using the same contours traced for volume measurement, neuronal profiles were counted in a 25 µmx25 µm counting frame with a 550 µmx550 µm grid size. These counts were then extrapolated to estimate the total number of neurons in the striatum. Hippocampal neuronal numbers were estimated by measuring the volume of the hippocampal cellular layer and dividing by the average volume of hippocampal neurons.

Nuclear localization of mutant huntingtin
Nuclear localization of mutant htt was examined in a series of coronal sections throughout the brain. Sections were stained with EM48 antibody as above at a concentration of 1:500 followed by an anti-rabbit secondary antibody, ABC amplification and DAB detection. Photographs were taken using Metamorph software with an exposure time of either 2 or 63 ms for pictures taken with the 10x or 100x objective, respectively. For quantification of EM48 staining intensity, pictures from each region were taken using the 100x objective. Subsequently, 20 individual neurons in each picture were outlined and the average intensity was measured by the Metamorph software. The intensity for each region was averaged and subtracted from the average intensity of a background region containing no cells. The presence of mutant htt in the nucleus was confirmed by subcellular fractionation of cortical lysates from WT and YAC128 mice using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA) followed by western blotting with bkp1 antibody.

Statistical analysis
Data are given as the mean±standard error of the mean (SEM). The regional volumes between YAC128 and WT mice were compared using a two-way ANOVA (genotypexregion) to demonstrate a significant effect of genotype. Differences between genotypes were assessed using a Student's t-test. Differences in htt expression levels and EM48 staining intensity were assessed by one-way ANOVA.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We would like to thank Dr Xiao-Jiang Li for providing EM48 antibody. This work was supported by grants from the Canadian Institutes of Health Research, the Huntington's Disease Society of America and the High Q Foundation. J.V.R. is supported by the Huntington Society of Canada. B.R.L. and M.R.H. are supported by the Canadian Institutes of Health Research, the Huntington Society of Canada, the Hereditary Disease Foundation and the Canadian Genetic Diseases Network. M.R.H. is a Killam University Professor and holds a Canada Research Chair in Human Genetics.

Conflict of Interest statement. None declared.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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