Human Molecular Genetics, 2002, Vol. 11, No. 17 1939-1951
© 2002 Oxford University Press
Increased huntingtin protein length reduces the number of polyglutamine-induced gene expression changes in mouse models of Huntington's disease
1Center for Molecular Medicine and Therapeutics, Department of Medical Genetics, Children's and Women's Hospital, University of British Columbia, Vancouver, British Columbia, Canada, V5H 4H4, 2Center for Aging, Genetics and Neurodegeneration, Massachusetts General Hospital, Charlestown, MA 02129-4404, USA, 3Fred Hutchinson Cancer Research Center, Seattle, WA 98195, USA, 4Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA and 5Department of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA
Received March 12, 2002; Accepted June 7, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Both transcriptional dysregulation and proteolysis of mutant huntingtin (htt) are postulated to be important components of Huntington's disease (HD) pathogenesis. In previous studies, we demonstrated that transgenic mice that express short mutant htt fragments containing 171 or fewer N-terminal residues (R6/2 and N171-82Q mice) recapitulate many of the mRNA changes observed in human HD brain. To examine whether htt protein length influences the ability of its expanded polyglutamine domain to alter gene expression, we conducted mRNA profiling analyses of mice that express an extended N-terminal fragment (HD46, HD100; 964 amino acids) or full-length (YAC72; 3144 amino acids) mutant htt transprotein. Oligonucleotide microarray analyses of HD46 and YAC72 mice identified fewer differentially expressed mRNAs than were seen in transgenic mice expressing short N-terminal mutant htt fragments. Histologic analyses also detected limited changes in these mice (small decreases in adenosine A2a receptor mRNA and dopamine D2 receptor binding in HD100 animals; small increases in dopamine D1 receptor binding in HD46 and HD100 mice). Neither HD46 nor YAC72 mice exhibited altered mRNA levels similar to those observed previously in R6/2 mice, N171-82Q mice or human HD patients. These findings suggest that htt protein length influences the ability of an expanded polyglutamine domain to alter gene expression. Furthermore, our findings suggest that short N-terminal fragments of mutant htt might be responsible for the gene expression alterations observed in human HD brain.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Huntington's disease (HD) gene (IT15) encodes a 3144-amino-acid protein of unknown function (huntingtin, htt). In HD, expansion of the polyglutamine [poly(Q)] tract, which begins 18 amino acid residues from the N terminus, leads to cellular dysfunction and neuronal death (1). Neurodegeneration in HD is cell-type-specific and medium spiny neurons of the striatum are most severely vulnerable (2–4). Much evidence supports the hypothesis that proteolysis of htt is an important step in HD pathogenesis (5–9), including the detection of htt fragments in HD patient brains (10–13). htt Can be cleaved into N-terminal fragments by caspases at particular sites, and caspase cleavage products may be hydrolyzed further by calpains and the 26S proteosome (12–16). In HD brain tissues, both wild-type and expanded forms of htt are proteolytically processed (11–13). Since caspase-cleaved wild-type htt fragments are also detectable in control brain tissues, proteolysis could be a normal part of htt function or turnover (12,13,17). In addition, caspase-cleaved htt has been observed in brain tissues from the YAC72 HD mouse model before onset of neurodegeneration (13).
Cleavage of mutant htt is hypothesized to enhance the toxicity of its expanded poly(Q) tract. In support of this theory, shorter N-terminal fragments of mutant htt have the greatest effects on cell viability in cell culture systems (6,18), and generally produce the most severe phenotypes in transgenic mice (19,20). Full-length htt is mostly cytosolic, but short N-terminal fragments of mutant htt appear to become concentrated over time in neuronal nuclei (6,21–25). Recently, full-length htt or N-terminal htt fragments have been shown to interact with transcriptional regulators such as N-CoR, p53, cAMP response element-binding protein (CREB)-binding protein (CBP), mSin3a, CA150, p300, p300/CBP-associated factor (P/CAF), C-terminal-binding protein transcriptional co-repressor (CtBP), TAFII130 and Sp1 (26–33). These proteins can affect transcription directly as transactivators (or repressors) or indirectly through histone acetylation. Thus, proteolytic processing of htt could promote the entry of short poly(Q)-containing htt fragments into the nucleus, where they could more readily interact with transcriptional regulators and perturb gene expression.
Alterations in gene expression have been investigated to gain insight into the pathogenic mechanisms of HD. Decreased levels of neurotransmitter receptor and preproenkephalin mRNAs have been observed in HD patient postmortem striatal tissues, with neuropathology ranging from grade 0 (no macroscopic atrophy) to grade 3 (significant atrophy) (34,35). Also, decreases in dopamine receptors can be observed in vivo by positron emission tomography early in the disease process (36). Some of the mRNA alterations seen in human HD brain are recapitulated in R6/2 transgenic mice that express a short exon 1-encoded N-terminal fragment of htt (37–39). N-terminal fragments of htt can also produce gene expression changes in cell culture systems (40–42) and yeast (43). Since these alterations in mRNA levels can be detected on an mRNA per cell basis, they appear to arise from cellular changes in gene expression rather than from gross neuronal loss.
Recently, we identified a set of gene expression changes common to five mouse models of poly(Q) disease (44). These shared mRNA changes suggested to us that some poly(Q)-related changes in gene expression could occur independently of protein context. Surprisingly, these changes were not observed in corresponding brain samples from transgenic mice expressing full-length mutant htt. This finding suggested that length of the htt protein could modulate the ability of its expanded poly(Q) domain to alter gene expression. Here, we investigated whether htt protein context can reduce the effects of expanded poly(Q) on gene expression by assessing mRNA levels in transgenic mice that express the N-terminal one-third or full-length version of mutant htt.
The lines of mice employed in the current study have been described previously. HD46 mice (lines 14 and 17) and HD100 mice (line 49) express the N-terminal one-third (964 amino acids) of htt with either 46 or 100 glutamine residues (45). HD46 and HD100 mice display htt accumulation and aggregates in the striatum and cerebral cortex. Striatal neurons in HD100 mice demonstrate impaired responsiveness to cortical stimulation. Behaviorally, these animals have impaired rotarod performance, exhibit clasping and demonstrate gait abnormalities. We also analyzed mice YAC72 transgenic mouse (line 2511) that expresses full-length htt with a 72 residue poly(Q) tract (22). By 6–7 months of age, YAC72 mice display behavioral hyperactivity and enhanced long-term potentiation in the hippocampus. By 9 months of age, decreased levels of brain-derived neurotrophic factor (BDNF) are observed in the striatum, cortex and hippocampus of YAC72 mice (46). By 12 months of age, YAC72 mice display striatal neuronal pathology, including nuclear htt accumulation, hyperchromasia, chromatin condensation, mitochondrial swelling and neurodegeneration. In addition, striatal medium spiny neurons in YAC72 mice of ages ranging from 6 months to 2 years display enhanced responses to NMDA (47,48). The relative sizes of the htt transproteins investigated in this study are shown in Figure 1.
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Here, we report that brain tissues of HD46, HD100 and YAC72 mice exhibit fewer gene expression changes than corresponding tissues of mice that express shorter N-terminal mutant htt fragments. Further, mRNAs that are consistently decreased in multiple poly(Q) disease mouse models or human HD patients were generally present at normal levels in HD46, HD100 and YAC72 mice. Thus, htt protein context appears to have a dramatic influence on the poly(Q)-related disruption of mRNA expression. Taken together with previous studies, our results suggest that the effects of mutant htt on gene expression in the brain occur after its proteolysis to shorter N-terminal fragments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Microarray analysis of striata from HD46 and YAC72 mice
To explore altered gene expression globally, striatal mRNAs of YAC72 and HD46 transgenic mice were analyzed using oligonucleotide microarrays. These studies were performed within the Hereditary Disease Array Group (HDAG), which utilized a single core microarray facility and single microarray platform. This facilitated comparisons between different poly(Q) disease models by standardizing experimental design, quality control and data analysis (50).
RNA samples were prepared from striatal tissues of 12-month-old YAC72 and 10- to 12-month-old HD46 transgenic mice (with Mild or Severe symptoms as defined in Materials and Methods) in addition to age- and strain-matched wild-type controls. Microarray data were analyzed using custom-designed statistical algorithms that incorporate error modeling (51). For each experiment, we determined the number of differentially expressed mRNAs that passed the P<0.001 threshold. Fifteen to twenty mRNAs passed this threshold in analyses of HD46 versus control and YAC72 versus control striata (Table 1). These numbers of mRNAs are only slightly higher than the predicted number of false-positive determinations (i.e. 1:1000, as defined by the P-value criterion). With the use of the same statistical algorithm, 147 mRNAs were differentially detected in 12-week-old R6/2 striatum versus control. These data clearly demonstrate that striatal gene expression changes are significantly more subtle in YAC72 and HD46 mice than in R6/2 mice—at least in mice of these particular ages.
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Differentially detected mRNAs meeting the P<0.001 threshold in 12-month-old YAC72 striatum are listed in Figure 2A. There was no overlap between the mRNAs that were differentially detected in YAC72 striatum by microarray (P<0.001) and those differentially detected in 12-week-old R6/2 mice or 4-month-old N171-82Q HD mice, which express amino acid residues 1–171 of mutant htt with an 82-residue poly(Q) tract (23). Microarray analyses of striata from symptomatic R6/2 and N171-82Q mice were performed within the HDAG and are reported in full elsewhere (39,44,49).
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To increase the statistical power of our microarray analysis, we increased the number of replicate HD46 samples to six per group. Lists of mRNAs with altered levels (P<0.001) in HD46 Mild and Severe mice are provided in supplemental data (www.neumetrix.info). HD46 microarray data for mRNAs corresponding to those identified in analyses of YAC72 striata are also shown in Figure 2A. The only mRNAs that meet the P<0.001 threshold in analyses of striatum from YAC72 and either Mild or Severe HD46 mice are N10 (increased in YAC72 and HD46 Mild, decreased in R6/2 and N171-82Q) and cholecystokinin (decreased in YAC72, trend toward decrease in R6/2, increased in HD46 Mild and N171-82Q). As described below, the confirmation rate of the N=6 microarray analysis was low, suggesting that the inability to detect mRNA changes was a true reflection of the HD46 molecular phenotype and not due to limited statistical power.
Cerebellar gene expression changes in YAC72 mice
The results above indicated that mRNA changes were subtle in striata of mice expressing a large N-terminal portion or full-length form of mutant htt. To determine whether this finding was unique to the striatum, we performed microarray analyses of cerebella from 12-month-old YAC72 and control mice. For reference, we drew comparisons with cerebellar data gathered from N171-82Q HD mice. The cerebellar N171-82Q microarray data set was gathered with similar power (N=4–5 per group) (44). The YAC72 cerebellum displayed fewer mRNA alterations at any statistical threshold than the N171-82Q cerebellum (e.g. at P<0.001, YAC72=84 genes; N171=165 genes, predicted false-positives=13 for each). Furthermore, the magnitudes of mRNA change (average fold change) were much smaller in YAC72 than in N171-82Q or R6/2 mice. Named mRNAs that showed altered levels in YAC72 cerebellum compared with control are summarized in Figure 2B. Comparison of mRNA alterations in YAC72 cerebellum with other poly(Q) disease models revealed that most of the transcripts that were differentially detected in YAC72 mice did not show similarly altered levels in cerebella of R6/2, N171-82Q and 12-month-old At-65Q (atrophin-1 transgenic) (52) mice (data from ref. 44). Conversely, the expanded poly(Q)-mediated cerebellar mRNA alterations observed in the R6/2 and N171-82Q mice (44) were not observed in the cerebellum of YAC72 mice. Thus, the mRNA alterations present in YAC72 cerebellum appear to be both more restricted and distinct from those caused by expanded poly(Q) within the short htt transproteins of R6/2 and N171-82Q mice.
Confirmation of microarray data in YAC72 and HD46 mice
Selected mRNAs that were differentially detected in microarray analyses were evaluated by northern analysis or quantitative RT–PCR using the original RNA samples assayed by microarray. All confirmation data are summarized in Table 2. In YAC72 striatal samples, RT–PCR confirmed increased levels of N10 and Krox 20 mRNAs (P<0.02; Fig. 3A). A putative decrease in pentraxin-1 mRNA in striatum could not be confirmed. In YAC72 cerebellar samples, RT–PCR confirmed a statistically significant decrease in cytochrome c synthase mRNA levels (P<0.0001), while levels of brain-derived neurotrophic factor (BDNF) and cathepsin mRNAs were not significantly different between YAC72 and control mice (Fig. 3B).
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In striatal samples from HD46 mice, candidate mRNA changes could not be confirmed for creatine kinase, dopamine D2 receptor or neuronal protein NP25 (Fig. 3C). Overall, the low confirmation rate observed for the HD46 and YAC72 microarray data is consistent with the high predicted false-discovery rate.
Anatomic analyses of candidate mRNAs in HD46, HD100 and YAC72 mice
As stated above, gene expression changes that are observed in the HD R6/2 and N171-82Q mouse models were not detected in HD46, HD100 or YAC72 mice. Because this finding was unexpected, we wished to rule out possible causes of false-negative results (e.g. regional heterogeneity within striatum). Therefore, we extended our studies to include radioligand-binding assays and in situ hybridization histochemistry, both of which retain anatomic integrity. We focused on neurotransmitter receptors, adenylyl cyclase, DARPP-32, the 11S proteasome activator 
subunit (PA28) and preproenkephalin because these were shown previously to be decreased in multiple mouse and/or human HD tissues (34–39). HD46, HD100 and YAC72 mice were studied at ages when neurologic phenotypes were detectable. No significant changes in the levels of DARPP-32, PA28, preproenkephalin, NMDA receptor or adenosine A2a receptor mRNAs could be detected in the striatum of 12-month-old YAC72 mice as compared with age-matched wild-type controls (Fig. 4A and Table 3). Likewise, no significant changes in binding to adenylyl cyclase, group I or group II metabotropic glutamate receptors or muscarinic acetylcholine receptors were detected in the striatum of YAC72 mice (Fig. 4B and Table 3).
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We similarly analyzed brain sections of HD46 and HD100 mice at 10–13 months of age. We observed no significant differences in preproenkephalin mRNA levels within the striatum of HD46 or HD100 mice as compared with wild-type controls (Fig. 4C and Table 3). The only abnormalities observed in these animals that recapitulated findings in other HD mice and HD patients were decreased levels of adenosine A2a receptor mRNA and decreased dopamine D2 receptor binding, which were selectively observed in the HD100 but not the HD46 mice. Unexpectedly, we detected increased mRNA levels for dopamine D1 receptor in the striatum of HD46 and HD100 mice—a finding opposite to that observed in HD patients and mice expressing shorter N-terminal fragments of mutant htt. These observations demonstrate that the gene expression changes in HD46 and HD100 mice are lower in magnitude and scope as compared with the R6/2 N-terminal htt transgenic mice. Importantly, however, the observed decreases in adenosine and dopamine receptors indicate that 10- to 13-month-old HD100 mice may be starting to manifest the transcriptional dysregulation evidenced in HD brain. Overall, the absence of mRNA or neurotransmitter receptor binding changes in YAC72 brain is highly consistent with microarray findings, supporting the hypothesis that htt protein length in excess of 171 N-terminal residues limits the ability of expanded poly(Q) to alter gene expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Overall, HD46, HD100 and YAC72 mice expressing extended N-terminal or full-length forms of mutant htt display subtler and qualitatively different gene expression changes than mouse models that express shorter N-terminal fragments of mutant htt. Messenger RNA (or corresponding protein) levels for specific molecular markers such as neurotransmitter receptors were analyzed in HD46, HD100 and YAC72 mice using standard assays established previously in our laboratories (37–39). Except for modest decreases in dopamine D2 receptor binding and A2a receptor mRNA in HD100 mice, even the most dramatic gene expression changes observed in other HD transgenic mice were absent in mice expressing the relatively longer and full-length mutant htt transproteins. Lower levels of transcriptional dysregulation in HD46 and YAC72 mice were also detected by microarray profiling. Although it is possible that gene expression changes may be present in a small subset of cells within HD46 and YAC72 brain tissues that eluded detection by the methods employed, it is clear that the majority of cells do not show large shifts in mRNA levels.
We examined HD46, HD100 and YAC72 mice at ages when poly(Q) disease signs were already present (22,45,46,48). Thus, our results show that large-scale perturbations in mRNA levels are not required for the development of the neurologic phenotypes observed in these HD animal models. A reasonable interpretation of this finding is that large-scale gene expression changes in the striatum are not an integral component of the early stages in HD pathogenesis. Data from young R6/2 mice (<6 weeks of age) also support this conclusion (44). In addition, our data suggest that the behavioral, electrophysiologic and neuropathologic abnormalities identified in YAC72, HD46 and HD100 mice represent earlier disease stages which precede overt transcriptional dysregulation. Since most neurotransmitter receptor decreases identified in grade 0 HD postmortem brain were not present in HD46, HD100 and YAC72 mice, the stages of disease progression in these animals (up to 
12 months of age) may best model HD brain with neuropathology less severe than grade 0.
Based on this projected timeline, we predict that more pronounced gene expression changes will take place in the striatum of HD46, HD100 and YAC72 mice at ages older than those studied here (limitations of a mouse's lifespan notwithstanding). The mild decreases in dopamine D2 and adenosine A2a receptors that already present in 10- to 13-month-old HD100 mice are consistent with a possible time and poly(Q) length-dependent pathogenic progression, since these receptor subclasses were described to comprise some of the earliest receptor decreases in grade 0 postmortem HD brain (53).
Multiple reports have demonstrated that R6/2 mice recapitulate many mRNA changes seen in HD patients (37–39). Here, we conclude that increased length of the htt transproteins leads to decreased transcriptional dysregulation in HD46, HD100 and YAC72 mice as compared with R6/2 and N171 mice. Taken together, the results suggest that short N-terminal fragments of mutant htt may be the true culprit underlying the abnormal gene expression observed in HD patient brain. Given the limited huntingtin context sequences in the N-terminal htt fragments of the R6/2 and N171 transproteins, it is plausible that some of these gene expression changes result from generalized poly(Q) effects (44,49).
There are several possible explanations for the apparent inverse relationship between htt protein length and transcriptional dysregulation. Mutant htt is believed to cause gene expression changes through direct interactions with transcriptional regulators, and most of the transcription factors known to associate with htt involve domains present in the exon 1-encoded N-terminal htt fragment (26–29,33). While both full-length and truncated huntingtin proteins can bind to these interacting proteins, other unique properties of the N-terminal fragment may explain its potency in dysregulating gene expression. First, short N-terminal htt fragments have an enhanced nuclear distribution (6). Although, nuclear htt immunoreactivity has been documented in HD46, HD100 (45) and YAC72 mice (22), the nuclear concentrations reached in these cases are likely to be lower than those found in R6/2 mice that express solely the short N-terminal fragment. Enhanced nuclear concentration of htt would increase its access to transcription factors at or near their site of action, possibly even while bound to DNA. Second, shorter fragments of mutant htt have a higher rate of aggregation in vitro (54) and in vivo (21). Thus, short N-terminal htt fragments may also have an enhanced capacity to entrap transcriptional regulators into aggregates.
Conversely, one may consider protective effects afforded by htt protein context. The simplest would invoke steric hindrance; C-terminal regions of htt could ‘hide’ the toxic poly(Q) stretch. Alternatively, a protective function or interaction requiring the unique C-terminal sequences of htt could be lost after the protein is cleaved. Full-length wild-type htt has been shown to protect against poly(Q)-mediated toxicity in mice (55), and cell culture data have indicated that domains with some protective function reside even within the N-terminal 548 amino acid residues of htt (56). Possibly, loss of a protective effect provided by htt sequences C-terminal to amino acid 171 could, thus, result in secondary changes that lead to altered gene expression.
Transcription-related abnormalities may be one of several parallel pathways involved in htt-mediated cellular dysfunction. The results of the present study suggest that short N-terminal fragments of mutant htt are important molecular intermediates for HD-related disruptions in gene expression. Further, our results suggest that large-scale alterations in gene expression occur later in disease progression than other abnormalities that have already been identified in mouse models expressing ‘large’ or full-length versions of mutant htt. Based on our conclusions, we predict that inhibition of htt proteolysis into shorter more toxic fragments might slow the later stages of progression and represent a potential HD therapeutic strategy. This approach would only be viable, however, if proteolytic processing of htt could be inhibited in a way that did not interfere with its normal function in the cell.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice
HD46 and HD100 animals, which express amino acid residues 1–964 of htt under control of the neuron-specific enolase (NSE) promoter, were assessed for severity of disease phenotype and assigned a hazard score as described previously (45). Animals defined as showing a ‘severe’ phenotype exhibited clasping and rotorod impairment, whereas these deficits were absent in mice defined as having a ‘mild’ phenotype.
YAC72 mice and sex-matched wild-type FVB/N mice were analyzed at 12 months of age. For each YAC72 mouse, genotypes and equivalent expression of mutant htt transprotein were confirmed using cortical tissue (see supplemental data at www.neumetrix.info).
Mice were sacrificed by cervical dislocation and brains were removed immediately. Whole brains for histologic studies were frozen in an isopentane bath on dry ice. For RNA extractions, striatal and/or cerebellar tissues were dissected and snap-frozen on dry ice. Within each experiment (YAC72 or HD46), all dissections were performed by the same investigator. Tissues were stored at -80°C until processed.
RNA isolation and preparation of microarray samples
Total RNA was isolated from striata and cerebella by extraction with TRI-Reagent (Sigma-Aldrich; HD46 samples) or Trizol (Invitrogen; YAC samples). For striatal samples, total RNA was purified further over RNeasy columns (Qiagen). For the striatal microarray analyses, 15 µg of total RNA was pooled for each sample. For YAC striatal samples (N=2 transgenic, N=2 wild-type), 7.5 µg of total RNA from each of two mice was pooled; for HD46 samples [N=6 wild-type, N=6 mild (4 from line 17, 2 from line 14), and N=6 severe (4 from line 17, 2 from line 14)], RNA was taken from one or two animals. For striatal samples, labeled cRNA probes were generated from total RNA samples according to the Affymetrix GeneChip protocol.
For the YAC72 cerebellar samples, poly(A)+ RNA was purified from total RNA (extracted from a single YAC72 or wild-type cerebellum) using the Oligotex mRNA isolation system (Qiagen). Cerebella from N=5 YAC72 and N=5 wild-type mice were analyzed. Labeled cRNA probes were generated from half of each poly(A)+ RNA sample according to the manufacturer's protocol (Affymetrix).
Biotinylated cRNA probes were hybridized to Murine 11K oligonucleotide microarrays (chip B first and then chip A) using the Affymetrix Fluidics Station 400 according to the manufacturer's standard protocol.
In situ hybridization and ligand-binding histochemistry
ISHH assays were performed as described in (37,38) for the adenosine A2a receptor and NMDA receptors and in (39) for preproenkephalin, DARPP32 and PA28. In YAC72 studies, ISHH signals for target mRNAs were normalized to actin mRNA measured in adjacent brain sections. Ligand binding assays were carried out as described in (39) for adenylyl cyclase and in (37,38) for neurotransmitter receptors.
Northern blotting
Northern analyses were used to quantitate mRNA levels in HD46 samples as described previously (39). To control for variations in sample loading, target mRNA intensities were normalized to ß-actin mRNA (the sequence and use of the actin probe are described in 49).
Real-time quantitative RT–PCR
First-strand cDNA was prepared from 2 µg of total RNA using Superscript II (Invitrogen) in a final volume of 20 µl according to the manufacturer's protocol. After incubation of first-strand reaction at 42°C for 1 h, samples were heated at 100°C for 3 min and brought to a volume of 200 µl total with water. These RT reactions (2 µl) were used as template in real-time PCR reactions. Real-time PCR was performed by SYBR green two-step RT–PCR (Roche Biochemical and Applied Biosystems). Serially diluted cDNA samples were used for standard curve calibration. For all primer sets, we confirmed that less than 0.5% of the quantitative signal was detected when negative control RT reactions (without Superscript enzyme) were substituted for cDNA template. Expression changes were normalized to ß-actin mRNA levels measured in parallel. Primary data analysis was performed using system software from Roche Biochemical or Applied Biosystems as recommended by the manufacturer. We confirmed in preliminary control experiments that differences in mRNA levels detected by real-time PCR were comparable to differences documented in the same samples using northern blotting (see supplemental data at www.neumetrix.info).
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ACKNOWLEDGEMENTS |
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The authors thank R.E. Hughes, K. Kegel, G. Yohrling and E. Signer for critical reading of the manuscript. They thank Y. Kaneko, K. Blankson, L. Farrell, J. Kuster and N. Peters for excellent technical support. The authors would also like to thank the Hereditary Disease Array Group, the Microarray Facility at the Fred Hutchinson Cancer Research Center and Affymetrix for assistance and support with microarray experiments. The data repository at www.neumetrix.info was generously developed by 3rd Millenium (Cambridge, MA). Data for N171-82Q and At-65Q mice were graciously shared with us by G. Schilling, C.A. Ross and D.R. Borchelt.
This work was supported by grants from the US National Institutes of Health to A.B.Y. (AG13617), R.L.-C. (NS10800), J.-H.J.C. (NS38106), M.D. (NS35711 and NS16367) N.A. (NS38194) and J.M.O. (NS42157). This work was also supported by grants from the US Department of Defense to N.A. (USAMRMC 98292059).
E.Y.W.C. is supported by fellowships from the Canadian Institutes of Health Research (CIHR), Huntington Society of Canada and the Michael Smith Foundation for Medical Research. B.R.L. is supported by a CIHR Clinician–Scientist award. M.R.H. is supported by the CIHR and Canadian Networks of Centers of Excellence, and is a holder of a Canada Research Chair. A.B.Y. and J.-H.J.C. are recipients of awards from the Glendorn Foundation. R.L.-C., A.B.Y., J.M.O., M.D., N.A., B.R.L. and M.R.H. are recipients of awards from the Cure HD Initiative of the Hereditary Disease Foundation. M.R.H., M.D. and J.-H.J.C. are recipients of awards from the Huntington's Disease Society of America Coalition for the Cure. N.A. is a recipient of the University of Massachusetts institutional award.
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FOOTNOTES |
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The authors wish it be known that, in their opinion the first two authors should be regarded as joint First Authors. 
* To whom correspondence should be addressed at: Fred Hutchinson Cancer Research Center, D4-100, 1100 Fairview Ave N, Seattle, WA 98109, USA. Tel: +1 2066677955; Fax: +1 2066672917; Email: jolson{at}fhcrc.org
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