Human Molecular Genetics, 2002, Vol. 11, No. 17 1911-1926
© 2002 Oxford University Press
Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain
1Center for Aging, Genetics and Neurodegeneration, Massachusetts General Hospital, Charlestown, MA 02129-4404, USA, 2Fred Hutchinson Cancer Research Center, Seattle WA 98109, USA and 3Center for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada V5H 4H4.
Received March 12, 2002; Accepted June 24, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Previous analyses of gene expression in a mouse model of Huntington's disease (R6/2) indicated that an N-terminal fragment of mutant huntingtin causes downregulation of striatal signaling genes and particularly those normally induced by cAMP and retinoic acid. The present study expands the regional and temporal scope of this previous work by assessing whether similar changes occur in other brain regions affected in Huntington's disease and other polyglutamine diseases and by discerning whether gene expression changes precede the appearance of disease signs. Oligonucleotide microarrays were employed to survey the expression of
11 000 mRNAs in the cerebral cortex, cerebellum and striatum of symptomatic R6/2 mice. The number and nature of gene expression changes were similar among these three regions, influenced as expected by regional differences in baseline gene expression. Time-course studies revealed that mRNA changes could only reliably be detected after 4 weeks of age, coincident with development of early pathologic and behavioral changes in these animals. In addition, we discovered that skeletal muscle is also a target of polyglutamine-related perturbations in gene expression, showing changes in mRNAs that are dysregulated in brain and also muscle-specific mRNAs. The complete dataset is available at www.neumetrix.info. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntington's disease is a neurodegenerative disorder caused by a CAG repeat expansion in the IT15 gene, which encodes a protein known as huntingtin (1–3). Huntingtin is expressed widely throughout the body and central nervous system (4–6). The initiation of the HD process requires the translation of the variable CAG repeat, placing HD into the category of polyglutamine [poly(Q)] diseases (7,8). The etiologic cascade of HD and the other poly(Q) diseases remains unclear.
As limited information can be gathered about a neurodegenerative process from non-invasive in vivo procedures and postmortem studies of human cases, the elucidation of poly(Q) pathogenesis relies heavily on disease models. The R6/2 mouse created by Bates and colleagues (9) has thus far been the most widely studied HD model system, and the pathology and phenotypic characteristics of these animals are well documented. R6/2 mice express an N-terminal portion of human huntingtin with a poly(Q) stretch of 150 under control of the human IT15 promoter. Interestingly, small N-terminal fragments of huntingtin appear to be generated by proteolysis in vivo (10–13), raising the possibility that the effects of such fragments are relevant to human disease.
Behaviorally, R6/2 animals are first distinguished from their wild-type counterparts by a spatial learning deficit [at 3–4 weeks of age (14)]. Attention learning deficits, abnormal performance in motor tests (swimming and high speed rotorod) and reduced long-term potentiation (LTP) appear at 5–6 weeks of age (14–16), followed by the development of a resting tremor, gait disturbances, visual learning deficits and impaired prepulse inhibition at 8–9 weeks of age (14,15). The neurologic status of the animals continues to decline over time, and they die at 13–15 weeks of age.
Pathologically, neuronal intranuclear inclusions are visible by 3–4 weeks of age in the cortex and by 4–6 weeks of age in the striatum and cerebellum of the R6/2 animal (17,18, and R.L.-C. and A.B.Y., unpublished observations). Although general atrophy of the cortex and striatum has been noted by 12 weeks of age, neither cell loss nor signs of enhanced cell death are detectable at this time point (19). [Signs of dark cell degeneration can be detected only in animals beyond 14 weeks of age, and this occurs only in limited regions of striatum and cortex (19).] The R6/2 mouse is thus a reasonable model in which to study changes of gene expression in the absence of cell loss caused by overt neuronal degeneration. The degree to which R6/2 animals have been characterized, combined with their relatively consistent timeline of biochemical and behavioral changes, provide an opportunity to determine where a particular biochemical change falls within the cascade of their disease process.
Regional specificity is the rule in Huntington's disease, with the striatum and cerebral cortex being affected early in the disease course (20,21). It is not clear whether region-specific effects exist in the R6/2 model, however. In fact, evidence to date suggests that multiple brain regions and peripheral tissues in which the transgene is expressed are subject to poly(Q) toxicity (16,22–24). In this study, we have explored the relative effects of the R6/2 transprotein on gene expression in various brain regions and skeletal muscle. These experiments allow us to address whether changes in steady-state mRNA populations caused by a mutant N-terminal huntingtin fragment show an inherent regional specificity. Further, since the IT15 (huntingtin) promoter directs the expression of the R6/2 transgene, we were able to examine the extent to which the HD gene promoter might convey regionally restricted effects on gene expression.
In a previous study, we found evidence of specific effects on gene regulation in the striata of R6/2 mice at 6 and 12 weeks of age, representing early and late symptomatic states, respectively (25). These experiments showed the general downregulation of striatal signaling genes and the general upregulation of genes indicative of stress and inflammation. In examining known regulatory mechanisms of the differentially expressed genes, we determined that many of these genes were positively regulated by cAMP, retinoids or interferons. Other studies have also provided strong evidence that transcription may be a major target of poly(Q) disease pathogenesis (26–32).
The current study builds on our previous work in several important ways. To explore the extent to which other tissues are subject to transcriptional perturbation by an expanded poly(Q) protein, we compared poly(Q)- related changes in gene expression in other brain regions and skeletal muscle in R6/2 mice with those observed in striatum. Specifically, we tested whether gene expression changes in R6/2 mice would be more severe in brain tissues affected in HD (striatum and cerebral cortex) than in other tissues (cerebellum and muscle). Also, we assessed the extent of overlap in the mRNAs showing differential expression in one or more regions. Another major aim of our study was to determine where changes in gene expression fall temporally along the continuum of well-characterized behavioral and pathologic signs of poly(Q) disease observed in R6/2 animals. To address this question, we used a combination of microarray and northern analyses to assess the timing of mRNA changes before and throughout the development of pathologic and behavioral disease parameters.
These studies demonstrate that the N-terminal huntingtin fragment can have dramatic effects on gene expression, and suggest that no regional selectivity of this effect is imparted by the IT15 promoter or N-terminal huntingtin polypeptide sequences. Further, mRNA changes appear early in the disease process in R6/2 mice [approximately coincident with changes in LTP (16)], but do not precede the formation of neuronal inclusions or the development of spatial learning deficits.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Regional gene expression profiles in control and R6/2 mouse brain
R6/2 mice develop learning, motor and coordination deficits suggesting that multiple brain regions are adversely affected by the poly(Q) expansion (9,14,15). In order to place transcriptional changes in each brain region into the appropriate context, we first established which genes were present in each tissue. Figure 1A shows the number of genes that were called ‘Present’ (detectable) by Affymetrix GeneChip software (version 4.0) in at least 50% of the samples assayed. Nearly half of all probes on the arrays detected transcripts in all three regions by these criteria. Of the mRNAs that were preferentially detected in one brain region, many are consistent with in situ hybridization and northern analysis results reported in the literature. The complete region-specific data set is provided in the supplemental datafile "Region_Specific_Data.xls" at www.neumetrix.info. A similar list of genes that meet the criteria of being called ‘Present’ at a threshold of 75% is also provided at this site. These brain region-specific transcript expression maps provide information for a broad array of uses, including the generation of mice with region-specific transgene expression. It also provides information about probes that do not reliably detect particular mRNAs, providing a basis for interpreting negative results.
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To better understand the regional scope of transcriptional dysregulation in this animal model, we compared gene expression profiles from 12-week R6/2 striatum, cortex and cerebellum with those of wild-type controls. The error model described in (33) revealed that a small fraction (1–2% at P<0.001) of assayed transcripts were differentially detected in each brain region (Table 1). In each case, the predicted false-discovery rate was less than 10% of the number of mRNAs that met the P<0.001 threshold at this age (Table 1). Very few transcripts (only 34) were differentially detected in all three brain regions of R6/2 compared with wild-type mice (Fig. 1B). Transcripts that were differentially detected in three of three regions or in two of three regions are shown in Figure 2A. Selected mRNAs that were differentially detected in only cortex or cerebellum are listed in Figure 2B, and the complete dataset is available at www.neumetrix.info.
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The Mu11K GeneChips used for this study assay the expression of
5000 more mRNAs than those used in a previous analysis (Mu6500) (25); therefore, we obtained additional information on striatal gene expression. Also, some changes in gene expression in R6/2 animals were observed uniquely in cerebral cortex and/or cerebellum (whereas no changes in striatal expression were detected, often because these particular mRNAs were absent or of low abundance in striatum). Since more mRNAs and additional brain regions were examined in the present study, many novel changes were detected. Data for striatal mRNAs assayed on both Mu11K and Mu6500 arrays (i.e. those examined in both the previous and present analyses) showed good concordance between studies.
As seen in our previous analysis of R6/2 striatum, mRNAs that were decreased in cerebral cortex and cerebellum were largely comprised of those that encode neuropeptides [e.g. preprosomatostatin, enkephalin, neuropeptide Y, prodynorphin and cholecystokinin (CCK)] and other proteins involved in neurotransmission [e.g. the GABA receptor 
subunit and protein kinase C (PKC)] and proteins involved in ion regulation (the ryanodine receptor and potassium channel proteins). Newly apparent were genes related to growth factor signal transduction [e.g. brain-derived neurotrophic factor (BDNF), bone morphogenetic protein 1, the growth factor-inducible immediate early gene 3CH134 and insulin-like growth factor-binding protein 5 (IGFBP5)]. Messenger RNAs that were increased in multiple brain regions of R6/2 mice were consistent with a cellular response to stress induced by the poly(Q)-containing transprotein (e.g. the proteasome PA28 subunit and DNA-repair enzymes). Newly appreciated mRNA increases included those encoding RNA polymerases and tRNA synthetic enzymes. A subset of the differentially expressed mRNAs was further assessed by northern analysis (Fig. 3).
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Time course of gene expression changes in R6/2 animals
We extended our array analyses to 2-, 4- and 6-week striatum and 2- and 6-week cerebral cortex. Application of the error model (33) revealed that the numbers of mRNAs detected at the 2- and 4-week time points were within the range of predicted false-positives. We conclude that when using this technology in homogenate samples of striatum and cortex, no transcription dysregulation can reliably be detected in the 2- and 4-week and samples (Table 1). To assess whether the GeneChips simply lacked sensitivity to detect changes in these samples, we conducted time-course studies by northern analysis for six mRNAs that were changed in R6/2 striatum at the 6- or 12-week time points. None of these mRNAs was differentially detected in 2- or 4-week samples by phospoimage analysis after normalization for sample loading. In 6-, 8-, 10- and 12-week samples, northern analysis confirmed the changes detected by microarray (Fig. 4A and 5). We similarly extended microarray findings in cerebellum and cerebral cortex by conducting a time-course study using northern analyses. As in striatum, gene expression changes in these regions were first detected at 6 weeks, and uniformly confirmed microarray-based findings (Fig. 4B, 4C and 5).
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Changes in the expression of Sp transcription factor-regulated genes
For mRNAs that were differentially detected in R6/2 mice compared with controls, we analyzed the corresponding promoter sequences for known transcription factor motifs. We found that all of the promoters that had been characterized contained putative binding sites for Sp transcription factors within 500 bp of the transcription start site (Table 2). Table 2 lists genes of which the promoter has been formally studied and in which an Sp site was present. Consistent with our previous study, we again noted many retinoid- and cAMP-responsive genes among those downregulated in R6/2 brain.
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Comparisons of RNA and corresponding protein levels in R6/2 brain
In a recent study, mRNA levels detected by microarray analysis showed limited correlation with protein levels detected by mass spectrometry (34). To determine whether protein levels determined by western analysis corresponded to RNA levels in R6/2 mice, we conducted western analysis of brain homogenates from R6/2 and control mice at 8 and 12 weeks of age. Protein levels were altered in the same direction as mRNA levels in all but one case (RNA polymerase II in cerebellum, see below). In addition, protein levels were more uniformly changed across the three regions of brain that were assayed (cerebellum, cerebral cortex and striatum) than were mRNA levels. By western analysis, calbindin, glutamic acid decarboxylase and neurofilament medium chain were decreased in all three brain regions examined, and excitatory amino acid transporter EAAT4, c-kit, and neuron-specific enolase (NSE) were decreased in selected brain regions. Also, glial fibrillary acidic protein (GFAP) was increased in the cerebellum and cerebral cortex (Fig. 6). These results confirmed that changes in mRNA levels predicted corresponding changes in protein levels in this model system. They also confirm that changes in gene expression in a particular brain area may significantly affect protein levels, and thus neuronal function, in other regions to which it projects.
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RNA polymerase II large subunit (RPIILS) was increased by microarray analysis in all three brain regions of R6/2 mice at 12 weeks compared with controls. Because this molecule plays a critical role in transcription, we conducted further studies of its expression. Northern analysis confirmed the microarray results in all three brain regions (Fig. 7A). In addition, western analysis confirmed that RPIILS protein levels were increased in striatum and cerebral cortex of R6/2 mice (Fig. 7B). The same blots showed that RPIILS protein levels were decreased in R6/2 cerebellum compared with wild-type, despite opposite findings for mRNA levels. We also assessed whether RPIILS protein is sequestered into poly(Q) aggregates. Indirect immunofluorescence microscopy revealed the presence of RPIILS in both nuclear and cytoplasmic aggregates of an SY5Y neuroblastoma cell line that overexpresses an expanded repeat huntingtin fragment (35). RPIILS co-localized with aggregates in the majority, but not all, of the cells that contained aggregates. The presence of RPIILS in aggregates was not sufficient to deplete the cells of normal, diffuse nuclear protein, as shown in Figure 7C. Additional studies will be required to learn whether sequestration of the RNA polymerase II large subunit in in vivo models of HD or in HD patient samples is sufficient to influence transcription of key neuronal genes.
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mRNA changes in muscle of R6/2 compared with control mice
There is evidence that mutant huntingtin may have detrimental effects on skeletal muscle (37), and that R6/2 mice have poly(Q) aggregate pathology in this tissue (22). Further, recent reports indicate that expanded poly(Q) aggregates sequester histone acetyltransferases and other transcriptional proteins in brain and in cultured cells (26–29). Based on these results, we predicted that huntingtin would alter gene expression in muscle as well as in brain. We compared gene expression profiles of muscle from 12-week-old R6/2 mice with those of age-matched controls. As seen in brain,
2% of mRNAs assessed were differentially detected between R6/2 and control muscle and more mRNAs were decreased than increased (Table 3 and neumetrix.info supplemental data). Also as in brain, mRNAs that were increased in muscle were consistent with a cellular stress response to the expanded poly(Q) transprotein (e.g. ubiquitin-conjugating enzymes and multiple DNA-repair enzymes). Messenger RNAs that were decreased in R6/2 compared with control muscle included markers of terminal muscle differentiation (e.g. myoD, actin, myosin light chain and troponin), mRNAs that encode metabolic enzymes (e.g. creatine kinase, enolase and aldolase) and mRNAs that encode signal transduction molecules (e.g. phosphatidylinositol glycan, cAMP-specific phosphodiesterase and Neu-associated kinase).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The R6/2 transgenic mouse is the most widely used model of Huntington's disease. In this study, we demonstrate that R6/2 mice have reproducible and widespread gene expression changes in brain and muscle. In 12-week-old R6/2 mice, the number and magnitude of mRNA changes caused by the expanded htt exon 1 transprotein were similar in all three brain regions examined. These results indicate that the mutant N-terminal huntingtin fragment carried in these mice conveys no inherent regional selectivity, at least at the level of gene expression. Likewise, these results suggest that poly(Q) effects on gene expression in HD are not regionally selective by virtue of the IT15 promoter, which also directs the expression of the R6/2 transprotein. Because the transprotein expressed by R6/2 mice comprises only a small portion of the full-length amino acid sequence of huntingtin, they are sometimes considered to be a general poly(Q) disease model rather than a model specific for HD. Considered as such, it is possible that many of the gene expression effects observed in R6/2 mice are generalizable to other poly(Q) diseases. We address general poly(Q) effects in a companion paper (38).
The specific mRNA changes that were detected were disproportionately high in single regions compared with the region-selective transcript expression maps (compare Figs. 1A and B). We believe that the latter is due to the fact that some gene expression changes remain below the level of sensitivity of the microarray analysis. This is based on a higher rate of detection of changes across multiple brain regions in northern analyses when the same mRNAs were differentially detected in only one or two of the brain regions by microarray. As shown in other studies (33,38,39), it is possible that some of these gene expression changes could be detected through the use of additional replicates.
Gene expression changes prior to 6 weeks of age were not reliably detected by microarray or northern analyses of R6/2 mice. Because gene expression changes were reliably detected within 24–48 hours in yeast cultures and mammalian cell lines that overexpress mutant poly(Q)-encoding transgenes (31,40,41), it is possible that transcriptional changes in young R6/2 mice simply fall below the level of detection. Explanations include asynchronous gene expression changes among subsets of cells in homogenate samples, homeostatic correction of mRNA levels and changes that are very small in magnitude. Alternatively, gene expression changes may truly occur later than other disease signs in these animals, which may differ from the cell lines because of a lower transgene dose.
One common pathologic feature of poly(Q) diseases is the formation of multimolecular aggregates that contain the disease protein (42,43). Recently, it has been shown that transcription factor proteins can be recruited away from their normal subcellular compartments and into these aggregates (26–29, 44,45). One consequence of such recruitment may be loss of transcriptional regulation normally controlled by these specific transcription factors (46–50). In this study, we demonstrated for the first time that the RNA polymerase II large subunit co-localizes with aggregates in a cellular model of poly(Q) disease. Future studies are indicated to establish whether an abnormal RPIILS protein interaction is responsible for some of the gene expression changes described in this study. It will also be important to determine whether poly(Q)-containing proteins interact directly with RPIILS or whether the latter is sequestered by virtue of its interaction with other transcriptional proteins that are sequestered. Consistent with data showing sequestration of transcriptional proteins by intranuclear inclusions, gene expression changes become robust in R6/2 brain shortly after large neuronal aggregates form. On the other hand, inclusions form earlier in the cerebral cortex (at 3–4 weeks) than in the striatum or cerebellum (4–6 weeks), yet mRNA changes appear at approximately the same time in all three regions (4–6 weeks), suggesting that gene expression changes could arise from an independent process, such as soluble protein–protein interactions (32). Given the limited time resolution of the present data and the heterogeneous formation of inclusions in the affected regions during this early period of disease, future studies will be required to define more precisely the relationship between aggregate formation and changes in gene expression.
Our data are also consistent with the hypothesis that Sp1-mediated gene regulation may be altered by mutant huntingtin (32,51). We have recently shown that Sp1 interacts with huntingtin directly, and that mutant huntingtin has a higher affinity for this transcriptional regulator than does the wild-type protein (32). Further, we have shown that Sp1-dependent transcription is disrupted by mutant huntingtin in vitro and that Sp1's DNA-binding activity is compromised in early-grade HD brain. Additional factors or mechanisms likely contribute to huntingtin-related gene expression, however, since other genes that contain functional Sp1 response elements are not differentially detected in HD patients or mice. Recent data from our group and other laboratories suggest that the poly(Q) interactor protein TAFII130 may play also an important role in huntingtin's disruption of transcription at Sp1-regulated promoters (29,32).
This study also demonstrated mRNA changes that are consistent with several other established hypotheses related to poly(Q)-mediated neurodegeneration, including disruption of cAMP response element binding protein (CREB)-binding protein (CBP), retinoid and BDNF activites, altered neurotransmission and cellular metabolism, and induction of stress response systems (16,23–31,40,41,52–55). Numerous genes that are known to be positively regulated through cAMP-responsive or retinoid-response elements were diminished in R6/2 brain, including several genes that were uniformly decreased across multiple brain regions (e.g. preproenkephalin and preprosomatostatin). BDNF levels were decreased in both cerebellum and cerebral cortex, consistent with the hypothesis that reduced trophic support from corticostriatal neurons contributes to striatal medium spiny neuron degeneration (52). The expression of multiple inflammation- and stress-related genes was increased in all regions, and the cerebellum additionally demonstrated evidence of gliosis (GFAP increase) and caspase activation [increased interleukin-1ß-converting enzyme (ICE, caspase-1)]. These results are consistent with previous assessments of R6 animals, including pathologic studies showing inflammatory changes in brain (18,56) and mRNA profiling by cDNA array and northern analyses showing increases in cell-death-related mRNAs (56,57). Because northern and western data confirmed positive gene expression changes detected with the use of an error model (33), it should now be possible to generate and test new hypotheses related to HD pathogenesis. With further study, these gene expression changes should aid in the elucidation of the specific molecular mechanisms causing poly(Q)-dependent disruptions in transcription and/or other RNA-processing events.
The R6/2 mouse gene expression profiles provide new targets for analysis in human tissues. Limited comparisons between gene expression changes in R6/2 mice and previous mRNA and protein studies of HD material suggest that R6/2 mice recapitulate many of the earliest biochemical changes observed in HD brain, including decreases in particular neurotransmitter receptors (25,58–60). Ongoing studies will address the extent to which mRNA changes in these mice predict transcriptional abnormalities in human poly(Q) disease.
We tested the hypothesis that neuronal gene expression changes would be mirrored in muscle of R6/2 mice. The functional relationship between the changes in gene expression in nerve and muscle were striking. The tissue-specific forms of creatine kinase, enolase and intermediate filaments were decreased in both brain and muscle, and evidence of cellular stress related to poly(Q) expression was evident in both. The demonstration that poly(Q)-mediated gene and protein level changes can be detected in tissue outside the central nervous system raises the possibility that biomarkers of disease progression will be detectable in skin, muscle, blood or other peripherally accessible tissue. Because current clinical trial endpoints for HD remain unsatisfactory, it is hoped that biomarkers can serve as surrogates in clinical trials, much like reductions of cholesterol levels are used to assess the efficacy of drugs that ultimately reduce cardiac arrest from coronary artery disease (http://www.hdfoundation.org/workshop.htm).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Animals
R6/2 animals (9) were obtained from Jackson Laboratories (Bar Harbor, ME). Age- and sex-matched groups of mice were sacrificed in parallel at the following ages: 12±1 days (
2 weeks), 25±2 days (4 weeks), 40±2 days (6 weeks), 56±4 days (8 weeks), 70±4 days (10 weeks), and 84±4 days (12 weeks). Tissues were dissected rapidly, frozen quickly on dry ice and stored at -80°C until processed.
RNA
RNA was extracted using Tri-Reagent (Sigma-Aldrich, St Louis, MO) according to the manufacturer's instructions. Striatal microarray samples from 2-week-old animals (comprising of total RNA) were additionally purified over RNeasy columns (Qiagen). Poly(A)+ RNA was extracted from 30–80 µg of total RNA using an Oligotex mRNA isolation kit (Qiagen) for other microarray samples.
Microarray analyses
The microarray analyses were conducted using Mu11K A+B GeneChip sets (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations. RNA samples for 2-week striatal microarray analyses comprised 15 µg of total RNA pooled from four animals (two male and two female). For other microarray samples, poly(A)+ RNA was extracted as described above from total RNA pooled from two to six female R6/2 or wild-type animals. Subsequent cRNA preparation and hybridization was conducted as described in (25). Pairwise comparisons between array samples were conducted using Affymetrix GeneChip Microarray Suite 3.3 or 4.0 software. Data were analyzed using the Difference Call Decision Matrix (61) and as described in (33). Muscle specimens were analyzed on cDNA spotted arrays that were generated in the Fred Hutchinson Cancer Center Microarray Facility. Samples were prepared, hybridized, scanned and analyzed as described previously (62).
Promoter analysis
We searched the Medline database for promoter motifs present in the genes that showed altered expression in the R6/2 transgenic compared with wild-type mice. We found that all genes which had promoter characterization contained putative binding sites for Sp transcription factors (Table 2). Criteria for selection of Sp sites were based on the presence of the consensus sequence (GGGCGGG) within the first 500 bp upstream from the transcription start site. Analysis of more distal transcription factor response elements will be reported separately.
Northern blot analyses
RNA samples (2 µg of total RNA per lane from age- and sex-matched cohorts of animals) were electrophoretically separated through a 1.2% agarose/3% formaldehyde gel in 1xMOPS buffer and transferred electrophoretically to GeneScreen II membranes 1xTAE buffer (NEN Life Sciences, Boston, MA). The resultant blots were hybridized to 32P-labeled cDNA probes in ExpressHyb hybridization medium (Clonetech, Palo Alto, CA) at 68°C for 3–16 h. cDNA probes were labeled by random priming using a Prime-It II kit (Stratagene, Cedar Creek, TX) according to the manufacturer's instructions (high-specific-activity protocol) with [32P]dATP (6000 Ci/mmol; NEN Life Sciences). Blots were washed twice for 15 min with 2xSSC followed by two sequential high-stringency washes (15 and 30 min) with either 0.2xSSC+0.1% SDS (for human cDNA probes: ß-actin and hippocalcin) or 0.1xSSC+ 0.1% SDS (for mouse cDNA probes: all others). The ß-actin probe (corresponding to bases 658–936 of GenBank accession no. X00351) was a gift from Dr David W. Miller (Massachusetts General Hospital), and the proenkephalin probe (comprising bases 182–655 of GenBank accession no. M13227) was cloned by RT–PCR. Other cDNA probes comprised the following sequence-verified IMAGE clones, obtained from ResGen/Invitrogen: BDNF (1397218), Cathepsin S (596534), CCK (874832), GC-BP (3153027), Hippocalcin (1540054), IGFBP5 (585964), MARCKS (637962), myosin light chain (318218), NAP22 (3971850), neurogranin (445656), NFYC (3325912), PCP4/PEP19 (403628), PDEB1 (557945), PKC (1066943), PTZ-17 (ID4160989), RPIILS (1383243), RSE (4017458), SNFK (1229791), somatostatin (480070) and 3CH134 (4235972). Sequence information on these clones is available at www.resgen.com/resources/apps/cdna/index.php3. Autoradio-grams were analyzed using a Molecular Dynamics phosphoimager and ImageQuant software v 1.2 (Sunnyvale, CA). To correct for variations in sample loading, hybridization signal for each target mRNA was expressed as a ratio to ß-actin signal on the same blot.
Western blotting
Tissue samples from the cerebellum, cortex or striatum were homogenized (1 : 10, w/v) in 0.25 M Tris–HCl, pH 6.8, 1% sodium dodecylsulfate (SDS), 5 mM EGTA, 5 mM EDTA, and 10% glycerol, supplemented with protease inhibitors (Roche). The samples were then sonicated for 15 s and centrifuged for 10 min at 10 000 g at 4°C, and the protein concentration of the supernatant was determined using the BCA assay. Samples were diluted to a final concentration of 2 mg/ml in 2xstop buffer (0.25 M Tris–HCl, pH 6.8, 2% SDS, 25 mM dithiothreitol, 5 mM EGTA, 5 mM EDTA, 10% glycerol and 0.01% bromophenol blue as tracking dye), and incubated in a boiling water bath for 10 min. Then samples were run on 5% SDS–polyacrylamide gels, and then transferred to nitrocellulose. The membranes were blocked for 60 min in Tris-buffered saline/Tween-20 (TBST) (137 mM NaCl, 20 mM Tris–HCl, pH 7.5, 0.1% Tween-20) containing 5% non-fat dried milk. The membranes were incubated overnight at 4°C with RPIILS antibody (Covance) in the blocking buffer. The membranes were then washed three times with TBST and incubated for 2 h with HRP-conjugated secondary antibody and the immunoreactive proteins were detected using a chemiluminescent substrate (ECL, Amersham).
Immunocytochemistry
Cells that express highly truncated mutant huntingtin (35) were seeded on poly-D-lysine-coated cover slips in 24-well plates. Cells were fixed in 90% methanol and 50 mM EGTA, pH 6.0 for 5 min at -20°C (44), incubated for 10 min with 0.2% Triton X-100 in PBS, and rinsed three times with PBS, prior to incubation with 5% bovine serum albumin in PBS for 90 min to reduce the background. Cells were then incubated at room temperature for 90 min with a monoclonal RNA polymerase II antibody (1 : 100, Covance) and a polyclonal N-terminal huntingtin antibody (1 : 20 000) (36) in 5% bovine serum albumin in PBS. Cells were then rinsed with PBS, and incubated for 60 min at room temperature with FITC-conjugated anti-mouse IgG (1 : 100) and Texas Red-conjugated anti-rabbit IgG (1 : 100) in 5% bovine serum albumin in PBS. Coverslips were then washed extensively in PBS and mounted. Cells were viewed using fluorescence microscopy (Zeiss). The digitally stored images were combined and displayed with the accompanying software and Adobe Photoshop 7.0.
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ACKNOWLEDGEMENTS |
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The authors wish to thank Jang-Ho Cha and George Yohrling for critical evaluation of this manuscript, Jeffrey Delrow and the FHCRC Microarray Facility for technical assistance and Sharon Melanson and John Kuster for help in preparing the manuscript. This work was funded by The Hereditary Disease Foundation Cure HD Initiative (R.L.C., J.M.O., D.K. and A.B.Y.), the National Institutes of Health (NS10800 to R.L.C., AG13617 to A.B.Y., NS42157 to J.M.O., NS023174 to D.K., and NS36086 to S.J.T.).
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FOOTNOTES |
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* 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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REFERENCES |
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A. D. Strand, A. K. Aragaki, D. Shaw, T. Bird, J. Holton, C. Turner, S. J. Tapscott, S. J. Tabrizi, A. H. Schapira, C. Kooperberg, et al. Gene expression in Huntington's disease skeletal muscle: a potential biomarker Hum. Mol. Genet., July 1, 2005; 14(13): 1863 - 1876. [Abstract] [Full Text] [PDF]
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N. K. Mazarakis, A. Cybulska-Klosowicz, H. Grote, T. Pang, A. Van Dellen, M. Kossut, C. Blakemore, and A. J. Hannan Deficits in Experience-Dependent Cortical Plasticity and Sensory-Discrimination Learning in Presymptomatic Huntington's Disease Mice J. Neurosci., March 23, 2005; 25(12): 3059 - 3066. [Abstract] [Full Text] [PDF]
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B. Zucker, R. Luthi-Carter, J. A. Kama, A. W. Dunah, E. A. Stern, J. H. Fox, D. G. Standaert, A. B. Young, and S. J. Augood Transcriptional dysregulation in striatal projection- and interneurons in a mouse model of Huntington's disease: neuronal selectivity and potential neuroprotective role of HAP1 Hum. Mol. Genet., January 15, 2005; 14(2): 179 - 189. [Abstract] [Full Text] [PDF]
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A. J. Morton, N. I. Wood, M. H. Hastings, C. Hurelbrink, R. A. Barker, and E. S. Maywood Disintegration of the Sleep-Wake Cycle and Circadian Timing in Huntington's Disease J. Neurosci., January 5, 2005; 25(1): 157 - 163. [Abstract] [Full Text] [PDF]
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C. A. Colton, Q. Xu, J. R. Burke, S. Y. Bae, J. K. Wakefield, A. Nair, W. J. Strittmatter, and M. P. Vitek Disrupted Spermine Homeostasis: A Novel Mechanism in Polyglutamine-Mediated Aggregation and Cell Death J. Neurosci., August 11, 2004; 24(32): 7118 - 7127. [Abstract] [Full Text] [PDF]
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G. Schilling, A. V. Savonenko, A. Klevytska, J. L. Morton, S. M. Tucker, M. Poirier, A. Gale, N. Chan, V. Gonzales, H. H. Slunt, et al. Nuclear-targeting of mutant huntingtin fragments produces Huntington's disease-like phenotypes in transgenic mice Hum. Mol. Genet., August 1, 2004; 13(15): 1599 - 1610. [Abstract] [Full Text] [PDF]
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Q. Ruan, M. Lesort, M. E. MacDonald, and G. V.W. Johnson Striatal cells from mutant huntingtin knock-in mice are selectively vulnerable to mitochondrial complex II inhibitor-induced cell death through a non-apoptotic pathway Hum. Mol. Genet., April 1, 2004; 13(7): 669 - 681. [Abstract] [Full Text] [PDF]
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T. L. Spires, H. E. Grote, N. K. Varshney, P. M. Cordery, A. van Dellen, C. Blakemore, and A. J. Hannan Environmental Enrichment Rescues Protein Deficits in a Mouse Model of Huntington's Disease, Indicating a Possible Disease Mechanism J. Neurosci., March 3, 2004; 24(9): 2270 - 2276. [Abstract] [Full Text] [PDF]
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K. L. Sugars, R. Brown, L. J. Cook, J. Swartz, and D. C. Rubinsztein Decreased cAMP Response Element-mediated Transcription: AN EARLY EVENT IN EXON 1 AND FULL-LENGTH CELL MODELS OF HUNTINGTON'S DISEASE THAT CONTRIBUTES TO POLYGLUTAMINE PATHOGENESIS J. Biol. Chem., February 6, 2004; 279(6): 4988 - 4999. [Abstract] [Full Text] [PDF]
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K. Obrietan and K. R. Hoyt CRE-Mediated Transcription Is Increased in Huntington's Disease Transgenic Mice J. Neurosci., January 28, 2004; 24(4): 791 - 796. [Abstract] [Full Text] [PDF]
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R. J. Ferrante, J. K. Kubilus, J. Lee, H. Ryu, A. Beesen, B. Zucker, K. Smith, N. W. Kowall, R. R. Ratan, R. Luthi-Carter, et al. Histone Deacetylase Inhibition by Sodium Butyrate Chemotherapy Ameliorates the Neurodegenerative Phenotype in Huntington's Disease Mice J. Neurosci., October 15, 2003; 23(28): 9418 - 9427. [Abstract] [Full Text] [PDF]
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E. Marcora, K. Gowan, and J. E. Lee Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2 PNAS, August 5, 2003; 100(16): 9578 - 9583. [Abstract] [Full Text] [PDF]
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T. Okuda, H. Hattori, S. Takeuchi, J. Shimizu, H. Ueda, J. J. Palvimo, I. Kanazawa, H. Kawano, M. Nakagawa, and H. Okazawa PQBP-1 transgenic mice show a late-onset motor neuron disease-like phenotype Hum. Mol. Genet., April 1, 2003; 12(7): 711 - 725. [Abstract] [Full Text] [PDF]
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E. Hockly, V. M. Richon, B. Woodman, D. L. Smith, X. Zhou, E. Rosa, K. Sathasivam, S. Ghazi-Noori, A. Mahal, P. A. S. Lowden, et al. Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington's disease PNAS, February 18, 2003; 100(4): 2041 - 2046. [Abstract] [Full Text] [PDF]
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C. Cepeda, R. S. Hurst, C. R. Calvert, E. Hernandez-Echeagaray, O. K. Nguyen, E. Jocoy, L. J. Christian, M. A. Ariano, and M. S. Levine Transient and Progressive Electrophysiological Alterations in the Corticostriatal Pathway in a Mouse Model of Huntington's Disease J. Neurosci., February 1, 2003; 23(3): 961 - 969. [Abstract] [Full Text] [PDF]
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A. D. Strand, J. M. Olson, and C. Kooperberg Estimating the statistical significance of gene expression changes observed with oligonucleotide arrays Hum. Mol. Genet., September 15, 2002; 11(19): 2207 - 2221. [Abstract] [Full Text] [PDF]
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R. Luthi-Carter, A. D. Strand, S. A. Hanson, C. Kooperberg, G. Schilling, A. R. La Spada, D. E. Merry, A. B. Young, C. A. Ross, D. R. Borchelt, et al. Polyglutamine and transcription: gene expression changes shared by DRPLA and Huntington's disease mouse models reveal context-independent effects Hum. Mol. Genet., August 15, 2002; 11(17): 1927 - 1937. [Abstract] [Full Text] [PDF]
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E. Y.W. Chan, R. Luthi-Carter, A. Strand, S. M. Solano, S. A. Hanson, M. M. DeJohn, C. Kooperberg, K. O. Chase, M. DiFiglia, A. B. Young, et al. Increased huntingtin protein length reduces the number of polyglutamine-induced gene expression changes in mouse models of Huntington's disease Hum. Mol. Genet., August 15, 2002; 11(17): 1939 - 1951. [Abstract] [Full Text] [PDF]
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