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Keywords:

  • gene expression;
  • human;
  • in situ hybridization;
  • neurodegeneration;
  • R6/ 1;
  • striatum

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We have identified and cataloged 54 genes that exhibit predominant expression in the striatum. Our hypothesis is that such mRNA molecules are likely to encode proteins that are preferentially associated with particular physiological processes intrinsic to striatal neurons, and therefore might contribute to the regional specificity of neurodegeneration observed in striatal disorders such as Huntington's disease (HD). Expression of these genes was measured simultaneously in the striatum of HD R6/1 transgenic mice using Affymetrix oligonucleotide arrays. We found a decrease in expression of 81% of striatum-enriched genes in HD transgenic mice. Changes in expression of genes associated with G-protein signaling and calcium homeostasis were highlighted. The most striking decrement was observed for a newly identified subunit of the sodium channel, beta 4, with dramatic decreases in expression beginning at 8 weeks of age. A subset of striatal genes was tested by real-time PCR in caudate samples from human HD patients. Similar alterations in expression were observed in human HD and the R6/1 model for the striatal genes tested. Expression of 15 of the striatum-enriched genes was measured in 6-hydroxydopamine-lesioned rats to determine their dependence on dopamine innervation. No changes in expression were observed for any of these genes. These findings demonstrate that mutant huntingtin protein causes selective deficits in the expression of mRNAs responsible for striatum-specific physiology and these may contribute to the regional specificity of degeneration observed in HD.

Abbreviations used
ACV

adenylate cyclase V

Ct

threshold cycle

DARPP32

dopamine and c-AMP regulated phosphoprotein of 32 kDA

GPR22

G protein coupled receptor 22

HD

Huntington's disease

5HT2

5-hydroxytryptamine receptor 2

IRSp53

insulin receptor substrate protein-53

ISL1

islet activating factor 1

KCNIP1

Kv channel-interacting protein 2

MBA

mouse brain anatomy

6-OHDA

6-hydroxydopamine

OSBPL-8

oxysterol binding protein like-8

PMI

postmortem interval

PD

Parkinson's disease

PDE

phosphodiesterase

RGS9

regulator of G-protein signaling-9

Rhes

ras homolog enriched in striatum

RXR

retinoid X receptor

SCNβ4

sodium channel subunit beta 4

SSC

saline-sodium citrate buffer

STEP61

striatal enriched phosphatase 61

The striatum (consisting of the caudate nucleus and putamen) is the largest and major receptive component of the basal ganglia, a group of subcortical nuclei that include the substantia nigra, globus pallidus and the subthalamic nucleus. The basal ganglia act via multiple intrinsic and extrinsic circuits to control motor and cognitive functions (Parent 1990; Graybiel 1995). The striatum exhibits complex patterns of neurochemical and neuroanatomical connectivity and compartmentalization, and is critically involved in the generation of directed motor behaviors through highly specialized output pathways (Graybiel 1995). Proper dynamic regulation of cellular signaling in the striatum is the key to its specific functions. Disruption of this complex balance can have detrimental consequences, as observed in Huntington's disease (HD) and Parkinson's disease (PD), two devastating neurodegenerative disorders affecting intrinsic striatal neurons, and the dopaminergic input to the striatum, respectively.

HD is a progressive neurodegenerative disorder characterized by motor, psychiatric, and cognitive disturbances (Ross et al. 1997). HD is caused by an abnormal expansion of a CAG trinucleotide repeat region in exon 1 of the HD gene (IT15) resulting in an abnormally long stretch of glutamine residues in the encoded huntingtin protein (Huntington's Disease Collaborative Research Group 1993). Huntingtin, a 3144 amino acid protein with no strong homology to other known proteins, exhibits widespread expression in both brain and peripheral tissues (DiFiglia et al. 1995). A core pathological feature of HD is severe atrophy and neurodegeneration of the striatum, and to a lesser extent, cortex (Vonsattel et al. 1985). However, how the disease protein causes specific neurodegeneration in the striatum, despite its ubiquitous expression, is unknown. Several transgenic mouse models for HD have been generated with the mutant HD gene. The first to be developed were the R6/1 and R6/2 mice expressing exon 1 of the human HD gene with an expanded number of CAG trinucleotide repeats: 144 CAG repeats for the R6/2 line and 116 CAG repeats for the R6/1 line (Mangiarini et al. 1996). The R6/1 transgenic mice, which have a later age of motor impairment onset and progression of symptoms (> 20 weeks) compared with the R6/2 line (9–11 weeks) (Mangiarini et al. 1996), are a useful model to study adult-onset HD, which represents a majority of cases.

Our hypothesis is that mRNAs with restricted expression in the striatum are likely to encode proteins that are preferentially associated with particular physiological or behavioral processes in the striatum compared with molecules with more generalized patterns of expression. Hence, their functional roles might be especially relevant to pathological conditions of the striatum and may account for the regional specificity of neurodegeneration observed in HD. Therefore, we have identified and cataloged a list of 54 striatal-enriched genes and have examined their expression in rodent models of Huntington's and Parkinson's diseases. Simultaneous measurement of expression of all 54 genes in mice was possible with the use of Affymetrix oligonucleotide microarrays. We found a decrease in expression of 81% of striatum-enriched genes in HD transgenic mice and similar decreases when caudate from human HD patients was examined. In contrast, 6-hydroxydopamine (6-OHDA) lesioning of the nigrostriatal pathway in rats resulted in no differences in expression in any of the striatal-enriched genes we analyzed, indicating independence from dopamine innervation. These results demonstrate that the presence of the mutant huntingtin protein causes selective deficits in the expression of mRNAs responsible for striatum-specific physiology.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

HD R6/1 transgenic mice [generated by Professor Gillian Bates (Mangiarini et al. 1996)], which express exon 1 of human HD gene carrying 116 CAG repeats were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) (‘B6CBACa-Tg(HDexon1)61Gpb/J’). The mice were bred using original R6/1 breeders obtained from Jackson Laboratories and reared in a colony at The Scripps Research Institute. At the age of 4 weeks, mice were genotyped according to the Jackson Laboratories' protocol to determine which of them were hemizygous for the HD transgene and to verify CAG repeat length. Transgenic mice (4 weeks to 9 months) and their age-matched littermate controls were killed, brains removed and the striata dissected out and frozen immediately for isolation of total RNA or were intracardially perfused with 4% paraformaldehyde for use in in situ hybridization analysis.

Male Sprague–Dawley rats (225–250 g) were housed in pairs in a temperature controlled environment and maintained on a normal 12-h light/dark cycle with lights on at 07.00 h. Food and water were available throughout the experiment ad libitum. Animals were allowed to adjust to their environment in the housing facility at least 1 week prior to surgery. All procedures were in strict accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Human subjects

Human postmortem caudate samples were obtained from Harvard Brain Tissue Resource Center (McLean Hospital, Belmont, MA, USA). Caudate samples consisted of two HD samples, grade 3, a female 53 years old, postmortem interval (PMI) = 22.33 h and a male 46 years old, PMI = 19.6 h, and two controls, a male 61 years old, PMI = 17.0 h and a male 22 years old, PMI = 24.2 h. The pH values of the samples ranged from 6.1 to 6.5. All samples were stored at −70°C until use.

Medial forebrain bundle lesion

Thirty minutes prior to surgery, rats received desipramine (25 mg/kg, i.p.) to block the uptake of 6-OHDA into noradrenergic neurons. Rats were then anesthetized with Nembutol (50 mg/kg) and placed in a stereotaxic apparatus. 6-OHDA (6 µg free base; Sigma, St. Louis, MO, USA) or vehicle (0.02 mg/mL ascorbic acid in 0.9% saline) was infused in a 3 µL volume in the medial forebrain bundle at the following coordinates from bregma: antero-posterior (AP) −4.2 mm, mediolateral (ML) −1.7 mm and dorsoventral (DV) −8.0 mm. Infusions were made with a 26-gauge Hamilton syringe and a microinfusion pump (Stoelting, Wood Dale, IL, USA) delivering the neurotoxin at 0.6 µL/min. After cessation of infusion, the syringe was left in place an additional 5 min to allow for diffusion of 6-OHDA. After surgery, rats were returned to their home cage and allowed to recover. Animals were intracardially perfused with 4% paraformaldehyde at 7 days (n = 5) or 28 days (n = 5) postlesion.

RNA preparations

Total RNA was prepared from the striata of presymptomatic (8–10 weeks) and symptomatic (> 6 months) HD transgenic mice and age-matched wild-type littermate controls using NucleoSpin RNA Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Three independent sets of RNA were isolated from each time point using n = 4 mice/experiment in the first preparation and n = 2 mice/experiment in the following two preparations, which were pooled to include a 50:50 ratio of males to females. RNA from ∼100 mg human postmortem caudate was isolated from each subject using Versagene RNA Purification System (GENTRA Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. All the samples were treated with DNAseI to eliminate genomic DNA contamination. RNA quantification was determined by spectrophotometer readings. The ratio of OD260/OD280 was used to evaluate the purity of the nucleic acid samples and the quality of the extracted total RNA was determined using agarose gel electrophoresis and checked with an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). RNA yields were comparable across all diseased and control samples.

Microarray analysis

RNA from the triplicate preparations was labeled and hybridized to the MOE430 2.0 Affymetrix arrays. One chip per condition was used (wild-type and HD, presymptomatic and symptomatic, three replicates each: 12 chips from 32 mice in total). Arrays were scanned using Affymetrix ScanArray 3000 and standard Affymetrix protocols as described previously (Lockhart et al. 1996). (Protocol available at http://affymetrix.com).

Present (P) and Absent (A) calls were determined with the Affymetrix algorithm (GCOS, Affymetrix, Santa Clara, CA, USA). All marginal (M) calls were treated as absent (A). Conditions were divided into four groups: HD presymptomatic, wild-type control for presymptomatic, HD symptomatic, and wild-type control for symptomatic. Probe sets were considered present overall for a given condition if they had been assigned a present call in at least two of the three replicate samples for that condition. The resulting lists identified as present in at least one of the two conditions (‘Present list’) totaled 20381 genes in the MOE430 2.0 array. Expression signal values were generated using the RMA algorithm (Irizarry et al. 2003), which models the performance of the perfect match probe sets using all chips from this study. This model is then used to calculate corrected expression values for all probe sets. (For more information, see http://www.stat.berkeley.edu/~bolstad/RMAExpress/RMAExpress.html). Using the ‘Present list’ and their respective RMA expression signal values, hierarchical clustering by sample was performed with BRB-ArrayTools software (http://linus.nci.nih.gov/BRB-ArrayTools.html) using default settings for correlation and linkage metrics.

Class comparisons were performed in BRB-ArrayTools to identify specific genes differentially expressed among groups. This class comparison test was conducted using a randomized variance model, a univariate threshold of either 0.01 or 0.05, and a multivariate permutation-based false discovery rate calculation. The predicted proportion of false discoveries was preset at 10% and the confidence level was set at of 80%, resulting in lists consisting of: HD presymp vs. HD symp, 413 genes; HD symp vs. wild-type symp, 1204 genes; HD presymp vs. wild-type presymp, 140 genes. From these lists, striatal-enriched genes were recognized using Affymetrix probe sets, identified from UniGene cluster ID numbers for each striatal gene. The 54 striatal genes were represented by 88 probe sets in total. For genes that were represented by more than one probe set, all probe sets showed similar patterns of gene expression regulation; however, the probe set giving the lowest p-value is reported.

We calculated the likelihood that 39 from 48 known striatum-specific genes that gave signals on the chip would be differentially expressed by chance, given that 1617 (1204 + 413 genes) out of 20 381 brain genes were differentially expressed on the MOE430 2.0 array. We determined the p-value of this chance based on a hypergeometric distribution given by eqn 1, where g is the number of brain genes on the array (given by the number of present calls of the brain samples) m is the number of known striatum-specific genes on the chip, and k is the number of genes that were significantly differentially expressed. For this calculation the values used for g, m, and k were 20,381, 52, and 1617, respectively. The likelihood that 39 out of 48 striatum-specific genes would be differentially expressed by chance is 5 × 10−119.

  • image(1)

Quantification of mRNA by real-time PCR analysis

For cDNA synthesis, 1 µg total RNA from the mice striatum was reverse transcribed using First Strand Synthesis kit (Amersham, Piscataway, NJ, USA) using a NotI/(dT)18 primer. For human samples, 5 µg total RNA were reverse transcribed in 50-µL reactions using oligodT primer and the Stratascript First-Strand Synthesis System (Stratagene, La Jolla, CA, USA) as per the manufacturer's protocol.

Specific primers for each studied sequence and for mouse and human endogenous controls were designed using Primer Express 1.5 software and their specificity to bind the desired sequence was searched against NCBI database. Standard curves were generated for each gene of interest using serial dilutions of mouse or human cDNAs. All primers used showed efficiencies between 90% and 118%and R2 values greater than 0.97, parameters calculated by linear regression analysis of the threshold cycle (Ct) vs. log[template] blots using Graph Pad Prism 3.0 software.

Real-time PCR experiments were performed using the ABI PRISMs 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Amplification was performed on a cDNA amount equivalent to 25 ng total RNA with 1 × SYBR® Green universal PCR Master mix (Applied Biosystems) containing deoxyribonucleotide triphosphates, MgCl2, AmpliTaq Gold DNA polymerase, and forward and reverse primers. PCR reactions were performed on two independent sets of template (n = 6 mice per condition). Experimental samples and no-template controls were all run in duplicate. The PCR cycling parameters were: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 94°C for 15 s, 60°C for 1 min. Finally, a dissociation protocol was also performed at the end of each run to verify the presence of a single product with the appropriate melting point temperature for each amplicon. To further ascertain the specificity and size of the PCR products, the products were run alongside molecular weight markers on a 2% agarose gel in 1 × tris-acetate EDTA (TAE). The amount of studied cDNA in each sample was calculated using SDS2.1 software by the comparative threshold cycle (Ct) method and expressed as 2exp(Ct) using hypoxanthine guanine phosphoribosyl transferase (HPRT) as an internal control for mice sequences, whereas β-2-microglobulin (B2M) was used for the human sequences. For calculations applying the Ct method, age-matched wild-type littermate control mice, or control, non-affected human individuals were used as calibrator samples. 100% of the genes we tested were validated by real-time PCR.

Statistics

One-way anova and Student's t-test (unpaired; two-tailed) were performed to assess significant differences between expression of striatal genes in wild-type vs. transgenic mice and in human postmortem samples. Statistical analyses were performed using Prism software (Graph Pad, San Diego, CA, USA).

In situ hybridization analysis

Perfused brains were postfixed in 4% paraformaldehyde for 12 h, cryoprotected in 30% sucrose/4% paraformaldehyde overnight before being rapidly frozen on dry ice. In situ hybridization was performed on coronal free-floating sections (25-µm thick) from brains of HD transgenic mice, wild-type controls and 6-OHDA-lesioned rats. Brain sections were hybridized at 55°C for 16 h with a 35S-labeled, single-stranded antisense cRNA probe against each striatal clone at 107 cpm/mL. Excess probe was removed by washing with 2 × saline-sodium citrate buffer (SSC; 1 × SSC =0.15 m NaCl/0.015 m sodium citrate) containing 14 mmβ-mercaptoethanol (30 min), followed by incubation with 4 g/mL ribonuclease in 0.5 m NaCl/0.05 m EDTA/0.05 m Tris-HCl (pH 7.5) for 1 h at 37°C. High-stringency washes were carried out at 55°C for 2 h in 0.5 × SSC/50% formamide/0.01 mβ-mercaptoethanol, and then at 68°C for 1 h in 0.1 × SSC/0.01 mβ-mercaptoethanol/0.5% sarkosyl. Slices were mounted onto gelatin-coated slides and dehydrated with ethanol and chloroform before autoradiography. Slides were exposed for 1–4 days to Kodak X-AR film and then dipped in Ilford K-5 emulsion. After 4 weeks, slides were developed with Kodak D19 developer, fixed, and counterstained with Richardson's blue. Sense strand probes gave only background hybridization signals.

Immunohistochemistry

Immunohistochemical experiments were performed using a mouse monoclonal antibody to rat tyrosine hydroxylase (1:500 dilution; Immunostar Incorporated, Hudson, WI, USA). The immunoreaction was detected with Vectastain ABC kit (Vector Laboratory Inc., Burlingame, CA, USA) according to the instructions of the manufacturer. Briefly, free-floating sections were incubated with Blocking Solution (4% bovine serum albumin in 0.1% Triton X-100/phosphate-buffered saline) for 2 h at 25°C followed by incubation with primary antibody in Blocking Solution for 16–20 h at 4°C. Sections were then washed with 0.1% Triton X-100/phosphate-buffered saline, incubated with secondary biotinylated antibody (1:200 dilution in Blocking Solution) for 2 h at room temperature, washed with 0.1% Triton X-100/phosphate-buffered saline, incubated for 1 h with ABC reagent (1:1 in Blocking Solution), and then washed finally with 0.1% Triton X-100/phosphate-buffered saline. Enzymatic development was performed in 0.05% diaminobenzene in phosphate-buffered saline containing 0.003% hydrogen peroxide for 3–5 min.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We have compiled a list of 54 genes that exhibit predominant expression in the striatum (Table 1). We designated these genes as ‘striatal enriched’ as they exhibit predominant expression in the striatum and related areas (nucleus accumbens, olfactory tubercle and islands of Calleja), with some genes being expressed at lower levels in additional regions (usually cortex, hippocampus or thalamus). These represent sequences previously published from our laboratory, genes previously described in the literature and unpublished sequences we recently identified in a mouse brain anatomy (MBA) expression study using the method TOGA® (Sutcliffe et al. 2000) (Table 1).

Table 1.  Compiled list of striatal-enriched genes and expression fold-ratios in R6/1 Huntington's disease transgenic mice
Gene descriptionAccession no.Striatum referenceFold-changeFold-change
10 weeksp-value6 monthsp-value
  1. Expression fold-changes are shown with negative values representing decreases in expression in HD mice relative to wild-type.

  2. Accession numbers show Reference Sequence Collection IDs, when available. MBA clones refer to ‘mouse brain anatomy’. A, Absent call; or NP, not present on Affymetrix array. p-values are shown for significant differences < 0.05.

5-HT4 receptorNM_008313Ullmer et al. 1996AA  
5-HT6 receptorNM_021358Ruat et al. 1993−1.471.45
Actinin, alpha 2NM_033268Dunah et al. 2000−1.55−3.034.50E-05
ARPP-16/19NM_021548Girault et al. 1990−1.690.005−3.111.60E-05
ARPP-21NM_033264Hemmings and Greengard 1989−1.24−1.960.0006
Adenylate cyclase type VXM_156060Glatt and Snyder 1993−1.400.001−2.645.50E-05
Adenosine A2 receptorNM_009630Svenningsson et al. 1997−1.640.003−3.558.80E-05
B3gnt1NM_016888Unpublished−1.500.037−2.230.0005
B-cell RAG ass'd proteinNM_029935Unpublished1.05−1.67
Bcl11bNM_021399Unpublished−1.23−2.060.002
BTE binding proteinNM_010638Usui et al. 19941.35−1.540.0044
CalDAG GEFNM_011242Kawasaki et al. 1998−1.560.001−4.631.10E-06
Copine VNM_153166Unpublished1.13−1.520.04
Dopamine D1 receptorNM_010076Fremeau et al. 1991−1.500.007−3.015.10E-05
Dopamine D2 receptorNM_010077Meador-Woodruff et al. 1989−1.490.008−3.420.0001
Dopamine D3 receptorNM_007877Stanwood et al. 1997AA  
DARPP-32NM_144828Ouimet et al. 1984−1.470.002−2.723.70E-05
Delta opioid receptorNM_013622Le Moine et al. 1994−1.39−1.19
Diacylglycerol kinaseNM_019505Goto and Kondo 19991.291.22
DRRFNM_078477Hwang et al. 2001−1.04−2.380.0003
EnkephalinNM_001002927Shivers et al. 1986−1.870.005−3.690.0001
Ephrin A4NM_007936Janis et al. 19991.381.14
FoxP1NM_053202Takahashi et al. 2003−1.27−2.220.0017
FoxP2NM_053242Takahashi et al. 20031.17−1.06
G protein, γ7 subunitNM_010319Watson et al. 1994−1.11−1.810.0008
G protein, αolfNM_177137Herve et al. 1993AA  
GPR88NM_022427Mizushima et al. 2000−1.28−1.970.002
HippocalcinNM_010471de Chaldee et al. 2003−1.26−2.040.0001
Insulin rec. substrate p53NM_130862Thomas et al. 2001−1.33−2.026.72E-05
ISL-1NM_021459Wang and Liu 20011.370.019−1.02
Kappa opioid receptorNM_011011Mansour et al. 1994−1.07−1.650.0005
KCNIP2NM_030716de Chaldee et al. 2003−1.24−2.642.60E-05
MBA51AI836640Unpublished−1.640.002−3.721.40E-06
MBA52BB313069Unpublished−1.540.001−3.851.50E-05
MBA55NM_177057UnpublishedNPNP  
mPPP1R16BNM_153089Magdaleno et al. 2002−1.31−2.110.0001
Mu-opioid receptorNM_011013Mansour et al. 1994AA  
NeurotensinNM_024435Smits et al. 20041.361.430.02
nGEFNM_019867Rodrigues et al. 2000−1.26−2.100.0005
Nolz-1NM_145459Chang et al. 2004NPNP  
OSBPL-8NM_001003717Thomas et al. 2003−1.36−1.780.0002
PDE10ANM_011866Fujishige et al. 1999−1.690.008−3.890.0001
PDE1BNM_008800Polli and Kincaid 1992−1.420.007−2.690.0003
Protein phosphatase 3NM_008913de Chaldee et al. 2003−1.19−1.650.0003
RAR beta receptor 1NM_011243Krezel et al. 1999−1.17−1.760.0002
RGS9NM_011268Thomas et al. 1998−1.550.007−3.531.00E-05
RhesXM_204287Falk et al. 1999−1.36−3.020.0001
RXR gamma receptorNM_009107Krezel et al. 1999−1.36−2.448.96E-05
Sodium channel, β4BK001031Yu et al. 2003−2.110.002−7.753.40E-05
STEP61NM_013643Lombroso et al. 1993−1.18−1.850.009
StriatinNM_133789Castets et al. 1996−1.42−4.690.0003
Substance PNM_009311Goedert and Hunt 19871.08−1.12
SynaptoporinNM_028052Marqueze-Pouey et al. 1991−1.05−1.44
TescalcinNM_021344de Chaldee et al. 2003−1.33−3.641.00E-05

To measure simultaneously the expression of these striatal genes in HD transgenic mice, we used Affymetrix oligonucleotide array analysis, focusing on those probe sets representing the striatum genes on our list. The Affymetrix mouse 430 2.0 chip, which contains 39 000 mouse nucleotide sequences, contained probe sets for all but two of the striatal enriched sequences on our list, one novel striatal sequence, MBA55 and Nolz-1. Arrays were hybridized with three biological replicates for each condition: striata from R6/1 and wild-type littermate controls at two different stages of illness: presymptomatic (10 weeks) and symptomatic (6 months). Using the Affymetrix detection call algorithm, 48 of the 52 striatum genes on the array were called as Present in two out of three of the experimental replicates. Class comparisons were performed in BRB-ArrayTools to identify specific genes differentially expressed between classes (see methods for details). Of these 48 genes, and to our surprise, 81% (n = 39) showed statistically significant differential expression in symptomatic HD transgenic mice compared to littermate controls. The likelihood that 39 out of 48 striatum-specific genes would be selectively affected by chance is 5 × 10−119 (see Methods). The expression of 38 of these genes was significantly decreased, and one gene significantly increased, in the striatum of HD transgenic mice (Table 1). When considering all genes screened on the array, 1.2% and 3.6% were found to be expressed at significantly altered levels at the p < 0.01 and p < 0.05 levels, respectively, and these data showed a normal distribution (Fig. 1). Comparing the expression fold-change ratios of the striatum genes to all genes expressed, HD transgenic mice differed in the distribution of striatal genes that were decreased over the 95% confidence level. The striatal gene group exhibited a pronounced decrease (shift to the left) in transcript expression in the HD transgenic mice (Fig. 1).

image

Figure 1. Gene expression differences in striatum of symptomatic Huntington's disease (HD) transgenic mice and wild-type littermate controls. All expressed genes were classified into signal intensity difference intervals (0.2 bins) according to their fold-change ratios. Transcripts in a ‘1’ bin had identical signal intensities. Positive values (to the right) on the x axis denote higher hybridization signal in HD mice, negative values (to the left) correspond to higher hybridization signal intensity in the wild-type mice. The y axis reports the percentage of expressed genes. White bars denote distribution of all expressed genes and shaded bars denote distribution of striatum-enriched genes. WT, wild-type.

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Among the striatal genes, the novel subunit, beta 4, for the voltage-gated sodium channel showed the most striking difference in expression (7.75-fold decrease) in 6-month-old HD brains. Eight of these 39 genes have been reported in an earlier microarray experiment to be decreased in the striatum of symptomatic HD R6/2 mice (Luthi-Carter et al. 2000), which have an accelerated HD phenotype compared to the R6/1 strain. These are the adenosine A2a receptor, dopamine D1 and D2 receptors, dopamine and cAMP regulated phosphoprotein of 32 KDa (DARPP-32), enkephalin, adenylyl cyclase V, alpha-actinin and the retinoid × (RXR) gamma receptor. Only one gene, neurotensin, showed significantly elevated expression in symptomatic HD transgenic mice, while another gene encoding the islet activating factor 1 (ISL1) transcription factor was increased in presymptomatic transgenic mice. Other striatal genes, including diacylglycerol kinase, ephrin A4 and the 5HT6 receptor, showed higher levels of expression in presymptomatic or symptomatic transgenic mice, however, these differences were not statistically significant in this experiment. Other genes encoding proteins related to those striatum-enriched species, but whose expression is not limited to the striatum, were found to be significantly increased in the striatum of symptomatic mice. These include G-protein γ4 and γ3 subunits, regulator of G-protein signaling-19, Rho-guanine nucleotide exchange factor, protein phosphatase 6, 5-hydroxytryptamine receptor 2c (5HT2c), orphan G protein coupled receptor 22 (GPR22) and several cell adhesion molecules (data not shown). Besides the striatal-enriched genes, additional sequences found to be highly significantly decreased in expression were also related to G-protein and intracellular signaling (data not shown).

In order to validate these findings, we performed real-time PCR on striatal RNA isolated from two independent sets of pooled wild-type and transgenic R6/1 mice at presymptomatic (8–10 weeks) and symptomatic time points (6 months old) for eight striatal sequences we recently isolated or previously identified: FoxP1, Bcl11b, sodium channel subunit beta 4 (SCNβ4), regulator of G-protein signaling-9 (RGS9), insulin receptor substrate protein-53 (IRSp53), copine V, G-protein γ7 subunit and MBA52 (Fig. 2). Relative abundance values correlated with the initial array data for both presymptomatic and symptomatic time points for all eight selected mRNAs.

image

Figure 2. Decrease of striatal transcripts levels in Huntington's disease (HD) transgenic mice. Real-time PCR amplification of the indicated genes from striatum of presymptomatic (10 weeks) symptomatic (6 months) wild-type (WT) and HD R6/1 transgenic mice was performed as described in Methods. Values are averages ± SEM of duplicate determinations from two independent experiments (n = 6 mice per condition). Amplification of the hypoxanthine guanine phosphoribosyl transferase (HPRT) housekeeping gene was used as an internal reference and the expression level from the age-matched wild-type was used as calibrator in the ΔCt method. Asterisks denote significant differences in gene expression as determined first by one-way anova and then Student's t-test (unpaired; two-tailed) to get exact p-values: *p < 0.05; **p < 0.001; ***p < 0.0001.

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In situ hybridization analysis was performed on brains from different litters of symptomatic R6/1 mice with probes for RGS9, Bcl11b, SCNβ4, MBA51, MBA52, the ras homolog enriched in striatum (Rhes), adenylate cyclase V (ACV) and FoxP1. In all cases, decreases in expression in the striatum of symptomatic mice were confirmed (Figs 3 and 4). Oxysterol binding protein like-8 (OSBPL-8), Golf and IRSp53 also showed decreases in expression in HD mice (data not shown). In addition to the striatum, decreases for these genes were observed in other brain regions, such as cortex, thalamus and hippocampus. For striatal genes exhibiting decreases in expression in presymptomatic time points in the array experiment, we examined expression in 4 week-old HD mice by in situ hybridization to determine how early diminished expression could be detected. None of the gene showed substantial decrements at 4 weeks of age, suggesting that its expression levels are not affected directly by the presence of the transgene (data not shown).

image

Figure 3. In situ hybridization of striatal transcripts in symptomatic Huntington's disease (HD) transgenic mouse brain. In situ hybridization analysis was performed on free-floating coronal sections from control (wild-type) and symptomatic HD transgenic mice brains (n = 3–4 pairs of wild-type and HD transgenic mice). Antisense 35S-labeled riboprobes against the indicated striatal genes were used as described in Methods. CPu, caudate putamen; Cx, cortex; HD-Tg, Huntington's disease transgenic mouse brain; Hipp, hippocampus; Thal, thalamus; WT, wild-type.

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image

Figure 4. High magnification views of striatal transcripts in symptomatic Huntington's disease (HD) transgenic mouse brain. Darkfield photomicrographs are shown for the indicated genes in wild-type (WT) and R6/1 mice. Antisense 35S-labeled riboprobes against the indicated striatal genes were used as described in Methods. Scale bar = 80 µm.

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To determine if the changes we observed in the R6/1 mouse model mimic the pathology in human HD, we performed real-time PCR on postmortem caudate samples from HD patients and controls with eight striatal-enriched genes that showed changes in the mouse transgenic model: SCNβ4, FoxP1, RGS9, Kv channel-interacting protein 2 (KCNIP1) (homologous to mouse KCNIP2), OSBPL-8, G-protein γ7 subunit, RAS, guanyl releasing protein 2 (CalDAG GEF) and neurotensin (Fig. 5). Alterations in expression matching those measured in transgenic mice were observed for all eight genes and the differences in expression were statistically significant for four of these, despite the n of only two. The increase in neurotensin expression is important, because despite the severe loss of medium spiny neurons in the caudate of HD patients (grade 3), there is no decrease in expression of this neuronally expressed gene.

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Figure 5. Alterations of striatal-enriched transcripts in postmortem caudate of Huntington's disease (HD) subjects and controls. Real-time PCR amplification of the indicated genes was performed on human postmortem caudate samples as described in Methods. Values are averages ± SEM of duplicate determinations from two independent experiments. HD brains were a pathological grade 3. Amplification of the beta-2-microglobulin housekeeping gene was used as an internal reference and the expression level in the control brain was used as calibrator in the ΔCt method. Asterisks denote significant differences in gene expression as determined first by one-way anova and then Student's t-test (unpaired; two-tailed) to get exact p-values: *p < 0.05; **p < 0.001; ***p < 0.0001.

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We next compared the expression of selected striatal genes in a rodent model of PD. Although there is no spontaneous occurrence of parkinsonism in animals, several experimental animal models of PD have been developed to mimic clinical features of this disorder in animals, with the first and most frequently used being the 6-OHDA-lesioning model (Mendez and Finn 1975; Mokry 1995). Because simultaneous measurements of the 54 striatal genes was not possible in rats (Affymetrix whole genome arrays for rat were not available at the time of the study), we investigated the expression of randomly selected striatal genes using in situ hybridization. Rats received unilateral injections of 6-OHDA resulting in a loss of dopaminergic input to the striatum with neuronal death occurring in a biphasic manner: an initial phase beginning 12 h postinjection to about 7 days postinjection in which most of the cell loss occurs, and a prolonged phase that extends ∼1 month. Tyrosine hydroxylase staining confirmed a decrease in dopamine content in the striatum and substantia nigra on the ipsilateral side of the injected toxin at both 7 and 28 days postinjection (Fig. 6). In situ hybridization was performed on brains from rats at 7 days and 1 month postinjection. No changes in the ipsilateral vs. contralateral sides of the injection were observed for any of the 15 striatal genes we tested at either 7 days or 1 month postinjection (Fig. 6; Table 2). Representative images for four striatal genes from rats at 7 day postinjection are shown in Fig. 6. Previously published studies have shown that expression of the genes for DARPP-32 and the adenosine A2a receptor are unchanged by destruction of the nigrostriatal pathway (Ehrlich et al. 1990; Kaelin-Lang et al. 2000); however, elevations in the expression of neurotransmitter and neuropeptides receptors in the striatum has been reported (see Table 2). Striatal neuropeptides, enkephalin and neurotensin, and the striatal enriched phosphatase, STEP61, have also been reported to be altered in the 6-OHDA model (Smith et al. 1993; Hanson and Keefe 1999; Napolitano et al. 2002). These results indicate that dopaminergic striatal denervation does not modify the expression of a majority of striatal-enriched genes.

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Figure 6. 6-Hydroxydopamine (6-OHDA) treatment results in a decrease in tyrosine hydroxylase (TH) staining in the striatum (a vs. b) and no change in mRNA expression of select striatal genes (c–f). Immunohistochemistry was performed on free-floating rat brain sections from rats 7 days postinjection of 6-OHDA into the medial forebrain bundle using an anti-TH antibody as described in Methods. In situ hybridization images are shown for the regulator of G-protein signaling 9 (RGS9), sodium channel beta 4 subunit (SCNβ4), ras homolog enriched in striatum (Rhes) and adenylyl cyclase V (ACV) at 7 days postinjection. Arrows designate side of unilateral 6-OHDA lesion.

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Table 2.  Comparisons of expression of striatal genes in rodent models for Huntington's and Parkinson's diseases
Gene nameHD R6/1 micePD 6-OHDA rats
Adenylate cyclase V[DOWNWARDS ARROW]NC
B-cell rag ass'd proteinNCNC
Bcl11b[DOWNWARDS ARROW]NC
BTE binding protein[DOWNWARDS ARROW]NC
Copine V[DOWNWARDS ARROW]NC
FoxP1[DOWNWARDS ARROW]NC
Gamma 7[DOWNWARDS ARROW]NC
Golf[DOWNWARDS ARROW]NC
IRSp53[DOWNWARDS ARROW]NC
MBA51[DOWNWARDS ARROW]NC
MBA52[DOWNWARDS ARROW]NC
OSBP-9[DOWNWARDS ARROW]NC
RGS9[DOWNWARDS ARROW]NC
Rhes[DOWNWARDS ARROW]NC
SCNβ4[DOWNWARDS ARROW]NC
previous studies:
Gene nameRegulation in HD R6/1 miceRegulation in 6-OHDA ratsReference
  1. Expression regulation for genes in response to 6-hydroxydopamine (6-OHDA) treatment reflects changes in protein or mRNA expression. HD, Huntington's disease; PD, Parkinson's disease; NC, no change.

A2a receptor[DOWNWARDS ARROW]NCKaelin-Lang et al. 2000
D1 receptor[DOWNWARDS ARROW]NC or [UPWARDS ARROW]Fornaretto et al. 1993; Brene et al. 1994
D2 receptor[DOWNWARDS ARROW][UPWARDS ARROW]Soghomonian 1993; Levesque et al. 1995
D3 receptor[DOWNWARDS ARROW][UPWARDS ARROW]Levesque et al. 1995
Mu-opioid receptor[DOWNWARDS ARROW][UPWARDS ARROW]Smith et al. 1993
Delta opioid receptor[DOWNWARDS ARROW][UPWARDS ARROW]Smith et al. 1993
Kappa opioid receptor[DOWNWARDS ARROW][UPWARDS ARROW]Smith et al. 1993
Enkephalin[DOWNWARDS ARROW][UPWARDS ARROW]Sivam et al. 1987
Neurotensin[DOWNWARDS ARROW][UPWARDS ARROW]Hanson and Keefe 1999
DARPP-32[DOWNWARDS ARROW]NCEhrlich et al. 1990
RXRβ receptor[DOWNWARDS ARROW][DOWNWARDS ARROW]Napolitano et al. 2002
STEP61[DOWNWARDS ARROW][DOWNWARDS ARROW]Napolitano et al. 2002

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Several studies have investigated gene expression in HD mouse models using microarray analysis and have discovered a wide variety of genes altered in their expression (Luthi-Carter et al. 2000, 2002a,b; Chan et al. 2002). Our approach was to focus specifically on those genes with enriched expression in the striatum, as we have previously hypothesized that these are among the most relevant candidates for involvement in neurological disorders that selectively involve the striatum and may provide a mechanism for the specific neurodegeneration observed in this brain region in HD (Usui et al. 1994; Watson et al. 1994; Thomas et al. 1998, 2001; Falk et al. 1999). Combining our own findings with those in the literature, we have cataloged 54 genes deemed ‘striatum-enriched’. In this study, we demonstrate that a large majority (81%) of the mRNA products of these genes are decreased in their levels in HD R6/1 transgenic mice, indicating a selective dysfunction of their transcription or stability within the striatum. An important issue is how well the results from the animal model correlate to the human disease. We found that alterations in expression of eight genes observed in the R6/1 HD mouse were also observed in postmortem caudate samples from HD patients. As the availability of high quality human HD caudate samples is very limited, we consider that the results presented here, despite being derived from only two subjects, are relevant findings. They suggest not only that striatal gene expression deficits are important to disease pathology, but also that the R6/1 transgenic mouse replicates the molecular aspects of HD pathology well.

Co-dysregulation of a large number of genes normally coregulated, suggests that one or more yet unknown transactivating factors are responsible for global transcriptional deficits in the striatum, although coordinated regulation of mRNA stability has not been ruled out. Transgenic R6/1 mice show no massive cell death, neuronal degeneration or reactive gliosis (Mangiarini et al. 1996; Hansson et al. 1999; Naver et al. 2003), hence the generalized decrease in striatal function implicated in this study occurs in the absence of neuronal degeneration and in many instances, prior to the onset of motor symptoms. We did not observe a decrease in the expression of the neuronal marker, neurofilament, or increases in the glial markers, glial fibrillary acidic protein and myelin basic protein, in our overall array study. This is consistent with postmortem studies in humans (Vonsattel et al. 1985; Myers et al. 1988) that suggest that both motor and cognitive symptoms appear in the absence of neuronal cell loss, suggesting that impairment in cognition is likely to be caused by a cellular dysfunction rather than a consequence of neuronal cell death. The data presented here indicate that the physiological deficit in HD is a result of diminished ability of striatal neurons to perform tasks for which they are uniquely programmed.

Previous studies identifying gene expression differences in HD mice have also reported changes in the expression of some striatal-enriched species. Early studies showed decreases in adenosine A2a, dopamine D1 and D2 receptor mRNA expression and levels of radioligand binding in the R6/1, R6/2 and R6/5 HD transgenic mouse lines (Cha et al. 1998, 1999). One of the first oligonucleotide microarray studies performed on the HD transgenic R6/2 mice demonstrated a reduction in the expression of some genes involved in striatal signaling pathways (Luthi-Carter et al. 2000). Of the 6000 genes screened on that array and ∼70 genes altered in the transgenic model, only eight had specific or enriched expression in the striatum: the adenosine A2a receptor, the dopamine D2 receptor, DARPP-32, enkephalin, adenylyl cyclase V, PDE1B1, alpha-actinin and the RXR gamma receptor. Five of these genes had similar altered expression in a second mouse model of HD, the N171-82Q transgenic line (Luthi-Carter et al. 2000). Candidate gene expression studies have shown decreases in the mRNA and/or protein expression of DARPP-32, ARPP-16, ARPP-21, enkephalin, PDE1B and PDE10A in R6/1 and/or R6/2 mice (Bibb et al. 2000; Menalled et al. 2000; van Dellen et al. 2000; Hebb et al. 2004). Importantly, similar changes in dopamine D1 and D2 receptors, enkephalin and substance P have also been observed in the striatum of Huntington's patients (Richfield et al. 1995; Augood et al. 1996, 1997). Our studies reveal the extent of such alterations.

We have previously noted that among the set of striatal-enriched transcripts, those related to G-protein signaling are particularly prominent (Usui et al. 1994; Thomas et al. 1998). Alterations in G protein-related striatal genes found in this study in HD include RAS, guanyl releasing protein 2 (also known as CalDAG GEF), guanine nucleotide binding protein, alpha olfactory type (Golf), γ7 subunit, RGS9, ACV, phosphodiesterase 1 B (PDE1B), phosphodiesterase 10A (PDE10A), DARPP32 61, protein phosphatase 1, regulatory subunit 16B (PPP1R16B), neuronal guanine nucleotide exchange factors (nGEF), Dopamine receptor regulating factor (DRRF), Rhes and striatal enriched phosphatase (STEP61). Of these, RGS9, ACV, PDE1B, PDE10A, CalDAG GEF, DARPP-32 were significantly decreased as early as 8–10 weeks of age in our Affymetrix analysis, far prior to the onset of motor symptoms, which do not occur until after 26 weeks of age in our colony, suggesting importance to the pathogenesis of the illness.

This study highlights another area of striatal dysfunction in HD: calcium homeostasis. Several genes related to calcium signaling were altered in symptomatic HD mice, including copine V, striatin, SCNβ4 and alpha-actinin 2, with the latter three showing diminished expression levels in presymptomatic mice. The potassium channel-interacting protein 2 (KCNIP2) may also be related, as potassium channels are key determinants of neuronal excitability. The gene demonstrating the highest level of expression changes was a novel subunit of the sodium channel, SCNβ4. It exhibits abundant expression in control striatum and an eight-fold decrease in expression in symptomatic R6/1 mice. Expression of SCNβ4 was not changed in mice at 4 weeks of age; however, significantly decreased expression was detected in mice 8–10 weeks of age, again prior to the onset of symptoms. Voltage-gated sodium channels are composed of pore-forming alpha subunits and auxiliary beta subunits that modify channel function. Sodium levels in the cell are directly related to intracellular calcium levels by means of the sodium–calcium exchanger, an ion transporter that exchanges Na+ and Ca2+ depending on membrane potential and transmembrane ion gradients (Blaustein and Lederer 1999). Therefore, alterations in expression or function of sodium channels can have dramatic consequences to intracellular calcium accumulation. These findings are consistent with previous studies reporting dysfunction in calcium signaling/currents in HD transgenic mice based on gene expression abnormalities and biological assessments (Luthi-Carter et al. 2000; Panov et al. 2002, 2003). Of direct relevance, a recent finding demonstrates that huntingtin-induced disturbances in calcium signaling leads to apoptosis of medium spiny neurons (Tang et al. 2005).

Major inputs to the striatum include the glutamatergic corticostriatal pathway and the dopaminergic nigrostriatal pathway. The substantia nigra (pars compacta) functions most prominently via massive projections to the striatum, thereby constituting the dopaminergic input to this region. The irreversible loss of dopamine-mediated control of striatal function is thought to cause a complex rearrangement of neuronal activity which involves specific dopamine-regulated cellular functions (Napolitano et al. 2002). A close interaction between the dopaminergic and glutamatergic systems exists in the striatum (Hersch et al. 1995; Starr 1995) and complex expression changes of glutamate receptor subunits have been reported in response to 6-OHDA (Ulas and Cotman 1996; Dunah et al. 2000). Accordingly, an array screen of 1756 genes showed that 6-OHDA treatment alters the expression of genes involved in several cellular functions, primarily alterations in genes related to glutamatergic neurotransmission (Napolitano et al. 2002). Only a handful of genes previously identified, however, show enriched striatal expression (see Table 2). In this study, none of the striatal-enriched genes we tested were altered in expression in 6-OHDA-treated rats, indicating that their expression is independent of dopaminergic innervation. Consistent with these findings, previous studies have also demonstrated no change in expression of striatal genes, DARPP-32 and adenosine A2a receptors, in 6-OHDA-lesioned rats (Ehrlich et al. 1990; Kaelin-Lang et al. 2000). This suggests that 6-OHDA treatment, which disrupts dopaminergic input to the striatum, does not affect the intrinsic functions of striatal neurons.

In contrast to PD, in Huntington's disease, although there is normal dopamine innervation of the striatum, the striatal neurons lose their intrinsic functionality and ultimately suffer selective neuronal death. The deficits may be initiated by deficient transcriptional regulation of a set of genes responsible for intrinsic striatal neuronal function. This may account for the selective striatal pathology caused by the widely expressed polyQ-expanded huntingtin protein.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by NIH grants #NS44169 (EAT), MH069696 (EAT) and GM32355 (JGS). The gene microarray analysis was conducted by the Gene Microarray Core of The Consortium for Functional Glycomics funded by the National Institute of General Medical Sciences grant GM62116. Human brain tissue was provided by Harvard Brain Tissue Resource Center, which is supported by PHS grant #R24-MH 068855. We thank Lana Shaffer for statistical analysis on striatum-enriched genes.

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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