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Physiol. Genomics 33: 170-179, 2008. First published February 5, 2008; doi:10.1152/physiolgenomics.00125.2007
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Received 8 June 2007; accepted in final form 1 February 2008.
Physiological Genomics 33:170-179 (2008)
1094-8341/08 $8.00 © 2008 American Physiological Society

Quantitative gene expression profiling of mouse brain regions reveals differential transcripts conserved in human and affected in disease models

Camille Brochier1, Marie-Claude Gaillard1, Elsa Diguet2, Nicolas Caudy1, Carole Dossat3, Béatrice Ségurens3, Patrick Wincker3, Emmanuel Roze4, Jocelyne Caboche4, Philippe Hantraye2, Emmanuel Brouillet2, Jean-Marc Elalouf1 and Michel de Chaldée1

1 Commissariat à l'Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay, Service de Biologie Intégrative et Génétique Moléculaire, Gif-sur-Yvette
2 Commissariat à l'Energie Atomique, Institut d'Imagerie Biomédicale, Molecular Imaging Research Center, Orsay
3 Commissariat à l'Energie Atomique, Institut de Génomique, Genoscope-Centre National de Séquençage, Evry
4 Université Pierre et Marie Curie-Paris 6, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche 7102, Paris, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Using serial analysis of gene expression, we collected quantitative transcriptome data in 11 regions of the adult wild-type mouse brain: the orbital, prelimbic, cingulate, motor, somatosensory, and entorhinal cortices, the caudate-putamen, the nucleus accumbens, the thalamus, the substantia nigra, and the ventral tegmental area. With >1.2 million cDNA tags sequenced, this database is a powerful resource to explore brain functions and disorders. As an illustration, we performed interregional comparisons and found 315 differential transcripts. Most of them are poorly characterized and 20% lack functional annotation. For 78 differential transcripts, we provide independent expression level measurements in mouse brain regions by real-time quantitative RT-PCR. We also show examples where we used in situ hybridization to achieve infrastructural resolution. For 30 transcripts, we next demonstrated that regional enrichment is conserved in the human brain. We then quantified the expression levels of region-enriched transcripts in the R6/2 mouse model of Huntington disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease and observed significant alterations in the striatum, cerebral cortex, thalamus and substantia nigra of R6/2 mice and in the striatum of MPTP-treated mice. These results show that the gene expression data provided here for the mouse brain can be used to explore pathophysiological models and disclose transcripts differentially expressed in human brain regions.

biological markers; neurodegenerative diseases; serial analysis of gene expression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE MAMMALIAN BRAIN IS ORGANIZED as a complex network involving a large number of structures. Each of them is primarily identified by its anatomical localization, general morphology, and cytoarchitecture and exhibits a characteristic developmental course and wiring pattern. The establishment and maintenance of these features rely largely on a tight control of gene expression. In the past few years, several large-scale efforts have benefited from the availability of genomic data and technical advances to explore the expression profile of adult mouse brain regions. A variety of strategies have been developed, such as microarray analysis of dissected regions (32, 35, 40) or voxels (5), high-throughput in situ hybridization (21, 25), or replacement of coding sequences by enhanced green fluorescent protein reporter gene in bacterial artificial chromosome transgenic vectors (17).

Here we performed serial analysis of gene expression (SAGE) (37) on 11 regions accurately dissected from the fore- and midbrain of the adult mouse, which have not been considered in earlier SAGE projects (3, 33), and included territories that are rarely collected due to their small size (e.g., areas of the prefrontal cortex, nucleus accumbens, ventral tegmental area). With >1.2 million cDNA tags sequenced, the present work provides a comprehensive quantitative expression database for adult brain regions.

The SAGE method has the advantage of precisely quantifying each transcript detected in a given sample. We could therefore apply stringent comparison criteria to identify genes preferentially expressed in each brain region, and, within each region, we could sort the enriched transcripts by expression level. The resulting lists, curated using data from the literature and complementary experimental approaches, comprise a total of 315 genes, many of them with no established function. For a subset of 50 transcripts, we additionally measured expression levels in human brain regions.

Transcriptomic studies of brain dysfunction usually compare expression profiles of brain regions between patients and unaffected individuals (2, 19, 28) or model and control animals (24, 27). Alternatively, to get insight into the pathogenesis of disorders like Huntington and Parkinson diseases, which primarily affect specific populations of neurons, the genes preferentially expressed in these cells could be analyzed as a priority (6, 12). We therefore measured the expression levels of region-enriched transcripts in brain structures of R6/2 transgenic mice and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, two widely used models of Huntington and Parkinson diseases, respectively.

The present study provides large-scale, knowledge-independent, quantitative gene expression data across a significant number of mouse brain regions. The expression level of any gene can be searched in this database and accurately compared with that of other genes in the same brain region or between different brain regions or tissues for which SAGE data are available. Here we illustrate the usefulness of this resource to find region-enriched transcripts of unknown function with possible relevance to human pathophysiology.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Collection of mouse brain samples.
All experiments on mice were supervised by an investigator licensed by the Ministère de l'Agriculture, in conformity with French regulations. Mice were deeply anesthetized by intraperitoneal injection of pentobarbital sodium (180 µg per gram of body wt) before decapitation. The brains were immediately removed, and all subsequent steps were carried out on ice. The cingulate, motor, and somatosensory cortices, caudate-putamen, and thalamus were scalpel-dissected from 1-mm-thick fresh brain slices cut in an ice-cold coronal matrix (Pelco International, Redding, CA). The orbital, prelimbic, entorhinal cortices, and the substantia nigra were scalpel-dissected from 0.3-mm-thick fresh brain coronal slices cut using a refrigerated Oxford vibratome (Vibratome, St. Louis, MO). The nucleus accumbens was punched from 1-mm-thick coronal slices using 1.1–1.2-mm internal diameter glass capillaries (Propper Manufacturing, Long Island City, NY). The ventral tegmental area was sampled from 0.3-mm-thick coronal slices using a 0.53-mm diameter punch (Stoelting, Wood Dale, IL). All dissections were performed under stereomicroscopic observation with reference to a mouse brain atlas (30). The methods used to dissect each brain region are further depicted in Supplemental Fig. S1.1

Mouse models of neurodegenerative disorders.
R6/2 transgenic mice express a human gene fragment containing the mutation responsible for Huntington disease. They develop a progressive neurological phenotype beginning at two mo of age (26). The striatum, cortex, thalamus, and substantia nigra were sampled from four or five 10- to 12-wk-old R6/2 mice (The Jackson Laboratory, Bar Harbor, ME) and four to six age-matched wild-type controls.

Administration of the neurotoxin MPTP was used to model Parkinson disease (34). Sixteen-week-old male C57BL/6 mice received four intraperitoneal injections of MPTP (20 mg/kg per injection) at 2-h intervals. Six MPTP-treated mice and five PBS-injected controls were killed 24 h after the last injection, and six MPTP-treated mice and four PBS-injected controls were killed 7 days after the last injection. Each brain was divided into two parts. The right caudate-putamen was immediately processed for total RNA extraction, while the left caudate-putamen was stored until measurement of tyrosine hydroxylase immunoreactivity using a rabbit polyclonal antibody (Institut de Biotechnologies Jacques Boy, Reims, France) at a 1:10,000 dilution. Degeneration of the nigrostriatal pathway was indirectly estimated by the relative immunostaining intensity determined from the optical density in the caudate-putamen and the adjacent sensorimotor cortex, using a method adapted from Georgievska et al. (16).

Construction of SAGE libraries.
Brain regions were sampled from 8- to 12-wk-old wild-type male C57BL/6 mice (Charles River Laboratories, L'Arbresle, France). Samples from three to 15 animals, depending on the size of the dissected region, were pooled and Dounce-homogenized in Lysis/Binding Buffer from Dynabeads mRNA DIRECT kit (Invitrogen, Carlsbad, CA). SAGE libraries were constructed according to a modified protocol adapted to small amounts of starting material (38). Sau3AI was used as the anchoring enzyme, and either BsmFI (for libraries from the somatosensory and entorhinal cortices, caudate-putamen, nucleus accumbens and thalamus) or MmeI (for libraries from the whole brain, orbital, prelimbic, cingulate, and motor cortices, substantia nigra, and ventral tegmental area) served as the tagging enzyme. BsmFI produces 10-bp-long tags, whereas MmeI generates 15-bp-long tags. We used MmeI tags to facilitate the annotation process (31). For comparisons between libraries, MmeI tags were converted to 10-bp-long tags.

Automated DNA sequencing was carried out on plasmid minipreparations either in our laboratory (for libraries from the somatosensory cortex, caudate-putamen, nucleus accumbens and thalamus) or at the Genoscope (Evry, France) (for all other libraries). Sequence analysis, tag extraction, and counting were performed as described (4). All SAGE data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database under accession nos. GSM183639, GSM183640, GSM183644-GSM183653.

Selection and annotation of region-enriched tags.
In each library, tags occurring at a frequency ≥5 in 80,000 and showing a sevenfold enrichment compared with six other libraries were selected (e.g., n = 7 vs. n = 0 or 1; n = 6 or 5 vs. n = 0; 80,000 tags per library). Monte-Carlo simulations indicate that the probability to observe such an enrichment by chance is still <0.05 for low tag counts (41). For libraries constructed from cortical areas, this criterion was broadened to include tags showing a sevenfold enrichment compared with five other libraries. This allowed retaining pancortical transcripts.

Each selected tag was manually annotated as previously described (10). Only the most abundant tag from each transcript was used for interregional comparisons. The resulting list of transcripts was curated using data from the literature and real-time quantitative RT-PCR (qRT-PCR) experiments. When the distribution of a transcript within the brain as reported in the literature was incompatible with the observed tag abundances, the annotation was considered improper and the tag was discarded from the list. Furthermore, the expression levels of 120 poorly documented genes were measured in mouse brain regions using qRT-PCR. For each gene, SAGE and qRT-PCR data were expressed as percentages, and the value was set to 100% in the region where the SAGE tag count is maximum. The mean absolute difference between both techniques was then calculated across all regions except the region set to 100% (Supplemental Fig. S2A). When plotting the cumulative distribution curve of this variable, a population of genes exhibiting a mean value <40% clearly stands out (Supplemental Fig. S2B). However, higher mean values can still be compatible with proper tag identification. In particular Creg1 transcripts, though exhibiting a mean value of 46.8%, have been independently demonstrated to be enriched in mouse thalamus (21). Slc44a1 transcripts (mean value = 49.1%) proved to be enriched in human substantia nigra (GoTable 2). To prevent undue rejection of these genes, the validation threshold was set to 50%. When the mean value was above this threshold, the annotation of the SAGE tag was considered incorrect, and the tag was removed from the list. Seventy-eight genes (65%) had a mean <50% and were retained (Supplemental Table S3), ten of which, including Creg1 and Slc44a1, had a mean between 40 and 50%. Forty-two genes (35%) had a mean >50% and were discarded.


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  		Table 1. Examples of brain region-enriched transcripts detected by SAGE

 

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  		Table 2. Transcripts whose regional enrichment is conserved between human and mouse

 
Functional classification of region-enriched transcripts.
Functional classification was performed using Database for Annotation, Visualization, and Integrated Discovery (DAVID) tools (11). The full list of mouse region-enriched transcripts (315 genes) was analyzed, as well as sublists of transcripts enriched in gross anatomical divisions: the cerebral cortex, including all the cortical areas investigated (86 genes), the striatum (caudate-putamen and nucleus accumbens, 125 genes), the thalamus (65 genes), and the ventral midbrain (substantia nigra and ventral tegmental area, 67 genes). The 30 human region-enriched transcripts were also analyzed by DAVID. Overrepresentation of a functional class in a gene list compared with the whole genome was assessed by the Expression Analysis Systematic Explorer (EASE) score (20).

Real-time qRT-PCR.
Mouse brain samples were pooled between animals, with the exception of striata, cortices, thalami, and substantiae nigrae from R6/2 mice and corresponding controls and striata of MPTP-treated mice and corresponding controls, which were processed independently for each animal. Total RNAs were extracted using a guanidinium thiocyanate-based method. Human RNA samples were purchased from commercial sources (Ambion, Austin, TX; Clontech Laboratories, Mountain View, CA).

qRT-PCR was performed as previously described (8). The ubiquitously expressed peptidylprolyl isomerase A gene was used for normalization. All experiments were carried out on two replicates of each sample. Primer sequences are available from the authors upon request.

Combined in situ hybridization and immunofluorescence.
In situ hybridization was performed as previously described (8), with the following modifications. cDNA fragments of A930038C07Rik (nucleotides 494-2313 of AK029210), Slc10a4 (nucleotides 81-1506 of AK087479), and Agpat4 (nucleotides 187-1347 of AK005139) were cloned into pCR4Blunt-TOPO or pCR2.1-TOPO vectors (Invitrogen). One microgram of each linearized plasmid served to perform in vitro transcription using T3 (antisense probes) or T7 (sense probes) RNA polymerase. Rabbit anti-tyrosine hydroxylase (Chemicon International, Temecula, CA) was added at a 1:1,000 dilution to the solution containing the alkaline phosphatase-conjugated Fab fragments. Before nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate staining, washed slides were incubated 3 h at room temperature with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen) diluted 1:1,000 in Tris-buffered saline supplemented with 0.1% Tween 20, 0.5% gelatin, and 20% normal goat serum, then washed extensively. Slides were coverslipped with FluorSave Reagent (Merck, Darmstadt, Germany) and examined using a Leica DMRXA fluorescence microscope (Leica, Wetzlar, Germany). Tyrosine hydroxylase-positive neurons were visualized under dark field with appropriate filter, while in situ hybridization-positive cells were visualized using bright-field illumination. Images were acquired with MetaMorph 4.6 software (Molecular Devices, Sunnyvale, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Generation of a gene expression database from mouse brain regions.
SAGE libraries were generated from the whole brain and 11 brain regions of the adult wild-type mouse (Supplemental Fig. S1). At least 60,000 tags were sequenced from each library, yielding a total of 1,269,681 tags in the whole project with a median value of ~84,000 tags (Supplemental Table S1). In the present paper, all tag counts were normalized to a total number of 80,000 tags per library.

While the most abundant transcripts are sufficient to identify the brain tissue (9), a deeper analysis is required to detect differential transcripts. Tyrosine hydroxylase messenger, the most abundant region-enriched transcript, accounts for only 0.2% of the total poly(A)+ RNA mass of the ventral tegmental area (tag count = 174 in 80,000).

Identification of region-enriched transcripts.
Tags enriched in brain regions were selected according to criteria calibrated on known regional markers (typically a sevenfold enrichment in one library compared with six others). We annotated 315 different tags meeting these criteria (Supplemental Table S2), a selection of which is displayed in Table 1. A wide range of expression levels was explored, down to transcripts representing only 0.006% of the poly(A)+ RNA mass (tag count = 5 in 80,000), like RIKEN cDNA 2010001M06 gene transcript in the caudate-putamen. The list is punctuated by known regional markers [e.g., dopamine receptor D1A (29); tyrosine hydroxylase (39)] but also contains genes with uncharacterized function, like most of those shown in Table 1, and even isolated expressed sequence tags (ESTs) (CK383136).

All region-enriched transcripts were functionally classified using Gene Ontology vocabulary (1). For 20% of them, the biological process and molecular function were either not mentioned or annotated only as unknown. Many genes involved in signal transduction were identified in all regions, while genes associated with locomotory behavior were significantly overrepresented in the striatum (caudate-putamen and nucleus accumbens) and ventral midbrain (substantia nigra and ventral tegmental area), compared with the whole genome (Supplemental Fig. S3). These structures are indeed known to be involved in the control of voluntary movement.

Distribution of region-enriched transcripts in mouse and human brains.
For each differentially expressed transcript, the literature was carefully reviewed, and a reference confirming regional enrichment was provided whenever available (Supplemental Table S2). The expression level of a number of poorly documented genes was further explored in mouse brain regions using qRT-PCR (Supplemental Table S3). This analysis was extended to the hippocampus and the cerebellum. For each gene, the mean absolute difference between SAGE and qRT-PCR data was calculated across brain regions. The agreement between both techniques is all the better as this mean value is low.

We investigated the expression of some region-enriched transcripts at higher resolution using in situ hybridization to mouse brain sections. Figure 1 shows the distribution of three mRNA species detected by digoxigenin-labeled riboprobes in the ventral midbrain. In these three examples, positive cells were located in the substantia nigra pars compacta and the ventral tegmental area, but no signal was observed in the substantia nigra pars reticulata. Co-detection of tyrosine hydroxylase, visualized by immunofluorescence, suggests that the cells expressing these transcripts are dopaminergic neurons.


Figure 1
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  		Fig. 1. Distribution of three mRNA species in mouse ventral midbrain and co-detection of tyrosine hydroxylase. mRNAs were evidenced by in situ hybridization using digoxigenin-labeled riboprobes whereas tyrosine hydroxylase (TH) was visualized by immunofluorescence. SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area.

 
To address whether regional enrichment was conserved in man, we measured the expression level of 50 genes in human whole brain and eight brain regions using qRT-PCR. These 50 candidates were sampled among poorly documented transcripts enriched in different mouse brain regions and spanning a wide range of expression levels. The human transcripts for which qRT-PCR evidenced a 2.5-fold enrichment in one region compared with three others (including the whole brain) were retained as region enriched. We considered that regional enrichment was conserved between man and mouse when a transcript enriched in human cerebral cortex (motor cortex, parietal lobe, or temporal cortex), caudate or putamen, thalamus, or substantia nigra was enriched, respectively, in mouse cerebral cortex, striatum (caudate-putamen or nucleus accumbens), thalamus, or ventral midbrain (substantia nigra or ventral tegmental area). Overall, 30 transcripts met these criteria (Table 2, Fig. 2 and Supplemental Fig. S4). Among them, transcripts coding for membrane proteins (12 genes, EASE score = 0.009), enzymes with transferase activity (7 genes, EASE score = 0.004), proteins involved in intracellular signaling cascades (5 genes, EASE score = 0.013), or in cellular lipid metabolism (3 genes, EASE score = 0.068) are significantly over-represented compared with the human genome.


Figure 2
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  		Fig. 2. mRNA levels of region-enriched transcripts in human brain assessed by quantitative (q) RT-PCR. Mouse gene symbols are provided in parenthesis when different from their human orthologs. For each transcript, the expression level was set to 100% in the whole brain. Note that the parietal lobe contains the somatosensory cortex, and the temporal cortex includes the entorhinal area.

 
Altered gene expression in mouse models of neurodegenerative disorders.
Region-enriched transcripts might be selectively dysregulated in pathophysiological conditions affecting the structures where they are predominantly expressed. Huntington disease is characterized by severe striatal atrophy due to the loss of medium spiny neurons. Other regions are also affected, though less dramatically (18). Using qRT-PCR, we measured gene expression in the R6/2 transgenic mouse model of Huntington disease (26). As recapitulated in Table 3, we analyzed transcripts enriched in the striatum (caudate-putamen and nucleus accumbens), thalamus, substantia nigra or cerebral cortex as well as pancerebral transcripts (listed in Supplemental Table S4). Ten- to twelve-week-old R6/2 mice (n = 3–5) were compared with age-matched wild-type controls. Among the 27 striatal transcripts quantified, 14 were significantly less abundant in R6/2 than in wild-type striata and only one (Rnf13) was more abundant (Fig. 3A). No significant difference was observed for the 12 others, arguing against a general dysregulation of striatal transcription. A large proportion of the thalamic (Fig. 3B) and nigral (Fig. 3C) candidates exhibited a significantly lower expression level in R6/2 than in wild-type animals, indicating that transcriptional changes are not confined to the striatum. The proportion of affected cortical transcripts was smaller (Fig. 3D). None of the 12 pancerebral transcripts tested was significantly altered in more than two brain regions of R6/2 mice (Fig. 3E). Both up- and downregulations were observed among them, whereas all affected region-enriched transcripts except Rnf13 were downregulated.


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  		Table 3. Proportion of transcripts affected in the R6/2 mouse model of Huntington disease

 

Figure 3
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  		Fig. 3. qRT-PCR analysis of gene expression in R6/2 mouse brain regions. A: quantification of striatum-enriched transcripts in striata of R6/2 mice. B: quantification of thalamus-enriched transcripts in thalami of R6/2 mice. C: quantification of substantia nigra-enriched transcripts in substantiae nigrae of R6/2 mice. D: quantification of cortex-enriched transcripts in cortices of R6/2 mice. E: quantification of pancerebral transcripts in striata, thalami, substantiae nigrae, and cortices of R6/2 mice. The expression level of each transcript is displayed as percent of the wild-type control value ± standard error of the mean (SE). Significant differences were assessed by Student's unpaired t-test: *P < 0.05; **P < 0.01; ***P < 0.001. Measurements were performed on the striata and cortices of 4 R6/2 mice and 4 controls, and on the thalami and substantiae nigrae of 5 R6/2 mice and 6 controls, except for Tmem90a, BC072620, Spock3, Slc41a1, St8sia3, Dclk3, which were analyzed on the striata of 3 R6/2 mice and 3 controls, and Brp44l, Dstn, Hprt1, Bex1, B2m, which were analyzed on the substantiae nigrae of 4 R6/2 mice and 5 controls.

 
Parkinson disease results from the selective degeneration of the dopaminergic neurons projecting from the substantia nigra to the caudate nucleus and putamen. This phenotype can be recapitulated in mice by MPTP intoxication (34). In the present study, measurement of tyrosine hydroxylase immunoreactivity in the caudate-putamen indicated a severe decrease in dopamine synthesis 24 h after the last MPTP injection. Tyrosine hydroxylase depletion was still evident seven days after the last injection (Fig. 4A). Using qRT-PCR, we thus quantified 26 striatum-enriched and 12 pancerebral transcripts in the caudate-putamen of MPTP-lesioned animals (n = 6) and PBS-injected controls at both time points. Three types of response to MPTP were observed among striatum-enriched transcripts (Fig. 4B): an early, transient downregulation (four genes); an early, transient upregulation (four genes); and a delayed upregulation (three genes). All six affected pancerebral transcripts exhibited a delayed upregulation (Fig. 4C).


Figure 4
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  		Fig. 4. Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication on mouse striatum. Brains were removed either 24 h or 7 days after the last MPTP injection. A: striatal tyrosine hydroxylase immunoreactivity. Left: representative sections. Right: specific optical density (OD) measurements were performed on 11 sections per animal and compared between MPTP-lesioned mice (n24 h = 6; n7 days = 6) and PBS-injected controls (n24 h = 5; n7 days = 4) using ANOVA (***P < 0.001). B: qRT-PCR quantification of striatum-enriched transcripts in the caudate-putamen of MPTP-treated mice. The following striatum-enriched transcripts were not affected by MPTP treatment at any time point: Sh2d5, Tesc, St8sia3, Tmem90a, Gm705, BC072620, Tbl1x, Mgat5b, Chchd3, Myt1l, Rnf13, Hs6st2, Mbd2, Crym, 1110018G07Rik. C: qRT-PCR quantification of pancerebral transcripts in the caudate-putamen of MPTP-treated mice. The following pancerebral transcripts were not affected by MPTP treatment at any time point: Bex2, Brp44, Bex1, Ppp2r5a, Dstn, Hprt1. The expression level of each transcript is displayed as a percentage of the corresponding PBS-injected control value ± SE. Significant differences were assessed by Student's unpaired t-test: *P < 0.05; **P < 0.01; ***P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present SAGE resource provides an in-depth analysis of gene expression in mouse fore- and midbrain structures. From this dataset, we found 315 transcripts that are preferentially expressed in discrete brain regions and, for 78 of them, we present independent expression level measurements in mouse brain. For 30 of them, we demonstrated that regional enrichment is conserved in man, and further analysis of the list of 315 candidates is expected to reveal additional conserved region-enriched transcripts. This work offers a number of advantages over the existing literature and databases. In particular, SAGE tag counts provide a quantitative measure of expression level, allowing accurate comparisons both across brain regions and between transcripts. For instance, in situ hybridization signals for striatin and tescalcin mRNAs have comparable intensities in the caudate-putamen (21), whereas SAGE data show that tescalcin messengers are five times more abundant. This aspect is of particular interest when selecting promoters to direct efficient expression in specific brain regions. Furthermore, the regional specificity of any transcript can be easily checked from our data. As an example, protein tyrosine phosphatase, nonreceptor type 5 (originally termed "striatal enriched phosphatase"), though considered as a marker of the caudate-putamen (23), is also expressed at significant levels in frontal areas of the cortex (tag count = 51 in the caudate-putamen; 20 in the prelimbic cortex; 17 in the orbital, cingulate, and motor cortices; 80,000 tags per library). It has not passed our criteria for regional enrichment. By contrast, adenosine A2a receptor transcript turned out to be enriched in the caudate-putamen. Its abundance is reputed to be low (13). Thus, we achieved a high sensitivity that could still be increased by sequencing more tags from the existing SAGE libraries. The libraries from the whole brain, the cingulate, motor, and entorhinal cortices have been extensively sequenced (Supplemental Table S1), and may already be used to detect rare transcripts.

The number of enriched transcripts varies from one brain region to another. They are especially numerous in the caudate-putamen and scarce in cortical areas, which may reflect greater cellular heterogeneity in the cortex (36). Furthermore, we observed partial overlap between related regions. In particular, both SAGE and qRT-PCR data suggest that several transcripts are pancortical (e.g., RIKEN cDNA 3110035E14 gene transcript). However, we also identified transcripts enriched in restricted cortical territories, such as C1q-like 3 transcript in frontal areas and nephroblastoma overexpressed gene transcript in the entorhinal cortex. Moreover, within a given structure, enriched transcripts may be synthesized in distinct subregions. As an example, in the substantia nigra, we were able to record genes specifically expressed in the pars compacta, as demonstrated for RIKEN cDNA A930038C07 gene by in situ hybridization, as well as genes expressed in the pars reticulata, like protein inhibitor of activated STAT4 (15).

Differentially expressed transcripts may be involved in pathophysiological mechanisms affecting specific brain structures. In the R6/2 mouse model of Huntington disease, 56% of the striatum-enriched transcripts that we analyzed were significantly dysregulated. For five of them (Scn4b, Tesc, St8sia3, Tmem90a, Gm705), we corroborate previous data obtained on striata of R6/2 (24) or R6/1 (12) mice. These five genes, as well as three others (Spock3, Gpr155, Crym), have recently been reported to be downregulated in the caudate nucleus of Huntington disease patients (19), thus confirming the reliability of the R6/2 model. Additionally, we show a significant downregulation for genes (SH2 domain containing 5; RIKEN cDNA 2010001M06 gene) whose expression has not been previously measured in mouse models or patients. Still, 44% of the striatal candidates were unchanged, suggesting that particular transcription pathways are selectively affected.

Furthermore, we provide evidence for multiple transcriptional alterations specific to the thalamus, substantia nigra, and, to a lesser extent, cerebral cortex of R6/2 mice. All these regions are connected to the striatum and participate in the control of voluntary movement. So far, gene expression had not been specifically analyzed in the thalamus and substantia nigra of models or patients of Huntington disease, though imaging of patients has revealed abnormalities in these structures, including in early stages of the disease (14). Interestingly, one of the transcripts that we found to be downregulated in the cortex encodes kalirin, a specific interactor of huntingtin-associated protein 1 (HAP1) (7). HAP1 binds to huntingtin, the protein encoded by the gene mutated in Huntington disease (22).

In the MPTP model of Parkinson disease, 45% of the transcripts analyzed in the striatum were significantly altered. Different patterns of regulation were observed: among the striatum-enriched transcripts, most changes were detected 24 h after the last injection and did not persist after 7 days, while all affected pancerebral transcripts were dysregulated mostly after seven days. Although using a different protocol of MPTP intoxication, Miller et al. (27) have reported widespread transcriptional effects in the striatum of MPTP-treated mice, in particular 7 days after the last injection. Noteworthy, we evidenced five striatum-enriched transcripts (Scn4b, Spock3, Slc41a1, Gpr155, Lypd1) that were also affected in the R6/2 model, indicating that common pathways may be mobilized in the striatum of both R6/2 and MPTP-intoxicated mice.

Many of the region-enriched transcripts provided here are poorly characterized, and 20% were classified as functionally unknown. Since regional enrichment is conserved in man in a majority of cases, and because brain regions were partly selected on the basis of their involvement in human neuropsychiatric disorders, the present work provides a reservoir of candidates to investigate brain pathophysiology.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
C. Brochier was the recipient of a fellowship from the Ministère de l'Education Nationale, de la Recherche et de la Technologie. E. Diguet and E. Brouillet were supported by CNRS, CEA, and High Q Foundation. The Genoscope received financial support from the Consortium National de Recherche en Génomique. We also received financial support from IMAGEN European integrated project (EUR617037286).


   ACKNOWLEDGMENTS
 
The authors thank Jean-Christophe Aude and Arnaud Martel for informatics support, Françoise Condé for help in dissection of brain regions, and Peggy Baudouin-Cornu for helpful discussion on statistical matters.

Expression data reported in this paper have been deposited in the GEO database under accession nos. GSM183639, GSM183640, GSM183644-GSM183653.


   FOOTNOTES
 
Address for reprint requests and other correspondence: J.-M. Elalouf, CEA Saclay, Bâtiment 144, F-91191 Gif-sur-Yvette Cedex, France (e-mail: jean-marc.elalouf{at}cea.fr) or M. de Chaldée, CEA Saclay, Bâtiment 144, F-91191 Gif-sur-Yvette Cedex, France (e-mail: michel.de.chaldee{at}cea.fr).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

1 The online version of this article contains supplemental material. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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