Neural system-enriched gene expression: relationship to biological pathways and neurological diseases

Jianhua Zhang1, Amy Moseley2, Anil G. Jegga3, Ashima Gupta3, David P. Witte4, Maureen Sartor5, Mario Medvedovic5, Sarah S. Williams3, Cathy Ley-Ebert6, Lique M. Coolen1, Gregory Egnaczyk7, Mary Beth Genter5, Michael Lehman1, Jerry Lingrel2, John Maggio7, Linda Parysek1, Ryan Walsh1, Ming Xu1 and Bruce J. Aronow3,6

1 Departments of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati 45267
2 Molecular Genetics, Microbiology and Biochemistry, University of Cincinnati College of Medicine, Cincinnati 45267
5 Environmental Health, University of Cincinnati College of Medicine, Cincinnati 45267
7 Pharmacology and Cellular Biophysics, University of Cincinnati College of Medicine, Cincinnati 45267
3 Division of Pediatric Informatics, Children’s Hospital Research Foundation, Cincinnati, Ohio 45229
4 Division of Pathology, Children’s Hospital Research Foundation, Cincinnati, Ohio 45229
6 Division of Molecular and Developmental Biology, Children’s Hospital Research Foundation, Cincinnati, Ohio 45229


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To understand the commitment of the genome to nervous system differentiation and function, we sought to compare nervous system gene expression to that of a wide variety of other tissues by gene expression database construction and mining. Gene expression profiles of 10 different adult nervous tissues were compared with that of 72 other tissues. Using ANOVA, we identified 1,361 genes whose expression was higher in the nervous system than other organs and, separately, 600 genes whose expression was at least threefold higher in one or more regions of the nervous system compared with their median expression across all organs. Of the 600 genes, 381 overlapped with the 1,361-gene list. Limited in situ gene expression analysis confirmed that identified genes did represent nervous system-enriched gene expression, and we therefore sought to evaluate the validity and significance of these top-ranked nervous system genes using known gene literature and gene ontology categorization criteria. Diverse functional categories were present in the 381 genes, including genes involved in intracellular signaling, cytoskeleton structure and function, enzymes, RNA metabolism and transcription, membrane proteins, as well as cell differentiation, death, proliferation, and division. We searched existing public sites and identified 110 known genes related to mental retardation, neurological disease, and neurodegeneration. Twenty-one of the 381 genes were within the 110-gene list, compared with a random expectation of 5. This suggests that the 381 genes provide a candidate set for further analyses in neurological and psychiatric disease studies and that as a field, we are as yet, far from a large-scale understanding of the genes that are critical for nervous system structure and function. Together, our data indicate the power of profiling an individual biologic system in a multisystem context to gain insight into the genomic basis of its structure and function.

microarray; nervous system; global context


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE MAMMALIAN NERVOUS SYSTEM consists of a complex network of neurons and supporting cells that are able to integrate internal and external signals and coordinate responses. To gain a comprehensive molecular description for its development and function, an increasing number of studies have used the genomics/microarray approach to compare gene expression in the nervous system during its development and aging (5, 25, 37, 53), among its subregions (6, 57, 68, 91, 92), or in its particular regions under different experimental or diseased conditions (50, 52, 59, 61, 64, 78). However, few studies addressed the critical question of how nervous tissues differ in their gene expression repertoire from peripheral tissues.

Obtaining an overview of nervous system genomic anatomy is critical for better understanding both normal nervous system function as well as the impact of gene expression on diseases. Although numerous single gene mutations, genetic polymorphisms, and alterations of gene expression have been found in various nervous system diseases (9, 30, 65, 85, 94), the molecular mechanisms of how genetic abnormalities lead to pathological consequences in the nervous system are poorly understood. One primary reason for the lack of understanding of disease mechanisms is the existence of and extensive interactions among nervous system-expressed gene products in influencing disease processes.

Warrington et al. (84) compared the expression of about 7,000 genes in 11 different human adult and fetal tissues and provided a glimpse of how normal tissues differ in their genetic constituents, especially regarding the expression of housekeeping genes. In another study, Penn et al. (58) discovered that 30% open reading frames from genome sequencing are novel genes and 29% are similar but not identical to known sequences using mRNA from 10 human tissues and cell types. Both the Warrington and Penn studies treated the brain as one single tissue. Because of the heterogeneity of brain regions, genes highly expressed in small regions of the brain would be masked and may not appear to be highly expressed in the whole brain. Treating the entire brain as one single tissue is therefore limited in providing comprehension of nervous tissue differentiation.

Miki et al. (51) provided a more comprehensive comparison of 49 adult and embryonic mouse tissues and provided evidence of neurogenesis and remodeling in the embryonic brain and postnatal cerebellum. The same group further described a detailed examination of expression profiles of enzymes in metabolic pathways and particularly glycolysis and illustrated differences in energy utilization among tissues such as muscle, liver, testes, kidney, and whole brain (51). A report by Su et al. (75) provided an elegant analysis of genes expressed in different brain regions as well as peripheral tissues focusing on G protein-coupled receptors and kinases, genes containing a pituitary response element, and genes highly expressed in human prostate cancer compared with other normal tissues.

Although those studies described above did include individual brain regions as well as some peripheral tissues, the analyses were focused on very specific biological questions, for example, cancer or energy metabolism. To address the more broadly based question of how nervous tissues differ in genetic composition from peripheral tissues, and how genes abundantly expressed in the nervous tissues cater to particular need of the nervous system and influence susceptibility to nervous system diseases, we have here compared the gene expression profiles of 10 distinct mouse nervous system tissues vs. 72 other mouse tissues representing 30 developing and adult stage organs. We identified genes that are abundant in one or more nervous tissues, including genes of diverse functional categories and genes known to cause neurological diseases. Our data suggest that this global-context approach provides a powerful tool and a large-scale resource for nervous system gene repertoire profiling, pathway analyses, and identification of candidate genes for neurological diseases.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparing poly(A)+ RNA from mouse tissues.
All animal procedures were approved by the IACUC. We obtained C57BL/6 male mice of 8 mo of age from the Jackson Laboratory (Bar Harbor, ME) for all the adult tissues. In-house timed breeding was carried out to provide embryonic day 18.5 (denoted E18 for all text and figures for simplicity) and postanatal day 2 (P2) brains. We dissected the 10 adult nervous tissues from 3–10 mice and immediately homogenized them in TRIzol reagent (Life Technologies, Rockville, MD). The 10 nervous tissues were: hippocampus (HC), nucleus accumbens (NAc), striatum (Str), hypothalamus (HT), cerebellum (Cb) and olfactory cortex (OlCt), spinal cord (SC), dorsal root ganglia (DRG), and the whole brain at E18 and at P2. All 10 nervous tissues were dissected within clearly defined boundaries immediately after euthanizing the mice. We dissected all cervical, thoracic, and lumbar DRGs from 10 mice, HC from 4 mice, HT, NAc and Str from 6 mice, and other nervous tissues from 3 mice each. We then isolated total RNA following the manufacturer’s protocol (Life Technologies) and purified poly(A)+ RNA using Oligotex resin (Qiagen, Valencia, CA). RNA concentration was quantified using RiboGreen dye (Molecular Probes, Eugene, OR) and profiled for size distribution and ribosomal RNA contamination using an Agilent Bioanalyzer 2100. We submitted in duplicate 600-ng samples of poly(A)+ RNA at 50 ng/µl per tissue to Incyte Genomics for cDNA labeling and microarray hybridization.

Microarray hybridization.
Incyte Genomics (Palo Alto, CA) prepared labeled cDNA from poly(A)+ RNAs using the GEMBright random primer reverse-transcription labeling kit (5' dye-terminated random primers) and competitive hybridization to mouse GEM1 microarrays (14). We also confirmed the microarray data from Incyte with our in-house microarray facility using the identical Incyte mouse GEM1 clone set (http://microarray.uc.edu/). Duplicate arrays were hybridized for each poly(A)+ RNA sample. Each hybridization was performed with Cy5-labeled poly(A)+ RNA from nervous tissues in competition with the "universal reference," which was Cy3-labeled poly(A)+ RNA from whole postnatal day 1 (P1) mouse.

Data analyses.
We used Incyte GEMTools software to analyze the quality of each hybridization using the parameters of signal to background fluorescence, spot geometry, the relative intensities of control genes "spiked" into the labeling reactions, and an assessment of dynamic range exhibited by each fluorescence channel. Spike controls exhibited low variation across the microarray series (data not shown). Data manipulation including normalization, filtering, and clustering was carried out using GeneSpring software (Silicon Genetics, Redwood City, CA). All selection and cutoff filters were applied to the mean expression ratio values based on the two replicate hybridizations (14).

We performed three types of normalization of the data. First, a per-array "whole mouse normalized" expression value for each gene was derived from the simple ratio of the sample to the whole mouse reference poly(A)+ RNA. For each array, a single linear correction, "balance coefficient," was used to multiply the Cy5 channel to correct for the median Cy3-to-Cy5 intensity value ratios. We found no significant bias in using Cy3 and Cy5 (P < 0.05 for 10 random selected genes between dye reversal experiments), and we eliminated all dye preference by using the next normalization. Second, a "each gene normalized" value for each gene in each tissue was derived from dividing the "whole mouse normalized" expression value for each gene in that tissue by the median of all "whole mouse normalized" expression values for that gene in all 82 sampled tissues. The 82 sampled tissues include the 10 nervous tissue and 72 peripheral tissues representing 30 organs, such as skeletal muscle, male and female reproductive organs, and different regions of the gastrointestinal tract, as well as organs in a developmental context including lung, liver, kidney, and heart. Genes highly expressed in the nervous system were then selected according to their abundance in the nervous tissues over the median values across all 82 tissues.

Genes highly expressed in the nervous system were annotated by putative functions of encoded proteins based on classification data provided from GenBank (http://www.ncbi.nlm.nih.gov), GeneCards (http://bioinformatics.weizmann.ac.il/cards; Ref. 63), TIGR (http://www.tigr.org), and the MGI resource version 2.7 (http://www.informatics.jax.org). The functional categorization of these genes was verified from searching PubMed.

Statistic analysis.
For analysis of genes expressed in the nervous tissues vs. peripheral tissues, analysis of variance was used based on Welch ANOVA (Benjamini and Hochberg false discovery rate, P < 0.05) provided by the GeneSpring software. We identified 1,361 genes that are significantly highly expressed in nervous tissues compared with peripheral tissues.

In situ hybridization.
cDNA clones were purchased from Incyte Genomics. In situ hybridization was performed as previously described (89). Brains from C57BL/6 mice were dissected, fixed in 4% paraformaldehyde in PBS, pH 7.4, overnight at 4°C, saturated in 30% sucrose, and frozen in liquid nitrogen. 35S-labeled UTP riboprobes to detect specific mRNA transcripts were synthesized from each cDNA cloned into BlueScript transcription vectors (Stratagene). Cryostat sections of the mouse brains cut at 8 to 10 µm were digested in proteinase K (0.1%) for 5 min at room temperature, acetylated in acetic anhydride, and dehydrated before being hybridized overnight at 45°C with 1 x 106 counts per slide. Following hybridization, the sections were digested with 50 µl/ml of RNase A and RNase T1, then stringently washed with 50% formamide in 0.1x SSC at 50°C. Slides were then dehydrated, dipped in NTB2 emulsion, exposed for periods ranging from 2–3 wk, and developed. Controls for riboprobe specificity included use of sense probe, as well as predigestion with RNases. Slides were counterstained with hematoxylin and eosin.

Data archive.
Gene identities and expression data of the 381 genes that are highly expressed in the nervous tissues are available on our microarray database web server (http://genet.cchmc.org) in the mouse GEM1 genome, in the ZhangEtalBrain2004 subdirectory.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Identification of genes highly expressed in mouse adult nervous tissues.
To gain a global view of the spectrum of genes highly expressed in the nervous system, we constructed a gene expression database in which we compared gene profiles in nervous tissues in relation to diverse mouse tissues using the C57BL/6 mouse strain. We have taken an approach based on a two-channel cDNA microarray technology in which a universal reference poly(A)+ RNA was used to intercompare relative expression profiles of widely different mouse tissue samples. This approach allows for direct comparison of multiple tissues and the ability to add more tissues and experimental conditions to the database. The 8,734 cDNAs on the microarray were clustered according to their expression patterns using self-organizing map with Pearson correlation (76) as implemented in GeneSpring (Fig. 1A).



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Fig. 1. Molecular signature of the mammalian nervous system. A: self-organizing map and Experiment Tree clustering of the expression of 8,734 cDNAs in all 82 tissues normalized against the median level of expression of each gene over all 82 tissues. Of these, 1,361 genes are highly expressed in the 10 nervous tissues vs. other tissues by Welch ANOVA. Six hundred genes are expressed at threefold or higher in at least one nervous tissue over their median expression in all 82 tissues. We found 1,580 are either in the 1,361-gene set or in the 600-gene set. We found 381 genes are in both the 1,361-gene set and the 600-gene set. B: self-organizing map and Experiment Tree clustering of the expression of 1,580 cDNAs in all 82 tissues normalized against the median level of expression of each gene over all 82 tissues. The red color indicates high expression, the yellow color indicates average expression, and the blue color indicates low expression. C: Venn diagram of the 1,361 genes highly expressed by nervous tissues according to Welch ANOVA and the 600 genes expressed at threefold or higher in at least one nervous tissue over the median of all 82 tissues. We found 980 genes are highly expressed in nervous tissue with statistical significance, while their expression in any nervous tissue is less than threefold over the median of all 82 tissues. We found 381 genes are both highly expressed in all nervous tissues over other tissues with statistic significance and expressed at threefold or higher in at least one nervous tissue over the median of all 82 tissues. We found 219 genes are expressed at threefold or higher in at least one nervous tissue over the median of all 82 tissues, while their expression are statistically higher in all nervous tissues vs. other tissues.

 
We selected genes that are highly enriched in the nervous system by two approaches. First, we used a Welch-ANOVA (Benjamini and Hochberg false discovery rate) analysis of the 10 nervous tissues vs. other tissues with a cutoff of P < 0.05. We identified 1,361 genes that are statistically higher in the 10 nervous tissues compared with other tissues. These genes represent 13.7% of all genes arrayed and may participate in common functions in nervous tissues. Second, from 8,734 genes, we identified 600 genes that exhibited threefold or greater expression levels in at least 1 of the 10 nervous tissues (E18, P2, DRG, Cb, HC, HT, SC, NAc, Str, and OlCt) over median values across all nervous and peripheral tissues. Therefore, a total of 1,580 genes fit the first or the second identification criteria. We clustered these 1,580 cDNAs according to their expression patterns using self-organizing map with Pearson correlation (76) and "Experiment Tree" analyses as implemented in GeneSpring (Fig. 1B). The 1,361 and the 600 genes have 381 in common, and represent a primary set of nervous tissue-enriched genes (Fig. 1C). The 219 genes left from the 600-gene list that also fail to be included in the 1,361-gene list may be genes exhibiting region-specific functions in the nervous system. See Supplemental Table S1 for detailed description of these gene lists, available at the Physiological Genomics web site.1

Data verification.
To verify the quality of our results, we sampled 12 genes in our gene expression profile data to compare with work published in the literature (Fig. 2). We found outstanding agreements in all cases with genes encoding: synaptogyrins 2 and 3, {alpha}-synuclein and {gamma}-synuclein, Na+-K+-ATPase {alpha}1 and {alpha}2, pirin, retinoid X receptor-{gamma} (RXR{gamma}), Slit1, Fabp7, calcium ATPase 2A (Cacna), and Jak2 (10, 12, 15, 17, 28, 47, 56, 87, 91). For example, synaptogyrin 3 is highly expressed in the nervous tissues in our study, whereas synaptogyrin 2 is expressed at a lower level in the nervous tissues compared with its expression in peripheral tissues. This finding agrees perfectly with and expands published work with Northern analysis that synaptogyrin 3 is highly expressed in the brain but almost nondetectable in heart, lung, liver, skeletal muscle, and kidney, whereas synaptogyrin 2 is highly expressed in the peripheral tissues and with very low levels in the brain (28). Similarly, we found that RXR{gamma} is highly expressed in the P2 brain, Str, and OlCt, a result in general agreement with the observation by in situ hybridization that RXR{gamma} is highly expressed in the striatum and olfactory tubercle, but low in the hippocampus, hypothalamus, cerebellum, and spinal cord (91), consistent with the previous studies that RXR{gamma} is important for dopamine D2 receptor expression (67). Furthermore, RXR{gamma} is important in modulating locomotion and response of rodents to the addictive drug cocaine, functions related to the striatum, and dopamine receptor expression (33).



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Fig. 2. The expression patterns of 12 selected genes in nervous and peripheral tissues exhibit outstanding correlation with those published in the literature. Synaptogyrin 3, {alpha}-synuclein, Na+-K+-ATPase {alpha}2, Slit1, Fabp7, and Jak2 are statistically highly expressed in the nervous tissues and are within the 381-gene list. Pirin, RXR{gamma}, and Cacna are within the 600-gene list with high expression in at least one but not all nervous tissues and thus are not shared with the 381-gene list. Synaptogyrin 2, {gamma}-synuclein, and Na+-K+-ATPase {alpha}1 are not highly expressed in the nervous tissues. N sys, nervous system; S/E, skin and epithelial tissues; Sy, synovial tissues; Im, immune system tissues; M, male reproductive system; F, female reproductive system; Ma, mammary gland; Mu, muscles; Ht, heart; K, kidney; GI, gastrointestinal tissues; E18, E18 brain; D2, postnatal day 2 brain; DRG, dorsal root ganglia; SC, spinal cord; Cb, cerebellum; HC, hippocampus; HT, hypothalamus; NAc, nucleus accumbens; Str, striatum; OlCt, olfactory cortex.

 
To further verify the quality of our microarray results and to provide neuroanatomical localization of some of the genes that are highly expressed in the nervous system, we confirmed the expression of three selected genes by in situ hybridization. All three genes were chosen because they are likely to be involved in signal transduction and neuroplasticity, and because they are highly expressed in the hippocampus as demonstrated by our microarray analyses. Two of the three genes were previously uncharacterized. These two genes have domains similar to G protein-coupled receptors, and a SH3 domain, respectively, but their localization and function are unknown.

Dickkopf family of secreted proteins is involved in Wnt signaling, which is critical in many developmental processes (27). The SH3 domain-containing proteins and G protein-coupled receptors are involved in intracellular signal transduction (38, 69). We performed in situ hybridization with mouse dickkopf homolog 3 (Dkk3, NM_015814, Fig. 3, AE), a putative G protein-coupled receptor (AB041649, Fig. 4, AG), and an SH3 domain-containing protein (W29432, Fig. 5, AI). Dkk3 and the putative G protein-coupled receptor are in the 381-gene list, but the SH3 domain-containing protein is in the 600-gene list but not in the 381-gene list. Our in situ hybridization results verified our microarray results and provided additional neuroanatomical description regarding the distribution of these gene products in the brain. Of particular interest, although all three genes express highly in the hippocampal formation, their expression patterns in the subregions of the hippocampal formation are different. Dkk3 expression is high in CA1–CA3 and low in dentate gyrus. The expression of the putative G protein-coupled receptor gene is high in the CA1 and the dentate gyrus, while low in CA3. The SH3 domain-containing protein gene is high in all regions of the hippocampal formation. Moreover, our results that Dkk3 is highly expressed in the cortex, hippocampus, and brain stem are consistent with a previously published work (34) and suggest a role of Dkk3 in neuroplasticity in the cortex and hippocampus. The putative G-protein-coupled receptor is also highly expressed in the cortex, hippocampus, striatum, and midbrain. The SH3 domain-containing protein is also expressed in the cortex and hippocampus, but in addition, it is expressed in the striatum and amygdala. Its exclusion from the 381-gene list may due to its low expression in the DRG and high expression in the testis as shown by our microarray study and previous Northern analyses (26).



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Fig. 3. Verification of Dkk3 (NM_015814) expression in the nervous system by in situ hybridization. Significant expression of Dkk3 was found in cortex, hippocampus, and brain stem. A and D: dark-field pictures of in situ hybridization signals in cortex and hippocampus, as well as in brain stem. B, C, and E: bright-field pictures of in situ hybridization signals in cortex, hippocampus, and brain stem. F: a dark-field picture of an overview of the brain. Scale bars: A = 100 µm; D = 20 µm; B, C, and E = 10 µm; F = 400 µm.

 


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Fig. 4. Verification of a putative G protein-coupled receptor (AB041649) expression in the nervous system by in situ hybridization. Significant expression of this gene was found in cortex, hippocampus, striatum, and midbrain. A, D, and F: dark-field pictures of in situ hybridization signals in cortex and hippocampus, as well as in striatum and midbrain. B, C, E, and G: bright-field pictures of in situ hybridization signals in cortex, hippocampus, striatum, and midbrain. H: a dark-field picture of an overview of the brain. Scale bars: A = 100 µm; D and F = 40 µm; B, C, E, and G = 10 µm; H = 400 µm. DG are from different brain sections as H.

 


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Fig. 5. Verification of a novel SH3-containing protein (W29432) gene expression in the nervous system by in situ hybridization. Significant expression of the novel gene was found in cortex, hippocampus, striatum, brain stem, and amygdala. A, D, F, and H: dark-field pictures of in situ hybridization signals in cortex and hippocampus, as well as in striatum, brain stem and amygdala. B, C, E, G, and I: bright-field pictures of in situ hybridization signals in cortex, hippocampus, striatum, brain stem, and amygdala. J: a dark-field picture of an overview of the brain. Scale bars: A, D, and H = 100 µm; F = 40 µm; B, C, E, G, and I = 10 µm; J = 400 µm. DI are from different brain sections as J.

 
Functional classification of genes highly expressed in adult nervous tissues.
Neurons and supporting tissues in the nervous system are specialized in integrating internal and external stimuli and coordinating response. Neuronal excitability and synaptic transmission are important aspects of nervous system functions and require extensive signaling events and structural support, as well as constant synthesis of various intracellular and extracellular molecules. To investigate genomic commitment to nervous tissue functions, we classified known genes from the 381-gene list according to their involvement in different biological functions (Tables 1 6). Diverse functional categories were represented in the 381 genes, including those important in intracellular signaling (Table 1); cytoskeleton function (Table 2); enzymatic activities (Table 3); RNA metabolism and transcription, and membrane structure and function (Table 4); cell differentiation, death, proliferation, and division, protein metabolism, extracellular message, secretory and secretion (Table 5); brain function and disease, prion and regulation, and others (Table 6) (also see DISCUSSION). Many of the 381 genes have not been previously recognized as genes having nervous system-specialized functions. Finding the high levels of expression of these genes in the nervous system may help in identifying the roles they engage in nervous system differentiation, maintenance, and plasticity.


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Table 1. Functional categories of genes highly expressed in the nervous system: Intracellular signaling

 

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Table 6. Functional categories of genes highly expressed in the nervous system: Related to brain function and/or diseases; Prion and regulation; and Others

 

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Table 2. Functional categories of genes highly expressed in the nervous system: Cytoskeleton and cytoskeleton binding protein

 

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Table 3. Functional categories of genes highly expressed in the nervous system: Enzymatic activities

 

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Table 4. Functional categories of genes highly expressed in the nervous system: RNA metabolism and transcription; and Membrane structure and function

 

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Table 5. Functional categories of genes highly expressed in the nervous system: Cell differentiation, death, proliferation, and division; Protein metabolism; Extracellular message; Secretory and secretion

 
Abnormalities of genes that are highly expressed in the nervous tissues contribute to neurological diseases development.
Determining tissue-specific and developmental stage-specific gene expression profiles in normal, healthy organisms is important for elucidation of mechanisms of pathogenesis of these diseases. We searched Online Mendelian Inheritance in Man (OMIM), University of California at San Francisco (UCSF), and the Human Gene Mutation Database (HGMD) sites and identified 110 known genes related to mental retardation, neurological disease, and neurodegeneration published in literature (Tables 7 and 8). These 110 genes were divided into four categories according to their expression patterns as shown in the Venn diagram in Fig. 1C (Tables 7 and 8).


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Table 7. Of 110 genes relevant to nervous system diseases: 21 common to the 381-gene list; 10 common to the 1,361-gene list but not in the 600-gene list; and 5 common to the 600-gene list but not in the 1,361-gene list

 

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Table 8. Of 110 genes relevant to nervous system diseases: 74 are common to the 7,154-gene list that are neither in the 1,361-gene list nor the 600-gene list

 
We found that many of these 110 genes are highly expressed in nervous tissues. Twenty-one of the 110 genes are within the 381-gene list, much more than the predicted 5 that would be found purely by random sampling (Fig. 6). Because of the high representation of disease-relevant genes in the 381-gene list, and because high levels of expression infer relevance in function, the other genes in the 381-gene list may also be involved in aspects of neurological and psychiatric disease development and modulation.



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Fig. 6. Genes highly expressed in the nervous system are likely to be relevant to nervous system diseases. The color is in accordance with that in Fig. 1C. Of a total of 110 genes suggested to be relevant to nervous system diseases, 21 are in the group of 381 (red). Five genes were expected to be within the 110-gene list for 381 random genes. Ten of the 110 disease-relevant genes are within the 980 gene group (blue). Twelve genes were expected to be within the 110-gene list for 980 random genes. Five of the 110 disease-relevant genes are within the 219 gene group (green). Three genes were expected to be within the 110-gene list for 219 random genes. Not shown in the charts are 74 of the 110 disease-relevant genes that are not in the nervous system-enriched gene list. Ninety genes were expected to be within the 110-gene list for 7,154 random genes.

 
The relatively large number of the genes expressed in the microarray also allowed us to identify genes that are coordinately expressed with those genes implicated in neurological diseases. For example, we found that genes for huntingtin-associated protein 1 (HAP1) and discs large homolog 4 are coordinately expressed. A few uncharacterized ESTs are coordinately expressed with genes implicated in diseases (AK018148 and amyloid-ß precursor protein, AK013636 and ubiquitin carboxy-terminal hydrolase L1, AK018316 and {alpha}-synuclein). Since coordinately expressed genes may modulate one another’s functions in the same tissues, the information provided by our database lays a foundation for further investigation of how the general and tissue-specific expression of genes in the nervous system contribute to the development of neurological diseases.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Using microarray technology, we have analyzed the expression profiles of 8,734 genes in 10 regions of the nervous system in the context of 30 peripheral organs. The large database of gene expression profiles in the nervous system and 30 peripheral organs is part of a continuing consortium effort of the University of Cincinnati and Children’s Hospital Research Foundation to develop a microarray database in which all poly(A)+ RNA samples were obtained and analyzed under identical experimental conditions and normalized to a single reference sample. Other studies using the University of Cincinnati-Children’s Hospital Medical Center Mouse Tissue Specific Gene Expression Database described gene expression in liver development and regeneration (29), in anatomical segments of the gastrointestinal tract (2), and in the olfactory mucosa (14).

Gene expression varies among individuals and strains (8). We have used pools of mice to carry out the microarray experiments. We also used biological duplicate, i.e., poly(A)+ RNA isolated from different sets of mice, for the duplicated microarray studies. Therefore, the genes expression differences we observe are not due to individual variations. In addition, we used C57BL/6 strains for all our analyses, thus eliminating strain variations. Our result may not be identical to results from other strains of mice.

We established a working group of 381 genes that are the most highly expressed in the nervous system based on statistical significance between nervous and peripheral tissues and based on their levels of expression in at least 1 nervous tissue over the mean expression levels. The entire set of 8,734 genes can be found in Data archive in MATERIALS AND METHODS. Of all the genes in the NIH Mouse Brain Molecular Anatomy Project (BMAP), 9,237 genes had RefSeq or RepAccNum identification numbers that can be used to compare with the Incyte cDNA microarray gene set. Of the 9,237 genes, 1,683 are present in the mouse GEM1 Incyte cDNA microarray used in this study. Of those 1,683 genes, 370 of these are expressed at a level that is at least twofold or higher in at least one nervous tissue, and 124 are in the 381-gene list. The lists of 1,683, 370, and 124 genes can be found in our microarray database, ZhangEtalBrain2004 subdirectory. Of note, many of the BMAP genes are not nervous system enriched. And many of the genes in the Incyte cDNA mouse GEM1 array that are highly enriched in the nervous system are not contained in the BMAP. Compared with the BMAP genes, we have identified 257 new genes (381 minus 124) that are both statistically highly expressed in the nervous system compared with other organs and expressed at least threefold in one or more regions of the nervous system compared with other organs.

Genes from diverse functional categories are represented in the nervous system. Within the group of genes encoding intracellular signaling, RhoN, Rab3a, Rab3b, Rab6, and RabJ are highly expressed in the nervous system, consistent with a role of Ras signaling pathway in neuronal development, synaptic plasticity, and growth and differentiation of oligodendrocyte progenitors, as well as amyloid protein secretion (4, 31, 46). Protein kinase A subunits (Cß, R{alpha}, and Rß) are highly expressed in the nervous system, and are involved in synaptic plasticity, neurite initiation, and ligand and voltage-gated channel function, as well as regulation of apoptosis (11, 22, 45, 60, 83). Frequenin homolog is a neuronal calcium sensor protein that may be involved in synaptic plasticity (13).

In the family of cytoskeleton and cytoskeleton binding proteins, adducin, drebrin, tau, Marcks, profilin, and tubulin are highly expressed in the nervous system, consistent with a crucial role in neuroplasticity (1, 21, 32, 43, 48, 66, 71, 74). Furthermore, these are particularly highly expressed in postnatal day 2 brains (Tables 16), suggesting that these gene products are involved in rapid expansion of neurons in the nervous system at early developmental stages (24). Neurofilament light chain protein is particularly highly expressed in the DRG, consistent with a role in nerve regeneration after injury (3, 81).

In the family of genes involved in protein metabolism, Von Hippel-Lindau binding protein (Vbp1) participates in the formation of an active E3 ubiquitin ligase complex, which is important for hypoxia-inducible factor-1 protein degradation (23, 49). Vbp1 may be involved in neuronal differentiation of central nervous system progenitor cells (Moseley A, Jegga AJ, Gupta A, Sartor M, Williams SS, Ley-Ebert C, Coolen L, Egnaczyk G, Genter MB, Lehman M, Lingrel J, Maggio J, Parysek L, Walsh R, Xu M, Zhang J, and Aronow BJ; unpublished observations), as well as oxygen sensing in ischemia/hypoxia-induced brain conditions (7, 72, 80).

In the category of genes encoding secreted extracellular messengers, growth differentiation factor 1 (Gdf1), neuropeptide Y, pleiotrophin, and somatostatin are highly expressed in the nervous system. Gdf1 is a member of the transforming growth factor-ß (TGF-ß) superfamily capable of regulating cell growth and differentiation and was previously shown to be highly expressed in the nervous system (39, 62). Neuropeptide Y and somatostatin are neuropeptides important for modulating neurotransmission (82). Pleiotrophin is a heparin binding protein important for neurite outgrowth, neuroplasticity, and neuronal survival (20, 42).

Genes that are highly conserved in evolution may be required for viability or other fundamental functions of the cell. We identified 17 of the 381 genes that have highly conserved homologs in nonmammalian organisms and show higher levels of expression in the nervous system compared with most peripheral tissues in mammals (Table 9). Consistent with their high conservation, these genes encode proteins of fundamentally important biological functions, such as signal transduction, RNA metabolism, cell differentiation, intercellular communication, and transcription. The expression profiles of these genes in the nervous system, along with their possible cellular functions open up new avenues for studying the functions of these evolutionarily conserved genes. For example, Gcn5l2 is also known as p300/CBP-associated factor (P/CAF), which has a lower eukaryotic homolog. P/CAF has an intrinsic histone acetylase activity, binds to E1A and Twist, and plays a role in regulation of gene expression (18). Furthermore, P/CAF is required for mouse embryogenesis (90). Whether histone acetylase activity is required for various adult neuronal activities in the mammalian nervous system will be interesting to investigate.


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Table 9. Evolutionarily conserved genes within the 381-genes list that are highly expressed in nervous tissues

 
We observed high representation of genes involved in neurological diseases in the 381-gene list. Twenty-one genes of the 381 were within the 110-gene list known to cause neurological disease symptoms, whereas the expected number from the 381-gene list to overlap with the 110-gene list is only 5. The 21 genes include amyloid-ß precursor protein and amyloid-ß precursor binding protein, which are involved in Alzheimer disease (70); huntingtin-associated protein 1, which is involved in Huntington disease (40, 41); and ubiquitin carboxy-terminal hydrolase L1 and {alpha}-synuclein, which are involved in Parkinson disease (36, 44). Furthermore, many genes in the 381-gene group may physically and functionally interact with those genes whose mutation or mal-expression directly predispose to diseases, thus contributing to disease mechanisms. Alternatively, a dysregulation of genes in the 381 list may provide new understanding of mechanisms in disease development and progression. For example, genes in the Ras signaling pathway are involved in development of neurofibroma by regulating neurofibromatosis type 1 (NF1) function (4). The cytoskeletal drebrin and the secreted pleiotrophin are involved in Alzheimer disease and Down syndrome by regulating synaptic plasticity (19, 20, 42, 73, 89). Maged2 is in a chromosomal hot spot for involvement in mental retardation (35). Acupuncture-induced gene 1 may be involved in regulation of acute and chronic pains. Analysis of the remaining of the 381 genes will help identify more neurological disease-associated genes and enhance our understanding of disease development.

Some of the genes implicated in neurological diseases are expressed in multiple tissues, including some in olfactory epithelium or peripheral blood (77, 78, 79). For example, we have confirmed that {alpha}-synuclein and Thy1 are two proteins expressed highly both in nervous tissues and in peripheral blood. Profiling genes implicated in nervous system diseases in multiple nervous tissues and in various peripheral tissues where easier sampling and gene profiling can be obtained will aid the development of better strategies for diagnosis.

Our study provides a unique opportunity to examine genes that are highly expressed in nervous tissues compared with other peripheral tissues. We provide in this study a foundation of information for many researchers to use to investigate many diverse neurobiological problems. This is particularly relevant to the study of neurological disease in both humans as well as in animal models. Further study is warranted to seek the identification of region-specific gene regulation in the nervous system and transcription regulatory elements involved in region-specific gene expression.


    GRANTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by the Howard Hughes Medical Institute, the Children’s Hospital Research Foundation, the center for environmental genetics grant, and the National Institutes of Health (to J. Zhang, L. Coolen, M. B. Genter, M. Lehman, J. Lingrel, J. Maggio, L. Parysek, M. Xu, and B. J. Aronow).


    ACKNOWLEDGMENTS
 
We thank the many contributors to the Mouse Gene Expression Database at Children’s Hospital Medical Center and the University of Cincinnati.

A. S. Greene served as the review editor for this manuscript submitted by Editor B. J. Aronow.


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

Address for reprint requests and other correspondence: J. Zhang, Dept. of Cell Biology, Neurobiology and Anatomy, Univ. of Cincinnati College of Medicine, Cincinnati, OH 45267 (E-mail: Jianhua.Zhang{at}uc.edu); or B. J. Aronow, Division of Pediatric Informatics, Children’s Hospital Research Foundation, Cincinnati, OH 45229 (E-mail: Bruce.Aronow{at}chmcc.org).

10.1152/physiolgenomics.00220.2003.

1 The Supplementary Material for this article (Supplemental Table S1) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00220.2003/DC1. Back


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