Comparative analysis of gene expression
In an effort to further understand the function and complexity of the mammalian choroid plexus, we examined global gene expression using an 18k rat microarray and compared the CP gene profile with the cortex, the hippocampus and the kidney. Shown in is a comparative CP gene expression overlap with the kidney, cortex and hippocampus. The results show a strikingly higher similarity of the CP with the kidney than either brain region. Functional classification of CP genes shows that it expresses molecules with diverse cellular functions, including multiple categories of receptors, transporters and carrier proteins.
Figure 1 Choroid plexus (CP) gene expression. (a) Venn diagram shows comparative gene expression overlap of the kidney, cortex and hippocampus with CP. Microarray analysis was performed by dual-channel experiments, where CP and other brain-region RNA were simultaneously (more ...)
Secondary validation using in situ hybridization
To obtain anatomical expression profiles of several CP genes that were indicated as significantly expressed by the array experiments, we performed in situ hybridization (ISH) using radiolabeled riboprobes. We examined a cross section of genes that had a broad range of CP expression levels and diverse molecular function classes (): channel protein—chloride intracellular channel (CLIC 6); transporter—organic anion transporting polypeptide (OATP 14); enzymes—Klotho, catechol-O-methyltransferase (COMT), superoxide dismutase (SOD1); carrier protein—transthyretin; receptors—insulin-like growth factor receptor (IGFR1), estrogen receptor 1; cytoskeleton protein—ezrin. Transthyretin had the highest level of expression of all the examined targets and showed exclusive CP expression, similar to the klotho expression pattern. Highest brain expression of CLIC6, OATP 14 and ezrin was detected in the CP. COMT was prominently expressed in the CP but was also detected in the cortex and caudate. Although EGFR1, IGFR1 and SOD 1 were clearly discernible in the CP, expression level was only moderately elevated in comparison to the caudate and cortex.
Figure 2 In situ hybridization analysis of choroid plexus (CP) gene expression. Radiolabeled riboprobes were used to examine CP expression of a subset of genes from array data. CLIC6, chloride intracellular channel protein 6; COMT, catechol-O-methyl transferase; (more ...)
Protein profiling, identification and genes overlap
Multiple fractionation methods, including fast protein liquid chromatography (FPLC), 2DLC and SDS-polyacrylamide gel electrophoresis, were employed in this study to increase the coverage and confidence of proteins identified. A collective total of 1400 proteins were identified in the CP proteome. Shown in is the FPLC elution profile of CP homogenate and a partial list of proteins identified in particular peaks. It is interesting to note that both dopamine-and cAMP-regulated phosphoprotein-32 (DARPP-32) and its substrate, protein phosphatase 1 (PP1) eluted in B8. This could be due to either shared hydrophobicity or potential protein–protein interactions. Multiple growth factor receptors, insulin-like growth factor (IGF2) and fibroblast growth factor (FGFR2) eluted in B1. Although the peaks were sharp and collected in individual tubes, it is clear from the MS data that each peak contains several different proteins. In the taller peaks, B1 and C2, 80 and 90 proteins were identified, respectively. In peaks B10, B8 and C6, 17, 19 and 20 proteins were identified, respectively. Additional proteins were identified from FPLC separations by individually (40 collection tubes) and collectively (pools of cytosolic and membrane fractions) processing the elution peaks. The pie chart () shows the proportion of CP proteins associated with various categories of biological processes. The high percentage (33.5%) of the proteins being mapped to metabolism is due to the identification of several enzymes, including 83 hydrolases, 75 oxidoreductases and 67 transferases (). We analyzed the overlap between the identified CP proteins and genes (). To obtain a comparable number of genes and proteins, we utilized the full list of proteins and limited the gene number by using the kidney–CP overlap set. Only genes that had the same annotation as that of the protein were used for comparison. This resulted in an overlap of 211 genes and proteins. The list of proteins that were identified in this are listed in Supplement 1
. The list of representative peptides are in Supplement 2
and are hyperlinked to the Uniprot database.
Figure 3 FPLC elution profile of choroid plexus (CP) homogenate. A representative elution profile is shown along with a short list of major proteins that were identified by mass spectrometry from specific numbered peaks. CP tissue (n=3) was pooled after rinsing (more ...)
Figure 4 Choroid plexus (CP) proteome. (a) Proteins were identified by the International Protein Index (IPI) identifiers and then converted to gene names and mapped to Gene Ontology (GO) terms. Mouse genome institute (MGI) GO slim 2 schema for biological processes (more ...)
Comparative analysis of cerebellum, lateral ventricle and the kidney
Shown in is a comparison of proteome profiles from the CP, the kidney tubules, cerebellum and lateral ventricle glia, analyzed for enriched gene ontology (GO) molecular function terms. Proteomes other than CP were obtained from the human protein atlas database and analyzed by Metacore. Only molecular function classes that exhibited substantial differences in target representation are shown (). The total numbers of targets in each tissue were CP—1403, kidney tubules—2448, cerebellum—2288 and lateral ventricle—884. Transcription regulators and nucleic acid binding proteins were represented at low levels in the CP, at 3.2% and 6.7%, respectively. In comparison, nucleic acid binding proteins in the kidney, cerebellum and lateral ventricle (LV) were represented at 26.2%, 27.7% and 29%. Electron carrier and transporter proteins exhibited higher representation in the CP, and the most striking difference in transporter expression was between CP and LV. Although represented by only 21 distinct proteins, the percentage of antioxidant molecules (1.6%) identified in the CP is at least twofold higher than the other tissues used in the comparison ().
Comparison of molecular functional classes of proteins represented in multiple tissue types
Immunohistochemical analysis of CP protein expression
We examined five CP genes for expression at the protein level by immunohistochemistry. Multiple isoforms of keratin were identified by the MS analysis. We chose an antibody that recognizes keratins 5 and 8 to confirm expression in the CP. Cytokeratin 5/8 was exclusively expressed in the CP () and no signal was noted in any other brain regions. Very low levels of expression were observed in the ependyma. The cytoskeletal and filamentous expression pattern is discernible at higher magnification (). Klotho was also strongly expressed only in the CP with low expression in the ependyma (). However, the pattern of klotho expression was distinctly different from cytokeratin, exhibiting strong signal polarity (). Rab7 was predominantly expressed in the CP (), followed by substantial expression in vasculature and low levels in the ependyma. Matrix metalloprotease 9 (MMP 9) has emerged as a key molecule in matrix remodeling and regulation of barrier integrity during inflammation. We therefore investigated the expression of MMP 9 in the CP and colocalization with cellular markers. Shown in is double immunohistochemical staining to detect GFAP and MMP 9 expression. Prominent MMP 9 staining was observed in the CP, whereas GFAP signal was restricted to the ependyma and absent in the CP (). MMP 9 was also expressed in the ependyma but signal colocalization with GFAP was not detected (). Rat endothelial cell antigen (RECA) staining was seen only in the vascular core of the CP and did not appear to colocalize with the epithelial cell expression of MMP 9 (). TIMP 1 expression was sparse in the CP and more strongly noted in the ependyma (). Although some TIMP 1 expression was in proximity to MMP 9 positive cells in the CP, it appeared to be limited to the basement membrane ().
Figure 5 Immunohistochemical analysis of choroid plexus (CP) protein expression. CP expression of cytokeratin, klotho and Rab 7 are shown in a–c, respectively ( × 150). Higher magnification images of the same proteins are shown in the corresponding (more ...)
Effects of chronic stress on CP gene regulation
In order to investigate the effects of chronic stress on choroid plexus gene expression, we performed a preliminary microarray analysis of gene expression using a pooled sample (N=3) of CP tissue from rats exposed to the chronic unpredictable stress (CUS) paradigm compared with home cage control animals. This analysis indicated that 35 days of CUS influenced CP gene regulation and enabled us to focus our attention on a subset of dysregulated genes. Using an independent cohort of rats (N=4), we examined stress-induced CP gene regulation by quantitative PCR analysis. CUS animals showed substantially increased expression of the 5HT2A receptor but reduced expression of 5HT2C and the glucocorticoid receptor (6A). CUS also influenced the CP expression of several trophic factors. Brain-derived neurotrophic factor (BDNF) was prominently elevated by 65% followed by IGF1 (6B). Erythropoietin was significantly decreased, whereas there was no change in vascular endothelial growth factor levels (6B). CUS reduced the expression of intraflagellar transport protein 88 (IFT88), smoothened (smo), platelet-derived growth factor receptor alpha (PDGFRa) and klotho (). IFT88, smo and PDGFRa are known to be expressed by cilia and could represent stress-induced alteration of CP cilia. In contrast to the decrease in expression of cilia genes, CUS elevated the expression of the proinflammatory cytokines interleukin 1b (IL-1b) and tumor necrosis factor α (TNFα). There was a 40% elevation in IL-1b and MMP 9, and a 30% increase in TNFα expression ().
Figure 6 Stress-induced gene regulation in the choroid plexus (CP). (a) Real-time PCR quantitation of CP gene reguation after chronic unpredictable stress. (b) BDNF, brain-derived neurotrophic factor; EPO, erythropoietin; IGF1, insulin-like growth factor 1; VEGF, (more ...)