A molecular characterization of the choroid plexus and stress-induced gene regulation

Abstract

The role of the choroid plexus (CP) in brain homeostasis is being increasingly recognized and recent studies suggest that the CP has a more important role in physiological and pathological brain functions than currently appreciated. To obtain additional insight on the CP function, we performed a proteomics and transcriptomics characterization employing a combination of high resolution tandem mass spectrometry and gene expression analyses in normal rodent brain. Using multiple protein fractionation approaches, we identified 1400 CP proteins in adult CP. Microarray-based comparison of CP gene expression with the kidney, cortex and hippocampus showed significant overlap between the CP and the kidney. CP gene profiles were validated by in situ hybridization analysis of several target genes including klotho, CLIC 6, OATP 14 and Ezrin. Immunohistochemical analyses were performed for CP and enpendyma detection of several target proteins including cytokeratin, Rab7, klotho, tissue inhibitor of metalloprotease 1 (TIMP1), MMP9 and glial fibrillary acidic protein (GFAP). The molecular functions associated with various proteins of the CP proteome indicate that it is a blood-cerebrospinal fluid (CSF) barrier that exhibits high levels of metabolic activity. We also analyzed the gene expression changes induced by stress, an exacerbating factor for many illnesses, particularly mood disorders. Chronic stress altered the expression of several genes, downregulating 5HT2C, glucocorticoid receptor and the cilia genes IFT88 and smoothened while upregulating 5HT2A, BDNF, TNFα and IL-1b. The data presented here attach additional significance to the emerging importance of CP function in brain health and CNS disease states.

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 hybridized. Data shown are from three independent replicates for each region. (b) Major functional categories of genes that were expressed in both the CP and the kidney. Only functional classes that had a minimum of 12 genes are shown.

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; IGFR1, insulin-like growth factor receptor 1; SOD1, superoxide dismutase; OATP 14, organic anion-transporting polypeptide.

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 in cold PBS. An elution gradient of increasing acetonitrile concentration is indicated by the background line. DARPP32, dopamine- and cAMP-regulated neuronal phosphoprotein; FGFR2, fibroblast growth factor receptor 2; GDI, GDP dissociation inhibitor; IGF2, insulin-like growth factor; MMP9, matrix metalloprotease; NBP, nucleotide binding protein; PBS, phosphate buffered saline.

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 was used to classify the CP proteome based on the biological role of the molecules. Pie chart shows the number of proteins represented in the GO molecular function term categories. (b) The enzymes that were identified by the proteomics analysis were classified using the UniProt database. Pie chart shows the number of proteins represented in the enzymatic function categories. (c) Venn diagram showing overlap between genes from the kidney–CP overlap set (blue circle) and the identified CP proteins (salmon circle).

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 lower panel d–f. Double immunohistochemical analysis of matrix metalloprotease 9 (MMP 9) (red) and GFAP (green) is shown in g and j at low and high magnification, respectively. Arrow in g denotes GFAP expression in the ependyma. Arrow in j denotes MMP 9 expression in ependymal cells. Double immunohistochemical analysis of rat endothelial cell antigen (RECA) (green) as a vascular marker and MMP 9 (red) is shown in h and k. Arrow in h denotes MMP 9 expression in the ependyma. Double immunohistochemical analysis of MMP 9 (red) and TIMP 1 (green) is shown in i and l.

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, vascular endothelial growth factor. (c) IFT88, intraflagellar transport; Smo, smoothened; PDGFRa, platelet-derived growth factor receptor. (d) IL-1b, interleukin; TNFα, tumor necrosis factor. ^*P⩽0.05, error bars represent s.e.m. from n=5.