Transcriptional profiling of transport mechanisms and regulatory pathways in rat choroid plexus

This study conformed to the European guidelines and ethical regulations for use of experimental animals. The study utilizes 9-week-old Sprague Dawley rats (Janvier Labs, France) of male and female sex. The rats were housed with 12:12 light cycle with access to water and food ad libitum in accordance with the guidelines of the Danish Veterinary and Food administration (Ministry of Environment and Food) and approved by the animal facility at the Faculty of Health and Medical Sciences, University of Copenhagen. The rats were anaesthetized with intraperitoneal injection of xylazine and ketamine (6 mg/ml and 60 mg/ml in sterile water, 0.17 ml per 100 g body weight (ScanVet, Fredensborg, Denmark)) prior to decapitation and tissue collection.

Choroid plexus (from lateral and 4th ventricles) were isolated from five male and five female rats, pooled respectively and stored in RNAlater® (Sigma-Aldrich, St. Louis, Missouri, USA) at − 80 °C. Kidney tissue was collected from the male rats, minced, and subsequently digested for 25 min at 37 °C in a table shaker at 850 rpm in collagenase solution containing 1 mg/ml collagenase (type II, Gibco®, Grand Island, NY, USA) and 1 mg/ml pronase (Roche, Mannheim, Germany) in buffer solution containing (in mM): 140 NaCl, 0.4 KH₂PO₄, 1.6 K₂HPO₄, 1 MgSO₄, 10 Na-acetate, 1 α-ketoglutarate, 1.3 Ca-gluconate, 5 glycin, in addition to 48 mg/l aprotinin (trypsin inhibitor, Sigma-Aldrich, St. Louis, Missouri, USA) and 25 mg/l DNase I (grade II, Roche, Mannheim, Germany), pH 7.56. In five-minute intervals, 1 ml of the solution containing the kidney tissue was transferred to an eppendorf tube containing 1 ml cold buffer solution with 0.5 mg/ml bovine serum albumin (Sigma-Aldrich, St. Louis, Missouri, USA) and replaced by 1 ml collagenase solution. Proximal tubules were manually collected under a microscope, centrifuged at 600×g for 5 min, and the pellet stored in RNAlater® at − 80 °C.

Choroid plexus was isolated from 10 male rats, minced, and digested in collagenase (15 mg/ml collagenase (type II, Gibco®, Grand island, NY, USA) in artificial CSF (aCSF)-HEPES containing (in mM): 120 NaCl, 2.5 KCl, 3 CaCl₂, 1.3 MgSO₄, 1 NaH₂PO₄, 10 glucose, 17 Na-HEPES, pH 7.56 for 30 min at 37 °C in a table shaker at 800 rpm. The supernatant was removed after 5 min centrifugation (600×g) and the pelleted cells were resuspended in aCSF-HEPES. The cells were triturated 20 times with a 1000 µl pipette and filtered through a 70 μm filter (pluriStrainer, Mini 70 µm, PluriSelect, Leipzig, Germany), prior to incubation with an anti-NKCC1 antibody with an extracellular epitope (1:200 in aCSF-HEPES, #ANT-071, Alomone Labs™, Jerusalem, Israel) for 30 min at 4 °C. The cells were pelleted (600 × g, 5 min) and resuspended in secondary antibody (1:500 in aCSF-HEPES, Alexa Fluor® 647—A-21245, Invitrogen™, Carlsbad, California, USA), in which it was kept for 20 min at 4 °C prior to centrifugation (600×g, 5 min) and resuspension in cold aCSF-HEPES. Cells were analyzed and sorted on a FACSAria Fusion flow cytometer (BD Biosciences, Lyngby, Denmark).

The RNA extraction and library preparation were performed by Novogene Company Limited, UK with NEB Next® Ultra™ RNA Library Prep Kit (NEB, USA) prior to their RNA sequencing (paired-end 150 bp, with 12 Gb output) on an Illumina NovaSeq 6000 (Illumina, USA).

All program parameter settings for library building and mapping, together with all scripts for the gene annotation and analysis are available at https://​github.​com/​Sorennorge/​MacAulayLab-RNAseq2.

Although it remains unknown whether a certain transcript level cut-off represents physiological relevance, all analyses exclude genes transcribed at levels below a cut-off at 0.5 transcripts per million (TPM) [13]. Raw data are available at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (accession number: GSE194236).

The 150 base paired-end reads were mapped to reference genome (Rattus norvegicus Rnor_6.0 v.103) using Spliced Transcripts Alignment to a Reference (STAR) RNA-seq aligner (v. 2.7.2a) [14]. The mapped alignment by STAR was normalized to TPM with RSEM (RNA-Seq by Expectation Maximization v. 1.3.3) [15]. Gene information was gathered with mygene (v3.1.0) python library [16‐18], from which gene symbol, alias, and Gene Ontology (GO) terms [19‐21] were extracted. Mitochondrial genes (seqname: MT) were omitted from the reference genome prior to comparison of the kidney proximal tubule transcriptional profiles with that of the choroid plexus, since the greater abundance of these genes in the proximal tubule would skew the plasma membrane transporter expression comparison.

The human choroid plexus transcriptome was obtained from GEO database (GSE137619, SRR10134643-SRR10134648) [22‐24] and the mouse choroid plexus transcriptome was obtained from GEO (GSE66312, SRR1819706-SRR18197014) [25]. All samples were quality controlled with fastqc [26] and trimmed with Trimmomatic [27] (Slidingwindow 4:20, minimum length of 35 bp). The human and mouse samples, together with rat sample 3 (male), were mapped to the human reference genome (Homo sapiens GRCh38 v.104), the mouse reference genome (Mus musculus GRCm39 v.104), and rat reference genome (Rattus norvegicus Rnor_6.0 v.103) with STAR (v. 2.7.2a). The reference genome for rat, mouse, and human for the cross-species analysis only contained  gene/transcripts of biotype ‘protein coding’. Mapped alignments were normalized to TPM with RSEM (v. 1.3.3.) and the mean from the samples for human and mouse, respectively, were used for further analysis. Transcribed genes sharing gene name between the compared species (or their orthologue, collected from ensemble.org via biomart martview) were included in the cross-species comparison.

Gene lists of transporters, pumps, water and ion channels, and G protein-coupled receptors (GPCR) were collected from the ‘target and family list’ from Guide to Pharmacology [28‐31]. Genes annotated as ‘transporters’ were employed to generate the list of membrane transporters and pumps whereas genes annotated as ‘voltage-gated ion channels’, ‘ligand-gated ion channels’, and ‘other ion channels’ were employed to generate the list of water and ion channels. To filter for plasma membrane proteins, the transporter and pump gene list was initially filtered to exclude the mitochondrial and vacuolar transport families SLC25, ATP5, and ATP6V, after which the transporter and channel lists were filtered based on associated GO terms; ‘integral component of plasma membrane’ or ‘plasma membrane’, but only included genes annotated as ‘integral component of membrane’ or ‘transmembrane’, but not annotated as ‘lysosome’, ‘endosome membrane’, ‘lysosomal’, ‘mitochondrion’, ‘mitochondrial’, ‘golgi apparatus’, ‘vacuolar’, or ‘endoplasmic’. Genes annotated as ‘GPCR’ were employed to generate the list containing GPCRs. Receptor tyrosine kinases (RTK) were gathered from Human Genome Organisation (HUGO) Gene Nomenclature Committee (HGNC) database [32], with the annotations ‘receptor tyrosine kinase’ including sub group ‘ephrin receptors’ and ‘ErbB family’. The list of kinases was obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [33‐36]for entries of ‘EC 2.7.10.2’ (non-specific protein-tyrosine kinase), ‘EC 2.7.12’ (Dual-specificity kinases) with the two sub-categories, and ‘EC 2.7.11’ (Protein-serine/threonine kinases) with the 33 sub-categories. These three entries were collected with organism specific “rno” (Rattus norvegicus) filter. These kinases were filtered for protein kinases by GO terms: ‘protein kinase activity’, ‘protein serine/threonine kinase activity’, ‘protein serine kinase activity’, ‘protein threonine kinase activity’, ‘protein tyrosine kinase activity’, ‘map kinase activity’. Kinases involved solely with transcription or cell cycle modulation were subsequently excluded. Phosphatases were gathered from the KEGG database with entries EC 3.1.3 (Phosphoric Monoester Hydrolases) with 108 subcategories. The phosphatases were filtered for protein-interacting phosphatases by GO terms: ‘phosphoprotein phosphatase activity’, ‘protein serine/threonine phosphatase activity’, ‘protein serine phosphatase activity’, ‘protein threonine phosphatase activity’, and ‘protein tyrosine phosphatase activity’. Phosphodiesterases (PDE) were collected based on gene name starting with ‘PDE’. Cyclases were obtained from the KEGG database [33‐36] entry numbers ‘EC 4.6.1.1’ (adenylate cyclase) and ‘EC 4.6.1.2’ (guanylate cyclase).

The network analysis was generated from protein–protein association tables from the String-database [37, 38] as a plugin for Cytoscape (v. 3.8.2) [39]. Firstly, full interaction tables were generated through a full protein query of every protein in the lists of transporters and pumps, water and ion channels, GPCRs, RTKs, kinases, phosphatases, PDEs and cyclases. Secondly, the tables were filtered for interaction between the ‘transporters and pumps’ and all the regulatory genes (lists of GPCRs, RTKs, kinases, phosphatases, PDEs, and cyclases). All interactions between regulatory proteins were discarded. The same was done for ‘water and ion channels’. We only included protein–protein associations that were curated in a database or were demonstrated to interact experimentally. Thirdly, these interaction tables were loaded back into Cytoscape (v. 3.8.2) and modulated: Confidence score (from string-db) of 0.6–1 was used for genes from the list ‘transporter and pumps’ interactions and 0.7–1 was used for genes from the list ‘water and ion channels’.

To determine the species similarity of the choroid plexus transcriptomic profile, we compared that obtained from the rat (Additional file 1, sheet 1) to published versions of these obtained from human [22] and murine [25] choroid plexus. Notably, this species comparison illustrates whether or not the gene is present but does not confer quantitative comparison of the expression levels. The rat shares 89% of its choroidal protein-coding genes with that of the human choroid plexus, and 91% with that of mice (Fig. 1). These numbers may represent an underestimate, as some of the remaining genes might not be annotated with the shared name or as an orthologue, or may fall just above or below the cut-off level of 0.5 TPM. However, the 11% non-shared expressed genes in humans only account for 3.3% of the total transcripts and only 5.5% of these non-shared genes were annotated with ‘transport activity’, meaning that only 0.1% are transporters not shared with the rat. The 9% non-shared expressed genes for mouse account for 2.8% of the total transcripts, and only 0.1% was annotated as transporters. Such transcriptomic similarity suggests that the functional integrity is conserved among these organisms, and that the rat is an applicable animal model in which to determine physiological aspects, especially those that are membrane transport-related, of the choroid plexus.

To obtain a complete list of transport proteins expressed in the choroid plexus and their transcriptomic abundance, the RNAseq data were filtered for genes encoding transport proteins located in the plasma membrane (see Methods). Each gene was associated with its alias, and a description of the type of membrane transport protein. Some protein names and their function were well established, while other transport mechanisms expressed in choroid plexus appeared not fully characterized with no general agreement on a given alias or function. The lists were therefore manually curated according to the Universal Protein Resource (Uniprot) [40] and the Human Gene Database (Genecards) [41] to ensure that the widest accepted alias and function were associated with each gene name. The transport proteins are divided into (i) coupled transporters and ATP-driven pumps and (ii) water and ion channels, and sorted according to their transcript abundance (Additional file 1, sheets 2 and 3). Tables 1 and 2 illustrate the 20 highest expressers in each category. Several of the transport proteins implicated in CSF secretion are found amongst these highly expressed transport proteins; different subunits of the Na⁺/K⁺-ATPase, the Na⁺,K⁺,2Cl⁻ cotransporter (NKCC1), aquaporin 1 (AQP1), and the HCO₃⁻ transporters NBCe2, NCBE, and AE2 [12, 42]. In addition, the lists present some additional transport proteins that could be envisaged to partake in CSF secretion, i.e. cation, anion and sulfate transporters (BOCT, BSAT1, SNAT1, SUT1, and MCT8, Table 1) in addition to various K⁺ and Cl⁻ ion channels (Table 2) with no prior association to CSF secretion. With such high relative expression in this tissue, these transport proteins are likely to serve a physiologically important function in choroid plexus, and could potentially be involved in CSF secretion.

The choroid plexus is a feather-like structure containing a monolayer of epithelial cells with centrally located vasculature, stroma, and immune cells. Most RNAseq studies of this tissue take advantage of the fact that the majority of the cells in the tissue are choroid plexus epithelial cells [43, 44] and thus include the entire structure into the RNAseq procedure [45‐49]. In this manner, some of the transcripts are anticipated to arise from other cell types than that of the choroid plexus epithelium. To resolve a putative discrepancy between a pure fraction of choroid plexus epithelial cells and that of the entire tissue, dissociated cells from acutely isolated choroid plexus were captured by FACS. In the choroid plexus, NKCC1 appears to be expressed exclusively on the luminal side of the epithelial cells [50‐52] and FACS with the anti-NKCC1 antibody targeted to an extracellular epitope on   this choroidal transport protein produced a fraction of highly fluorescent and large cells (Fig. 2A), which was absent in the FACS conducted in the absence of antibody (Fig. 2B) or with only the secondary antibody (Fig. 2C). Intact choroid plexus epithelial cells captured by FACS expressed AQP1 (Fig. 2D), which in the choroid plexus is expressed solely in the epithelial cells [53], demonstrating the epithelial origin of the captured cells. RNAseq analysis of the epithelial cells captured by FACS retrieved 97% of the genes detected in the entire choroid plexus structure (Fig. 2E). The 3% of the genes that were absent from the choroid plexus epithelial cells captured by FACS, were largely (> 95% of the genes with cell type annotation) annotated primarily to cell types other than epithelial cells [54, 55], such as cells of mesenchymal, endothelial, neuronal, immune, or glial origin, which reside in the choroid plexus structure [43]. The cell population obtained from the FACS procedure thus essentially represents choroid plexus epithelial cells. Choroid plexus epithelial cells captured by FACS displayed nearly identical transcriptomic profiles to that of the entire choroid plexus of choroidal transport proteins (97%, Fig. 2F) and channels (92%, Fig. 2G). The non-shared genes place around the cut-off TPM of 0.5 for both transporters and channels. More specifically, the transporters/pumps found amongst the top 20 highest expressers in the choroid plexus were all detected within the top 26 highest expressed transport proteins in the pure fraction of choroid plexus epithelial cells (Table 1). Such similarity was reproduced for choroid plexus membrane channels (except for the two ion channels K_ir2.4 and K_v1.1 that placed further down the list), Table 2. Complete lists of transporters and membrane channels detected in the purified choroid plexus epithelial cells are included as Additional file 1, sheets 4 and 5. We conclude that RNAseq analysis of the complete choroid plexus structure provides a representative quantitative identification of the membrane transport proteins expressed in the choroid plexus epithelial cells.

The solute carriers (SLC) represent a large group of membrane transport proteins containing more than 400 members divided amongst 66 families [56]. The 66 family names and transport functions were collected from the Bioparadigms SLC database [57, 58]. Of the 66 existing SLC families, transcripts of 52 of these gene families were detected in choroid plexus, and 44 of these coding for SLC family members residing in the plasma membrane (representing 63% of all choroid plexus transporter and pump transcripts in the plasma membrane). To obtain an overview of choroidal plasma membrane transport functions, the 44 SLC families detected in the choroid plexus plasma membrane were grouped into 11 supercategories according to similarities in transported substrates (Fig. 3, Additional file 1, sheet 6). The most abundantly expressed supercategory consisted of 31 electrolyte and bicarbonate transporters (six families, 2602 TPM) followed by seven organic an/cationic transporters (one family, 2581 TPM) and 19 large anion transporters (five families, 1929 TPM). These are followed by amino acid and neurotransmitter transporters (36 genes), various sugar transporters (17 genes), and vitamin transporters (6 genes), (Fig. 3 and Additional file 1, sheet 6). Notably, the choroid plexus expresses a wealth of metal transporters (dispersed among 22 genes from five families, 928 TPM, with zinc transporters as the most prominent (15 genes)), (Fig. 3 and Additional file 1, sheet 6). With such abundance and variety in transporter types, the choroid plexus obviously serves important physiological roles in transporting solutes across this epithelial layer, besides that serving as the CSF secreting machinery.

To verify the sex-specific transcriptomic profile of female versus male rat choroid plexus, we compared the expression profiles for choroid plexus obtained from each age-matched sex. The female choroid plexus shared 98% of the overall expression profile with the male choroid plexus (Fig. 4A), suggesting high functional similarity of the choroid plexus in the two sexes. Notably, the non-shared genes account for less than 0.1% of the total transcripts. To reveal potential sex-specific differences within the CSF secreting machinery, we compared the expression profile for membrane transport mechanisms. Choroid plexus obtained from the female rats shared 98% of the transcripts encoding transporters (Fig. 4B) and 95% of those encoding channels (Fig. 4C) with the male rat. The male 20 highest expressed genes were included in the top 21 highest expressed transporter genes in the female transporter category (Table 1) and in the top 22 highest expressed channel genes in the female channel category (Table 2). Choroid plexus obtained from the two sexes thus shares high degree of expression profile similarity, with near-identical expression of abundant transport proteins. These data suggest a comparable choroid plexus CSF secreting machinery in female and male rats.

To reveal possible transport protein candidates involved in fluid secretion across the choroid plexus, we obtained the transcriptomic profile of an epithelium of comparable isotonic fluid-transporting capacity to that of choroid plexus; the kidney proximal tubule [2]. The majority (91%) of the transcribed genes obtained in the proximal tubule were detected in the choroid plexus. However with the larger number of transcribed genes found in the choroid plexus, this common pool of transcribed genes amounted to only 41% of the choroidal expression profile (Fig. 5A). 64% of the transporter-encoding transcribed genes obtained from the proximal tubule were retrieved in the choroidal samples (in which proximal tubule transcribed genes represented 30%, Fig. 5B). In the channel category, the equivalent numbers were 52% of the proximal tubule transcribed genes being retrieved in the choroid plexus samples and 15% of the choroidal transcribed genes detected in the proximal tubule (Fig. 5C). Of the 20 highest expressed transporters/pumps (Table 3) and channels (Table 4) in the proximal tubule (see Additional file 1, sheets 7 and 8 for the complete lists), only 10 (transporters and pumps) and 10 (water and ion channels) genes were found in the choroid plexus (Tables 3 and 4). The Na⁺/K⁺-ATPase α1β1, AQP1, TRP channels, and some voltage-gated Cl⁻ channels present themselves as common transport mechanisms in the two tissues. Although the proximal tubule joins the choroid plexus in the ranks of high-capacity fluid-transporting epithelia, the molecular machinery driving the fluid secretion appears, at least in part, to rely on distinct transport mechanisms.

It is anticipated that CSF secretion is tightly regulated to ensure stable brain fluid dynamics and thus intracranial pressure, although the regulatory control of CSF secretion is largely unresolved. To reveal which plasma membrane receptors are expressed in choroid plexus, all G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) expressed in choroid plexus were filtered from the RNAseq data set and listed according to their expression levels (Additional file 1, sheets 9 and 10). Each gene was associated with its alias and a description of the type of receptor (if known). All receptors were manually curated [40, 41] to ensure that the widest accepted alias and function were associated with each gene name. Of the 20 highest expressed GPCRs (Table 5), the serotonin receptor 2C (HTR2C) figures at the top, and is accompanied by the endothelin receptor type B, the GABA_B receptor, corticotrophin releasing hormone receptor 2, several adhesion-associated receptors, and those belonging to the family of frizzled GPRCRs. Notably, the list contains several orphan GPCRs (GPR146, GPR175, GPR107) without well-established ligands and functions, although GPR146 has been proposed as a receptor for insulin C-peptide [59], which in turn may regulate the Na⁺/K⁺ -ATPase [60]. Dominant amongst the receptor tyrosine kinase top expressers (Table 5) are receptors for growth factors (GF) of various kinds; fibroblast GF, platelet derived GF, vascular endothelial GF, and insulin/insulin-like receptors, in addition to various immune-related RTKs.

The receptors generally exert their function via intracellular signaling cascades promoting phosphorylation (kinases) or dephosphorylation (phosphatases) of target proteins such as transport proteins and transcriptional factors potentially promoting expression of select transport proteins. To reveal such regulatory proteins expressed in choroid plexus, the RNAseq data were filtered for kinases and phosphatases, and the data manually curated to obtain alias and function (complete lists found as Additional file 1, sheets 11 and 12). The list of the 20 highest expressed kinases (Table 6) encompasses a large variety of kinases including MAP kinases, AKT kinases, and casein kinases. Interestingly, the Stk39 kinase (SPAK), which is the second highest expressed kinase in choroid plexus, is directly implicated in CSF secretion via its ability to activate NKCC1 [6]. The list of the 20 highly expressed phosphatases covers a variety of phosphatases encompassing both serine/threonine and tyrosine phosphatases (Table 6).

Some kinases are activated by cyclic nucleotides, such as cAMP required for PKA activation and cGMP required for PKG activation. The abundance of these cyclic nucleotides is regulated by cyclases and phosphodiesterases, the presence and activity of which could well modulate the CSF secretion in the choroid plexus [61‐63]. The RNAseq data set was therefore filtered for the presence of these, and the 10 highest expressers amongst cyclases and phosphodiesterases are listed in Table 7, with the complete lists of manually curated (for alias and function) genes included as Additional file 1, sheets 13 and 14.

To obtain insight into potential physiologically relevant regulatory properties of the choroid plexus transport machinery, we built association networks of transporters/pumps (Fig. 6) and channels (Fig. 7) with the various choroidal receptors (GPCRs and RTKs) and intracellular messengers (kinases, phosphatases, PDEs, and cyclases). Such networks are obtained with a string database and provide links between the transport mechanisms and a regulatory factor, if such has been implied experimentally or in curated databases [64] in published work on any cell type or tissue. Most notably, various isoforms of protein kinase A (PKA) and MAP kinases (MAPK) associate with phosphorylation of different subunits of the Na⁺/K⁺-ATPase and the plasma membrane Ca²⁺-ATPase (PMCA), whereas the facilitative glucose transporter (GLUT4) and the Na⁺/H⁺ exchanger (NHE1) associate with a variety of different regulatory candidates (Fig. 6). The SPAK-mediated regulation of NKCC1 in choroid plexus [6, 65] is evidenced in the network association of these proteins along with the with-no-lysine kinases (WNK1-4), oxidative stress responsive kinase 1 (OSR1), and other cation-Cl⁻ cotransporters (Fig. 6). The different PKA isoforms associate with various ion channels as well, most prominently with voltage-gated K⁺ channels, while PKC isoforms associate with the transient receptor potential vanilloid channel 4 (TRPV4) (Fig. 7), which modulates the rate of CSF secretion [66] and has been implicated in hydrocephalus development in a rat model of genetically-induced hydrocephalus [67]. In addition, various highly expressed phosphatases associate with different ligand- and voltage-gated ion channels (Fig. 7). Altogether, such network analysis indicated a plethora of potential regulatory pathways, some of which could be implicated in regulation of CSF secretion, or other physiological processes.