Choroid plexus LAT2 and SNAT3 as partners in CSF amino acid homeostasis maintenance

Male and female 8- to 16 weeks old wild type and LAT2 (Slc7a8) KO (knockout) [24] C57BL/6J mice were used (Charles River (Crl), Germany and in-house breeding). Animals were kept in standard cages under 12-h light/dark cycle (7:00 h/19:00 h) with free access to food and tap water. All animal experiments were conducted in accordance with the Swiss federal and cantonal law and performed with the approval of the Swiss Veterinary Council, Approval number 205/2016.

CSF samples were obtained as previously described [25]. After CSF collection blood was carefully withdrawn by cardiac puncture, transferred to an Eppendorf tube with heparin and kept on ice. As soon as the last sample was obtained all blood samples were centrifuged for 10 min at 10,000g (4 °C) to separate plasma.

Deeply anaesthetized mice were transcardially perfused with ice-cold PBS (pH 7.4), brains were removed, cut into two halves and fixed in 4% PFA at 4 °C overnight. The next day the right half of each brain was washed in PBS, incubated in 30% sucrose and afterwards frozen in OCT embedding matrix (CellPath Ltd, Newtown, UK) on dry ice. The left half of each brain was washed in PBS, stepwise incubated in ethanol of 20%, 40% and 60%, stored in 70% till paraffinization on Microm spin tissue processor STP-120 (Microm International GmbH, part of Thermo Fischer Scientific, Walldorf, Germany) and subsequently embedded in paraffin. Saggital 10 µm thick cryosections were cut on a cryostat (Leica CM1850, Leica Biosystems Nussloch GmbH, Nussloch, Germany) and mounted on SuperFrost Plus adhesion slides (J1800BMNZ, Thermo Scientific, Thermofisher Scientific AG, Reinach, Switzerland) and kept at − 20 °C till staining procedure. Paraffin blocks were cut sagitally in 5 µm thick slices using a microtome (RM 2235, Leica biosystems Nussloch GmbH, Nussloch, Germany). For most amino acid transporters staining was performed on cryosections with antigen retrieval using sodium citrate buffer (pH 6.0) for 20 min at 98 °C in the rapid microwave histoprocessor (HistoPRO SW 2.0.0, Milestone medical, Kalamazoo, USA). The sections were incubated for 1 h at room temperature in blocking buffer containing 5% donkey serum (D9663, Sigma-Aldrich Chemie GmbH by Merck, Buchs, Switzerland) and 0.3% Triton X-100. Blocked specimens were then incubated for 1 h at room temperature in incubation buffer (PBS, 1% BSA, 0.3% Triton X-100) containing primary antibodies diluted as indicated in Additional file 2: Table S1. Secondary antibody incubation was performed with donkey anti-mouse DyLight 488 (96875, Abcam, Cambridge Science Park, Milton, Cambridge, UK) and anti-rabbit DyLight 594 (96921, Abcam, Cambridge Science Park, Milton, Cambridge, UK) for 1 h at RT. PBS was used for washes between incubation with primary and secondary antibodies. Nuclear counterstaining was performed by incubation with 2 μg/mL of diamidine-2-phenylindole-dihydrochloride (DAPI) for 10 min at room temperature. Brain sections were mounted with DAKO-Glycergel (C0563, DAKO North America, Carpinteria, USA) and examined under a confocal laser scanning upright microscope Leica TCS SP8 (Leica Microsystems CMS GmbH, Mannheim, Germany) using a 63× objective (oil, numerical aperture of 1.4, pinhole set to 1.0 airy unit). Images were processed and merged by Imaris software (version 7.5.1; bitplane). For LAT2 transporter staining in specimens obtained from LAT2 KO and corresponding age-matched wild-type animals paraffin sections were subjected to deparaffinization (Pathisto AS-2, Pathisto GmbH, Garbsen, Germany), followed by extensive wash in PBS. Antigen retrieval in this case was performed by incubation in 0.1% SDS/PBS for 5 min and subsequent wash in running tap water and PBS. Then sections were blocked for 1 h at room temperature in PBS solution with 5% donkey serum (D9663, Sigma-Aldrich Chemie GmbH by Merck, Buchs, Switzerland) and subsequently incubated overnight in solution containing anti-LAT2 antibodies (1:1000), 1% BSA and 0.02% Triton-X 100. Specimens were washed twice in hyper-PBS (doubled concentration of NaCl, 274 mM) and once in PBS followed by incubation in solution containing secondary anti-rabbit DyLight 488 antibodies diluted 1:500 and DAPI. Afterwards samples were mounted with DAKO-Glycergel and staining was analyzed on a Leica TCS SP8 confocal laser scanning microscope (Leica) using a 63× objective lens (pinhole 1.0, numerical aperture 1.4). Typically stacks of 4 to 8 images (512 × 512) were taken and analyzed at 122 nm intervals through z axis of a section. Alternatively a Nikon Eclipse TE300 epifluorescence microscope (Nikon Instruments Inc, Melville, NY) equipped with a DS-5M Standard charge-coupled device camera (Nikon Instruments Inc) was used. Confocal images were processed using the software Imaris (Bitplane, Zurich, Switzerland). Images with LAT2 staining in CPs of wild-type animals vs LAT2 KO were merged using overlay function in Photoshop 9.

Animals were anesthetized with a ketamine (100 mg/kg) and xylazine (10 mg/kg) cocktail administrated IP, and choroid plexuses were rapidly removed from four ventricles of each animal under stereomicroscope Olympus (SZX10, Volketswil, Switzerland) as described by Bowyer [26]. The rest of each brain (cerebrum and cerebellum separately; referred as total brain) was cut into small pieces (~ 30 mg) and these samples were used later as purity control of isolated CPs. All samples were snap frozen in liquid nitrogen and stored at − 80 °C till further analysis.

Total RNA from individual CPs and total brain was isolated with Trizol (15596026, Thermofisher Scientific AG, Reinach, Switzerland) according to the manufacturer protocol followed by purification on RNeasy Micro (74004) or Mini columns (74106, Qiagen AG, Hombrechtikon, Switzerland). Total RNA was quantified using NanoDrop ND 1000 spectrophotometer (Thermo Fisher Scientific Wilmington, USA) and quality was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only samples with RIN values ≧ 8.0 were used for reverse transcription. The cDNA was synthesized from 100 ng (5 ng/μL) of total RNA using qScript cDNA Synthesis Kit (95047-100, Quantabio, Beverly, USA) according to the manufacturer’s protocol. Quantitative real-time PCR reactions (qRT-PCR) with 10 ng of cDNA as template were performed using the Taq-Man Universal PCR master mix (4304447, Thermofisher Scientific AG, Reinach, Switzerland) in triplicates. In each reaction mixture eukaryotic 18S rRNA endogenous control (4310893E, Thermofisher Scientific AG, Reinach, Switzerland) was included, while cDNA produced without RT enzyme were used as the negative control for each gene. All reactions were carried out in MicroAmp Fast Optical 96-Well Reaction Plates (4346906 Thermofisher Scientific AG, Reinach, Switzerland) using the Fast Real Time PCR System 7500 (Applied Biosystems) with the following parameters: an initial step at 50 °C for 2 min, denaturation at 95 °C for 10 min for polymerase activation followed by 45 cycles with denaturation step at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Primers and probes were either previously described or designed at Universal probe Library Assay Design Center Roche [27] and listed in Additional file 2: Table S2. Prior to usage, the specificity of all newly designed primers was tested on cDNA samples obtained from several different organs and in each case a single product of expected size was observed. Probes were labeled with reporter dye VIC or FAM at 5′ end and quencher dye TAMRA no dye at 3′ end. Relative expression of each gene of interest was calculated based on the comparative ΔC_T method according to the formula: relative expression = 2^−ΔCT, where ΔCT = average C_T value of gene of interest − average C_T value of housekeeping gene, where 18S rRNA was used as housekeeping gene. C_T values of 18S rRNA were between 7.2 and 12.5. The ones of amino acid transporter mRNAs with a relative expression > 2 × 10⁶ ranged from 24.1 (Slc38a3) to 30.3 (Slc1a3).

Measurements of AA concentrations were performed at the Functional Genomic Centre Zurich.

Amino acid concentrations were determined in samples using Mass Track Amino Acid Analysis Application Solution (Waters, Milford, USA) by ACQUITY UPLC according to the manufacturer’s protocol. CSF samples were analyzed directly and for plasma samples deproteinization 1:1 with 10% SSA (sulfosalicylic acid) was performed prior to AA measurements. Plasma samples after precipitation with 10% SSA were diluted 10 times with borate buffer (500 mM, pH 9), precipitated with methanol (5 times) and then analyzed.

To study the expression of specific amino acid transporters in CP, we tested first their mRNA levels, although they are known not to correlate with protein expression. However, the presence of an mRNA is per se a prerequisite for the expression of its protein product. We tested initially the purity of the CPs isolated from the four ventricles of each individual animal by measuring the mRNA level of four cell specific markers by qPCR, transthyretin (Ttr) as choroidal marker, glial fibrillary acidic protein (Gfap) for astrocytes, platelet endothelial cell adhesion molecule-1 (Pecam 1 or Cd31) for brain endothelial cells and synaptophysin (Syp) for neurons (Fig. 1a). The level of Gfap and Syp mRNAs were strongly decreased in choroid plexuses when compared to cerebral samples isolated from the same animals (by ~ 91% and ~ 99%. respectively), while the level of Cd31 mRNA was only halved (~ 46%), reflecting the expected presence of vascular endothelial cells in the choroid plexus samples. Since Ttr mRNA was increased ~ 330-fold in isolated choroid plexus compared with cerebral samples, we considered that the enrichments was sufficient and proceeded with a first experiment in which a set of 32 selected Slc transcripts encoding AATs (out of 66 known amino acid transporters including intracellular ones [28]) were tested in three animals (Additional file 1: Figure S1). Based on expression values calculated relative to the endogenous reference 18S rRNA, the tested gene products were arbitrarily assigned into three groups with different expression levels: 22 with low (0–2 * 10⁻⁶ relative to 18S), 5 with moderate (2–10 * 10⁻⁶ relative to 18S) and 5 with high (> 10 * 10⁻⁶ relative to 18S) expression level. Taking into account data available in the literature [18‐20, 29] and the results of our first experiment, we selected 14 amino acid transporter mRNAs (Slc1a1, Slc1a3, Slc6a20b, Slc7a5, Slc7a6, Slc7a7, Slc7a8, Slc7a10, Slc7a11, Slc38a1, Slc38a2, Slc38a3, Slc38a5 and Slc38a6) for a detailed investigation that involved ten different animals measured in three independent experiments (Fig. 1b). In agreement with previous studies we confirmed a significant mRNA expression for Slc6a20b (SIT1), Slc7a10 (ASC1) and Slc38a3 (SNAT3) [18‐20]. Additionally we found highest mRNA expression levels for two other amino acid transporters, namely Slc7a8 (LAT2) and Slc38a1 (SNAT1), actually in contrast to a previous study that had reported lower than average levels [20]. The mRNAs of the Slc38 family members Slc38a2 (SNAT2) and Slc38a6 (SNAT6), the y⁺L system member Slc7a6 (y⁺LAT2) and the Glu transporter Slc1a3 (EAAT1 or GLAST) were found moderately expressed, while the mRNAs of Slc1a1 (EAAT3), Slc7a5 [large neutral amino acid 1 (LAT1)], Slc7a7 (y⁺LAT1), Slc7a11 (xCt) and Slc38a5 (SNAT5) were expressed at a low level.

Next, we aimed to localize amino acid transporters highly expressed at the mRNA level, on choroid epithelial cell membranes using immunofluorescence.

We chose to use only custom-made antibodies the specificity of which had been previously validated in transfected cells or mouse tissue (brain, kidney and cochlea), specifically anti-SNAT3 (Fig. 2a, j), anti-LAT2 (Fig. 2d, m) and anti-SNAT1 (Fig. 2g, p) antibodies [30‐34]. The fact that we localized only these three amino acid transporters may be considered as a limitation in view of the larger number of transporters detected at the mRNA level, but since protein localization studies are per se prone to artefacts (cross-reactivity etc.), including only these three increased the reliability of our results.

As a marker for the luminal, CSF-facing membrane of the choroid plexus epithelial cells we used an antibody recognizing the Na⁺, K⁺-ATPase α subunit (isoforms α1–3) and for the basolateral, blood-facing side an antibody recognizing the anion exchanger 2 (AE2). These localizations correspond to the so-called inverse polarization of the choroid plexus epithelium [2, 35, 36]. Staining of adult mouse brain sections revealed in CP clear SNAT3 colocalization with the Na⁺, K⁺-ATPase α subunit (Fig. 2a–c), but not with AE2 (Fig. 2j–l) and interestingly, the same localization pattern was demonstrated for LAT2 (Fig. 2d–f, m–o). While SNAT1 transporter was visualized solely on the basolateral membrane co-localizing with AE2 and resulting in an evident yellow staining (Fig. 2g–i and m, q, r). Unfortunately, we have not been able to observe any reliable signal for the two other amino acid transporters highly expressed in choroid plexus at the mRNA level, ASC1 and SIT1, using commercially available or in-house produced antibodies.

Considering the high level of LAT2 expression in CP, we examined the impact of LAT2 ablation on AA concentrations in CSF of Lat2 KO animals [33]. We confirmed ablation of LAT2 transporter in CP on mRNA and protein levels (Additional file 1: Figure S2A, B) and measured amino acid levels in plasma and CSF samples. Previously Braun et al. had reported elevated levels of 8 amino acids (Ala, Ser, Gly, Thr, Glu, Asp and Lys) for the serum of LAT2 knockout (KO) animals [37], however these alterations were not reproduced in our experiments using another LAT2 knock-out model (Additional file 2: Table S3) [32, 33]. Therefore, we compared the CSF/plasma ratio of each out of 19 detected amino acids [18 proteinogenic AA (all except Cys and Ile) and Tau] between wild type and LAT2 KO animals. Raised CSF/plasma ratios were detected for at least six amino acids (other possible increases were not significant): the large neutral branched chain and aromatic amino acids Leu (p < 0.01), Val (p < 0.01) and Trp (p < 0.05), the inhibitory neurotransmitter Gly (p < 0.001), the imino acid proline (Pro) (p < 0.01), and the excitatory amino acid Glu (p < 0.05) (Fig. 3). Interestingly, the latter three amino acids are not influx substrates of LAT2 [38], suggesting a possible functional cooperation of LAT2 with other amino acid transporters.