Role of serotonin in human placental cytotrophoblast differentiation and gene expression

Serotonin (5-hydroxytryptamine; 5-HT) is transported into the human placenta through the serotonin transporter (SERT/SLC6A4) on the surface of the syncytiotrophoblast, where it ultimately makes its way to the circulation within the conceptus. During this transit a significant amount of 5-HT becomes concentrated in the cytotrophoblast nucleus. We used immunochemistry, inhibitors of SERT and transglutaminase 2 (TGM2; the enzyme that mediates the covalent linkage of 5-HT to protein glutamine residues—a process known as serotonylation), and RNA sequencing to elucidate the mechanism and consequences of this nuclear localization. Exogenous 5-HT recapitulated the uptake of 5-HT into the trophoblasts and its preferential concentration in cytotrophoblast nuclei we observed in the intact placenta. Inhibiting SERT with escitalopram or TGM2 with cystamine blocked cytotrophoblast differentiation in vitro; namely, flattening, aggregation and forming syncytia. Cystamine eliminated the staining of the nuclei in placental explants by exogenous 5-HT, suggesting that serotonylation mediated this phenomenon. This was confirmed by western blots and immunoprecipitation which identified histone 3 (H3), and specifically the 5th glutamine residue in H3, as a site of serotonylation. Blocking serotonylation led to marked changes in RNA expression. Of the 38,524 mRNAs identified in these trophoblasts, cystamine changed the expression of 1,986 and escitalopram significantly altered 374. Both treatments altered the expression of 155 mRNAs either positively or negatively. In general, the genes that were downregulated were involved with cell proliferation, morphogenesis, motility, and growth—while genes that were upregulated controlled cell survival and protection pathways. These findings suggests that maternal 5-HT promotes placental, embryonic/fetal, and organismal development through histone serotonylation and consequent alterations in gene expression. They raise the possibility that alterations in 5-HT flux in the placenta affect placental and fetal growth, as well as organismal somatic and neurologic developmental trajectories.

Methods
Specimens

Term placentas were collected anonymously from patients undergoing elective repeat Cesarian sections who delivered healthy babies. All the women who provided term placental samples signed an informed consent (Yale IRB protocol 1208010742).

Trophoblast Purification

Cytotrophoblast were prepared from fresh human term placentas by trypsin digestion and Percoll gradient fractionation, with the addition of an anti-CD9 and CD45 magnetic bead purification step, as previously described (1,2). During the initial steps of the digestions and up to the magnetic purification step, 10% fetal bovine serum (FBS, Gemini Bio-Products, Broderick, CA, 100-106) diluted in DMEM/F12 (Gibco, ThermoFisher Scientific, Waltham, MA, 11330-032) with 4 mM L-glutamine, 1% penicillin/streptomycin was utilized. After the magnetic purification step, no FBS was added to the culture media, unless specifically noted. Cytotrophoblasts were then diluted to 1x106 cells per ml in DMEM/F12 with 4 mM L-glutamine, 1% penicillin/streptomycin and either: 1) pelleted immediately by centrifugation for a zero-time point; 2) cultured in two- or four-chamber Nunc™ Lab-Tek™ II Chamber Slide™ System slides (ThermoFisher Scientific) or six-wells plates (CELLSTAR® #657160 6-well cell culture plates) for 24-96 hours in humidified 5% CO2-95% air at 37˚C; or 3) and used for additional studies as described below.

RNA sequencing

RNA Seq Quality Control: Total RNA quality was determined by estimating the A260/A280 and A260/A230 ratios by Nanodrop (Nanodrop, ND-1000, ThermoFisher Scientific). RNA integrity was determined by running an Agilent Bioanalyzer 2100 gel, which measures the ratio of the ribosomal peaks.

RNA Seq Library Prep: Using the Kapa RNA HyperPrep Kit with RiboErase (KR1351, Roche Diagnostics, Indianapolis, IN), rRNA was depleted starting from a normalized input of total RNA by hybridization of rRNA to complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed toDNA. Samples were then fragmented using heat and magnesium. 1st strand synthesis was performed using random priming. 2nd strand synthesis incorporated dUTPs into the 2nd strand cDNA. Adapters were then ligated, and the library was amplified. Strands marked with dUTPs were not amplified allowing for strand-specific sequencing. Indexed libraries that meet appropriate cut-offs for both quantity and quality were quantified by qRT-PCR using a commercially available kit (KAPA Library Quant Kit, Roche Diagnostics, 07960298001) and insert size distribution determined with an Agilent Bioanalyzer (Agilent TapeStation 4200). Samples with a yield of ≥0.5 ng/µl were used for sequencing.

Flow Cell Preparation and Sequencing: Sample concentrations were normalized to 2.0 nM and loaded onto an Illumina NovaSeq (San Diego, CA) flow cell at a concentration that yields 35 million passing filter clusters per sample. Samples were sequenced using 100bp paired-end sequencing on an Illumina NovaSeq according to Illumina protocols. The 10bpunique dual index was read during additional sequencing reads that automatically follow the completion of read 1. Data generated during sequencing runs were simultaneously transferred to the Yale Center for Genomic Analysis (New Haven, CT) high-performance computing cluster. A positive control (prepared bacteriophage Phi X library) provided by Illumina was spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time.

Data Analysis and Storage: Signal intensities were converted to individual base calls during a run using the system's Real Time Analysis (RTA) software. Base calls were transferred from the machine's dedicated personal computer to the Yale High Performance Computing cluster via a 1 Gigabit network mount for downstream analysis. Primary analysis—sample de-multiplexing and alignment to the human genome—was performed using Illumina's CASAVA 1.8.2 software suite. The data were returned to the user if the sample error rate was less than 2% and the distribution of reads per sample in a lane was within reasonable tolerance. Data was retained on the cluster for at least 6 months, after which it was transferred to a tape backup system.

Following RNA sequencing, low quality reads were trimmed, and adaptor contamination were removed using Trim Galore (v0.5.0; Babraham Bioinformatics, Cambridge, UK). Trimmed reads were mapped to the human reference genome (mm10) using HISAT2 (v2.1.0) (3). Gene expression levels were quantified using StringTie (v1.3.3b) (4) with gene models (v27) from the GENCODE project (5). Differentially expressed genes were identified using DESeq2 (v 1.22.1) (6). Transcripts per million (TPM) were calculated as previously described (7), while adjusted p values were calculated with DESeq2 using the Benjamini and Hochberg method (8).

References

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