Domesticated HERV-W env contributes to the activation of the small conductance Ca2+-activated K+ type 2 channels via decreased 5-HT4 receptor in recent-onset schizophrenia

Abstract

The human endogenous retroviruses type W family envelope (HERV-W env) gene is located on chromosome 7q21-22. Our previous studies show that HERV-W env is elevated in schizophrenia and HERV-W env can increase calcium influx. Additionally, the 5-HTergic system and particularly 5-hydroxytryptamine (5-HT) receptors play a prominent role in the pathogenesis and treatment of schizophrenia. 5-hydroxytryptamine receptor 4 (5-HT4R) agonist can block calcium channels. However, the underlying relationship between HERV-W env and 5-HT4R in the etiology of schizophrenia has not been revealed. Here, we used enzyme-linked immunosorbent assay to detect the concentration of HERV-W env and 5-HT4R in the plasma of patients with schizophrenia and we found that there were decreased levels of 5-HT4R and a negative correlation between 5-HT4R and HERV-W env in schizophrenia. Overexpression of HERV-W env decreased the transcription and protein levels of 5-HT4R but increased small conductance Ca²⁺-activated K⁺ type 2 channels (SK2) expression levels. Further studies revealed that HERV-W env could interact with 5-HT4R. Additionally, luciferase assay showed that an essential region (-364 to -176 from the transcription start site) in the SK2 promoter was required for HERV-W env-induced SK2 expression. Importantly, 5-HT4R participated in the regulation of SK2 expression and promoter activity. Electrophysiological recordings suggested that HERV-W env could increase SK2 channel currents and the increase of SK2 currents was inhibited by 5-HT4R. In conclusion, HERV-W env could activate SK2 channels via decreased 5-HT4R, which might exhibit a novel mechanism for HERV-W env to influence neuronal activity in schizophrenia.

Keywords: 5-Hydroxytryptamine receptor 4 (5-HT4R); Env; Human endogenous retroviruses type W (HERV-W); Schizophrenia; Small conductance Ca(2+)-activated K(+) type 2 channels (SK2).

Fig. 1

The expression levels of 5-HT4R and the correlation between 5-HT4R and HERV-W env in recent-onset schizophrenia. (A) and (B) The concentration of 5-HT4R in healthy controls (N ​= ​31) and schizophrenia (N ​= ​33) by ELISA. (C) and (D) The concentration of HERV-W env in healthy controls (N ​= ​31) and schizophrenia (N ​= ​33) by ELISA. E Linear regression correlation between HERV-W env and 5-HT4R expression levels in schizophrenia. X-axis: the concentration of HERV-W env; Y-axis: the concentration of 5-HT4R. The line represents the calculated “best-fit” equation of values within the boxed area, with correlation value indicated on the top (R² ​= ​0.76). P ​< ​0.01, P ​< ​0.001 by median and nonparametric analysis. Note: each point generally represents a single patient, but a few are overlapping and cannot be separated on the graph.

Fig. 2

HERV-W env could down-regulate the expression of 5-HT4R and directly interact with 5-HT4R in human neuroblastoma SH-SY5Y cells. A HERV-W env phylogenetic tree. Phylogenetic tree constructed using amino acid sequences of the envelope proteins consensuses region of HERVs and other representative retroviruses (alpha-, beta-, gamma-, deltaretroviruses, and lentiviruses). HERVs family contained class I (HERV-H env, HERV9 env, HERV-W env, HERVADP env, HERV-T env, HERVIP env, HERVFA_env, HERVFB_env, HERV-FC_env, hervh48_env, prima41 env, HERV1 env, HERV3 env, HERV4_env, HERVI env, HERV-E env, PABL env, and HUERSP3_env), class II (HERVL_env and HML2_env), and class III (HERVL32_env and HERVS_env). Alpharetrovinus: ALV (Chicken), EVA_HP (Chicken), Cja_BASJ02003037.1 (Coturnix japonica), RSV (Chicken), Tcu_MOXI01007493.1 (Tympanuchus cupido), and Csq_MCFN01002646.1 (Callipepla squamata). Betaretrovirus: MMTV (mouse). Gammaretrovirus: F MuLV (mouse), R-MuLV (mouse), MDEV (mouse), KoRV (Phascolarctos cinereus), Mab3_env (Mabuya), Mab4_env (Mabuya), and GALV (Gibbon). Deltaretrovirus: HTLV1 (human), HTLV3 (human), and HTLV4 (human). Lentivirus: HIV1 (human) and HIV2 (human). Unclassifiable: LTR46_env (human). The red triangle is the HERV-W env. The env tree is rooted in betaretrovirus and lentiretrovirus. B–C SH-SY5Y cells were transfected with pCMV-HERV-W env or empty vector pCMV. The mRNA and protein levels of 5-HT4R were determined by real-time quantitative PCR (B) and Western blotting (C). D Coimmunoprecipitation assays (COIP) were performed with anti-HA-HERV-W env or anti-Flag-5-HT4R antibodies by Western blotting in SH-SY5Y cells. E STED images of HERV-W env-TM (green) and 5-HT4R (red). All experiments were repeated three times. Data are presented as the mean ​± ​SD. Statistical analysis was performed by one-way analysis of variance (ANOVA). ∗P ​< ​0.05.

Fig. 3

HERV-W env positively regulated the SK2 expression by enhancing its promoter activity. (A, B) SH-SY5Y cells were transfected with pCMV-HERV-W env or empty vector pCMV. The mRNA and protein levels of SK2 were determined by real-time quantitative PCR (A) and Western blotting (B). C Schematic illustration of SK2 promoter constructs. SK2 full-length promoter region from −572 to +90. T1-T2, serial 5′ truncated SK2 promoter constructs. T3-T4, two special promoter fragments. (D–F) Luciferase activity for SK2 promoter. D SH-SY5Y cells were cotransfected with full-length SK2 promoter-luciferase reporter construct and HERV-W env expression vector or empty vector pCMV. (E–F) Reporter plasmids containing different lengths of putative SK2 promoters were cotransfected with pCMV-HERV-W env or empty vector pCMV in SHSY5Y cells. Each experiment was repeated three times. Data shown are mean ​± ​SD. Statistical analysis was performed by one-way analysis of variance (ANOVA). ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001, ^NSP ​> ​0.05.

Fig. 4

5-HT4R mediated the up-regulation of SK2 induced by HERV-W env. SH-SY5Y cells were transfected with HERV-W env and pENTER-5-HT4R. The expression of the SK2 mRNA was examined by RT-qPCR (A). The expression of SK2 protein was detected by Western blotting (B). SH-SY5Y cells were transfected with HERV-W env and then treated by 5-HT4R agonist RS67333. The expression level of the SK2 mRNA and protein was determined by RT-qPCR (C) and Western blotting (D). E Assessment of 5-HT4 receptor function in regulating the increased SK2 promoter activity via tri-transfection of pGL3/pGL3-SK2-promoter-full, pCMV/pCMV-HERV-W env plasmid, and pENTER/pENTER-5-HT4R. Data shown are mean ​± ​SD from three independent experiments. Statistical analysis: one-way ANOVA (∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001).

Fig. 5

HERV-W env triggered SK2 channel activation in SH-SY5Y. A Representative current recorded in pIRES2-EGFP (control cell) and in pIRES2-EGFP-env (HERV-W env expression cell) using whole-cell patch, voltage steps ranging from −120 mV to +70 ​mV in the presence of 100 ​nmol/L Apamin or 50 ​μmol/L Lei-dab7 at 500 ​nmol/L Ca²⁺ free. B The currents of SK2 in SH-SY5Y cell were measured by whole-cell patch experiments, 100 ​nmol/L Apamin and 50 ​μmol/L Lei-dab7 were separately added to the chamber solution. C Histograms showed the average current density (pA/pF) of SK2 channel in SH-SY5Y from pIRES2-EGFP controls, pIRES2-EGFP-HERV-W env-transfected, pIRES2-EGFP-HERV-W env-transfected ​+ ​Apamin or Lei-dab7 at 50 ​mV, 60 ​mV and 70 ​mV. Group data are mean ​± ​SD. ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001; one-way ANOVA. Each experiment was repeated three times.

Fig. 6

HERV-W env induced SK2 channel activation by inhibiting the 5-HT4 receptor signal pathway. (A, B) Representative SK2 channel current traces were obtained in SH-SY5Y cells from pIRES2-EGFP (control cell), pIRES2-EGFP-HERV-W env (HERV-W env-expression cell), pIRES2-EGFP-HERV-W env in the presence of RS67333, pIRES2-EGFP-HERV-W env in the presence of RS67333 and Apamin (A, right), or Lei-dab7 (B, right). (C, D) The currents of SK2 in SH-SY5Y cells were measured by whole-cell patch experiments. (C) 100 ​nmol/L Apamin, (D) 50 ​μmol/L Lei-dab7. (E, F) Histograms showed the average current density (pA/pF) of SK2 channel in SH-SY5Y from pIRES2-EGFP control, pIRES2-EGFP-HERV-W env-transfected, pIRES2-EGFP-HERV-W env-transfected ​+ ​RS67333, pIRES2-EGFP-HERV-W env-transfected ​+ ​RS67333+Apamin or Lei-dab7 at 50 ​mV, 60 ​mV and 70 ​mV. (E) 100 ​nmol/L Apamin, (F) 50 ​μmol/L Lei-dab7. Data are presented as mean ​± ​SD. Data represent three independent experiments. ∗P ​< ​0.05, ∗∗P ​< ​0.01, ^NSP ​> ​0.05; one-way ANOVA.

Fig. 7

A possible hypothesis is that HERV-W env may influence excitability to participate in the etiology of schizophrenia. There is a negative correlation between HERV-W env and 5-HT4R in schizophrenia. HERV-W env can directly interact with 5-HT4R and downregulate 5-HT4R expression levels. Additionally, 5-HT4R participates in the regulation of SK2 promoter activity induced by HERV-W env. Hence, there is an increased level of SK2 expression and activating SK2 channels in the cells. The opening of the SK2 channel leads to hyperpolarization and increases K⁺ current, which affects the excitability of the cell membrane to participate in the development of schizophrenia.