Fig 2: HERV-W ENV enhances the sodium influx through DRD2/PP2A/AKT1/GSK3 in the DA neurons: (A) a representative set of sodium current traces from SH-SY5Y cells that were elicited by depolarizing steps from -60 to 20 mV in 10-mV increments from a holding potential of -100 mV. (B) The sharp contrast of sodium currents in the control (the red line), HERV-W ENV (the black line), and the HERV-W ENV and Droperidol coexistence (the blue line) groups. (C) The average sodium currents alteration from -130 mV to 60 mV in the three groups. (D) The sodium currents were voltage dependent and were different in -20 mV, -10 mV, 0 mV, 10 mV, and 20 mV among the three groups. (E) Activation-voltage relationships obtained in the presence or absence of HERV-W ENV and Droperidol. There was a significant rightward shift of the activation-voltage relationship in the presence of HERV-W ENV (p < 0.01). The 1/2-activation voltage was recovered in the absence or presence of Droperidol (p < 0.01). (F) Illustration of the signaling pathway by which HERV-W ENV regulates dopaminergic system in schizophrenia. Each experiment was performed three times with a representative result shown. ns p > 0.05, *** p < 0.001.

Fig 3: HERV-W ENV facilitates the synthesis and transport of DA: (A) DA concentration in the culture supernatants of SH-SY5Y cells reached the highest peak 24 h after transfection and the concentration in the HERV-W ENV overexpressed group was greater than the control. (B) The mRNA levels of TH and DAT in the HERV-W ENV overexpression group were higher than the control group in the DA neurons. (C) The protein level of TH and DAT in the HERV-W ENV overexpression group was higher than the control group in the DA neurons. (D) Cellular immunofluorescence assays of TH in the DA neurons. (E) Representative Western blots for the synaptic vesicle-related proteins and GAPDH in the DA neurons. (F) SYP expressed higher in the HERV-W ENV overexpression group by cellular immunofluorescence. (G) STED images of FM 1-43 in the primary neurons between the pCMV group (1) and pCMV-HERV-W ENV group (2). The OD value and number of SVs increased after HERV-W ENV overexpression. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: HERV-W ENV promotes synaptic proteins and regulates the PP2A/AKT1/GSK3 signal pathway: (A,B) representative Western blots for the presynaptic and postsynaptic proteins, GAPDH, and TUJ1 in the SH-SY5Y cells and the primary neurons. (C) Overexpression of HERV-W ENV in the DA neurons increased PP2A and AKT1 and regulated the downstream protein. (D) PP2A activity detected by biochemical kit assay increased in the pCMV-HERV-W ENV group in SH-SY5Y cells. (E) The PP2A was downregulated and its downstream proteins were upregulated after using LB-100 (inhibitor of PP2A) in the DA neurons. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 5: HERV-W ENV increases DRD2 expression by colocalizing and interacting with it: (A) the mRNA level of DRD2 in the HERV-W ENV overexpression group was higher than the control group in the DA neurons. (B) The protein level of DRD2 in the HERV-W ENV overexpression group was higher than the control group in the DA neurons. (C) Luciferase assays of pGL3-DRD2 promoter co-transfected with pCMV-HERV-W ENV or control vector in SH-SY5Y cells. (D) Three-color STED images of Hoechst, which specifically stain the nuclei (D1), HERV-W ENV-TM (D2), and DRD2 (D3) in HEK293T cells (D4). Three-color overlay STED images of Hoechst, pEGFP-HERV-W ENV-TM, and pENTER-DRD2 in HEK293T cells. (E) Three-color STED images performed as in Figure 4D in SH-SY5Y cells. (F) Co-IP results shown in the DRD2-Flag-IP group could show the HERV-W ENV-HA band but could not show such in the negative control, which demonstrated the interactions between each other. * p < 0.05, ** p < 0.01, *** p < 0.001.