Implication of the env gene of the human endogenous retrovirus W family in the expression of BDNF and DRD3 and development of recent-onset schizophrenia

Abstract

Objective: Retrovirus has been suggested as one of agents involved in the development of schizophrenia. In the present study, we examined the role of the human endogenous retrovirus W family (HERV-W) env gene in the etiopathogenesis of recent-onset schizophrenia, using molecular and epidemiological approaches.

Methods: Nested RT-PCR was used to detect the messenger RNA (mRNA) of the HERV-w env gene in plasmas. Quantitative real-time polymerase chain reaction (PCR) was employed to detect the viral reverse transcriptase activity in human sera. Human U251 glioma cells were used to study the potential role of the HERV-W env gene in the etiopathogenesis of recent-onset schizophrenia.

Results: We identified genes with mRNA sequences homologous to HERV-W env gene from plasmas of 42 out of 118 individuals with recent-onset schizophrenia but not from any of 106 normal persons (P < .01, t test). Quantitative real-time PCR showed a significantly increase in the reverse transcriptase activity in the sera of patients (by 35.59%) compared with controls (by 2.83%) (P < .05, t test). Overexpression of HERV-w env in human U251 glioma cells upregulated brain-derived neurotrophic factor (BDNF), an important schizophrenia-associated gene, neurotrophic tyrosine kinase receptor type 2 (NTRK2, also called TrkB), and dopamine receptor D3 and increased the phosphorylation of cyclic adenosine monophosphate response element-binding (CREB) protein. BDNF promoter reporter gene assays showed that the HERV-W env triggers BDNF production in human U251 glioma cells. Using gene knockdown, we found that CREB is required for the expression of BDNF that is regulated by env.

Conclusion: Our data revealed that the transcriptional activation of HERV is associated with the development of schizophrenia in some patients and indicated that HERV-W env regulates the expression of schizophrenia-associated genes. This report is the first to elucidate the signaling pathway responsible for the upregulation of HERV-W env-triggered BDNF. Our study provides new evidence for the involvement of HERV-W in the central nervous system, which will benefit the diagnosis and treatment of the devastating schizophrenia and related disorders.

Fig. 1.

Detection of Human Endogenous Retrovirus (HERV) env Sequence in the Blood of Recent-Onset Schizophrenia. (A) No contamination of genomic DNA in total RNA from blood samples. RT-PCR using glyceraldehyde-3-phosphate dehydrogenase–specific primer pairs have been conducted on total RNAs from patients (lane 3) and from normal individual (lane 4) and complementary DNA (cDNA) reverse transcripted from patients (lane 1) and normal individual (lane 2). Lane 5 is standard DNA markers (DL2000, from up to low 2000, 1000, 750, 500, 250, and 100 bp). (B) Detection of HERV env sequence in the cDNA of leukocytes by nested RT-PCR. Lanes 1–5, plasmas of 5 individuals with recent-onset schizophrenia; lanes 6, no template controls; lane 7, DNA markers (DL2000, up to down: 2000, 1000, 750, 500, 250, 100).

Fig. 2.

Sequence Comparison of Amplified Sequence From Patients’ Plasma With ERVW1 (AY101583), Human Endogenous Retrovirus (HERV)-W (AF072506), and Multiple Sclerosis–Associated Retrovirus (MSRV) (AF331500) sequence.

Fig. 3.

Mapping and Phylogenetic Analysis. (A) Ideogram of human chromosome 7q11.23–7q22.1 with the hypothesized location of env gene (shadow marks, positions of the mapped env sequences). (B) Phylogenetic tree for the pol genes obtained by the neighbor-joining method. Sequences used in phylogenetic tree are as follows: Human endogenous retrovirus W (AF075206), H. sapiens isolate 21 endogenous retrovirus Human Endogenous Retrovirus (HERV)-W, ERVWE1 (AY101583), multiple sclerosis–associated retrovirus element clone pV14 (AF331500), env-related transmembrane protein (S52616), Pan troglodytes similar to ERV-BabFc env provirus (XM_526964), P. troglodytes LOC470416 (LOC470416) (XM_525797), Canis familiaris similar to myocardin (LOC489509)(XM_546628), chimpanzee endogenous retrovirus proviral envH3 gene (AJ862649), HERV-H/env59 proviral copy (AJ289711), RD114 retrovirus env gene(X87829), P. troglodytes similar to ERV-BabFcenv provirus (XM_528924), porcine endogenous retrovirus C(EU440732), human T-lymphotropic virus 2 (NC_001488), HIV-1 isolate 02CM-A1394 from Cameroon (DQ845388), Mus dunni endogenous virus complete genome (AF053745), H. sapiens HERV-FRD provirus ancestral env polyprotein (HERV-FRD) (NM_207582), human endogenous retroviral sequence 3 (includes zinc finger protein H-plk/HPF9) (ERV3)(NM_001007253), H. sapiens endogenous virus HERV-K68 (DQ112096), human endogenous virus HERV-Pb1 (DQ247958), Mason-Pfizer monkey virus (NC_001550). HIV, human immunodeficiency virus.

Fig. 4.

Comparison of Schizophrenia Case and Healthy Control Sera by Real-Time Polymerase Chain Reaction. (A) Standard curve for reverse transcriptase activity quantification. The threshold cycle values were plotted against the reverse transcriptase activity. (B) Reverse transcriptase activities for individual samples from both groups are designated. The bottom and top boundaries of the boxes indicate the 25th and 75th percentiles of the values. The bottom and top horizontal bars indicate the 5th and 95th percentiles of the values, respectively. The notch indicates the 95 confidence limits around the median.

Fig. 5.

Human Endogenous Retrovirus (HERV)-W env Upregulates the Expression of Messenger RNA (mRNA) of Brain-Derived Neurotrophic Factor (BDNF), NTRK2, and DRD3 in Human U251 Glioma Cells. (A) BDNF, NTRK2, and DRD3 gene expression in the human U251 glioma cells. A 363-bp Complementary DNA fragment for BDNF mRNA, a 384-bp fragment for NTRK2 mRNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (B) Histograms show BDNF, NTRK2, and DRD3 mRNA levels quantified; each bar represents the mean ± standard error of the mean of 3 values of IL-8 amplification products normalized to the starting total RNA volumes and referred to the corresponding GADPH values. n = 3 (*P < .005) (Student t test). (C) BDNF, NTRK2, and DRD3 mRNA levels using quantitative RT-PCR; each bar represents the mean ± standard error of the mean of 3 values of IL-8 amplification products normalized to the starting total RNA volumes and referred to the corresponding GADPH values. n = 3 (*P < .005) (Student t test).

Fig. 6.

Human Endogenous Retrovirus (HERV)-W env Upregulates the Expression of protein of Brain-Derived Neurotrophic Factor (BDNF), NTRK2, and DRD3 in human U251 glioma Cells. (A) Western blot analysis for the expression of BDNF, NTRK2, and DRD3 after 48 h of transfection. (B) Bars represent the intensity of the bands, quantitated by densitometry. Data are expressed as the mean ± SD of triplicate determination in a representative experiment from 2 independent experiments with similar results. n = 3 (*P < .005).

Fig. 7.

Human Endogenous Retrovirus (HERV)-W env–Induced Brain-Derived Neurotrophic Factor (BDNF) Promoter Activation. Human U251 glioma cells were transfected with a BDNF promoter luciferase vector along with env plasmid. Luciferase activity served as a marker of BDNF promoter activity. Forty-eight hours later, luciferase activity was measured and standardized. Data are mean ± SD. Statistically different as compared with control cultures. All data are expressed as fold induction of normalized BDNF luciferase activity (mean ± standard error of the mean) relative to uninfected cells (n = 3). And Infection with HERV-W env for 48 h also significantly increased BDNF luciferase activity by >2-fold as compared with control cells transfected with an empty vector.

Fig. 8.

Cyclic Adenosine Monophosphate Response Element (CRE) on Brain-Derived Neurotrophic Factor (BDNF) Promoter Is Required for the Expression of BDNF Regulated by env. Assessment of CRE function using luciferase reporter construct. Wild-type (pGL3-PIII) and mutant promoter III (pGL3-PIII_CREm) plasmid constructs were cotransfected with plasmid pSV-ß-gal into human U251 glioma cells using lipofectamine method. Cells were lysed and analyzed for luciferase activity (as described in the “Materials and Methods”) assay. Transfection efficiency of each sample was normalized by β-galactosidase activity. The data represent the mean ± SE from 3 independent experiments.

Fig. 9.

env Increases the Phosphorylated Cyclic Adenosine Monophosphate Response Element–Binding (CREB) Protein in Human U251 Glioma Cells. (A) Western blot analysis for the expression of the phosphorylated CREB after 48 h of transfection. (B) Bars represent the intensity of the bands, quantitated by densitometry. Data are expressed as the mean ± SD of triplicate determination in a representative experiment from 2 independent experiments with similar results. n = 3 (*P < .005).

Fig. 10.

Silencing of Cyclic Adenosine Monophosphate Response Element–Binding (CREB) by Using Specific Small Interfering RNA (siRNA) Prevents the Expression of Brain-Derived Neurotrophic Factor (BDNF) Regulated by env. (A) Cells were transfected for 4 h with 100 nM siRNA. Western blot analysis for the expression of BDNF after 48 h of transfection. (B) Bars represent the intensity of the bands, quantitated by densitometry. Data are expressed as the mean ± SD of triplicate determination in a representative experiment from 2 independent experiments with similar results. n = 3 (*P < .005).

Fig. 11.

A Hypothesis for Human Endogenous Retrovirus (HERV) Contribute to the Pathogenesis of Recent-Onset Schizophrenia. Infectious agents (such as virus, etc), environmental factors (such as UV, etc), and genetic variation could trigger the expression of HERV-W env. The expression of HERV proteins (such as env) increases the promoter activation of some genes, such as BDNF, NTRK2, and DRD3, etc (figure 7), and increases the expression of these genes (figures 5 and 6) in neuroglia cells. The expression of these genes contributes to the pathogenesis of the recent-onset schizophrenia.,– BDNF, brain-derived neurotrophic factor.