CPEB1, a novel risk gene in recent-onset schizophrenia, contributes to mitochondrial complex I defect caused by a defective provirus ERVWE1

Figure 1 Cytoplasmic polyadenylation element-binding protein 1 (CPEB1) and NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) deficiency and the correlation among ERVWE1, CPEB1, NDUFV2, and NDUFV2 pseudogene (NDUFV2P1) in schizophrenia patients. A and C: Respectively represent the protein expression of cytoplasmic polyadenylation element-binding protein 1 (CPEB1) and NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) in the schizophrenia patients (n = 41) and in the control groups (n = 38) using enzyme-linked immunosorbent; B, D, and E: Respectively represent the mRNA levels of NDUFV2, NDUFV2 pseudogene (NDUFV2P1), and ERVWE1 in schizophrenia patients (n = 19) and the control groups (n = 38) using quantitative polymerase chain reaction; F: Correlation between ERVWE1 mRNA levels and CPEB1 protein levels in patients with schizophrenia and control groups (n = 57), where Y is the protein expression for CPEB1 and X is ERVWE1 mRNA value for each sample; G: Correlation between ERVWE1 mRNA levels and NDUFV2 protein levels in patients with schizophrenia and control groups (n = 57), where Y is the protein expression for NDUFV2 and X is ERVWE1 mRNA value for each sample; H: Correlation between ERVWE1 and NDUFV2P1 mRNA levels in patients with schizophrenia and control groups (n = 57), where Y is the mRNA expression for NDUFV2P1 and X is ERVWE1 mRNA value for each sample; I: Correlation between NDUFV2P1 mRNA levels and NDUFV2 protein levels in patients with schizophrenia and control groups (n = 57), where Y is the protein expression for NDUFV2 and X is NDUFV2P1 mRNA value for each sample. ^aP < 0.05; ^bP < 0.01.

Figure 2 Overexpression of ERVWE1 elevated NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) pseudogene (NDUFV2P1) and reduced NDUFV2 levels. A and C: Respectively represent the mRNA levels of NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) pseudogene (NDUFV2P1) and NDUFV2 in ERVWE1-transfected SH-SY5Y cells using quantitative polymerase chain reaction; B: Luciferase assays of pGL3-NDUFV2P1 promoter co-transfected with pCMV-ERVWE1 or control vector in SH-SY5Y cells; D: Western blots of NDUFV2 and GAPDH in SH-SY5Y cells; E: Luciferase assays of pGL3-NDUFV2 promoter co-transfected with pCMV-ERVWE1 or control vector in SH-SY5Y cells. Each bar represents the mean ± SD of three independent experiments. ^aP < 0.05; ^bP < 0.01.

Figure 3 Role of NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) pseudogene (NDUFV2P1) in ERVWE1-induced downregulation of NDUFV2. A: The mRNA levels of NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) in NDUFV2 pseudogene (NDUFV2P1)-overexpressing SH-SY5Y cells using quantitative polymerase chain reaction (qPCR); B: Western blots of NDUFV2 and GAPDH in SH-SY5Y cells; C: The mRNA levels of NDUFV2 in SH-SY5Y cells after co-transfection with ERVWE1 and shNDUFV2P1 using qPCR; D: Western blots of ERVWE1, NDUFV2, and GAPDH in SH-SY5Y cells; E: Luciferase assays of pGL3-NDUFV2 promoter co-transfected with pCMV-NDUFV2P1 or control vector in SH-SY5Y cells; F: Luciferase assays of pGL3-NDUFV2 promoter co-transfected with pCMV-ERVWE1 + shNDUFV2P1 or control in SH-SY5Y cells. Each bar represents the mean ± SD of three independent experiments. ^aP < 0.05; ^bP < 0.01.

Figure 4 ERVWE1 elevated NADH dehydrogenase ubiquinone flavoprotein 2 pseudogene (NDUFV2P1) expression by suppressing cytoplasmic polyadenylation element-binding protein 1 (CPEB1). A: The mRNA levels of cytoplasmic polyadenylation element-binding protein 1 (CPEB1) after overexpression of ERVWE1 in SH-SY5Y cells using quantitative polymerase chain reaction (qPCR); B: Western blots of CPEB1 and GAPDH in SH-SY5Y cells; C: Luciferase assays of pGL3-CPEB1 promoter co-transfected with pCMV-ERVWE1 or control vector in SH-SY5Y cells; D: Luciferase assays of different lengths of pGL3-CPEB1 promoter co-transfected with pCMV-ERVWE1 or control vector in SH-SY5Y cells; E: The mRNA levels of NADH dehydrogenase ubiquinone flavoprotein 2 pseudogene (NDUFV2P1) after overexpression of CPEB1 in SH-SY5Y cells using qPCR; F: Represent the mRNA levels of NDUFV2P1 in SH-SY5Y cells after co-transfection with ERVWE1 and CPEB1 using qPCR. Each bar represents the mean ± SD of three independent experiments. ^aP < 0.05; ^bP < 0.01; NS: Not significant.

Figure 5 ERVWE1 reduced NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) expression by suppressing cytoplasmic polyadenylation element-binding protein 1 (CPEB1). A: The mRNA levels of NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) after overexpression of cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in SH-SY5Y cells using quantitative polymerase chain reaction (qPCR); B: Western blots of NDUFV2 and GAPDH in SH-SY5Y cells; C: Represent the mRNA levels of NDUFV2 in SH-SY5Y cells after co-transfection with ERVWE1 and CPEB1 using qPCR; D: Western blots of ERVWE1, CPEB1, NDUFV2, and GAPDH in SH-SY5Y cells. Each bar represents the mean ± SD of three independent experiments. ^aP < 0.05; ^bP < 0.01; NS: Not significant.

Figure 6 ERVWE1 contributed to complex I deficits through the cytoplasmic polyadenylation element-binding protein 1 (CPEB1)/ NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) pseudogene (NDUFV2P1)/NDUFV2 signaling pathway. A: Complex I activity in SH-SY5Y cells transfected with pCMV-ERVWE1 or control; B: Complex I activity in SH-SY5Y cells co-transfected with pCMV-ERVWE1 + shNDUFV2P1 or control; C: Complex I activity in SH-SY5Y cells co-transfected with pCMV-ERVWE1 + pcDNA3.1-CPEB1 or control. Each bar represents the mean ± SD of three independent experiments. ^aP < 0.05.

Figure 7 The proposed signaling pathways for ERVWE1 contributes to mitochondrial energy metabolism deficiency in schizophrenia. 1: ERVWE1 suppressed cytoplasmic polyadenylation element-binding protein 1 (CPEB1) mRNA and protein expression by regulating its promoter activity; 2: ERVWE1 upregulated the expression of NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) pseudogene (NDUFV2P1) through enhancing its promoter activity. CPEB1 downregulated NDUFV2P1 expression and functioned as a mediator of NDUFV2P1 transcript induced by ERVWE1; 3: NDUFV2P1 reduced NDUFV2 expression through inhibiting its promoter activation. Additionally, ERVWE1 decreased the mRNA and protein expression of NDUFV2 and suppressed NDUFV2 promoter through increasing NDUFV2P1 transcript. Moreover, CPEB1 was involved in regulating NDUFV2P1/NDUFV2 signaling pathway triggered by ERVWE1; 4: ERVWE1 reduced complex I activity via regulating NDUFV2 and its retrocopies expression. Decreased CPEB1 was involved in the complex I activity deficiency induced by ERVWE1; 5: Deficient complex I activity might result in mitochondrial metabolic dysregulation and contributed to the development of schizophrenia.