Figure 1.
Effects of COMT Val/Met genotype and disease on NRG1-stimulated Ser-473 phosphorylation of AKT1in B lymphoblasts derived from controls and patients with schizophrenia.
(A) COMT Val/Meet genotype and NRG1-stimulated Ser-473 phosphorylation of AKT1. NRG1-stumulated increases in AKT1 phosphorylation in B lymphoblasts were significantly decreased in Val homozygotes (n = 18), compared with Met homozygotes (n = 12) (p = 0.0024, t-test). The Y-axis shows the peak fold increases in the phosphorylated-AKT1/total AKT1 ratio during one hour stimulation. (B) Disease and and NRG1-stimulated phosphorylation of AKT1. There was no difference in NRG1-stumulated increases in AKT1 phosphorylation in B lymphoblasts between controls (n = 16) and patients with schizophrenia (n = 14). (C) COMT Val homozygotes had significantly lower NRG1-stimulated phosphorylation of AKT1 in both controls (P = 0.029) and patients with schizophrenia (p = 0.042, t-test).
Figure 2.
Effects of AKT1 rs1130233 on AKT1 protein expression and NRG1-stimulated Ser-473 phosphorylation of AKT1in B lymphoblasts derived from controls and patients with schizophrenia.
(Upper panel; A, B and C) Effects of AKT1 rs1130233 on AKT1 protein expression. The minor rs1130233 allele was associated with lower expression of AKT1 protein (AKT1/β-actin ratio, mean±SE). p = 0.006, rs1130233 G/A vs. G/G; p = 0.0406. The associations were preserved in both control and patient groups (B), similarly in COMT Val/Val and Met/Met groups (C). Within the rs1130233 G/A group (C), AKT1protein expression was significantly decreased in individuals with Val carriers compared with Met carriers. p = 0.0371, rs1130233 G/A, Val vs. G/A, Met. The number of subjects carrying [G/G, Val], [G/G, Met], [G/A, Val] and [G/A, Met] genotype was 18, 12, 10 and 16, respectively. (Lower panel; D, E and F) Effects of AKT1 rs1130233 on NRG1-stimulated phosphorylation of AKT1. AKT1 rs1130233 had no effect on this phenotype in the total population (D) and no interaction with disease on the phenotype (E). Two-way ANOVA showed significant main effects of AKT rs1130233 genotype (P = 0.0066), significant main effect of COMT genotype (p = 0.0003) and significant interactions of these genotypes (p = 0.0234) on NRG1-induced phosphorylation. In individuals who were also COMT Met/Met homozygotes, individuals carrying the AKT1 S rs1130233 minor A allele, showed significantly lower phosphorylation than G/G carriers (P = 0.0182). This effect was not appeared in Val/Val individuals because of the maximum reductions in AKT1 phosphorylation.
Figure 3.
Effects of COMT transfection on COMT activity and NRG1-stimulated Ser-473 phosphorylation of AKT1 in SH-SY5Y cells.
SH-SY5Y cells were transfected with COMT-GFP or with the control GFP vector. After 48 hrs, cells were analyzed for enzyme activity and expression of COMT and tested for Ser-473 phosphorylation of AKT1 in response to NRG1α. (A) GFP-detection after transfection of GFP-tagged COMT or control GFP vector showed 70–80% transfection efficiency. (B) Expression of the transfected COMT. Western blotting showed expression of a 60-kDa band, representing GFP-tagged human MB-COMT in cells transfected with COMT-GFP (left three lanes). Endogenous 30-kDa MB- and 25-kDa S- COMT proteins were expressed under both transfection conditions. (C) Effects of COMT transfection on COMT enzyme activities. The COMT enzyme assay indicated a significant five-fold increase in COMT activity (dpm per mg total protein) in cells transfected with COMT-GFP compared to cells transfected with control vector. *p<0.0001, 3 samples per group. The specificity of the assay was confirmed by a complete attenuation of the enzyme activity by 100 µM of the specific COMT inhibitor tolcapone. (D) Effects of COMT transfection on NRG1α-stimulated Ser-473 phosphorylation of AKT1 in SH-SY5Y cells. At 48 hrs after transfection, the transfected cells were stimulated with NRG1α (100 ng/ml) for the time points indicated. Protein isolated from the cells was analyzed by Western blotting. To quantify the level of phosphorylation, the immunoblots were stained with antibodies specific to phosphorylated AKT1- at Ser-473 and were then stripped and reprobed with antibodies to total AKT1. The bar graphs represent changes in the ratio of phosphorylated form to total AKT1 (means±SEM, from 3 transfection experiments). A repeated measure ANOVA showed a significant main effect of COMT transfection on AKT1 phosphorylation (P = 0.0017).
Figure 4.
Effects of COMT Val/Met genotype on PHD-AKT1translocation and phospholipid levels in B lymphoblasts derived from controls and patients with schizophrenia.
(A) There was a positive correlation between NRG1-stimulated Ser-473 phosphorylation of AKT1 and NRG1-simulated PHD-AKT translocation. B lymphoblasts from control subjects were transfected with pEYFP-ph-AKT. At 24 hrs after transfection, PHD-AKT1 translocation was stimulated with NRG1 for 15 min and measured under the fluorescence microscope. Translocation indexes from 8 control subjects were positively correlated with NRG1-stimulated peak fold increases in Ser-473 phosphorylation of AKT1 (phophorylated-AKT1/total AKT1 ratio) that were separately obtained from the same individuals (r = .832, p = 0.0075). (B) Representative fluorescence microscopic images of PHD-AKT1 fluorescence. Single cell images of PHD-AKT1fluorescence before and after NRG1 stimulation in typical COMT Val/Val and Met/Met homozygotes are shown. The bar graph represents PHD-AKT1 tranlocation index (means±SEM). *p = 0.029, individuals with Val/Val genotype (n = 5) vs. Met/Met genotype (n = 5). (C) There was a correlation between COMT enzyme activity and phosphatidylserine (PS) synthesis ability. Y-axis indicates relative COMT activity (dpm per mg total protein) in the B lymphoblasts. X-axis indicates PS synthesis ability (the ratio of total PS fluorescence intensity of B lymphoblast after exposure to serum free medium for 24 hrs/PS fluorescence intensity after culturing in normal culture media containing 15% FBS, which reflects a cell's ability to maintain PS levels) (see Materials and Methods). COMT enzyme activity was significantly correlated with PS synthesis ability in total 25 subjects (12 patients and 13 controls) (Total; R = −.622, p = 0.0006, patients; R = −.667, p = 0.0156 and controls; R = −.634, p = 0.0180).
Figure 5.
Effects of high COMT activity on NRG1-stimulated PHD-AKT1 translocation and PIP3 generation in transfected SH-SY5Y cells.
(A) Effects of high COMT activity on PHD-AKT1translocation. SH-SY5Y cells were double-transfected with either pDsRed-ph-AKT plus COMT or pDsRed-ph-AKT plus control vector. After 48 hrs, cells were analyzed for PHD-AKT1 translocation in response to NRG1α. The bar graph represents percent positive cells indicating PHD-AKT1 translocation (means±S.E.) from four transfection experiments. Two-way ANOVA, interaction between NRG1α treatment and COMT transfection, *p<0.05, **p = 0.0074. Lower inset shows representative fluorescence microscopic images of PHD-AKT1 fluorescence in a single cell. From left, untranslocated homogenous distribution of PHD-AKT1, translocated PHD-AKT1 as spots, clusters and broad membranous distributions. (B) Effects of high COMT activity on NRG1-stimulated PIP3 generation. SY5Y cells were transfected with either COMT or control vector. The cells were analyzed for NRG1-stimulated PIP3 generation using flow cytometry. Top panel indicates a summation of positive and negative changes in PIP3 during a 30 min observation period and lower panel indicates peak folds of PIP3 during a 30 min period.
Figure 6.
Effects of SAM on NRG1-stimulated AKT1 phosphorylation and PS amount in transfected SH-SY5Y cells.
(A) Effects of SAM treatment on NRG1-stimulated Ser-473 phosphorylation of AKT1 in SH-SY5Y cells overexpressing COMT. At 48 hrs after transfection with COMT-GFP, cells were treated with either 1 mM SAM or vehicle for 60 minutes and stimulated with NRG1α (100 ng/ml). The immunoblot shows a representative time course of Ser-473 phosphorylation of AKT1and expression of total AKT1 after NRG1α. The bar graph represents the ratio of phosphorylated- to total AKT1 at 60 min after the stimulation (means±SEM, from 3 transfection experiments). p = 0.0413, vehicle vs. SAM treatment. (B) Effects of COMT transfection on total PS amount in SH-SY5Ycells. SH-SY5Y cells were transfected with COMT-GFP or with control GFP vector. After 48 hrs, cells were and treated with either 1 mM SAM or vehicle control for 60 min and analyzed to estimate total PS amount of cells by flow cytometry. A graph indicates PS fluorescence intensity (geometric mean) from two independent transfection experiments. A repeated measure of ANOVA showed significantly lower PS fluorescence intensity in COMT-transfected cells than that in cells transfected with control vector (*P = 0.0087) and a significant reversal of the transfection effect by SAM treatment (*p = 0.0004).
Figure 7.
Effects of COMT transfection on Ser-473 phosphorylation of AKT1 after stimulation with various ligands in transfected SH-SY5Y cells.
At 48 hrs after transfection, the transfected SH-SY5Y cells were stimulated with NRG1β (100 ng/ml), cannabinoid CB1 agonist ACEA (14 nM), SDF1β (50 ng/ml) or BDNF (50 ng/ml). The bar graphs represent maximum changes in the ratio of phosphorylated- to total AKT1 during the 60-minutes observation period (means±SEM, from 3–4 transfection experiments per ligand).
Figure 8.
Effects of COMT transfection and SAM treatment on NRG1-stimulated migrataion in SH-SY5Y cells.
SH-SY5Y cells were transfected with COMT-GFP or control vectors (empty GFP) and cultured in media containing 2%FBS. After 48 hrs, cells were tested for NRG1-stimulated migration. A graph indicates the chemotaxis index (mean± S.E.) from three transfection experiments (n = 4–5 per group). ANOVA showed a significant a SAM treatment effect (p = 0.0193) and transfection-treatment interaction (P = 0.0144).