Unknown,Transcriptomics,Genomics,Proteomics

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Marked Reduction of AKT1 Expression and Deregulation of AKT1-associated Pathways in Peripheral Blood Mononuclear Cells of Schizophrenia Patients

ABSTRACT: Recent studies have suggested that deregulated AKT1 signaling is associated with schizophrenia. We hypothesized that if this is indeed the case, we should observe both decreased AKT1 expression as well as deregulation of AKT1 regulated pathways in Peripheral Blood Mononuclear Cells (PBMCs) of schizophrenia patients. We therefore examined PBMC expression levels of AKT1 in schizophrenia patients versus controls, and examined whether functional biological processes in which AKT1 plays an important role are deregulated in schizophrenia patients. We performed a case-control study, investigating whole-genome PBMC gene expression in male, recent onset (<5 years) schizophrenia patients (N=43) as compared to controls (N=29). Genes, differentially expressed between patients and controls were identified using ANOVA with Benjamini-Hochberg correction (false discovery rate (FDR)= 0.05). Functional aspects of the deregulated set of genes were investigated with the Ingenuity Pathway Analysis (IPA) Software Tool. From each participant 30 ml of blood was drawn into heparinized tubes. All blood samples were obtained between 10.00 and 11.00 am to minimize diurnal variation. PBMC‚Äôs were isolated by Ficoll gradient separation, started within 20 minutes after the drawing of blood and performed with minimum time variation. Cells were subsequently disrupted (Qiashredder kit; Qiagen), and RNA was isolated (RNeasy minikit; Qiagen) with an additional DNAse digestion step (RNase-free DNase set; Qiagen), all according to the manufacturer‚Äôs protocol, diluted in nuclease free water, and frozen at ‚Äì80 C before use. Before freezing, RNA purity and quantity was assessed with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). After thawing the isolated RNA was biotinylated into cRNA using the One-Cycle Target Labeling and Control Reagents Kit (Affymetric Co) according to the manufacturer‚Äôs protocol. Before hybridisation RNA quality and integrity was assessed using the Agilent 2100 BioAnalyzer (Agilent). Biotinylated cRNA was hybridized to the Affymetrix Human Genome U133 plus 2.0 GeneChip¬© microarray containing 54,675 probe sets (Affymetrix Co). Each sample was individually biotinylated and hybridized to an individual microarray. Biotinylation was performed in batches with randomisation of patients and controls according to their ratios across the batches. The arrays were scanned and analyzed using Affymetrix Microarray Suite 4.2 software. Scanning was performed in three batches with randomization of patients and controls according to their ratios across the batches. A case-control study, investigating whole-genome PBMC gene expression in male, recent onset (<5 years) schizophrenia patients (N=43) as compared to controls (N=29). Patients consisted of two subgroups: acutely admitted, severely psychotic (N=22), and remitted patients (N=21). Genes, differentially expressed between patients and controls were identified using ANOVA with Benjamini-Hochberg correction (false discovery rate (FDR)= 0.05).

ORGANISM(S): Homo sapiens

SUBMITTER: nico van beveren

PROVIDER: E-GEOD-27383 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Recent studies have suggested that deregulated AKT1 signaling is associated with schizophrenia. We hypothesized that if this is indeed the case, we should observe both decreased AKT1 expression as well as deregulation of AKT1 regulated pathways in Peripheral Blood Mononuclear Cells (PBMCs) of schizophrenia patients. We therefore examined PBMC expression levels of AKT1 in schizophrenia patients versus controls, and examined whether functional biological processes in which AKT1 plays an important role are deregulated in schizophrenia patients. We performed a case-control study, investigating whole-genome PBMC gene expression in male, recent onset (<5 years) schizophrenia patients (N=43) as compared to controls (N=29). Genes, differentially expressed between patients and controls were identified using ANOVA with Benjamini-Hochberg correction (false discovery rate (FDR)= 0.05). Functional aspects of the deregulated set of genes were investigated with the Ingenuity Pathway Analysis (IPA) Software Tool. From each participant 30 ml of blood was drawn into heparinized tubes. All blood samples were obtained between 10.00 and 11.00 am to minimize diurnal variation. PBMC’s were isolated by Ficoll gradient separation, started within 20 minutes after the drawing of blood and performed with minimum time variation. Cells were subsequently disrupted (Qiashredder kit; Qiagen), and RNA was isolated (RNeasy minikit; Qiagen) with an additional DNAse digestion step (RNase-free DNase set; Qiagen), all according to the manufacturer’s protocol, diluted in nuclease free water, and frozen at –80 C before use. Before freezing, RNA purity and quantity was assessed with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). After thawing the isolated RNA was biotinylated into cRNA using the One-Cycle Target Labeling and Control Reagents Kit (Affymetric Co) according to the manufacturer’s protocol. Before hybridisation RNA quality and integrity was assessed using the Agilent 2100 BioAnalyzer (Agilent). Biotinylated cRNA was hybridized to the Affymetrix Human Genome U133 plus 2.0 GeneChip© microarray containing 54,675 probe sets (Affymetrix Co). Each sample was individually biotinylated and hybridized to an individual microarray. Biotinylation was performed in batches with randomisation of patients and controls according to their ratios across the batches. The arrays were scanned and analyzed using Affymetrix Microarray Suite 4.2 software. Scanning was performed in three batches with randomization of patients and controls according to their ratios across the batches.

Project description:Akt1 plays a protective role in the postnatal C57BL6 mouse testis following lactational exposure to the neonatal goitrogen, propylthiouracil (PTU). To elucidate the transcriptional profile mediating this phenotypic effect, we monitored changes in testicular gene expression at postnatal days (PNDs) 15 and 25 in Akt1+/+, Akt1+/-, and Akt1-/- testes following exposure to 0.01% PTU allowing us to determine changes in gene expression due to 1.) genotype effects; 2.) exposure effects; and 3.) genotype-by-exposure interactions. Early PTU-dependent gene changes included genes involved in lipid metabolism, spermatid differentiation, meiosis and adhesion. Early Akt1-dependent effects were associated with germ cell development, spermatid development and differentiation, and sperm motility. By PND25, the Akt1 gene-environment interaction had pronounced effects on genes associated with Sertoli cell (SC) differentiation and claudin-associated junctional formation suggesting delayed formation of the blood-testis barrier (BTB). To confirm these observations, biotin tracer experiments demonstrated a permeable blood-testis barrier in PTU-exposed PKB/Akt1-/- tubules as late as PND25 compared to PTU-exposed Akt1+/+ seminiferous tubules. Transmission electron microscopy demonstrated altered SC morphology, aberrant SC localization, and disorganized actin bundle formation. Taken together, loss of Akt1 coupled with postnatal exposure to the neonatal goitrogen, PTU, in the testis contributes to a transcriptional profile associated with impaired integrity of the blood-testis barrier. In summary, the Akt1-/- mouse represents a potentially important model to study BTB formation and reassembly in response to male reproductive toxicants and the various signaling networks which mediate these responses. The microarray analysis employed a balanced factorial design, with one chip created for each of three mice under each experimental condition: mouse genotype (Akt1+/+, Akt1+/-, and Akt1-/-), exposure (PTU and Control), and age (sacrificed at PND15 and 25), with a total of 36 mice and 36 chips. MoGene 1.0 st v1 arrays were used for the samples from PND 15 and Mouse Genome 430 2.0 arrays were used for the samples from PND 25. One chip, PTU-exposed, Akt1-/-, PND25, was determined to not be of high quality and was not included in the analysis or provided here.

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Marked Reduction of AKT1 Expression and Deregulation of AKT1-associated Pathways in Peripheral Blood Mononuclear Cells of Schizophrenia Patients

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