Lower antioxidant capacity in the prefrontal cortex of individuals with schizophrenia.

OBJECTIVE: The glutathione (GSH) pathway is the main antioxidant system to protect against oxidative stress in the human brain. In this study, we tested whether molecular components of the GSH antioxidant system are changed in dorsolateral prefrontal cortex tissue from people with schizophrenia compared to controls. METHOD: The levels of total glutathione and reduced GSH were determined by fluorometric assay via quantifying thiols in extracts from frontal cortex of 68 people. Immunoblotting was used to measure levels of enzymes responsible for maintaining GSH, the glutamyl-cysteine ligase (GCL) catalytic subunit (GCLC) and the GSH peroxidase (GPx)-like protein ( n = 74). Quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to measure GCLC messenger RNA (mRNA) expression. RESULTS: Both total glutathione ( t(66) = 2.467, p = 0.016) and reduced GSH ( t(66) = 3.001, p = 0.004) levels were significantly less in people with schizophrenia than in controls. However, there were no significant differences in either GCLC-like protein ( t(72) = -1.077, p = 0.285) or GCLC mRNA expression ( t(71) = -0.376, p = 0.708) between people with schizophrenia and control subjects. There was also no significant difference of GPx-like protein levels between schizophrenia and controls ( t(72) = -0.060, p = 0.952). Moreover, no significant correlations of putative confounding factors with GSH changes were detected. DISCUSSION: These results suggest that people with schizophrenia have impaired GSH antioxidant capacity, alongside normal levels of key regulatory proteins.

Increased expression of astrocyte markers in schizophrenia: Association with neuroinflammation.

OBJECTIVE: While schizophrenia may have a progressive component, the evidence for neurodegenerative processes as indicated by reactive astrocytes is inconclusive. We recently identified a subgroup of individuals with schizophrenia with increased expression of inflammatory markers in prefrontal cortex, and hypothesized that this subgroup would also have reactive astrocytes. METHOD: We measured glial fibrillary acidic protein (GFAP) mRNA by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) and protein levels by immunoblotting in grey matter homogenate from 37 individuals with schizophrenia and 37 unaffected controls. We examined the morphology of GFAP-positive astrocytes in immunostained sections of middle frontal gyrus. We tested if GFAP expression or astrocyte morphology were altered in people with schizophrenia with increased expression of inflammatory markers. We used RNA-Seq data on a subset of patients and controls (n=20/group) to ascertain whether mRNA transcripts associated with astrogliosis were elevated in the individuals with active neuroinflammation. RESULTS: GFAP (mRNA and protein) levels and astrocyte morphology were not significantly different between people with schizophrenia and controls overall. However, individuals with schizophrenia with neuroinflammation had increased expression of GFAP mRNA (t(33)=2.978, p=0.005), hypertrophic astrocyte morphology (χ(2)(2)=6.281, p=0.043), and statistically significant elevated expression of three mRNA transcripts previously associated with astrogliosis. CONCLUSIONS: We found clear evidence of astrogliosis in a subset of people with schizophrenia. We suggest that the lack of astrogliosis reported in previous studies may be due to cohort differences in aetiopathology, illness stage, treatment exposure, or a failure to examine subsets of people with schizophrenia.

Apoptosis and schizophrenia: a pilot study based on dermal fibroblast cell lines.

INTRODUCTION: The aim of this study was to investigate whether there is an increased susceptibility to apoptosis in cultured fibroblasts from patients with schizophrenia. METHOD: Dermal fibroblasts were collected and cultured from three groups: patients with schizophrenia, patients with non-schizophrenic psychosis, and healthy comparison subjects. Susceptibility to apoptosis was measured at the level of degradation product (proportion of cells in the sub-G0 cell cycle fraction in which apoptotic bodies accumulate), pro-apoptotic effector (activated caspase-3), and molecular regulators (P53, Bax and Bcl-2). Cell lines were studied under both basal culture and cycloheximide (an apoptotic inducer) exposure conditions. RESULTS: Consistent with increased susceptibility to apoptosis, the proportion of sub-G0 cells under basal conditions was significantly larger in the schizophrenia group, compared to the non-schizophrenic psychosis group. However when apoptosis was stimulated with cycloheximide, the schizophrenia group showed an attenuated caspase-3 response. The pattern of correlations between regulators, caspase-3 and the proportion of sub-G0 cells was different in the schizophrenia group, consistent with group-specific apoptotic pathway dysregulation. CONCLUSION: The study demonstrated anomalous apoptotic mechanisms in schizophrenia, which appear not to affect non-schizophrenia psychosis patients. The detection of these anomalies in fibroblasts suggests that altered apoptosis may be observable in all somatic cell types in schizophrenia.

A microarray study of post-mortem mRNA degradation in mouse brain tissue.

BACKGROUND: Although there is evidence that post-mortem interval (PMI) is not a major contributor to reduced overall RNA integrity, it may differentially affect a subgroup of gene transcripts that are susceptible to PMI-related degradation. This would particularly have ramifications for microarray studies that include a broad spectrum of genes. METHOD: Brain tissue was removed from adult mice at 0, 6, 12, 18, 24, 36 and 48 h post-mortem. RNA transcript abundance was measured by hybridising RNA from the zero time point with test RNA from each PMI time point, and differential gene expression was assessed using cDNA microarrays. Sequence and ontological analyses were performed on the group of RNA transcripts showing greater than two-fold reduction. RESULTS: Increasing PMI was associated with decreased tissue pH and increased RNA degradation as indexed by 28S/18S ribosomal RNA ratio. Approximately 12% of mRNAs detected on the arrays displayed more than a two-fold decrease in abundance by 48 h post-mortem. An analysis of nucleotide composition provided evidence that transcripts with the AUUUA motif in the 3' untranslated region (3'UTR) were more susceptible to PMI-related RNA degradation, compared to transcripts not carrying the 3'UTR AUUUA motif. Consistent with this finding, ontological analysis showed transcription factors and elements to be over-represented in the group of transcripts susceptible to degradation. CONCLUSION: A subgroup of mammalian mRNA transcripts are particularly susceptible to PMI-related degradation, and as a group, they are more likely to carry the 3'UTR AUUUA motif. PMI should be controlled for in human and animal model post-mortem brain studies, particularly those including a broad spectrum of mRNA transcripts.

Postsynaptic density levels of the NMDA receptor NR1 subunit and PSD-95 protein in prefrontal cortex from people with schizophrenia.

BACKGROUND: There is converging evidence of involvement of N-methyl-d-aspartate (NMDA) receptor hypofunction in the pathophysiology of schizophrenia. Our group recently identified a decrease in total NR1 mRNA and protein expression in the dorsolateral prefrontal cortex in a case-control study of individuals with schizophrenia (n=37/group). The NR1 subunit is critical to NMDA receptor function at the postsynaptic density, a cellular structure rich in the scaffolding protein, PSD-95. The extent to which the NMDA receptor NR1 subunit is altered at the site of action, in the postsynaptic density, is not clear. AIMS: To extend our previous results by measuring levels of NR1 and PSD-95 protein in postsynaptic density-enriched fractions of prefrontal cortex from the same individuals in the case-control study noted above. METHODS: Postsynaptic density-enriched fractions were isolated from fresh-frozen prefrontal cortex (BA10) and subjected to western blot analysis for NR1 and PSD-95. RESULTS: We found a 20% decrease in NR1 protein (t(66)=-2.874, P=0.006) and a 30% decrease in PSD-95 protein (t(63)=-2.668, P=0.010) in postsynaptic density-enriched fractions from individuals with schizophrenia relative to unaffected controls. CONCLUSIONS: Individuals with schizophrenia have less NR1 protein, and therefore potentially fewer functional NMDA receptors, at the postsynaptic density. The associated decrease in PSD-95 protein at the postsynaptic density suggests that not only are glutamate receptors compromised in individuals with schizophrenia, but the overall spine architecture and downstream signaling supported by PSD-95 may also be deficient.

Gene expression analysis implicates a death receptor pathway in schizophrenia pathology.

An increase in apoptotic events may underlie neuropathology in schizophrenia. By data-mining approaches, we identified significant expression changes in death receptor signaling pathways in the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia, particularly implicating the Tumor Necrosis Factor Superfamily member 6 (FAS) receptor and the Tumor Necrosis Factor [ligand] Superfamily member 13 (TNFSF13) in schizophrenia. We sought to confirm and replicate in an independent tissue collection the noted mRNA changes with quantitative real-time RT-PCR. To test for regional and diagnostic specificity, tissue from orbital frontal cortex (OFC) was examined and a bipolar disorder group included. In schizophrenia, we confirmed and replicated significantly increased expression of TNFSF13 mRNA in the DLPFC. Also, a significantly larger proportion of subjects in the schizophrenia group had elevated FAS receptor expression in the DLPFC relative to unaffected controls. These changes were not observed in the bipolar disorder group. In the OFC, there were no significant differences in TNFSF13 or FAS receptor mRNA expression. Decreases in BH3 interacting domain death agonist (BID) mRNA transcript levels were found in the schizophrenia and bipolar disorder groups affecting both the DLPFC and the OFC. We tested if TNFSF13 mRNA expression correlated with neuronal mRNAs in the DLPFC, and found significant negative correlations with interneuron markers, parvalbumin and somatostatin, and a positive correlation with PPP1R9B (spinophilin), but not DLG4 (PSD-95). The expression of TNFSF13 mRNA in DLPFC correlated negatively with tissue pH, but decreasing pH in cultured cells did not cause increased TNFSF13 mRNA nor did exogenous TNFSF13 decrease pH. We concluded that increased TNFSF13 expression may be one of several cell-death cytokine abnormalities that contribute to the observed brain pathology in schizophrenia, and while increased TNFSF13 may be associated with lower brain pH, the change is not necessarily causally related to brain pH.

Evidence of aberrant DNA damage response signalling but normal rates of DNA repair in dividing lymphoblasts from patients with schizophrenia.

OBJECTIVES: Cancer incidence in schizophrenia is not increased commensurate with higher rates of risk exposures. Here we report an investigation of the DNA damage response, an anti-tumorigenic defence, in immortalised lymphoblasts from patients with schizophrenia. METHODS: Unirradiated and irradiated (5Gy) lymphoblasts from schizophrenia patients (n = 28) and healthy controls (n = 28) were immunostained for the phosphorylated histone variant H2AX (γH2AX), an index of DNA double-strand breaks. Flow cytometry was used to assess cell cycle distribution and γH2AX immunofluorescence. Rate of DNA repair was quantified by determining the temporal change in γH2AX values following irradiation. RESULTS: In unirradiated lymphoblasts, γH2AX levels were significantly increased in the schizophrenia group compared with controls (effect size = 0.86). This increase was most evident in patients with cognitive deficits. In irradiated lymphoblasts, peak radiation-induced γH2AX levels were significantly reduced in patients. No differences between patients and controls were found in the rate of DNA repair or in cell cycle distribution. CONCLUSIONS: The significant differences in DNA damage response signalling observed involve modification of histone variant H2AX and thereby implicate regulatory processes determining chromatin structure in dividing lymphoblasts from patients with schizophrenia. The role that aberrant DNA damage response signalling plays in protecting patients from cancer is unclear.