Background Innate immune gene expression is regulated in part through high mobility group box 1(HMGB1), an endogenous proinflammatory cytokine, that activates multiple members of the interleukin-1/Toll-like receptor (IL-1/TLR) family associated with danger signaling. We investigated expression of HMGB1, TLR2, TLR3 and TLR4 in chronic ethanol treated mouse brain, post-mortem human alcoholic brain, and rat brain slice culture to test the hypothesis that neuroimmune activation in alcoholic brain involves ethanol activation of HMGB1/TLR danger signaling. Methods Protein levels were assessed using Western blot, ELISA, immunohistochemical immunoreactivity (+IR), and mRNA levels were measured by real time PCR in ethanol-treated mice (5 g/kg/day, i.g., 10 days + 24 hr), rat brain slice culture, and post-mortem human alcoholic brain. Results Ethanol treatment of mice increased brain mRNA and +IR protein expression of HMGB1, TLR2, TLR3, and TLR4. Post-mortem human alcoholic brain also showed increased HMGB1, TLR2, TLR3, and TLR4+IR cells that correlated with lifetime alcohol consumption as well as each other. Ethanol treatment of brain slice culture released HMGB1 into the media and induced the proinflammatory cytokine, IL-1β. Neutralizing antibodies to HMGB1 and small inhibitory mRNA to HMGB1 or TLR4 blunted ethanol induction of IL-1β. Conclusions Ethanol-induced HMGB1/TLR signaling contributes to induction of the proinflammatory cytokine, IL-1β. Increased expression of HMGB1, TLR2, TLR3, and TLR4 in alcoholic brain and in mice treated with ethanol suggests that chronic alcohol-induced brain neuroimmune activation occurs through HMGB1/TLR signaling.
Objective To conduct postmortem human brain research into the neuropathological basis of schizophrenia, it is critical to establish cohorts that are well-characterised and well-matched. Our objective was to determine if specimen characteristics, including: diagnosis, age, postmortem interval (PMI), brain acidity (pH), and/or the agonal state of the subject at death related to RNA quality, and to determine the most appropriate reference gene mRNAs. Methods We selected a matched cohort of 74 cases (37 schizophrenia / schizoaffective disorder cases and 37 controls cases). Middle frontal gyrus tissue was pulverised, tissue pH was measured, RNA isolated for cDNA from each case, and RNA integrity number (RIN) measurements were assessed. Using RT-PCR, we measured nine housekeeper genes and calculated a geomean in each diagnostic group. Results We found that the RINs were very good (mean 7.3) and all nine housekeeper control genes were significantly correlated with RIN. Seven of nine housekeeper genes were also correlated with pH, and two clinical variables, agonal state and duration of illness did have an effect on some control mRNAs. No major impact of PMI or freezer time on housekeeper mRNAs was detected. Our results show that people with schizophrenia had significantly less PPIA, and SDHA and tended to have less GUSB and B2M mRNA suggesting that these control genes may not be good candidates for normalisation. Conclusions In our cohort, less than 10% variability in RIN values was detected and the diagnostic groups were well matched overall. Our cohort was adequately powered (0.80–0.90) to detect mRNA differences (25%) due to disease. Our study suggests that multiple factors should be considered in mRNA expression studies of human brain tissues. When schizophrenia cases are adequately matched to control cases subtle differences in gene expression can be reliably detected.
The genetic, epigenetic and environmental factors may influence the risk for neuropsychiatric disease through their effects on gene transcription. Mechanistically, these effects may be integrated through regulation of methylation of CpG dinucleotides overlapping with single-nucleotide polymorphisms (SNPs) associated with a disorder. We addressed this hypothesis by analyzing methylation of prodynorphin (PDYN) CpG-SNPs associated with alcohol dependence, in human alcoholics. Postmortem specimens of the dorsolateral prefrontal cortex (dl-PFC) involved in cognitive control of addictive behavior were obtained from 14 alcohol-dependent and 14 control subjects. Methylation was measured by pyrosequencing after bisulfite treatment of DNA. DNA binding proteins were analyzed by electromobility shift assay. Three PDYN CpG-SNPs associated with alcoholism were found to be differently methylated in the human brain. In the dl-PFC of alcoholics, methylation levels of the C, non-risk variant of 3′-untranslated region (3′-UTR) SNP (rs2235749; C > T) were increased, and positively correlated with dynorphins. A DNA-binding factor that differentially targeted the T, risk allele and methylated and unmethylated C allele of this SNP was identified in the brain. The findings suggest a causal link between alcoholism-associated PDYN 3′-UTR CpG-SNP methylation, activation of PDYN transcription and vulnerability of individuals with the C, non-risk allele(s) to develop alcohol dependence.
Cortical grey matter volume deficits and neuro-inflammation exist in patients with schizophrenia, although it is not clear whether elevated cytokines contribute to the cortical volume reduction. We quantified cortical and regional brain volumes in fixed postmortem brains from people with schizophrenia and matched controls using stereology. Interleukin (IL)-6, IL-1β, IL-8 and SERPINA3 messenger RNAs (mRNAs) were quantified in the contralateral fresh frozen orbitofrontal cortex. We found a small, but significant reduction in cortical grey matter (1.3% F(1,85)=4.478, P=0.037) and superior frontal gyrus (6.5% F(1,80)=5.700, P=0.019) volumes in individuals with schizophrenia compared with controls. Significantly reduced cortical grey matter (9.2% F(1,24)=8.272, P=0.008) and superior frontal gyrus (13.9% F(1,20)=5.374, P=0.031) volumes were found in cases with schizophrenia and ‘high inflammation' status relative to schizophrenia cases with ‘low inflammation' status in the prefrontal cortex. The expression of inflammatory mRNAs in the orbitofrontal cortex was significantly correlated with those in dorsolateral prefrontal cortex (all r>0.417, all P<0.022), except for IL-8. Moreover, average daily and lifetime antipsychotic intake negatively correlated with cortical grey matter and superior frontal gyrus volumes (all r<−0.362, all P<0.05). The results suggest that the reduction in cortical grey matter volume in people with schizophrenia is exaggerated in those who have high expression of inflammatory cytokines. Further, antipsychotic medication intake does not appear to ameliorate the reduction in brain volume.
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