Elevated synaptophysin I in the prefrontal cortex of human chronic alcoholics.

Convergent lines of evidence suggest potentiation of glutamatergic synapses after chronic ethanol exposure, and indicate that the presynaptic effect hereof is on modulators of synaptic strength rather than on executors of glutamate release. To address this hypothesis in the context of ethanol dependence in humans, we used semiquantitative immunoblotting to compare the immunoreactivities of synaptophysin I, syntaxin 1A, synaptosome-associated protein 25, and vesicle-associated membrane protein in the prefrontal and motor cortices between chronic alcoholics and control subjects. We found a region-specific elevation in synaptophysin I immunoreactivity in the prefrontal cortex of alcoholics, but detected no significant differences between the groups in the immunoreactivities of the other three proteins. Our findings are consistent with an effect of repeated ethanol exposure on modulators of synaptic strength but not on executors of glutamate release, and suggest a role for synaptophysin I in the enduring neuroplasticity in the prefrontal cortical glutamate circuitry that is associated with ethanol dependence.

Validation of endogenous controls for quantitative gene expression analysis: application on brain cortices of human chronic alcoholics.

Real-time PCR is frequently used for gene expression quantification due to its methodological sensitivity and reproducibility. The gene expression is quantified by normalization to one or more reference genes, usually beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPD) or to ribosomal RNA (18S). However, different environmental or pathological conditions might also influence the expression of normalizing genes, which could severely skew the interpretation of quantitative results. This study evaluates whether 16 genes frequently used as endogenous controls in expression studies, can serve as such for comparison of human brain tissues of chronic alcoholics and control subjects. The prefrontal and motor cortices that are affected differently by chronic alcohol consumption were analyzed. The reference genes that have no or small differences in expression in alcoholics and control subjects, were found to be specific for each region: beta-actin (ACTB) and ribosomal large P0 (RPLP0) for the prefrontal cortex while importin 8 (IPO8) and RNA polymerase II (POLR2A) for the motor cortex. Four out of sixteen analyzed genes demonstrated significant differences in expression between alcoholics and controls: phosphoglycerate kinase (PGK1), hypoxanthine phosphoribosyl transferase (HPRT1) and peptidylprolyl isomerase A (PPIA) in the motor cortex and beta-2-microglobulin (B2M) in the prefrontal cortex. Our study demonstrates the importance of validation of endogenous control genes prior to real-time PCR analysis of human brain tissues. Prescribed and non-prescribed drugs, pathological or environmental conditions along with alcohol abuse may differentially influence expression of reference genes.

Dysregulated postsynaptic density and endocytic zone in the amygdala of human heroin and cocaine abusers.

BACKGROUND: Glutamatergic transmission in the amygdala is hypothesized as an important mediator of stimulus-reward associations contributing to drug-seeking behavior and relapse. Insight is, however, lacking regarding the amygdala glutamatergic system in human drug abusers. METHODS: We examined glutamate receptors and scaffolding proteins associated with the postsynaptic density in the human postmortem amygdala. Messenger RNA or protein levels were studied in a population of multidrug (seven heroin, eight cocaine, seven heroin/cocaine, and seven controls) or predominant heroin (29 heroin and 15 controls) subjects. RESULTS: The amygdala of drug abusers was characterized by a striking positive correlation (r > .8) between α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptor subunit 1 (GluA1) and postsynaptic density protein-95 (PSD-95) mRNA levels, which was not evident in control subjects. Structural equation multigroup analysis of protein correlations also identified the relationship between GluA1 and PSD-95 protein levels as the distinguishing feature of abusers. In line with the GluA1-PSD-95 implications of enhanced synaptic plasticity, Homer 1b/c protein expression was increased in both heroin and cocaine users as was its binding partner, dynamin-3. Furthermore, there was a positive relationship between Homer 1b/c and dynamin-3 in drug abusers that reflected an increase in the direct physical coupling between the proteins. A noted age-related decline of Homer 1b/c-dynamin-3 interactions, as well as GluA1 levels, was blunted in abusers. CONCLUSIONS: Impairment of key components of the amygdala postsynaptic density and coupling to the endocytic zone, critical for the regulation of glutamate receptor cycling, may underlie heightened synaptic plasticity in human drug abusers.

Dysregulation of cell death machinery in the prefrontal cortex of human alcoholics.

In human alcoholics, the cell density is decreased in the prefrontal cortex (PFC) and other brain areas. This may be due to persistent activation of cell death pathways. To address this hypothesis, we examined the status of cell death machinery in the dorsolateral PFC in alcoholics. Protein and mRNA expression levels of several key pro- and anti-apoptotic genes were compared in post-mortem samples of 14 male human alcoholics and 14 male controls. The findings do not support the hypothesis. On the contrary, they show that several components of intrinsic apoptotic pathway are decreased in alcoholics. No differences were evident in the motor cortex, which is less damaged in alcoholics and was analysed for comparison. Thus, cell death mechanisms may be dysregulated by inhibition of intrinsic apoptotic pathway in the PFC in human alcoholics. This inhibition may reflect molecular adaptations that counteract alcohol neurotoxicity in cells that survive after many years of alcohol exposure and withdrawal.

Neuroadaptations in human chronic alcoholics: dysregulation of the NF-kappaB system.

BACKGROUND: Alcohol dependence and associated cognitive impairments apparently result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. Here we investigated whether transcription factors of Nuclear Factor-kappaB (NF-kappaB) family, controlling neuronal plasticity and neurodegeneration, are involved in these adaptations in human chronic alcoholics. METHODS AND FINDINGS: Analysis of DNA-binding of NF-kappaB (p65/p50 heterodimer) and the p50 homodimer as well as NF-kappaB proteins and mRNAs was performed in postmortem human brain samples from 15 chronic alcoholics and 15 control subjects. The prefrontal cortex involved in alcohol dependence and cognition was analyzed and the motor cortex was studied for comparison. The p50 homodimer was identified as dominant kappaB binding factor in analyzed tissues. NF-kappaB and p50 homodimer DNA-binding was downregulated, levels of p65 (RELA) mRNA were attenuated, and the stoichiometry of p65/p50 proteins and respective mRNAs was altered in the prefrontal cortex of alcoholics. Comparison of a number of p50 homodimer/NF-kappaB target DNA sites, kappaB elements in 479 genes, down- or upregulated in alcoholics demonstrated that genes with kappaB elements were generally upregulated in alcoholics. No significant differences between alcoholics and controls were observed in the motor cortex. CONCLUSIONS: We suggest that cycles of alcohol intoxication/withdrawal, which may initially activate NF-kappaB, when repeated over years downregulate RELA expression and NF-kappaB and p50 homodimer DNA-binding. Downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently resulted in derepression of kappaB regulated genes. Alterations in expression of p50 homodimer/NF-kappaB regulated genes may contribute to neuroplastic adaptation underlying alcoholism.