Rd Mayfield

Identifying Changes in the alcoholic synaptic proteome

MicroRNAs (miRNAs) represent a large class of small single-stranded non-coding RNAs that are thought to regulate diverse functions by post-transcriptional gene silencing. Recently, miRNAs are have been shown to be crucial regulators of gene expression, affecting a wide variety of cellular functions. In brain, miRNAs have been shown to play an important role during development and in regulation of synaptic plasticity, and have been implicated in human brain diseases. In previous studies, we and others have used cDNA and oligonucleotide microarrays to identify and classify genes with altered expression following long-term alcohol consumption; however, the potential role of miRNA regulation of these genes has not been investigated. In the present study, we used miRNA arrays (Ambion+Invitrogen human, mouse, and rat probes) to classify miRNAs with altered expression in postmortem prefrontal cortex following long-term alcohol consumption. miRNAs profiles were obtained from the identical samples (14 well characterized alcoholics and 13 matched controls) that were used in a previous cDNA microarray study (Neuropsychopharm 31, 1574-82, 2006). We found that ~20 miRNAs were significantly up-regulated (p<0.002; 20-45%) in the alcoholic group compared to controls. Interestingly, miRNA down-regulation was not observed at this level of significance. Functional classification of the predicted target genes of the regulated miRNAs demonstrated a large degree of overlap with published mRNA data. For example, genes involved in ubiquitination, apoptosis, cell adhesion, neurogenesis, and neural disease are predicted targets of the regulated miRNAs. In addition, approximately one third of the significantly regulated mRNAs from the previous microarray study were identified as potential targets of the significantly changed miRNAs. miRNAs are key regulators of gene expression but the functions of many human miRNAs are yet to be discovered. This study provides the first analysis of miRNA levels in human alcoholism and raise the possibility that the reduced of expression of brain genes seen in human alcoholism may be due, at least in part, to increased levels of miRNAs. Supported by NIH AA12404.

MICRORNA PROFILING IN POSTMORTEM BRAIN OF HUMAN ALCOHOLICS

MicroRNAs (miRNAs) represent a large class of small single-stranded non-coding RNAs that are thought to regulate diverse functions by post-transcriptional gene silencing. Recently, miRNAs are have been shown to be crucial regulators of gene expression, affecting a wide variety of cellular functions. In brain, miRNAs have been shown to play an important role during development and in regulation of synaptic plasticity, and have been implicated in human brain diseases. In previous studies, we and others have used cDNA and oligonucleotide microarrays to identify and classify genes with altered expression following long-term alcohol consumption; however, the potential role of miRNA regulation of these genes has not been investigated. In the present study, we used miRNA arrays (Ambion+Invitrogen human, mouse, and rat probes) to classify miRNAs with altered expression in postmortem prefrontal cortex following long-term alcohol consumption. miRNAs profiles were obtained from the identical samples (14 well characterized alcoholics and 13 matched controls) that were used in a previous cDNA microarray study (Neuropsychopharm 31, 1574-82, 2006). We found that ~20 miRNAs were significantly up-regulated (p<0.002; 20-45%) in the alcoholic group compared to controls. Interestingly, miRNA down-regulation was not observed at this level of significance. Functional classification of the predicted target genes of the regulated miRNAs demonstrated a large degree of overlap with published mRNA data. For example, genes involved in ubiquitination, apoptosis, cell adhesion, neurogenesis, and neural disease are predicted targets of the regulated miRNAs. In addition, approximately one third of the significantly regulated mRNAs from the previous microarray study were identified as potential targets of the significantly changed miRNAs. miRNAs are key regulators of gene expression but the functions of many human miRNAs are yet to be discovered. This study provides the first analysis of miRNA levels in human alcoholism and raise the possibility that the reduced of expression of brain genes seen in human alcoholism may be due, at least in part, to increased levels of miRNAs. Supported by NIH AA12404.

Genomic and proteomic analysis of synaptic protein expression in the brains of human alcoholics

RODGER BROUGH: Next we have a joint presentation Coralie Ober who currently works as a research fellow, Queensland Alcohol and Drug Research and Education Centre at the University of Queensland School of Population Health. She has a very broad experience in clinical nursing through to teaching, advocacy and advisory roles. Her work with Indigenous communities is widely recognised inside and outside this country and her role with the WHO is testimony to that.Alcoholism results in changes in the human brain which reinforce the cycle of craving and dependency, and these changes are manifest in the pattern of expression of mRNA and proteins in key cells and brain areas. Long-term alcohol abuse also results in damage to selected regions of the cortex. We have used cDNA microarrays to show that less than 1% of mRNA transcripts differ signifi cantly between cases and controls in the susceptible area and that the expression profi le of a subset of these transcripts is suffi cient to distinguish alcohol abusers from controls. In addition, we have utilized a 2D gel proteomics based approach to determine the identity of proteins in the superior frontal cortex (SFC) of the human brain that show differential expression in controls and long term alcohol abusers. Overall, 182 proteins differed by the criterion of > 2-fold between case and control samples. Of these, 139 showed signifi cantly lower expression in alcoholics, 35 showed signifi cantly higher expression, and 8 were new or had disappeared. To date 63 proteins have been identifi ed. The expression of one family of proteins, the synucleins, has been further characterized using Real Time PCR and Western Blotting. The expression of alpha-synuclein mRNA was signifi cantly lower in the SFC of alcoholics compared with the same area in controls (P = 0.01) whereas no such difference in expression was found in the motor cortex. The expression of beta- and gamma- synuclein were not signifi cantly different between alcoholics and controls. In contrast, the pattern of alphasynuclein protein expression differs from that of the corresponding RNA transcript. Because of the key role of synaptic proteins in the pathogenesis of alcoholism, we are developing 2-D DIGE based techniques to quantify expression changes in synaptosomes prepared from the SFC of controls and alcoholics.Chronic alcohol misuse leads to both widespread and localized damage in human cerebral cortex. The latter, as neuronal loss, is marked in superior frontal cortex (SFC) but milder in primary motor cortex (PMC) and elsewhere. Quantitative morphometry by Harper et al showed that neuronal loss is greater in alcoholics with comorbidity (Wernicke Korsakoff syndrome, liver cirrhosis). Previous work revealed a paradox: the marked differences in GABAA receptor density, pharmacology, and expression between alcoholics without cormorbidity and controls are muted or absent in cirrhotic alcoholics. This concurs with work by the Butterworth group on hepatic encephalopathy cases — most of whom had an alcoholic aetiology — who show only minor differences from controls. Glutamate receptor differences are muted in many autopsy studies, though we have evidence that NMDA site pharmacology may vary in cirrhotic alcoholics. Here we used Real-Time PCR normalized to GAPDH deltaCT to quantify NMDA NR1, NR2A and NR2B subunit expression in SFC and PMC samples obtained at autopsy from alcoholics with and without comorbid cirrhosis and matched controls. Overall subunit transcript expression was signifi cantly lower in alcoholic cirrhotics than in either of the other groups (F2,42 = 12.942, P < 0.001). The effect was most marked for the NR1 subunit; males differed from females, particularly in SFC. The data suggest that if excitotoxicity mediates neuronal loss in SFC, it may be implemented differently: passively in uncomplicated alcoholics, by altered GABAergic transmission; actively in cirrhotic alcoholics, by altered glutamatergic transmission. We also subdivided cases on a panel of genetic markers. Different genotypes interacted with NMDA and GABAA pharmacology and expression. Cirrhotic and uncomplicated alcoholics may differ pathogenically because of inherent characteristics in addition to possible neurotoxic sequelae to the liver damage.