Fernando Leonardi-Essmann

Genome-wide Association Study of Alcohol Dependence

CONTEXT Alcohol dependence is a serious and common public health problem. It is well established that genetic factors play a major role in the development of this disorder. Identification of genes that contribute to alcohol dependence will improve our understanding of the mechanisms that underlie this disorder. OBJECTIVE To identify susceptibility genes for alcohol dependence through a genome-wide association study (GWAS) and a follow-up study in a population of German male inpatients with an early age at onset. DESIGN The GWAS tested 524,396 single-nucleotide polymorphisms (SNPs). All SNPs with P < 10(-4) were subjected to the follow-up study. In addition, nominally significant SNPs from genes that had also shown expression changes in rat brains after long-term alcohol consumption were selected for the follow-up step. SETTING Five university hospitals in southern and central Germany. PARTICIPANTS The GWAS included 487 male inpatients with alcohol dependence as defined by the DSM-IV and an age at onset younger than 28 years and 1358 population-based control individuals. The follow-up study included 1024 male inpatients and 996 age-matched male controls. All the participants were of German descent. MAIN OUTCOME MEASURES Significant association findings in the GWAS and follow-up study with the same alleles. RESULTS The GWAS produced 121 SNPs with nominal P < 10(-4). These, together with 19 additional SNPs from homologues of rat genes showing differential expression, were genotyped in the follow-up sample. Fifteen SNPs showed significant association with the same allele as in the GWAS. In the combined analysis, 2 closely linked intergenic SNPs met genome-wide significance (rs7590720, P = 9.72 x 10(-9); rs1344694, P = 1.69 x 10(-8)). They are located on chromosome region 2q35, which has been implicated in linkage studies for alcohol phenotypes. Nine SNPs were located in genes, including the CDH13 and ADH1C genes, that have been reported to be associated with alcohol dependence. CONCLUSIONS This is the first GWAS and follow-up study to identify a genome-wide significant association in alcohol dependence. Further independent studies are required to confirm these findings.

The dopamine D3 receptor plays an essential role in alcohol-seeking and relapse

Our study aimed to identify new candidate genes, which might be involved in alcohol craving and relapse. To find changes in gene expression after long‐term alcohol consumption, we studied gene expression profiles in the striatal dopamine system by using DNA microarrays of two different alcohol‐preferring rat lines (HAD and P). Our data revealed an up‐regulation of the dopamine D3 receptor (D3R) after 1 yr of voluntary alcohol consumption in the striatum of alcohol preferring rats that was confirmed by qRT‐polymerase chain reaction. This finding was further supported by the finding of up‐regulated striatal D3R mRNA in nonselected Wistar rats after long‐term alcohol consumption when compared with age‐matched control animals. We further examined the role of the D3R in mediating alcohol relapse behavior using the alcohol deprivation effect (ADE) model in long‐term alcohol drinking Wistar rats and the model of cue‐induced reinstatement of alcohol‐seeking behavior using the selective D3R antagonist SB‐277011‐A (0, 1, 3, and 10 mg/kg) and the partial agonist BP 897 (0, 0.1, 1, and 3 mg/kg). Both treatments caused a dose‐dependent reduction of relapse‐like drinking in the ADE model as well as a decrease in cue‐induced ethanol‐seeking behavior. We conclude that long‐term alcohol consumption leads to an up‐regulation of the dopamine D3R that may contribute to alcohol‐seeking and relapse. We therefore suggest that selective antagonists of this pharmacological target provide a specific treatment approach to reduce alcohol craving and relapse behavior.—Vengeliene, V., Leonardi‐Essmann, F., Perreau‐Lenz, S., Gebicke‐Haerter, P., Drescher, K., Gross, G., Spanagel, R. The dopamine D3 receptor plays an essential role in alcohol‐seeking and relapse. FASEB J. 20, 2223–2233 (2006)

Effects of the Circadian Rhythm Gene Period 1 (Per1) on Psychosocial Stress-Induced Alcohol Drinking

OBJECTIVE Circadian and stress-response systems mediate environmental changes that affect alcohol drinking. Psychosocial stress is an environmental risk factor for alcohol abuse. Circadian rhythm gene period 1 (Per1) is targeted by stress hormones and is transcriptionally activated in corticotropin releasing factor-expressing cells. The authors hypothesized that Per1 is involved in integrating stress response and circadian rhythmicity and explored its relevance to alcohol drinking. METHOD In mice, the effects of stress on ethanol intake in mPer1-mutant and wild-type mice were assessed. In humans, single nucleotide polymorphisms (SNPs) in hPer1 were tested for association with alcohol drinking behavior in 273 adolescents and an adult case-control sample of 1,006 alcohol-dependent patients and 1,178 comparison subjects. In vitro experiments were conducted to measure genotype-specific expression and transcription factor binding to hPer1. RESULTS The mPer1-mutant mice showed enhanced alcohol consumption in response to social defeat stress relative to their wild-type littermates. An association with the frequency of heavy drinking in adolescents with the hPer1 promoter SNP rs3027172 and with psychosocial adversity was found. There was significant interaction between the rs3027172 genotype and psychosocial adversity on this drinking measure. In a confirmatory analysis, association of hPer1 rs3027172 with alcohol dependence was shown. Cortisol-induced transcriptional activation of hPer1 was reduced in human B-lymphoblastoid cells carrying the risk genotype of rs3027172. Binding affinity of the transcription factor Snail1 to the risk allele of the hPer1 SNP rs3027172 was also reduced. CONCLUSIONS The findings indicate that the hPer1 gene regulates alcohol drinking behavior during stressful conditions and provide evidence for underlying neurobiological mechanisms.

Glycine Transporter-1 Blockade Leads to Persistently Reduced Relapse-like Alcohol Drinking in Rats

BACKGROUND Residual dysfunction of multiple neurotransmitter systems due to chronic alcohol use is likely responsible for the occurrence of compulsive alcohol seeking during abstinence and relapse behavior. There is increasing evidence that glycine, which activates both glycine and N-methyl-D-aspartate receptors, contributes to excessive alcohol consumption. We therefore hypothesized that the blockade of glycine transporter 1 might interfere with compulsive alcohol consumption and relapse behavior. METHODS We used our animal model of alcoholism--long-term alcohol consumption with repeated deprivation phases in rats--to study the effects of a selective blocker of glycine transporter 1 Org25935. The abstinence-promoting drug acamprosate was used as a reference compound. Subsequently, we examined alterations in dorsal striatal gene expression caused by chronic ethanol (EtOH) consumption, focusing on glycinergic and glutamatergic signaling-related genes. Gene expression profiles of Org25935-treated EtOH-drinking rats were compared with vehicle-treated EtOH-drinking versus age-matched EtOH-naive rats. RESULTS We found that repeated treatment with Org25935 reduced compulsive relapse-like drinking without the development of tolerance. Importantly, these antirelapse properties were maintained for at least 6 weeks in a treatment-free period. This persistent effect was paralleled by a reversal of altered expression levels of a set of glycinergic and glutamatergic signaling-related genes to the levels found in EtOH-naive control rats. CONCLUSIONS This study shows that treatment of rats with Org25935 leads to a reduction of compulsive alcohol consumption and relapse-like drinking behavior--an effect that persists into treatment-free periods. This long-term antirelapse effect might result from a restoration of normal glycinergic and glutamatergic signaling function.

Regulation of immune-modulatory genes in left superior temporal cortex of schizophrenia patients: a genome-wide microarray study

Abstract Objectives. The role of neuroinflammation in schizophrenia has been an issue for long time. There are reports supporting the hypothesis of ongoing inflammation and others denying it. This may be partly ascribed to the origin of the materials (CSF, blood, brain tissue) or to the genes selected for the respective studies. Moreover, in some locations, inflammatory genes may be up-regulated, others may be down-regulated. Methods. Genome-wide microarrays have been used for expression profiling in post-mortem brains of schizophrenia patients. Array data have been analyzed by gene set enrichment analysis (GSEA) and further confirmed with selected genes by real-time PCR. Results. In Brodman Area 22 of left superior temporal cortex, at least 70 genes (19%) out of 369 down-regulated genes (P < 0.05) belonged to the immune system. 23 from those 70 genes were randomly selected for real-time PCR. Six reached significance level at P < 0.05. Conclusions. The present data support a brain-specific view of the role immune-modulatory genes may play in the left superior temporal cortex in schizophrenia, because immune functions in the patients are not disturbed. In keeping with comparable, previous studies supporting the notion that schizophrenia is a disease of the synapse, we hypothesize that dysregulation of immune-related genes modifies synaptic functions and stability in this region.

Microarrays ‐ The Challenge of Preparing Brain Tissue Samples

Microarray experiments allow researchers to collect an amazing amount of gene expression data that have the potential to provide unique information to help interpretation of the biological functions of the central nervous system. These experiments are, however, technically demanding and present unique difficulties when used in the context of neuroscience research, in particular. Success or failure of microarray experiments are highly dependent on reproducible target preparations. This involves a relatively long chain of preparation steps, such as removal of tissue from experimental animals or from post‐mortem human brains, storage, selection, and excision of brain regions. This is followed by RNA extraction, reverse transcription, and labeling of target cDNAs or cRNAs. Additionally, it is emphasized that the quality of microarray data largely relies on the proper handling of animals throughout experiments and the time of the day when experiments are stopped. This article tries to provide hints for some basic rules to be observed in preparation of samples for expression profiling studies.

Structural synaptic elements are differentially regulated in superior temporal cortex of schizophrenia patients

Inaccurate wiring and synaptic pathology appear to be major hallmarks of schizophrenia. A variety of gene products involved in synaptic neurotransmission and receptor signaling are differentially expressed in brains of schizophrenia patients. However, synaptic pathology may also develop by improper expression of intra- and extra-cellular structural elements weakening synaptic stability. Therefore, we have investigated transcription of these elements in the left superior temporal gyrus of 10 schizophrenia patients and 10 healthy controls by genome-wide microarrays (Illumina). Fourteen up-regulated and 22 downregulated genes encoding structural elements were chosen from the lists of differentially regulated genes for further qRT-PCR analysis. Almost all genes confirmed by this method were downregulated. Their gene products belonged to vesicle-associated proteins, that is, synaptotagmin 6 and syntaxin 12, to cytoskeletal proteins, like myosin 6, pleckstrin, or to proteins of the extracellular matrix, such as collagens, or laminin C3. Our results underline the pivotal roles of structural genes that control formation and stabilization of pre- and post-synaptic elements or influence axon guidance in schizophrenia. The glial origin of collagen or laminin highlights the close interrelationship between neurons and glial cells in establishment and maintenance of synaptic strength and plasticity. It is hypothesized that abnormal expression of these and related genes has a major impact on the pathophysiology of schizophrenia.

Development of morphine-induced tolerance and withdrawal: Involvement of the clock gene mPer2

The present study has been designed to assess specifically the involvement of the clock gene mPer2 in morphine-induced tolerance and withdrawal. At first, we checked the absence of initial differences in the expression of several gene transcripts involved in the development of morphine dependence in Per2(Brdm1) mutant mice and in their respective wild-type (WT) control littermates. Morphine-induced tolerance as well as precipitated withdrawal was then assessed in these mice. The Per2(Brdm1) mutant mice clearly developed less tolerance and showed attenuated withdrawal signs compared to WT. These results show that mPER2 is involved in morphine-induced tolerance and withdrawal.

Gene expression in superior temporal cortex of schizophrenia patients

We investigated gene expression pattern obtained from microarray data of 10 schizophrenia patients and 10 control subjects. Brain tissue samples were obtained postmortem; thus, the different ages of the patients at death also allowed a study of the dynamic behavior of the expression patterns over a time frame of many years. We used statistical tests and dimensionality reduction methods to characterize the subset of genes differentially expressed in the two groups. A set of 10 genes were significantly downregulated, and a larger set of 40 genes were upregulated in the schizophrenia patients. Interestingly, the set of upregulated genes includes a large number of genes associated with gene transcription (zinc finger proteins and histone methylation) and apoptosis. We furthermore identified genes with a significant trend correlating with age in the control (MLL3) or the schizophrenia group (SOX5, CTRL). Assessments of correlations of other genes with the disorder (RRM1) or with the duration of medication could not be resolved, because all patients were medicated. This hypothesis-free approach uncovered a series of genes differentially expressed in schizophrenia that belong to a number of distinct cell functions, such as apoptosis, transcriptional regulation, cell motility, energy metabolism and hypoxia.

Interactive molecular networks obtained by computer-aided conversion of microarray data from brains of alcohol-drinking rats.

Lists of differentially expressed genes in a disease have become increasingly more comprehensive with improvements on all technical levels. Despite statistical cutoffs of 99% or 95% confidence intervals, the number of genes can rise to several hundreds or even thousands, which is barely amenable to a researchers understanding. This report describes some ways of processing those data by mathematical algorithms. Gene lists obtained from 53 microarrays (two brain regions (amygdala and caudate putamen), three rat strains drinking alcohol or being abstinent) have been used. They resulted from analyses on Affymetrix chips and encompassed approximately 6 000 genes that passed our quality filters. They have been subjected to four mathematical ways of processing: (a) basic statistics, (b) principal component analysis, (c) hierarchical clustering, and (d) introduction into Bayesian networks. It turns out, by using the p-values or the log-ratios, that they best subdivide into brain areas, followed by a fairly good discrimination into the rat strains and the least good discrimination into alcohol-drinking vs. abstinent. Nevertheless, despite the fact that the relation to alcohol-drinking was the weakest signal, attempts have been made to integrate the genes related to alcohol-drinking into Bayesian networks to learn more about their inter-relationships. The study shows, that the tools employed here are extremely useful for (a) quality control of datasets, (b) for constructing interactive (molecular) networks, but (c) have limitations in integration of larger numbers into the networks. The study also shows that it is often pivotal to balance out the number of experimental conditions with the number of animals.