Differential gene regulation in the anterior cingulate cortex and superior temporal cortex in schizophrenia: A molecular network approach.

The closely connected anterior cingulate cortex (ACC) and superior temporal cortex (STC) are important for higher cognitive functions. Both brain regions are disturbed in schizophrenia, i.e., functional and structural alterations have been reported. This postmortem investigation in brains from patients with schizophrenia and controls compared gene expression in the left ACC and left STC. Most differentially expressed genes were unique to each brain region, but some clusters of genes were equally dysregulated in both, giving rise to a more general disease-specific pattern of gene regulation. The data was used to construct a molecular network of the genes identically expressed in both regions as primary nodes and the metabolically connected genes as secondary nodes. The network analysis identified downregulated clusters of immune-associated gene products and upregulated clusters belonging to the ubiquitin-proteasome system. These findings could help to identify new potential therapeutic targets for future approaches.

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.

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.

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

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.

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.

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.

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.

Transcriptional changes in insulin- and lipid metabolism-related genes in the hippocampus of olfactory bulbectomized mice.

Affymetrix chips were used to perform a hypothesis-free large-scale screening of transcripts in the hippocampus of olfactory bulbectomized mice, an established animal model of depression. Because only 11 transcripts were significantly changed, the statistically subsequent 25 transcripts below the significance level were additionally included in a first round of qRT-PCR evaluations. Furthermore, all 36 genes were then tested for mutual interactions or interactions with other molecules in a physiological context using PathwayArchitect software. Thirty of them were displayed in a network interacting with at least one partner molecule from the list or with other partner molecules known from the literature. All partner molecules from the most prominent 10 molecules of this network were then identified and put together into a new list. On those grounds, the hypothesis was made that metabolic network components of the insulin signaling pathway are perturbed in the disease. This pathway was subsequently tested by a second round of qRT-PCR, adding also a few additional candidate molecules belonging to this pathway. It turned out that the key target -- FABP7 -- fell into the group of transcripts not significantly regulated within the chip data, and another key target -- IRS1 -- did not show up in the chip experiments at all. In conclusion, our data reveal a problem with adhering to statistical significances in microarray experiments, insofar as molecules important for the disease may fall into the range of statistical noise. This approach may also be useful to find new targets for pharmacotherapy in affective disorders.

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.

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.

Fractalkine-upregulated milk-fat globule EGF factor-8 protein in cultured rat microglia.

Fractalkine is the only known member of the CX(3)C-chemokine family, and so is its receptor CX(3)CR1. Fractalkine, typically is expressed by neurons where it is inserted in the plasma membrane ("chemokine on a stalk"). It can, however, be clipped off by a specific enzyme and diffuse into the extracellular space. CX(3)CR1 is primarily expressed by microglia, the phagocytes of the brain. This study was aimed at studying gene expression changes in cultured rat microglia upon fractalkine stimulation using gene chip technology. Six genes turned out to be upregulated, amongst which milk-fat globule EGF factor-8 protein (MFG-E8) was the most surprising, but also the most revealing one. We hypothesize that it serves as a bridging molecule between apoptotic cells (neurons) and microglia. Since the docking to microglia is, in part, mediated by members of the integrin family, six of these molecules have been-post hoc-included in real-time PCR confirmations of chip results. Two of them-integrin alpha(2) and integrin beta(5)-were upregulated as well. These data provide a much closer look into molecular mechanisms involved in apoptosis of neurons and their removal by microglia.