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Background: The alpha7 neuronal nicotinic acetylcholine receptor subunit gene (CHRNA7) is localized in a chromosomal region (15q14) linked to schizophrenia in multiple independent studies. CHRNA7 was selected as the best candidate gene in the region for a well-documented endophenotype of schizophrenia, the P50 sensory processing deficit, by genetic linkage and biochemical studies.Methods: Subjects included Caucasian-Non Hispanic and African-American case-control subjects collected in Denver, and schizophrenic subjects from families in the NIMH Genetics Initiative on Schizophrenia. Thirty-five single nucleotide polymorphisms (SNPs) in the 5'-upstream regulatory region of CHRNA7 were genotyped for association with schizophrenia, and for smoking in schizophrenia.Results: The rs3087454 SNP, located at position -1831 bp in the upstream regulatory region of CHRNA7, was significantly associated with schizophrenia in the case-control samples after multiple-testing correction (P=0.0009, African American; P=0.013, Caucasian-Non Hispanic); the association was supported in family members. There was nominal association of this SNP with smoking in schizophrenia.Conclusions: The data support association of regulatory region polymorphisms in the CHRNA7 gene with schizophrenia. [ABSTRACT FROM AUTHOR]

Schizophrenia is a common mental illness with a large genetic component. Three genome-wide association studies have implicated the major histocompatibility complex gene region on chromosome 6p21.3-22.1 in schizophrenia. In addition, nicotine, which is commonly abused in schizophrenia, affects the expression of central nervous system immune genes. Messenger RNA levels for genes in the 6p21.3-22.1 region were measured in human postmortem hippocampus of 89 subjects. The effects of schizophrenia diagnosis, smoking and systemic inflammatory illness were compared. Cell-specific expression patterns for the class I major histocompatibility complex gene HLA-A were explored utilizing in situ hybridization. Expression of five genes was altered in schizophrenic subjects. Messenger RNA levels for the class I major histocompatibility complex antigen HLA-B were increased in schizophrenic nonsmokers, while levels for smokers were indistinguishable from those of controls. β2 microglobulin, HLA-A and Notch4 were all expressed in a pattern where inflammatory illness was associated with increased expression in controls but not in subjects with schizophrenia. Schizophrenia was also associated with increased expression of Butyrophilin 2A2. HLA-A was expressed in glutamatergic and GABAergic neurons in the dentate gyrus, hilus, and the stratum pyramidale of the CA1-CA4 regions of the hippocampus, but not in astrocytes. In conclusion, the expression of genes from the major histocompatibility complex region of chromosome 6 with likely roles in synaptic development is altered in schizophrenia. There were also significant interactions between schizophrenia diagnosis and both inflammatory illness and smoking., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The α7 nicotinic acetylcholine receptor is known to regulate a wide variety of developmental and secretory functions in neural and non-neural tissues. The mechanisms that regulate its transcription in these varied tissues are not well understood. Epigenetic processes may play a role in the tissue-specific regulation of mRNA expression from the α7 nicotinic receptor subunit gene, CHRNA7. Promoter methylation was correlated with CHRNA7 mRNA expression in various tissue types and the role of DNA methylation in regulating transcription from the gene was tested by using DNA methyltransferase (DNMT1) inhibitors and methyl donors. CHRNA7 mRNA expression was silenced in SH-EP1 cells and bisulfite sequencing PCR revealed the CHRNA7 proximal promoter was hypermethylated. The proximal promoter was hypomethylated in the cell lines HeLa, SH-SY5Y, and SK-N-BE which express varying levels of CHRNA7 mRNA. Expression of CHRNA7 mRNA was present in SH-EP1 cells after treatment with the methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza-CdR), and increased in SH-EP1 and HeLa cells using another methylation inhibitor, zebularine (ZEB). Transcription from the CHRNA7 promoter in HeLa cells was increased when the methyl donor methionine (MET) was absent from the media. Using methylation-sensitive restriction enzyme analysis (MSRE), there was a strong inverse correlation between CHRNA7 mRNA levels and promoter DNA methylation across several human tissue types. The results support a role for DNA methylation of the proximal promoter in regulation of CHRNA7 transcription.

Objective: The chromosomal region, 15q13-q14, including the α7 nicotinic acetylcholine receptor gene, CHRNA7, is a replicated region for schizophrenia. This study fine-mapped genes at 15q13-q14 to determine whether the association is unique to CHRNA7., Methods: Family-based and case-control association studies were performed on Caucasian-non-Hispanic and African-American individuals from 120 families as well as 468 individual patients with schizophrenia and 144 well-characterized controls. Single-nucleotide polymorphism (SNP) markers were genotyped, and association analyses carried out for the outcomes of schizophrenia, smoking, and smoking in schizophrenia., Results: Three genes were associated with schizophrenia in both ethnic populations: TRPM1, KLF13, and RYR3. Two SNPs in CHRNA7 were associated with schizophrenia in African-Americans, and a second SNP in CHRNA7 was significant for an association with smoking and smoking in schizophrenia in Caucasians., Conclusion: Results of these studies support association of the 15q13-q14 region with schizophrenia. The broad positive association suggests that more than one 15q gene may be contributing to the disorder, either in combination or through a regulatory mechanism.

The alpha7 neuronal nicotinic receptor gene (CHRNA7) has been implicated in the pathophysiology of schizophrenia by genetic and pharmacological studies. Expression of the alpha7* receptor, as measured by [(125)I]alpha-bungarotoxin autoradiography, is decreased in postmortem brain of schizophrenic subjects compared to non-mentally ill controls. Most schizophrenic patients are heavy smokers, with high levels of serum cotinine. Smoking changes the expression of multiple genes and differentially regulates gene expression in schizophrenic hippocampus. We examined the effects of smoking on CHRNA7 expression in the same tissue and find that smoking differentially regulates expression of both mRNA and protein for this gene. CHRNA7 mRNA and protein levels are significantly lower in schizophrenic nonsmokers compared to control nonsmokers and are brought to control levels in schizophrenic smokers. Sufficient protein but low surface expression of the alpha7* receptor, seen in the autoradiographic studies, suggests aberrant assembly or trafficking of the receptor.

Multiple genetic linkage studies support the hypothesis that the 15q13-14 chromosomal region contributes to the etiology of schizophrenia. Among the putative candidate genes in this area are the alpha7 nicotinic acetylcholine receptor gene (CHRNA7) and its partial duplication, CHRFAM7A. A large chromosomal segment including the CHRFAM7A gene locus, but not the CHRNA7 locus, is deleted in some individuals. The CHRFAM7A gene contains a polymorphism consisting of a 2 base pair (2 bp) deletion at position 497-498 bp of exon 6. We employed PCR-based methods to quantify the copy number of CHRFAM7A and the presence of the 2 bp polymorphism in a large, multi-ethnic population. The 2 bp polymorphism was associated with schizophrenia in African Americans (genotype p=0.005, allele p=0.015), and in Caucasians (genotype p=0.015, allele p=0.009). We conclude that the presence of the 2 bp polymorphism at the CHRFAM7A locus may have a functional significance in schizophrenia.

The alphaN-catenin (CTNNA2) gene represents a promising candidate gene for schizophrenia based upon previous genetic linkage, expression, and mouse knockout studies. CTNNA2 is differentially regulated by smoking in schizophrenic patients. In this report, the genomic structure of a primate-specific alphaN-catenin splice variant (alphaN-catenin III) is described. A comparison of alphaN-catenin III mRNA expression across postmortem hippocampi from schizophrenic and non-mentally ill smokers and non-smokers revealed a significant decrease in expression among patient non-smokers compared to all other groups. The recent evolutionary divergence of this gene, as well as the differences in gene expression in postmortem brain of schizophrenic non-smokers, supports the role of alphaN-catenin III as a novel disease susceptibility gene., (2007 Wiley-Liss, Inc.)

The hypothesis that the 15q13-15 region of chromosome 15 contains a gene that contributes to the etiology of schizophrenia is supported by multiple genetic linkage studies. The alpha7 neuronal nicotinic acetylcholine receptor (CHRNA7) gene was selected as the best candidate gene in this region for molecular investigation, based on these linkage findings and biological evidence in both human and rodent models. CHRNA7 receptors are decreased in expression in postmortem brain of schizophrenic subjects. A dinucleotide marker, D15S1360, in intron two of the CHRNA7 gene is genetically linked to an auditory gating deficit found in schizophrenics and half of the first-degree relatives of patients. Single strand conformation polymorphism (SSCP) and sequence analyses of DNA from schizophrenic and control individuals identified 33 variants in the coding region and intron/exon borders of the CHRNA7 gene and its partial duplication, dupCHRNA7; common polymorphisms were mapped. Twenty-one variants were found in the exons, but non-synonymous changes were rare. Although the expression of CHRNA7 is decreased in schizophrenia, the general structure of the remaining receptors is likely to be normal., (Copyright 2003 Wiley-Liss, Inc.)

Background: The alpha7 neuronal nicotinic acetylcholine receptor subunit gene (CHRNA7) has been implicated as a candidate gene for schizophrenia, and for an auditory sensory processing deficit found in the disease, by both genetic linkage at 15q14 and biochemical data. The expression of CHRNA7 is reduced in several brain regions in schizophrenic subjects compared with control subjects. This study presents DNA sequence analysis of the core promoter region for CHRNA7 in schizophrenic and control subjects., Methods: Single-strand conformation polymorphism analysis and DNA sequencing were used for mutation screening of the core promoter in the CHRNA7 gene. The sample included subjects from 166 schizophrenic families and 165 controls. Controls had no evidence of current or past psychosis and had auditory evoked potentials recorded., Results: Multiple polymorphic patterns were identified in the CHRNA7 core promoter in both schizophrenic and control subjects. Functional analysis of polymorphisms indicated that transcription was reduced. The prevalence of functional promoter variants was statistically greater in schizophrenic subjects than in the controls. Presence of an alpha7 promoter polymorphism in controls was associated with failure to inhibit the P50 auditory evoked potential response., Conclusions: Although linkage disequilibrium with other genetic alterations cannot be excluded, the CHRNA7 core promoter variants, found in this study, may contribute to a common pathophysiologic feature of schizophrenia.

Previous studies have suggested that an abnormality in neuronal nicotinic acetylcholine receptor expression or function may be involved in the neuropathophysiology of schizophrenia. [(3)H]-nicotine and [(3)H]-epibatidine binding were compared in postmortem brain from control and schizophrenic subjects with varying smoking histories. In control subjects, increased receptor binding was seen in hippocampus, cortex, and caudate with increasing tobacco use. In contrast, schizophrenic smokers had reduced nicotinic receptor levels in these brain regions compared to control smokers. Chronic haloperidol and nicotine treatment, in the rat, was used to assess neuroleptic effects on receptor up-regulation by nicotine. A significant increase in cortical nicotinic receptors was seen in both nicotine treated as well as haloperidol and nicotine co-treated animals, suggesting that the abnormal regulation of high affinity neuronal nicotinic receptors in schizophrenics following nicotine use was not related to chronic neuroleptic treatment.

The human alpha7 neuronal nicotinic acetylcholine receptor gene (HGMW-approved symbol CHRNA7) has been characterized from genomic clones. The gene is similar in structure to the chick alpha7 gene with 10 exons and conserved splice junction positions. The size of the human gene is estimated to be larger than 75 kb. A putative promoter 5' of the translation start in exon 1 has been cloned and sequenced. The promoter region lacks a TATA box and has a high GC content (77%). Consensus Sp1, AP-2, Egr-1, and CREB transcription factor binding sites appear to be conserved between bovine and human genes. The alpha7 nAChR gene was found to be partially duplicated, with both loci mapping to the chromosome 15q13 region. A yeast artificial chromosome contig was constructed over a genetic distance of 5 cM that includes both alpha7 loci and the region between them. Four novel exons are described, located in genomic clones containing the partially duplicated gene. The duplicated sequences, including the novel exons, are expressed in human brain., (Copyright 1998 Academic Press.)

Neuronal nicotinic acetylcholine receptors are expressed in the human central nervous system. A specific subtype of this receptor family, the alpha7 nicotinic acetylcholine receptor, is thought to be the principal alpha-bungarotoxin (alphaBTX)-binding protein in mammalian brain. Although the expression of this receptor subtype has been characterized in rat, no study has specifically compared the expression of both the alpha7 gene and the localization of BTX binding sites in human brain. Expression of alpha7 mRNA and receptor protein in human postmortem brain tissue was examined by in situ hybridization and [125I]-alpha-bungarotoxin autoradiography, respectively, with particular emphasis on regions associated with sensory processing. Regions with high levels of both alpha7 gene expression and [125I]-alphaBTX binding include the nucleus reticularis of the thalamus, the lateral and medial geniculate bodies, the basilar pontine nucleus, the horizontal limb of the diagonal band of Broca, the nucleus basalis of Meynert, and the inferior olivary nucleus. High-to-moderate levels of alpha7 probe hybridization were also seen in the hippocampus and the cerebral cortex; however, there was a reduced or variable degree of [125I]-alphaBTX binding in these regions compared with the level of probe hybridization. In most brain regions, [125I]-alphaBTX binding was localized to neuronal cell bodies similar in morphology to those that exhibited alpha7 hybridization, suggesting that the high-affinity [125I]-alphaBTX binding sites in the human brain are likely to be principally composed of alpha7 receptor subtypes.

Chronic nicotine administration in animal models evokes a dose-dependent increase in brain nicotinic receptor numbers. Genetically determined variability in nicotinic receptor number in different mouse strains has also been reported, which is thought to affect sensitivity to nicotine, as well as the development of tolerance. Humans self-administer nicotine principally in the form of cigarettes and other tobacco products. The present study compared [3H]nicotine binding in human postmortem brain from thalamus and hippocampus of nonsmoking subjects, subjects who had variable life-long smoking histories and subjects who had quit smoking. A significant increase was seen in [3H]nicotine binding in both hippocampus and thalamus of subjects with life-long smoking histories. In the hippocampus, this change resulted from a change in total receptor number (Bmax), with no change in receptor affinity (Kd). There was also a positive correlation between the degree of smoking, as measured by the average reported packs smoked per day, and the number of nicotine binding sites found in both the hippocampus and thalamus, showing that humans exhibit a dose-dependent increase in brain nicotinic receptor binding. Receptor levels in these brain regions after smoking cessation were at or below those found in the control population, which indicated that smoking-induced changes are reversible after cessation of nicotine treatment. These results suggest that increases in nicotinic receptor levels in the human brain may underlie nicotine tolerance and addiction in smokers.

Although glutamatergic receptors are localized throughout the mammalian central nervous system (CNS), the specific cellular localization of the various glutamatergic receptor subtypes throughout human brain remains largely unknown. PCR fragments to human GluR1, GluR2, and GluR3 receptor subtypes were cloned and used as probes for in situ hybridization in order to examine the anatomical and cellular localization of glutamate receptor subtype gene expression in dissected regions of human postmortem brain tissue. Although hybridization was observed throughout the CNS, results indicated that the highest levels of hybridization were in the hippocampus, with localization primarily to cells in the pyramidal cell layer of the CA1-CA3 region, and the granular cells of the dentate gyrus. Prominent hybridization also was observed in the medium to large neurons of the cingulate cortex, temporal lobe, septum, and amygdala, as well as in scattered neurons in the thalamus, cerebral cortex, and medulla. A striking pattern of differential hybridization was observed within the cerebellum. GluR1 demonstrated light hybridization along the Purkinje/Bergmann glia layer, with GluR2 and GluR3 demonstrating hybridization to Purkinje cells, and GluR3 also to cells within the molecular layer, previously identified as stellate-basket cells. Changes in glutamate receptor function have been shown to be important in the pathogenesis of a number of neurological disorders. Therefore, an examination of glutamatergic receptor expression in human postmortem brain tissue may provide important information on the molecular basis of a variety of neurological and psychiatric disorders of the CNS.

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF), the two best characterized members of a growing family of heparin-binding growth factors, have been shown to affect both survival of cultured neurons and regeneration of nerve terminals when applied exogenously. The endogenous expression of these growth factors in response to brain injury is not well understood. We have utilized the Swiss-Webster mouse, treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and a quantitative polymerase chain reaction assay to examine changes in endogenous synthesis of mRNA for both aFGF and bFGF in the striatum and substantia nigra. We have found that MPTP treatment results in a loss of 95% of dopaminergic function and is accompanied by an increase in expression of both aFGF and bFGF in the striatum at 1 week post-lesion. After 5 weeks, the terminals appear to be regenerating and FGF mRNA expression has returned to control levels. These results suggest that cellular reaction to chemical lesion in the brain may involve changes in growth factor expression, including both aFGF and bFGF.

We have compared RNA polymerase promoter activities in PCR-generated DNA fragments for use in the in vitro transcription of cRNA probes. Sense oligonucleotide primers, specific for the mouse acidic fibroblast growth factor gene, were synthesized with 5' extensions containing promoter sequences for the T7, T3 and SP6 RNA polymerase promoters. A common antisense primer was used with each of the promoter/aFGF primers to prepare PCR-generated DNA fragments (minigenes). In vitro transcription efficiency for each of these constructs was evaluated by incorporation of radioactivity into the cRNA products. We find that both the T7 and T3 promoters can direct the synthesis of cRNA probes of high specific activity from a PCR-generated DNA fragment, but that SP6 cannot. No detectable cRNA product was obtained using either T7 polymerase on the T3/minigene or T3 on the T7/minigene. Antisense cRNA probes, transcribed from minigene constructs were used for both Northern and in situ hybridization studies. A PCR-generated DNA fragment with RNA polymerase promoter sequences at each end provides a single template for synthesis in vitro of either sense or antisense cRNA probes.

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.

A somatic cell mutant has been isolated which is resistant to killing and growth inhibition by mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The resistance phenotype is dominant with respect to the wild-type cell and can largely be ascribed to a 6-7-fold lowering of the KM for HMG-CoA. We thus conclude that mevinolin resistance can be utilized to obtain a genetic marker for the structural gene encoding HMH-CoA reductase.

Previous studies on a somatic cell mutant auxotrophic for mevalonate (Mev-1) have shown that these cells rapidly lose viability when deprived of mevalonic acid in culture medium supplemented with serum cholesterol. Testing of all known end products of mevalonate metabolism in cultured mammalian cells has been conducted to determine the basis for this mevalonate requirement. It has been found that the recently discovered mevalonate metabolite 24(S),25-epoxycholesterol produces a partial restoration of viability of Mev-1 cells starved for mevalonate, whereas other structurally similar oxysterols do not. It appears that 24(S),25-epoxycholesterol has a specific, vital cellular function in CHO-K1 cells.