Preliminary evidence for association between schizophrenia and polymorphisms in the regulatory Regions of the ADRA2A,DRD3 and SNAP-25 Genes

The results of linkage and candidate gene association studies have led to a range of hypotheses about the pathogenesis of schizophrenia. We limited our study to polymorphisms in candidate genes involved in dopaminergic and noradrenergic systems, and in the 25 KDa synaptosomal-associated protein (SNAP-25) gene that is related to neurotransmitter exocytosis. Eight single nucleotide polymorphisms (SNPs) in regulating or coding regions of genes for the alpha-2A adrenergic receptor (ADRA2A), dopamine receptors D1 and D3 (DRD1 and DRD3), dopamine β-hydroxylase (DBH) and SNAP-25 were genotyped in male patients with schizophrenia (n=192) and in healthy controls (n=213). These polymorphisms were previously associated with schizophrenia. The allelic association between schizophrenia and ADRA2A rs1800544 polymorphism, SNAP-25 rs1503112 polymorphism, and DRD3 rs6280 polymorphism was found in our study. However, only observations for rs1503112 survived correction for multiple testing. Association was also evaluated by considering the polymorphisms as interactions; in this case, a likelihood ratio test (LRT) revealed evidence for association with schizophrenia in four polymorphism combinations: two DRD3*SNAP-25 combinations (rs6280*rs3746544 and rs6280*rs3746544, P=0.02), one ADRA2A*SNAP25 combination (rs1800544*rs3746544) and one ADRA2A*DBH combination (rs1800544*rs2519152). Our results are in agreement with the previously proposed role of DNA polymorphisms involved in dopaminergic, noradrenergic and synaptic functions in the pathogenesis of schizophrenia. Further relevant studies including larger sample size and more markers are needed to confirm our results.     

Elevated cerebrospinal fluid SNAP-25 in schizophrenia.

BACKGROUND: Research suggests an association between abnormal exocytosis and schizophrenia. We previously demonstrated increased synaptosomal associated protein, 25 kDa (SNAP-25), a member of the exocytotic mechanism, in the cerebrospinal fluid (CSF) of schizophrenia subjects. In this study, we explored SNAP-25 level and clinical variables in a new group of subjects. METHODS: Twenty-five haloperidol-treated subjects with chronic schizophrenia and twenty-five healthy control subjects participated in the study. Subjects received haloperidol treatment for at least 3 months and then had a lumbar puncture (n = 19). Medication was replaced by placebo, and the lumbar puncture was repeated (n = 25) after 6 weeks or sooner if limited psychotic symptoms occurred. We measured the level of SNAP-25 in the CSF and symptoms with the Brief Psychiatric Rating Scale (BPRS). RESULTS: In both haloperidol (p =.001) and placebo (p =.001) treatment conditions, SNAP-25 was elevated. There was no significant difference in SNAP-25 level between conditions. We identified significant positive correlations among SNAP-25 and the BPRS total score and psychosis and thinking disturbance subscales in subjects on haloperidol. CONCLUSIONS: These observations confirm our previous report of elevated CSF SNAP-25 and suggest that synaptic pathology may be linked with the pathophysiology of schizophrenia.     

An association study between polymorphisms in three genes of 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein) family and paranoid schizophrenia in northern Chinese population.

We performed an association study between three SNPs in the genes of 14-3-3 family and paranoid schizophrenia. SNP rs983583 G/A in the YWHAZ gene showed significant association with paranoid schizophrenia. Our study indicated that the YWHAZ gene was a potential susceptibility gene for paranoid schizophrenia in the population studied.     

14-3-3 protein is a component of Lewy bodies in Parkinson's disease-mutation analysis and association studies of 14-3-3 eta

Mutations in alpha-synuclein have been identified in some rare families with autosomal dominant Parkinson's disease (PD). The synuclein gene family shares physical and functional homology with 14-3-3 proteins and binds to 14-3-3 proteins and to its ligands. We therefore investigated whether 14-3-3 proteins are also involved in the pathogenesis of PD. Here we demonstrate that 14-3-3 proteins are colocalized with Lewy bodies in PD. We investigated the 14-3-3 eta (YWHAH) gene by mutation analysis and association studies as it maps to human chromosome 22q12.1-q13.1, a region which has been recently implicated in PD and carried out immunohistochemical studies of Lewy bodies with two different 14-3-3 eta antibodies. In 358 sporadic and familial PD patients, disease causing mutations were not identified. Furthermore, association studies with intragenic polymorphisms do not provide evidence for an involvement of 14-3-3 eta in the pathogenesis of PD. In accordance with these findings, there was no staining of substantia nigra Lewy bodies with antibodies specific for the 14-3-3 eta subunit.     

CNS 14-3-3zeta: changes with sex but not psychiatric diagnoses or psychotropic drug treatment

mRNA for 14-3-3zeta, an abundant signalling protein in human CNS, is reported as decreased or unchanged in cortex from subjects with schizophrenia. Addressing this dichotomy, using Western blot analyses, we measured levels of 14-3-3zeta proteins in cortex and caudate nucleus from subjects with schizophrenia, bipolar disorder, age/sex matched controls and in analogous CNS regions from rats treated with psychotropic drugs. Anti-14-3-3zeta antibody bound to three proteins (molecular weights: 27, 54 and 70 kDa), in all CNS tissue. Levels of all proteins did not vary with diagnoses (27 kDa: F(2,42.0)=0.35, p=0.71; 54 kDa: F(2,42.1)=0.62, p=0.54; 70 kDa: F(2,41.0)=2.43, p=0.10). By contrast, independent of diagnoses, there were significant increases in the levels of the 27 kDa protein (+32%; p<0.001) and 54 kDa protein (51%; p=0.001) in the caudate nucleus from males compared to females. In addition, there was a trend (-25%; p=0.06) to decreased levels of the 70 kDa protein in BA 9 in males compared to females. Treating with haloperidol, olanzapine, lithium or a combination thereof did not alter 14-3-3zeta levels in rat cortex or striatum. Therefore, this study suggests that 14-3-3zeta proteins are not altered in the cortex or caudate nucleus in schizophrenia, bipolar disorder or in analogous regions in psychotropic drug treated rats. By contrast, our study suggests that levels of 14-3-3zeta in some regions of the human CNS may be modulated by some sex-specific mechanism.     

Down-regulation of 14-3-3 eta gene expression by IGF-I in mouse cerebellum during postnatal development

Insulin-like growth factor I (IGF-I) overexpression in the postnatal cerebellum of transgenic (Tg) mice results in remarkable cerebellar overgrowth characterized by a near doubling of granule cell number that is predominantly due to inhibition of apoptosis. Using this Tg model we set out to investigate IGF-I anti-apoptotic mechanisms by defining the influence of IGF-I on gene expression. Using a cDNA array technique, we screened a total of 243 mouse apoptosis-related genes, and found that 14-3-3 eta gene expression was significantly reduced in the cerebella of Tg mice compared with their wild-type (Wt) littermates. Using Northern blot analysis to corroborate our microarray finding, we showed that 14-3-3 eta mRNA abundance was decreased from postnatal day P5 through P17. Nonetheless, the expression pattern of 14-3-3 eta in Tg mice followed the same pattern observed in Wt mice, and was indistinguishable from that in Wt mice at P20 and P23. 14-3-3 eta protein abundance, as determined by Western immunoblot analyses, showed similar decreases in the cerebella of Tg mice. In situ hybridization demonstrated that 14-3-3 eta was predominantly, if not exclusively, expressed and regulated in Purkinje cells. 14-3-3 proteins have multiple functions, including participation in pathways that favor cell survival. Our finding of IGF-I-induced down-regulation of 14-3-3 eta expression in Purkinje cell at a time when IGF-I promotes granule cell survival leads us to speculate that down-regulation of 14-3-3 eta may: (a) serve a negative feedback role to modulate Purkinje cell survival, i.e. limit Purkinje cell number, and/or (b) function as part of a distinct signaling mechanism, perhaps one that augments the capacity of Purkinje cells to promote granule cell survival.     

Selective regulation of 14-3-3eta in primary culture of cerebral cortical neurons and astrocytes during development

The 14-3-3 proteins exist predominantly in the brain and may play regulatory roles in cellular processes of growth, differentiation, survival, and apoptosis. The biological functions, however, of the various 14-3-3 isoforms (beta, epsilon, eta, gamma, and zeta) in the brain remain unclear. We have reported previously upregulation of 14-3-3gamma in ischemic astrocytes. In the present study, we report selective regulation of 14-3-3eta in cultured cerebral cortical neurons and astrocytes during in vitro development. In cultured neurons, gene expression levels of 14-3-3eta increase with culture age (0-10 days). Brain-derived neurotrophic factor and neurotrophin-3 upregulate 14-3-3eta gene expression. In cultured astrocytes, 14-3-3eta is downregulated with culture age (1-5 weeks). The gene expression level of 14-3-3eta is not affected by scratch injury in astrocytes or by ischemia in neurons. These data suggest a possible role of 14-3-3eta in growth and differentiation of neurons and astrocytes, indicating an intricate mechanism governing coordinated and well-controlled developmental events in the brain to ensure normal neural functions.     

Differential effects of antipsychotics on hippocampal presynaptic protein expressions and recognition memory in a schizophrenia model in mice

We compared the effects of subchronic clozapine and haloperidol administration on the expression of SNAP-25 and synaptophysin in an animal model of schizophrenia based on the glutamatergic hypothesis. Mice were first treated with a non-competitive NMDA antagonist MK-801 (0.3 mg/kg/day) or saline for 5 days, and then clozapine (5 mg/kg/day), haloperidol (1 mg/kg/day) or saline was administered for two weeks. The locomotion test, as a behavioral model of the positive symptoms of schizophrenia, was applied after MK-801/saline administration on day 6 for acute effects and after antipsychotic/saline administration on day 19 for enduring effects on mice activity. Memory function was assessed by the Novel Object Recognition (NOR) test, one day after the last day of antipsychotic/saline administration (day 20). Western Blotting technique was used to determine SNAP-25 and synaptophysin expressions in the hippocampus and frontal cortex. Both antipsychotics reversed the enhanced locomotion effects of MK-801. MK-801 and haloperidol decreased recognition memory performance. On the other hand, clozapine did not compromise memory. It also did not reverse the negative effects of MK-801 on memory performance. MK-801 did not change SNAP-25 and synaptophysin expressions in the hippocampus and frontal cortex. Clozapine increased hippocampal SNAP-25, decreased hippocampal synaptophysin expression, whereas frontal SNAP-25 and synaptophysin expressions remained unchanged. Haloperidol had no effects on levels of SNAP-25 and synaptophysin in the frontal cortex and hippocampus. These findings support the idea that the differential effects of clozapine might be related to its plastic effects and synaptic reorganization of the hippocampus.     

Associations of SNAP-25 polymorphisms with cognitive dysfunctions in Caucasian patients with schizophrenia during a brief trail of treatment with atypical antipsychotics

The synaptosomal-associated protein of 25 kDa (SNAP-25) is part of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment receptor (SNARE), which mediates synaptic neurotransmission. In earlier studies a possible involvement of this protein in schizophrenia has been shown. As neurocognitive impairment is a core feature in the pathology of schizophrenia and considered to be a putative endophenotype according to genetic studies we investigated the influences of different SNAP-25 polymorphisms on neuropsychological test results before and during treatment with atypical antipsychotics. A total of 104 schizophrenic patients treated with atypical antipsychotics were genotyped for three different polymorphisms of the SNAP-25 gene (MnlI, TaiI and DdeI in the 3'-UTR). Cognitive function was assessed at baseline, week 4 or 6 and week 8 or 12. Results of individual neuropsychological tests were assigned to six cognitive domains (reaction time and quality; executive function; working, verbal and visual memory) and a general cognitive index. The MnlI and TaiI polymorphisms showed no associations to deficits on neuropsychological test results. In contrast, we observed a significant relation between the DdeI polymorphism of the SNAP-25 gene and cognitive dysfunctions. Homozygote T/T allele carriers of the DdeI polymorphism showed significant better neuropsychological test results in cognitive domains verbal memory and executive functions than those with the combined T/C and C/C genotypes (P < 0.01) at all three time points, but no differences in response to treatment with atypical antipsychotics. Additionally, TT carriers exhibited significantly better results in a general cognitive index (P < 0.05). As we observed an association between the DdeI polymorphism of the SNAP-25 gene and cognitive dysfunctions of schizophrenic patients our finding suggests that the SNAP-25 gene could play a role in the pathophysiology of neurocognitive dysfunctions in schizophrenia but is not predictive for treatment response with atypical antipsychotics.     

Reduced thyroid hormones with increased hippocampal SNAP-25 and Munc18-1 might involve cognitive impairment during aging

The mechanism underlying the decline of age-related learning and memory remains unclear. Brain-region-specific changes of synaptic proteins and decreased thyroid hormones (THs) have been implied involving this decline. During normal aging, however, the relationships among synaptic proteins, THs and abilities of learning and memory remain to be elucidated. In this study, the age-related spatial learning and memory ability of 41 Kunming mice (KM) (14 mice aged 6 months, 13 mice aged 11 months, 14 mice aged 22 months) was measured with radial six-arm water maze. The levels of SNAP-25 and Munc18-1 in brain regions were semi-quantified by Western blotting and the serum THs were detected by radioimmunoassay. Our results showed the old Kunming mice had marked impairment of spatial learning and memory, with decreased serum free triiodothyronine (FT3) and increased SNAP-25 and Munc18-1 in dorsal hippocampus (DH), ventral hippocampus (VH) and frontal lobe (F). The Pearson's correlation test showed the impairment of spatial learning ability positively correlated with SNAP-25 in DH and Munc18-1 in DH and VH. While, the levels of SNAP-25 (DH, VH and F) and Munc18-1 (DH) negatively correlated with the serum FT3 level, and the spatial memory decline marginal negatively correlate with serum THs. These results suggested that increased hippocampal SNAP-25 and Munc18-1 which seemingly result from decreased serum THs might involve the age-related impairment of spatial learning and memory.     

Polymorphism of the 5'-upstream region of the human SNAP-25 gene: an association analysis with schizophrenia

Recent studies have suggested that synaptic abnormalities may be part of the pathophysiology of schizophrenia. SNAP-25 (synaptosomal-associated protein of 25 kD) is one of the synaptic proteins responsible for presynaptic neurotransmission, axonal elongation and synaptogenesis. Genetic variation in the 5'-upstream region of the SNAP-25 gene was analyzed in 87 unrelated schizophrenic patients and 100 healthy controls. A novel polymorphic (TAAA)(n) tandem repeat was identified in the 5'-upstream region. There were no significant differences between the patient and the control groups in the distribution of repeat numbers of alleles or genotypes. In addition, no associations were found between the polymorphism for subtypes, longitudinal courses or positive family history of the patients. Our results suggest that polymorphisms in the 5'-upstream region of the SNAP-25 gene have no association with schizophrenia.     

Convergent functional genomics of schizophrenia: from comprehensive understanding to genetic risk prediction

We have used a translational convergent functional genomics (CFG) approach to identify and prioritize genes involved in schizophrenia, by gene-level integration of genome-wide association study data with other genetic and gene expression studies in humans and animal models. Using this polyevidence scoring and pathway analyses, we identify top genes (DISC1, TCF4, MBP, MOBP, NCAM1, NRCAM, NDUFV2, RAB18, as well as ADCYAP1, BDNF, CNR1, COMT, DRD2, DTNBP1, GAD1, GRIA1, GRIN2B, HTR2A, NRG1, RELN, SNAP-25, TNIK), brain development, myelination, cell adhesion, glutamate receptor signaling, G-protein-coupled receptor signaling and cAMP-mediated signaling as key to pathophysiology and as targets for therapeutic intervention. Overall, the data are consistent with a model of disrupted connectivity in schizophrenia, resulting from the effects of neurodevelopmental environmental stress on a background of genetic vulnerability. In addition, we show how the top candidate genes identified by CFG can be used to generate a genetic risk prediction score (GRPS) to aid schizophrenia diagnostics, with predictive ability in independent cohorts. The GRPS also differentiates classic age of onset schizophrenia from early onset and late-onset disease. We also show, in three independent cohorts, two European American and one African American, increasing overlap, reproducibility and consistency of findings from single-nucleotide polymorphisms to genes, then genes prioritized by CFG, and ultimately at the level of biological pathways and mechanisms. Finally, we compared our top candidate genes for schizophrenia from this analysis with top candidate genes for bipolar disorder and anxiety disorders from previous CFG analyses conducted by us, as well as findings from the fields of autism and Alzheimer. Overall, our work maps the genomic and biological landscape for schizophrenia, providing leads towards a better understanding of illness, diagnostics and therapeutics. It also reveals the significant genetic overlap with other major psychiatric disorder domains, suggesting the need for improved nosology.     

cDNA cloning and chromosome assignment of the gene for human brain 14-3-3 protein eta chain.

We present the nucleotide sequence of a cDNA clone of mRNA encoding human 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases and an endogenous inhibitor of protein kinase C. The 1,730-nucleotide sequence of the cloned cDNA contains 191 bp of a 5'-noncoding region, the complete 738 bp of coding region, and 801 bp of a 3'-noncoding region containing three canonical polyadenylation signals. The 14-3-3 protein eta chain cDNA encoded a polypeptide of 246 amino acids with a predicted molecular weight 28,196. The predicted amino acid sequence of human 14-3-3 protein eta was highly homologous to that of previously reported bovine and rat 14-3-3 proteins with only two amino acid differences. The sequence carries structural features as putative regions responsible for activation of tyrosine and tryptophan hydroxylases and for inhibition of Ca2+/phospholipid-dependent protein kinase C. Northern blot analysis demonstrated widespread expression of the 14-3-3 protein eta chain in cultured cell lines derived from various human tumors. These findings suggest the conservative functions of the 14-3-3 protein among species. Spot blot hybridization analysis with flow-sorted chromosomes showed that the human 14-3-3 protein eta chain gene is assigned to chromosome 22.     

Association analysis of serotonin 2A receptor gene T102c polymorphism and schizophrenia.

The serotonin neurotransmitter has been associated with the pathogenesis of mood disorders and schizophrenia. Serotonin receptors genes may therefore be candidate genes for the study of the genetics of these disorders. In this study, patients with schizophrenia (n=235) and controls (n=344) were analysed to determine the correlation between the 5HT(2A) receptor gene T102C polymorphism and schizophrenia. No association was found between the studied polymorphism and schizophrenia (p=0.854 for alleles and p=0.945 for genotypes). Results were also not significant when analysed by gender (for male p=0.861-allele frequency and p=0.467-genotype frequency, for female p=0.857-allele frequency and p=0.833-genotype frequency). Subgroups with regard to schizophrenia subtypes, age of onset and clinical course of schizophrenia were analysed with negative results.     

Association of SNAP-25 Gene Ddel and Mnll Polymorphisms with Adult Attention Deficit Hyperactivity Disorder

Objective

The synaptosomal-associated protein of 25 kDa (SNAP-25) gene is a presynaptic plasma membrane protein and an integral component of the vesicle docking and fusion machinery mediating secretion of neurotransmitters. Previously, several studies reported association between SNAP-25 and attention deficit hyperactivity disorder (ADHD). We investigated whether these SNAP-25 polymorphisms (MnlI T/G and DdelI T/C) were also associated with ADHD in the Turkish population.

Methods

Our study comprised unrelated 139 subjects who met DSM-IV criteria for ADHD and 73 controls and all were of Turkish origin. Genetic analyses were performed and patients were evaluated with Wender-Utah Rating Scale and Adult ADD/ADHD DSM IV-Based Diagnostic Screening and Rating Scale.

Results

SNAP-25 DdelI polymorphism was not associated with ADHD but there was a statistically significant difference between ADHD patients and controls for SNAP-25 MnlI polymorphism. For SNAP-25 MnlI polymorphism patients with G/G genotype of the SNAP-25 gene MnlI polymorphism had higher Wender-Utah scores and higher scores in the 1st and 3rd parts of adult ADD/ADHD Scale.

Conclusion

We detected a significant association of the MnlI polymorphism in our ADHD sample which was similar to previous findings. Our study also revealed that SNAP-25 MnlI polymorphism was also associated with symptom severity of ADHD. This study is also, the first report on the association of SNAP-25 with ADHD in the Turkish population.     

Biased paternal transmission of SNAP-25 risk alleles in attention-deficit hyperactivity disorder

Attention-deficit hyperactivity disorder (ADHD) is the most common childhood psychiatric disorder, affecting 5-10% of school-age children. Although the biological basis of this disorder is unknown, twin and family studies provide strong evidence that ADHD has a genetic basis involving multiple genes. A previous study found an association between ADHD and two polymorphisms in the 3' untranslated region (UTR) of SNAP-25, a gene encoding a synaptic vesicle docking protein known to play a role in the hyperactivity observed in the Coloboma mouse strain. In this paper, we test biased transmission of the 3' UTR SNAP-25 haplotype using a larger ADHD sample of 113 families with 207 affected children. Using the transmission disequilibrium test (TDT), we found a trend consistent with biased transmission of the TC haplotype of SNAP-25 in all transmissions and detected a significant distortion (P=0.027) when paternal transmissions were evaluated.