Gabriela Novak

Association of polymorphisms in the BDNF, DRD1 and DRD3 genes with tobacco smoking in schizophrenia

Emerging evidence indicates that the DRD1‐BDNF‐DRD3 cluster plays an important role in nicotine addiction. We have performed an association analysis of 42 SNPs within these genes with cigarette consumption in a group of 341 schizophrenia patients. The ACCG haplotype consisting of four BDNF markers (Val66Met (rs6265), rs11030104, rs2049045 and rs7103411) showed an association with the risk of smoking (p = 0.0002). Both DRD1 markers tested (rs4532 and rs686) and the DRD3 marker (rs1025398) showed association with quantity of tobacco smoked (p = 0.01, 0.005 and 0.002, respectively). Our findings are preliminary; however, they support the involvement of the DRD1, BDNF and DRD3 genes in smoking behaviour.

Schizophrenia: elevated mRNA for dopamine D2Longer receptors in frontal cortex

Because dopamine D2 receptors are the primary targets for antipsychotic drugs, including clozapine and quetiapine, and because some studies have found D2 receptors to be elevated in schizophrenia, we examined the mRNA of three forms of the D2 receptor, particularly the new form of the dopamine D2 receptor, D2(Longer), in post-mortem brains from patients who died with schizophrenia. Using quantitative competitive RT-PCR (reverse transcriptase-polymerase chain reaction), the D2(Longer) mRNA was higher in the frontal cortex, compared to control tissues. The mRNA concentration of D2(Long) and D2(Short) was also higher in the frontal cortex, compared to control tissues. Although most of the schizophrenia patients had received different antipsychotic drugs for varying periods of time, the mRNA of D2(Longer), as well as that for D2(Long) and D2(Short), in such medicated tissues was similar to that in a frontal cortex tissue from a patient who had reliably never received antipsychotic drugs. It is possible, therefore, that the elevation of the mRNAs for D2(Longer), D2(Long) and D2(Short) in the frontal cortex may be related to the disease of schizophrenia itself.

Nogo A, B and C expression in schizophrenia, depression and bipolar frontal cortex, and correlation of Nogo expression with CAA/TATC polymorphism in 3'-UTR

Schizophrenia may result from altered gene expression leading to abnormal neurodevelopment. In a search for genes with altered expression in schizophrenia, our previous work on human frontal cerebral cortex found the mRNA of Nogo, a myelin-associated protein which inhibits the outgrowth of neurites and nerve terminals, to be overexpressed in schizophrenia. Because those earlier results did not examine tissues for the separate Nogo A, B and C isoforms from age- and sex-matched individuals, we repeated the study for all three isoforms, using a new set of tissues from matched individuals, and using the more accurate method of quantitative real-time PCR (polymerase chain reaction). We found Nogo C to be overexpressed by 26% in the schizophrenia tissues, which is in accordance with our earlier results. The expression of Nogo B was statistically significantly reduced by 17% in the frontal cortices from individuals who had been diagnosed as having had severe depression. Furthermore, we show that there is a direct correlation between the expression of Nogo A and C and the presence of alleles with a CAA insert, irrespective of disease status. While upregulation of Nogo C expression may play a role in schizophrenia, altered Nogo B may contribute to the clinical condition of depression. Nogo A showed a statistically non-significant increase in expression in schizophrenia.

Hyperactive mice show elevated D2(High) receptors, a model for schizophrenia: Calcium/calmodulin-dependent kinase II alpha knockouts

The cerebral frontal cortex of patients who had schizophrenia shows elevated levels of RNA for calcium/calmodulin‐dependent protein kinase II beta (CaMKIIβ). In addition, recent research shows that animal models for schizophrenia, such as amphetamine‐sensitized rats, consistently show elevated levels of D2 receptors in their high‐affinity state (D2High), the major target for antipsychotic medication. The present study was done, therefore, to examine whether an alteration in the levels of CaMKIIβ could lead to altered levels of D2High receptors. We found that the CaMKII inhibitor, KN‐93, markedly reduced D2High states in rat striatum. In addition, we studied heterozygous CaMKIIα knock‐out mice that show features analogous to schizophrenia. The striata of these mice revealed a 2.8‐fold increase in D2High receptors. In frontal cortex of the heterozygous CaMKIIα knock‐out mice, CaMKIIα mRNA levels were reduced by 50%, while CaMKIIβ mRNA levels were unaltered. In striatum, CaMKIIβ mRNA levels were increased by 29%, suggesting the presence of a new CaMKIIβ regulatory pathway not previously described. The elevated levels of CaMKIIβ mRNA in the striatum suggest that this enzyme may increase D2High in animals and possibly in schizophrenia itself. Synapse 64:794–800, 2010.

Exposure to nicotine produces an increase in dopamine D2(High) receptors: a possible mechanism for dopamine hypersensitivity.

ABSTRACT Dopamine D2 receptors exist in both low- and high-affinity states (D2High), the latter being the functionally relevant state. Cocaine self-administration produces an increase in D2High, a phenomenon that could explain why cocaine administration results in hypersensitivity to dopamine, even though drug addicts were found to have a decreased number of striatal dopamine D2 receptors. As nicotine acts through the same mesocortical dopaminergic signaling pathways as other stimulant drugs, which are known to increase the levels of D2High, we hypothesized that nicotine exposure could produce an increase in D2High levels. We determined D2High levels in rats after nicotine administration (1.5 mg/kg/day; 14 days), in rats voluntarily self-administering nicotine using an intravenous self-administration (IVSA) protocol (mean dose 0.5 mg/kg/day; 14 days), as well as after a prolonged withdrawal. An increase in the levels of D2High was found in rats who had nicotine administered at a uniform dose, as well as in rats who self-administered nicotine via IVSA, but these changes appear to normalize over time, as indicated by lower D2High levels in rats after a prolonged withdrawal period. We suggest that nicotine-induced elevation in D2High levels could be participating in hypersensitivity to dopamine following nicotine exposure.

Schizophrenia: elevated mRNA for calcium-calmodulin-dependent protein kinase IIβ in frontal cortex

Because amphetamine releases two to three times more dopamine in schizophrenia patients than in control subjects, and because calcium-calmodulin-dependent protein kinase II has a key role in the enhanced action of amphetamine-induced dopamine release in rats, the synaptic content of calcium-calmodulin-dependent protein kinase IIbeta mRNA was measured (by quantitative competitive RT-PCR; reverse transcriptase-polymerase chain reaction) in seven frontal cerebral cortices of post-mortem brains from patients who had schizophrenia and in seven control tissues. The results indicate that the mRNA of this kinase is elevated in the schizophrenia frontal cortex.

Striatal development involves a switch in gene expression networks, followed by a myelination event: Implications for neuropsychiatric disease

Because abnormal development of striatal neurons is thought to be the part of pathology underlying major psychiatric illnesses, we studied the expression pattern of genes involved in striatal development and of genes comprising key striatal‐specific pathways, during an active striatal maturation period, the first two postnatal weeks in rat. This period parallels human striatal development during the second trimester, when prenatal stress is though to lead to increased risk for neuropsychiatric disorders. To identify genes involved in this developmental process, we used subtractive hybridization, followed by quantitative real‐time PCR, which allowed us to characterize the developmental expression of over 60 genes, many not previously known to play a role in neuromaturation. Of these 12 were novel transcripts, which did not match known genes, but which showed strict developmental expression and may play a role in striatal neurodevelopment. An additional 89 genes were identified as strong candidates for involvement in this neurodevelopmetnal process. We show that during the first two postnatal weeks in rat, an early gene expression network, still lacking key striatal‐specific signaling pathways, is downregulated and replaced by a mature gene expression network, containing key striatal‐specific genes including the dopamine D1 and D2 receptors, conferring to these neurons their functional identity. Therefore, before this developmental switch, striatal neurons lack many of their key phenotypic characteristics. This maturation process is followed by a striking rise in expression of myelination genes, indicating a striatal‐specific myelination event. Such strictly controlled developmental program has the potential to be a point of susceptibility to disruption by external factors. Indeed, this period is known to be a susceptibility period in both humans and rats. Synapse, 2013.

Replicated association of the NR4A3 gene with smoking behaviour in schizophrenia and in bipolar disorder.

Schizophrenia and bipolar disorder are associated with dopamine neurotransmission and show high comorbidity with tobacco dependence. Recent evidence indicates that the family of the NR4A orphan nuclear receptors, which are expressed in dopamine neurons and in dopaminoceptive brain areas, may play a role in dopamine‐mediated effects. We have, therefore, analysed the association of six single nucleotide polymorphisms (SNPs) within the three genes belonging to the NR4A orphan nuclear receptor family, NR4A1 (rs2603751, rs2701124), NR4A2 (rs12803, rs834835) and NR4A3 (rs1131339, rs1405209), with the degree of smoking in a sample of 204 unrelated schizophrenia patients, which included 126 smokers and 78 non‐smokers. SNPs within the NR4A3 gene (rs1131339 and rs1405209) were significantly associated with heavy smoking in this cohort, using a stepwise analysis of the escalated number of cigarettes smoked per day (P = 0.008 and 0.006, respectively; satisfying the Nyholt significance threshold of 0.009, an adjustment for multiple testing). We then repeated the association analysis of the NR4A3 markers (rs1131339 and rs1405209) in a larger cohort of 319 patients with bipolar disorder, which included 167 smokers and 152 non‐smokers. We have replicated the positive association with smoking of the NR4A3 SNP rs1131339 in this group (P = 0.04), providing an important confirmation of the involvement of the NR4A3 gene in nicotine addiction in patients with mental health disease, a population significantly at risk for nicotine addiction.

Association of the orphan nuclear receptor NR4A1 with tardive dyskinesia.

Recent evidence has identified the NR4A1 (NUR77, NGFI-B) gene as a strong candidate for involvement in tardive dyskinesia (TD). We have investigated the association of six single nucleotide polymorphisms within the NR4A family of genes with TD in a sample of 171 patients with schizophrenia of Caucasian descent. The NR4A1 single nucleotide polymorphism (SNP) marker rs2603751 showed a nominal association with the risk of TD, as well as with the extent of TD based on the Abnormal Involuntary Movements Scale (AIMS) scores. The haplotype generated by the markers rs2603751 and rs2701124 also showed association with TD and, after adjustment for multiple testing, both the NR4A1 marker rs2603751 and the haplotype continued to show a trend toward association with TD. Although the results of this study are limited by a small sample size, it presents important pilot data and warrants further investigation of the involvement of NR4A1 variants in TD.

Postnatal maternal deprivation and pubertal stress have additive effects on dopamine D2 receptor and CaMKII beta expression in the striatum.

The goal of this study was to determine whether two stressors commonly used to model aspects of neuropsychiatric disease in rats have an additive effect on striatal dopamine type 2 receptor (D2R) expression, a key player in the etiology of neuropsychiatric disease.