Catrin Wernicke

Allelic variants of the functional promoter polymorphism of the human serotonin transporter gene is associated with auditory cortical stimulus processing.

The loudness dependence (LD) of the auditory-evoked N1/P2 component has been shown to be related to the central serotonergic neurotransmission. Allelic variants in the promoter region of the 5-hydroxytryptamine transporter (5-HTT) gene were shown to modulate serotonergic activity. It was hypothesized that the three genotypes (l/l, s/l, s/s) differ with respect to LD. Allelic variants of the 5-HTT promoter region and LD at the Cz electrode were determined in 185 healthy subjects prospectively. A significant association was found between LD and genotype (ANOVA: F=4.172, p=0.017). Individuals homozygous for the l allele exhibited a weaker LD compared to heterozygous subjects. The results are consistent with the reported association between 5-HTT genotype and serotonin transport capacity in lymphoblasts, and indicate that auditory stimulus processing is associated with genetic variants of the brain serotonergic system. The LD may serve as endophenotype in human serotonin research.

Polymorphisms in the N-methyl-D-aspartate receptor 1 and 2B subunits are associated with alcoholism-related traits.

BACKGROUND This study examined the hypothesis that allelic variants of the ionotropic glutamatergic N-methyl-D-aspartate receptor (NMDAR) are associated with vulnerability to alcoholism and some related traits. METHODS We investigated the silent G2108A and C2664T polymorphisms of the NMDAR1 and the NMDAR2B genes, respectively. The case control study included 367 alcoholic and 335 control subjects of German origin. The family-based study comprised 81 Polish alcoholic patients and their parents using the transmission disequilibrium test. RESULTS The genotype frequencies of the NMDAR1 polymorphism differed significantly between control and alcoholic subjects. This difference was also observed in more homogenous subgroups of alcoholic subjects with vegetative withdrawal syndrome and Cloninger type 1. Patients with a history of delirium tremens or seizures during withdrawal showed a significantly increased prevalence of the A allele. Genotyping of the NMDAR2B polymorphism revealed a significantly reduced T allele in Cloninger type 2 alcoholics and in patients reporting an early onset compared with control subjects. Our family-based study for NMDAR2B, revealed a trend to a preferred transmission of the C allele by the fathers, and families with early-onset patients contributed most to this trend. CONCLUSIONS These results suggest that variants in NMDAR genes are associated with alcoholism and related traits.

Association of the dopamine D2 receptor gene with alcohol dependence: haplotypes and subgroups of alcoholics as key factors for understanding receptor function.

Objectives The dopamine D2 receptor (DRD2) plays an important role in the reinforcing and motivating effects of ethanol. Several polymorphisms have been reported to affect receptor expression. The amount of DRD2, expressed in a given individual, is the result of the expression of both alleles, each representing a distinct haplotype. We examined the hypothesis that haplotypes composed of polymorphisms, associated with reduced receptor expression, are more frequent in alcoholics compared with healthy individuals. Methods The polymorphisms −141ins/del, C957T, A1385G, and TaqIA were genotyped in a case–control sample comprising 360 alcoholics and 368 controls, and in a family-based sample of 65 trios. To investigate more homogenous groups, we constructed two subgroups with respect to age at onset and antisocial personality disorder. In addition, a subgroup with positive family history of alcoholism was investigated. Results The haplotypes I-C-G-A2 and I-C-A-A1 occurred with a higher frequency in alcoholics [P = 0.026, odds ratio (OR): 1.340; P = 0.010, OR: 1.521, respectively]. The rare haplotype I-C-A-A2 occurred less often in alcoholics (P = 0.010, OR: 0.507), and was also less often transmitted from parents to their affected offspring (1 vs. 7). Among the subgroups, I-C-G-A2 and I-C-A-A1 had a higher frequency in Cloninger 1 alcoholics (P = 0.083 and 0.001, OR: 1.917, respectively) and in alcoholics with a positive family history (P = 0.031, OR: 1.478; P = 0.073, respectively). Cloninger 2 alcoholics had a higher frequency of the rare haplotype D-T-A-A2 (P<0.001, OR: 4.614) always compared with controls. In patients with positive family history haplotype I-C-A-A2 (P = 0.004, OR: 0.209), and in Cloninger 1 alcoholics haplotype I-T-A-A1 (P = 0.045, OR: 0.460) were less often present. Conclusion We confirmed the hypothesis that haplotypes, which are supposed to induce a low DRD2 expression, are associated with alcohol dependence. Furthermore, supposedly high-expressing haplotype weakened or neutralized the action of low-expressing haplotypes.

Association of human hippocampal neurochemistry, serotonin transporter genetic variation, and anxiety.

The impact of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) on anxiety-related behavior and related cerebral activation has facilitated the understanding of neurobiological mechanisms of anxiety. However, the influence of the 5-HTTLPR genotype on hippocampal neuronal development and neurochemistry, which is relevant to anxiety behavior, has not been investigated. In 38 healthy subjects, absolute concentrations of N-acetylaspartate (NAA) were measured as a main surrogate parameter for hippocampal neurochemistry on a 3-T scanner. A significantly lower hippocampal NAA concentration in s allele carriers was observed as compared to l/l genotype. Other metabolites (choline, creatine + phosphocreatine, glutamate) were unaffected by genotype. The hippocampal NAA concentration was negatively correlated with trait anxiety scores (STAI). Metabolites measured in the anterior cingulate cortex (reference region) were not associated with genotype. The results are in accordance with the recently reported relationship between hippocampal neuronal development and anxiety behavior in adult animals and show an association between human limbic neurochemistry and genetically driven serotonergic neurotransmission relevant to anxiety.

No Association of a Functional Polymorphism in the Serotonin Transporter Gene Promoter and Anxiety-Related Personality Traits

Serotonergic neurotransmission, which is involved in many psychiatric disorders, is mediated by the serotonin transporter protein. Gene coding for the serotonin transporter protein is designated SLC6A4, which has been differentially associated with anxiety-related behavioral traits and neuroticism in healthy subjects. To confirm the association between the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) and anxiety-related personality traits, we examined 228 healthy unrelated participants (age 38.6 ± 12.8 years; 115 male, 113 female) of German origin, who were carefully examined with respect to psychiatric health. The self-ratable State-Trait Anxiety Inventory (STAI) and the NEO Five Factor Inventory (NEO-FFI) were performed. No significant association was observed between the 5-HTTLPR genotype and STAI 1 (state, χ2 = 0.82, p < 0.66, d.f. = 2), STAI 2 (trait, χ2 = 2.7, p < 0.25, d.f. = 2) and NEO-FFI scores of any of the 5 major axes, including neuroticism (χ2 = 3.35, p < 0.18, d.f. = 2) in all subjects. Given the small effect of this 5-HTT polymorphism on behaviour in previous studies, a lack of significant genotype differences in these tests could be due to considerable individual variability in these measures.

Raf kinase inhibitor protein enhances neuronal differentiation in human SH-SY5Y cells.

Neuronal differentiation has evolved as an essential process even in the adult brain, once disturbed being associated with the pathogenesis of several psychiatric disorders. To study the effects of Raf kinase inhibitor protein (RKIP) on neuronal differentiation, we generated neuroblastoma cell lines overexpressing RKIP (RKIP+) and expressing RKIP-directed short hairpin RNA for downregulation of RKIP (RKIP–). During a 4-week time course of continuous differentiation by retinoic acid (RA), expression of neuronal and glial markers, intracellular cyclic adenosine monophosphate (cAMP) levels, protein kinase C (PKC) signal transduction to extracellular signal-regulated kinase 1/2 (ERK-1/2) and cellular morphology were investigated in relation to RKIP levels. RKIP+ cells showed accelerated neurite outgrowth, formation of elaborated neuronal networks and increased neuronal marker expression both in RA-induced differentiation and to some extent even in non-RA-treated cells. RKIP– cells showed glial-like cell bodies and increased glial fibrillary acidic protein, suggesting a shift from neuronal to glial phenotype. With respect to differentiation-inducing signal pathways, PKC-mediated ERK-1/2 activation significantly correlated with RKIP levels. Furthermore, basal and forskolin-stimulated intracellular cAMP was potently increased in RKIP+ cells versus controls. In conclusion, the conserved signal transduction modulator RKIP was shown to enhance several aspects of neuronal differentiation via enhanced crosstalk from PKC to ERK-1/2 and enhancement of G-protein-coupled receptor signaling.

9-Methyl-β-carboline up-regulates the appearance of differentiated dopaminergic neurones in primary mesencephalic culture

beta-Carbolines (BCs) derive from tryptophan and its derivatives. They are formed endogenously in humans and mammals and occur inter alia in cooked meat and tobacco smoke. They have been detected in human brain, cerebrospinal fluid, and plasma. Up to now they were predominantly identified as compounds exhibiting neurotoxic actions. Since significantly higher amounts are present in parkinsonian patients, they are regarded as potential pathogenetic factors in Parkinsons disease. We identified for the first time a BC (9-methyl-BC; 9-me-BC) exerting neuroprotective and neuron-differentiating effects. Treatment of primary mesencephalic dopaminergic cultures with 9-me-BC inhibited the basal release of lactate dehydrogenase and reduced the number of cells stained with propidium iodide. Caspase-3 activity was decreased, the total protein content was unchanged and ATP content was increased. Furthermore, the expression of inflammation-related genes was reduced. The number of differentiated dopaminergic neurones was significantly increased and a wide array of neurotrophic/transcription factors (Shh, Wnt1, Wnt5a, En1, En2, Nurr1, Pitx3) and marker genes (Th, Dat, Aldh1a1) decisive for dopaminergic differentiation was stimulated. Consistently, the dopamine content was slightly, although non-significantly, increased and the dopamine uptake capacity was elevated. An anti-proliferative effect was observed in human neuroblastoma SH-SY5Y cells which is consistent with a reduced incorporation of bromodesoxyuridine into the DNA of primary mesencephalic cells. Whether the additional dopaminergic neurones in primary culture derive from dopaminergic precursor cells, previously tyrosine hydroxylase negative dopaminergic neurones or are the result of a transdifferentiation process remains to be established.

Norepinephrine transporter gene polymorphism is not associated with susceptibility to alcohol dependence

Abnormalities in monoamine neurotransmission have been implicated in the pathogenesis of alcoholism, mood disorders and schizophrenia. Murine norepinephrine transporter gene (NET) has been mapped to a region on chromosome 8 where a quantitative trait locus for ethanol sensitivity. Therefore we tested whether norepinephrine transporter (NET) gene variants confer susceptibility to either alcohol dependence or severe alcohol withdrawal symptoms. There is a highly polymorphic silent G1287A mutation in the NET gene. In our study 157 alcoholics and 185 healthy unrelated matched control subjects were analyzed for a silent G1287A mutation. No significant differences in allele and genotype distribution between control subjects f(A)=0.33 and alcoholics f(A)=0.29 were found. No significant results were found in more homogenous subgroups, i.e. alcoholics with severe alcohol withdrawal (seizures, delirium), early onset age<26 nor dependent patients with positive familial history of alcoholism. These results suggest that the NET gene polymorphism in exon 9 accession number: mRNA: NM_001043, genomic contig.: NT_019610, is unlikely to be involved in the susceptibility to alcoholism and severe alcohol withdrawal.

Haplotypes of dopamine and serotonin transporter genes are associated with antisocial personality disorder in alcoholics.

Introduction A different genetic background is postulated for alcoholics with early onset and with antisocial personality disorder (type 2 alcoholics) compared with those with late onset and without antisocial personality disorder (type 1 alcoholics). The dopamine transporter (DAT) and the serotonin transporter (SERT) are involved in endophenotypes that are associated with these subtypes. Our study was aimed at investigating whether distinct haplotypes, defined by polymorphisms associated with the expressions of DAT and SERT, were associated with subgroups of alcohol dependence. Methods Intron 8 variable number of tandem repeats (VNTR), exon 15 rs27072 and VNTR (DAT), promoter VNTR and rs25531, and intron 2 VNTR (SERT) were genotyped in a case–control sample comprising 360 alcoholics and 368 controls, and in a family-based sample of 65 trios, all of German origin. Results DAT: The haplogenotypes 6-A-10/6-G-10 and 5-G-9/5-G-9 were more often present in type 2 alcoholics as compared with type 1 alcoholics [odds ratio (OR): 2.8], and controls (OR: 5.8), respectively. The daily ethanol consumption was associated with haplogenotypes. SERT: haplotypes SA-10 (OR: 2.3) and LG-12 (OR: 2.5) were more often present in type 2 alcoholics compared with controls. Haplotype LA-10 was less often present in type 2 alcoholics (OR: 0.5), and was more often transmitted, in families, to the affected offspring (transmission disequilibrium test: OR: 5.2; family-based association test: Z: 1.9). The haplotype LA-12 was significantly undertransmitted to affected offspring in the whole group (transmission disequilibrium test: OR: 0.216; family-based association test: Z: −2.2). A gene by environment interaction was observed with respect to the time course of the depression score after alcohol withdrawal and with respect to the positive family history of alcohol dependence. Conclusion Haplotype analysis, sub-grouping with respect to more homogeneous endophenotypes, and inclusion of quantifiable characteristics are sensible strategies to untangle the genetic background of such a complex disorder like alcohol dependence.

Long-Term Ethanol Exposure Impairs Neuronal Differentiation of Human Neuroblastoma Cells Involving Neurotrophin-Mediated Intracellular Signaling and in Particular Protein Kinase C

BACKGROUND Revealing the molecular changes in chronic ethanol-impaired neuronal differentiation may be of great importance for understanding ethanol-related pathology in embryonic development but also in the adult brain. In this study, both acute and long-term effects of ethanol on neuronal differentiation of human neuroblastoma cells were investigated. We focused on several aspects of brain-derived neurotrophic factor (BDNF) signaling because BDNF activates the extracellular signal-regulated kinase (ERK) cascade, promoting neuronal differentiation including neurite outgrowth. METHODS The effects of ethanol exposure on morphological differentiation, cellular density, neuronal marker proteins, basal ERK activity, and ERK responsiveness to BDNF were measured over 2 to 4 weeks. qRT-PCR and Western blotting were performed to investigate the expression of neurotrophin receptor tyrosin kinase B (TrkB), members of the ERK-cascade, protein kinase C (PKC) isoforms and Raf-Kinase-Inhibitor-Protein (RKIP). RESULTS Chronic ethanol interfered with the development of a neuronal network consisting of cell clusters and neuritic bundles. Furthermore, neuronal and synaptic markers were reduced, indicating impaired neuronal differentiation. BDNF-mediated activation of the ERK cascade was found to be continuously impaired by ethanol. This could not be explained by expressional changes monitored for TrkB, Raf-1, MEK, and ERK. However, BDNF also activates PKC signaling which involves RKIP, which finally leads to ERK activation as well. Therefore, we hypothesized that ethanol impairs this branch of BDNF signaling. Indeed, both PKC and RKIP were significantly down-regulated. CONCLUSIONS Chronic ethanol exposure impaired neuronal differentiation of neuroblastoma cells and BDNF signaling, particularly the PKC-dependent branch. RKIP, acting as a signaling switch at the merge of the PKC cascade and the Raf/MEK/ERK cascade, was associated with neuronal differentiation and significantly reduced in ethanol treatment. Moreover, PKC expression itself was even more strongly reduced. In contrast, members of the Raf-1/MEK/ERK cascade were less affected and the observed changes were not associated with impaired differentiation. Thus, reduced RKIP and PKC levels and subsequently reduced positive feedback on ERK activation provide an explanation for the striking effects of long-term ethanol exposure on BDNF signal transduction and neuronal differentiation, respectively.