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P.1.027 Modulation of the neurotrophin brain-derived neurotrophic factor in serotonin transporter mutant rats
Molecular neuropsychopharmacology Reference(s) [1] Sanacora, G., Zarate, C.A., Krystal J.H., Manji, H.K., 2008 Targeting the glutamatergic system to develop novel, improved therapeutics for mood disorders. Nature Review Drug Discovery 7, 426–437. [2] Vollmayr, B., Henn, F.A., 2001 Learned helplessness in the rat: improvements in validity and reliability. Brain Research Protocols 8, 1−7. [3] Musazzi, L., Milanese, M., Farisello, P., Zappettini, S., Tardito, D., Barbiero, V.S., Bonifacino, T., Mallei, A., Baldelli, P., Racagni, G., Raiteri, M., Benfenati, F., Bonanno, G., Popoli, M., 2010 Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the dampening action of antidepressants. PloS ONE 5, e8566. P.1.026 Dopamine-related gene polymorphisms in schizophrenia and in therapeutic response A. Feher1 ° , A. Juh´asz1 , A. Riman´oczy1 , G. Szekeres1 , Z. Janka1 . 1 University of Szeged, Department of Psychiatry, Szeged, Hungary Genetic epidemiological studies have demonstrated that schizophrenia (SCZ) has a high heritability, indicating a significant genetic component to its aetiology. In light of the dopamine hypothesis of SCZ, genes encoding dopaminergic receptors and enzymes related to the metabolism of dopamine might be regarded as candidate genes [1]. The aim of our study was to evaluate the possible role of the dopamine transporter gene (SLC6A3) 40 bp VNTR, the dopamine receptor 2 gene (DRD2) TaqIA and the dopamine beta-hydroxylase gene (DBH) C-1021T polymorphisms in SCZ and in therapeutic response either alone or in genetic interaction. A total of 303 Hungarian Caucasian subjects were enrolled in the study: 120 SCZ patients and 183 healthy control (HC) probands. The SCZ patients received atypical antipsychotics (amisulpride, olanzapine, risperidone) for 12 weeks before the evaluations. Seventy-three percent of the SCZ patients were responder (R) and 27 percent of them were non-responder (NR) subjects. A patient was considered to be responder if there was an improvement of at least 20 points in the Global Assessment of Functioning scale during the treatment period. DNA was extracted from peripheral blood leukocytes, the genetic analyses were performed by PCR amplifications. Fisher’s exact and Pearson’s c2 tests were used to compare allele and genotype frequencies between the investigated groups. A logistic regression model was used to test for the interaction between the investigated polymorphisms and to calculate odds ratios (ORs) with 95% confidence intervals (CI).
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Comparison of SLC6A3 and DRD2 genotype frequencies between SCZ and HC groups did not reach statistically significant difference (SLC6A3: p = 0.742; DRD2: p = 0.069). The frequencies of the DBH C/T and T/T genotypes were significantly higher in SCZ as compared to HC group (C/T: SCZ: 38.3%, HC: 27.9%; T/T: SCZ: 5.0%, HC: 1.6%; p = 0.025). Given the relatively low occurrence of the T/T genotype both in SCZ cases and in controls, the analysis was also conducted by presence or absence of the T allele in the genotypes. The T+ genotypes had a significantly increased risk for SCZ (OR = 2.29, 95%CI: 1.18–4.45; p = 0.014) considering C/C genotype as reference category (OR = 1). The distributions of the SLC6A3, DRD2 and DBH genotypes did not differ significantly between the R and NR groups (SLC6A3: p = 0.751; DRD2: p = 0.218; DBH: p = 0.422). Logistic regression analysis revealed no interaction between the investigated polymorphisms affecting the therapeutic response in SCZ. According to our results, there is no association between the SLC6A3 40 bp VNTR or the DRD2 TaqIA polymorphisms and SCZ. We also failed to detect a significant correlation of these polymorphisms and the therapeutic response in SCZ. The results suggest however that the DBH T allele carriers may have an increased risk for developing SCZ, but the DBH C-1021T polymorphism does not have any influence on therapeutic response, although further investigations are required. This work was supported by a grant from the Hungarian Ministry of Health (ETT 052−07/2009). Reference(s) [1] Di Forti, M., Lappin, J.M., Murray R.M., 2007 Risk factors for schizophrenia − All roads lead to dopamine. European Neuropsychopharmacology 17, S101-S107. P.1.027 Modulation of the neurotrophin brainderived neurotrophic factor in serotonin transporter mutant rats F. Macchi1 ° , R. Molteni1 , F. Calabrese1 , A. Cattaneo2 , G. Racagni1 , B.A. Ellenbroek3 , M.A. Riva1 . 1 University of Milan, Center of Neuropharmacology Dept. of Pharmacological Sciences, Milan, Italy; 2 IRCCS Centro San Giovanni di Dio Fatebenefratelli, Genetic Unit, Brescia, Italy; 3 University of Nijmegen, Psychoneuropharmacology, Nijmegen, The Netherlands It is well established that depression is associated with a background of genetic vulnerability. One of these genes encodes for the serotonin transporter (SERT), which plays a key role in controlling the synaptic levels of serotonin and interacts with adverse life events to determine a depressive phenotype [1]. Animals with genetic defects
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Molecular neuropsychopharmacology
of SERT can therefore be used to investigate in great detail the molecular and functional mechanisms that may be associated with heightened susceptibility for mood disorders. On this basis, we used SERT mutant rats to investigate whether a vulnerable genotype can be associated with deficits of neuronal plasticity, which appear to be relevant for the psychopathologic risk. Specifically, we analyzed the expression of brain-derived neurotrophic factor (BDNF), a neurotrophin involved in neuronal plasticity and mood disorders [2], in rats with partial and total deletion of the SERT gene [3]. By real time RT-PCR, we measured mRNA levels of the total form of the neurotrophin and its different transcripts. In addition, we have examined if alterations on BDNF expression were also present in human leukocytes from healthy individuals with different 5-HTTLPR genotypes. Finally, based on the importance of the gene x environment interaction in the etiology of depression, we evaluated whether the rapid modulation of the BDNF system under an acute stressful challenge (5 min of forced swim) was affected by deletion of the SERT gene. Total BDNF (isoform IX) gene expression was significantly reduced in the hippocampus of SERT knockout (−14%, p < 0.05) but not in heterozygous rats, whereas in prefrontal cortex a significant decrease was found in both genotypes (SERT KO: −34% and SERT HET: −16%, p < 0.001). These changes were due to a significant decrease of specific BDNF isoforms, primarily exon IV and VI. Interestingly, in the prefrontal cortex of SERT KO rats, these changes appear to be sustained by epigenetic mechanisms, with reduced H3 histone acetylation at the promoter IV and increased DNA methylation at the promoter VI. BDNF gene expression was also significantly reduced in leukocytes from healthy subjects carrying the S allele of the 5-HTTLPR (−22% in LS, p < 0.001 and −38% in SS, p < 0.001) as compared to non-carriers (LL). Finally, we found that SERT genotype influenced the stress-induced regulation of BDNF in hippocampus. Indeed, total BDNF mRNA levels were up-regulated only in stressed-KO rats (+24%, p < 0.001 vs. KO/No stress) without any change in wild-type (WT) animals. A similar profile was observed for isoform VI (+31%, p < 0.001 vs KO/No stress) whereas the isoform IV expression was increased only in stressed-WT rats (+24%, p < 0.001 vs. WT/No stress). In summary, our results provide support for a link between genetic susceptibility to mood disorders and impaired neuronal plasticity suggesting that the reduced expression of BDNF may contribute to the pathologic phenotype shown by SERT KO, which may also be relevant for individuals carrying the short variant of the 5-HTTLPR promoter.
Reference(s) [1] Caspi, A., K. Sugden, et al. (2003). “Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene.” Science 301, 386–389. [2] Martinowich, K., et al. (2007). New insights into BDNF function in depression and anxiety. Nat. Neurosci. 10, 1089–1093. [3] Smits, B. M., J. B. Mudde, et al. (2006). “Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis.” Pharmacogenet Genomics 16, 159–169. P.1.030 Epigenetic modification of the glucocorticoid receptor gene predicts women’s salivary cortisol following a threat to the social self S. Edelman1 ° , I. Shalev1 , F. Uzefovsky2 , S. Israel2 , 1 The Hebrew A. Knafo2 , D. Mankuta3 , R.P. Ebstein4 . University of Jerusalem, Neurobiology, Jersualem, Israel; 2 The Hebrew University of Jerusalem, Psychology, Jersualem, Israel; 3 Hadassah Medical Organization, Labor and Delivery, Jersualem, Israel; 4 National University of Singapore, Psychology, Singapore, Singapore Background: Evidence suggests that the reactivity of the hypothalamus-pituitary-adrenal axis (HPAA), a major pathway for regulating stress response, is modulated by both genetic and environmental variables. Epigenetic mechanisms are ideal candidates for mediating the effects of environmental signals on the HPAA reactivity. A key target of the HPAA release is the glucocorticoid receptor (GR, NR3C1). Methylation of CpG islands in the GR exon 1F promoter, mediated by the nerve growth factor-inducible protein A (NGFI-A) transcription factor, are important in regulating this gene’s expression [1]. Additionally, both in animal and human studies, the estrogen receptor alpha (ESR1) and the serotonin transporter (5-HTTLPR) partially modulate HPAA reactivity. In the current study we investigated the role of GR methylation levels and the contribution of the 5-HTTLPR and the ESR1 promoter repeat polymorphisms in explaining gender differences in cortisol response to stress. Methods: Student subjects from the Hebrew University (46 males and 46 females, average age 25.29, S.D. = 3.6) participated in the Trier Social Stress Test (TSST). The TSST consists of public speaking followed by a mental arithmetic task performed in front of 3 ‘judges’ and a camera [2]. Salivary cortisol was sampled eight times. DNA was extracted from mouthwash samples and methylation levels were determined by pyrosequencing
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Comparison of SLC6A3 and DRD2 genotype frequencies between SCZ and HC groups did not reach statistically significant difference (SLC6A3: p = 0.742; DRD2: p = 0.069). The frequencies of the DBH C/T and T/T genotypes were significantly higher in SCZ as compared to HC group (C/T: SCZ: 38.3%, HC: 27.9%; T/T: SCZ: 5.0%, HC: 1.6%; p = 0.025). Given the relatively low occurrence of the T/T genotype both in SCZ cases and in controls, the analysis was also conducted by presence or absence of the T allele in the genotypes. The T+ genotypes had a significantly increased risk for SCZ (OR = 2.29, 95%CI: 1.18–4.45; p = 0.014) considering C/C genotype as reference category (OR = 1). The distributions of the SLC6A3, DRD2 and DBH genotypes did not differ significantly between the R and NR groups (SLC6A3: p = 0.751; DRD2: p = 0.218; DBH: p = 0.422). Logistic regression analysis revealed no interaction between the investigated polymorphisms affecting the therapeutic response in SCZ. According to our results, there is no association between the SLC6A3 40 bp VNTR or the DRD2 TaqIA polymorphisms and SCZ. We also failed to detect a significant correlation of these polymorphisms and the therapeutic response in SCZ. The results suggest however that the DBH T allele carriers may have an increased risk for developing SCZ, but the DBH C-1021T polymorphism does not have any influence on therapeutic response, although further investigations are required. This work was supported by a grant from the Hungarian Ministry of Health (ETT 052−07/2009). Reference(s) [1] Di Forti, M., Lappin, J.M., Murray R.M., 2007 Risk factors for schizophrenia − All roads lead to dopamine. European Neuropsychopharmacology 17, S101-S107. P.1.027 Modulation of the neurotrophin brainderived neurotrophic factor in serotonin transporter mutant rats F. Macchi1 ° , R. Molteni1 , F. Calabrese1 , A. Cattaneo2 , G. Racagni1 , B.A. Ellenbroek3 , M.A. Riva1 . 1 University of Milan, Center of Neuropharmacology Dept. of Pharmacological Sciences, Milan, Italy; 2 IRCCS Centro San Giovanni di Dio Fatebenefratelli, Genetic Unit, Brescia, Italy; 3 University of Nijmegen, Psychoneuropharmacology, Nijmegen, The Netherlands It is well established that depression is associated with a background of genetic vulnerability. One of these genes encodes for the serotonin transporter (SERT), which plays a key role in controlling the synaptic levels of serotonin and interacts with adverse life events to determine a depressive phenotype [1]. Animals with genetic defects
S24
Molecular neuropsychopharmacology
of SERT can therefore be used to investigate in great detail the molecular and functional mechanisms that may be associated with heightened susceptibility for mood disorders. On this basis, we used SERT mutant rats to investigate whether a vulnerable genotype can be associated with deficits of neuronal plasticity, which appear to be relevant for the psychopathologic risk. Specifically, we analyzed the expression of brain-derived neurotrophic factor (BDNF), a neurotrophin involved in neuronal plasticity and mood disorders [2], in rats with partial and total deletion of the SERT gene [3]. By real time RT-PCR, we measured mRNA levels of the total form of the neurotrophin and its different transcripts. In addition, we have examined if alterations on BDNF expression were also present in human leukocytes from healthy individuals with different 5-HTTLPR genotypes. Finally, based on the importance of the gene x environment interaction in the etiology of depression, we evaluated whether the rapid modulation of the BDNF system under an acute stressful challenge (5 min of forced swim) was affected by deletion of the SERT gene. Total BDNF (isoform IX) gene expression was significantly reduced in the hippocampus of SERT knockout (−14%, p < 0.05) but not in heterozygous rats, whereas in prefrontal cortex a significant decrease was found in both genotypes (SERT KO: −34% and SERT HET: −16%, p < 0.001). These changes were due to a significant decrease of specific BDNF isoforms, primarily exon IV and VI. Interestingly, in the prefrontal cortex of SERT KO rats, these changes appear to be sustained by epigenetic mechanisms, with reduced H3 histone acetylation at the promoter IV and increased DNA methylation at the promoter VI. BDNF gene expression was also significantly reduced in leukocytes from healthy subjects carrying the S allele of the 5-HTTLPR (−22% in LS, p < 0.001 and −38% in SS, p < 0.001) as compared to non-carriers (LL). Finally, we found that SERT genotype influenced the stress-induced regulation of BDNF in hippocampus. Indeed, total BDNF mRNA levels were up-regulated only in stressed-KO rats (+24%, p < 0.001 vs. KO/No stress) without any change in wild-type (WT) animals. A similar profile was observed for isoform VI (+31%, p < 0.001 vs KO/No stress) whereas the isoform IV expression was increased only in stressed-WT rats (+24%, p < 0.001 vs. WT/No stress). In summary, our results provide support for a link between genetic susceptibility to mood disorders and impaired neuronal plasticity suggesting that the reduced expression of BDNF may contribute to the pathologic phenotype shown by SERT KO, which may also be relevant for individuals carrying the short variant of the 5-HTTLPR promoter.
Reference(s) [1] Caspi, A., K. Sugden, et al. (2003). “Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene.” Science 301, 386–389. [2] Martinowich, K., et al. (2007). New insights into BDNF function in depression and anxiety. Nat. Neurosci. 10, 1089–1093. [3] Smits, B. M., J. B. Mudde, et al. (2006). “Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis.” Pharmacogenet Genomics 16, 159–169. P.1.030 Epigenetic modification of the glucocorticoid receptor gene predicts women’s salivary cortisol following a threat to the social self S. Edelman1 ° , I. Shalev1 , F. Uzefovsky2 , S. Israel2 , 1 The Hebrew A. Knafo2 , D. Mankuta3 , R.P. Ebstein4 . University of Jerusalem, Neurobiology, Jersualem, Israel; 2 The Hebrew University of Jerusalem, Psychology, Jersualem, Israel; 3 Hadassah Medical Organization, Labor and Delivery, Jersualem, Israel; 4 National University of Singapore, Psychology, Singapore, Singapore Background: Evidence suggests that the reactivity of the hypothalamus-pituitary-adrenal axis (HPAA), a major pathway for regulating stress response, is modulated by both genetic and environmental variables. Epigenetic mechanisms are ideal candidates for mediating the effects of environmental signals on the HPAA reactivity. A key target of the HPAA release is the glucocorticoid receptor (GR, NR3C1). Methylation of CpG islands in the GR exon 1F promoter, mediated by the nerve growth factor-inducible protein A (NGFI-A) transcription factor, are important in regulating this gene’s expression [1]. Additionally, both in animal and human studies, the estrogen receptor alpha (ESR1) and the serotonin transporter (5-HTTLPR) partially modulate HPAA reactivity. In the current study we investigated the role of GR methylation levels and the contribution of the 5-HTTLPR and the ESR1 promoter repeat polymorphisms in explaining gender differences in cortisol response to stress. Methods: Student subjects from the Hebrew University (46 males and 46 females, average age 25.29, S.D. = 3.6) participated in the Trier Social Stress Test (TSST). The TSST consists of public speaking followed by a mental arithmetic task performed in front of 3 ‘judges’ and a camera [2]. Salivary cortisol was sampled eight times. DNA was extracted from mouthwash samples and methylation levels were determined by pyrosequencing