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437. Differential expression of newly identified gene with RING-H2 finger motif after chronic antidepressant treatment in rat brain
134S
Saturday Abstracts
BIOL PSYCHIATRY 2000;47:1S–173S
pathological dissociation in BPD which seems unrelated to childhood traumatic experiences. Our preliminary genetic findings on all personality disorders suggest that genetic factors may influence pathological dissociation.
437. DIFFERENTIAL EXPRESSION OF NEWLY IDENTIFIED GENE WITH RING-H2 FINGER MOTIF AFTER CHRONIC ANTIDEPRESSANT TREATMENT IN RAT BRAIN M. Yamada (1), M. Yamada (2), S. Yamazaki (2), Y. Kiuchi (3), H. Ozawa (4), K. Momose (2), K. Kamijima (1), T. Higuchi (5) (1) Dept. of Psychiatry, School of Medicine; (2) Dept. of Pharmacology and (3) Pathophysiology, Showa University, Shinagawa, Tokyo, 142-866; (4) Dept. of Psychiatry, Sapporo Medical University, Hokkaido, 060-8556; (5) National Center of Neurology and Psychiatry, Kohnodai Hospital, Chiba 272-8516 Japan Depression is one of the major neuropsychiatric diseases. Although blockade by antidepressants of monoamine uptake is one of the cornerstones of the monoamine hypothesis of depression, the exact mechanism of action of antidepressants remains uncertain. In the present study, we used RNA fingerprinting technique to identify the common biochemical changes induced after chronic treatment with imipramine or sertraline. One of the cDNA fragments which was extracted from differentially expressed band on acrylamide gel was subcloned to TA cloning vector and sequenced. Homology analysis of the fragment with the EMBL-Gene Bank database showed significant matches to mouse kf-1 gene. This clone, ADRG-34 might be a rat homologue of the kf-1 gene. We determined the full length of cDNA sequence encoding 685 amino acid residues with a calculated molecular mass of 79 kDa protein. This protein contains a RING-H2 finger motif, involves in the protein sorting machinery, at the carboxy-terminus. Treatment with the antidepressants imipramine or sertraline induced the expression of ADRG-34 in the mRNA levels in frontal cortex and hippocampus when compared to controls. On the other hand, the mRNA level was not affected by these treatments in hypothalamus. Immunoblot analysis revealed that ADRG-34 protein is in the nucleus fraction and the expression of ADRG-34 was induced after antidepressant treatments in hippocampus. Our data suggest that ADRG-34 may be one of the common molecules induced after chronic antidepressant treatment.
438. IMPASE-2 EXPRESSION AND ALTERED CALCIUM HOMEOSTASIS PHENOTYPES IN BIPOLAR I DISORDER I-S. Yoon (1,6), P.P. Li (1,4,5), J.L. Kennedy (2,4,6), R. Cooke (3,4), J.J. Warsh (1,3,4,5,6) Sections of (1) Biochemical Psychiatry and (2) Neurogenetics, and (3) Bipolar Clinic Centre for Addiction and Mental Health, and Departments of (4) Psychiatry, (5) Pharmacology and (6) Institute of Medical Science, University of Toronto, Toronto, IN, Canada, M5T 1R8 Building evidence implicates altered calcium (Ca2⫹) homeostasis and phosphoinositide (PI) signaling in bipolar affective disorder (BD).
Inositol monophosphatase (IMPase) is a key enzyme in the PI cycle and a target of lithium action. Altered IMPase expression and elevated basal intracellular calcium levels ([Ca2⫹]i) in B lymphoblasts from bipolar I (BP-I) patients may reflect cellular endophenotypes of BD. We examined whether these two putative endophenotypes are related. Using an RT-PCR assay, mRNA levels were estimated for IMPase-2, which is located in the region 18p11.2 that may harbor a susceptibility gene(s) for BD, in B lymphoblast cell lines phenotyped on [Ca2⫹]i, from patients with a DSM-IV diagnosis of BP-I disorder and from age- and sexmatched healthy subjects. Two-way ANOVA revealed a significant effect of sex (F ⫽ 5.32, df ⫽ 1,30, p ⫽ 0.03) and an interaction between sex and phenotype (F ⫽ 8.41, df ⫽ 2,30, p ⫽ 0.001). Simple contrasts revealed significantly lower IMPase-2 expression (mean relative units ⫾ S.E.M, 0.10 ⫾ 0.02, p ⫽ 0.02) in B lymphoblasts from male BP-I patients with the high Ca2⫹ phenotype ([Ca2⫹]i ⱖ 66.6 nM) compared with values from the normal Ca2⫹ BP-I phenotype (0.19 ⫾ 0.02) or healthy subjects (0.18 ⫾ 0.02). IMPase-2 mRNA levels were also significantly inversely correlated with [Ca2⫹]i in cells from male (r ⫽ ⫺0.616, p ⫽ 0.006) but not female BP-I patients. However, considerably more male (5/6) than female (1/6) patients were receiving lithium treatment in the BP-I group with the high Ca2⫹ phenotype. Thus, an association of reduced IMPase-2 expression with response to lithium must also be considered to explain these findings. Supported by NARSAD.
439. THE IDENTIFICATION AND LOCALIZATION OF ER STRESS PROTEINS AS VALPROATE REGULATED GENES C.D. Bown, B. Chen, J.-F. Wang, L.T. Young Mood Disorders Program, McMaster University, Hamilton, ON, Canada, L8N 3Z5 Identifying lasting changes in gene expression following pharmacological treatment may help understand the pathophysiologic changes that are responsible for mood disorders. However, few studies have tested these notions empirically. Using differential display PCR from cerebral cortex of rats treated with chronic valproate we identified 78-kilodalton glucoseregulated protein (GRP78) as a valproate regulated gene. Studies in rat C6 glioma cells showed that carbamazepine was also capable of upregulating GRP78 expression. In addition, the expression of other endoplasmic reticulum (ER) stress proteins, GRP94 and calreticulin, were also increased following valproate treatment in rat C6 glioma cells. All the ER stress proteins showed a dose- and time-dependent increase in expression following valproate treatment. Protein expression of these three ER stress proteins were also shown to be increased in a dosedependent manner after 7 days of treatment. Following removal of the drug from the medium, expression levels of the ER stress proteins all returned to basal levels within 6 hours. This suggests that valproate has a direct effect on the transcription of these genes and does cause the observed changes in mRNA levels by damaging the cell. Immunohistochemistry showed that chronic treatment of rats with valproate also increased expression of all the ER stress proteins in the frontal cortex, parietal cortex and CA1 region of the hippocampus. The ER stress proteins, GRP78, GRP94 and calreticulin, function as calcium binding proteins and molecular chaperones to assist in the regulation of protein folding, both of which have been shown to have neuroprotective effects. Regulation of ER stress proteins by chronic treatment with mood stabilizing drugs suggests that this family of proteins may be involved in the pathophysiology of mood disorders.
Saturday Abstracts
BIOL PSYCHIATRY 2000;47:1S–173S
pathological dissociation in BPD which seems unrelated to childhood traumatic experiences. Our preliminary genetic findings on all personality disorders suggest that genetic factors may influence pathological dissociation.
437. DIFFERENTIAL EXPRESSION OF NEWLY IDENTIFIED GENE WITH RING-H2 FINGER MOTIF AFTER CHRONIC ANTIDEPRESSANT TREATMENT IN RAT BRAIN M. Yamada (1), M. Yamada (2), S. Yamazaki (2), Y. Kiuchi (3), H. Ozawa (4), K. Momose (2), K. Kamijima (1), T. Higuchi (5) (1) Dept. of Psychiatry, School of Medicine; (2) Dept. of Pharmacology and (3) Pathophysiology, Showa University, Shinagawa, Tokyo, 142-866; (4) Dept. of Psychiatry, Sapporo Medical University, Hokkaido, 060-8556; (5) National Center of Neurology and Psychiatry, Kohnodai Hospital, Chiba 272-8516 Japan Depression is one of the major neuropsychiatric diseases. Although blockade by antidepressants of monoamine uptake is one of the cornerstones of the monoamine hypothesis of depression, the exact mechanism of action of antidepressants remains uncertain. In the present study, we used RNA fingerprinting technique to identify the common biochemical changes induced after chronic treatment with imipramine or sertraline. One of the cDNA fragments which was extracted from differentially expressed band on acrylamide gel was subcloned to TA cloning vector and sequenced. Homology analysis of the fragment with the EMBL-Gene Bank database showed significant matches to mouse kf-1 gene. This clone, ADRG-34 might be a rat homologue of the kf-1 gene. We determined the full length of cDNA sequence encoding 685 amino acid residues with a calculated molecular mass of 79 kDa protein. This protein contains a RING-H2 finger motif, involves in the protein sorting machinery, at the carboxy-terminus. Treatment with the antidepressants imipramine or sertraline induced the expression of ADRG-34 in the mRNA levels in frontal cortex and hippocampus when compared to controls. On the other hand, the mRNA level was not affected by these treatments in hypothalamus. Immunoblot analysis revealed that ADRG-34 protein is in the nucleus fraction and the expression of ADRG-34 was induced after antidepressant treatments in hippocampus. Our data suggest that ADRG-34 may be one of the common molecules induced after chronic antidepressant treatment.
438. IMPASE-2 EXPRESSION AND ALTERED CALCIUM HOMEOSTASIS PHENOTYPES IN BIPOLAR I DISORDER I-S. Yoon (1,6), P.P. Li (1,4,5), J.L. Kennedy (2,4,6), R. Cooke (3,4), J.J. Warsh (1,3,4,5,6) Sections of (1) Biochemical Psychiatry and (2) Neurogenetics, and (3) Bipolar Clinic Centre for Addiction and Mental Health, and Departments of (4) Psychiatry, (5) Pharmacology and (6) Institute of Medical Science, University of Toronto, Toronto, IN, Canada, M5T 1R8 Building evidence implicates altered calcium (Ca2⫹) homeostasis and phosphoinositide (PI) signaling in bipolar affective disorder (BD).
Inositol monophosphatase (IMPase) is a key enzyme in the PI cycle and a target of lithium action. Altered IMPase expression and elevated basal intracellular calcium levels ([Ca2⫹]i) in B lymphoblasts from bipolar I (BP-I) patients may reflect cellular endophenotypes of BD. We examined whether these two putative endophenotypes are related. Using an RT-PCR assay, mRNA levels were estimated for IMPase-2, which is located in the region 18p11.2 that may harbor a susceptibility gene(s) for BD, in B lymphoblast cell lines phenotyped on [Ca2⫹]i, from patients with a DSM-IV diagnosis of BP-I disorder and from age- and sexmatched healthy subjects. Two-way ANOVA revealed a significant effect of sex (F ⫽ 5.32, df ⫽ 1,30, p ⫽ 0.03) and an interaction between sex and phenotype (F ⫽ 8.41, df ⫽ 2,30, p ⫽ 0.001). Simple contrasts revealed significantly lower IMPase-2 expression (mean relative units ⫾ S.E.M, 0.10 ⫾ 0.02, p ⫽ 0.02) in B lymphoblasts from male BP-I patients with the high Ca2⫹ phenotype ([Ca2⫹]i ⱖ 66.6 nM) compared with values from the normal Ca2⫹ BP-I phenotype (0.19 ⫾ 0.02) or healthy subjects (0.18 ⫾ 0.02). IMPase-2 mRNA levels were also significantly inversely correlated with [Ca2⫹]i in cells from male (r ⫽ ⫺0.616, p ⫽ 0.006) but not female BP-I patients. However, considerably more male (5/6) than female (1/6) patients were receiving lithium treatment in the BP-I group with the high Ca2⫹ phenotype. Thus, an association of reduced IMPase-2 expression with response to lithium must also be considered to explain these findings. Supported by NARSAD.
439. THE IDENTIFICATION AND LOCALIZATION OF ER STRESS PROTEINS AS VALPROATE REGULATED GENES C.D. Bown, B. Chen, J.-F. Wang, L.T. Young Mood Disorders Program, McMaster University, Hamilton, ON, Canada, L8N 3Z5 Identifying lasting changes in gene expression following pharmacological treatment may help understand the pathophysiologic changes that are responsible for mood disorders. However, few studies have tested these notions empirically. Using differential display PCR from cerebral cortex of rats treated with chronic valproate we identified 78-kilodalton glucoseregulated protein (GRP78) as a valproate regulated gene. Studies in rat C6 glioma cells showed that carbamazepine was also capable of upregulating GRP78 expression. In addition, the expression of other endoplasmic reticulum (ER) stress proteins, GRP94 and calreticulin, were also increased following valproate treatment in rat C6 glioma cells. All the ER stress proteins showed a dose- and time-dependent increase in expression following valproate treatment. Protein expression of these three ER stress proteins were also shown to be increased in a dosedependent manner after 7 days of treatment. Following removal of the drug from the medium, expression levels of the ER stress proteins all returned to basal levels within 6 hours. This suggests that valproate has a direct effect on the transcription of these genes and does cause the observed changes in mRNA levels by damaging the cell. Immunohistochemistry showed that chronic treatment of rats with valproate also increased expression of all the ER stress proteins in the frontal cortex, parietal cortex and CA1 region of the hippocampus. The ER stress proteins, GRP78, GRP94 and calreticulin, function as calcium binding proteins and molecular chaperones to assist in the regulation of protein folding, both of which have been shown to have neuroprotective effects. Regulation of ER stress proteins by chronic treatment with mood stabilizing drugs suggests that this family of proteins may be involved in the pathophysiology of mood disorders.