- Email: [email protected]
Reverse transcriptase activity in human lymphocytes and brain tissue
54
hippocampus are reduced by 38% and 22% respectively, (p<0.05, Students t-test). In comparison, levels of GluRA 'flop' mRNA are entirely unchanged in the dentate gyrus (hybridisation levels: schizophrenics 226n Ci/g; controls 256n Ci.g). This study provides further evidence that nonNMDA receptor abnormalities occur in the hippocampus in schizophrenia, adding weight to the glutamate theory of schizophrenia. Whether the more specific abnormality in the level of expression of the developmentally early ('flip') isoforms of glutamate receptor mRNAs is relevant to developmental theories of schizophrenia will require further study.
THE EXPRESSION OF NMDA RECEPTOR SUBUNITS IN SCHIZOPHRENIC POST-MORTEM HUMAN HIPPOCAMPUS J.P. Beckwith*, N.C. Stefanis, D.P. McLaughlin, R.W. Kerwin
Department of Neuroscience, Institute of Psychiatry, London SE5 8AF, UK Interest in EAAS developed only after the nature and function of the glutamate receptor subtypes were identified. Cloning has identified mRNA species encoding NMDA receptor subunits, termed NMDAR2A, 2B, 2C and 2D which have 55-70% identity (Monyer et al., 1992; Kutsuwada et al., 1992). These are structurally related with less than 20% peptide sequence identity (including the NMDA receptor subunit NMDAR1). Phencyclidine (PCP), a potent psychotropic drug which causes symptoms similar to schizophrenia is a noncompetitive antagonist of NMDA receptors. This has led to the development of an aetiologic hypothesis of schizophrenia which links PCP-sensitive sites on NMDA receptors to the disease. In this study, we have investigated the expression of the mRNA coding for NMDA receptor subunits (NR1, 2A, 2B, 2C and 2D) in the hippocampus of 10 schizophrenics and 10 age- and sex-matched controls post mortem. In situ hybridisation and quantitative autoradiography using specific oligonucleotide probes for each subunit revealed that all subunits (with the exception of NR2C and NR2D) were expressed in the hippocampus. Quantitative analysis of film densities indicated that in the CA1 hippocampal subfield and granule cell layer of the dentate gyrus, an increase of 48% and 27% respectively in expression of mRNA coding for the NR1 NMDA subunit is observed in schizophrenics. An increase in expression of NR2A mRNA was observed in all subfields of the hippocampus in schizophrenics (p < 0.05, Students t-test), e.g., in dentate gyrus, hybridization values in controls were 197 nCi/g and in schizophrenics were 319 nCi/g. In comparison, levels of expression of mRNAs for NR2B subunits appear to be unchanged in all hippocampal regions in schizophrenia. These findings suggest that an upregulation of NMDA receptors may occur in schizophrenia.
COMPARISON ANALYSIS OF ALU-POLYMERASE CHAIN REACTION ON SAMPLES BETWEEN PATIENTS WITH SCHIZOPHRENIA AND MATCHED CONTROLS L. Bobo*, N. Jafari, E.F. Torrey, S. Morris, P. Campbell, R. Yolken
Stanley Laboratory for the Study of Schizophrenia and Bipolar Disease, Department of Pediatrics, Johns Hopkins University, Baltimore, MD 21287-4933, USA Alu repetitive elements are dispersed throughout the genome, and may provide a means to localize markers of interest using PCR with an Alu-specific primer (Alu-PCR) and other primers of interest. Alu-PCR was performed on schizophrenic cases and matched controls, and results were analyzed by gel electrophoresis and capillary zone electrophoresis (CZE). Lymphocyte genomic DNAs from 2 homozygous twins discordant for schizophrenia and 2 normal twins were subject to PCR using an Alu primer with either a (CAG)lo or (CGG)lo primer. Alu X CGG PRC indicated that a 450 bp was present in the ill twin. Alu X Alu PCR was also performed on genomic and eDNA from temporal pole samples from normal and schizophrenic individuals. An 850 bp band was seen only from eDNA from the schizophrenic individual. In addition, Alu X CGG PCR yielded a 450 bp product from enterorhinal cortex from 2 individuals with schizophrenia but not from normal controls. PCR products are being analyzed in order to define the diseaseassociated sequences. AIu-PCR combined with CZE analyses may facilitate the localization and characterization of novel neuropsychiatric disease markers.
REVERSE TRANSCRIPTASE ACTIVITY HUMAN LYMPHOCYTES AND BRAIN TISSUE
IN
L. Jones Brando*, C. Ojeh, M.M. Herman, J.E. Kleinman, T.H. Hyde, R. Yolken
Stanley Laboratory for the Study of Schizophrenia and Bipolar Disease, Department of Pediatrics, Johns Hopkins University, Baltimore, MD 2128~4933, USA Several aspects of the natural history of schizophrenia suggest the involvement of a neurotropic retrovirus in the pathogenesis of the disease. Previous efforts to detect retrovirus growth [by testing for reverse transcriptase (RT) activity] from the lymphocytes of schizophrenic patients employed a traditional yet relatively insensitive method and yielded negative results. We have employed a recently reported ultrasensitive method of detection for particle-associated RT activity to examine basal ganglia from a brain collection which includes brains from schizophrenics as well as control individuals. This method measures RT activity using product amplification by PCR followed by detection of the target DNA in silver stained
55
polyacrylamide gels. We found RT activity in brains of schizophrenic patients as well as in individuals without a defined history of schizophrenia. Additionally we have detected activity in the supernatant of cells that have been co-cultured with lymphocytes from a cohort of monozygotic twins discordant for schizophrenia or bipolar disease. Efforts are underway to quantitate and characterize the RT activity and to determine the relationship between this activity and the disease process.
5-HT1A AND 5-HT2A RECEPTOR GENE EXPRESSION IN SCHIZOPHRENIA P.W.J. Burnet*, P.J. H a r r i s o n
Department of Neuropathology, Radcliffe Infirmary, OxJbrd, UK 5-HT (serotonin) and its receptors may be involved in schizophrenia and in the actions of neuroleptics. We show by in situ hybridization and RT-PCR that 5-HT1AR and 5-HT2AR mRNAs are reliably detectable in human brain post mortem. Knowledge of the distribution and abundance of these mRNAs extends information from autoradiography using ligands selective for each binding site ([3H]-8OHDPAT for 5-HT1AR; [3H]ketanserin for 5-HT2AR). Moreover, levels of each mRNA correlate with densities of the encoded binding sites. Using these complementary methods we have also studied the receptors and their mRNAs in the left temporal lobes of 7 schizophrenics and 7 matched controls. Neuropathological examinations of all brains were unremarkable. 5-HTIAR binding sites decreased with age but were unaltered in all subfields in schizophrenics. In contrast, in schizophrenics, 5-HT2AR binding sites were significantly reduced in parahippocampal gyrus (12.8 + 1.5 vs 20.2+ 1.4 fmol/mg tissue; p<0.01 ) but not in hippocampus (CA1). We will also present data concerning 5-HTzAR mRNA in the same cases to determine whether decreased gene expression underlies the reductions. Other investigations in progress include extending the analysis to include temporal neocortex, and measuring the same parameters in rats treated chronically with neuroleptics. The data indicate a selective loss of 5-HT2AR expression in the parahippocampal gyrus in schizophrenia. These data extend the evidence for serotonergic dysfunction in the disease and are consistent with suggestions that drugs acting upon the 5-HTEA receptor may be of value.
CHARACTERIZATION OF CALCIUM CURRENTS IN NEURONS FROM THE NUCLEUS ACCUMBENS D. Churchill, B.A. MacVicar*
Neuroseience Research Group, University of Calgary, Alberta, Canada, T2N 4N1 The nucleus accumbens (NA) Is involved in motivational and attentional processes and is implicated in schizophrenia.
Calcium (Ca) channels are important in determining the firing patterns of many neurons. Since NA Ca channels are a possible target of the modulatory action of dopamine and thus a potential secondary target of antipsychotic medications, we are studying Ca currents in these neurons using both biophysical and pharmacological methods. We have characterized, using whole-cell recording, the Ca-current subtypes present both in cultured and acutely-isolated NA neurons isolated from 5-7 day old and 4 week old rats. In both cultured and acutelyisolated neurons a rapidly inactivating low-voltage-activated current similar to other T-type Ca currents was observed along with sustained and inactivating high-voltage-activated (HVA) Ca currents. These probably correspond to N- and L-type Ca currents, respectively, since the HVA current was almost completely blocked by the combined application of nimodipine, an L-type Ca-channel blocker and ~-conotoxin-GVIA, a specific N-type Ca-channel blocker. Thus, NA neurons express distinct T-, L- and N-type Ca channels. Their modulation by dopamine and other neuromodulators is currently under investigation. Supported by CIBA-Geigy and MRC Canada.
CIRCULATING L-ARGININE METABOLITES AND PLATELET NITRIC OXIDE SYNTHASE IN SCHIZOPHRENICS I. Das*, N.S. K h a n , M. C a n t h a b o o , B.K. Puri, S.R. S o o r a n n a , J. de Belleroche, S.R. Hirsch
Departments of Psychiatry, Obstetrics & Gynaecology & Biochemistry, Charing Cross & Westminster Medical School London W6 8RP, UK Nitric oxide (NO), a neuromodulatory messenger molecule functionally coupled to the glutamatergic neurotransmitter system, is implicated in schizophrenia. Nitric oxide and Lcitrulline are produced from L-arginine by the enzyme nitric oxide synthase (NOS). L-Arginine is also the precursor of polyamines. In view of the central role of the L-arginine : nitric oxide pathway in normal and diseased states, the present study was undertaken to determine the dynamics of nitric oxide and polyamines production from their precursor L-arginine in plasma samples from drug naive patients meeting DSM IIIR criteria for schizophrenia and the NOS activity in platelets from these patients and healthy control subjects. Our results showed that the plasma citrulline/arginine ratio was higher than plasma polyamine/arginine ratio in drug naive schizophrenic patients. This ratio imbalance indicates formation of NO in favour of polyamine synthesis from any circulating Larginine. Platelet NOS activity was also significantly increased in schizophrenic subjects compared to control subjects. Elevated platelet NOS activity and plasma citrulline/arginine ratio may represent hyperactive NOS in the brain which could be involved in the pathogenesis of schizophrenia.
hippocampus are reduced by 38% and 22% respectively, (p<0.05, Students t-test). In comparison, levels of GluRA 'flop' mRNA are entirely unchanged in the dentate gyrus (hybridisation levels: schizophrenics 226n Ci/g; controls 256n Ci.g). This study provides further evidence that nonNMDA receptor abnormalities occur in the hippocampus in schizophrenia, adding weight to the glutamate theory of schizophrenia. Whether the more specific abnormality in the level of expression of the developmentally early ('flip') isoforms of glutamate receptor mRNAs is relevant to developmental theories of schizophrenia will require further study.
THE EXPRESSION OF NMDA RECEPTOR SUBUNITS IN SCHIZOPHRENIC POST-MORTEM HUMAN HIPPOCAMPUS J.P. Beckwith*, N.C. Stefanis, D.P. McLaughlin, R.W. Kerwin
Department of Neuroscience, Institute of Psychiatry, London SE5 8AF, UK Interest in EAAS developed only after the nature and function of the glutamate receptor subtypes were identified. Cloning has identified mRNA species encoding NMDA receptor subunits, termed NMDAR2A, 2B, 2C and 2D which have 55-70% identity (Monyer et al., 1992; Kutsuwada et al., 1992). These are structurally related with less than 20% peptide sequence identity (including the NMDA receptor subunit NMDAR1). Phencyclidine (PCP), a potent psychotropic drug which causes symptoms similar to schizophrenia is a noncompetitive antagonist of NMDA receptors. This has led to the development of an aetiologic hypothesis of schizophrenia which links PCP-sensitive sites on NMDA receptors to the disease. In this study, we have investigated the expression of the mRNA coding for NMDA receptor subunits (NR1, 2A, 2B, 2C and 2D) in the hippocampus of 10 schizophrenics and 10 age- and sex-matched controls post mortem. In situ hybridisation and quantitative autoradiography using specific oligonucleotide probes for each subunit revealed that all subunits (with the exception of NR2C and NR2D) were expressed in the hippocampus. Quantitative analysis of film densities indicated that in the CA1 hippocampal subfield and granule cell layer of the dentate gyrus, an increase of 48% and 27% respectively in expression of mRNA coding for the NR1 NMDA subunit is observed in schizophrenics. An increase in expression of NR2A mRNA was observed in all subfields of the hippocampus in schizophrenics (p < 0.05, Students t-test), e.g., in dentate gyrus, hybridization values in controls were 197 nCi/g and in schizophrenics were 319 nCi/g. In comparison, levels of expression of mRNAs for NR2B subunits appear to be unchanged in all hippocampal regions in schizophrenia. These findings suggest that an upregulation of NMDA receptors may occur in schizophrenia.
COMPARISON ANALYSIS OF ALU-POLYMERASE CHAIN REACTION ON SAMPLES BETWEEN PATIENTS WITH SCHIZOPHRENIA AND MATCHED CONTROLS L. Bobo*, N. Jafari, E.F. Torrey, S. Morris, P. Campbell, R. Yolken
Stanley Laboratory for the Study of Schizophrenia and Bipolar Disease, Department of Pediatrics, Johns Hopkins University, Baltimore, MD 21287-4933, USA Alu repetitive elements are dispersed throughout the genome, and may provide a means to localize markers of interest using PCR with an Alu-specific primer (Alu-PCR) and other primers of interest. Alu-PCR was performed on schizophrenic cases and matched controls, and results were analyzed by gel electrophoresis and capillary zone electrophoresis (CZE). Lymphocyte genomic DNAs from 2 homozygous twins discordant for schizophrenia and 2 normal twins were subject to PCR using an Alu primer with either a (CAG)lo or (CGG)lo primer. Alu X CGG PRC indicated that a 450 bp was present in the ill twin. Alu X Alu PCR was also performed on genomic and eDNA from temporal pole samples from normal and schizophrenic individuals. An 850 bp band was seen only from eDNA from the schizophrenic individual. In addition, Alu X CGG PCR yielded a 450 bp product from enterorhinal cortex from 2 individuals with schizophrenia but not from normal controls. PCR products are being analyzed in order to define the diseaseassociated sequences. AIu-PCR combined with CZE analyses may facilitate the localization and characterization of novel neuropsychiatric disease markers.
REVERSE TRANSCRIPTASE ACTIVITY HUMAN LYMPHOCYTES AND BRAIN TISSUE
IN
L. Jones Brando*, C. Ojeh, M.M. Herman, J.E. Kleinman, T.H. Hyde, R. Yolken
Stanley Laboratory for the Study of Schizophrenia and Bipolar Disease, Department of Pediatrics, Johns Hopkins University, Baltimore, MD 2128~4933, USA Several aspects of the natural history of schizophrenia suggest the involvement of a neurotropic retrovirus in the pathogenesis of the disease. Previous efforts to detect retrovirus growth [by testing for reverse transcriptase (RT) activity] from the lymphocytes of schizophrenic patients employed a traditional yet relatively insensitive method and yielded negative results. We have employed a recently reported ultrasensitive method of detection for particle-associated RT activity to examine basal ganglia from a brain collection which includes brains from schizophrenics as well as control individuals. This method measures RT activity using product amplification by PCR followed by detection of the target DNA in silver stained
55
polyacrylamide gels. We found RT activity in brains of schizophrenic patients as well as in individuals without a defined history of schizophrenia. Additionally we have detected activity in the supernatant of cells that have been co-cultured with lymphocytes from a cohort of monozygotic twins discordant for schizophrenia or bipolar disease. Efforts are underway to quantitate and characterize the RT activity and to determine the relationship between this activity and the disease process.
5-HT1A AND 5-HT2A RECEPTOR GENE EXPRESSION IN SCHIZOPHRENIA P.W.J. Burnet*, P.J. H a r r i s o n
Department of Neuropathology, Radcliffe Infirmary, OxJbrd, UK 5-HT (serotonin) and its receptors may be involved in schizophrenia and in the actions of neuroleptics. We show by in situ hybridization and RT-PCR that 5-HT1AR and 5-HT2AR mRNAs are reliably detectable in human brain post mortem. Knowledge of the distribution and abundance of these mRNAs extends information from autoradiography using ligands selective for each binding site ([3H]-8OHDPAT for 5-HT1AR; [3H]ketanserin for 5-HT2AR). Moreover, levels of each mRNA correlate with densities of the encoded binding sites. Using these complementary methods we have also studied the receptors and their mRNAs in the left temporal lobes of 7 schizophrenics and 7 matched controls. Neuropathological examinations of all brains were unremarkable. 5-HTIAR binding sites decreased with age but were unaltered in all subfields in schizophrenics. In contrast, in schizophrenics, 5-HT2AR binding sites were significantly reduced in parahippocampal gyrus (12.8 + 1.5 vs 20.2+ 1.4 fmol/mg tissue; p<0.01 ) but not in hippocampus (CA1). We will also present data concerning 5-HTzAR mRNA in the same cases to determine whether decreased gene expression underlies the reductions. Other investigations in progress include extending the analysis to include temporal neocortex, and measuring the same parameters in rats treated chronically with neuroleptics. The data indicate a selective loss of 5-HT2AR expression in the parahippocampal gyrus in schizophrenia. These data extend the evidence for serotonergic dysfunction in the disease and are consistent with suggestions that drugs acting upon the 5-HTEA receptor may be of value.
CHARACTERIZATION OF CALCIUM CURRENTS IN NEURONS FROM THE NUCLEUS ACCUMBENS D. Churchill, B.A. MacVicar*
Neuroseience Research Group, University of Calgary, Alberta, Canada, T2N 4N1 The nucleus accumbens (NA) Is involved in motivational and attentional processes and is implicated in schizophrenia.
Calcium (Ca) channels are important in determining the firing patterns of many neurons. Since NA Ca channels are a possible target of the modulatory action of dopamine and thus a potential secondary target of antipsychotic medications, we are studying Ca currents in these neurons using both biophysical and pharmacological methods. We have characterized, using whole-cell recording, the Ca-current subtypes present both in cultured and acutely-isolated NA neurons isolated from 5-7 day old and 4 week old rats. In both cultured and acutelyisolated neurons a rapidly inactivating low-voltage-activated current similar to other T-type Ca currents was observed along with sustained and inactivating high-voltage-activated (HVA) Ca currents. These probably correspond to N- and L-type Ca currents, respectively, since the HVA current was almost completely blocked by the combined application of nimodipine, an L-type Ca-channel blocker and ~-conotoxin-GVIA, a specific N-type Ca-channel blocker. Thus, NA neurons express distinct T-, L- and N-type Ca channels. Their modulation by dopamine and other neuromodulators is currently under investigation. Supported by CIBA-Geigy and MRC Canada.
CIRCULATING L-ARGININE METABOLITES AND PLATELET NITRIC OXIDE SYNTHASE IN SCHIZOPHRENICS I. Das*, N.S. K h a n , M. C a n t h a b o o , B.K. Puri, S.R. S o o r a n n a , J. de Belleroche, S.R. Hirsch
Departments of Psychiatry, Obstetrics & Gynaecology & Biochemistry, Charing Cross & Westminster Medical School London W6 8RP, UK Nitric oxide (NO), a neuromodulatory messenger molecule functionally coupled to the glutamatergic neurotransmitter system, is implicated in schizophrenia. Nitric oxide and Lcitrulline are produced from L-arginine by the enzyme nitric oxide synthase (NOS). L-Arginine is also the precursor of polyamines. In view of the central role of the L-arginine : nitric oxide pathway in normal and diseased states, the present study was undertaken to determine the dynamics of nitric oxide and polyamines production from their precursor L-arginine in plasma samples from drug naive patients meeting DSM IIIR criteria for schizophrenia and the NOS activity in platelets from these patients and healthy control subjects. Our results showed that the plasma citrulline/arginine ratio was higher than plasma polyamine/arginine ratio in drug naive schizophrenic patients. This ratio imbalance indicates formation of NO in favour of polyamine synthesis from any circulating Larginine. Platelet NOS activity was also significantly increased in schizophrenic subjects compared to control subjects. Elevated platelet NOS activity and plasma citrulline/arginine ratio may represent hyperactive NOS in the brain which could be involved in the pathogenesis of schizophrenia.