Naoya Murakami

Increase in [3H]cAMP binding sites and decrease in Giα and Goα immunoreactivities in left temporal cortices from patients with schizophrenia

To search for possible alterations in second messenger systems in the temporal cortex (Brodmans area 22) of patients with schizophrenia, we measured the binding activities of [3H]adenosine 3′,5′-cyclic monophosphate ([3H]cAMP) and [3H]4β-phorbol 12,13-dibuytrate ([3H]PDBu) which can label the regulatory subunit of cAMP-dependent protein kinase (protein kinase A) and the regulatory domain of Ca2+/phospholipid-dependent protein kinase (protein kinase C), respectively. We also immunoquantified the variable subunits of guanine nucleotide binding proteins (G-proteins), using specific polyclonal antisera against Gsα, Giα and Goα. Brains were obtained at autopsy on 10 patients with schizophrenia and 10 age-matched control subjects. Representative Scatchard plots for specific [3H]cAMP bindings to the soluble fraction consisted of a single component with high affinity (Kd = 2.36 nM, Bmax = 737fmol/mg protein. Among the tested adenyl and and guanyl nucleotides, or neuroleptics, cAMP alone potently inhibited the binding (Ki = 4.95 nM). The binding sites for [3H]cAMP were discretely localized, and were in the order of: cerebral cortex = hypothalamus= amygdala > hippocampus = neostriatum = thalamus = nucleus accumbens > globus pallidus = cerebellum. Specific [3H]cAMP binding to the soluble fractions were about 30% greater in the left temporal cortices of schizophrenic patients, as compared to findings in the right side of the patients and the left side of the control subjects, no control brain showed this asymmetry. The specific [3H]PDBu binding in schizophrenic and control groups did not change. Giα and Goα immunoreactivities in the crude membranes were decreased by about 30% in the left temporal cortices of schizophrenic patients, as compared to findings in the controls, on the left side, while Gsα immunoreactivities were unchanged between the two groups, on either side. Our findings suggest the enhanced responsiveness of adenylate cyclase to receptor stimulation through relative preponderance of Gsα over Giα, which in turn would facilitate the generation of regulatory subunits of protein kinase A. The significance of decreased Goα immunoreactivities in schizophrenia is the subject of ongoing study. Our observations support the hypothesis of left (‘dominant’) temporal lobe dysfunction in schizophrenia.

Synapse-Associated Protein 90/Postsynaptic Density- 95-Associated Protein (SAPAP) is Expressed Differentially in Phencyclidine-Treated Rats and is Increased in the Nucleus Accumbens of Patients with Schizophrenia

Phencyclidine (PCP) induces a psychotomimetic state that closely resembles schizophrenia. Therefore, PCP-treated animals can provide a model for schizophrenia. Using differential display, we identified a gene regulated by the delayed action of PCP in rat nucleus accumbens (NAcs). Sequence analysis showed that the cDNA clone obtained was identical to rat synapse-associated protein 90/postsynaptic density-95-associated protein 1 (SAPAP1). Quantitative reverse transcriptase (RT)-PCR analysis showed that SAPAP1 mRNA had increased significantly in rat NAc (P<0.0001) and hippocampus (P<0.01) 24u2009h after a PCP (10u2009mg/kg) injection as compared to the controls. Immunoquantification using an anti-SAPAP1 antibody indicated that immunoreactivity for SAPAP1 increased significantly (P<0.05) in the NAcs of unmedicated patients with schizophrenia, as compared to the control subjects and medicated patients with schizophrenia. Our findings support the hypothesis that there is abnormal glutamatergic neurotransmission in schizophrenia and show evidence of abnormalities in the intracellular signal transduction via N-methyl-D-aspartate (NMDA) receptors.

Asymmetrical changes in the fodrin α subunit in the superior temporal cortices in schizophrenia

Background: We examined possible abnormalities in neural structural proteins that may underlie morphometric changes reported in the left superior temporal cortices (Brodmanns area 22) of schizophrenics. Methods: Particulate proteins of the superior temporal cortices taken at autopsy from 11 schizophrenic and 9 control brains were fractionated by gel electrophoresis. Target proteins, identified by reading their amino acid sequences, were immunoquantified using the specific antibody. Results: Amino acid sequences of the 150-kDa proteins on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, which were significantly increased on the left side of schizophrenic superior temporal cortices, revealed that they were proteolytic fragments of the α subunit of fodrin, a major cytoskeletal protein underlying the plasma membrane. Immunoquantification using the specific antibodies against α and β subunits of fodrin indicated that there exist concomitant decreases in the full-length 240-kDa form and increases in the 150-kDa, form of α fodrin with no changes of the 235-kDa form of β-fodrin in the left superior temporal cortices of the schizophrenic brains. Conclusions: The findings may be a possible molecular basis for linking morphometric changes to neurochemical pathophysiology in schizophrenia.

Potassium and calcium channel involvement in induction of long-lasting synaptic enhancement by calyculin A, a protein phosphatase inhibitor, in rat hippocampal CA1 region.

In the Schaffer collateral-CA1 pathway in rat hippocampal slices, exposure to calyculin A induced a long-lasting potentiation of the extracellular field potentials with a transient increase in glutamate release. The synaptic enhancement produced by calyculin A was blocked by staurosporine and nicardipine, but not by D,L-2-amino-5-phosphonovalerate. In dissociated CA1 pyramidal cells, calyculin A blocked the action-potential repolarization and fast after-hyperpolarization, and increased spike frequency. These results suggest that calyculin A-induced long-lasting potentiation is triggered by the blockade of Ca(2+)-activated K+ channels, the transient increase of glutamate release and the consequent activation of voltage-gated Ca2+ channels, and is maintained by increases in protein kinases activities.

Association study of a polymorphism of nonerythroid α‐spectrin gene with schizophrenia

The specificity of cytoarchitectural abnormalities in limbic structures of patients with schizophrenia and their contributions towards the etiology of schizophrenia remain unknown. We have recently reported an increased breakdown of nonerythroid alpha-spectrin (fodrin), a major component of neuronal cytoskeletal proteins, in schizophrenic left superior temporal cortices [Kitamura et al., 1998: Biol Psychiatry 43:254-262], suggesting that polymorphisms of the alpha-spectrin gene might contribute to the vulnerability to schizophrenia. We screened for genetic variations associated with schizophrenia through the C-terminus sequences of the human nonerythroid alpha-spectrin gene (SPTAN1) spanning two EF-hands and also tested a possible contribution of the polymorphism to the development of schizophrenia by an association study. We found a polymorphic region of an intron located in the second EF-hand of SPTAN1 gene. There was no significant difference between patients with schizophrenia and controls in allele frequencies or genotype distribution. There is evidence that the Psh BI SPTAN1 gene polymorphism does not play a major role in the genetic component of schizophrenia.

820 Translocation of protein kinase C subspecies in living cells — Visualization using fusion protein with green fluorescent protein (GFP)

NORIFUMI YAMASHITA’, CLAUS W. HEIZMANN”, TOSHIO KOSAKA3 The distribution of a specific calcium binding protein of the SlOO protein family, SlOOA6 ( Calcyclin ), in the rat nervous system was examined, using antisera against human recombinant SlOOA6. The main SlOO A6 immunoreactive elements were 1) neuronal somata and dendrites in some specific regions of the limbic system, 2) olfactory nerve fibers and axon terminals in the olfactory bulb, 3) some tracts of the hindbrain and spinal cord, as well as some sensory neurons of their origins ( in dorsal root and trigemminal ganglia ), 4) some glial cells in the white matter of the forebrain and around the ventricles, 5) some ependymal cells, especially in the spinal cord, and 6) Schwann cells. Thus, the SlOOA6 immunoreactivity was contained in only some subpopulations of neurons, glial cells and ependimal cells. Furthermore, our preliminary observations of the mouse nervous system indicated prominent species differences in the SlOOA6 immunoreactivity.

504 Regulation of serotonin transporter (set) activity by phosphorylation /dephosphorylation norio sakai

A considerable number of pharmacological, biochemical and structural studies have strengthened the supposition that serotonin (%-IT) is a neurotransmitter in the intestine, however the 5HTimmunoreactive enteric neurons have only demonstrated in the guinea pig but not in the rat. In the present study, the SIlTcontaining nerve cells and axons were demonstrated immunohistochemically in the rat gastrointestinal(G1) tracts. %-IT-containing nerve cells were localized at the myenteric plexus of cardia, antrum, duodenum, jujunum, ileum and colon of rats. The cell bodies had short and broad processes and projected very long axons spread widely in the wall of GI tracts. In the submucosa, the SHT-immunoreactive fibers were entwined around the blood vessels and they projected into the lamina propria of the mucosa.

Alteration of [3H] Ins (1,4,5) P3 and [3H] PDBu Binding Sites in Brains of Patients with Parkinson’s Disease and Multiple System Atrophy

The roles of membrane inositol phospholipid turnover in intracellular signal transduction have been vigorously researched. Stimulation of several receptors by neurotransmitters, hormones, and growth factors leads to accelerated turnover of polyphosphoinositides. Hydrolysis of phosphatidylinositol 4,5-bisphosphate by activated phospholipase C generates inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and 1,2-diacylglycerol, both of which act as second messengers. The former increases intracellular Ca2+ and the latter activates protein kinase C (PKC) (Nishizuka, 1986).