Yasuo Kajimoto

Differential changes in serotonin 5-HT1A and 5-HT2 receptor binding in patients with chronic schizophrenia.

Serotonin 5-HT1A and 5-HT2 receptors were examined in the postmortem brains of controls and patients with chronic schizophrenia. In the prefrontal cortex from patients with schizophrenia, 5-HT1A receptor binding was increased, while 5-HT2 receptor binding was decreased, when compared to controls. The increased 5-HT1A receptor binding or the decreased 5-HT2 receptor binding was observed in both the patients who had been medicated with neuroleptics at time of death and those who had not, at least 2 months prior to death. Thus, abnormalities of 5-HT receptor subtypes seem to exist in the brains of patients with chronic schizophrenia. 5-HT related agents might be beneficial for the treatment of schizophrenia.

Molecular Cloning of Two Additional Members of the Neural Visinin‐Like Ca2+‐Binding Protein Gene Family

Abstract: We have isolated a rat cDNA clone encoding a neural visinin‐like Ca2+‐binding protein (NVP), which we designate NVP‐1. To identify additional molecular forms of NVP, a rat brain cDNA library was screened for their presence using an NVP‐1 cDNA probe under low‐stringency hybridization conditions. Two types of cDNA clones encoding structurally related proteins, designated NVP‐2 and NVP‐3, have been isolated. The deduced amino acid sequences of NVP‐2 and NVP‐3 are 89.0% and 68.6% identical to that of NVP‐1, respectively, and contain consensus sequences for EF‐hand Ca2+‐binding sites. Northern blot analysis shows that NVP‐1, NVP‐2, and NVP‐3 mRNAs are most highly expressed in brain and are differentially expressed in various regions of rat brain. These results suggest that NVP‐2 and NVP‐3 are additional members of the NVP gene family.

Decreased histamine H1 receptors in the frontal cortex of brains from patients with chronic schizophrenia

Involvement of histamine H1 receptor in the brains of schizophrenic patients was investigated using 3H-mepyramine as a ligand. The specific 3H-mepyramine binding in the frontal cortex was saturable with the dissociation constant (Kd) of about 0.6 nM and the maximum number of binding sites (Bmax) of 64 fmol/mg protein. Specific H1 antagonists, mepyramine (Ki = 1.4 nM), promethazine (Ki = 1.4 nM), diphenylpyraline (Ki = 4.1 nM), triprolidine (Ki = 5.3 nM), diphenylhydramine (Ki = 35 nM), but not the specific H2 antagonist, cimetidine (Ki greater than 10(5) nM), strongly inhibited the 3H-mepyramine binding. Regional distribution of the specific 3H-mepyramine binding was in the order of: frontal cortex greater than hippocampus greater than cerebellum greater than hypothalamus greater than thalamus, putamen, and pallidum. The specific 3H-mepyramine binding in schizophrenic brains was reduced by 56% in the frontal cortex. Representative Scatchard analyses of the specific 3H-mepyramine binding revealed changes resulting from a decrease in receptor density but not in receptor affinity. Down-regulation of the histamine H1 receptor in the frontal cortex may be involved in the pathophysiology of schizophrenia.

cDNA cloning of a neural visinin-like Ca2+-binding protein

A 21,000-dalton Ca(2+)-binding protein (Walsh, M.P., Valentine, K.A., Ngai, P.K., Carruthers, C.A., and Hollengerg, M.D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca(2+)-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca(2+)-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain.

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) 24 h after a PCP (10 mg/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.

Mutation and association analysis of the DAP‐1 gene with schizophrenia

Glutamate dysfunction has been hypothesized to be involved in the pathophysiology of schizophrenia. The human homolog of Drosophila discs large protein (hDLG) and post‐synaptic density‐95‐associated protein‐1 (DAP‐1) is one of the major proteins that are involved in intracellular signal transduction via N‐methyl‐d‐aspartate receptors. In the present study 33 Japanese patients with schizophrenia were screened for mutations in the DAP‐1 gene. A single nucleotide polymorphism was identified in the DAP‐1 gene (1618A/G). A case–control study using a larger sample of unrelated patients and controls did not reveal a significant association between this polymorphism and schizophrenia. The results do not provide evidence that the DAP‐1 gene is involved in vulnerability to schizophrenia.

Delayed changes in neural visinin-like calcium-binding protein gene expression caused by acute phencyclidine administration

Phencyclidine (PCP) induces a psychotomimetic state that closely resembles schizophrenia, and PCP-treated animals can serve as a model for schizophrenia. The effects of PCP on the gene expression of NVP-1, a novel Ca2+-binding protein, were studied in rats. After 24 hours, the NVP-1 mRNA level in the nucleus accumbens showed a significant decrease of 42%. This result suggests that alterations in Ca2+-binding protein may be involved in the pathology of PCP-induced psychosis and, presumably, schizophrenia.

Increased glutamate transporter (GLT-1) immunoreactivity in the rat striatum after repeated intermittent administration of methamphetamine.

The corticofugal glutamatergic system, which is linked to mesencephalic dopaminergic neurons both anatomically and functionally,l is now thought to be associated with schizophrenia.* Repeated intermittent administration of a psychostimulant methamphetamine (MAP) to rats produces a progressive and enduring enhancement in stereotyped behavior such as stereotyped sniffing and repetitive head movement, i .e., behavioral sensitization. A sensitized rat can serve as a model of lasting susceptibility to exacerbation of MAP-induced delusional psychosis in nonschizophrenic subjects as well as a model of vulnerability to relapse of schizophrenic sympto rn~ .~ Most attempts to identify a neural correlate of behavioral sensitization have focused on the mesencephalic dopaminergic system, but few have focused on the glutamatergic system. Recently, four distinct glutamate transporters, EAATl (GLAST),4 EAAT2 (GLT-l),S EAAT3 (EAAC1),6 and EAAT4’ were cloned and detected in the brain (TABLE 1). Among these, GLT-1 is specific to brain and is localized to astroglia. Any treatment that modulates glutamate release should induce similar changes in glial responses through regulation of glutamate transporters including GLT1. Accordingly, the present study was conducted to examine the effect of a single or repeated intermittent administration of MAP on glial glutamate transporter responses in the rat brain, using GLT-1 immunoreactivity as a probe.