Yoshiharu Momota

Characterization of recombinant and brain neuropsin, a plasticity-related serine protease.

Activity-dependent changes in neuropsin gene expression in the hippocampus implies an involvement of neuropsin in neural plasticity. Since the deduced amino acid sequence of the gene contained the complete triplet (His-Asp-Ser) of the serine protease domain, the protein was postulated to have proteolytic activity. Recombinant full-length neuropsin produced in the baculovirus/insect cell system was enzymatically inactive but was readily converted to active enzyme by endoprotease processing. The activational processing of prototype neuropsin involved the specific cleavage of the Lys32-Ile33 bond near its N terminus. Native neuropsin that was purified with a purity of 1,100-fold from mouse brain had enzymatic characteristics identical to those of active-type recombinant neuropsin. Both brain and recombinant neuropsin had amidolytic activities cleaving Arg-X and Lys-X bonds in the synthetic chromogenic substrates, and the highest specific activity was found against Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. The active-type recombinant neuropsin effectively cleaved fibronectin, an extracellular matrix protein. Taken together, these results indicate that this protease, which is enzymatically novel, has significant limbic effects by changing the extracellular matrix environment.

Blockade of neuropsin, a serine protease, ameliorates kindling epilepsy

The behavioural and electrographical abnormalities associated with seizures in epileptic (kindled) mice correspond with those of human epilepsy. In kindled mice, neuropsin was markedly increased in the hippocampus and cerebral cortices. A single intraventricular injection of monoclonal antibodies specific to neuropsin reduced or eliminated the epileptic pattern noted on electroencephalograms and, as a result markedly inhibited the progression of kindling. Therefore, neuropsin appears to be a key protein controlling pathogenic events in the hippocampus, and thus neuropsin inhibitors might be useful for treatment of epilepsy.

Kindling induces neuropsin mRNA in the mouse brain

Neuropsin mRNA expression was analyzed and mapped in the mouse brains after kindling epileptogenesis by using in situ hybridization histochemistry. Dynamic increases of the neuropsin mRNA were observed in the layer II of prelimbic, somatosensory, auditory, perirhinal, entorhinal, and piriform cortices in an activity-dependent manner, though no neuropsin gene was expressed in these areas in control mice. In addition to the confirmation of our previous studies showing increases of mRNA in the hippocampus and amygdaloid complex, there were also remarkable increases of the neuropsin mRNA in the limbic areas, such as the accessory olfactory nucleus, the medial and lateral septal nucleus, the nucleus of diagonal band, the substantia innominata and the zona incerta. The dynamic activity-dependent changes of the gene expression and the site-specificity of neuropsin localization are suggesting that this molecule is implicated in cortical- and limbic-specific neuronal reorganization.

Ontogeny of neuropsin mRNA expression in the mouse brain

In our previous study, we found a novel gene encoding a serine protease termed neuropsin (NP) which exhibited activity-dependent gene expression or repression in the mouse hippocampus (Chen et al., 1995). In the present study, we examined the ontogeny of NP mRNA expression by in situ hybridization in the brain. Weak hybridization signals were also observed in the choroid plexus at this stage in addition to neuronal labeling. The signals continued to show this localization pattern until postnatal day 12. After embryonic day 18, the number of hybridization signals localized in the neurons of the forebrain limbic area were more predominant than those in the lower brainstem. NP gene expression spread in the anterior olfactory nucleus, hippocampus, septal nuclei, diagonal band of Broca, amygdala and limbic cortex successively from early embryonic to adult stage, though signals in the other brain regions were gradually decreased after birth. Thus, the widespread localization and two types of expression pattern, constitutive or transient, suggest that NP is a multiple functional protein involved in development, neuronal plasticity and cerebrospinal fluid production.

Expression of Neuropsin mRNA in the Mouse Embryo and the Pregnant Uterus

Neuropsin is a novel serine protease whose mRNA is expressed in the mouse central nervous system. We examined the expression of neuropsin mRNA during embryonic development using Northern and in situ hybridization in non-neural tissues. The pregnant uterus showed strong expression of neuropsin mRNA, whereas the nonpregnant uterus did not express this mRNA. Expression was first detected in the primary decidual zone at 5.5 days post coitum and was maximized at 10 days post coitum, decreasing remarkably thereafter. During mouse organogenesis, neuropsin expression was observed in the developing heart, lung, thymus, pituitary, choroid plexus, and epithelial linings of the skin, oral cavity, tongue, esophagus, and forestomach. In adult mouse organs, neuropsin mRNA was expressed in epithelial tissues covered by keratinocytes with moderate density, whereas low expression was observed in lung, thymus, and spleen. Neuropsin mRNA expression in developing organs and adult keratinocytes suggests that neuropsin is associated with extracellular matrix modifications and cell migrations.

Basal magnocellular and pontine cholinergic neurons coexpress FGF receptor mRNA

By in situ hybridization histochemistry, fibroblast growth factor receptor gene (flg)-expressing neurons were newly identified in the basal magnocellular nuclei (the vertical and horizontal limbs of the diagonal band, and Meynerts nucleus). The present study also confirmed flg localization in the laterodorsal tegmental nucleus and the pedunculopontine tegmental nucleus of the pons. Immuno- and in situ hybridization histochemistry on the same sections demonstrated that choline acetyltransferase and flg were colocalized in single neurons of the diagonal band, Meynerts nucleus and the pontine tegmental areas. The results suggest that a significant number of the basal magnocellular and a majority of the mesopontine cholinergic neurons are directly affected by fibroblast growth factors (FGF) via FGF receptor gene.

Decidualization Induces the Expression and Activation of an Extracellular Protease Neuropsin in Mouse Uterus

Abstract Uterine decidualization is accompanied by the remodeling of the cell-matrix and cell-cell interactions around the endometrial stromal cells to allow an appropriate invasion of trophoblasts. This remodeling is thought to require the proteolysis of extracellular matrix proteins or cell adhesion molecules; however, the molecular mechanism remains poorly understood. In this study, decidualization induced the expression and activation of an extracellular serine protease neuropsin in the mouse uterus. Although nonpregnant uteri contained little neuropsin, the protein content and enzymatic activity increased markedly and peaked at the midgestational period in pregnant uteri. Neuropsin expression and activity was also upregulated in artificially induced deciduomata but not in nondecidualized pseudopregnant uteri. Neuropsin is the first extracellular protease to show the evident induction of expression and activity by decidualization and might contribute to the remodeling of extracellular components after decidualization.

1323 The physiological role of a serine protease, neuropsin, on the neural plasticity in hippocampus

Mayumi Nishi, Mitsuhiro Kawata Brain-derived neurotrophic factor (BDNF) is known to exert a multitude of trophic effects on diverse neuronal populations, including central serotonergic(5HT) neurons. In vivo, the action of BDNF persists for many weeks after the end of treatment,which may involve activation of glial cells by BDNF. BDNF affects 5HT neurons through trkB receptor and glial cells through truncated trkB receptor. Glial cells release SlOOb which has trophic effects on 5HT neurons. Are some of the trophic effects by BDNF mediated via SlOOb? The effects of BDNF and SlOOb on various properties of 5HT neurons were investigated in fetal rat raphe primary cultures. We also examined the effects of BDNF on SlOOb expression. Finally the effects of anti-SlOOb antibody and trkB tyrosine kinase inhibitor were analyzed. Our results suggest that BDNF and SlOOb are active growth factors on 5HT neurons and BDNF may require trophic interactions with SlOOb to exert its full neurotrophic potential.

Ontogeny of plasminogen activators in the mouse brain

In mammals, two isozymes of topoisomerase II (Topo II) have been identified. Topo IIcl plays a key role in DNA replication. Physiological functions of Topo IIP are still unclear and its expression level is independent of cellular proliferation profiles. We have reported previously that expression of Topo IIP transiently increased in developing cerebellum. Analysis of genomic regions recognized by Topo I@ would certainly facilitate the understanding of its functional significance. To clone the genomic regions targeted by Topo I@, libraries were constructed from DNA fragments recovered by Topo IIP-specific antibodies from the rat cerebellum (10 days after birth) which had been incubated with VP16. Topo II inhibitors such as VP16 are well known to stabilize the covalent complex between Topo II and DNA (cleavable complex). Nucleotide sequencing of 42 clones at the both ends showed that two-thirds of the clones were multicopy repetitive sequences such as LINE (long interspersed nuclear sequence), satellite I and ID. The remaining one-third appeared to be single copy sequences which were derived mostly from noncoding regions. Detailed mapping of the Topo II cleavage sites is under progress.

1307 Neuropsin is involved in epileptogenesis

Masumi Wakita It has been previously reported that bilateral damage in the auditory cortex resulted in a severe deficit in the acquisition of the auditory pattern discrimination if it was incurred at adulthood while animals with comparative lesion on the day of birth could learn the discrimination. In the present report, a critical period for sparing of auditory pattern discrimination was surveyed behaviorally in rats sustaining neonatal lesion in the auditory cortex. Consequently, rats with such lesions before weaning could learn the tasks as intact animals. On the contrary, rats who sustained comparative lesions after weaning could not learn this discrimination. These findings imply that auditory pattern discrimination was spared from early brain damage if the brain is injured before cortical function and circuitry are under development.