Takuya Oka

Role of Loop Structures of Neuropsin in the Activity of Serine Protease and Regulated Secretion

Neuropsin involved in neural plasticity in adult mouse brain is a member of the S1 (clan SA) family of serine proteases and forms characteristic surface loops surrounding the substrate-binding site (Kishi, T., Kato, M., Shimizu, T., Kato, K., Matsumoto, K., Yoshida, S., Shiosaka, S., and Hakoshima, T. (1999)J. Biol. Chem. 274, 4220–4224). Little, however, is known about the roles of these loops. Thus, the present study investigated whether surface loop structures of neuropsin were essential for the generation of enzymatic activity and/or secretion of the enzyme via a regulated secretory pathway. The loops include those stabilized by six disulfide bonds or a loop C (Gly69–Glu80) and anN-glycosylated kallikrein loop (His91–Ile103) not containing a site linked by a disulfide bond. First, among the six disulfide bonds, only SS1 in loop E (Gly142–Leu155) and SS6 in loop G (Ser185–Gly197) were necessary for the catalytic efficiency of neuropsin. Second, disruptions of loop C and the N-linked oligosaccharide chain on the kallikrein loop affected the catalytic efficiency and P2 specificity, respectively. Alternatively, disruptions of loop C and the kallikrein loop enhanced the regulated secretion, whereas there was no one disruption that inhibited the secretion, indicating that there was no critical loop required for the regulated secretion among loops surrounding the substrate-binding site.

Dendritic aberrations in the hippocampal granular layer and the amygdalohippocampal area following kindled-seizures

Amygdaloid kindling is a model of human temporal lobe epilepsy, in which excitability in limbic structures is permanently enhanced by repeated stimulations. We report here dendritic aberrations occurring in mice following kindled-seizures. Adult mice received a biphasic square wave pulse [495+/-25.5 (S.E.M.) microA 60 Hz, 200 micros duration, for 2 s] unilaterally in the basolateral amygdaloid complex once a day and mice with electrophysiologically and behaviorally verified seizures were used in the experiments. The hippocampus and amygdaloid complex contralateral to the lesions were observed by immunofluorescence histochemistry with a somatodendritic marker, microtubule-associated protein 2 (MAP2), showing that kindled-seizures caused hypertrophy of proximal dendrites in the granule cells of the dentate gyrus and in neurons of the amygdalohippocampal area. To further characterize the morphological changes of the dendrites, electron micrographic analysis was performed on the contralateral side. (1) In the granular layer of the dentate gyrus and the amygdalohippocampal area, kindled-seizures generated an increase in the number of dendrites containing polymerized microtubules and width of dendritic profiles showing the increase was in the range 0.2-3.0 and 0.2-1.4 microm, respectively. (2) In the granular layer, bundles between dendrites separated by the puncta adhaerentia increased. (3) In the granular layer, the seizure-induced dendritic aberration was more severe in the rostral than the caudal region. These results suggested that growth of dendrites with enriched-stable microtubules is part of the structural plasticity in response to seizure activity in specific areas of the adult brain.

Extracellular serine protease neuropsin (KLK8) modulates neurite outgrowth and fasciculation of mouse hippocampal neurons in culture.

A serine protease neuropsin expressed in the hippocampus of adult brain has been implicated in synaptic plasticity. We report here that endogenous neuropsin was localized extracellularly in neuronal cell bodies and their neurites in mouse hippocampal cultures. Furthermore, we found that, in cultured mouse hippocampal neurons, recombinant neuropsin enhanced neurite projection from soma after 14 h of culture and neuronal aggregation with neurite fascicles at 48 h. This suggests that neuropsin is involved in neurite outgrowth and fasciculation during the development of the nervous system.

Characterization and partial purification of neuropsin-biding protein

We demonstrated that theta burst stimulation produces NMDA receptor-dependent ‘fast’ long-term potentiation (fLTP) and NMDA receptor-independent L-type voltage gated calcium channel-dependent ‘slow’ LTP (sLTP) of field potentials in layer 4 of developing rat visual cortical slices evoked by white matter stimulation. fLTP, showing a fast time course, is independent of protein or RNA synthesis whereas sLTP, demonstrating a slow time course, depends upon the macromolecular syntheses (Kurotani et al., 1996). In the present study, we examined whether protein kinases and phosphatases are involved in the induction of these LTPs. fLTP and sLTP were studied separately in the presence of an Ltype voltage gated calcium channel antagonist, nimodipine, and an NMDA receptor antagonist, m-APV, respectively. Bath application of either a protein phosphatase 2B inhibitor, FK-506, or a protein phosphatase li2A inhibitor, Calyculin A, reduced the incidence of fLTP. In contrast, FK-506 increased the incidence of sLTP. On the other hand, a bath application of serine/threonine kinase inhibitor staurosporine reduced the incidence of sLTP in a dose-dependent manner but did not affect fLTP. Neither FK-506 or staurosporine, if applied after theta burst stimulation, showed any significant effects on either of the LTPs. These results suggest that fLTP requires the activation of protein phosphatases for induction while sLTP requires that of serineithreonine kinases instead. Supported by a Grant-in-Aid for Ecncouragement of Young Scientists (09780771).

The effect of the addition of carbohydrate to neuropsin on the enzyme activity

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 active-type recombinant neuropsin had amidolytic activities cleaving Arg-Xand Lys-Xbonds in the synthetic chromogenic substrates, and the highest specific activity was found against Boc-Val-Pro-Arg-MCA. The active-type recombinant neuropsin effectively cleaved tibronectin, 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.