Reduction in BDNF from Inefficient Precursor Conversion Influences Nest Building and Promotes Depressive-Like Behavior in Mice.

We generated a knock-in mouse line in which the gene encoding brain-derived neurotrophic factor (Bdnf) was replaced with a sequence for proBDNF containing human single nucleotide polymorphisms encoding arginines proximal to the cleavage site (R125M and R127L). The ratio of the mature form of BDNF (mBDNF) to precursor BDNF (proBDNF) in hippocampal tissue lysates was decreased in a manner dependent on the number of copies of the mutant gene, indicating that the mutations inhibited proteolytic conversion of proBDNF into mBDNF. Although homozygous mice had a proBDNF/mBDNF ratio of ~9:1, they survived until adulthood. The levels of mBDNF were reduced by 57% in heterozygous mutant mice, which exhibited a depressive-like behavior in the tail suspension test and weight gain when housed in social isolation, showing that impaired proBDNF cleavage contributes to stress-induced depressive-like phenotypes. Furthermore, socially isolated heterozygous mice displayed a pronounced deficit in daily nest-building behaviors. These findings suggest that the decreased production of mBDNF by impaired proBDNF cleavage disturbs daily activities in mice.

BDNF pro-peptide actions facilitate hippocampal LTD and are altered by the common BDNF polymorphism Val66Met.

Most growth factors are initially synthesized as precursor proteins and subsequently processed into their mature form by proteolytic cleavage, resulting in simultaneous removal of a pro-peptide. However, compared with that of mature form, the biological role of the pro-peptide is poorly understood. Here, we investigated the biological role of the pro-peptide of brain-derived neurotrophic factor (BDNF) and first showed that the pro-peptide is expressed and secreted in hippocampal tissues and cultures, respectively. Interestingly, we found that the BDNF pro-peptide directly facilitates hippocampal long-term depression (LTD), requiring the activation of GluN2B-containing NMDA receptors and the pan-neurotrophin receptor p75(NTR). The BDNF pro-peptide also enhances NMDA-induced α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor endocytosis, a mechanism crucial for LTD expression. Thus, the BDNF pro-peptide is involved in synaptic plasticity that regulates a mechanism responsible for promoting LTD. The well-known BDNF polymorphism valine for methionine at amino acid position 66 (Val66Met) affects human memory function. Here, the BDNF pro-peptide with Met mutation completely inhibits hippocampal LTD. These findings demonstrate functional roles for the BDNF pro-peptide and a naturally occurring human BDNF polymorphism in hippocampal synaptic depression.

BDNF Binds Its Pro-Peptide with High Affinity and the Common Val66Met Polymorphism Attenuates the Interaction.

Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function and assists the folding of the BDNF protein. However, our results suggest a new role for the BDNF pro-domain (or pro-peptide) following proteolytic cleave of precursor BDNF, and provide insight into the Val66Met polymorphism.

Neurobiological actions by three distinct subtypes of brain-derived neurotrophic factor: Multi-ligand model of growth factor signaling.

Brain-derived neurotrophic factor (BDNF) is one of the most active members of the neurotrophin family. BDNF not only regulates neuronal survival and differentiation, but also functions in activity-dependent plasticity processes such as long-term potentiation (LTP), long-term depression (LTD), learning, and memory. Like other growth factors, BDNF is produced by molecular and cellular mechanisms including transcription and translation, and functions as a bioactive molecule in the nervous system. Among these mechanisms, a particular post-translational mechanism, namely the conversion of precursor BDNF into mature BDNF by proteolytic cleavage, was not fully understood. In this review, we discuss the manner through which this post-translational mechanism alters the biological actions of BDNF protein. In addition to the initially elucidated findings on BDNF, the biological roles of precursor BDNF and the BDNF pro-peptide, especially synaptic plasticity, will be extensively discussed. Recent findings on the BDNF pro-peptide will provide new insights for understanding the mechanisms of action of the pro-peptides of growth factors.

Ganglioside contained in the neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) fraction prepared from the detergent-resistant membrane microdomain of rat brain inhibits the phosphatase activity of calcineurin.

Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant membrane microdomain fraction (DRM). Neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) is one of the major protein components of neuronal DRM. To determine the cellular function of NAP-22, interacting proteins were screened with an immunoprecipitation assay, and calcineurin (CaN) was detected. Further studies with NAP-22 prepared from DRM and CaN expressed in bacteria showed the binding of these proteins and a dose-dependent inhibitory effect of the NAP-22 fraction on the phosphatase activity of CaN. On the other hand, NAP-22 expressed in bacteria showed low binding to CaN and a weak inhibitory effect on phosphatase activity. To solve this discrepancy, identification of a nonprotein component that modulates CaN activity in the DRM-derived NAP-22 fraction was attempted. After lyophilization, a lipid fraction was extracted with chloroform/methanol. The lipid fraction showed an inhibitory effect on CaN without NAP-22, and further fractionation of the extract with thin-layer chromatography showed the presence of several lipid bands having an inhibitory effect on CaN. The mobility of these bands coincided with that of authentic ganglioside (GM1a, GD1a, GD1b, and GT1b), and authentic ganglioside showed an inhibitory effect on CaN. Treatment of lipid with endoglycoceramidase, which degrades ganglioside to glycochain and ceramide, caused a diminution of the inhibitory effect. These results show that DRM-derived NAP-22 binds several lipids, including ganglioside, and that ganglioside inhibits the phosphatase activity of CaN.

Structures of septin filaments prepared from rat brain and expressed in bacteria.

Septin forms a conserved family of cytoskeletal GTP-binding proteins that have diverse roles in protein scaffolding, vesicle trafficking and cytokinesis. There are 14 mammalian septin isoforms and these isoforms assemble into hetero-oligomeric rod-shaped complexes and these short filaments are the basal units to construct higher-order structures such as longer filaments, rings, gauzes or hourglasses. Septin expressed in a eukaryotic expression system forms various structures such as bundles, sheets, helixes, and rings. Septin expressed in bacteria formed hexameric short filaments and single or parallel long filaments, but no such higher order structures were observed so far. In a previous study, we showed maturation-dependent localization of septin isoforms to the lipid raft fraction of rat brain. In this study, we attempted further purification of raft-localized septin isoforms. Repeated cycles of extraction with high MgCl(2) solution and precipitation under low ionic solution were combined with several column procedures. The obtained fraction contained several septin isoforms and showed rings of bundled filaments with a diameter of ~0.4µm. Several non-septin proteins were also detected in the fraction. We also attempted expression of septin isoforms in bacteria and found that the expressed septin complexes formed bundles of filaments. In addition to linear and curled filaments, circular bundles of thin filaments with a diameter of ~0.6µm were also observed. These results suggest that the curvature of the bundles of septin filaments may be regulated by the regulatory factor(s) in the lipid raft.

Inhibitory effect of NAP-22 on the phosphatase activity of synaptojanin-1.

Endocytosis of the synaptic vesicle is a complicated process, in which many proteins and lipids participate. Phosphatidylinositol 4,5-bisphosphate (PIP(2) ) plays important roles in the process, and the dynamic regulation of this lipid is one of the key events. Synaptojanin is a PIP(2) phosphatase, and dephosphorylation of PIP(2) of the clathrin coated-vesicle results in the uncoating of the vesicle. NAP-22 is one of the major proteins of the neuronal detergent-resistant membrane microdomain and localizes in both the presynaptic plasma membrane and the synaptic vesicle. To elucidate the role of NAP-22 in synaptic function, a screening of the NAP-22 binding proteins through pull-down assay was performed. In addition to CapZ protein, synaptojanin-1 was detected by LC-MS/MS, and Western blotting using antisynaptojanin-1 confirmed this result. The interaction seems to be important in the course of synaptic vesicle endocytosis, because NAP-22 inhibited the phosphatase activity of synaptojanin in a dose-dependent manner. The inhibitory region for 5-phosphatase and the binding region for PIP(2) overlapped in the amino acid sequence of NAP-22, so elucidation of the regulatory mechanism of the PIP(2) binding ability of NAP-22 could be important in understanding the membrane dynamics at the presynaptic region.

Biochemical interaction of an actin-capping protein, CapZ, with NAP-22.

NAP-22 is a neuronal protein localized in the presynaptic membrane and synaptic vesicles and recovered in a Triton-insoluble low-density microdomain fraction after biochemical fractionation of the synaptic plasma membrane. NAP-22 organizes membrane microdomains through binding to membrane lipids such as cholesterol, phosphatidylethanolamine, and phosphatidylinositol 4,5-bisphosphate. In this study, NAP-22-binding proteins were screened through the pull-down assay using brain-derived NAP-22 bound to Sepharose 4B. An actin-capping protein, CapZ, was identified in the precipitate through mass spectrometry and Western blotting. CapZ was then expressed in E. coli and the purified protein-bound NAP-22 directly. Because bacterially expressed NAP-22 bound CapZ, it was determined that the N-terminal myristoyl moiety of NAP-22 is not necessary for the binding. The binding of NAP-22 showed no effect on the actin nucleation activity of CapZ measured with centrifugation and viscometric assays. Hence, the CapZ-NAP-22 complex could work as the nucleation site of actin polymerization or as the actin filament-anchoring site on the membrane microdomain.

Balanced expression of various TrkB receptor isoforms from the Ntrk2 gene locus in the mouse nervous system.

A brain-derived neurotrophic factor (BDNF) receptor TrkB involves three spliced variants, namely the tyrosine kinase domain (TK) intact (+) and two TK(-) isoforms T1 and T2, yet their precise roles are largely unknown. Here we extensively map the mRNA expression patterns of BDNF and TrkB variants, further to gain insights in TK(-) specific functions during mouse development. Consequently, we found that TK(+), T1 and T2 were expressed in distinct regions of the mouse nervous system at the embryonic and postnatal stages, implicating separable functions of TK(-) forms. Additionally we uncovered five expressed segments in the intron between T2 and T1 specific exons, and one of these segments was revealed to code novel TK(-) receptors with unique responsiveness in vitro. These results suggest dynamic modes of expression from the Ntrk2 gene locus and multiple roles of TK(-) forms in the developing mouse nervous system.

Biochemical characterization of membrane-associated septin from rat brain.

Within the cell membrane there exist various microdomains (lipid rafts) in which specific lipids and proteins are assembled and these microdomains are recovered in the detergent-resistant low-density membrane fraction (DRM). Septin is a novel GTP-binding, cytoskeletal protein having various isoforms that assemble into homo- and heterooligomers and filaments. As the localization of septin 3 in DRM was found through a proteomics analysis of brain-derived DRM, the presence of other septin isoforms in DRM was studied. Western blotting analysis showed maturation-dependent enrichment of several septin isoforms in DRM prepared from synaptic plasma membrane (SPM). These isoforms were solubilized with high MgCl2 solution and recovered as the precipitate after dialysis to low ionic solution. Three times cycling of the extraction-dialysis process resulted in the partial purification of septin complex and electron microscopic observation of this fraction revealed rod-like structures in which building units were observed. The presence of heterooligomers was shown with western blotting after the separation of the MgCl2 extract with blue-native polyacrylamide gel electrophoresis. Immunoprecipitation assay using monoclonal anti-septin11 antibody also showed the presence of heterooligomers. These results show that septin localizes in the membrane microdomains of the SPM in adult brain and may have important roles in the membrane dynamics of neurons.

Morphological analysis on the distribution of membrane lipids and a membrane protein, NAP-22, during neuronal development in vitro.

Specific localization of membrane proteins based on the interactions with membrane lipids at various microdomains (MDs) is under active investigation, since the elucidation of the molecular mechanism of the interactions could reveal a novel concept of cell organization. Due to the strong interactions not only between lipids but also between lipids and proteins, these MDs are considered to be recovered in a detergent-resistant low-density membrane fraction (DRM) after detergent extraction and density-gradient centrifugation. Neurons take well-developed membrane systems during maturation and specific localization of various membrane components, not only proteins but also lipids, is essential for the establishment of the nervous system. In previous studies, we showed that NAP-22 is a major protein of neuronal DRM and binds liposomes in a cholesterol-dependent manner. In this study, we analyzed the localization of membrane lipids during neuronal maturation in vitro and compared their distribution with that of NAP-22. In an attempt to detect DRM-associated lipids, we observed the staining patterns of neurons treated with Triton-X-100 at 4 degrees C before fixation. Our results showed the less staining patterns of cholesterol and sphingomyelin at the axonal tips and a different staining pattern of two gangliosides, GM(1) and GD(3). The enrichment of cholesterol at the NAP-22 localizing spots was observed after the treatment of the detergent. Since the application of maitotoxin, a calcium ion channel, caused the diminution of NAP-22 and cholesterol positive spots, the distribution of these molecules are considered under the calcium regulation.

Molecular interaction of neurocalcin alpha with alsin (ALS2).

Membrane microdomains (MDs), or lipid rafts, are recently identified dynamic membrane domains on which various signal-transductions are performed. Intracellular Ca(2+)-binding proteins participate in the Ca(2+) signaling through interaction with various proteins. Neurocalcin alpha (NCalpha) is a member of neuronal calcium sensor (NCS) protein family and shows Ca(2+)-dependent binding to the cell membrane through N-terminal myristoyl moiety. Since NCalpha was identified as a Ca(2+)-dependent binding protein to neuronal MDs, its binding proteins may participate in the signal-transduction on the MDs. In an immunoprecipitate using anti-NCalpha antibody, alsin (ALS2), a protein product of one of the responsive genes for amyotrophic lateral sclerosis, was detected through LC-MS/MS. Specific antibody to alsin was produced and immunoprecipitation using this antibody showed co-sedimentation of NCalpha. Some part of alsin bound to brain-derived MD fraction in the presence of Ca(2+) ions and eluted out by the chelation of Ca(2+) ions, as in the case of NCalpha. Immunostaining of cultured neurons showed broad distribution of alsin and NCalpha, and membrane association of these proteins were increased through Ca(2+) loading by maitotoxin. These results suggest that alsin binds cell membrane in a Ca(2+)-dependent manner through NCalpha and regulates membrane dynamics.

Myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction prepared from rat pituitary.

Pituitary gland is a well-known endocrine tissue. The hypothalamo-neurohypophysial system, containing arginine vasopressin and oxytocin, shows a reversible morphological reorganization of both neurons and glial cells during chronic physiological stimulations. Since many signal transducing and cell adhesion molecules (CAMs) are recovered in membrane microdomain (MD) fractions, MDs are considered as signaling platforms of cells. In order to know the molecular background for these endocrine systems, we characterized MD-components derived from rat pituitary and found specific enrichment of several proteins in the fraction. One of them was identified as myelin protein zero (P0) with mass analysis and this result was further confirmed by a result that a specific antibody to this protein reacted to the authentic P0 protein in the myelin fraction of rat sciatic nerve. P0 is one of type-I transmembrane CAMs and a major structural component of mammalian peripheral nerve myelin. In mammals, expression of P0 has been considered to be restricted to peripheral nervous system. This result however indicates that P0 expresses more widely and its enrichment in the MD-fraction from rat pituitary suggests the participation in cell-cell communications.

Ouabain-induced isoform-specific localization change of the Na+, K+-ATPase alpha subunit in the synaptic plasma membrane of rat brain.

Na+, K+-ATPase is one of major membrane proteins that has two subunits, alpha and beta. The alpha subunit has the ATPase activity and the ouabain binding site. Among four isoforms of the alpha subunit, expression of alpha1, alpha2, and alpha3, but not alpha4, is observed in matured rat brain. Ouabain is one of cardiac glycosides, and endogenous ouabain-like compounds have been recognized as a new class of steroid hormone. The alpha subunit is considered as their endogenous receptor. Recent studies envisaged the importance of membrane microdomains (MDs) as signaling platforms, which are recovered as a detergent-resistant membrane microdomain fraction (DRM). Although this ATPase has been considered as a non-DRM protein, some amount of the alpha subunit was found to be a component of the DRM prepared from the synaptic plasma membrane fraction (SPM) of rat brain. Ouabain treatment increased the amount of alpha3 isoform, but not alpha1, in the DRM derived from synaptosome fraction and SPM. These results suggest that the localization of the alpha subunit of Na+, K+-ATPase is regulated with isoform-specific mechanisms and the physiological importance of DRM in the signal transduction of the endogenous ouabain-like steroid hormone in neurons.

A truncated tropomyosin-related kinase B receptor, T1, regulates glial cell morphology via Rho GDP dissociation inhibitor 1.

Through tropomyosin-related kinase B (TrkB) receptors, brain-derived neurotrophic factor (BDNF) performs many biological functions such as neural survival, differentiation, and plasticity. T1, an isoform of TrkB receptors that lacks a tyrosine kinase, predominates in the adult mammalian CNS, yet its role remains controversial. In this study, to examine whether T1 transduces a signal and to determine its function, we first performed an affinity purification of T1-binding protein with the T1-specific C-terminal peptide and identified Rho GDP dissociation inhibitor 1 (GDI1), a GDP dissociation inhibitor of Rho small G-proteins, as a signaling protein directly associated with T1. The binding of BDNF to T1 caused Rho GDI1 to dissociate from the C-terminal tail of T1. Astrocytes cultured for 30 d expressed only endogenous T1 among the BDNF receptors. In 30 d cultured astrocytes, Rho GDI1, when dissociated in a BDNF-dependent manner, controlled the activities of the Rho GTPases, which resulted in rapid changes in astrocytic morphology. Furthermore, using 2 d cultured astrocytes that were transfected with T1, a T1 deletion mutant, or cyan fluorescent protein fusion protein of the T1-specific C-terminal sequence, we demonstrated that T1-Rho GDI1 signaling was indispensable for regulating the activities of Rho GTPases and for the subsequent morphological changes among astrocytes. Therefore, these findings indicate that the T1 signaling cascade can alter astrocytic morphology via regulation of Rho GTPase activity.

Identification of V-ATPase as a major component in the raft fraction prepared from the synaptic plasma membrane and the synaptic vesicle of rat brain.

Cholesterol is important in the maintenance and remodeling of the synapse. Since membrane cholesterol participates in the formation of the membrane microdomain (raft), the characterization of raft components within membrane structures in the synaptic region could be a good approach to understand the role of cholesterol in the synaptic function. In this study, protein complexes in the raft prepared from synaptic plasma membrane and the synaptic vesicle were analyzed with blue-native polyacrylamide gel electrophoresis and vacuolar H(+)-pump (V-ATPase) was identified as a major raft component using mass spectrometry. The ATPase activity was reduced through cholesterol deprivation with methyl-beta-cyclodextrin. Since the H(+) -gradient is used to transport synaptic transmitters or their precursors into the vesicle, this result suggests the essential role of cholesterol and raft in the synaptic function.

Identification of dynamin as a septin-binding protein.

Lipid rafts (detergent-resistant low-density membrane microdomain: DRM) are signal-transducing membrane platforms. In a previous study, we showed maturation-dependent localization of septin in the DRM fraction of rat brain. Mammalian septin is composed with 13-14 isoforms and these isoforms assemble to form rod-shaped hetero-oligomeric complexes. End-to-end polymerization of these complexes results in the formation of higher order structures such as filamentous sheets or bundles of filaments that restrict the fluid-like diffusion of the membrane proteins and lipids. Considering the function of septin as the membrane scaffold, elucidation of the molecular interaction of septin in DRM could be a breakthrough to understand another role of lipid rafts. In order to identify septin-binding proteins in DRM, solubilization and fractionation of septin from DRM was attempted. Several proteins were co-fractionated with septin and LC-MS/MS analysis identified one of these proteins as dynamin and Western blotting using anti-dynamin confirmed this result. Immunoprecipitation of septin11 in a crude supernatant showed co-precipitation of dynamin and dynamin fraction prepared from brain contained several septin isoforms. Within bacterially expressed septin isoforms, septin5 and septin11 bound dynamin but septin9 did not. These results suggest that some septin isoforms participate in the dynamin-related membrane dynamics.

Interaction of NAP-22 with brain glutamic acid decarboxylase (GAD).

NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched protein localized mainly in the synaptic vesicles and the synaptic plasma membrane. Biochemically, it is recovered in the lipid raft fraction. In order to understand the physiological function of the neuronal lipid raft, NAP-22 binding proteins were screened with a pull-down assay. Glutamic acid decarboxylase (GAD) was detected through LC-MS/MS, and Western blotting using a specific antibody confirmed the result. Two isoforms of GAD, GAD65 and GAD67, were expressed in bacteria as GST-fusion forms and the interaction with NAP-22 was confirmed in vitro. Partial co-localization of NAP-22 with GAD65 and GAD67 was also observed in cultured neurons. The binding showed no effect on the enzymatic activity of GAD65 and GAD67. These results hence suggest that NAP-22 could participate in the transport of GAD65 and GAD67 to the presynaptic termini and their retention on the synaptic vesicles as an anchoring protein.

Localization of phospholipase C beta 1 on the detergent-resistant membrane microdomain prepared from the synaptic plasma membrane fraction of rat brain.

The membrane microdomain (MD), such as detergent-resistant low-density membrane microdomain fraction (DRM), has been paid much attention because many signal-transducing molecules are recovered in this fraction, although precise localization and interactions of these molecules are largely unclear. To identify neuronal MD-localized proteins, monoclonal antibodies (mAbs) against the DRM-components of synaptic plasma membrane fraction (SPM) were produced and the antigens were characterized. One of the antigens reacted with two closely positioned bands of about 140 kDa in SDS-PAGE and the antigen showed age-dependent localization on DRM. The antigen was immunoprecipitated with the mAb after partial solubilization with 0.6 M NaCl from SPM-derived DRM and identified as phospholipase C beta 1 through mass analysis. The identity was further confirmed with Western blotting using a specific polyclonal antibody. The enzyme purified from the DRM was activated by the alpha subunit of trimeric G protein, Gq, expressed in HEK293 cells. The lipid composition of the liposomes affected the enzymatic activity and the addition of NAP-22, a neuronal DRM-localized protein, inhibited the activity. These results suggest that there exists a signal-transducing MD that performs important roles in neuronal functions through PIP(2) signaling and Ca(2+) mobilization.

Regulation of lipocalin-type prostaglandin D synthase gene expression by Hes-1 through E-box and interleukin-1 beta via two NF-kappa B elements in rat leptomeningeal cells.

The promoter function of the rat lipocalin-type prostaglandin D synthase (L-PGDS) gene was characterized in primary cultures of leptomeningeal cells prepared from the neonatal rat brain. Luciferase reporter assays with deletion and site-directed mutation of the promoter region (-1250 to +77) showed that an AP-2 element at -109 was required for activation and an E-box at +57, for repression. Binding of nuclear factors to each of these cis-elements was demonstrated by an electrophoretic mobility shift assay. Several components of the Notch-Hes signaling pathway, Jagged, Notch1, Notch3, and Hes-1, were expressed in the leptomeningeal cells. Human Hes-1 co-expressed in the leptomeningeal cells bound to the E-box of the rat L-PGDS gene, and repressed the promoter activity of the rat L-PGDS gene in a dose-dependent manner. The L-PGDS gene expression was up-regulated slowly by interleukin-1 beta to the maximum level at 24 h. The reporter assay with deletion and mutation revealed that two NF-kappa B elements at -1106 and -291 were essential for this up-regulation. Binding of two NF-kappa B subunits, p65 and c-Rel, to these two NF-kappa B elements occurred after the interleukin-1 beta treatment. Therefore, the L-PGDS gene is the first gene identified as the target for the Notch-Hes signal through the E-box among a variety of genes involved in the prostanoid biosynthesis, classified to the lipocalin family, and expressed in the leptomeninges. Moreover, the L-PGDS gene is a unique gene that is activated slowly by the NF-kappa B system.