Makoto Hamanoue

Neurotrophic effect of hepatocyte growth factor on central nervous system neurons in vitro

Although the expression of hepatocyte growth factor (HGF) and its receptor, proto‐oncogene c‐met, has been demonstrated in the central nervous system (CNS), the function of HGF in the CNS was not fully understood. In the present studies, we determined the effects of HGF on neuronal development in neocortical explant and mesencephalic neurons obtained from embryonic rat brain. HGF clearly enhanced neurite outgrowth in neocortical explants. In the mesencephalic culture, the number of tyrosine hydroxylase (TH)‐positive neurons was significantly higher in the HGF‐treated wells and the neurites of the TH‐positive neurons appear to be more developed. Moreover, the dopamine uptake into mesencephalic neurons was also enhanced by HGF treatment, indicating that HGF promotes the survival and/or maturation of mesencephalic dopaminergic neurons. In both neocortical explants and mesencephalic neurons, c‐met autophosphorylation was induced by HGF and MAP kinase activation was also detected in the neocortical explant. Furthermore, Western blot analysis of the cultured CNS cells revealed that HGF was expressed mainly in microglia. These results suggest that HGF from microglia has neurotrophic activity on the CNS neurons and plays significant roles in the development of the CNS.

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

Abstract: In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7‐8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 μM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N‐methoxysuccinyl‐Ala‐Ala‐Pro‐Val‐methyl chloride, eluted at a position corresponding to a molecular mass of ∼ 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase‐like protease. This suggestion was confirmed by the finding that the 25‐kDa protein band was stained with anti‐elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopoly‐saccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.

Microglia isolated from rat brain secrete a urokinase-type plasminogen activator

In a previous study, we found particular proteases which degrade myelin basic protein (MBP) in a conditioned medium of cultured rat brain microglia. The MBP degrading activity in microglial-conditioned medium (Mic-CM) increased markedly in the presence of plasminogen. By Sephadex G-150 column chromatography, plasminogen-dependent MBP degrading activity was eluted at the position of about 47 kDa and 28 kDa. Furthermore slight plasminogen-dependent protease activity in the presence of fibrin (tissue plasminogen activator activity) was detected at a molecular weight of about 68 kDa. The two molecular forms (47 kDa and 28 kDa) of plasminogen-dependent protease were demonstrated by casein-zymography, and it was suggested that they were urokinase type-plasminogen activators (uPA). This suggestion was confirmed by immunoblotting using anti-uPA antiserum. The unique 28 kDa type was considered to be produced from the 47 kDa form by limited proteolysis. Secretion of PA from microglia was demonstrated by cell zymography. In contrast, significant secretion of plasminogen activator inhibitor could not be detected in the Mic-CM. In addition, lipopolysaccharide significantly decreased the secretion of PA from microglia, while interleukin-1 and basic fibroblast growth factor enhanced the secretion.

Production of basic fibroblast growth factor in cultured rat brain microglia

The production of basic fibroblast growth factor (bFGF) in cultured rat brain microglia was investigated. Rat brain microglia were found to express mRNA of bFGF in analysis by polymerase chain reaction (PCR) technique. Basic FGF was also detected in microglial cell lysate by Western blot analysis. These results indicate that microglia produce bFGF and possibly contribute to the regulation of neuronal development and regeneration.

Plasminogen Binds Specifically to α-Enolase on Rat Neuronal Plasma Membrane

Abstract: Plasminogen (PGn) that we identified in microglial‐conditioned medium has a neurotrophic factor‐like effect on cultured neurons. We have also shown that PGn binds specifically to a protein with a molecular mass of 45 kDa in the neuronal plasma membrane. As a candidate PGn receptor‐like molecule on the neuronal surface, this 45‐kDa protein was purified from the plasma membrane of embryonic rat brain. Amino acid sequence analysis of polypeptides derived from the cleavage of the protein with cyanogen bromide and V8 protease revealed that the 45‐kDa protein is identical to rat α‐enolase. In fact, PGn was found to bind to purified rat α‐enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat α‐enolase. Physical properties of the 45‐kDa protein, such as molecular mass, isoelectric point, and the ability to form dimers, are quite similar to those of α‐enolase. The 45‐kDa PGn‐binding protein in the plasma membrane was also recognized by anti‐rat α‐enolase antibody, and pretreatment with α‐enolase antibody markedly diminished the PGn‐binding to the plasma membrane. In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that α‐enolase is present on the cell surface of a certain population of neurons. These results suggest that α‐enolase may function as a PGn‐binding molecule on the neuronal cell surface.

Microglia-Derived Elastase Produces a Low-Molecular-Weight Plasminogen that Enhances Neurite Outgrowth in Rat Neocortical Explant Cultures

In the course of analysis of plasminogen in microglial conditioned medium (Mic‐CM), novel low‐molecular‐weight (LMW) zymogen with a molecular mass of ∼36 kDa was detected by casein‐urokinase zymography. Because this form was produced when rat native plasminogen was incubated with Mic‐CM, a specific protease in the Mic‐CM was thought to be responsible for the production of LMW plasminogen. The production of LMW plasminogen was strongly inhibited by elastase inhibitors. Furthermore, elastase (pancreatic or leukocyte) was also found to produce LMW zymogen from native plasminogen. These results indicate that LMW plasminogen is produced through limited proteolysis by an elastase‐like protease in Mic‐CM. To determine the biochemical characteristics of LMW plasminogen, rat native plasminogen was cleaved by pancreatic elastase, and the fragments (LMW plasminogen and nonzymogen fragments) were purified by several kinds of column chromatography. Amino acid sequence analysis revealed that LMW plasminogen is a carboxy‐terminal region that contains the fifth kringle domain and a protease active site, and the amino acid sequence is identical to that of LMW plasminogen produced by MicCM. On the other hand, the nonzymogen fragment was the amino‐terminal region containing four kringle domains. The effects of native plasminogen and the fragments on neurite outgrowth of rat brain explant were examined. LMW plasminogen promoted neurite outgrowth as well as did native plasminogen, whereas nonzymogen fragments did not. These results suggest that LMW plasminogen, which is produced from native plasminogen by elastase, may be a physiologically active molecule that mediates the intercellular interaction between microglia and neurons.

Catechin, green tea component, causes caspase-independent necrosis-like cell death in chronic myelogenous leukemia

Management strategies of chronic phase chronic myelogenous leukemia (CML) have been revolutionized due to the discovery of a selective tyrosine kinase inhibitor, imatinib (Gleevec, STI571), which is substantially improving median survival. However, emergence of imatinib‐resistance has put up a serious problem that requires novel treatment methods. Catechins, polyphenolic compounds in green tea, are gathering much attention due to their potential antitumor effects. So far (–)‐epigallocatechin‐3‐gallate (EGCG), the most abundant component of catechin, has been shown to cause typical apoptosis in several tumor cell lines in most cases through activation of caspases. In this study, we showed that EGCG predominantly caused necrosis‐like cell death via a caspase‐independent mechanism in CML cells, K562 and C2F8, whereas imatinib induced the typical apoptotic cell death. Moreover, this caspase‐independent cell death partially mediated the release of apoptosis‐inducing factor, AIF, and serine protease, HtrA2/Omi, from the mitochondria to cytosol. In addition, EGCG enhanced the imatinib‐induced cell death (P < 0.01) resulting in additive cell death in K562 cells and EGCG alone, effectively reduced the viability of imatinib‐resistant K562 cells (P < 0.01). Catechin is a possible candidate for an antitumor agent that causes cell death in CML cells via a caspase‐independent mechanism. (Cancer Sci 2009; 100: 349–356)

Modulatory effect of plasminogen on NMDA-induced increase in intracellular free calcium concentration in rat cultured hippocampal neurons

The effect of plasminogen on the intracellular Ca2+ concentration ([Ca]i) was examined in cultured rat hippocampal neurons. Plasminogen (100 nM) did not increase [Ca]i in the presence of tetrodotoxin (TTX, 3 microM) but increased [Ca]i in the absence of TTX. In all the cells which responded to plasminogen (100 nM), NMDA (5 microM) also increased [Ca]i in the presence of TTX. Furthermore, plasminogen (100 nM) enhanced the NMDA-evoked [Ca]i increase, and the potentiation by plasminogen was blocked by an NMDA receptor blocker, 2-amino-phosphonovalerate (APV). These data suggest that plasminogen enhances glutamate-evoked [Ca]i increase through modulation of NMDA receptor in hippocampal neurons.

Schwann cell plasticity after spinal cord injury shown by neural crest lineage tracing

After spinal cord injury (SCI), various cell types are recruited to the lesion site, including Schwann cells, which originate in the neural crest and normally myelinate axons in the peripheral nervous system. Here, we investigated the differentiation states, migration patterns, and roles of neural crest derivatives following SCI, using two transgenic mouse lines carrying neural crest‐specific reporters, P0‐Cre/Floxed‐EGFP and Wnt1‐Cre/Floxed‐EGFP. In these mice, EGFP is expressed only in the neural crest cell lineage. Immunohistochemical analysis revealed that most of the EGFP+ cells that infiltrated the lesion site after SCI were Schwann cells. Seven days after SCI, the P0‐positive, mature Schwann cells residing at the nerve roots had dedifferentiated into P0−/p75+ immature Schwann cells, which proliferated and began migrating into the lesion site. The dedifferentiation of the Schwann cells was corroborated by their expression of phosphorylated c‐Jun, which promotes dedifferentiation and inhibits the expression of myelin‐associated genes in the peripheral nerves. Thereafter, the number of EGFP+/p75+ immature Schwann cells decreased and that of EGFP+/P0+ mature cells increased gradually, indicating that the cells redifferentiated into mature Schwann cells within the lesion site. This study draws on the advantages offered by transgenic mouse lines bearing a genetic cell‐lineage marker and extends previous work by describing the origins and behavior of the neural crest‐derived cells that contribute to endogenous repair after SCI. This process, involving Schwann cell plasticity, is a novel repair mechanism for the lesioned mammalian spinal cord.

Cell surface N-glycans mediated isolation of mouse neural stem cells.

The isolation of neural stem cells (NSCs) from the brain has been hampered by the lack of valid cell surface markers and the requirement for long‐term in vitro cultivation that may lead to phenotype deterioration. However, few suitable specific cell surface antigens are available on NSCs that could be used for their prospective isolation. The present study demonstrated that the expression of complex type asparagine‐linked oligosaccharide (N‐glycans) was detected on brain cells dissociated from embryonic and adult brain using Phaseolus vulgaris erythroagglutinating lectin (E‐PHA) which binds to biantennary complex type N‐glycans, and demonstrated that E‐PHA bound preferentially to purified NSCs, but not to neurons, microglia, or oligodendrocyte precursor cells. The labeling of dissociated mouse embryonic brain cells or adult brain cells with E‐PHA enabled the enrichment of NSCs by 25‐fold or 9‐fold of the number of neurosphere‐forming cells in comparison to that of unsorted cells, respectively. Furthermore, a lectin blot analysis revealed the presence of several glycoproteins which were recognized by E‐PHA in the membrane fraction of the proliferating NSCs, but not in the differentiated cells. These results indicate that complex type N‐glycans is a valuable cell surface marker for living mouse NSCs from both the embryonic and adult brain.