No abstract

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. © 1996 Wiley‐Liss, Inc.

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 microM 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 approximately 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 lipopolysaccharide, 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.

Cellular localization of platelet-activating factor (PAF) receptor in the rat brain was determined by (1) in situ hybridization, (2) Northern blot analysis in primary cell cultures of neurons, microglia, astrocytes, and fibroblasts, and (3) Ca2+ imaging in hippocampal culture. In situ hybridization revealed that the PAF receptor mRNA is expressed intensely in microglia and moderately in neurons. Northern blot analysis revealed that PAF receptor mRNA is the most abundant in microglia. In primary hippocampal cultures, PAF elevated intracellular Ca2+ concentration in microglia and also in neurons, but to a lesser extent. These results suggest predominant presence of PAF receptor in microglia. Cultured microglia also expressed cPLA2 mRNA the most intensely. PAF-activated microglia released arachidonic acid in a Ca(2+)-dependent manner and without conversion to its derivatives. We propose that microglia as well as neurons contribute to PAF-related events in the CNS by releasing arachidonic acid.

Plasminogen (PGn) that we identified in microglial-conditioned medium has a neurotrophic factorlike 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 a-enolase. In fact, PGn was found to bind to purified rat a-enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat a-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 a-enolase. The 45-kDa PGn-binding protein in the plasma membrane was also recognized by anti-rat a-enolase antibody, and pretreatment with aenolase antibody markedly diminished the PGn-binding to the plasma membrane . In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that a-enolase is present on the cell surface of a certain population of neurons. These results suggest that a-enolase may function as a PGn-binding molecule on the neuronal cell surface. Key Words: Plasminogen-Neurons-a-Enolase-Microglia-Neurotrophic factor.