Masato Shimojo

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.

Interleukin-2 enhances the viability of primary cultured rat neocortical neurons

We determined the neurotrophic activity of interleukin-2 (IL-2) on primary cultured neocortical neurons from embryonic rat brain. IL-2 clearly enhanced the viability of cultured neurons in a dose-dependent manner. The neurotrophic effect of IL-2 was completely neutralized by IL-2 antibody. Furthermore, expression of IL-2 receptor mRNA was more pronounced in neurons than in other cultured cells such as astroglia and microglia. These results strongly suggest that IL-2 plays certain roles in the central nervous system as a neurotrophic factor.

Generation of microglial cell lines by transfection with simian virus 40 large T gene

Microglial cells, which were isolated from a primary culture of neonatal rat brain, were transfected with temperature-sensitive simian virus 40 (SV40) large T gene by the calcium phosphate precipitation method. Four weeks after transfection, several colonies were generated, and cloned cells were characterized. One of the cloned cells (RBM129) proliferated actively at 37 degrees C and the dividing rate was significantly decreased at 40.5 degrees C. The expression of large T antigen was detected by western blotting in cells incubated at both 37 degrees C and 40.5 degrees C. The cell line showed high activity of non-specific esterase, isolectin B4 binding and phagocytosis. Also the cells were stained by ED 1 monoclonal antibody. These results indicate that these cells were derived from rat brain microglia, and immortalized by large T gene.

Neuroprotective action of a novel compound-M50463-in primary cultured neurons

The neuroprotective effects of a novel synthetic compound, M50463, have been determined by using embryonic rat neocortical neurons in various culture conditions. M50463 was initially characterized as a potent specific ligand for a voltage-dependent sodium channel by radioligand binding studies. In fact, M50463 inhibited neuronal cell death induced by veratrine and inhibited an increase of the intracellular calcium level in neurons evoked by veratrine. In addition to such expected effects, M50463 had the ability to prevent glutamate neurotoxicity, to promote the neuronal survival in serum-deprived medium and to prevent nitric oxide-induced neurotoxicity. These results suggested that M50463 is not a simple sodium channel blocker, but a neuroprotective agent which has some crucial mechanism of action on neuronal death occurring in various situations, and it is a novel, innovative candidate for neuroprotective therapy for various neurodegenerative disorders.