Study on the molecular mechanism by which microglia transform into phagocytes

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Little has been known about the mechanism by which resting microglia change into active phagocytes. Therefore, I aimed to identify the specific molecule(s) associated with the induction of phagocytic activity in activated microglia.In the first year, we tried to establish an animal model of neuronal cell death induced by the injection of neurotoxic lectin RCA6O into facial nerve. However, the experiment was not carried out because RCA60 was not available in Japan. Instead, we used hydrogen peroxide or peroxinitrite to induce neuronal cell death. These radicals were found to be effective for injuring motoneurons in facial nucleus.In the second year, the molecules associated with the induction of phagocytic activity were explored by subtraction method in the hydrogen peroxide-injected facial nucleus. The subtraction method revealed that some molecules are induced in hydrogen peroxide-injected facial nucleus, but the most of the molecules belongs to proteases, other enzymes and cytoskelet … More ons. Despite the repeated experiments, we could not find out candidate molecules for phagocytic activity-related proteins. Apart from the in vivo model, we tried to prepare non-phagocytic microglia in vitro, and finally succeeded to do it by treating microglia with phorbol myristate acetate (PMA). Thus, it became possible to compare the proteins between phagocytic microglia and non-phagocytic microglia.In the last year, we continued to analyze the phagocytes-related proteins by subtraction method. However, the results were not significant. On the other hand, the comparison of proteins between phagocytic microglia and non-phagocytic microglia indicated that a molecule with molecular weight of approximately 90 kDa and pl 4.8 is present in phagocytic microglia. Therefore, the molecule (p90) was tried to be isolated from the cultured microglia. The microglial homogenate was applied to DEAE Sephadex, Hydroxyapatite, Sephacryl S200 column chromatographies, and subsequently SDS polyacrylamide gel electrophoresis/transblotting. Finally, p90 on PVDF membrane was analyzed for N-terminal amino acid sequence. The resultant sequence was Asp-Asp-Glu-Val-----(not opened). These results including the identification and the amino acid sequence of p90 remain to be confirmed. Less