Microglial cell death induced by a low concentration of polyamines

Pathological activation of microglia, which reside quiescently in physiological CNS, contributes various neurodegenerative diseases. Endogenous polyamines, spermidine (SPD) and spermine (SPM) are known to be activators of cell proliferation and differentiation. We examined the effect of polyamines on microglial activation in culture. Cultured microglia prepared from the whole brains of newborn rats produced nitric oxide (NO) by the stimulation with lipopolysaccharide (LPS). LPS-induced NO production was markedly inhibited by SPD and SPM; half effective concentrations (EC(50)) of SPD and SPM were about 3 and 1 microM, respectively. Cell viability assessed by total mitochondrial activity decreased by the incubation with SPD and SPM for 24 h at similar concentration ranges. After the treatment with SPM for 24 h, the cells changed into small round morphology, and were strongly stained with propidium iodide. By the staining with bis-benzimide trihydrochloride, condensation and fragmentation of the nucleus were often observed. Semiquantitative analysis of fragmented DNA with enzyme-linked immunosorbent assay technique revealed that a large amount of fragmented DNA appeared in cytosol prior to disruption of the cell membrane. Fragmented DNA in the cytosol increased dose dependently with SPM; EC(50) was less than 10 microM. Furthermore, most of the cells after 24 h incubation with 10 microM SPD and SPM were positive for terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling. These results suggest that microglial cell death is induced by a low concentration of polyamines via an apoptotic process rather than necrotic one.