EXTERNALIZATION OF PHOSPHATIDYLSERINE MAY NOT BE AN EARLY SIGNAL OF APOPTOSIS IN NEURONAL CELLS, BUT ONLY THE PHOSPHATIDYLSERINE-DISPLAYINGAPOPTOTIC CELLS ARE PHAGOCYTOSED BY MICROGLIA

Earlier reports on nonneural cells have shown that the normally inner plasma membrane lipid, phosphatidylserine (PS), flip-flops out during the early stages of apoptosis, whereas DNA laddering and plasma membra ne permeabilization occur during the late stages. In this study, the a pplicability of these parameters to CNS-derived neuronal cells was tes ted using hippocampal HN2-5, cells that undergo apoptosis under anoxia . Because such insults on unsynchronized cells, e,g,, undifferentiated HN2-5 cells, result in both early and late apoptotic cells, we mechan ically separated these cells into three fractions containing (a) cells that had completely detached during anoxia, (b) cells that remained w eakly attached to the tissue culture dish and, once detached by tritur ation in serum-containing medium, did not reattach, and (c) cells that reattached in 2-3 h. Fractions a and b contained cells that showed pr onounced DNA laddering, whereas cells in fraction c did not show any D NA laddering. Double staining with fluorescein isothiocyanate-annexin V (which binds to PS) and propidium iodide (which stains the DNA in ce lls with a permeable cell membrane) revealed that all cells in fractio n a had a permeable cell membrane (propidium iodide-positive) and PS m olecules in the outer leaflet of the plasma membrane (fluorescein isot hiocyanate-annexin V-positive), By contrast, fractions b and c contain ed cells with no externalized PS molecules. Cells in fractions a-c als o showed, respectively, 50-, 21-, and 5.5-fold higher caspase-3 (CPP32 ) activity than that in healthy control cells. All these results show that fraction a contained late apoptotic cells, which also had the hig hest CPP32 activity; cells in fraction b were at an intermediate stage , when DNA laddering had already occurred; and fraction c contained ve ry early apoptotic cells, in which no DNA laddering had yet occurred. Therefore, in the neuronal HN2-5 cells, externalization of PS occurs o nly during the final stages of apoptosis when the cells have completel y lost their adhesion properties. Further experiments showed that ameb oid microglial cells isolated from neonatal mouse brain phagocytosed o nly the cells in fraction a. These results show that in CNS-derived HN 2-5 cells, (a) PS externalization is a late apoptotic event and is con comitant with a complete loss of surface adhesion of the apoptotic cel ls and (b) PS externalization is crucial for microglial recognition an d phagocytosis of the apoptotic HN2-5 cells. Thus, PS externalization could be causally linked to the final detachment of apoptotic neuronal cells, which in turn prepares them for rapid phagocytosis by microgli a.