Rapid annexin-V labeling in synaptosomes

Distal neuronal terminals may be the site of apoptotic events and early synapse loss in neurodegenerative disease. To examine apoptosis in synaptic regions, we established a cell-free assay using a rat brain crude synaptosomal preparation (P-2 fraction) as a model system. The apoptosis marker annexin-V was used to measure phosphatidylserine (PS) exposure, and to ensure that only intact terminals were assayed, synaptosomes were dual labeled with a viability marker (calcein AM). Fluorescence was quantified by flow cytometry analysis. Annexin-V labeling increased rapidly in synaptosomes, following a 1 min incubation with staurosporine. However, increased caspase-3-like activity was not measured until 30 min with a fluorometric assay. The addition of a peptide inhibitor of caspase-3-like activity (Ac-DEVD-CHO) during homogenization was not able to block the initial increase in annexin labeling, but resulted in a partial blockade of annexin labeling after 30 min. These data demonstrate that PS externalization and caspase activation occur rapidly in this widely used neurochemical preparation.