Abstract
Caspase activation and apoptotic events may take place in terminal regions far removed from the cell body and contribute to synapse loss in neurodegenerative diseases. For examination of events in terminals, we have developed a cell-free assay using quantitative flow cytometric analysis (fluorescence-activated cell sorting) of neuronal particles in a P2 synaptosomal preparation (P-2) from rat brain as a model system. Staurosporine-induced loss of neuronal particles was blocked by nonselective caspase inhibition (z-VAD-fmk) and by calpain inhibition (calpain inhibitor II [ALLM]). Phosphatidylserine exposure was increased in the P-2 by staurosporine treatment, and this increase was blocked by a peptide inhibitor of caspase-3-like activity (Ac-DEVD-CHO). Increased caspase activity in the crude synaptosomal fraction was confirmed by direct measurement with a fluorometric assay. These results indicate activation of both caspase and calpain in the P-2 fraction and suggest a role for these cysteine proteases in the in vitro degradation of nerve terminals.
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