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.
UCLA School of Nursing and Brain Research Institute, Box 956919, Factor Building, Los Angeles, CA 90095, USA. kgylys@sonnet.ucla.edu
Karen H
KH
Gylys
Jeffrey A
JA
Fein
Dorothy J
DJ
Wiley
Gregory M
GM
Cole
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism*; D001923:Brain Chemistry / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D004396:Coloring Agents; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000201:enzymology / Q000378:metabolism*
D017304:Annexin A5; D004396:Coloring Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D020169:Caspases; C007740:fluorexon