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.
Gylys
Karen H
KH
UCLA School of Nursing and Brain Research Institute, Box 956919, Factor Building, Los Angeles, CA 90095, USA. kgylys@sonnet.ucla.edu
Fein
Jeffrey A
JA
Wiley
Dorothy J
DJ
Cole
Gregory M
GM
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