ACTIVATION OF CASPASE-3 AND APOPTOSIS IN CEREBELLAR GRANULE CELLS

Caspase-3 activity increased dramatically in cytosolic extracts of rat cerebellar granule cells exposed to apoptotic conditions (basal mediu m Eagle (BME) containing 5 mM K+ without serum) when assayed with Ac-D EVD-amc, but not with Ac-YVAD-afc, a preferred substrate for caspase-1 , This provided a basis to examine relationships between enzyme activi ty and cell viability for purposes of selecting an optimal time for co mparing neuroprotective agents or strategies, Exposure of neurons to a n apoptotic medium containing 5 mM K+ in absence of serum led to a rap id 5- to 10-fold increase in caspase-3 within 2-4 hr but without signi ficant cell loss, or morphological alterations, Exposure to apoptotic medium followed by replacement with maintenance medium containing 25 m M K+ and serum led to a rapid fall in caspase-3 and prevention of cell death, This strategy was not effective after 13 hr exposure despite a large fall in enzyme activity. These temporal changes infer systems f or rapid enzyme turnover and/or activation of cytoplasmic components l inked to later DNA degradation, The effects of cycloheximide point to requirements for protein synthesis, and those of Glu exclude a caspase -3 dependent pathway for necrotic cell damage, Brief treatment with 10 mu M LIGA(20), an anti-necrotic agent, also attenuated cell loss and caspase-3 activity, indicating a broad spectrum of neuroprotection, Ra pid and long-lasting effects, together with its biophysical properties , suggest that this semisynthetic ganglioside acted upstream at or nea r a membrane site, As such, gangliosides provide useful agents to furt her probe pathways relevant to neuronal death in culture. (C) 1998 Wil ey-Liss, Inc.