INCREASED AMYLOIDOGENIC SECRETION IN CEREBELLAR GRANULE CELLS UNDERGOING APOPTOSIS

Some clues suggest that neuronal damage induces a secondary change of amyloid beta protein (A beta) metabolism, We investigated this possibi lity by analyzing the secretion of A beta and processing of its precur sor protein (amyloid precursor protein, APP) in an in vitro model of n euronal apoptosis, Primary cultures of rat cerebellar granule neurons were metabolically labeled with [S-35]methionine. Apoptosis was induce d by shifting extracellular KCI concentration from 25 mM to 5 mM for 6 h. Control and apoptotic neurons were then subjected to depolarizatio n-stimulated secretion, Constitutive and stimulated secretion media an d cell lysates were immunoprecipitated with antibodies recognizing reg ions of A beta, full-length APP, alpha- and beta-APP secreted forms, I mmunoprecipitated proteins were separated by SDS/PAGE and quantitated with a PhosphorImager densitometer, Although intracellular full-length APP was not significantly changed after apoptosis, the monomeric and oligomeric forms of 4-kDa A beta were 3-fold higher in depolarization- stimulated secretion compared with control neurons, Such increments we re paralleled by a corresponding increase of the beta-APP(s)/alpha-APP (s) ratio in apoptotic secretion, Immunofluorescence studies performed with an antibody recognizing an epitope located in the A beta sequenc e showed that the A beta signal observed in the cytoplasm and in the G olgi apparatus of control neurons is uniformly redistributed in the co ndensed cytoplasm of apoptotic cells, These studies indicate that neur onal apoptosis is associated with a significant increase of metabolic products derived from beta-secretase cleavage and suggest that an over production of A beta may be the consequence of neuronal damage from va rious causes.