Increased amyloidogenic secretion in cerebellar granule cells undergoing apoptosis.
Some clues suggest that neuronal damage induces a secondary change of amyloid beta protein (Abeta) metabolism. We investigated this possibility by analyzing the secretion of Abeta and processing of its precursor protein (amyloid precursor protein, APP) in an in vitro model of neuronal apoptosis. Primary cultures of rat cerebellar granule neurons were metabolically labeled with [35S]methionine. Apoptosis was induced by shifting extracellular KCl concentration from 25 mM to 5 mM for 6 h. Control and apoptotic neurons were then subjected to depolarization-stimulated secretion. Constitutive and stimulated secretion media and cell lysates were immunoprecipitated with antibodies recognizing regions of Abeta, full-length APP, alpha- and beta-APP secreted forms. Immunoprecipitated 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 Abeta were 3-fold higher in depolarization-stimulated secretion compared with control neurons. Such increments were paralleled by a corresponding increase of the beta-APPs/alpha-APPs ratio in apoptotic secretion. Immunofluorescence studies performed with an antibody recognizing an epitope located in the Abeta sequence showed that the Abeta signal observed in the cytoplasm and in the Golgi apparatus of control neurons is uniformly redistributed in the condensed cytoplasm of apoptotic cells. These studies indicate that neuronal apoptosis is associated with a significant increase of metabolic products derived from beta-secretase cleavage and suggest that an overproduction of Abeta may be the consequence of neuronal damage from various causes.
Galli
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Istituto di Neurobiologia, Consiglio Nazionale delle Ricerche, Via K. Marx 43, 00137 Rome, Italy. galli@biocell.irmkant.rm.cnr.it
Piccini
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Ciotti
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Castellani
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Calissano
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P
Zaccheo
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Tabaton
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D016229:Amyloid beta-Peptides / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D019084:Fluorescent Antibody Technique, Indirect; D009474:Neurons / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor