Calpains and calpastatin in SH-SY5Y neuroblastoma cells during retinoic acid-induced differentiation and neurite outgrowth: comparison with the human brain calpain system.

Calpains have importance in human neurodegenerative disease pathogenesis, but these mechanisms are difficult to study in postmortem tissues. To establish a cellular model of the human calpain and calpastatin system, we characterized calpain I, calpain II, and calpastatin in SH-SY5Y human neuroblastoma cells in relation to their counterparts in human brain and investigated their expression and activity after inducing cellular differentiation with retinoic acid (RA), a physiological effector of normal brain development. Calpain I in both SH-SY5Y cells and human brain existed in the cytosolic and particulate fractions as three isoforms (80, 78, and 76 kDa) and exhibited atypical isoelectric focusing behavior. Calpain II in SH-SY5Y cells, as in human brain, migrated as a single predominantly cytosolic 76-kDa protein with an isoelectric point ranging from 5.9 to 6.3. Calpastatin from both sources was also 90% cytosolic. In the cells it was composed of four discrete bands, ranging in molecular weight from 110 to 127 kDa. Levels of activated (76 and 78 kDa) and precursor (80 kDa) calpain I isoforms rose 54% (P < 0.0001) in the particulate fraction and 26% (P < 0.0001) in the soluble fraction after 3 days of RA exposure. Because levels and activity of calpastatin remain unchanged during the first 7 days of RA exposure, the increased abundance of calpain I implies a net activation of the calpain system during differentiation. Calpain I activation may contribute to the remodeling of cell shape and neurite extension/retraction associated with neuronal differentiation.