Calcium, calmodulin, and calcium/calmodulin-dependent kinase and phosphatase: Roles in neuronal cell death.

Abstract

The overall objective of this thesis is to investigate the effects of changes in intracellular calcium concentration ([Ca<super>2+</super>_i]) on calmodulin (CaM) localization, CM-dependent enzyme activation and cell death. Changes in [Ca<super>2+</super>_i] are an essential signaling mechanism for many neuronal processes. CaM acts as a calcium-sensing switch that binds to and activates diverse enzymes. In carbachol-treated SK-N-SH cells, CaM translocates from the membrane to the cytosol. I show that CaM translocation is due to release of intracellular Ca<super>2+</super>. Pre-treatment with the Ca<super>2+</super> chelator BAPTA, but not the Ca<super>2+</super>-channel antagonist Ni<super>2+</super> prevents carbachol-mediated CaM translocation. Thapsigargin, which empties intracellular Ca<super>2+</super> stores, induces CaM translocation. K<super>+</super>, which mediates influx of extracellular Ca<super>2+</super> (Ca<super>2+</super>_e), does not induce CaM translocation. Alterations in Ca<super>2+</super> homeostasis have been implicated in the onset of neurodegenerative-linked cell death. I show that exposing SH-SY5Y cells to 5 mM Ca<super>2+</super>_e leads to activation of the Ca<super>2+</super> dependent cysteine protease calpain and induces necrosis. Conversely, depletion of Ca<super>2+</super>_e with 2 mM EGTA activates caspase-3-like proteases and induces apoptosis. EGTA-mediated activation of caspase-3-like proteases was interrupted by restoration of Ca<super> 2+</super>_e. The fate of CaM-dependent kinase and phosphatase in neuronal cells undergoing apoptosis was also examined. CaM kinase inhibition potentiates thapsigargin-mediated cytotoxicity through a caspase-dependent pathway. Ca<super>2+</super> /CaM-dependent protein kinase IV (CaMK IV) is fragmented by calpain and caspase-3 during apoptosis at different sites within the catalytic domain. CaMK activity decreases in SH-SY5Y cells and cerebellar granule neurons undergoing apoptosis. The loss in activity precedes loss of cell viability. Conversely, inhibition of the CaM-dependent phosphatase calcineurin protects against thapsigargin-mediated apoptosis. Calcineurin is also cleaved during apoptosis. Digestion of purified calcineurin with purified caspase-3 produces a 45 kDa fragment comprised of the catalytic and calcineurin B-binding domains. The regulatory domain is cleaved off. The phosphatase activity of purified calcineurin increases as a result of caspase-3 digestion. The activity loses its CaM-dependency, consistent with the loss of the regulatory domain. Work in this thesis clarifies the effects of changes in Ca<super>2+</super> concentration in protease activation and the roles of CaM-dependent kinases and phosphatase in neuronal apoptosis.