RNAi-induced silencing of the plasma membrane Ca2+-ATPase 2 in neuronal cells:: effects on Ca2+ homeostasis and cell viability

Intraneuronal calcium ([Ca2+](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca2+ transporters. We previously reported that the levels of the plasma membrane Ca2+-ATPase (PMCA) and the V-max for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro- transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca2+](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca2+](i), slower recovery from KCl-induced Ca2+ transients, and incomplete return to pre-stimulation Ca2+ levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca2+ signaling and enhanced susceptibility to stresses that can alter the regulation of Ca2+ homeostasis.