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

Abstract

Intraneuronal calcium ([Ca 2+ ] i ) regulation is altered in aging brain, possibly because of the changes in critical Ca 2+ transporters. We previously reported that the levels of the plasma membrane Ca 2+-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.

Plasma membrane Ca2+-ATPases: Targets of oxidative stress in brain aging and neurodegeneration

“…Cells with elevated Ca 2+ levels would likely exhibit an increased vulnerability to various metabolic and oxidative insults. Consistent with this idea, PMCA 2 deficient cells are more susceptible to cellular stresses particularly those involving Ca 2+ overload [82] . Although experimental lowering of the PMCAs in cultured cells cannot be extrapolated to the in vivo situation that exists in brain, they greatly emphasize the importance of maintaining normal levels of PMCA protein in neuronal survival and growth.…”

“…PMCA activity is significantly reduced in response to cellular cholesterol depletion suggesting the possibility of local regulation of the pump activity in lipid rafts [78] . Antisense-(AS) plasmid-mediated reduction of specific isoforms has yielded valuable information on the role of individual PMCA subtypes in regulating the dynamics of cellular Ca 2+ handling and also their contribution to a diverse array of neuronal functions [80][81][82][83][84] . For example, blockade of PMCA 1 causes no change in the levels of resting free [Ca 2+ ]i or its release from intracellular stores but results in a significantly slower rate of Ca 2+ clearance following release from intracellular stores [80] .…”

“…We utilized an RNA interference strategy to experimentally lower PMCA 2 expression [82] . Short interference RNA treatment led to 80% reduction of PMCA 2 expression, which remained suppressed throughout a 6 d period.…”

“…Short interference RNA treatment led to 80% reduction of PMCA 2 expression, which remained suppressed throughout a 6 d period. siRNA-treated cells exhibit marked changes in total PMCA activity, with a shift from Michaelis-Menten kinetic properties, a three-fold increase in Kact for Ca 2+ and 22% suppression of Vmax [82] . Ca 2+ imaging studies using Fura 2 show that neurons with lowered PMCA 2 have (1) elevated resting [Ca 2+ ]i; (2) a significantly slower recovery following depolarization; and (3) inability to return to their own resting levels present prior to depolarization, signifying the importance of PMCA 2 in maintaining resting [Ca 2+ ]i.…”

Contribution of plasma membrane Ca2+ATPase to cerebellar synapse function

“…This makes the search for new approaches to study the physiological contribution of PMCA2 and its isoforms both timely and necessary. In vivo viral-based delivery of timed siRNA PMCA2 knockdown might offer a more useful approach [43,44] together with the development of transgenic mice approaches for knock-in or knock-out, especially if expression could be limited to specific cell types within the cerebellum, such as the PNs [45] . In addition, improvement of PMCA-specific pharmacological tools beyond the advances made by the development of the caloxins [46] would further aid physiological studies.…”