Our recent preliminary studies suggest that the commonly used general anesthetics (GAs), isoflurane and propofol, regulate autophagy via mTOR dependent pathway by activation of InsP3R Ca channel 2+ opening, which contribute to GAs effect on cell survival or death. The overarching goal of this R01 renewal is to investigate the mechanisms of GAs on autophagy activity via activation of InsP3Rs and the subsequent role on cell survival or death, synapse integrity and function and cognitive function. Thus, we hypothesize that GAs modulate autophagy through a dose- and time-dependent activation of InsP3 R, and the magnitude determines the fate of neuronal survival, synaptic integrity and function, and subsequent cognitive function. We have set up three specific aims (SA) to investigate this hypothesis: SA1). We will determine the role of GA-mediated activation of InsP3R subtypes on modulation of autophagy. Using the single channel nuclear patch-clamp technique in cell cultures with various levels of each subtype of InsP3R, we will characterize and compare the activating potency of four GAs (propofol, isoflurane, sevoflurane and desflurane) on each of the three subtypes of InsP3Rs, and correlate patch-clamp data with anesthetics-evoked changes in cytosolic, mitochondrial and ER Ca2+ concentrations. We will study the effects and mechanisms of GAs on autophagy via mTOR dependent pathway using various stimulators and inhibitors and controls of various autophagy regulatory sites in different cell cultures or hippocampus or cortex with different levels of baseline autophagy and InsP3R activity. SA2). We will determine the role of GA-modulated autophagy on apoptosis and synaptic integrity. We will study the effects of GAs on autophagy and apoptosis simultaneously in the same cell cultures and brain regions of mice with varying levels of autophagy and InsP3R baseline activity, in the presence or absence of various autophagy and apoptosis stimulators or inhibitors. We will study the GAs effects on synapse integrity by measuring changes of presynaptic synapsins and postsynaptic markers in primary neuronal cultures or hippocampus and cortex of mice with deficient autophagy or InsP3R-1. SA3). We will determine the role of GA-modulated autophagy on synaptic and cognitive function. We will study the long-term effects of GAs on basic synaptic transmission, long term potentiation (LTP) in hippocampal slices, and cognition in mice with deficient autophagy or InsP3R-1, in the presence or absence of various autophagy and apoptosis stimulators and inhibitors. We will correlate these GAs effects on synapse and cognitive function to their effects on activation of InsP3R, Ca release from the 2+ ER, autophagy activity and related apoptosis and synapse integrity.
Our recent preliminary studies suggest that the commonly used general anesthetics isoflurane and propofol can modulate autophagy activity via mTOR dependent pathway by activation of the type 1 and 3 InsP3R calcium channel opening, which may contribute to their effects of inducing apoptosis, synapse and cognitive dysfunction. We, therefore, intend to focus on studying the mechanisms through which general anesthetics regulate autophagy, an important cellular process to control cell survival or death, synapse integrity and function as well as cognitive function. Ultimately, we hope to apply these findings t utilize beneficial but minimizing the harmful effects of GAs and improvement of patient safety.
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