MECHANISMS OF GENERAL ANESTHETICS MEDIATED NEUROTOXICITY IN ALZHEIMER’S DISEASE: ROLE OF AUTOPHAGY DYSFUNCTION

Abstract

We hypothesize that general anesthetics propofol and isoflurane, induce cell death through impairment of lysosome and autophagy function via disruption of intracellular calcium homeostasis in Alzheimer's disease (AD). PC12 cells transfected with wild type (WT) or mutated presenilin-1 (L286V) were treated with different concentrations of propofol for 6 or 12 hr or 2.4% isoflurane for 24 hr. The effects of propofol or isoflurane on cell survival, cytosolic calcium concentration ([Ca2+]c) and autophagy induction and flux were investigated. Changes with [Ca2+]c were determined by Fura-2 AM dye. We evaluated [Ca2+]c after exposing to propofol in the presence or absence of the ryanodine receptor (RYR) antagonist (dantrolene), the inositol 1,4,5-trisphosphate (InsP3) receptor antagonist, (Xc), and intracellular calcium chelator (BAPTA-AM). We used LysoTracker probes to determine the effects of propofol on lysosome acidification. We examined the effects of isoflurane on autophagy induction and flux using a mRFP-GFP-LC3 construct and transfection procedure. VATPase on lysosome and ER was determined by immunostaining technique. Propofol dose- and time- dependently induced cytotoxicity in both PC12 cells, while bafilomycin significantly aggravated the propofol-mediated cell death only in L286V but not WT cells. BAPTA-AM, dantrolene and Xc significantly inhibited propofol-induced elevation of [Ca2+]c and cell damage. However, combined use of dantrolene and Xc, paradoxically and abnormally increased [Ca2+]c by calcium influx from the extracellular space and potentiated propofol induced cell damage. AD cells demonstrated significantly impaired lysosome acidification. Isoflurane significantly impaired the turnover of autolysosome in an InsP3R activity dependent manner, in L286V but not WT PC12 cells. AD cells significantly increased VATPase in ER but decreased VATPase in lysosome, resulting in significantly more impaired lysosome acidification. Our results show that propofol and isoflurane induce neurotoxicity in an FAD cell model with PS1 mutation through autophagy flux or functional impairment via excessive calcium release from the ER through over-activation of RYR or InsP3R, which may be associated with lysosome and autophagsome dysfunction.