Transcription Factor EB Probably Involved In Midazolam-Disturbed Lysosomal Homeostasis And Its Induced ?-Amyloid Accumulation

Background:Alzheimer's disease is one of the most common neurodegenerative diseases,of which the main pathological feature is the deposition of extracellular?-amyloid,the formation of intracellular neurofibrillary tangles and the reduction in the number of synapse and neurons.And ?-amyloid play a leading role in the pathogenesis of AD.Patients with Alzheimer's disease gradually show mild cognitive impairment,as well as decreased memory and learning ability,and in the late stages of the disease,the patient's brain will experience severe atrophy,loss of ability to live independently.Lysosomes,as important organelles for intracellular component degradation,signal transduction,cell secretion and energy metabolism,contain a variety of hydrolases,which can produce smaller functional proteins,it is closely related to neurodegenerative diseases by removing organelles or aggregated proteins that may cause disease damage.TFEB,a main regulating factor of autophagy and lysosome biosynthesis,is involved in the pathogenesis of AD by regulating autophagy-lysosomal pathways.Midazolam is a commonly used intravenous anesthetic widely used for clinical sedation and general anesthesia during surgery.To date,the choice of anesthetics during surgery in patients with neurodegenerative diseases and evaluation of the effects and underlying mechanisms in these patients have rarely been reported.Methods:In this study,HEK293-APP cells overexpressing APP,HeLa cells and EGFP-TFEB/HeLa cells transfected with TFEB green fluorescence were used.The cellswere treated with midazolam at different concentrations and different times,then lysosomes were stained by lysotracker and their morphology and number were observed under a fluorescence microscope.The number and size of lysosomes were analyzed using imagj j software.The levels of TFEB protein in the nucleus and APP-cleaved proteins intracellular CTF? and CTF? were detected by nuclear separation and Western blotting,the expression of TFEB in the nucleus and cytoplasm was detected by co-localization.Finally,ELISA was used to detect the levels of A?40 and A?42 in the cells after drug treatment.Results:We found that 30?M midazolam decreased the number of lysosomes and increased its size in HEK293 and HeLa cells.However,15?M midazolam transiently disturbed lysosomal homeostasis at 24 h and recovered it at 36 h.Notably,there was no significant difference in the extent to which lysosomal homeostasis was disturbed between treatments of different concentrations of midazolam at 24 hours.In addition,30?M midazolam prevents the transport of TFEB to the nucleus in either normal or starved cells.Finally,the intracellular CTF?,CTF?,A?40 and A?42 levels were all significantly elevated in 30?M midazolam-treated HKE293-APP cells.Conclusion:Collectively,the inhibition of TFEB transport to nucleus may be involved in midazolam-disturbed lysosomal homeostasis and its induced ?-amyloid accumulation in vitro.The results indicated the risk of accelerating the pathogenesis of AD by midazolam and suggested that TFEB might be a candidate target for reduction of midazolam-dependent neurotoxicity.