Study On The Mechanisms Of Acetaldehyde-induced Neuronal Toxicity Mediated By Mitochondrial Damages

Epidemiological studies have shown that alcohol abuse has deleterious effects on cognitive function,and increases the risk of neurodegenerative diseases such as Alzheimer's disease.Acetaldehyde is mainly generated in liver from ethanol by alcohol dehydrogenase after absorption.The major enzyme responsible for the detoxification and metabolism of acetaldehyde is aldehyde dehydrogenase(ALDH)2.Approximately 30%-50% Asian have defective aldh2 gene.Excessive alcohol consumption can lead to higher blood concentration of acetaldehyde in individuals with defective aldh2 gene in comparison to individuals with aldh2 wild-type gene.Acetaldehyde,the most toxic metabolite of alcohol,has been speculated to play important role in mediating the neurotoxicity induced by alcohol abuse,but the mechanism was still unclear.In this study,the cytotoxic effects of acetaldehyde and the underlying mechanisms were investigated in neuronal cells,having important implications for targeted drug discovery for neurotoxicity associated with alcoholism.Trypan blue assay and apoptosis assay were employed in the present study to explore the neuronal cytotoxicity induced by acetaldehyde,and the results showed that acetaldehyde decreased the cell viability and induced apoptosis of neuronal cells.Western blot assays were performed to study the effects of acetaldehyde on cell survival mechanisms.It was shown that acetaldehyde treatment led to a significant decrease in the levels of activated Akt and CREB.In addition,acetaldehyde inhibited the activation of ERK.Reactive oxygen species(ROS)probe was used to detect the level of intracellular ROS.It was found that acetaldehyde treatment caused an increase in the level of ROS.Antioxidant was found to inhibit the cytotoxicity induced by acetaldehyde.These results suggested that acetaldehyde might induce apoptosis and cytotoxicity via induction of oxidative stress.The results of MTT assay demonstrated that acetaldehyde significantly decreased mitochondrial activities of primary cultured rat cortical neurons and SH-SY5 Y cells.Acetaldehyde significantly decreased the mitochondrial membrane potential and the production of ATP,causing the dysfunction of mitochondria.Examination of mitochondrial morphology using Mito Tracker Green staining showed that acetaldehyde induced mitochondrial fragmentation.Further analyses revealed that acetaldehyde might induce mitochondrial fragmentation by promoting the activation of dynamin-related protein 1(Drp1)and the transfer of Drp1 from cytoplasm to mitochondria.Both JNK and p38 MAPK played important roles in the activation of Drp1 by acetaldehyde.Furthermore,acetaldehyde induced the increase of intracellular Ca2+ levels and the activation of Ca MKII.Antioxidant,BAPTA-AM,the chelator of intracellular Ca2+,and the inhibitor of Ca MKII,KN-93,could inhibit acetaldehyde-induced activation of Drp1 and cytotoxicity.These findings suggested that the elevation of ROS and Ca2+ caused mitochondrial fragmentation and dysfunction in acetaldehyde-treated cells.Meanwhile,the increase of intracellular Ca2+ levels and the activation of Ca MKII could also be inhibited by antioxidant,suggesting an interaction between ROS and Ca2+-mediated signaling.Excessive fission of mitochondria has been considered as a prerequisite that initiates mitophagy.The co-localization of mitochondria and lysosome in acetaldehydetreated cells indicated that acetaldehyde induced mitophagy.It was also found that the protein levels of key mitophagic proteins PINK1,Parkin,autophagy initiator Beclin1,autophagosome membrane formation proteins(Atg5 and Atg16L1)were increased in acetaldehyde-treated cells.Besides,antioxidant treatment attenuated the mitophagic responses induced by acetaldehyde and promoted the survival of cells,suggesting a close relation between oxidative stress and acetaldehyde-induced mitophagy.In this study,the cytotoxicity of acetaldehyde and the underlying mechanisms were explored using neuronal cell models.It was found that acetaldehyde disturbed the equilibrium of mitochondrial dynamic,damaged mitochondrial function and promoted mitophagy,and eventually induced apoptosis.The induction of oxidative stress and Ca2+ imbalance were mechanisms of acetaldehyde-induced mitochondrial dysfunction and cytotoxicity.This study has important implications for targeted drug discovery for neurotoxicity associated with alcoholism.