Protective Effects Of Trehalose On Manganese-induced Mitochondrial Dysfunction In Striatal Neurons In Mice

Objective:Manganese（Mn）is an essential trace element because it is required for proper immune function,antioxidant defense,energy metabolism.regulation of reproduction,digestion,bone growth,blood coagulation,and hemostasis.In addition,manganese is an important occupational toxicant and environmental pollutant.Chronic Mn exposure induces neurotoxicity,which is characterized by Parkinsonian-like symptoms resulting from impairment in the extrapyramidal motor system of the basal ganglia referred to as manganism.Throughout the world,manganese toxicity is due to environmental exposures,particularly airborne exposure.Typical airborne exposure routes are from automobile exhaust and occupational exposure（welders,ferroalloy industry workers）.Although dopamine depletion,mitochondrial damage,abnormal release of neurotransmitters,oxidative stress and autophagy dysregulation have been actively investigated,cell-level homeostatic mechanisms of Mn have not been identified.Over the past few decades,a number of the evidences suggested that mitochondrion is one of most important sites of Mn-induced cellular dysfunction.Excess accumulation of Mn in mitochondria disrupts mitochondrial homeostasis and impairs mitochondrial function.Mitochondria,the so-called‘powerhouses’of the cell,are double-membrane organelles that form highly dynamic and multifunctional networks in the cytoplasm of eukaryotic cells.Mitochondria provide the bulk of energy supply to the cell in the form of adenosine triphosphate（ATP）.Due to their high energy demand neurons are strongly affected by impairments of energy supply,which lead to necrotic or apoptotic cellular death.In this regard,a timely clearance of dysfunctional mitochondria induced by Mn may present a strategy for the cell to defend against or reduce the damage caused by Mn.Trehalose（Tre）,a natural disaccharide that presents in a diverse range of organisms including plants,bacteria,yeast and fungi.Trehalose exerts cell protective effects under various stress conditions such as oxidative damage,dehydration and temperature changes.Although several studies have reported that trehalose could alleviate nerve cell injury in vivo and in vitro,little evidence exist on trehalose involved in Mn-induced mitochondrial dysfunction,information that is critical for more fully evaluating the Mn-induced neurotoxicity.In this study,adult C57 mice were taken as the research object,and manganese poisoning mice model was established.Starting from mitochondrial damage,the relationship between mitochondrial damage and mitochondrial autophagy was analyzed.The specific molecular mechanism of trehalose-activated autophagy to alleviate manganese-induced mitochondrial damage was revealed,which provided an experiment evidence for the study of manganese-induced neurotoxicity mechanism.Methods:Total 70 C57/B16 mice were obtained from the laboratory animal center for the following experiments.The laboratory temperature is 21-24℃,the relative humidity is 30-40%,and the animal feed is provided by the laboratory animal center.Before the formal experiment,they were fed for 1 week.They were randomly divided into 7 groups according to their weight,with 10 animals in each group,half male and half female.Normal saline control group,50,100,200 mu/kg manganese chloride poisoning group,4%trehalose control group:4%（W/V）trehalose solution was provided as drinking water for mice to drink at will.High-dose manganese chloride group with trehalose intervention:intraperitoneally injected 200 mol/kgMnCl₂,and provided 2%（W/V）and 4%（W/V）trehalose solution as drinking water,which was freely drunk by mice.5 times a week,after 6 weeks of continuous exposure to the Mn,the relevant indicators were tested.Open-field test and gait test were used to detect behavioral changes in mice.the microwave digestion-manganese content in the atomic absorption method to detect the striatum,mitochondrial ultrastructure observation by transmission electron microscopy（SEM）,using flow cytometry instrument detection of mitochondrial ROS,and ROS cell,cell apoptosis and autophagy,MDC staining to observe autophagy vesicles,dyeing detection of mitochondrial membrane potential,JC-1 multifunctional enzyme mark detecting ATP levels,The colocalization and interaction of LC3 and TOM20 were evaluated by Proximity ligation assay（PLA）and Co-immunoprecipitation（CO-IP）.The levels of Beclin1,LC3 II/I,p62,Bcl-2,Bax,PARP,Cyt C,PINK1 and Parkin were detected by Western blot.Results:Manganese staining for 6 weeks resulted in neurobehavioral abnormalities in mice.The open-field experiment showed that the average velocity and the residence time in the central region decreased obviously compared with the control group.Gait retardation and decreased gait stability were detected in mice exposed to manganese by 200 mm/kg.The manganese content in the striatum of mice in the manganese staining group was significantly higher than that in the control group.Compared with the control group,ATP level and mitochondrial membrane potential in manganese staining group were significantly decreased.Compared with the control group,the formation of mitochondrial ROS and cell ROS increased significantly,and the level of SOD activity and MDA increased significantly.Group compared with control group,the dye manganese neural cell apoptosis（flow cytometry to detect apoptosis levels,Bax/Bcl-2 in the cytoplasm,PARP cleaved/Full and Cyt C protein expression increased）and the level of autophagy（flow cytometry to detect autophagy vesicle levels,Beclin1 in the cytoplasm and LC3 II/I protein expression increased,p62protein expression decreased,Parkin and PINK1 protein expression in mitochondria increased）were significantly increased;With the increase of manganese dose,the binding of TOMM20 and LC3 increased,and the mitochondrial autophagy was enhanced.Compared with the manganese staining group,the treatment with trehalose could alleviate apoptosis,reduce oxidative damage and mitochondrial damage,and further activate autophagy.Conclusion:Manganese can induce mitochondrial injury,while trehalose has the effect of anti-oxidation and activating mitochondrial autophagy,so as to reduce mitochondrial injury and alleviate the toxic effect of manganese on nerve cells.