The Effects And Mechanisms Of Mitochondrial Quality Control On Central Nervous System Nerve Cells Injury Induced By Lanthanum In Rats

Objective:Rare earth elements（REEs）is the general term for lanthanides,scandium and yttrium.REEs have unique physical and chemical properties and are widely used in crop fertilizers,livestock feeds,medicines and high-tech materials and other industries.With the extensive exploitation and application of REEs,REEs enter the human body through the digestive tract increasingly and accumulate in liver,bone,brain and other tissues.The brain is a target organ of REEs,and REEs have obvious damage to the central nervous system（CNS）of children.The results of epidemiological surveys in China’s rare-earth mining areas showed that children with only 2.18±1.08ng REEs per gram blood exhibited obviously lower intelligence quotient and cognition function than ones in the control area.As a representative of light REEs,lanthanum（La）is abundant in the earth’s crust and has strong biological activity.Therefore,it is often used as a representative substance for studying the neurotoxicity of REEs.Xiao et al.found that La can cross the placental barrier and enter the body of the offspring,reduce the level of acetylcholine,increase the level of glutamate,and induce learning and memory disorders in the offspring.Feng et al.found that La could interfere with the levels of neurotransmitter and trace elements in the brain of rats,impairing their learning and memory capabilities.The above studies show that La is a type of environmental factor that seriously affects the development of intelligence,but its neurotoxic mechanism has not been clarified,and further exploration is still needed.Nerve cells need to consume a lot of energy when performing learning and memory functions,and the energy they need is mainly provided by mitochondria.A healthy mitochondrial network is essential for cell survival.Mitochondria have evolved a sophisticated and complex mitochondrial quality control system（MQC）that regulates the balance of their own quantity,shape and function,ensuring the normal progress of various physiological functions in the mitochondria,in order to respond to external stimuli and meet the physiological needs of cells.Mitochondrial biogenesis promotes the replication of mitochondrial DNA（mt DNA）,synthesizes mitochondrial protein and membrane structure through the PGC-1α-NRF1-TFAM signaling pathway,and plays the function of repairing old mitochondria and generating new mitochondria.It is the renewal mechanism of MQC.Mitochondrial fission and fusion regulates the morphology and quantity of mitochondrial network through DRP1 and MFN1,2 and other proteins,maintains mitochondrial function and adapts to metabolic needs,which is an important way for MQC.Mitophagy isolates and removes damaged or senescent mitochondria and mitochondrial fragments through the PINK1-PARKIN signaling pathway,which is a key part of MQC.The three are coordinated with each other,while keeping healthy mitochondria to the greatest extent,remove irreversibly damaged mitochondria and maintain mitochondrial homeostasis.Our previous research results indicated that La exposure can cause large amounts of reactive oxygen species（ROS）to be produced in rat hippocampal neurons and primary neurons,mitochondrial depolarization is enhanced,mitochondria are significantly swollen,and cell apoptosis is enhanced.It suggestted that La may interfere with mitochondrial quality control affects the structure and function of nerve cells,thereby impairing learning and memory.This study uses a combination of animals and in vitro neuronal primary culture,and applies neurobehavioral,molecular biology and other experimental methods to explore the effects of La on mitochondrial biogenesis,mitochondrial fission and fusion,and mitophagy.Comprehensively elucidate the neurotoxicity mechanism of La and provide a new scientific basis for finding effective molecular targets for intervention.Methods:In the animal experiment,96 healthy adult Wistar rats（48 females and 48males,female weight 220±10g,male weight 240±10g）were provided by the Experimental Animal Center of China Medical University.They were reared adaptively in the animal room.After 7 days,the female rats were divided into 4 treatment groups according to the principle of randomization,with 12 rats in each group,then females and males mated at 1:1.It was recorded as gestational day 0 when the female vaginal plug was found.The maternal rats were treated with LaCl₃ in distilled drinking water under one of four doses:0%,0.25%,0.5%or 1.0%.After weaning,the pups were permitted free access to drinking water（containing 0,0.25%,0.5%and 1.0%LaCl₃）for 1 month.The hippocampus of pups from the four groups was used for subsequent experiments.Morris water maze test was used to detect the learning and memory ability of rats,NAO staining was used to detect cardiolipin content,ELISA method was used to detect the content of ATP and mitochondrial respiratory chain complex IV.The the m RNA transcription and protein expression levels of TOM20,TIM23,PGC-1α,NRF1,TFAM,DRP1,p-DRP1 Ser616,p-DRP1 Ser637,MFN1,MFN2,PINK1,PARKIN,LC3B and P62 in hippocampal nerve cells of each group were detected by Realtime PCR and Western Blot methods.Immunofluorescence was used to detect the co-localization of TOM20 and DRP1.The number of mitophagosomes and mitochondrial morphology in hippocampal neurons of rats were detected by transmission electron microscopy.In vitro experiments were performed on primary culture of cerebral cortical neurons using Wistar pups within 24 hours of birth.Neuron-specific enolase was used for neuron identification,TMRM staining was used to detect the change of mitochondrial membrane potential,Mito SOX staining was used to detect the generation of mitochondrial ROS.The morphology and quality of mitochondria was detected by Mito Tracker Green staining method.The protein expression levels of PGC-1α,NRF1,TFAM,DRP1,p-DRP1 Ser616,p-DRP1 Ser637,MFN1,MFN2,PINK1,PARKIN,LC3B and P62 in neurons were detected by Western Blot method.The relative content of mt DNA was detected by Realtime PCR method.Immuno-fluorescence was used to detect the co-localization of LC3B and mitochondria.The number of mitophagosomes in neurons was detected by transmission electron microscopy.After intervention with DRP1 inhibitor Mdivi-1 and PGC-1αagonist ZLN005,the relevant indicators were measured again to clarify its action and mechanism.Results:1.The effect of LaCl₃ on learning and memory ability of pups.The results of the place navigation test showed with the increasing dose of LaCl₃,the escape latency and total swimming distance increased gradually.And the paths of the LaCl₃-treated groups were messy and purposeless.The results of spatial probe test showed that the number of target quadrant crossings and the time in the target quadrant of the LaCl₃-treated groups were significantly lower than those of the control group.The trajectory thermography showed that the rats of the LaCl₃-treated groups swam aimlessly and seldom went into the target quadrant.2.The effect of LaCl₃ on the mitochondrial quality,morphology and function.LaCl₃can down-regulate the levels of cardiolipin and expression level of TOM20 and TIM23.And LaCl₃can down-regulate ATP content and mitochondrial respiratory chain complex IV activity.LaCl₃ can change mitochondrial morphology in neurons,reduce mitochondrial quality,down-regulate ATP content and mitochondrial membrane potential levels,and up-regulate ROS levels of mitochondria.3.The effect of LaCl₃ on mitochondrial biogenesis.LaCl₃ can down-regulate the expression level of PGC-1α,NRF1,TFAM and the relative content of mt DNA.Treatment with ZLN005 can activate the PGC-1α-NRF1-TFAM signaling pathway inhibited by LaCl₃,and increase the relative content of mt DNA.4.The effect of LaCl₃ on mitochondrial fission and fusion.LaCl₃ can up-regulate the expression of DRP1,p-DRP1Ser616,and down-regulate the expression of p-DRP1Ser637,MFN1,MFN2,and promote the fission of mitochondria.Treatment with Mdivi-1 can antagonize the enhancement of mitochondrial fission induced by LaCl₃.5.The effect of LaCl₃ on mitophagy.LaCl₃can up-regulate the protein expression of PINK1,LC3B-II and PARKIN in cells and mitochondrial components,down-regulate the expression of P62,and increase the production of mitophagosomes.Treatment with Mdivi-1 can antagonize the excessive mitophagy caused by LaCl₃ promoting mitochondrial fission.Conclusion:1.LaCl₃ can cause learning and memory impairment of rats.2.LaCl₃ can damage mitochondrial quality,morphology,and function.3.LaCl₃ can inhibit the PGC-1α-NRF1-TFAM signal pathway and affect the biogenesis of mitochondria.4.LaCl₃ can up-regulate the protein expression of DRP1 and p-DRP1 Ser616,and down-regulate the protein expression of MFN1 and MFN2,promote mitochondrial fission and disrupt the mitochondrial fission and fusion balance.5.LaCl₃ can activate the PINK1-PARKIN signaling pathway and induce excessive mitophagy.6.After the intervention of ZLN005 and Mdivi-1,it can up-regulate the level of mitochondrial biogenesis,down-regulate the level of mitochondrial fission and mitophagy,and antagonize the La-induced mitochondrial quality control disorder.