Effect And Mechanism Of Lanthanum Chloride On Mitochondrial Axon Transport In Hippocampal Neurons

Objective:In the periodic table,lanthanides,thorium and yttrium with atomic numbers between 57 and 71 are collectively referred to as rare earth elements?REEs?.It is widely used in medical,chemical industry,animal husbandry.When more and more REEs appear in the environment,the body will have more and more opportunities to contact it,and the harm caused by it will increase.Epidemiological survey results show that in the major rare earth mining areas in China,the average children's IQ?cognitive function,learning and memory ability,etc.?are obviously lower than those in the control area.Due to the stability of lanthanum itself,and its ability to accumulate in brain tissue and generate biological activity,it is used as a representative element to study the injure of REEs on the nervous system.Synaptic plasticity of the nervous system involves structural and morphological changes,such as the growth of dendritic spines and the occurrence of synapses.These modifications are the cell's response to adaptation in neuronal activity and are thought to be relevant for learning and memory.Mitochondria are found in axon terminals and dendrites of neurons and are deemed to play an crucial role in synaptic plasticity.Studies have shown that in neurons,newly synthesized protein complexes and organelles will be carried to axons and synaptic ends through anterograde transport.This mode of transport is very important.Any defects in mechanical movement based on microtubules will cause defects and many human nervous system diseases.Mitochondria,as organelles with high productivity,also need long-distance and directional axon transport to reach various energy demand points.Among them,the energy demand of synapses is important to maintain the functional activity of synapses.In this study,lanthanum chloride?LaCl₃?was used to treat primary cultured neurons to build an in vitro cell injury model.The effects of La on the transport and anchoring of mitochondria on axons and their functions in neurons were observed.From the perspectives of kinesin,adapter protein,orbital protein and anchoring protein of axon transport,the mechanism of the disorder of mitochondrial axon transport and anchoring induced by La was studied.Using1-Azakenpaullone to intervene in the corresponding action links,and providing new methods and scientific research ideas for further elucidating La's neurotoxic mechanism and finding effective molecular targets.Methods:60 adult Wistar rats?20 males,40 females?weighing 260±10 g were kept in animal room with a comfortable humidity and temperature?45-55%,17-23??.After raising another week,20 cages of rats were randomly grouped into different cages with female:male=2:1 for each one.Acoording to former experience,Vaginal secretions will be found in the tray as the 0th day of pregnancy.The pups were fetched within 24 h after birth.The hipocampus were taken from the head to perform primary neuron culture.The immunofluorescence was used to identify the purity of primary neurons in order to pursue the following experiences.First,the neuronal cells were treated with 0.025,0.05,0.1,0.2,0.5 mmol/L LaCl₃ for 24 h,respectively,and the cell survival rate was detected.After the dose is determined,the primary cultured neurons are treated with 0.025,0.05,and 0.1mmol/L LaCl₃ for 24 h,and the following indicators are detected:?1?neuron morphological observation;?2?neuron mitochondrial ATP detection;?3?Detection of neuron mitochondrial morphology and axon transport;?4?Detection of mRNA and protein expression levels of neuron KHC,KLC1,KLC2,SNPH and detection ofprotein expression levels of neuron SYNTABULIN,TRAK1,FEZ1,RHOT1,RHOT2,?-TUBULIN,?-TUBULIN,JIP1,DISC1.?5?The neurons were treated with control group,control plus 5mM 1-Azakenpaullone group,0.1 mmol/L LaCl₃ group and 0.1 mmol/L LaCl₃ plus 5mM 1-Azakenpaullone group,respectively,for 24 h.TAU and P-TAU'protein expression levels were detected,morphology of the neurons were observed,and neurons mitochondrial axon transport were measured.Results:Compared with the control group,neurons'survival rate in the LaCl₃ treatment group was remarkablely decreased,and the survival rate gradually reduced with the increase of the dose.Compared with the control group,the neurons among different LaCl₃treatment groups had fewer cell numbers,atrophy of cell body morphology,and network connection damage.LaCL₃ can reduce the mRNA and protein expression levels of KHC,KLC1,KLC2,SNPH,and decrease the protein expression levels of SYNTABULIN,TRAK1,FEZ1,RHOT1,RHOT2,?-TUBULIN,?-TUBULIN,JIP1,and up-regulate the protein expression of DICS1.Compared with the control group,the mitochondrial ATP level of the neurons in the LaCl₃ treatment group was arrestingly lower than that in the control group.There is a prominent statistical difference between the 0.05mM LaCl₃ or the0.1mM LaCl₃ group and the control group.At the same time,In the LaCl₃ treatment group,the mitochondrial movement rate on the neuron axons decreased,occurred separation,shortened length,decreased distribution,and decreased density.Compared with the LaCl₃treatment group,the intervention group recovered neuronal cell damage to a certain extent,the mitochondrial migration rate increased,and the proportion of P-TAU in the total TAU increased.Conclusions:1.La can have a detrimental effect on neurons,resulting in decreased mitochondrial function of primary cultured neurons,leading to a significant decrease in ATP levels.2.La can down-regulate motor proteins expression of?KHC,KLC1,and KLC2?,anchor proteins?SNPH?,adapter proteins?SYNTABULIN,TRAK1,FEZ1,RHOT1,and RHOT2?,and orbital proteins??-tubulin and?-tubulin?in neuron axons,causing abnormal expression of related regulatory factors,inhibiting mitochondrial transport efficiency on axons,and causing abnormal changes in mitochondrial morphology.3.1-Azakenpaullone,a specific interfering agent of TAU upstream regulator GSK3?,can to some extent antagonize the abnormal proportion of phosphorylated TAU in total TAU,mitochondrial axon transport disorder,and neuron injure attributed to lanthanum-induced neurons.It is indicated that the increased phosphorylation level of TAU plays a harmful role in La-induced neuron damage and mitochondrial axon transport disorder.