The Role And Mechanism Of Cadherin-catenin Complex In The Pruning Disorder Of Dendritic Spines Caused By Lanthanum

Objective:Rare earth elements(REEs)are the general term for 15 lanthanide elements with atomic numbers ranging from 57 to 71 in the periodic table of chemical elements,as well as scandium and yttrium with similar chemical properties.They are widely present in nature.REEs have low fluidity and persist in the environment.They can accumulate in foods,vegetables and other foods through soil,drinking water,etc.,and then enter the human body and accumulate through the food chain,causing health damage to the body.Studies have confirmed that REEs,especially the lanthanide elements,have a special affinity for nerve cells,which are easy to accumulate in the brain and can cause damage to the nervous system.The IQ and cognitive development levels of children living in rare earth mining areas are significantly lower than those of the same age in the control area.child.Lanthanum(Lanthanum,La),as a representative of light rare earth elements,is rich in reserves in the earth's crust and has relatively active chemical properties.It is often used as a representative material for the study of REEs.Hu et al.confirmed that exposure to La Cl3 through drinking water from the mother mouse's pregnancy to 1 month after birth can cause La accumulation in the hippocampus and cortex of the offspring.The previous research of our research group confirmed that La Cl₃ can affect the expression of NF-?B,c-fos,c-jun and BDNF and other proteins to reduce the learning and memory ability of rats.At the same time,La can also cause damage to the nervous system and abnormal morphology and structure of primary neurons and glial cells.Therefore,La has a clear neurotoxicity,but its toxicity mechanism is still very unclear,and further exploration is needed.Dendritic spines are functional tiny protrusions on the branches of neuron dendrites,which are widely found in mammalian brain tissues.As the original site of excitatory synaptic transmission in the central nervous system,dendritic spines rely on their enlarged head and contracted neck to accommodate the post-synaptic compacts and related organelles of excitatory synapses,which play a role in neurons.Important biochemical and electrical signal conduction.Dendritic spines undergo continuous dynamic changes during their development.Studies have shown that slender spines are more active,while mushroom and stubby spines are more stable and have greater postsynaptic density.In the process of neurodevelopment,dendritic spines will gradually transform from slender immature spines to stable mature spines.In addition to morphological changes,there are dynamic changes in the number of dendritic spines.Studies have confirmed that the dendritic spines develop rapidly in the early postnatal period,and the number continues to increase.After the individual enters puberty,in order to achieve the best information transmission and storage effect,the neural network will self-optimize after it has developed to a certain degree of complexity,which is mainly manifested as a tree.The pruning mechanism of the spine is specifically reflected in the reduction of the total number of dendritic spine.In this process,about40%of the dendritic spines will be selectively eliminated,and the remaining dendritic spines will mainly exist in the form of mature spines.In addition,in vivo imaging of the mouse sensory cortex showed that during brain maturation,the rate of dendritic spine elimination exceeds the rate of dendritic spine formation.This rapid dendritic spine elimination indicates that there is a tree in the process of neural circuit refinement and formation.The pruning mechanism of the spine.Studies have shown that there are abnormalities in dendritic spine pruning in many developmental neurological diseases such as autism spectrum disorder and schizophrenia.It has been confirmed that the key molecule that determines the fate of dendritic spines during the pruning process of dendritic spines is the cadherin-catenin cell adhesion complex.The cadherin-catenin complex is a type of cell adhesion complex that exists widely in the synaptic transmitter release area and the post-synaptic dense zone.It is mainly composed of neural cadherin(N-cadherin)and?-catenin(?-catenin).catenin)and?N-catenin(?N-catenin).Studies have confirmed that in the pruning mechanism of dendritic spine,the biological effect of cadherin-catenin complex on dendritic spine pruning is mainly realized by N-cadherin and?-catenin.N-cadherin is highly expressed in nerve tissues and participates in processes such as neuron migration,axonal guidance,dendritic spines and synaptic functions,and synaptic plasticity.?-catenin is a multifunctional protein with dual activities,which plays an important role in mediating cell adhesion and regulating signal transduction.On the one hand,?-catenin can combine with cadherin on the cell membrane to play a role in cell adhesion.On the other hand,it can also be an important functional protein in the classical Wnt signaling pathway to participate in signal pathway regulation.Studies have shown that?-catenin can play an important role in regulating dendritic morphology,dendritic spine density,postsynaptic structure and function through intercellular adhesion.Based on the above,this study uses a combination of whole animal experiments and in vitro primary neuron culture,starting from the Wnt/?-catenin signaling pathway,to explore the effect of La on the pruning of dendritic spines regulated by the cadherin-catenin complex.To clarify the neurotoxicity and mechanism caused by La to provide new theoretical reference and laboratory data.Methods:In vivo experiment.72 experimental Wistar rats provided by the Experime-ntal Animal Center of China Medical University,weighing 230±20 g,and the ratio of male to female is 1:1.The temperature in the experimental animal room is between 17?23?,and the corresponding humidity is maintained at 45-55%.The feed for the experimental animals is provided by the Qianmin Experimental Animal Feed Factory in Yuhong District,Shenyang.The animals were bred adaptively for a week to adapt to the environment,and then the female rats were randomly divided into cages according to the control group and the low,medium and high dose La-exposed groups,with 12 in each group,and then mated in a cage at a ratio of 1:1 between female and male rats.The rats were observed the next morning.If a vaginal plug or vaginal secretion was found to have sperm under microscope,the female rats could be judged to be pregnant,which was recorded as the 0 th day of pregnancy.The pregnant female rats were bred separately,the control group drank distilled water,and the low-dose and high-dose groups drank distilled water containing 0.125%La Cl₃ and 0.5%La Cl₃ respectively,and the poisoning was stopped on the 50th day after birth(postnatal day,PN50).The pups were stained with La by sucking breast milk before weaning,and after weaning(PN21)they took in the corresponding concentration of La Cl₃aqueous solution.The animals were taken on the 30th day(PN30),the 35th day(PN35),the 40th day(PN40),and the 50th day(PN50)of the pups respectively after birth to establish an animal model,and then measure various indicators.Golgi-Cox staining was used to observe the changes in the hippocampal neurons of the offspring at each time point;the blood La content of the mother and the offspring at each time point was detected by ICP-MS;Use Real time PCR,Western blot,etc.to detect hippocampal tissue WNT7A/B,LRP5,LRP6,Frizzled 7,DVL,GSK-3?,p-GSK-3?(Ser9),AXIN1,AXIN2,?-catenin,p-?-catenin(Ser33/37/Thr41),N-cadherin and?N-catenin m RNA transcription and protein expression levels.In vitro experiments.Primary neurons were cultured with pups within 24 hours after birth.After the neurons were cultured in vitro for 10 days,they were treated with serum-free medium containing 0,0.025,0.05 and 0.1 m M La Cl₃ respectively.After 24hours of culture,the changes in related indicators were detected.After neurons were identified by neuron-specific purified enzymes,the changes in dendritic spines in primary cultured neurons were observed by Dil staining.Western blot was used to detect WNT7A/B,LRP5,LRP6,Frizzled 7,DVL,GSK-3?,p-GSK-3?(Ser9),AXIN1,AXIN2,?-catenin,p-?-catenin(Ser33/37/Thr41)?N-cadherin and?N-catenin protein expression levels in primary neurons;After pretreatment with GSK-3?activity inhibitor SB-216763,the changes in dendritic spines and the expression levels of downstream related proteins were re-observed;After N-cadherin and?-catenin were overexpressed,the changes of dendritic spines were re-observed.Result:1.The content of La Cl₃ in the blood can increase with the time and dose of exposure.2.La Cl₃ can cause the dendritic spine density of PN30,PN35,PN40 and PN50 hippocampal neurons to decrease.During the period from PN30 to PN50,the dendritic spine density of each dose group decreased.Among them,the control group decreased by 54.53%,the 0.125%La Cl₃ exposure group decreased by 48.98%,and the0.5%La Cl₃ exposure group decreased by 46.17%.Among them,during the period from PN30 to PN50,the dendritic spine density of the control group decreased by 54.53%,the 0.125%La Cl₃exposure group decreased by 48.98%,and the 0.5%La Cl₃ exposure group decreased by 46.17%,which were significantly lower than the control group.In addition,mature mushroom and stubby dendritic spines showed a downward trend with increasing La Cl₃ exposure,while immature branched and thin dendritic spines showed no statistically significant changes.At each time point of PN30,PN35,PN40 and PN50,the proportion of immature dendritic spines in the total dendritic spines of young mice increased with the increase of the exposure dose.3.La Cl₃ exposure can induce down-regulation of WNT7A/B,LRP5,LRP6,Frizzled 7,DVL,GSK-3?,p-GSK-3?(Ser9),AXIN1,AXIN2,?-catenin,p-?-catenin(Ser33/37/Thr41),N-cadherin??N-catenin protein expression and m RNA transcription levels.4.SB-216763 pretreated neurons can antagonize the decrease of dendritic spines density and the decrease of p-GSK-3?(Ser9),?-catenin,p-?-catenin(Ser33/37/Thr41)and N-cadherin protein expression levels caused by La exposure.5.Overexpression of N-cadherin and?-catenin can antagonize the decrease of dendritic spine density caused by La exposure and increase the density of dendritic spine.Conclusion:1.La can accumulate in the blood of rats;2.La can cause obstacles to the pruning mechanism of dendritic spine in offspring rats,and slow down the rate of dendritic spine reduction of hippocampal neurons;3.In in vivo and in vitro experiments,La can down-regulate the expression of related molecules in the Wnt/?-catenin signaling pathway,resulting in a decrease in the expression of?-catenin and N-cadherin,and destroying the cadherin-catenin complex;4.SB216763 pretreatment can antagonize the decrease of dendritic spine density caused by La exposure,and up-regulate the decrease of p-GSK-3?(Ser9),?-catenin,p-?-catenin(Ser33/37/Thr41)and N-cadherin protein expression levels caused by La;in addition,overexpression of?-catenin and N-cadherin can antagonize the decrease of dendritic spine density of primary cultured neurons caused by La exposure;5.The dendritic spine pruning disorder caused by La exposure may be caused by the abnormal change of the cadherin-catenin complex mediated by the down-regulation of the Wnt/?-catenin signaling pathway.