Effects Of Lanthanum Chloride On The Protein Expression Related To Mitochondrial Axon Transport And Anchoring In Pup Rat Hippocampus

Objective:Rare earth metals,also called rare earth elements（REEs）,have received widespread attention in recent years.China is also known as a rare earth country.Rare earth is widely used and has potential impacts on human body due to environmental residues.Based on existing epidemiological research reported in China,children living in rare earth mining areas have lower cognitive development and cognitive abilities.Experimental studies have also confirmed that the accumulation of REEs in the body threatens human health.Lanthanum（La）,as a representative of REEs,can not be ignored due to its active biological properties and high accumulation level in the brain.Information transfer between neurons occurs at the synaptic site,and mitochondria power the synapse with high energy requirements.Therefore,mitochondrial axon transport and accurate anchoring are extremely important for maintaining synaptic function.In this study,through animal experiments,pathological,neurobehavioral,molecular biology,and co-immunoprecipitation techniques were used to explore the mechanism of La on the protein expression related to mitochondrial axon transport and anchoring,thereby cause learning and memory impairment in offspring rats.Methods:Thirty-two female and thirty-two male Wistar rats were used by the Experimental Animal Center of China Medical University.The weight range was 250±10 g.The experimental animal room temperature was maintained at 22±1°C and the relative humidity was maintained at 50±5%.After a one-week adaptation period,64adult Wistar rats were randomly divided into control group,low,medium,and high dose LaCl₃ group.Mating with male rats in the same cage at the ratio of 1:1.The pregnancy was judged to be the 0th day of pregnancy,when the vulva was found.Female rats in the control group,low,medium and high dose groups were given distilled water,0.125%,0.25%,0.5%LaCl₃ respectively,in distilled water.After the end of pregnancy and lactation,poisoning was ended,all kind of indexes were detected in the pups.Water maze and jumping test were used to test the learning and memory ability of the pups,HE staining and transmission electron microscopy were used to observe the pathological damage of the hippocampal neurons and ultrastructure of mitochondria and synapses in the neurons Abnormal changes.Immunofluorescence was used to detect the distribution of mitochondria at synaptic sites,and ELISA was used to detect hippocampal mitochondrial function indicators.Western blot was used to detect hippocampal Cyt c expression in mitochondria and cytoplasm.The expressions of hippocampal KIF5B,KLC1,KLC2,Syntabulin,FEZ1,TRAK1,RHOT1,αTubulin,SNPH were determined by Western blot and Realtime PCR.And use Western blot to detect RHOT2,JIP1,and DISC1 expressions.Co-immunoprecipitation method was used to detect the interaction between KIF5B and F E Z 1,S y n t a b u l i n,αT u b u l i n a n d S N P H p r o t e i n i n h i p p o c a m p u s.Results:1.The effect of LaCl₃ on the learning and memory ability of pups.With the increase of LaCl₃ dose,the learning and memory abilities of the pups in each dose group decreased compared with the control group.The results of the positioning navigation test of the Morris water maze experiment showed that compared with the control group,the escape latency of the young rats in each LaCl₃ dose group to find the underwater platform increased.The results of the space exploration test showed that compared with the control group,the cumulative stay time of the pups in the LaCl₃ dose group and the number of times the pups crossed the target quadrant decreased with the increase of the LaCl₃ dose.The results of the platform jump test showed that compared with the control group,as the LaCl₃ dose increased,the number of errors increased gradually,and the latency of the first jump off the platform gradually decreased.2.The effects of LaCl₃ on the morphological structure,mitochondria and synaptic ultrastructure of hippocampal neurons in offspring.In the control group,the neurons were arranged neatly and tightly as a whole,and the neuron cells were full in shape and intact in structure.With the increase of La-dosing dose,the neurons in each dose group had different degrees of structural damage,the pyknosis of the nucleus,the arrangement gap between cells became larger,and the tissue structure was scattered.In the control group,the mitochondrial membrane structure was clear and the mitochondria were round and full.When the dose of LaCl₃ was increased,the damage of the mitochondrial membrane structure gradually increased,the mitochondrial ridge became obscured,and the ultrastructure changed abnormally.3.LaCl₃ affects the expression and co-localization of synaptic vesicle marker protein SV2 and mitochondrial outer membrane protein TOM20 in the hippocampus of young rats.The co-expression of the two gradually weakened with the increase of the dose of LaCl₃.4.The effects of LaCl₃on mitochondrial function in hippocampus of young rats.Compared with the control group,the ATP concention COX IV,PDHC,and KGDHC activity were decreased with the increase of the dose.Compared with the control group,Cyt c protein level was decreased in mitochondria and increased in cytoplasm.5.The effects of LaCl₃ on mitochondrial axonal transport motor protein Kinesin-1（KIF5B、KLC1、KLC2）and its adaptors（Syntabulin,FEZ1,TRAK1,RHOT1,RHOT2）,orbital protein（αTubulin）,anchor protein（SNPH）related mRNA transcription and protein expression levels.KIF5B,KLC1,Syntabulin,FEZ1,TRAK1,RHOT1,αTubulin mRNA transcription and protein expression and KLC2,RHOT2,SNPH,protein expression levels were lower than the control group.6.The effects of LaCl₃ on the expression levels of regulatory factors JIP1and DISC1 in hippocampal axonal transport of young rats.Compared with the control group,the JIP1 protein level decreased and the DISC1 protein expression level increased in a dose-depenent manner.7.The effects of LaCl₃ on the interaction between KIF5B and FEZ1,Syntabulin,Tubulin,and SNPH proteins in the offspring.The data suggested that FEZ1,SNPH,Syntabulin,andαTubulin were combined with KIF5B,respectively,and all proteins’interaction levels were reduced in the 0.5%LaCl₃ group,when compared with the control group.Conclusion:1.LaCl₃ exposure during pregnancy and lactation can disrupt the learning and memory ability of pups.2.LaCl₃ exposure during pregnancy and lactation can damage the morphology,mitochondria and ultrastructure of neurons in hippocampal neurons of offspring.3.LaCl₃ exposure during pregnancy and lactation can reduce the distribution of mitochondria at synaptic sites.4.LaCl₃ exposure during pregnancy and lactation damaged hippocampus mitochondrial function of offspring rats.5.KIF5B,KLC1,KLC2,Syntabulin,FEZ1,TRAK1,RHOT1,RHOT2,alpha Tubulin,SNPH expression levels decreased,regulating factor JIP1 and DISC1 abnormal expression,interaction between KIF5B and FEZ1,Syntabulin,alpha Tubulin,SNPH damaged caused mitochondrial axon transport and anchor disordered.It may related to the disruption of mitochondrial distribution and function,and the disruption of mitochondrial and synaptic function.