Regulation Of RyR3 On Neurite Outgrowth And Memory Related Proteins In PC12 Cells Exposed To Low Dose Lead

Objective:Lead neurotoxicity has attracted much attention and become one of the hot topics of public health.It has been shown that lead can cause the increase of intracellular free calcium level in hippocampal neurons of rats and lead to intracellular calcium disorder,and then influence the development of neuron dendrites.While calcium(Ca²⁺)downstream learning and memory related Ca²⁺/calmodulin-dependent protein kinase II?CaMKII?and cAMP-response element binding protein?CREB?expressions are abnormal,which lead to learning and memory and cognitive impairment.However,it is not completely clear the pathway or mechanism of lead causes intracellular calcium disorders.Intracellular calcium stores are important calcium homeostasis regulatory systems in cells,including endoplasmic reticulum calcium stores and mitochondrial calcium stores.The ryanodine receptors?RyRs?in the Ca²⁺release channel of the endoplasmic reticulum are the largest Ca²⁺release channels ever discovered.More and more studies have found that RyRs-mediated calcium release plays a key role in the process of synaptic plasticity and memory.So,whether lead may affect the learning and memory process by affecting RyRs to cause intracellular calcium disorders?RyR3 is called brain type because it was first detected in the brain.It is mainly distributed in hippocampus and cortex and other areas that participate in learning and memory formation.In this study,the lead exposure model of rat adrenal medullary chromaffin cell line?PC12 cell?was established in vitro and used flow cytometry,F-actin staining,immunofluorescence,plasmid transfection and other research methods to explore the role of RyR3 in low-dose lead exposure causing intracellular calcium disorders,cell neurite outgrowth,and changes in memory-associated proteins,which provides a basis for further study of the mechanism of lead exposure neurotoxicity.Methods:?1?PC12 cells were treated with lead acetate?0?M,0.01?M,0.1?M,1?M,10?M?respectively for 24h,48h,and 72h.The effects of lead on cell viability,protuberance growth and the expression of alpha and protein were detected by CCK8,F-actin staining and immunofluorescence respectively.?2?PC12 cells were treated with lead acetate?0?M,0.01?M,0.1?M,1?M,10?M?respectively for 24h,48h and 72h.The effects of lead on the level of free calcium and the expression of RyR3 protein in PC12 cells were examined by flow cytometry and immunofluorescence.?3?PC12 cells were transfected with the RyR3-shRNA plasmid by using liposome transient transfection assays and then treated with 1?M lead acetate for 48h.And F-actin staining and immunofluorescence were used to detect the growth of PC12 cells and the expression level of RyR3,CaMKII?and CREB proteins.Results:?1?Effects of low-dose lead exposure on PC12 cell viability,cell neurite outgrowth,and expression of learning and memory-related proteins Ca MKII?and CREB.The CCK8 assay showed that after lead exposure PC12 cells 24h and 48h,there was no significant difference in cell viability between groups of lead exposure groups?P>0.05?.After lead exposure 72h,the activity of PC12 cells in only 10?M lead exposed group decrease?P<0.05?.When exposure to less than 10?M lead acetate,that is,lead exposure did not have a significant effect on cell viability,F-actin staining revealed that the average neurite length and tip end numbers of cells were reduced in a time-and concentration-dependent manner when the NGF-induced PC12cells were exposed to Pb for 24h,48h and 72h?P<0.05?;while immunofluorescence assay showed that the expression of CaMKII?and p-CaMKII?protein in PC12 cells was up-regulated and p-CREB protein levels were down-regulated in a time-and concentration-dependent manner when the PC12 cells were exposed to Pb for 24h,48h and 72h?P<0.05?;p-CaMKII?/CaMKII?and p-CREB/CREB ratio decreased after lead exposure for 48h and 72h?P<0.05?.?2?Low-dose lead exposure resulted in elevate intracellular free calcium levels and high RyR3 protein expression in PC12 cells.Flow cytometry showed that the level of free calcium in PC12 cells increased in a time-and concentration-dependent manner when the PC12 cells were exposed to Pb for 24h,48h and 72h?P<0.05?.While the immunofluorescence detection showed that the RyR2 and RyR3 protein expression level was up-regulated in a time-and concentration-dependent manner when the PC12 cells were exposed to Pb for 24h,48h and 72h?P<0.05?,RyR3protein expression was consistent with the changes of intracellular free calcium level.?3?Knocking down RyR3 reduce the effect of low dose lead exposure on PC12cell neurite outgrowth,learning and memory related protein CaMKII,CREB.PC12cells were transfected with RyR3-shRNA plasmid by liposome and exposed to 1?M lead acetate for 48h.Immunofluorescence assay showed that RyR3-shRNA transfection knocked down the high expression of RyR3 induced by lead exposure compared with Con-shRNA vector.Transfection of RyR3-shRNA Improves P-CaMKII?/CaMKII?and p-CREB/CREB Ratios decrease caused by lead exposure?P<0.05?;In addition,F-actin staining assay showed that the average neurite length and tip end numbers also be improved in cells transfected with RyR3-shRNA plasmid compared to transfect Con-shRNA cells?P<0.05?.Conclusions:Lead exposure may increase the level of free calcium in nerve cells through up regulation of RyR3 protein,which may lead to neurite growth disorder and changes in learning and memory related proteins.It is suggested that RyR3 may be a new target for intervening lead induce calcium disturbance and subsequent development of learning and memory impairment.