Mechanism Of Microwave Exposure Induced Abnormalities Of Dendritic Spines Plasticity Mediated By SNK-SPAR Pathway

Objective:Microwave technology has been widely used in the production,military and daily life.As is known to all,central nervous system is one of the most sensitive targets of microwave radiation.Epidemiological and experimental studies showed that microwave radiation could result in sleep disorder,abnormal EEG and deterioration of the memory.The hippocampus plays a key role in learning and memory.Synaptic plasticity is the neural basis of learning and memory,and the vast majority of excitatory synapses are composed of dendritic spines as their postsynaptic structure.Therefore,the related researches about synaptic plasticity,especially dendritic spines plasticity,have been widely concerned.SNK-SPAR pathway plays a key role in synaptic reconstruction depending on activation of neurons.In activated neurons,SNK and SPAR are phosphorylated and combined with each other,then identified and modified by ubiquitin ligase,degraded by proteasome pathway,causing loss of PSD-95,making mushroom shaped dendritic spines come into narrow ones.It is reported that the microwave radiation could induce abnormality in dendritic spines plasticity,however,the mechanism are rarely reported.Thus SNK-SPAR pathway was selected to explore the mechanism of abnormal dendritic spines plasticity caused by microwave radiation,thus to provide new candidate targets to prevent brain injury induced by microwave radiation.Materials and methods:(1)100 Wistar rats were radiated by microwave of 30mW/cm~2 at 2.856 GHz for one month,6 min/time,3 times/week.Morris water maze was proceeded to test the spatial learning and memory ability of rats,the navigation training was proceeded at 1~5 d after microwave radiation,the probe test was carried out at 14d after microwave radiation;at 6 h,7 d,14 d and 28 d after radiation,Golgi stain was applied to observe the distribution of dendrtic spines in hippocampus;TEM was adopted to detect the ultrastructure of neurons and dendritic spines in hippocampus;(2)The mRNA of SNK was detected by real time rt-PCR,the protein expression of SNK,SPAR and PSD-95 was detected by Western-blot;the colocalization of SPAR and PSD-95 was detected by double labled immunofluorescence;(3)Primary hippocampal neurons of rats were exposed to microwave of 30 mW/cm~2 at 2.856 GHz for 6 min×3 times,the interval of each exposure was 6 min.At 0 h,6 h,1 d,2 d and 3 d after microwave radiation,the cytomembrane was labeled by CM-Di I solution to detect dendrtic spine plasticity of neurons.The aminoacidtransmitters released by neurons were detected by HPLC.The mRNA of SNK was detected by real time rt-PCR,the protein expression of SNK,SPAR and PSD-95,other upstream moleculars such as CaN,CREB,p-CREB,NFATC4 and p-NFATC4,as well as the downstream moleculars such as Ras/Rap family were detected by Western-blot.The location and expression of p-CREB was detected by immunofluorescence;The phosphorylation and ubiquitylation modified levels/sites of SNK and SPAR were detected by co-immunoprecipitation and Nano LC MS/MS;(4)Aiming at the changed step,the inhibitor of SNK(BI2536)and Ca N(FK506)was added repectively,in order to find out the changes of the associated molecules in the following steps and the distribution of dendritic spines.Results:(1)Effects of microwave radiation on spatial learning and memory ability,distribution of dendritic spines and ultrastructure of hippocampal neurons of rats.(1)Spatial learning and memory:in navigation test,the AEL of rats in exposure group was significantly longer than that in control group(p<0.05 or p<0.01);in probe test,the percentage of time spent in target quadrant and the time of across the platform in exposure group were less than that in control group(p<0.05);(2)The distribution of dendritic spines in hippocampus:the density of dendritic spines in the granule cells of dentate gyrus was lower than control group at 6 h,14 d and 28 d after microwave exoposure(p<0.05),the perncentage of mature mushroom shaped spines in pyramidal cells was less than control group at 28 d after microwave exposure(p<0.05);(3)The ultrastructure of neurons/dendritic spines in hippocampus:expansion of endoplasmic reticulum,swelling and cavitation of mitochondria in neurons,spinule endocytosis at dendritic spines were observed in exposure group,the thickness and length of PSD were less than those in control group(p<0.05?p<0.01).(2)Influence of microwave radiation on SNK-SPAR pathway and its related moleculars in hippocampus of rats.(1)The mRNA of SNK in hippocampus was up-regulated compared to control group(p<0.01),while the expression of SNK was lower than control group at 6 h after microwave exposure and showed high expression at 14 d and 28 d(p<0.05 or p<0.01),the expression of SPAR was lower than control group at 6h and 14d(p<0.01),the expression of PSD-95 was lower than control group at 28 d after exposure(p<0.05);(3)The extent of interaction between SPAR and PSD-95 was significantly lower at 6 h and 28d(p<0.05 or p<0.01).(3)Effects of microwave radiation on distribution of dendritic spines and the related molecules of SNK-SPAR pathway in primary hippocampal neurons.(1)Both the density of spines and percentage of mature spines were decreased at 1d after microwave exposure(p<0.05)and came back to normal levels at 3 d;the glutamate released by primary neurons decreased at 6 h after exposure(p<0.01),the glycine higher than control groups except 6 h after exposure(p<0.05 or p<0.01);(2)The mRNA of SNK was decreased at 0 h after microwave exposure and then up-regulated compared to control group at 6 h~1 d after microwave exposure(p<0.01),the expression of SNK was lower than control group immediately after microwave exposure and showed high expression at 6 h and 1 d(p<0.05),the expression of SPAR was lower than control group at 0 h~1 d after exposure(p<0.05 or p<0.01),the expression of PSD-95 was lower than control group at 6 h~1 d after exposure(p<0.05 or p<0.01)(3)The phosphorylation sites in GKBD of SPAR which was the pecific binding domain with PSD-95 had changed from ser-1567 and ser-1472 to ser-1603 after exposure;the interaction of polyubiquitin-B with SNK and SPAR had increased in different degrees;there was interaction of SPAR and MAP2 after microwave exposure;(4)In Ras/Rap family,RapGEF2 and SynGAP decreased at 0 h~1 d after microwave exposure,RasGRF1decreased at 0 h~6 h after exposure(p<0.05 or p<0.01);(5)CaN,the upstream of SNK,was decreased at 0 h~3 d after microwave exposure(p<0.05 or p<0.01),p-CREB decreased at 1d after exposure(p<0.01)and p-CREB gathered to nucleus and showed high expression at 1 d after microwave exposure(p<0.05);the p-NFATC4 in nucleus was extremely low,NFATC4 showed no changes between exposure and control groups;(4)Intervention of SNK-SPAR pathway in primary hippocampal neurons.(1)After the addition of BI2536,the inhibitor of SNK,the expression of SNK in inhibited exposure group was lower than that in control group and exposure group(p<0.05 or p<0.01),the spine density in inhibited exposure group was significantly higher than that in control group and exposure group(p<0.05),the percentage of mature spines was significantly higher than exposure group(p<0.05)but showed no significance compared to control group;(2)After adding FK506,the inhibitor of CaN,the expression of CaN was significantly lower than exposure group(p<0.01),and p-CREB was significantly higher than exposure group(p<0.05);the SNK mRNA expression was significantly higher than exposure group;there was no difference in the protein expression of SNK between intervention and control group.Conclusions:30 mW/cm~2 microwave radiation could result in:1.Dysfunction of spatial learning and memory ability in rats,alteration of dendritic spines showed as spinule endocytosis,decrease in thickness and length of PSD,lower spine density in dentate gyrus and lower percentage of mature spines in pyramidal cells.2.Abnormality of SNK-SPAR related protein expresstion shown as the increase of SNK which might induce degradation of SPAR and loss of PSD-95,led to obstacle in spine maturation and debilitate synaptic connections which could injure the cognitive function of exposed rats.3.The density of dendritic spines and percentage of mature spines decreased in cultured primary hippocampal neurons,the excitatory amino acid released fewer.4.The homeostasis of SNK-SPAR pathway was disrupted:the low expression of CaN led to the increase and nuclear translocation of p-CREB,thus lifted the transcription and expression level of SNK;the interactiion between polyubiquitin-B and SNK/SPAR suggested the increase of phosphorylation of SNK and SPAR,proteins in Ras/Rap family,the downstream of SNK,showed disequilibrium expression after microwave exposure;5.SNK was one of the effective targets in SNK-SPAR pathway mediated abnormality dendritic spines plasticity induced by microwave radiation.6.The transformation of phosphorylated modification sites in SPAR might influenced the combination with PSD-95;SPAR interacted with MAP2,which might play a role in obstacle in spine maturation and debilitated synaptic connections.