Role Of Phosphorylated Synapsin Ⅰ And Related Kinases In The Impairment Of Amino Acid Neurotransmitter Release After Microwave Radiation

Background: Microwaves have become extremely important for many industries,particularly in communication and medical fields. With the increasing understandingof the latent health hazards of microwave exposure, it is becoming clear that effectivemethods for protection and treatment of those working in the presence of microwavesare urgently needed. Among potential environmental risk factors of learning andmemory ability, microwave exposures have received some attention. Central nervoussystem is one of the most sensitive targets of microwave radiation, but themechanisms of injury of learning and memory ability are still under investigation.Synaptic plasticity is the neurobiology foundation of learning and memory abilitywhich is known as the advanced function of brain. The essence of synaptic plasticityincludes the plasticity of synaptic junction and synaptic transmission which is realizedby neurotransmitter release. In presynaptic membrane, phosphorylated synapsin Iregulates the release and transmission of neurotransmitters. Abnormal release ofneurotransmitter after microwave radiation can cause the injury of learning andmemory ability, but the mechanisms are still unclear. So in the study, synapsin I as atarget, the potential mechanism of the injury of synaptic junction and synaptictransmission after microwave radiation were investigated.Materials and methods:(1) Wistar rats were placed in individual polypropylenecages under microwave exposure which the mean power density was30mW/cm2for5min. Assessment of behavior was performed by the Morris water maze task at6h,1d,2d,3d,4d,7d and14d after microwave exposure. The hippocampus structure wasdetermined by HE staining and mossy fiber sprouting was assessed by Timm staining.Immunofluorescence was used to detect the expression of synapsin I in hippocampus.Neurotransmitter release of synaptosomes was examined using high performance liquid chromatography. Immune electron microscopy and westernblot were usedrespectively to detect the location and expression of phosphorylated synapsin I (p-SynI) and vesicle neurotransmitter transporters.(2) PC12cell line was exposed bymicrowave under the mean power density of30mW/cm2for5min. Neurotransmitterrelease was examined by high performance liquid chromatography at1h,6h,12h,1d,2d, and3d after microwave exposure. Immunofluorescence and Westernblot wereused to detect the expression of p-SynI. Synapsin I RNAi and the inhibitors ofupstream kinases of phosphorylated synapsin I were used to intervene.Results:(1) Effect of microwave radiation on learning and memory ability andhippocampus structure. Decrease of learning and memory ability of rats aftermicrowave exposure. Compared to the sham group, the average escape latency (AEL)of rats were significantly longer in the exposure group at1d,2d,3d and7d (p <0.05or p <0.01) after exposure. Microwave radiation of30mW/cm2induced the injuryof hippocampal structure. Pycnosis and deep staining of pyramidal cells, granularcells and glial cells were observed at pyramidal layer and granular cell layer in theCA3and the dentate gyrus of the hippocampus. Vascular enlarged perivascular spaceswere detected in the same location of the hippocampus. The mossy fiber sproutingof30mW/cm2group were decreased compared to sham group at7d,14d,28d (p <0.01or p <0.05) after exposure. At3d after exposure, the synapsin I expressionwas significantly downregulated in the CA3and the dentate gyrus of hippocampuswhich was consistant with the inhibition of mossy fiber sprouting in its time and thelocation.(2) Effect of microwave radiation on synapsin I, phosphorylatedsynapsin I (p-syn I) and vesicle neurotransmitter transporter. After30mW/cm2microwave radiation in1d, synapsin I expression was found significantly increasedcompared to sham group(p <0.01). The expression of synapsin I was decreased at thegreatest scale at3d after exposure (p <0.01). P-Syn I (ser-62/67) proteinexpression was increased significantly at1d and3d after exposure (p <0.05and p <0.01, respectively), while there was no difference at7d between the exposed and shamgroups. Unlike ser-62/67, the expression of p-Syn I (ser-553) was decreased at3dafter the exposure (p <0.01) with subsequent elevated protein expression at7d and 14d (p <0.01). No protein expression variation was observed for p-Syn I (ser-603). Vesicular GABA transporter (VGAT) protein expression was significantly elevatedin the exposed group at6h,1d and7d (p <0.01). Vesicular glutamate transporter1(VGLUT1) was found to be down-regulated at the early time points (6h and1d, p <0.05and p<0.01) whereas significant up-regulation was observed in the latermeasurements (7d and14d, p<0.01). At7d after microwave exposure, numeroussmall clear synaptic vesicles gathered abnormally in presynaptic terminals.Additionally, the immunogold labeling of p-Syn I (ser-553) and VGAT was enrichedin these vesicles while VGLUT1and p-Syn I (ser-62/67) remained unchanged.(3)Effect of microwave radiation on the expression of Cdk5, Calpain and p35/p25inthe hippocampus of Wistar rats. Cdk5expression was significantly elevated in theexposed group at6h (p <0.05),3d (p <0.01) and7d (p <0.01) with subsequentdecreased protein expression at14d(p <0.01). Calpain expression was significantlyelevated in the exposed group at3d (p <0.01) with subsequent decreased proteinexpression at14d (p <0.05). p25expression was significantly decreased in theexposed group at1d (p <0.05) with subsequent elevated protein expression at3d and7d (p <0.01).(4) Effect of microwave radiation on the expression ofphosphorylated synapsin I (p-Syn I) in PC12cell line. The protein expression ofp-Syn I (ser-553) was greatly attenuated at6h and12h after microwave exposure (p <0.01) with subsequent elevated protein expression at48h(p <0.01). The proteinexpression of p-Syn I (ser-62/67) was greatly elevated at6h after microwave exposure(p <0.05) and back to normal expression level at1d.(5) Silencing p-Syn I resultedin the reduction of GABA release. Compared to control group, the expression of SynI and p-Syn I (ser-553, ser-62/67) were found to be significantly down-regulated aftershRNA transfection (p <0.01).(6) Effect of U0126and Roscovitine on theexpression of p-Syn I (ser-553, ser-62/67) and their related kinases in PC12cellsafter microwave exposure. The expression of p-ERK and p-Syn I (ser-62/67) wasgreatly elevated after exposure (p <0.05) while decreased expression was detectedafter using U0126with the most suppression effect under the combination of U0126and exposure (p <0.01). The expression of Cdk5was greatly elevated after exposure (p<0.05) while decreased expression was detected after using Roscovitine with themost suppression effect under the combination of Roscovitine and exposure (p <0.01).The expression of p-Syn I (ser-553) was greatly decreased after exposure (p <0.05)while increased expression was detected after using Roscovitine with the mostelevation effect under the combination of Roscovitine and exposure (p <0.01).(7)Effect of microwave radiation on the release of amino acids neurotransmitter inhippocampus of Wistar rats and PC12cell line. Compared to sham group, therelease of GABA was significantly decreased in the synaptosomes of hippocampusafter30mW/cm2microwave radiation (p <0.01). Compared to sham group, therelease of GABA and GLY were significantly decreased in PC12cell line after30mW/cm2microwave radiation (p <0.01). GABA release was decreased aftersynapsin I silencing or microwave exposure(p <0.01). Maximal reduction of GABArelease was found after synapsin I silencing combined with microwave exposeure(p <0.01). Glutamate and glycine levels were also found to be diminished as a result ofmicrowave exposure and/or synapsin I silencing. Combination of Roscovitine andradiation resulted in maximal elevation on the release of GABA (p <0.05) whilecombination of U0126and radiation showed no difference compared to the exposuregroup.Conclusions:30mW/cm2microwave radiation could result in:1. The decrease oflearning and memory ability of rats and the injury of hippocampal structure in theCA3and the dentate gyrus shown as the neuron impairment and decrease of mossyfiber sprouting. The synapsin I expression was significantly downregulated in theCA3and the dentate gyrus of hippocampus indicating that microwave radiation cansuppress the expression of synapsin I to cause the impairment of synaptic structure,the deficits of learning and memory.2. Abnormal release of GABA and significantlyelevated expression of VGAT was showed that synaptic veslcles intake of GABAincreased, and synaptic vesicles which contained GABA doking and fusion reduced atpresynaptic membrane after exposure.3. The elevated expression ofp-ERK-dependent phosphorylated synapsin I (ser-62/67), showed that increasedactivity of p-ERK positive regulate the expression of phosphorylated synapsin I (ser-62/67) after exposure.4. The decreased expression of Cdk5-dependentphosphorylated synapsin I (ser-553), elevated expression of Cdk5、Calpain and p25,enrichment of p-Syn I (ser-553) and VGAT in the presynaptic accumulated vesicleindicates that Cdk5/p25negative regulate phosphorylation of synapsin I at ser-553resulting in the decreased GABA release.