The Effects Of Glutamate Excitotoxicity On The Injury Of Motor Neurons Induced By Syncytin-1

Background: Motor neuron disease(MND)is a fatal degenerative disease of the nervous system.At present,the pathogeneses of MND is still unknown.The glycoprotein syncytin-1,which is encoded by the env gene of the human endogeneous retrovirus-W(HERV-W)family,had been detected elevated expression in the biopsied skeletal muscles in the patients suffered from MND.Our previous studies had shown that elevated level of syncytin-1 in the skeletal muscle tissue of the hind limb of mice can induce the injury of spinal cord motor neurons in the part of lumbar enlargement,however,the mechanisms were unknown.Objective: To study the effects of glutamate excitotoxicity in the injury of spinal cord and cortical motor neurons induced by the elevated expression of syncytin-1 in the hind limb skeletal muscle of mice.Methods: 8 weeks old C57BL/6J male mice were randomly divided into 3 groups,respectively,the experimental group 1,the experimental group 2 and the control group.With intraperitoneal injection for anesthesia,exposure of the left tibialis anterior muscle,the experimental group 1 and 2,respectively injected 100 μl,200 μl of the recombinant plasmids p CMV-tag2B-syncytin(0.8μg/μl),the control group with 200μl of empty vector plasmid p CMV-tag2B(0.8μg/μl).After 4 months,the weight of mice was measured and recorded.At the same time,the motor function was evaluated.Resting potentials,light contraction potentials and strong contraction potentials were measured in the gastrocnemius muscles.Then the mice were sacrificed,and the cerebral hemispheres,spinal cords and gastrocnemius muscles were collected.The morphological changes of the gastrocnemius muscles of mice were observed by HE staining.The anterior horn motor neurons of lumbar spinal cords were observed with cresyl violet Nissl staining.Glial Fibrillary Acidic Protein(GFAP)in lumbar spinal cords were observed by immunofluorescence.The protein levesl of glutamate transporter 1(GLT-1),α-amino-3-hydroxyl-5-methyl-4-isox-azolepropionic acid receptors(AMPA)Glu R2 subway type,syncytin-1 in the spinal cords were analyzed by western blot.The m RNA levels of GLT-1,Glu R2,syncytin-1 were measured by q RT-PCR.Results: Four months after the injection of plasmids,the weight of mice from the 3 groups(F=0.51,P﹥0.05)and climbing barbed wire test(F=3.37,P﹥0.05)had no significant differences.Nerve damage was measured by electromyography in the mice of the experimental group 1 and 2.Resting potentials showed as fibrillation potentials.Light contraction presented as increased amplitude and extended time limit.Strong contraction showed as typical denervation.By HE staining,experimental 1and 2 group showed that part of muscle fibers were atrophic,and some were triangular or polygonal which were manifestations of typical nerval damage,and the degree of atrophy was more obvious in experimental 2 group.In the experimental group1 and 2,the number of Nissl bodies in the spinal cord anterior horns was significantly lower than that in the control group by Nissl’s staining(F=23.37,P < 0.05).Compared with the experimental group 1,the reduction was more pronounced in the experimental group 2(q=3.27,P < 0.05).The number of Nissl bodies in the motor neurons of the experimental group 1 and 2 was decreased(F = 224.96;P < 0.01),the reduction was more pronounced in the experimental group 2(q= 12.76,P < 0.01).The relative expression of syncytin-1 protein and m RNA in the experimental group 1 and 2 was significantly higher than that in the control group(P < 0.05).The expression of GFAP in the lumbar spinal cords and motor cortex were increased by immunofluorescence in the experimental group1 and 2.Compared with the control group,the expressions of syncytin-1 in the 3 group were gradually increased by western blot(P ﹤0.05).q RT-PCR showed that the m RNA levels of syncytin-1 were also gradually increased(P ﹤0.05).Compared with the control group,the expression of GLT-1,AMPA receptor(Glu R2)was significantly decreased by western blot and q RT-PCR in the experimental group1 and 2.(P all ﹤0.05).Conclusion: 1.The elevated level of syncytin-1 in skeletal muscles of mouse hind limbs may induce the injury of motor cortex and spinal anterior horn motor neurons,and the degree of injury is positively correlated with the levels of syncytin-1 in the skeletal muscles.2.The glutamate excitotoxicity and astrocytes activation may be involved in the pathological process of motor neurons injury which induced by syncytin-1.The reduction of glutamate transporter GLT-1 which induced glutamate accumulation in synapses,and the increased calcium ion permeability caused by the reduction of the postsynaptic membrane Glu R2 may be the pathways of glutamate excitotoxicity.