Behavioral Effects And Mechanisms Of Combined Knockdown Of Ptbp1 And NgR In PTSD Mice

Objectives:Post-traumatic stress disorder(PTSD)is a serious mental illness that often appears after major traumatic events.Although the pathogenesis of PTSD is not fully understood,studies have confirmed that learning and memory impairments in PTSD patients are associated with reduced hippocampal volume,loss of hippocampal neurons,and impaired synaptic plasticity.As a co-receptor of myelin-associated inhibitors(MAIs),Nogo-66receptor(NgR)mediated MAIs signaling pathway is not only involved in the inhibition of axon regeneration,it is also involved in hippocampal synaptic plasticity regulation.In addition,polypyrimidine tract binding protein 1(Ptbp1),as an RNA binding protein,has been widely confirmed to be involved in the regulation of neuronal development as a splicing inhibitor.The up-regulated expression of Ptbp1 induces the alternative splicing of the pre-m RNA of synapse-related factors,and then the transition to the isoforms that are unfavorable to synaptic growth and inhibit the development of neurons.Studies have shown that Nogo-A is also one of the target genes for alternative splicing.However,existing studies are controversial on how Ptbp1 regulates the expression of Nogo-A.Our previous study showed that Nogo-A/NgR expression was significantly up-regulated after Ptbp1 knockdown.However,whether they have ameliorative effects on PTSD and their possible ameliorative mechanisms remain unclear.Therefore,to observe the behavioral changes of mice with PTSD after combined knockdown Ptbp1 and NgR,and to explore the potential mechanisms,in this study,the single prolonged stress(SPS)method was used to prepare mouse models of PTSD,and adeno-associated virus(AAV)was used to combined knockdown of Ptbp1 and NgR in PTSD mice.This study will provide new ideas for the prevention and treatment of PTSD and the related brain injury diseases.Methods:(1)Eighty-four SPF female C57BL/6 healthy mice aged 8-10 weeks were randomly divided into 6 groups with 14 mice in each group.Normal control group,AAV-Cas RxNgR/SPS group,AAV-Cas Rx-Ptbp1/SPS group,AAV-Cas Rx-Ptbp1+NgR/SPS group,AAV-Cas Rx-Lacz/SPS group,and PTSD group.PTSD mouse model was established by SPS method.The mice of AAV-Cas Rx-NgR/SPS group,AAV-Cas Rx-Ptbp1/SPS group,AAV-Cas Rx-Ptbp1+NgR/SPS group and AAV-Cas Rx-Lacz/SPS group were injected with AAV-Cas Rx-NgR virus,AAV-Cas Rx-Ptbp1 virus,AAV-Cas Rx-Ptbp1+NgR virus and AAV-Cas Rx-Lacz virus,respectively.The normal control group did not do any treatment,and the PTSD group underwent SPS modeling.(2)The level of fear,anxiety and spatial learning and memory ability of each group of mice was respectively evaluated by open field,elevated plus maze,and Morris water maze experiments.(3)The morphology of hippocampal neurons and the number of surviving neurons in each group were observed by HE and Nissl staining.(4)By using immunofluorescence,it was possible to see whether Neu N protein was expressed in the hippocampus of the mice in each group and how the combined knockdown of Ptbp1 and NgR affected the number of hippocampal neurons in PTSDaffected mice.(5)Using immunofluorescence or a western blot,the levels of the proteins GAP-43 and NF200 were expressed in the hippocampus of the mice in each group,and the effects of combined knockdown Ptbp1 and NgR on the axon regeneration of hippocampal neurons in PTSD mice were further verified.(6)Golgi-Cox staining was used to observe the changes of dendrites in the DG region of mice hippocampus.Immunofluorescence and western blot were used to detect the expression changes of Synapsin I,a marker of synaptic plasticity in the hippocampal DG region of mice in each group,and to explore the effects of combined knockdown Ptbp1 and NgR on hippocampal synaptic plasticity of mice with PTSD.Results:(1)The PTSD mouse model was effectively developed by SPS method.AAV-Cas RxNgR and AAV-Cas Rx-Ptbp1 viruses were constructed and packaged successfully,and the hippocampus was infected via intracerebroventricular.(2)The fear-anxiety-like behavior,impaired spatial learning and memory,and PTSDlike behavior in mice were all significantly improved by the combined knockdown of Ptbp1 and NgR.(3)Combined knockdown of Ptbp1 and NgR improved the morphology of hippocampal tissue and increased the number of surviving neurons in the hippocampal region of mice with PTSD,which had an important protective effect on hippocampal neurons.(4)Compared with the Control group,the fluorescence intensity of Neu N in the hippocampus of PTSD group and AAV-Cas Rx-Lacz/SPS group was significantly decreased,and the number of surviving neurons was significantly decreased.Combined knockdown of Ptbp1 and NgR reduced the loss of hippocampal neurons,and the expression of neuronspecific marker Neu N was significantly up-regulated,alleviating the pro-apoptotic effect induced by SPS.(5)Compared with the Control group,the fluorescence intensity of GAP-43 and NF200 in hippocampus of PTSD group and AAV-Cas Rx-Lacz/SPS group decreased.Combined knockdown of Ptbp1 and NgR reversed the inhibition of axon regeneration and significantly increased the expression of GAP-43 and NF200 markers of axon growth,alleviating the growth inhibition of SPS.(6)The hippocampal dendrites in PTSD group and AAV-Cas Rx-Lacz/SPS group were disordered and bald,with fewer branches and less density,and the expression of Synapsin I protein was down-regulated.Combined knockdown of Ptbp1 and NgR enhanced hippocampal synaptic plasticity,increased the number of dendrites,branch extension,dendrite spinous density,and significantly increased the expression of Synapsin I,a marker of synaptic plasticity,which improved the phenomenon of impaired plasticity caused by SPS.Conclusion:Combined knockdown of Ptbp1 and NgR can significantly alleviated the fear anxietylike behavior and spatial learning and memory impairment in PTSD mice,which may be achieved by reducing the apoptosis of hippocampal neurons,promoting the axon regeneration of hippocampal neurons,and enhancing hippocampal synaptic plasticity.