Study On The Molecular Mechanism Of RIN1 Regulating Fear Memory

Objectives: Posttraumatic stress disorder(PTSD)refers to the great suffering or shock caused by natural disasters,terrorist events,traffic accidents,tragic mental,mental and physical symptoms,or anxiety syndrome.Mental disorders that lead to delayed onset and long-term persistence.In recent years,with the increase of various sudden disaster events at home and abroad,the incidence of PTSD has been increasing year by year,which has become a serious psychological disorder caused by post-disaster trauma and the primary public concern problem.The clinical symptoms of PTSD are mainly fear memory disorder,that is,the pathological recurrence of traumatic events.PTSD patients may exhibit abnormal consolidation and recovery of traumatic memory,leading to traumatic reappearance.The hippocampus and amygdala are involved in the fear memory pathway and play a key role in the regulation of fear-related emotions.RIN1 gene(Ras interaction/interference1)is an effector of Ras protein and is highly expressed in hippocampus and amygdala.RIN1 is located in the cell body and dendrites of nerve cells.The expression of RIN1 is low in embryonic stage and gradually increased after birth,suggesting that RIN1 is only related to mature neurons.RIN1 regulates cellular endocytosis and cytoskeleton reconstruction by activating downstream ABL and binding downstream Rab5,and modulates fear memory by regulating Eph A4 endocytosis.Our previous study found that SPS caused phobic memory disorder,morphological and functional abnormalities of hippocampus and amygdala.However,the mechanism of PTSD causing fear and memory disorder is unclear.This study includes the following two aspects: 1)using RIN1 as the entry point,we used the PTSD animal model to study whether PTSD induced the changes of RIN1 expression in hippocampus and amygdala.2)RIN1knockout rat(RIN1-/-)model was used to study the molecular mechanism of RIN1 regulating fear memory.It is proved that RIN1 is involved in the regulation of fear memory caused by PTSD,and it also provides a new idea for finding potential drug targets for the treatment of PTSD.Methods: PTSD animal model was constructed by single continuous stimulation of(SPS).The fear memory of control rats,SPS rats,Wild rats and RIN1-/-rats,and the difference of fear memory between normal rats and SPS rats before and after plantar click were detected by conditioned fear box test(Conditional Fear Box).The expression of RIN1 and its pathway ABL1/2?Rab5 in hippocampus and amygdala of control rats,SPS rats,Wild rats and RIN1-/-rats were detected by Western blot method,and the difference of AMPAR?Caspase7?Caspase9?Caspase12 expression was also observed.The intracellular distribution and immunoreactivity of RIN1 and AMPAR in hippocampus and amygdala of control rats,SPS rats,wild rats and RIN1-/-rats were detected by immunofluorescence labeling and double immunofluorescence labeling.The expression of ABL1/2 ? Rab5 m RNA in hippocampus of Wild rats and RIN-/-1 rats was detected by PCR method.The apoptosis of hippocampal cells in Wild rats and RIN1-/-rats was detected by TUNEL assay.Results: Research content 1: 1.The results of conditioned fear box test showed that the fear memory in SPS group was significantly increased compared with that in control group.2.The expression of RIN1 protein in hippocampus of SPS group was significantly higher than that of control group,and the expression of RIN1 protein in amygdala of SPS group was consistent with that of RIN1 protein in hippocampus by)Western blot method.3.The results of immunofluorescence showed that RIN1 was expressed in neurons in hippocampus of rats,and the immunoreactivity of RIN1 in SPS group was higher than that in control group.Research content 2: 1.The results were as follows compared with Wild group,the fear memory of RIN1-/-rats in RIN1-/-group was decreased.2.Western blot,immunofluorescence and real-time PCR assay showed that the levels of protein,immunoreactivity and m RNA in hippocampus of RIN1-/-rats were significantly lower than those of Wild rats.3.The total number of hippocampal cells in RIN1-/-group was lower than that in Wild group,and the percentage of TUNEL positive cells in RIN1-/-group was significantly higher than that in Wild group.In RIN1-/-group,the levels of Caspase3 and Caspase7 increased significantly,but the level of Caspase12 did not change significantly.4.The results of immunofluorescence and western blot showed that the AMPAR in hippocampal neuron cytoplasm of RIN1-/-rats was significantly lower than that of Wild rats,but there was no significant change in AMPAR on the cell membrane of RIN1-/-rats.Conclusion: 1.The increased fear memory in SPS rats may be related to the increased expression of RIN1 in hippocampus and amygdala.2.The regulation of fear memory by RIN1 may be mediated by mitochondrial pathway apoptosis and decreased AMPAR expression in the cytoplasm.