| Background and Objectives Neurogenesis is the procession of generating new functional neurons in specific regions of the nervous system.In normal physiological states,neurogenesis in adult individuals is mainly focused on the dentate gyrus granule region of the hippocampus,which maintains an active neurogenic potential throughout life,and this property is also considered to be one of the important reasons for the maintenance of memory and cognition in the hippocampus.In the physiological state of disease,disruption of neurogenesis is also considered to be a key factor influencing the development and poor prognosis of neurological disorders.However,little is known about the mechanisms and regulation of adult neurogenesis;therefore,further study of the molecular mechanisms of adult neurogenesis is important for the development of brain science and neurobiology.Calcium homeostasis is an important basis for the maintenance of normal physiological functions of the nervous system,and imbalance in neuronal calcium homeostasis may also lead to disruption of various cellular physiological processes,including transcription factor dysfunction,cell cycle imbalance,and blocked cellular immune response,which in turn can cause a variety of diseases,including neurodevelopmental defects,neuronal differentiation disorders,and neurodegenerative pathologies.The development of these disorders is a major concern.Therefore,further investigation of the regulation of calcium homeostasis by calcium channels at different levels is a fundamental issue that needs to be addressed in calcium signaling research as well as in the brain science and neurobiology neighborhood.SUMOylation refers to a class of post-translational modifications of proteins that are similar and functionally complementary to ubiquitination modifications in eukaryotes,and SENP2,as an important type of de-SUMOylation enzymes,is essential for maintaining intracellular SUMOylation homeostasis,while SENP2-deficient mice exhibit a phenotype of massive hippocampal neuronal loss.In order to investigate the important role of SENP2 in the maintenance of neuronal function and neurogenesis,we investigated the neuronal types in the hippocampus of SENP2-deficient mice,aiming to provide a basis for exploring the molecular mechanism of SENP2 regulation of neurogenesis,and to provide a new biological basis for SUMOylation regulation of neurogenesis,which in turn may provide a new clinical basis for the treatment of neurogenic disorders.It is also expected to provide a new biological basis for the regulation of neurogenesis by SUMOylation,and thus provide a new medicinal target and theoretical premise for the treatment of neurogenesis disorders.Methods and Results 1.SENP2 deficiency causes neurogenesis defeat in the mice hippocampus: This study firstly used SENP2 wild-type and SENP2-deficient mice as the research models,and initially explored the regulation of SENP2 and the number and morphology in mice hippocampal neurons by performing Golgi staining as well as Nissl staining analysis on the brains of mice at p42;then we performed immunofluorescence analysis on the hippocampal tissues and isolated neurons in vitro from the hippocampal of mice to further confirmed that regulation role of SENP2.We investigated the mechanism of SENP2 in regulating the function role of hippocampal neurogenesis in adult mice.2.SENP2 regulates cytoplasmic-endoplasmic reticulum calcium homeostasis: Through cytoplasmic and endoplasmic reticulum specific calcium probes,explore the effect of SENP2 deficiency on neuronal calcium homeostasis;isolate and construct SENP1 knock-out MEF cells,SENP3 knocked-down MEF cells combined with calcium probes to clarify that SENP2 directly and specifically regulates calcium homeostasis.The specific calcium channel inhibitor/agonist of the endoplasmic reticulum and the PLC-specific inhibitor of the upstream effector of the calcium channel are used to determine the effector protein of SENP2 regulating calcium homeostasis;Mouse hippocampal tissue and MEF cells were analyzed to clarify the molecular mechanism of SENP2 regulating calcium channels;immunoblotting and transcriptional detection were used to clarify the regulatory relationship between SENP2 and endoplasmic reticulum calcium channel disorders.3.SENP2 regulates the SUMOylation of PLCβ4 and its protein stability: Considering that SENP2 has specific regulation role in the protein expression of PLCβ4 and strictly depends on the de SUMOylation catalytic activity of SENP2.To further explored the molecular mechanism of how SENP2 directive regulates the expression of PLCβ4,through in vitro cell experiments,we overexpress PLCβ4 and SUMO to modify key regulatory factors,as well as tissue samples from SENP2-deficient mice in vivo,and use immunoprecipitation to analyze the types of SUMOylation of PLCβ4;use immunoprecipitation and laser confocal to explore in cells SENP2 regulates the homeostasis of the SUMOylation of PLCβ4;combined with bioinformatics and in vitro Mapping experiments,the SUMOylation site of PLCβ4 is determined.4.SUMOylation promotes the ubiquitination and degradation of PLCβ4 through STUBL RNF4:The protein synthesis inhibitor cycloheximide was used to test the stability of PLCβ4,combined with the ubiquitin proteasome inhibitor MG132 and the lysosomal inhibitor chloroquine to preliminarily determine the mechanism of SUMOylation regulating PLCβ4 degradation;cell experiments combined with immunoprecipitation And laser confocal experiments to verify the molecular mechanism of SUMOylation regulating the stability of PLCβ4;and using in vitro ubiquitination experiments to determine the type of SUMOylation regulating the ubiquitination degradation of PLCβ4.Small interfering RNA interferes with RNF4 and RNF111 respectively,and further locks and regulates its ubiquitination then degraded by E3 ligase RNF4.5.Nup205 and Ran Bp2 mediate hyper-SUMOylation of PLCβ4 nucleus import: Through laser confocal subcellular localization analysis of wild-type and SUMO modified site mutant PLCβ4 and separation of nucleo-plasmic protein components to determine the regulation of SUMOylation on PLCβ4 subcellular localization;data analysis using co-immunoprecipitation combined with mass spectrometry SUMOylation regulates the regulation mechanism of PLCβ4 nuclear and cytoplasmic shuttling;real-time fluorescence quantification is used to preliminarily determine the nuclear function of PLCβ4.6.The imbalance of calcium homeostasis caused by SENP2 deficiency affects neurogenesis which depends on PLCβ4: By constructing a PLCβ4 knockdown cell line combined with calcium probe analysis,it was verified that the SUMOylation of PLCβ4 participates in the regulation of cytoplasmic-endoplasmic reticulum calcium homeostasis.In accordance with the self-renewal regulation mode of neural stem cells,the isolation of hippocampal neural stem cells further verified the potential of PLCβ4 to regulate the self-renewal of neural stem cells;secondly,the use of mouse hippocampal neural stem cells isolated in vivo and cell models constructed in vitro to verify exogenous calcium ion uptake indicated the rescue effect on the cytoplasmic-endoplasmic reticulum calcium homeostasis caused by SENP2 deficiency and the regulation of cell viability;in addition,the SENP2 knockdown cell line constructed in vitro and the hippocampal neural stem cells of SENP2-deficient mice are used as models.Exogenously overexpress PLCβ4 to rescue the imbalance of calcium homeostasis and down-regulation of cell viability caused by SENP2 deficiency,and verify the regulation of the SENP2-PLCβ4 signal axis on calcium homeostasis and neural stem cell viability;detect mouse-derived nerves by immunoblotting.The downstream effector of stem cell PLCβ4,preliminarily determined the signal pathway that SENP2-PLCβ4 regulates neural stem cell self-renewal.7.SENP2-PLCβ4 signal axis as a sensor to respond to hunger or oxidative stress: First,the in vitro glucose deprivation experiment was used to simulate the oxidative stress of neurons,and the key factors of the SUMOylation process and the effect elements of calcium homeostasis were detected by immunoblotting and real-time fluorescent quantitative PCR,and the physiological response of SENP2-PLCβ4 to neuronal oxidative stress was determined.Establish a neuronal oxidative stress model in vitro through glucose deprivation,hydrogen peroxide treatment,and NAC incubation with the peroxide scavenger,and combine western blotting and calcium probes to explore the SENP2-PLCβ4 signal axis in neuronal oxidative stress mode.Conclusions and significance This study used SENP2-deficient mice as model and demonstrated that SENP2 deficiency caused the disorder of neurogenesis phenotype in hippocampus.A series of experiments proved that SENP2 directly regulates the adult-neurogenesis procession in the granular dentate gyrus of the mice hippocampal.And the SENP2-PLCβ4 axis as a sensor of oxidative stress,by regulating the procession of cytoplasm-endoplasmic reticulum calcium homeostasis,then affects the self-renewal of neural stem cells,and ultimately leads to neurogenesis disorder in the granular dentate gyrus of the mouse hippocampus.Our results confirm the fundamental role of SENP2 in mediating the procession of adult-neurogenesis,and demonstrated that the SENP2-PLCβ4 axis regulates neuronal calcium homeostasis and thus affects the molecular mechanism of neurogenesis,providing a new horizon for the regulation and active targets of neuronal calcium homeostasis procession and adult-neurogenesis regulation. |
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