Effects And Mechanism Of SRPK2 On Nervous System Of Mouse

Alzheimer’s Disease(AD)is a common age-dependent neurodegenerative disease,and the specific pathogenic mechanism is still unclear.Current studies showed that the main pathological factors of Alzheimer’s disease are the abnormal phosphorylation of Tau protein in hippocampal neurons,which leads to neurotoxic neurofibrillary tangles and β-amyloid protein.Both of these substances are neurotoxic.Once it accumulates in a large amount in the nervous system,it will destroy the number and structure of neurons,and ultimately affect the normal function of the nervous system.However,the mechanisms underlying the Tau protein hyperphosphorylation still remain unclear.SRPKs(SR Protein-specific Proteins)is a kinase family that were discovered as SR proteins specific kinase.So far,three family memebers are identified:SRPK1,SRPK2 and SRPK3.Existing studies proved that SRPK2 shows specific and high expression in brain tissues,and its activity was significantly increased in AD patient mice and human brains;in addition,Tau protein was showed to be one of the substrates of SRPKs,suggesting SRPK2 is related to the occurrence and development of Alzheimer’s disease.Further study on the effects and possible mechanisms of SRPK2 in the hippocampus on the nervous system will help people understand how Alzheimer’s and other neurodegenerative diseases occur,and may provide new research directions for the treatment of those diseases.In this dissertation,we successfully constructed a heritable SRPK2 tissue-specific knockout mouse model in hippocampal CA1 region,by using the Cre-loxp recombinase system.Results from frozen section of brain tissue and Nissl staining showed that SRPK2 knockoutin the CA1 region of hippocampus significantly decreased the number of corpuscles or neurons indicating that SRPK2 is essential for the growth and development of neurons in the nervous system.The following behavioral analysis showed that SRPK2 KO mice had strong fear of new environments and the cognitive ability was significantly degenerated compared to the WT mice.These results indicated that SRPK2 KO in the CA1 region may result in a significant loss of the number of hippocampal neurons,therefore reaulting in disfunction of the nervous system in mice,which is mainly manifested in the obstacles of learning,memory and cognition.Afterwards,in order to further study the mechanisms of SRPK2 in the CA1 region at both gene and protein level,we conducted RNA-seq and phospho-proteomic analysis,and found that by knocking-down the expression of SRPK2,the gene expression in the CA1 region showed significant changes in alternative splicing.In the meanwhile,signal transduction,nervous system,and endocrine signaling pathways had also been affected,especially the MAPK signaling pathway,Ras signaling pathway,and calcium signaling pathway,both at the gene level and protein level.More importantly,Tau protein is hyperphosphorylated after SRPK2 knockout,and the functions of genes such as microtubule cytoskeleton and microtubule binding protein were also affected.This demonstrated that SRPK2 may affect the nervous system of mice by affecting the function of signaling pathways.Next,in order to elucidate the possible effects of SRPK2 on the development of mice,we generated a germ cells-specific SRPK2 knockout mice line.After several rounds of breeding between the heterozygotes,no homozygous knockout mice were generated,demonstrating that the SPRK2 KO in mouse germ cells is lethal.This further proved that SRPK2 is essential for the developnent of mice embryos.In order to further study the possible functions of SRPK2 at the cellular level,we isolated the mice embryonic fibroblast(MEF)cells,and knocked out SRPK2 by using the retrovirus infection system.Further cellular and biochemical analysis is still going on.The results from this dissertation demonstrated that,firstly,SRPK2 KO in the CA1 region can destroy the structure hippocampal neurons and significantly decrease their number.These in turn result in neuronal dysfunction,and therefore the dysfunction of learning and memory in mice.Secondly,After SRPK2 KO in the CA1 region the gene function and alternative splicing of the mouse hippocampus are all changed.In the meanwhile,the Tau protein was hyperphosphorylated,and The MAPK,Ras and calcium pathway showed significant changed both at the gene level and protein level,which indicates that SRPK2 may regulate the phosphorylation of Tau protein,and cause damage on the nervous system,mainly through affecting the function of those signal pathway.Thirdly,Maintaining the right level of SRPK2 in germ cells is essential for the mice development.These results provide a research direction for study on the possible functions and mechanisms of SRPK2 in the central nervous system and development.This will also provide a theoretical basis for further research on the occurrence and treatment of Tau protein-related diseases such as Alzheimer’s disease.