【作者】 魏晓红；

【导师】 王萍萍；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2022， 硕士

【摘要】 丝氨酸/精氨酸蛋白特异性激酶(Ser/Arg protein-specific kinases,SRPKs)是一种磷酸化剪接因子SR蛋白的激酶家族。目前,哺乳动物中已鉴定SRPKs家族成员有三个:SRPK1、SRPK2及SRPK3。SRPKs家族成员激酶区的氨基酸序列高度相似,但在体内呈现组织特异分布,说明该家族各成员在生长发育中可能具有独特的功能。其中,SRPK2在神经系统疾病如阿尔兹海默症中表达异常,暗示其在脑组织中的特异分布性可能对神经系统的正常发育具有重要意义。此外,研究表明,SRPK2还可以作为某些癌症联合药物的治疗靶点。因此,我们初步探究了SRPK2在神经系统发育及细胞正常生命活动中的可能作用机制,完善了其在生物体内的功能,为临床应用提供了新的治疗思路。为了研究SRPK2在神经系统正常发育中的功能,我们利用Cre-lox P系统成功构建了SRPK2海马体CA1区条件性敲除小鼠(SRPK2 CA1-c KO)。对条件性敲除小鼠和同周龄的野生型小鼠进行饲养观察,发现其行为有异常现象。旷场实验和Y迷宫实验分析发现,6月龄的SRPK2条件性敲除小鼠与同周龄的野生型小鼠相比,SRPK2在CA1区的敲除造成小鼠极度焦虑及空间认知能力出现障碍,表明SRPK2的缺失可能造成小鼠神经系统缺陷,导致行为异常。其次,CA1区海马体的透射电镜实验证明,与野生型小鼠相比,SRPK2 CA1-c KO小鼠的CA1区神经元线粒体明显膨大,边界模糊且内部出现空泡,证明SRPK2敲除使CA1区神经元超微结构受损。同时,对小鼠海马体内部突触后致密蛋白(PSD-95)的表达量进行检测,发现与野生型小鼠相比,SRPK2 CA1-c KO小鼠海马体内PSD-95蛋白表达量显著降低,表明SRPK2敲除导致海马体CA1区神经元突触可塑性降低。以上结果证明,海马体中SRPK2可能通过影响神经元结构及功能来影响神经系统的发育。为了进一步探究CA1区内SRPK2对神经系统发育的可能作用机制,我们进行了RNA-Seq转录组分析及TMT标记的磷酸蛋白组鉴定。RNA-Seq结果表明,SRPK2在CA1区的敲除造成与神经系统发育相关基因选择性剪接事件发生显著变化,随后进行了RT-PCR验证,与野生型小鼠相比,SRPK2 CA1-c KO小鼠中参与神经营养因子信号通路的Irak1基因发生外显子跳跃;参与神经元突触小泡循环的Tcirg1基因发生内含子保留等,这些结果表明SRPK2可能通过调控神经发育相关基因的选择性剪接来影响神经系统正常发育。此外,对Tau蛋白进行的Western blot和免疫组化实验也证明了SRPK2敲除可以造成海马体内Tau蛋白异常积累。同时,对小鼠海马体进行的磷酸蛋白组数据分析显示,敲除SRPK2后,海马体Tau蛋白内有6个位点的磷酸化水平显著上调,其中S648位于微管的结合区,与其功能密切相关。我们通过GO功能注释分析发现,细胞骨架、微管聚合、基于微管过程的调控等蛋白的磷酸化水平也有显著变化。以上研究结果表明,小鼠中SRPK2可能通过调控Tau蛋白积累和磷酸化水平及其他微管相关蛋白的磷酸化水平影响神经元正常功能。为研究SRPK2在细胞内的功能,我们从SRPK2基因flox杂合子小鼠的胚胎提取了胚胎成纤维细胞(MEF cells)。通过β-gal染色实验发现,SRPK2敲除导致原代MEF细胞发生早衰;同时,细胞划痕实验和Transwell侵袭实验发现永生化的SRPK2敲除MEF细胞的迁移率和侵袭率要显著高于对照组,说明SRPK2敲除可能导致细胞癌化。随后进行的Western blot实验证明,SRPK2敲除可激活Akt、p38 MAPK等细胞内的关键信号通路。综合上述实验结果,我们认为SRPK2敲除可能通过激活Akt、p38 MAPK信号通路导致细胞的癌化。综上所述,本论文证明:1.SRPK2在CA1区的敲除可以导致神经元超微结构受损、突触可塑性下降,进而造成小鼠行为异常;2.SRPK2可能通过调控神经发育相关基因的选择性剪接从而影响神经系统的正常发育;3.CA1区敲除SRPK2可以导致海马体内Tau蛋白积累及过度磷酸化,进而阻碍神经元微管相关过程,影响神经系统正常发育;4.MEF细胞中,敲除SRPK2可以激活Akt、p38 MAPK等信号通路,使细胞产生癌化表型。总之,本论文对SRPK2在神经系统及细胞内的可能作用机制的研究完善了SRPK2在生物体内的功能,为神经系统疾病和癌症治疗提供了新的思路。更多还原

【Abstract】 Serine/arginine protein-specific kinases(Ser/Arg protein-specific kinases,SRPKs)are a kinase family that phosphorylates splicing factor SR proteins.At present,three members of the SRPKs family in mammals have been identified: SRPK1,SRPK2,and SRPK3.These members of the SRPKs family are highly conserved in amino acids in their kinase domains,but they show tissue-specific expression in mammals,indicating they may have special functions for growth and development.Of them,the abnormal expression of SRPK2 in nervous system diseases,such as Alzheimer’s disease,suggested that its specific distribution in brain tissue seems to be of great significance to the development of nervous system and provides a new direction for the treatment of neurodegenerative diseases.In addition,SRPK2 can be used as a target for combined drug therapy in the treatment of some cancers.Therefore,we studied the potential mechanisms of SRPK2 in nervous system development and cellular activities,to further dissect its functions in vivo,and to provide therapeutic ideas for clinical applications.To dissect the potential functions of SRPK2 in the development of nervous system,first,the hippocampal CA1 region-SRPK2 conditional knockout mice(SRPK2 CA1-c KO)were successfully constructed by using the Cre-lox P system,and further behavior analyses between SRPK2 CA1-c KO mice and WT mice both at same ages revealed abnormal behavior of SRPK2 CA1-c KO mice.By open-field test and Y-maze test,we found that compared with WT mice,6-month-old SRPK2 CA1-c KO mice showed extreme anxiety and spatial cognitive impairment,indicating that loss of SRPK2 may cause nervous system defects and lead to abnormal behavior.Secondly,transmission electron microscope analyses of the hippocampal samples in CA1 area showed that compared with WT mice,the mitochondria of neurons in CA1 region of SRPK2 CA1-c KO mice were dilated,vacuoles appeared inside,and the boundary was blurred.These results confirmed that SRPK2 knockout damaged the ultrastructure of neurons in CA1 region.In addition,Western blot analyses showed that the expression of postsynaptic dense protein(PSD-95)in the hippocampus of SRPK2 CA1-c KO mice was lower than that of WT mice,indicating that SRPK2 knockout may regulate the development of nervous system by affecting the structures and functions of neurons in the hippocampus.To further explore the mechanisms of SRPK2 in the CA1 region on nervous system development,RNA-Seq transcriptome analysis and TMT labeled phosphoproteomic analysis were carried out.RNA-Seq results showed that SRPK2 knockout in the CA1 region caused significant changes in alternative splicing of genes related to nervous system development.Further RT-PCR verification of alternative splicing differential genes was carried out and results confirmed that Irak1,which involves in the neurotrophin signaling pathway,showed increased exon skipping in SRPK2 CA1-c KO mice compared with WT mice.Whereas Tcirg1 the gene involved in synaptic vesicle circulation showed increased intron retention.These results showed that SRPK2 may affect the development of nervous system by regulating the alternative splicing of genes involved in neural development.In addition,Western blot and immunohistochemical staining on Tau protein showed that SRPK2 knockout caused abnormal accumulation of Tau protein in hippocampus.Further phosphoproteomic analysis showed that six phosphorylation sites of Tau protein in hippocampus were significantly up-regulated in SRPK2 CA1-c KO mice compared with WT mice,of which S648 was located in the binding region of microtubules that is related to its function.GO analysis of proteins with significant changes in phosphorylation showed accumulation of proteins in biological processes such as cytoskeleton,microtubule polymerization,and regulation based on microtubule process.Our study showed that SRPK2 may affect the function of neurons by regulating the accumulation and phosphorylation of Tau protein and the phosphorylation of other microtubule-related proteins.To study the potential functions of SRPK2 in cells,we isolated mouse embryonic fibroblast cells(MEFs)from flox heterozygous mice with SRPK2 gene.β-gal staining on these primary cell lines confirmed that SRPK2 knockout caused the premature senescence of primary MEF cells.Further cell scratch test and Transwell invasion assay on immortalized MEF cells,showed that the cell migration and invasion rate of SRPK2 KO cells were significantly higher than those of the control group.Later,Western blot analyses indicated that SRPK2 knockout activated Akt and p38 MAPK signal pathways.All these results confirmed that SRPK2 knockout may lead to cell oncogenesis by activating Akt and p38 MAPK signaling pathways.The experiments in this dissertation prove that 1,SRPK2 knockout in CA1 region leads to the damage of neuronal ultrastructure and the decrease of synaptic plasticity,which leads to abnormal behavior in mice;2,SRPK2 affects the development of nervous system by regulating the alternative splicing of genes involved in neurodevelopment;3,conditional knockout of SRPK2 in CA1 region leads to the accumulation and hyperphosphorylation of Tau protein in hippocampus,which hinders the process of neuronal microtubule and affects the development of nervous system;4,in MEF cells,SRPK2 knockout activates Akt and p38 MAPK pathways.In a word,exploring the functions of SRPK2 in nervous system and cells in this dissertation perfects the study of SRPK2 functions in vivo and provides potential new ideas for the treatment of nervous system diseases and cancers.更多还原