【作者】 刘菲；

【导师】 王建枝； 杨莹；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2020， 博士

【摘要】 背景成年海马神经新生（Adult hippocampal neurogenesis,AHN）缺陷会加速衰老、阿尔茨海默病（Alzheimer’s disease,AD）等过程中认知功能的衰退,但其发生机制尚不清楚。糖原合酶激酶3β（Glycogen synthase kinase-3β,GSK-3β）在神经系统新生和神经退行性病变过程中均发挥着重要作用。本研究旨在探讨衰老过程中GSK-3β是否以及如何调节AHN。方法在3月和6月龄加速衰老小鼠（SAM-P8）和正常老化小鼠（SAM-R1）小鼠中,通过Brd U,DCX免疫染色和逆转录病毒标记的方法评估AHN,同时检测小鼠与AHN相关认知功能的改变,以及GSK-3β的表达和活性。使用腺相关病毒在Nestin-Cre ERT2小鼠海马神经干细胞（Neural stem cell,NSC）中选择性过表达人源性GSK-3β,或将Nestin-Cre ERT2小鼠与GSK-3β^flox/flox小鼠杂交以选择性敲除海马NSC内源性GSK-3β,以研究GSK-3β对成年小鼠AHN及相关认知功能的影响。最后,检测GSK-3β选择性抑制剂SB216763对SAM-P8小鼠AHN及相关认知功能障碍的挽救作用。结果1.相比于同龄SAM-R1小鼠,3月龄SAM-P8小鼠AHN数目增加,但新生神经元树突长度和复杂性减小;而6月龄SAM-P8小鼠AHN数目减少,新生神经元树突长度、复杂性和树突棘密度均显著减小,并伴随着突触相关蛋白表达,以及AHN相关模式分离、空间学习-记忆功能的改变。2.相比于同龄SAM-R1小鼠,3月龄SAM-P8小鼠齿状回区GSK-3β表达增加,同时GSK-3β活性均显著增强。而6月龄小鼠中GSK-3β表达量不再增加,其活性也未见增强。3.野生型小鼠的海马NSC中选择性过表达人源性GSK-3β后促进新生神经元的存活,短期（4周）内AHN数目增加,新生神经元树突复杂性增加、树突棘密度降低;但长期（8周）内AHN数目减少、新生神经元树突复杂性、树突棘密度均显著减小,其原因可能是GSK-3β过表达造成了NSC池的加速消耗。4.使用GSK-3β特异性抑制剂SB216763可以有效促进6月龄SAM-P8小鼠AHN,并改善模式分离功能障碍。5.选择性半敲除野生型小鼠NSC中的GSK-3β对AHN无显著影响,全敲除GSK-3β后AHN数目减少,新生神经元树突复杂性降低。结论NSC胞内GSK-3β的表达和激活在维持小鼠AHN中发挥着重要作用。加速衰老小鼠海马NSC中GSK-3β的增多和活性增强可以加速NSC池的损耗,造成短期内AHN数目增加,但长期内AHN数目减少、树突分化出现障碍。使用药物抑制GSK-3β的过度激活可以有效保护快速衰老小鼠中的AHN损伤,并改善相关认知功能。这些结果揭示在加速衰老过程中海马NSC胞内GSK-3β的过度激活是造成AHN损伤的重要机制,并为相关疾病的促神经新生治疗提供了新的分子靶标。更多还原

【Abstract】 Background Adult hippocampal neurogenesis(AHN)deficits contribute to the cognitive decline during aging,Alzheimer’s disease,and other processes,with the mechanistic cause poorly understood.Glycogen synthase kinase-3β(GSK-3β)plays an important role in the development of the nervous system and neurodegenerative diseases.This study aimed to investigate whether and how GSK-3β regulates AHN during accelerated aging.Methods AHN was assessed by Brd U,DCX immunostaining and retroviral labeling in 3 and 6 month-old senescene-accelerated mice prone8(SAM-P8)and senescence resistant 1 mice(SAM-R1).AHN-related cognitive functions,expression and activity of GSK-3β were also detected in these mice.GSK-3β was selectively overexpressed using adenoassociated virus,or knocked-out by crossbreeding with GSK-3β floxed mice in the neural stem cells(NSCs)of Nestin-Cre mice to study the effect of GSK-3β on AHN and related cognitive functions in adult mice.Finally,the salvage effect of GSK-3β selective inhibitor SB216763 on AHN and related cognitive dysfunction in SAM-P8 mice was tested.Results 1.Compared with SAM-R1 mice of the same age,the number of AHNs of 3-month-old SAM-P8 mice was increased,but the dendritic length and complexity of neonatal neurons were reduced.However,the number of AHNs of 6-month-old SAM-P8 mice was decreased,Neuron dendrite length,complexity,and dendritic spine density were significantly reduced,accompanied by changes in synapse-related protein expression,AHN-related pattern separation,and spatial learning-memory changes.2.Compared with SAM-R1 mice of the same age,the expression of GSK-3β in the dentate gyrus region of 3-month-old SAM-P8 mice increased,and the activity of GSK-3β was significantly enhanced.The expression and the activity of GSK-3β in the dentate gyrus region of 3-month-old SAM-P8 mice is same with SAM-R1 mice.3.Selective overexpression of human-derived GSK-3β in hippocampal NSCs of wildtype mice promotes the survival of neonatal neurons.The number of AHNs increases in the short term(4 weeks),together with the dendritic complexity of neonatal neurons increases,and dendritic ridge Density decreased.However,the number of AHNs decreased in the long term(8 weeks),the dendritic complexity of neonatal neurons,and the density of dendritic decreased significantly,due to the accelerated consumption of the NSC pool.4.GSK-3β-specific inhibitor SB216763 can effectively promote AHN in 6-month-old SAM-P8 mice and improve pattern separation dysfunction.5.The number of AHNs decreased and the complexity of neonatal neuron dendrites decreased after GSK-3β completely knocked out.Selective semi-knock-out of GSK-3β in wild-type mouse NSC had no significant effect on AHN.Conclusion The expression and activation of GSK-3β in NSC plays an important role in maintaining mouse AHN.In senescence-accelerated mice,the increase of GSK-3β expression and the enhancement of its activity can accelerate the depletion of the NSC pool,resulting in AHN increasing in the short term but decreasing in the long term accompanied by deteriorate dendritic differentiation.Inhibiting GSK-3β over-activation can effectively protect AHN damage in rapidly aging mice and improve related cognitive functions.These results reveal that the over-activation of intracellular GSK-3β in hippocampal NSCs during accelerated aging is an important mechanism that causes AHN damage and provides new molecular targets for the neuronal therapy of related diseases.更多还原