【作者】 张翔；

【导师】 雷鹏；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2023， 博士

【摘要】 铁是一种直接参与机体生命活动的微量元素;其在脑中的生理功能至关重要。更为重要的是,铁与神经退行性疾病（Neurodegenerative Diseases）中多种关键蛋白相互作用,并且直接参与疾病进程。例如在阿尔兹海默病（Alzheimer’s diseases,AD）中,一方面,铁促进胞内Tau蛋白过度磷酸化、胞外β淀粉样蛋白（Amyloidβ,Aβ）聚集形成淀粉样斑块;另一方面,胞内淀粉样前体蛋白（Amyloid precursor protein,APP）和Tau蛋白共同作用,调节铁转运,其功能性失调导致铁在脑部沉积,形成恶性循环。AD等疾病均与衰老密切相关,而大脑正常衰老的过程中,铁在特定脑区沉积,且沉积区域与神经退行性疾病患者主要被影响的脑区基本一致。因此,研究铁在脑衰老过程的调控机制及其毒性机制,对相关神经退行性疾病的病因机制探索具有启示作用。果蝇作为一种生命周期较短且进化保守的经典模式动物,且基因编辑较为便利,被广泛用于基因-表型筛选。本研究将以果蝇为研究对象,利用基因突变研究其脑部铁转运及毒性的分子机制,并开展细胞验证。本研究首先利用果蝇Tau编码基因（dTau）敲除及对照w1118果蝇,分别探索果蝇在dTau蛋白缺失或衰老条件下的基因及通路改变。利用蛋白质组学对不同年龄dTau敲除后果蝇脑部整体蛋白变化进行系统分析,并进一步进行表型验证和脑部金属浓度检测。随后,向培养基外源性添加高浓度金属对dTau敲除果蝇进行刺激,通过金属浓度变化、不同基因型果蝇的反应差异,结合蛋白组学进行分析,筛选得到dTau蛋白缺失引起的果蝇脑部相关蛋白及通路的变化。最后,针对筛选结果开展对应靶点功能的挽救实验。本研究同时分析了w1118衰老过程中整体蛋白组、转录组及功能变化,并利用多种二号染色体片段缺失果蝇进行铁离子毒性实验。在此基础上,结合测序分析,筛选调控铁毒性相关基因并进行功能验证,以期进一步明确果蝇衰老过程中铁稳态和毒性调节的机制。对dTau敲除后果蝇脑部蛋白质组学结果的分析发现,果蝇铁代谢相关蛋白显著变化。通过金属毒性试验,发现dTau敲除果蝇仅在衰老后（45天）对铁过载毒性有抵抗作用,减轻由铁过量导致的果蝇运动功能障碍和神经元死亡。转铁蛋白Tsf1上调将铁从大脑转运到外周,而在衰老dtau敲除果蝇脑内高表达,可能介导了铁毒性的抵抗。在dTau敲除的背景下构建Tsf1敲低的果蝇模型,发现其脑部铁水平回升;在铁过载刺激下,果蝇失去对于脑部神经元及运动能力损伤的挽救作用。这一系列结果表明,dTau蛋白在果蝇脑内通过调控Tsf1介导铁离子转运,其功能丧失在衰老过程中能够抵御铁离子毒性。另一方面,通过对w1118果蝇脑部蛋白质组学和转录组学的生物信息学分析,并结合染色体片段缺失的果蝇在高浓度铁处理后寿命的变化,筛选到果蝇脂质体蛋白Fbp2可能参与铁过载毒性。Fbp2的m RNA和蛋白水平在衰老过程中均显著下调,且Fbp2缺失显著促进果蝇对铁毒性敏感性。通过构建Fbp2过表达和敲除的果蝇模型,发现其降低导致高浓度铁处理的果蝇寿命减少及运动功能障碍,而过表达对铁毒性具有明显的抵抗作用。考虑到Fbp2在多种组织和细胞中均有表达,本研究利用小鼠乳腺癌细胞系4T1,构建了Fbp2在哺乳动物中的同源基因HPGD的过表达模型,发现HPGD表达量增加诱导胞内铁蛋白（Ferritin）减少,游离Fe²⁺也随之下降;外源性向HPGD过表达细胞中梯度给予铁刺激,发现HPGD显著减少细胞死亡。这一系列实验初步证明HPGD通过调节胞内铁离子代谢参与铁过载毒性。总之,本研究利用多组学联用,鉴定了影响果蝇脑内铁代谢及毒性的多个分子,并利用生物化学、细胞生物学等手段进行了实验验证。研究发现,在果蝇中dTau蛋白通过调节Tsf1表达,参与果蝇大脑铁代谢;同时筛选到Fbp2及其同源基因HPGD通过减少铁沉积而参与铁毒性。考虑到果蝇基因组进化高度保守,且在鼠源细胞中能够初步验证,这些新靶点的发现有助于进一步开展铁代谢调控及其毒性机制在哺乳动物中的探索,最终为相关疾病的治疗提供新的切入点。更多还原

【Abstract】 Iron is a trace element directly involved in life activities,which is crucial to the physiological function of brain.More importantly,iron can interact with key proteins related to neurodegenerative disease to directly participate in the progress of the disease.For example,in Alzheimer’s diseases（AD）,iron can promote the aggregation of both the intracellular hyperphosphorylatedTau and the extracellular Amyloidβ（Aβ）to form amyloid plaques.Meanwhile,the dysfunction of amyloid precursor protein（APP）andTau,both of which concurrently modify the transportation of iron,leads to the deposits of excessive iron in brain,resulting in a vicious circle.AD is closely related to aging.Researchers have found that during normal brain aging,iron would deposit in specific brain regions,which are consistent with the regions affected among the patients with neurodegenerative disease.Therefore,the study on the regulatory and toxic mechanism of iron in brain aging will enlighten the pathological process of associated neurodegenerative disease.Drosophila melanogaster,as a classic animal model with short life cycle and conserved evolution,is easy for gene editing and has been widely used for gene-phenotype screening.Therefore,we selected Drosophila as the research object and relied on gene mutation to explore the molecular mechanism of the transportation and toxicity of iron in the brain.Further cell verification was carried out.In this study,we used dTau knock-out and wild-type w1118 Drosophila models to investigate the alterations of genes,proteins and pathways related to loss of Tau and aging,respectively.We used mass spectrometry（MS）to analyze the relative changes of brain proteome among dTau knock-out Drosophila with different age stages,verified the corresponding phenotypic changes and tested the concentration of metal ions in the brains.Then,we added exogenous high concentration of metal to stimulate dTau knockout Drosophila.Based on the analysis of the changes of metal concentration distribution,the different responses to the stimuli among the drosophila with different genotypes,and proteome sequencing results,we further screened out the related proteins and pathways changes caused by dTau protein deficiency in the brain.Finally,according to the screening results,we carried out functional rescue experiments.This study also analyzed the expression and function changes of the whole proteome and transcriptome during normal aging among w1118 Drosophila and used Drosophila models with different deletion of chromosome 2 fragments to carry out iron toxicity experiments.We concurrently analyzed the sequencing results and the toxicity experiments to further screen targets related to iron toxicity,and conducted functional verification of the identified targets,with the aim of finding out the mechanism of iron homeostasis and toxicity regulation in the aging process of Drosophila.The MS analysis of dTau knock-out Drosophila revealed significant expression changes of key proteins related to iron metabolism in Drosophila.Through iron toxicity experiments,we found that dTau knockout Drosophila can resist excessive iron toxicity only at the advanced age stage（45 days）,alleviating motor dysfunction and neuronal death in the brain caused by excessive iron.The up-regulation of the transferrin,Tsf1,can export excessive iron out of the brain.Among aged dTau knockout Drosophila,the expression of Tsf1 significantly increased,which could mediate the resistance of iron toxicity.By constructing the Tsf1 knockdown Drosophila model under the background of dTau knockout,we found an increased iron level in the brain and when stimulated with excessive iron,the Drosophila lost the ability to rescue themselves from the impairments of motor function and neuronal death.The results above suggested that dTau can regulate the expression of Tsf1 to affect the transportation of iron in the brain of Drosophila and loss of dTau would lead to the resistance to iron toxicity during aging.By sequencing the proteome and transcriptome of the brain of w1118,combined with the analysis of the changes in the lifespan of drosophila with different chromosome fragment deficiency after the treatment of high concentration iron,we found out that the drosophila Fatbody protein Fbp2 may participate in iron overload toxicity.During aging,Fbp2 was down-regulation at both the m RNA and protein level and the deletion of Fbp2 could significant increase the sensitivity to iron toxicity in Drosophila.By constructing a drosophila model of Fbp2 over-expression and knockout,we demonstrated that reduced expression of Fbp2 led to shortened lifespan and motor deficit in drosophila,while the over-expression of Fbp2 showed a protective effect on lifespan and motor function.Given that Fbp2 is expressed in a variety of tissues and cells,this study used mouse breast cancer cell line 4T1 to construct an over-expression model of the homologous gene of Fbp2,HPGD,in mammals.The increased expression of HPGD would decrease the expression of intracellular Ferritin and intracellular free Fe²⁺.In addition,when iron was added to HPGD over-expression cell with gradient,the over-expression of HPGD significantly decreased the death of cell.These experiments preliminarily demonstrated that HPGD participated in iron overload toxicity by regulating intracellular iron metabolism.In conclusion,several genes which involved in iron metabolism and toxicity were identified via multiple bio-informatics methods in this research,and verified by means of biochemistry and cell biology.This study found that dTau protein can participate in iron metabolism of brain neurons by regulating Tsf1 expression in Drosophila.Meanwhile,we screened out Fbp2 and its homologous gene HPGD,which could protect cells from excessive iron toxicity by preventing iron accumulation.Because the evolution of drosophila genome is highly conserved and we preliminarily demonstrated these genes using mouse cells,the discovery of these new target genes will help further explore the regulation of iron metabolism and iron toxic mechanism in mammals,and finally provide a new entry point for the treatment of related diseases of human beings.更多还原