【作者】 王慧青；

【导师】 闫福岭；

【作者基本信息】 东南大学 ， 神经病学， 2025， 博士

【摘要】 研究背景与目的:帕金森病(Parkinson’s disease,PD)是以中脑黑质致密部多巴胺能神经元进行性丢失为核心病理特征的神经退行性疾病。铁代谢紊乱驱动的铁超载及铁稳态失衡是PD神经退行性病变的重要病理机制,通过激活铁死亡途径加速神经元丢失。铁自噬通过溶酶体降解铁蛋白释放游离亚铁离子,加剧铁死亡。然而,调控铁自噬的上游分子机制尚未系统阐明。既往研究指出针对铁死亡的抑制性治疗措施,可以有效改善PD模型小鼠的运动功能并且可以降低多巴胺能神经元的丢失。因此,寻找PD相关的铁死亡分子对于PD的治疗具有重要意义。基于上述科学问题,本研究整合GEO数据库中PD转录组数据与铁死亡相关基因集,通过生物信息学分析筛选出关键交叉基因NADPH氧化酶1(NADPH Oxidase 1,NOX1)。既往研究提示,NOX1可能通过调控活性氧(Reactive Oxygen Species,ROS)生成和α-突触核蛋白(α-synuclein,α-syn)异常聚集参与PD进展,但该分子是否通过调控铁自噬-铁死亡轴影响多巴胺能神经元尚未见报道。此外,PD病理状态下NOX1异常高表达的表观遗传调控机制仍有待探索。因此,本研究拟解决三个关键科学问题:(1)明确NOX1对多巴胺能神经元的影响;(2)解析NOX1下游通过RUNX2/STK32A通路介导铁自噬的分子机制;(3)揭示E3泛素连接酶FBXW7通过泛素化修饰影响NOX1的蛋白降解。本研究通过系统性阐明NOX1在PD铁死亡中的调控作用及其分子机制,将为靶向NOX1的PD治疗策略提供新的思路和理论依据。研究方法:1.(1)通过GEO数据库下载PD患者转录组学数据集(GSE7621和GSE20141),利用R语言分析筛选差异表达基因(Differentially Expressed Genes,DEGs)并与铁死亡基因集取交集,结合蛋白质网络互作(Protein-Protein Interaction Network,PPI)分析和LASSO回归模型分析筛选核心基因,并通过GSE20292外部数据集和MPP~+/鱼藤酮(Rotenone)诱导的PD细胞模型以及1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的PD小鼠模型进行实验验证,获得最终关键基因。(2)在整体水平,通过腺相关病毒(AAV-sh-NOX1)靶向敲低C57BL/6小鼠黑质部位NOX1,利用Western blotting和免疫组化实验检测酪氨酸羟化酶(Tyro-sine Hydroxylase,TH)的表达情况;通过旷场实验、疲劳转棒实验以及爬杆实验评估小鼠的运动功能。在离体细胞水平,利用慢病毒(sh-NOX1/OE-NOX1)敲低或过表达NOX1,通过细胞计数试剂盒8(Cell Counting Kit 8,CCK8)实验检测细胞活力以及乳酸脱氢酶(Lactate Dehydrogenase,LDH)实验检测细胞毒性。2.(1)在整体和离体细胞水平,利用Western blotting检测铁死亡相关指标(GPX4、FTH1、NQO1、HO-1)的表达情况;通过Ferro Orange探针检测亚铁离子含量;利用BODIPY 581/591 C11探针检测脂质过氧化水平以及通过DCFH-DA探针检测ROS生成,明确PD体内外模型中存在铁死亡。随后,利用腺相关病毒和慢病毒敲低NOX1后,再次检测上述指标,进而明确NOX1对铁死亡的调控作用。(2)通过转录组学测序筛选过表达NOX1后激活的下游信号通路,通过染色质免疫共沉淀(Chromatin Immunopre-cipitation,Ch IP)明确RUNX2作为STK32A的上游转录因子;利用Western blotting检测过表达NOX1后对RUNX2/STK32A通路以及铁自噬标志物(NCOA4、LC3II、Beclin1、p62)蛋白表达的影响。抑制RUNX2/STK32A通路后再次评估对铁自噬相关指标的影响;敲低NCOA4后,利用Western blotting检测FTH1的蛋白表达以及利用CCK8实验、MDA检测、BODIPY 581/591 C11以及DCFH-DA探针检测等实验来评估铁死亡相关指标的变化。3.(1)通过泛素在线网站预测NOX1上游的E3泛素连接酶,利用Western blotting检测FBXW7在PD体内外模型中的表达情况。(2)利用q PCR和Western blotting检测FBXW7敲低后NOX1的m RNA和蛋白水平。(3)通过内外源性的蛋白质免疫共沉淀(Co-Immunoprecipitation,Co-IP)与免疫荧光双染验证FBXW7与NOX1的相互作用。(4)通过多泛素化分析,明确K48连接的多泛素链介导FBXW7对NOX1的泛素化降解作用。研究结果:1.(1)生物信息学分析识别到NOX1、BECN1和NOS2为PD与铁死亡基因集共同关键基因,在PD体内外模型中NOX1和NOS2表达显著上调,BECN1表达显著降低。(2)NOX1敲低可显著增加MPTP诱导的小鼠TH的表达;NOX1敲低增加小鼠在旷场实验中行走的总距离和在中心区域的停留时间;延长小鼠在转棒仪上的停留时间;缩短爬杆到底部所用的时间。(3)NOX1敲低有效提高了PD细胞模型中的细胞活力,降低了细胞毒性。然而,过表达NOX1则呈现相反的趋势。2.(1)在PD的体内外模型中铁死亡相关指标GPX4、NQO1、FTH1以及HO-1的蛋白表达显著降低;在MPP~+/Rotenone诱导的PD细胞模型中亚铁离子含量增加;谷胱甘肽(Glutathione,GSH)水平降低而丙二醛(Malondialdehyde,MDA)含量增加;脂质过氧化积累增多;ROS产生增加。NOX1敲低可有效逆转PD体内外模型中铁死亡相关指标的变化。(2)转录组学测序结果发现,STK32A为NOX1下游激活的关键靶标分子;RUNX2作为STK32A的上游转录因子;与对照组相比,在NOX1过表达后STK32A、RUNX2、NCOA4、LC3II以及Beclin1表达上调而p62表达下降。抑制RUNX2或敲低STK32A可在一定程度上阻碍NOX1对铁自噬相关指标的激活作用。另外,NCOA4敲低后,FTH1表达增加,细胞活力增加,MDA含量降低,脂质过氧化和ROS产生减少。3.(1)在线网站预测FBXW7作为NOX1上游的E3泛素连接酶。(2)FBXW7可降低NOX1的蛋白表达,m RNA水平无显著差异。(3)FBXW7与NOX1具有相互作用。(4)FBXW7依赖于K48连接的多泛素链促进NOX1的蛋白降解。研究结论:1.NOX1在PD体内外模型中表达增加,NOX1敲低可延缓多巴胺能神经元丢失。2.NOX1通过激活RUNX2/STK32A通路,促进NCOA4介导的铁自噬,进而诱导铁死亡。3.FBXW7依赖于K48连接的多泛素链促进NOX1蛋白泛素化降解。更多还原

【Abstract】 Background and objectives:Parkinson’s disease(PD)is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain.Iron over-load and iron homeostasis imbalance driven by iron metabolic disorders represent critical pathological mechanisms in PD neurodegeneration,accelerating neuronal loss through the ac-tivation of the ferroptosis pathway.Ferritinophagy,which degrades ferritin via lysosomes to release free ferrous ions,exacerbates ferroptosis.However,the upstream molecular mecha-nisms regulating ferritinophagy remain poorly understood.Previous studies have shown that ferroptosis-targeted therapeutic interventions can effectively improve motor function and re-duce dopaminergic neuron loss in PD model mice,highlighting the importance of identifying PD-related ferroptosis genes for the treatment of PD.To address these scientific gaps,this study integrated PD transcriptomic data from the GEO database with ferroptosis-related gene sets and employed bioinformatics analysis to screen for key intersecting genes,identifying NADPH Oxidase 1(NOX1)as a critical candidate.Although NOX1 has been implicated in PD progression through regulating reactive oxygen species(ROS)production andα-synuclein(α-syn)aggregation,its role in modulating the ferritinophagy-fer-roptosis axis to affect dopaminergic neurons remains unreported.Besides,the epigenetic mech-anisms underlying NOX1 overexpression in PD require systematic exploration.This study aims to address three key questions:(1)to elucidate the role of NOX1 on dopa-minergic neurons;(2)to elucidate the molecular mechanisms by which the downstream RUNX2/STK32A pathway mediates ferritinophagy in the context of NOX1;and(3)to reveal the E3 ubiquitin ligase FBXW7 modulates NOX1 protein degradation through ubiquitination modification.By systematically clarifying the regulatory role and molecular mechanisms of NOX1 in PD-associated ferroptosis,this research provides novel insights and a theoretical basis for developing NOX1-targeted therapeutic strategies for PD.Methods:1.(1)Transcriptomic datasets from PD patients(GSE7621,GSE20141)were downloaded from the GEO database.Differentially expressed genes(DEGs)were identified by R language,followed by the intersection with a ferroptosis gene set.Core genes were screened via protein-protein interaction(PPI)network analysis and a least absolute shrinkage and selection operator(LASSO)regression model.Validation was performed using an external dataset(GSE20292),MPP~+/rotenone-induced PD cell models,and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced PD mouse models to identify key genes.(2)At the systemic level,targeted knockdown of NOX1 in the substantia nigra of C57BL/6 mice was achieved using adeno-asso-ciated virus(AAV-sh-NOX1).Tyrosine hydroxylase(TH)expression was assessed via Western blotting and immunohistochemistry.Motor functions were evaluated through open field tests,rotarod tests,and pole tests.At the cellular level,lentivirus-mediated NOX1 knockdown(sh-NOX1)or overexpression(OE-NOX1)was performed.Cell viability and cytotoxicity were as-sessed using CCK-8 and LDH assays,respectively.2.(1)At both systemic and cellular levels,ferroptosis-related markers(GPX4,FTH1,NQO1,HO-1)were analyzed via Western blotting.Ferrous iron levels were detected using Ferro Or-ange probes,lipid peroxidation was measured with BODIPY 581/591 C11 probes,and ROS generation was assessed via DCFH-DA probes,confirming the presence of ferroptosis in PD.Subsequently,adeno-associated virus(AAV)or lentivirus-mediated NOX1 knockdown was employed to re-evaluate the above indicators,clarifying the regulatory role of NOX1 in ferrop-tosis.(2)Transcriptome sequencing was employed to screen the downstream signaling path-ways activated by NOX1 overexpression,and chromatin immunoprecipitation(Ch IP)was used to identify RUNX2 as an upstream transcription factor of STK32A.Western blotting was uti-lized to assess the effects of NOX1 overexpression on the RUNX2/STK32A pathway and fer-ritinophagy markers(NCOA4,LC3II,Beclin1,p62).Besides,Inhibition of the RUNX2/STK32A pathway was followed by re-evaluation of ferritinophagy-related markers.Following NCOA4 knockdown,Western blotting was used to detect FTH1 protein expression,while Cell Counting Kit 8(CCK8)assays,malondialdehyde(MDA)detection kits,and BOD-IPY 581/591 C11 probe staining were used to assess changes in ferroptosis-related indicators.3.(1)Potential upstream E3 ubiquitin ligases for NOX1 were predicted using ubiquitin-re-lated online databases,and FBXW7 expression was detected by Western blotting in PD models both in vivo and in vitro.(2)Following FBXW7 knockdown,NOX1 m RNA and protein levels were measured via q PCR and Western blotting.(3)Endogenous and exogenous Co-Immuno-precipitation(Co-IP)as well as immunofluorescence co-staining were used to validate the in-teraction between FBXW7 and NOX1.(4)Polyubiquitination analysis was performed to con-firm that K48-linked polyubiquitin chains mediate FBXW7-dependent ubiquitin degradation of NOX1.Results:1.(1)Bioinformatics analysis identified NOX1,BECN1,and NOS2 as key common genes between PD and ferroptosis gene sets.NOX1 and NOS2 were significantly upregulated,while BECN1 was downregulated in PD models both in vitro and in vivo.(2)NOX1 knockdown significantly increased the expression of TH in MPTP-induced mice,improved motor function as reflected by increased total distance traveled and time spent in the central area in the open-field test,prolonged retention time on the rotarod,and shortened descent time in the pole test.(3)NOX1 knockdown effectively increased cell viability and decreased cytotoxicity in the PD cell model.However,the opposite trend was observed after overexpression of NOX1.2.(1)The protein levels of ferroptosis-related markers including GPX4,NQO1,FTH1,and HO-1 were significantly downregulated in PD models both in vivo and in vitro.In MPP~+/rote-none-induced PD cellular models,ferrous iron levels were elevated,accompanied by reduced glutathione(GSH)content,increased malondialdehyde(MDA)accumulation,enhanced lipid peroxidation,and elevated ROS production.Notably,NOX1 knockdown effectively reversed these ferroptosis-associated alterations in PD models.(2)Transcriptomic sequencing identified STK32A as a key downstream target gene activated by NOX1,with RUNX2 confirmed as an upstream transcription factor of STK32A.Compared with the control group,overexpression of NOX1 upregulated the expression of STK32A,RUNX2,NCOA4,LC3II,and Beclin1,while downregulating p62.Inhibition of RUNX2 or knockdown of STK32A partially attenuated NOX1-mediated activation of ferritinophagy-related markers.Additionally,NCOA4 knock-down increased the expression of FTH1,enhanced cell viability,reduced MDA levels,and de-creased lipid peroxidation and the production of ROS.3.(1)FBXW7 was identified as an upstream E3 ubiquitin ligase for NOX1 on the ubiquitin-related online databases.(2)FBXW7 reduced NOX1 protein levels without affecting m RNA expression.(3)Co-immunoprecipitation and immunofluorescence confirmed the direct interac-tion between FBXW7 and NOX1.(4)FBXW7 promoted NOX1 protein degradation via K48-linked polyubiquitin chains.Conclusions:1.NOX1 expression is significantly upregulated in PD models both in vivo and in vitro,and NOX1 knockdown attenuates dopaminergic neuron loss.2.NOX1 activates the RUNX2/STK32A pathway to promote NCOA4-mediated ferritinophagy,thereby inducing ferroptosis.3.FBXW7 promotes NOX1 protein degradation via K48-linked polyubiquitin chains.更多还原