CN115414485B - uN2CpolyG蛋白抑制剂的用途 - Google Patents
uN2CpolyG蛋白抑制剂的用途 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域，具体涉及uN2CpolyG蛋白抑制剂的用途，进一步涉及uN2CpolyG蛋白抑制剂、改善线粒体功能的试剂以及LRPPRC蛋白激动剂在制备治疗神经元核内包涵体病的药物中的用途。本发明为NIID的临床治疗提供了新的治疗方案。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及uN2CpolyG蛋白抑制剂的用途，进一步涉及uN2CpolyG蛋白抑制剂、改善线粒体功能的试剂以及LRPPRC蛋白激动剂在制备治疗神经元核内包涵体病的药物中的用途。
背景技术
神经元核内包涵体病(Neuronal intranuclear inclusion disease，NIID)是一种以中枢神经系统、外周神经系统以及多器官组织中出现细胞核内嗜酸性包涵体为主要病理特征的遗传性神经系统变性病/神经肌肉病。经多个团队研究发现，NIID的起因与NOTCH2NLC基因的5’非翻译区(untranslated region，UTR)CGG异常重复扩增突变相关，这是NIID研究的重要里程碑。另有研究发现，在其他多种神经系统疾病(包括成人遗传性脑白质病(Leukoencephalopathy)、特发性震颤(Essential tremor，ET)、痴呆(Dementia)、多系统萎缩(Multiple system atrophy，MSA)、帕金森病(Parkinson Disease，PD)、肌萎缩侧索硬化症(Amyotrophic lateral sclerosis，ALS)、眼咽远端肌病(Oculopharyngodistalmyopathy，OPDM)等)的低比例患者中也检测到NOTCH2NLC基因CGG重复扩增异常，提示NIID的疾病谱系涵盖了以上多种神经系统疾病。
尽管近年来针对NIID采取了多种治疗手段，但是能够用于NIID治疗的治疗靶点仍然较少，因此，寻找针对NIID治疗的新的治疗靶点、提供新的治疗方案对于NIID的临床治疗具有重要意义。
发明内容
因此，本发明要解决的技术问题在于提供针对NIID治疗的新的治疗靶点和治疗方案，从而提供uN2CpolyG蛋白抑制剂的用途，进一步提供uN2CpolyG蛋白抑制剂、改善线粒体功能的试剂以及LRPPRC蛋白激动剂在制备治疗神经元核内包涵体病的药物中的用途。
为此，本发明提供了uN2CpolyG蛋白抑制剂在制备改善线粒体功能的药物和/或治疗神经元核内包涵体病的药物中的用途。
可选地，所述uN2CpolyG蛋白抑制剂包括抑制uN2CpolyG蛋白基因表达的试剂、抑制uN2CpolyG蛋白活性的试剂或者降解uN2CpolyG蛋白的试剂中的至少一种。
uN2CpolyG蛋白(NOTCH2NLC-polyG)是NOTCH2NLC基因CGG重复扩增翻译产生的多聚甘氨酸多肽(polyGlycine，polyG)，能够介导细胞变性。本发明的发明人研究发现，NOTCH2NLC基因的CGG重复扩增突变，能够通过该基因上游的新开放阅读框(upstream openreading frame，uORF)中的ATG起始密码子，翻译产生uN2CpolyG蛋白，该蛋白具有毒性，能够引起果蝇模型多系统的进行性退化，证明uN2CpolyG蛋白是NOTCH2NLC基因动态突变引起NIID发病的主要原因，因此可将抑制uN2CpolyG蛋白作为NIID的潜在治疗方式。针对uN2CpolyG蛋白设计的小分子、基因药物、抗体等抑制剂均有可能用于治疗NIID。
uN2CpolyG蛋白的氨基酸序列可以表示为：MWICPGGG…GGGDREDARPAPLCCGRCWRSGCAARPPRMHCSVEMAMNPV，其中，GGG…GGG部分表示uN2CpolyG蛋白的氨基酸序列中可以有多个G，因NIID病人的NOTCH2NLC基因动态突变的CGG重复扩增次数不同，导致该基因突变表达的polyG长度不一，在致病范围内，GGG…GGG部分的长度可为40个G到300个G。
本发明还提供了改善线粒体功能的试剂在制备治疗神经元核内包涵体病的药物中的用途。
可选地，所述改善线粒体功能的试剂包括改善线粒体氧化磷酸化功能的试剂、促进线粒体ATP合成的试剂或改善线粒体复合物Ⅰ功能的试剂中的至少一种。
可选地，所述改善线粒体功能的试剂包括艾地苯醌或其盐，以及具备与艾地苯醌相似的治疗效果的试剂。
本发明的发明人通过构建NIID的转基因果蝇模型，发现NIID的转基因果蝇模型中多系统表达uN2CpolyG蛋白可以引起果蝇进行性的神经细胞丢失、运动障碍、寿命缩短等，以及形成uN2CpolyG蛋白包涵体，模拟了NIID重要的病理及临床特点。本发明的发明人进一步研究发现，在NIID的转基因果蝇模型中存在线粒体形态异常(肿胀)、功能异常(氧化磷酸化功能下降、ATP合成减少)、复合物Ⅰ受损等病理性改变，而且利用药物艾地苯醌改善线粒体的复合物I功能或者氧化磷酸化功能，能够有效治疗NIID果蝇模型的神经退化表型。因此，可将改善线粒体功能作为NIID的潜在治疗方式，而且，艾地苯醌及类似效果的药物可作为NIID的潜在治疗药物。
艾地苯醌(IDB)分子式为C19H30O5，为黄色结晶或结晶性粉末，无臭，极难溶于水，极易溶于氯仿、甲醇或无水乙醇，易溶于醋酸乙酯，难溶于正已烷。艾地苯醌对线粒体功能有激活作用，对脑功能代谢和脑功能障碍有改善作用，能提高脑内葡萄糖的利用率，促进ATP生成，能改善脑内神经递质5-羟色胺的代谢，具有较强抗氧化和清除自由基的作用。
本发明还提供了LRPPRC蛋白激动剂在制备改善线粒体功能的药物和/或治疗神经元核内包涵体病的药物中的用途。
可选地，所述LRPPRC蛋白激动剂包括促进LRPPRC蛋白基因表达的试剂、促进LRPPRC蛋白活性的试剂、过表达LRPPRC蛋白的基因试剂或者LRPPRC蛋白补充剂中的至少一种。
可选地，所述过表达LRPPRC蛋白的基因试剂包括编码LRPPRC蛋白的核酸，和/或所述核酸的载体，和/或携带所述载体的细胞；
所述LRPPRC蛋白补充剂包括LRPPRC蛋白。
本发明的发明人进一步研究发现，uN2CpolyG蛋白通过与线粒体RNA结合蛋白LRPPRC相互作用，介导线粒体基因表达下降，引起线粒体损伤，并且过表达或补充LRPPRC蛋白可以延缓NIID果蝇模型的神经退化。因此，LRPPRC蛋白的过表达或补充可以作为NIID的潜在治疗方式，具体可以通过腺相关病毒AAV等方式上调LRPPRC蛋白表达的方式开发基因治疗药物。
其中，LRPPRC蛋白的氨基酸序列的NCBI编号为：NP_573566.2，LRPPRC蛋白的编码信使RNA的核苷酸序列的NCBI编号为：NM_133259.4。
本发明还提供了艾地苯醌或其盐在制备改善线粒体功能的药物中的用途。
可选地，所述改善线粒体功能的药物包括改善线粒体氧化磷酸化功能的药物、促进线粒体ATP合成的药物或改善线粒体复合物Ⅰ功能的药物中的至少一种。
本发明技术方案，具有如下优点：
本发明提供了uN2CpolyG蛋白抑制剂、改善线粒体功能的试剂以及LRPPRC蛋白激动剂在制备治疗神经元核内包涵体病的药物中的用途，为NIID的临床治疗提供了新的治疗方案。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本发明实施例中uN2CpolyG蛋白在转基因果蝇模型中的致病作用的验证结果图；
图2是本发明实施例中uN2CpolyG蛋白在转基因果蝇模型中的多系统毒性验证结果图；
图3是本发明实施例中uN2CpolyG蛋白与线粒体共定位的验证结果图；
图4是本发明实施例中NIID中线粒体和分子改变情况的验证结果图；
图5是本发明实施例中艾地苯醌对NIID的治疗效果的验证结果图；
图6是本发明实施例中uN2CpolyG蛋白与LRPPRC蛋白相互作用的检测结果图。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明，并不局限于所述最佳实施方式，不对本发明的内容和保护范围构成限制，任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品，均落在本发明的保护范围之内。
实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
1、实验方法：
(1)转基因质粒与转基因果蝇构建
将人源的NOTCH2NLC(uN2C，uN2CpolyG)的上游开放阅读框亚克隆到attB-pUAST载体中，该载体在启动子区域包含UAS序列(图1A)。具有9个甘氨酸的uN2C为野生型uN2C，具有100个聚甘氨酸的uN2CpolyG为突变型uN2C。uN2C和uN2CpolyG蛋白都在其羧基末端与GFP(绿色荧光蛋白)融合。在验证了cDNA序列后，通过标准显微注射将具有GFP融合蛋白的质粒插入phiC31果蝇品系的attP2位点，产生了表达GFP载体对照、uN2C-GFP和uN2CpolyG-GFP的转基因果蝇。线粒体复合物I的RNAi果蝇由中国科学院生物物理研究所Jane Wu博士实验室赠送，UAS-Bsf果蝇由北京生命科学研究所王涛博士实验室慷慨提供。所有Gal4系统均来自Bloomington Drosophila Stock Center。所有果蝇均在25℃的标准玉米粉琼脂培养基上饲养，光照为12小时明暗循环。
(2)攀爬试验和寿命试验
攀爬试验和寿命试验均在培养瓶中进行，每个培养瓶内含有20-30只相同性别的果蝇。所有果蝇在羽化后1天内被挑出进行测试，并在整个测定期间每4天转移到新的培养瓶中。对于攀爬试验，将果蝇轻轻敲击到小瓶底部，然后记录15秒内爬到5厘米以上的果蝇数量。每次攀爬试验重复五次，计算平均值和标准误差。如上所述，在三个性别匹配和年龄匹配的组之间进行寿命测定，每5天记录一次攀爬试验和寿命试验。
(3)线粒体部分的提取
将50只活果蝇转移到装有500μL预冷缓冲液(220mM甘露醇、70mM蔗糖、20mM Hepes和1mM EDTA)的玻璃匀浆器中，并在冰上匀浆20次。将匀浆转移到1.5ml管中，在4℃下以300g离心5分钟。然后将上清液在4℃下以6000g离心10分钟以富集线粒体。线粒体沉淀在1ml洗涤缓冲液(250mM蔗糖、50mM Hepes、1mM EDTA(pH7.4))中洗涤，然后重悬于相同的缓冲液中，并在使用前储存在-80℃。
(4)蛋白酶K消化实验
将新鲜分离的线粒体样品与指定浓度的蛋白酶K(PK)在冰上孵育10分钟。然后用终浓度为2mM的苯甲基磺酰氟(PMSF)终止反应，然后在95℃加热10分钟。通过SDS/PAGE分离线粒体蛋白，并通过蛋白质印迹分析检测蛋白质。
(5)RNA-seq和生物信息学分析
使用mirVana miRNA分离试剂盒(Ambion)提取总RNA。使用Agilent2100Bioanalyzer(Agilent Technologies,Santa Clara,CA,USA)评估RNA完整性。对RNA完整性数(RIN)≥7的样品进行后续分析。使用TruSeq Stranded mRNA LTSample Prep Kit(Illumina,San Diego,CA,USA)构建文库。这些文库在Illumina测序平台(HiSeqTM 2500或Illumina HiSeq X Ten)上进行测序，并生成125bp/150bp双末端读数。使用DESeq R包函数estimateSizeFactors和nbinomTest鉴定了差异表达基因(DEG)。P值<0.05和foldchange>2或foldchange 0.5被设置为显着差异表达的阈值。对DEG进行层次聚类分析以探索基因表达模式。DEGs的KEGG通路富集分析是基于超几何分布使用R单独进行的。
(6)定量逆转录PCR(RT-qPCR)
使用TRizol试剂(Invitrogen)从患者的骨骼肌样品中分离总RNA。进行cDNA合成和qPCR。使用2-ΔΔCT方法计算RNA水平的倍数变化，并通过t检验进行分析。
(7)艾地苯醌的给药
艾地苯醌(IDB)购自齐鲁制药，粉末状，4℃保存，溶于DMSO中，保存浓度为1mM，-20℃保存。用果蝇玉米粉琼脂培养基或新鲜细胞培养基将药物稀释至工作浓度，每次实验前新鲜配制药物。IDB在卵子阶段给药，成虫每周3次被转移到装有药物的新鲜小瓶中。将IDB溶解在0.025％DMSO中，最终浓度为0μM、7μM、15μM和50μM。用不同浓度(0μM、1μM、2μM、3μM和4μM)的IDB处理表达uN2CpolyG的SH-SY5Y细胞24h，进行免疫荧光。
(8)电子显微镜(EM)
收集样品并在4℃下固定在2.5％戊二醛中过夜。在Leica EM UC6/FC6超薄切片机上对样品进行切片。切片用甲苯胺蓝染色以确保固定位置。然后将切片转移到铜网格上，在EM成像之前用乙酸双氧铀和乙酸铅进行复染。
(9)蛋白质印迹分析
样品用RIPA缓冲液[1％NP-40，0.5％脱氧胆酸钠，1％SDS(pH7.4)]含有蛋白酶抑制剂混合物(Roche)进行裂解。用相应的特异性抗体通过蛋白质印迹分析样品裂解物中相应蛋白的表达量。
(10)患者、肌肉活检和脑尸检
本研究经北京大学第一医院伦理委员会批准。所有方法均按照相关指南和规定进行。检查NIID患者和年龄匹配的对照受试者的肌肉活检样本。实验中使用的所有临床材料均在获得知情同意后获得用于诊断目的。所有样品之前都已通过常规组织学技术和电子显微镜检查。新鲜冷冻样品保存在-80℃直至使用。尸检NIID患者的脑标本用20％缓冲福尔马林固定，多个组织块包埋在石蜡中。
(11)SH-SY5Y细胞培养和转染
SH-SY5Y细胞在添加了10％胎牛血清(Gibco)、100单位/ml青霉素-链霉素的Dulbecco改良Eagle培养基(DMEM-F12；Gibco)中于37℃、5％CO2/95％空气的湿化培养箱中培养。使用表达GFP、uN2C-GFP和uN2CpolyG-GFP的腺病毒进行病毒感染。
(12)免疫荧光
肌肉样本进行连续冷冻切片(8μm)。将脑样本固定在福尔马林中，包埋在石蜡中并切片。用抗N2CpolyG(4D12)、COX IV(Proteintech Group 11242-1-AP；1:200)、泛素(Proteintech Group 10201-2-AP；1:200)和p62(Abcam ab56416,抗鼠；1:200)等一抗进行免疫荧光染色。用1×磷酸盐缓冲液(PBS)冲洗细胞，在转染GFP、uN2C-GFP和uN2CpolyG-GFP后24小时用1×PBS中的4％聚甲醛固定。线粒体用mitotracker Red(Invitrogen)染色。
(13)免疫电子显微镜
对于免疫电镜，NIID患者的脑组织在室温下固定在PBS中含有2％聚甲醛和0.2％戊二醛(pH7.2)的固定溶液中3小时。在冲洗和固定后处理之后，在4℃下在2.3M蔗糖中制备样品块。使用金刚石刀在120℃下切割超薄切片(70nm)。封闭后，用与6nm胶体金颗粒偶联的单克隆抗小鼠uN2CpolyG抗体(1:100)对切片进行免疫染色。
(14)免疫共沉淀
SH-SY5Y细胞用于蛋白质-蛋白质相互作用的转染和分析。实验在转染后24小时进行。收获的细胞用磷酸盐缓冲盐水(PBS)洗涤，并在冰上在含有混合蛋白酶抑制剂(罗氏)的裂解液中裂解。收集细胞裂解物的可溶部分并用于在4℃下与特异性抗GFP抗体和蛋白A-琼脂糖(Roche)进行免疫共沉淀。使用具有适当抗体的蛋白质印迹检查免疫沉淀物。
(15)线粒体耗氧率检测
对于每组，将五只活果蝇称重并转移到装有500μL预冷呼吸介质(3mM MgCl2、60mM乳糖酸、20mM牛磺酸、10mM KH2PO4、20mM HEPES、110mM D-蔗糖、1g/L BSA和0.5mM EGTA)并在冰上匀浆20次。在双通道滴定注射呼吸计(Oxygraph-2k；Oroboros Instruments，Innsbruck，Austria)中测定线粒体呼吸功能。组织匀浆分别转移到氧图室。经过短暂的稳定期后，关闭腔室，并使用DatLab软件5.2(Oroboros Instruments，Innsbruck，Austria)记录数据。为了评估果蝇线粒体呼吸功能，应用专门设计的底物-解偶联剂-抑制剂滴定。当呼吸稳定时测量常规呼吸(无添加剂，常规)。在没有ADP的情况下用谷氨酸(G，5mM)和苹果酸(M，2mM)滴定后检查复合物I(CI Leak)的呼吸泄漏状态。添加5mM ADP后测定复合物I(CIOXPHOS)的氧化磷酸化能力。添加琥珀酸盐(Suc，100mM)以检测CI和复合物II(CII，CI+IIOXPHOS)的最大OXPHOS能力。随后，通过滴定FCCP(逐步注入达到0.5μM)(CI+II ETS)获得电子传递系统(ETS)的最大解偶联呼吸能力。添加鱼藤酮(Rot，0.5μM)后测量CII支持的解偶联呼吸功能(CII ETS)。在添加抗霉素A(Ama，2.5μM)后评估剩余的耗氧量。
(16)ATP测定
使用 Luminescent Cell Viability Assay(Promega)测量总细胞ATP水平。简而言之，对于果蝇，将等量的果蝇匀浆与CellTiter-Glo底物在37℃孵育10分钟。将反应混合物转移到另一个不透明的96孔板中以测量发光。对于细胞，在测定前24小时，将表达GFP、N2C-GFP或N2CpolyG-GFP的SH-SY5Y细胞接种在96孔板中。除去培养基和细胞裂解后，将反应混合物转移到另一个不透明的96孔板中，在37℃下放置10分钟以测量发光。
2、测试内容及结果
基于上述实验方法中记载的各实验的操作过程，进行如下试验，试验结果如图1-图6所示。
(1)为了证明uN2CpolyG在转基因动物模型中的致病作用，按照上述方法，使用UAS-GAL4系统在果蝇中表达GFP、对照uN2C-GFP和uN2CpolyG-GFP(图1A)。通过蛋白质印迹检测到GFP、uN2C-GFP和uN2CpolyG-GFP蛋白在果蝇模型中具有相近的表达水平(图1B)。对各果蝇模型进行跟踪检查分析，发现uN2CpolyG的表达导致出生第5天的果蝇眼中出现视杆结构轻度丢失，而表达GFP或uN2C的果蝇眼则显示具有七个视杆的完整小眼结构(图1C-F)。为了研究表达uN2CpolyG是否会导致进行性的神经变性，在第30天检查了果蝇眼中的小眼结构，发现uN2CpolyG的表达导致30天的果蝇出现严重的小眼变性，而表达GFP或uN2C-GFP的果蝇具有完整的小眼结构体，统计结果表明，在第30天表达uN2CpolyG的果蝇视杆数量为3，而第5天为6(图1C-F)。并且，在表达uN2CpolyG的果蝇中发现了核内包涵体，但在表达对照uN2C蛋白的果蝇中没有发现(图1G)。这些数据表明uN2CpolyG蛋白在果蝇中的表达会导致进行性神经变性和核内包涵体形成，模拟了NIID的主要临床和病理特征。
(2)为了检测uN2CpolyG在转基因果蝇模型中的多系统毒性，按照上述方法，使用actin5C-GAL4在果蝇全身广泛表达GFP、uN2C-GFP和uN2CpolyG-GFP，并检测成虫的运动能力(攀爬试验)和寿命。检测结果显示，与GFP或uN2C-GFP组相比，表达uN2CpolyG-GFP的果蝇的运动能力在雄性和雌性组中均显著且逐渐减少(图2A为雄性，图2B为磁性)。此外，表达uN2CpolyG-GFP的果蝇的寿命显著缩短(图2C为雄性，图2D为雌性)。
(3)为了进一步证实uN2CpolyG与线粒体的共定位，在神经元细胞系SH-SY5Y中表达了GFP、uN2C-GFP或uN2CpolyG-GFP。结果显示，uN2CpolyG聚集体与线粒体部分共定位，而GFP或uN2C弥散分布在细胞质中(图3A和3B)。然后，从表达uN2CpolyG-GFP的SH-SY5Y细胞中分离出线粒体，蛋白质印迹实验表明，线粒体组分富含线粒体蛋白TIM23，而缺乏细胞质蛋白GAPDH或核蛋白组蛋白H3，说明线粒体组分较纯。在这些线粒体组分中始终检测到uN2CpolyG蛋白(图3C)。为了进一步探索线粒体内的uN2CpolyG定位，对分离的线粒体进行蛋白酶K(PK)消化，蛋白质印迹显示线粒体外膜(OMM)被PK处理(0.5至1μg/ml PK)破坏，并且uN2CpolyG对PK消化敏感，与OMM蛋白标记物TOM20的模式相同，而线粒体内膜标记TIM23保持完整(图3D)。这些数据表明，uN2CpolyG主要与线粒体外膜(OMM)相关，而少量的uN2CpolyG转运到线粒体膜间隙(IMS)。为了验证uN2CpolyG在体内的亚线粒体定位，使用NIID脑组织进行了免疫电子显微镜(immuno-EM)分析，发现uN2CpolyG位于OMM和IMS中(图3E)，表明uN2CpolyG与NIID脑中的线粒体共定位。随后，进行免疫共沉淀(co-IP)检测，发现线粒体RNA结合蛋白LRPPRC(富含亮氨酸的五肽重复基序蛋白)与uN2C和uN2CpolyG的相互作用(图3F)，提示LRPPRC是uN2CpolyG线粒体内靶点。
(4)为了进一步探索NIID中线粒体和分子改变，通过RNA测序分析了三名具有肌肉病理改变的NIID患者(N1-N3)和三名年龄匹配的对照(C1-C3)的骨骼肌活检样本。全基因组mRNA表达谱的火山图和热图揭示了对照和NIID样本之间的许多基因表达变化(图4A和4B)。重要的是，KEGG富集分析显示，最显着下调的表达基因对应于氧化磷酸化通路(图4C)，编码线粒体复合物I、III、IV和V的线粒体基因和核基因的表达显着降低(图4D和4E)。与三个对照个体相比，RT-qPCR证实了另外5名NIID患者骨骼肌样本中复合物I编码基因的转录下调，包括ND2、ND6、NDUFA3、NDUFA6、NDUFA7和NDUFV3 mRNAs(图4F)。为了进一步研究线粒体复合物I亚基在uN2CpolyG诱导的发病机制中的作用，检查线粒体复合物I的下调是否改变了uN2CpolyG诱导的毒性。线粒体复合物I编码基因(包括NDUFA6和NDUFS2)的敲低加剧了表达uN2CpolyG蛋白的果蝇视网膜变性，而对照果蝇中这些基因的敲低没有显示出任何可检测的改变(图4G)。最后，鉴于表达uN2CpolyG的果蝇线粒体形态和分子改变，进一步探索果蝇模型是否表现出线粒体呼吸能力的改变。使用Oroboro Oxygraph系统，观察到与表达对照uN2C蛋白的果蝇相比，表达uN2CpolyG蛋白的果蝇线粒体呼吸能力显着降低(图4H)，其中线粒体复合物I(CI OXPHOS)的氧化磷酸化显著下降(图4I)。此外，进一步测量了这两组果蝇全身的线粒体ATP合成水平。与表达对照uN2C蛋白的果蝇相比，表达uN2CpolyG蛋白的果蝇ATP合成水平显着降低(图4J)。这些数据表明，线粒体复合物I的变化可能是NIID的一个关键致病过程，可能是该疾病的治疗靶点。
(5)艾地苯醌(IDB)是辅酶Q的类似物，可促进沿呼吸链的电子转移并增加线粒体复合物I的活性。由于检测数据表明复合体I缺陷可能与uN2CpolyG发病机制有关，因此采用用递增浓度的IDB处理表达uN2CpolyG-GFP的SH-SY5Y神经元细胞。免疫荧光显示，2μM IDB的浓度显着降低了uN2CpolyG聚集体的形成(图5A和5B)。此外，表达uN2CpolyG的果蝇以7、15或50μM浓度的IDB喂养20天，显示出显著改善的运动能力(分别为p<0.0001、p<0.0001和p<0.01)(图5C)。重要的是，用15μM IDB处理显著延长了表达uN2CpolyG蛋白的果蝇寿命(P<0.0001)(图5D)。最后，测量了用IDB处理的25天大的表达uN2CpolyG的果蝇的全身线粒体ATP合成水平，与对照果蝇相比，用7μM、15μM或50μM IDB处理显著改善了ATP合成(分别为p<0.0001、p<0.0001和p<0.001)，最佳剂量为15μM(图5E)。这些数据表明，IDB减轻了NIID转基因果蝇模型中的线粒体功能障碍和神经变性，可作为该疾病的潜在治疗剂。
(6)本发明试验数据显示，uN2CpolyG蛋白通过与线粒体RNA结合蛋白LRPPRC相互作用，介导线粒体相关基因表达下降，导致线粒体损伤。通过果蝇遗传学方法，在表达uN2CpolyG蛋白的果蝇眼中过表达(补充)LRPPRC同源基因Bsf可以延缓神经细胞丢失。这些数据表明，LRPPRC可以作为NIID的干预靶点(图6)。
图1和图2的实验结果说明，uN2CpolyG毒性蛋白在动物模型表达，引起细胞毒性和神经退化，证明了uN2CpolyG蛋白的致病性；图5实验结果说明NIID疾病模型中利用艾地苯醌减少uN2CpolyG毒性蛋白形成的包涵体(既阻止或降解uN2CpolyG毒性蛋白包涵体)可以有效缓解疾病模型的神经退行性表型。这两部分结果说明抑制uN2CpolyG毒性蛋白，能够治疗NIID。
图4实验结果证明uN2CpolyG毒性蛋白引起NIID患者骨骼肌中线粒体相关基因表达下降，导致NIID转基因果蝇模型的线粒体氧化磷酸化能力下降和ATP合成减少，说明uN2CpolyG毒性蛋白引起线粒体功能异常；图5实验结果说明通过艾地苯醌改善线粒体功能，增加ATP合成，有效改善NIID果蝇模型的运动能力和生存能力。这两部分结果说明改善线粒体功能，能够治疗NIID。
图3实验结果证明uN2CpolyG毒性蛋白与线粒体RNA结合蛋白LRPPRC相互作用；图6实验结果说明，NIID果蝇模型中过表达(补充)LRPPRC蛋白(果蝇中同源基因Bsf)后，可以有效延缓神经退化。这两部分实验结果说明过表达或补充LRPPRC蛋白，能够治疗NIID。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (1)
1.艾地苯醌或其盐在制备治疗神经元核内包涵体病的药物中的用途。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP3533455A1 (en) * | 2018-03-01 | 2019-09-04 | International Institute of Molecular and Cell Biology in Warsaw | Inhibition of proteolytic activity in the treatment of mitochondriopathies |
| CN111349697A (zh) * | 2018-12-24 | 2020-06-30 | 中国科学院生物物理研究所 | Tdp-43蛋白病诊断和治疗的新靶点 |
| WO2020234454A1 (en) * | 2019-05-23 | 2020-11-26 | Deutsches Krebsforschungszentrum | Combination treatment of cancer targeting energy metabolism and intracellular ph |
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| CN111349697A (zh) * | 2018-12-24 | 2020-06-30 | 中国科学院生物物理研究所 | Tdp-43蛋白病诊断和治疗的新靶点 |
| WO2020234454A1 (en) * | 2019-05-23 | 2020-11-26 | Deutsches Krebsforschungszentrum | Combination treatment of cancer targeting energy metabolism and intracellular ph |
Non-Patent Citations (2)
| Title |
|---|
| CGG repeat expansion in NOTCH2NLC causes mitochondrial dysfunction and progressive neurodegeneration in Drosophila model;Jiaxi Yu et al.;NEUROSCIENCE;第119卷;第1-11页 * |
| 神经元核内包涵体病临床特点及尿细胞沉渣病理检查;黄招君等;中国神经精神疾病杂志;第48卷;第405-412页 * |
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