防治Alzheimer病药物作用候选基因靶标的初步验证
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摘要
阿尔茨海默病(Alzheimer's Disease,AD)是一种严重危害人类健康的神经退行性疾病。由于其发病机制复杂,迄今尚不十分清楚,亦无理想的治疗药物问世,因此,给其防治带来了很大的困难,近期很难取得突破性进展。目前临床用于治疗AD的主要药物如胆碱酯酶抑制剂只能减轻部分症状,对患者脑内的主要病理变化并无明显改善作用,也不能延缓病程或控制病情的发展。因此,研究开发疗效好、毒副作用小的新型防治AD药物变得极为迫切。
创新药物研究和开发中最重要的环节之一就是药物靶标的发现。AD是典型的多基因复杂疾病,其发生发展过程涉及多系统、多环节结构和功能的异常,因此,针对单一靶点的防治策略和措施难以取得满意疗效,多靶点综合干预才是提高AD防治水平的重要策略。因此,寻找并确立一组或几组与AD脑内病理改变及认知功能障碍密切相关的、相互之间具有补充与协同关系的“组合靶标”,将为AD防治药物的筛选和发现提供新的筛选靶标体系,将大大提高AD防治药物的研究和开发水平。
中药复方具有通过多途径、多环节和多靶点发挥作用的特点,因此其在防治AD这类多基因复杂疾病方面,与作用靶点相对单一的化学药物相比,具有明显的优势和特色。我国和日本等国家在防治AD中广泛使用的中药复方如六味地黄汤(Liu-Wei-Di-Huang decoction,LW)、八味地黄汤(Ba-Wei-Di-Huang decoction,BW)、黄连解毒汤(Huang-Lian-Jie-Du decoction,HL)、当归芍药散(Dang-Gui-Shao-Yao-San,DSS)、调心方(Tiao-Xin-Fang decoction,TXF)等,不但在治疗AD中取得了良好的临床疗效,而且在实验药理学研究中也被证明对于AD模型动物或学习记忆功能障碍模型动物具有良好治疗作用。从中医药分类看,这些中药复方的功能主治不尽相同,如补肾、活血化瘀、清热解毒等,提示它们发挥药效的靶点和作用机理可能有所不同,但对AD病人或模型动物具有良好疗效是其共同特点。因此,从这些中药中选择几种疗效明确的代表方剂做为工具,研究和探索它们发挥益智作用的环节或靶点有望为防治AD药物筛选与研究新靶标的发现提供线索,同时还可能为从药理学角度研究和阐述AD的病理生理学过程和发病机制提供新的线索。
基于上述原因,本实验室在国家重点基础研究发展规划(973)项目的资助下,选择包括六味地黄汤、八味地黄汤、当归芍药散、调心方及黄连解毒汤在内的几种功能主治不同的中药复方作为工具药物,以与AD具有相似病理特点的快速老化模型小鼠(senescence-accelerated mouse,SAM)P8亚系(SAMP8)为模型,通过实验治疗并结合抑制消减杂交、基因芯片和蛋白质组学等技术,从与学习记忆或认知功能密切相关的脑区,即海马和大脑皮层,寻找药物反应基因和蛋白,并通过系统的分析与比较,初步提出了一组可能用于AD防治药物筛选和评价的候选“组合基因靶标”。本课题在上述研究的基础上进行,重点在于对上述所提出的“组合基因靶标”进行初步验证。
本研究主要从所提出的“组合基因靶标”中选择了2个与AD病理变化密切相关的分子APP和Tau,和5类9个具有其它功能代表性的基因进行研究,其中包括与异常蛋白降解相关的分子AMFR和STUB1、细胞骨架相关分子NF-L、线粒体功能相关分子NDUFS2、信号转导相关分子CAMK2A、EphB6、CSTN1以及神经发生生长相关分子NRXN1、NGRN基因等。主要开展了以下几方面工作:首先观察了所选基因及其蛋白在SAMP8及其正常对照SAM R1亚系(SAMR1)海马和皮层中的增龄性表达和分布变化,进而观察了防治AD有效的5种中药复方LW、BW、HL、DSS、TXF以及1种天然产物石杉碱甲(Hupzine A,HupA)对这些基因及其蛋白表达的影响,最后,采用生物信息学方法对所选基因之间的相互关系及其与已知的几种AD关键病理分子之间的可能关系等进行了分析。
一、AD关键病理分子APP和tau蛋白表达的动态变化及其对防治AD药物的反应
β淀粉样蛋白(βamyloid protein,Aβ)沉积形成老年斑(senile plaques,SP)、tau蛋白异常聚集形成神经原纤维缠结(neurofibrillary tangles,NFT)和神经元丢失是AD的主要病理学特征,其中,Aβ和tau蛋白被认为是AD形成和发展的关键因素。因此本研究首先对实验所采用的与AD具有相似病理学特征的模型动物SAMP8海马和皮层中β-淀粉样前体蛋白(β-amyloid precursor protein,APP)和tau蛋白的表达情况进行了探讨。
1.快速老化模型小鼠增龄过程中APP表达的动态变化及其对防治AD药物的反应
采用实时定量PCR技术,观察了SAMP8和SAMR1增龄过程中海马和皮层APP mRNA表达的变化。结果表明,增龄过程中,SAMR1海马和皮层中APP mRNA的表达维持在相对恒定水平,而SAMP8中的表达随增龄逐渐增加,其中,12月龄时的表达水平显著高于2月龄和6月龄。与同龄SAMR1相比,6和12月龄SAMP8皮层中、12月龄SAMP8海马中APP表达水平均显著升高。上述结果提示SAMP8海马和皮层中APP mRNA表达水平随增龄明显增高,这可能是其学习记忆功能随增龄进行性衰退的重要原因。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,除BW外,其余药物均能不同程度地纠正或逆转SAMP8皮层中APP mRNA表达的升高;BW和DSS能逆转海马中其表达的升高。表明所选中药复方和天然药物对SAMP8APP基因的异常表达具有不同程度的改善或纠正作用,提示APP基因对所选的防治AD药物具有明显反应性。
2.快速老化模型小鼠增龄过程中tau蛋白表达的动态变化及其对防治AD药物的反应
观察SAMP8和SAMR1增龄过程中海马和皮层中tau蛋白mRNA表达变化的结果表明,增龄过程中,SAMR1皮层中tau蛋白mRNA的表达无明显变化,而SAMP8皮层中其表达随增龄增加,12月龄时表达水平最高,且显著高于同龄SAMR1。在海马中,tau蛋白mRNA的表达虽然在二亚系动物中均随增龄进行性增高,但在各月龄SAMP8中,其表达水平均明显高于同龄SAMR1。提示tau蛋白基因表达水平的增高可能也是SAMP8学习记忆功能衰退的重要原因之一。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,均明显下调SAMP8皮层tau蛋白基因表达的升高;而在海马中,只有HL未表现出对该基因表达的下调作用,其余药物均表现出对其的下调作用。表明所选药物对SAMP8tau蛋白mRNA的异常表达具有不同程度的改善作用,提示tau蛋白基因对所选防治AD药物亦具有明显反应性。
结合文献和我们以往的研究结果,可以认为,APP与tau的表达随增龄在SAMP8海马和皮层中的进行性增高可能是SAMP8学习记忆功能进行性衰退的重要原因,药物对其表达异常的改善或纠正正是药物改善其认知功能的重要原因。上述研究结果进一步说明SAMP8是一个可用于AD发生机理和药物筛选的良好动物模型,从而为我们选择此模型开展防治AD药物作用候选“组合基因靶标”的验证提供了新的实验依据。
二、AMFR等9个候选基因表达的动态变化及其对防治AD药物的反应
(一)与异常蛋白降解相关的分子AMFR和STUB1
1.AMFR
AMFR(Autocrine motility factor receptor,自分泌运动因子受体)是一个G-蛋白偶联受体,具有E3泛素-蛋白连接酶活性,参与滑面型内质网介导的异常蛋白的降解,AMFR与其配体AMF结合后,能够产生刺激细胞运动、促进神经元的生长与存活等作用。为了研究AMFR是否参与AD的发病,本研究观察了随增龄,AMFR及其配体AMF在SAM脑中的表达以及AMFR对防治AD药物的反应。
1.1快速老化模型小鼠增龄过程中AMFR及其配体表达的动态变化
采用实时定量PCR技术,观察了SAMP8和SAMR1增龄过程中海马和皮层AMFR mRNA表达的变化。结果表明,增龄过程中,在SAMR1的皮层和海马中,AMFR表达水平在2月龄最低,在6月龄最高;而在SAMP8的上述脑区,其表达于12月龄时达最低水平,且显著低于同龄SAMR1。
进而,采用Western blot技术,观察了SAMP8和SAMR1增龄过程中海马和皮层中AMFR蛋白表达的变化。结果表明,SAMR1皮层和海马中AMFR蛋白的表达随增龄显著增加,6月龄时已基本达表达高峰,此后基本无显著变化;而在SAMP8中,其表达则随增龄显著下降,至15月龄降至最低。与同龄SAMR1比较,2月龄,即幼龄时,SAMP8海马和皮层中AMFR的表达均显著增高,而15月龄,即老龄时,SAMP8中其表达则显著降低。由于AMFR具有参与内质网介导的异常蛋白降解的功能,因此,我们推测,老龄SAMP8脑中包括Aβ在内的异常蛋白沉积的增多可能与AMFR表达水平的降低有关。
AMF(Autocrine motility factor,自分泌运动因子)是AMFR的配体,具有神经营养作用。接下来,又观察了SAMP8和SAMR1增龄过程中海马和皮层该蛋白表达的变化。结果表明,在SAMR1皮层中,AMF蛋白在10、15月龄的表达较2、6月龄显著增加;而在SAMP8皮层中,其表达在6月龄最高,其后随增龄逐渐降低,至15月龄降至最低。两亚系之间比较的结果表明,除6月龄外,各月龄SAMP8皮层中AMF的表达均显著低于同龄SAMR1。在海马中,该蛋白的表达在各月龄SAMR1间无显著差别,而在SAMP8中,其表达于10月龄时最高,于2月龄时最低。两亚系之间进行比较发现,该蛋白在各月龄SAMP8海马中的表达也显著低于同龄SAMR1。结果提示,AMF水平的显著低下可能也是SAMP8表现有增龄性脑神经病理损害和认知功能障碍的重要原因。
1.2防治AD药物对AMFR表达的影响
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,LW和HupA显著上调SAMP8皮层中AMFR mRNA异常低表达;而在海马中,该基因表达的降低可被除LW之外的所有受试药物显著上调。进一步观察上述药物对AMFR蛋白表达影响的结果表明,无论在皮层还是海马中,LW、BW、HupA均对SAMP8AMFR蛋白表达的降低显示出了显著的上调作用。上述结果表明所选药物对SAMP8 AMFR基因和蛋白的异常表达具有不同程度的改善作用,提示AMFR对所选防治AD药物具有明显反应性。
2.STUB1
STUB1(STIP1 homology and U-Box containing protein 1,STIP1同源和U-Box域包含蛋白)兼有辅助分子伴侣功能和E3泛素-蛋白连接酶活性,是异常蛋白降解的泛素-蛋白酶系统和溶酶体系统二者间转换的分子开关,能够协调细胞内蛋白质量控制系统的平衡和稳定,在蛋白质量控制中起关键作用。
2.1快速老化模型小鼠增龄过程中STUB1表达的动态变化
分别采用实时定量PCR和Western blot技术,观察了SAMP8和SAMR1增龄过程中海马和皮层STUB1 mRNA和蛋白表达的变化。结果表明,增龄过程中,SAMR1皮层和海马中STUB1 mRNA表达均随增龄进行性增加,12月龄时表达最高;而在SAMP8中,其在皮层和海马中的表达规律有所不同,其中,在皮层中的表达为2月龄时最高,在海马中,则为6月龄时表达最高。两亚系之间比较的结果表明,2月龄SAMP8皮层中STUB1表达水平明显高于同龄SAMR1,12月龄SAMP8皮层和海马中STUB1 mRNA表达水平均明显低于同龄SAMR1。STUB1蛋白表达的增龄性变化的趋势与mRNA基本一致。上述结果提示老龄SAMP8STUB1表达水平的低下可能造成该蛋白参与的异常蛋白降解功能降低,从而使其蛋白质量控制作用下降,这可能与文献报道的异常蛋白在老龄SAMP8中的累积有关。
2.2防治AD药物对STUB1表达的影响
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,观察药物对STUB1基因表达的影响,结果表明,皮层中除TXF外,海马中除DSS外的其余5个药物均对SAMP8 STUB1 mRNA表达水平的低下表现出了明显的上调作用。观察药物对STUB1蛋白表达的影响,结果表明,皮层中除LW外,海马中除LW和HupA外,其它药物均显著上调了SAMP8中STUB1蛋白的异常低表达。结果表明所选药物对SAMP8 STUB1基因和蛋白的异常表达具有不同程度的改善作用,说明STUB1对所选防治AD药物具有明显反应性。
(二)细胞骨架相关分子NF-L
神经微丝是神经元特有的微丝蛋白,NF-L(Neurofilament protein,light chain,神经丝蛋白-轻链)是神经微丝三联体蛋白的聚合骨架,可通过调节神经生长、轴突管径以及轴突转运,来维持和调节着神经元细胞骨架的可塑性,在神经系统正常发育和正常发挥其功能中具有重要作用。
1.快速老化模型小鼠增龄过程中NF-L表达的动态变化
采用实时定量PCR和Western blot技术,观察了SAMP8和SAMR1增龄过程中海马和皮层NF-L mRNA和蛋白表达的变化。结果表明,增龄过程中,SAMR1皮层中,NF-L mRNA和蛋白表达均维持在相对恒定的水平:而海马中,其表达均在6月龄最高;与SAMR1相比较,10月龄后SAMP8皮层和海马中NF-L基因和蛋白表达均显著减少。上述结果提示NF-L的异常低表达可能造成神经元细胞骨架受损,而诱发神经元丢失。
2.防治AD药物对NF-L表达的影响
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,除LW和BW外,其余药物均能够显著上调12月龄SAMP8皮层中NF-L基因和蛋白的异常低表达;而海马中,各给药组对SAMP8中NF-L基因和蛋白的异常低表达均有上调效应。表明所选药物对SAMP8 NF-L的异常表达具有不同程度的改善作用,提示调节NF-L表达,改善由于其下降导致的神经元骨架受损而保护神经元可能是上述防治AD药物发挥AD治疗作用的共有机制之一。
(三)线粒体相关分子NDUFS2
NDUFS2(NADH dehydrogenase(ubiquinone)Fe-S protein 2,还原型烟酰胺二核苷酸脱氢酶铁硫蛋白2)是线粒体复合体Ⅰ铁硫蛋白的一个亚基,具有氧化还原酶活性,参与能量代谢通路,可以反映出机体的氧化磷酸化水平。
采用实时定量PCR技术,观察了SAMP8和SAMR1增龄过程中海马和皮层NDUFS2 mRNA表达的变化。结果表明,SAMR1中,NDUFS2 mRNA的表达均为6月龄最高,而2月龄最低;而SAMP8中,该基因的表达均在6月龄最低;与SAMR1相比较,SAMP8中,NDUFS2表达均在2月龄显著增加,而6月龄则显著减少。表明SAMP8早期就存在氧化磷酸化功能的异常。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,所有药物均能显著下调12月龄SAMP8皮层中NDUFS2 mRNA的异常高表达;而海马中,除LW和HupA外,其余四种药物对SAMP8中海马中NDUFS2 mRNA的异常低表达均有上调作用。提示上述防治AD有效的药物能够调节NDUFS2的异常表达从而改善SAMP8中的氧化磷酸化异常,这可能是这些防治AD药物发挥AD防治作用的共有途径之一。
(四)信号转导相关分子CAMK2,EphB6和CSTN1
1.CAMK2A
CaMKⅡ(Calcium/calmodulin-dependent protein kinase typeⅡ,钙/钙调蛋白依赖性蛋白激酶Ⅱ型α亚基)在神经递质释放、神经突触可塑性、学习和记忆等方面发挥着重要作用。其α亚基即CAMK2A(CaMKⅡalpha chain,CAMK2α)是CaMKⅡ在大脑皮层和海马中表达最为丰富的一个亚基,它不仅参与了tau蛋白的磷酸化,而且也可能参与了APP的磷酸化,在AD的发病过程中发挥重要作用。观察SAMP8和SAMR1增龄过程中海马和皮层CAMK2A表达变化的结果表明,增龄过程中,在SAMR1皮层和海马中,CAMK2A基因和蛋白均呈现出递减的趋势;而SAMP8中,其表达则均呈现出递增的趋势;SAMP8与SAMR1相比,10月龄后SAMP8中CAMK2A的表达均显著增加。结果提示CAMK2A表达的增龄性降低可能是导致SAMR1正常老化过程中学习记忆功能衰退的原因;而其表达的异常增加则可能导致SAMP8加速老化过程中学习记忆功能衰退。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,所有药物均能够显著下调12月龄SAMP8皮层和海马中CAMK2A mRNA的异常高表达。除LW外,上述药物均能显著下调12月龄SAMP8皮层中该蛋白的异常高表达;而在海马中,除HL和DSS外,上述药物对SAMP8中该蛋白的异常高表达也具有显著的下调作用。提示上述防治AD药物能够调节CAMK2A的异常表达从而改善SAMP8中的认知功能障碍,这也可能是六种药物改善SAMP8学习记忆功能的共同机制之一。
2.EphB6
EphB6(erythropoietin-producing hepatocyte receptor,促红细胞生成素源肝细胞受体B6)是一种跨膜受体蛋白,归属于酪氨酸激酶,具有独特的双向信号转导系统,能通过调节细胞骨架而发挥重要的生物学功能,在某些细胞株上其家族蛋白可调节Tau蛋白的磷酸化。
观察SAMP8和SAMR1增龄过程中海马和皮层EphB6表达的变化,结果表明,增龄过程中,SAMR1 EphB6 mRNA和蛋白表达均在6-10月间最高;而SAMP8中,其表达则在10月龄后最高;SAMP8与SAMR1相比较,10月龄后其表达均显著升高。结果提示EphB6早期的异常低表达可能造成了SAMP8的细胞免疫功能的缺陷,而后期其表达的异常增加则可能是加速了SAMP8的异常老化。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,LW、HL、TXF和DSS显著下调12月龄SAMP8皮层中该基因的异常高表达;而LW、BW、TXF和DSS则能够下调12月龄SAMP8海马中该基因的异常高表达。提示上述防治AD药物能够通过调节EphB6的异常表达而起到一定防治AD作用,但如早期给药,可能会收到更好效果。
3.CSTN1
CSTN1(Calsyntenin-1,钙同线蛋白-1)是一个能够将胞外蛋白水解活性与胞内Ca2+信号转导相关联的独特的突触后膜蛋白,能与钙离子结合,参与信号转导和细胞交流,能通过胞外蛋白水解来调节突触后钙的动力,同时,APP和CSTN1共同作用可增加Aβ的分泌,在AD的发生过程中发挥作用。
观察SAMP8和SAMR1增龄过程中海马和皮层CSTN1 mRNA表达变化的结果表明,增龄过程中,SAMR1 CSTN1 mRNA表达均在6月龄最高,2月龄最低;而在SAMP8中,其表达则有增龄性增加的趋势,其中以皮层的变化较为显著;与SAMR1相比,其表达在12月龄SAMP8中均显著增加。结果提示CSTN1的异常高表达可能造成SAMP8中Aβ释放的增加,从而加速AD的进程。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和HupA一个月,LW、BW、HL和DSS显著下调12月龄SAMP8皮层中该基因的异常高表达;而BW、HL、DSS和HupA也能够显著下调12月龄SAMP8海马中该基因的异常高表达。提示上述防治AD药物可能通过调节CSTN1的异常表达而减少Aβ的释放,起到一定的防治AD作用。
(五)神经发生生长相关分子Neurexin1和NGRN
1.Neurexin 1
NeurexinⅠ(Neurexin蛋白-1)可作为突触后受体与黑寡妇蜘蛛毒素α-latrotoxin结合,激发神经递质的释放,可作为细胞黏附分子和受体而发挥其作用,并与突触发生紧密相关。
本研究还观察了SAMP8和SAMR1增龄过程中海马和皮层NRXN1的mRNA表达的变化。结果表明,增龄过程中,SAMR1中,NRXN1 mRNA的表达在皮层中在6月龄最低,而海马中则在6月龄最高;在SAMP8中,其在皮层中增龄性递减,而在海马的表达则维持在不变的低水平;与SAMR1相比,其表达均在12月龄SAMP8中显著减少。结果提示NRXN1的异常表达可能造成SAMP8中细胞的营养不良环境,从而加速AD的进程。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,除LW、HL外的其它药物均显著上调12月龄SAMP8皮层中该基因的异常低表达;同样,除LW外的其它药物均显著上调12月龄SAMP8海马该基因的异常低表达。提示上述防治AD药物能够通过上调NRXN1的异常低表达而部分改善SAMP8神经细胞的营养环境而发挥一定AD防治作用。
2.NGRN
NGRN(Neurite outgrowth associated protein,神经突生长相关蛋白)可能与细胞生长和维持有关,并可能在神经元分化过程中起重要作用。
观察SAMP8和SAMR1增龄过程中海马和皮层NGRN的mRNA表达变化的结果表明,增龄过程中,SAMR1中,NGRN mRNA的表达在皮层随增龄递减,在海马中则相反;在SAMP8中,其在皮层和海马的表达均为6月龄最低;与SAMR1相比,其在6月龄SAMP8的皮层和12月龄的海马中均显著减少。结果提示NGRN的异常表达可能造成SAMP8中细胞的营养不良环境,从而加速AD的进程。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,LW、HL、DSS和HupA显著上调SAMP8中该基因的异常低水平,而海马中,除TXF外,其它药物对SAMP8中该基因的异常低水平表达也均有上调效应。提示上述防治AD药物能够通过上调NRXN1的异常低表达而部分改善SAMP8神经细胞的营养环境而发挥一定的抗AD作用。
三、候选分子AMFR等与AD关键病理蛋白的关系研究
1.AD关键病理蛋白在快速老化模型小鼠脑中的组织分布变化
用免疫组织化学的方法研究了AD关键病理蛋白APP、Tau和P-Tau在SAM鼠脑中的组织分布变化,结果表明,随SAMP8增龄,这三种AD关键病理蛋白在海马齿状回、海马CA3区以及颞叶皮层中均呈现出表达增加的趋势。
2.AMFR在快速老化模型小鼠脑中的组织分布变化
用免疫组织化学的方法研究了AMFR蛋白在SAM鼠脑中的组织分布变化,结果表明,随SAMP8增龄,AMFR蛋白在海马齿状回、海马CA3区以及颞叶皮层中均呈现出表达减少的趋势。
3.AMFR、STUB1与APP在快速老化模型小鼠额叶皮层的共定位
免疫荧光共定位研究结果显示:AMFR、STUB1与APP能够共表达于SAMP8的额叶皮层,在6月龄的SAMP8小鼠大脑的额叶皮层,AMFR与APP、STUB1与APP存在共定位关系。
四、候选基因靶标间的相互关系及其与AD关键病理分子关系的生物信息学分析
采用Osprey软件,首先考察了能够与AD关键病理蛋白及能够与所选9个分子直接相互作用的分子情况,结果表明,能够与APP发生直接相互作用的分子共49个;能够与Tau发生直接相互作用的分子共16个;能够与AMFR和STUB1发生直接相互作用的分别有3个和8个分子;能够与NF-L发生直接相互作用的分子共4个;未发现与NDUFS2和NGRN直接作用的分子;能够与CAMK2A、EphB6和CSTN1发生直接相互作用的分子分别为14个、4个和3个。
其次,分析了所选9个分子间及他们与APP、Tau、PS1、PS2和ApoE的直接相互作用。结果表明,所选9个分子间不存在直接的相互作用;只有CSTN1可以和APP、PS1发生直接相互作用,而其余分子间则不发生直接作用。
最后,分析了所选9个分子及与之相互作用的分子和AD关键病理分子的可能关系,预测结果表明它们中的80%能够与AD关键病理分子APP、Tau发生间接相互作用,而且是通过多条通路。其中,AMFR可以通过EphB6与APP、Tau发生联系;STUB1、EphB6、NRXN1均能与APP、Tau发生联系;NF-L与Tau联系密切,但同时也能够与APP发生联系;CaMK2A与APP关系较近,但也可以通过APP与Tau发生联系。结果提示从候选“组合基因靶标”中所选的各分子不是孤立在发挥作用,而是与AD关键病理蛋白形成相互作用网络来参与AD的病理生理过程。这一结果支持我们的假说,即它们不是单个基因的作用,而是形成一个作用网络,来参与AD的病理。
上述结果表明,从候选“组合基因靶标”中选择的5类9个分别代表不同功能的分子在SAMP8海马和皮层中的表达水平随增龄出现不同程度的异常,尤其在老龄SAMP8中表达异常最为明显,抗AD有效药物对其异常表达具有改善或纠正作用。同时,研究还表明,AMFR和STUB1在海马和皮层中与AD关键病理分子之一APP存在共定位关系,提示其可能对APP代谢生成Aβ产生影响。生物信息学分析提示所选的候选基因及其蛋白可通过多种途径与AD关键病理分子发生相互作用,从而发挥干预AD病理生理过程及改善认知功能的作用。上述研究结果提示,所选9个候选基因及其蛋白表达的异常可能是SAMP8随增龄脑内出现多种神经病理损害,并导致其学习记忆功能进行性衰退的重要原因,改善这些基因和蛋白的异常表达可能是降低AD相关神经病理损害、改善认知功能障碍的重要途径。因此,上述这9个分子有成为抗AD药物筛选潜在靶标的可能,有待进一步深入研究。
Alzheimer's disease(AD),one of the most prevalent neurodegenerative diseases, geatly threatens the health of the elderly and has been the fourth killing disease.AD holds about 60-70%of the totally 32 kinds of dementia.Mainly due to the uncertain aetiology of AD,the complexity of this disease,limited therapeutic drugs,it is hard to make great progress on the prevention and cure of AD in the near future.Cholinesterase inhibitors are most widely used drugs for the treatment of AD now,but their effects are only to ameliorate symptoms but do not achieve any permanent improvement.Therefore, to develop more effective AD therapeutic drugs with lower toxicity and lower side-effect remains an urgent task.The definite targets for anti-AD drugs restrict the development of new promising therapeutic drugs for AD and the breakthrough in this field is urgently calling for discovery and verification of the new targets for anti-AD drugs.
However,for any of the compounds,aiming at a sole target for AD,it is indeed a difficulty to obtain the satisfied therapeutic effects since AD is with such a complex pathogenesis which is still unclear till now,and also partly due to the serious consequences of AD,which are linked to abnormal construction and anormal functions of multi-systems.This fact suggests that intervention of multiple targets for AD is more important and which may be a feasible strategy to make great progress in prevention and cure of this stubborn disease.Thus,we propose a hypothesis that "combination targets" may be of great value to design,screen and further evaluate new drugs for AD.
Traditional Chinese medicine(TCM)are composed of several herbs and have many ingredients,their effects have the characteristics of multi-mechanisms, multi-pathway and multi-targets,likewise AD is a complex,multi-factorial and genetically heterogeneous syndrome.So the character of the whole traditional Chinese medicinal prescription is consistent with AD,the complicated disease,It is well known that many widely used traditional Chinese medicinal(TCM)prescriptions,including Liu-Wei-Di-Huang decoction(LW),Ba-Wei-Di-Huang decoction(BW), Huang-Lian-Jie-Du decoction(HL),Dang-Gui-Shao-Yao decoction(DSS), Tiao-Xin-Fang decoction(TXF)and Huperzine A(HupA),play important roles in the clinical therapy of AD patients,and also have beneficial effects on cognitive abilities in animal models.Senescence-accelerated mouse(SAM)prone/8(SAMP8)is a good animal model to investigate the fundamental mechanisms of age-related learning and memory deficits such as Alzheimer's disease(AD)at the gene and protein levels,and SAM resistant/1(SAMR1)is its normal control.It has been proved that all of the above-mentioned five TCM,including LW,BW,HL,DSS and TXF,can improve the functional impairments of learning and memory in SAMP8 in our previous studies. Although these five TCM are initially designed for different diseases,they do show the common therapeutic effects on AD patients and animal models.However,they may indeed have the different characters and through the different action mechanisms on the cognitive functions.
Having influences on gene expression is one of the mechanisms on drug action. Cerebral cortex and hippocampus are the key regions which are closely linked to learning and memory.Since these five TCM can improve the ability of learning and memory in AD cases,gene expression pattern of SAMP8 should be changed after SAMP8 were given to these TCM.To study these different expressed genes in SAMP8, drug reaction genes could be picked out.Then,to further compare the difference and identity of these drug reaction genes,the common genes responding to all these drugs and also the special genes responding to certain drug could be discovered.The common responding genes may reflect the similar action mechanism of all these drugs while the specific responding genes may represent the distinct action mechanism of the special drug.Thus,it will be very helpful to investigate the changed genes during the degenerative process of cognitive ability in SAMP8,which will not only give cues to find new targets for anti-AD drags but also be an important way to study and search the molecular targets for AD.
Consequently,the distinct anti-AD drugs such as TCM LW,BW,HL,DSS,TXF and natural product Hupzine A(HupA)were used as tools and experimental therapy had been conducted to investigate their effects on cognitive ability of SAMP8.Meantime,by Suppression subtractive hybridization(SSH)and cDNA microarray technique,our team bad successfully screened out the candidate "combination targets" for anti-AD drugs, which are composed by various molecules,such as protein degradation related genes AMFR and STUB1,neural cystoskeleton related gene NF-L,mitochondria function related gene NDUFS,signal transduction related genes CAMK2A,EphB6 and CSTN1, and neurogenesis related genes NRXN1 and NGRN.
However,these results are mainly based on the cDNA technique,which has a relatively higher false positive rate(FPR).So the necessary verification procedure is needed for the further validation of these targets for anti-AD drugs.
In order to preliminary verification of the candidate "combination targets" for anti-AD drugs,from which,this paper selected totally 9 representative genes belonging to five types and used SAMP8 as an animal model for AD.By the methods such as real time Quantitative fluorescence RT-PCR,Western blot,Immunohistochemistry and Immunofluorescence.the auther firstly investigated the age-related expression changes of these 9 molecules in SAMP8 and SAMR1 with aging.Then,the expression changes of these 9 molecules in 12-month SAMP8 treated with anti-AD drugs LW,BW,HL, DSS,TXF and natural product HupA were also investigated.Finally,the respective networks of these 9 molecules,the interaction of these 9 molecules and their possible roles in AD process had also been studied by bioinformatics.This paper have provided the evidences for supporting the hypothesis that "'combination targets",based on the bio-regulative network of targets for anti-AD drugs,may be of great value to design, screen and further evaluate new drugs for AD.
Part one.Expression changes of APP and Tau in the cerebral cortex and hippocampus of SAMR1 and SAMP8 and their responses to anti-AD drugs
The main pathological feature of AD is neuron loss,senile plaques(SP)and neurofibrillary tangles(NFT)in the key brain regions.SP and NFT are formed by the intercellular and intracellular deposition and aggregation byβamyloid protein(Aβ)and hyperphosphorylation form of Tau(HP Tau),respectively.Thus,Aβand Tau has been considered as the key factors that lead to the onset and progress of AD.Aβis produced byβ-amyloid precursor protein(APP).Consequently,this paper firstly investigated the expression changes of APP and Tau in the key brain regions related to cognitive functions in SAM.
1.Age-related expression changes of APP and its responses to anti-AD drugs With aging process,the expression of APP mRNA is maintained at certain level in the hippocampus and cerebral cortex from SAMR1,while it was increased after 2-month-old in the hippocampus and cerebral cortex from SAMP8.Compared with SAMR1,the expression of APP is increased significantly in SAMP8;The results from immunohistochemistry(IHC)showed that the number of positive cells is increased with aging in the cortex and hippocampus in SAMP8.
After SAMP8 was given anti-AD drugs LW,BW,HL,DSS,TXF and HupA for a month,the abnormal high mRNA expression of APP was significantly down-regulated in cerebral cortex of 12-month-old SAMP8 treated with LW,HL,DSS,TXF and HupA. In hippocampus of SAMP8,only BW and DSS treated groups showed down-regulative effects on mRNA expression of APP.The results indicated that these anti-AD drugs could regulate the abnormal mRNA expression of APP and meantime they indicated that APP could actively respond to these anti-AD drugs.
2.Age-related expression changes of Tau and its responses to anti-AD drugs
With aging process,the expression of Tau mRNA is maintained at certain level in the cerebral cortex from SAMR1,while it was significantly increased in cerebral cortex in 12-month-old SAMP8.Compared with SAMR1,the expression of Tau is increased significantly in the cerebral cortex in 12-month-old SAMP8.In the hippocampus from both SAMP8 and SAMR1,Tau mRNA was increased with aging.Compared with SAMR1,the expression of Tau is significantly increased in hippocampus in every month of SAMP8.The different expression pattern for Tau in SAMP8 indicated that the abnormal higher expression of Tau may result in the degeneration ability in learning and memory.
After SAMP8 was given to anti-AD drugs LW,BW,HL,DSS,TXF and HupA for a month,the abnormal high mRNA expression of Tau was significantly down-regulated in cerebral cortex of 12-month-old SAMP8 treated with all these anti-AD drugs.In hippocampus of SAMP8,only HL-treated group didn't show down-regulative effects on mRNA expression of Tau.The results indicated that these anti-AD drugs could regulate the abnormal mRNA expression of Tau and meantime they indicated that Tau could actively respond to these anti-AD drugs.
Many studies have indicated that,there were age-related defects in cognitive abilities in SAMP8.Our studies showed that in SAMP8,both APP and Tau were increased with aging process both in the cerebral cortex and hippocampus,the key brain regions closely linked to learning and memory.At the same time,they showed the apparent responses to these anti-AD drugs.These results further indicated that SAMP8 is an excellent animal model and which could be used to preliminarily verify the candidate obtained "combination targets" for anti-AD drugs.
Part two.Expression changes of the 9 molecules from the candidate "combination targets" for anti-AD drugs and their responses to anti-AD drugs
1.Abnormal protein degradation related molecules AMFR and STUB1
1.1 AMFR
AMFR,a G-protein-coupled receptor,shows an ubiquitin protein ligase(E3) activity and participates in degradation of abnormal proteins from the smooth endoplasmic reticulumits-associated degradation(ERAD).Studies showed that after its binding with its endogenous ligand Autocrine motility factor(AMF),AMFR played various roles such as stimulating cell migration,facilitating the cell survival.To study whether AMFR involves in the AD process or not,this paper had studied the age-related mRNA and protein expression of AMFR and its distribution in SAM with aging.
1.1.1 Age-related expression of AMFR and its ligand AMF in the cerebral cortex and hippocampus of SAM with aging
In both cerebral cortex and hippocampus,AMFR mRNA increased significantly after 2-month-old in SAMR1 while it decreased significantly in both cerebral cortex and hippocampus from SAMP8.Compared with SAMR1,the expression of AMFR mRNA decreased significantly in both the cerebral cortex and hippocampus in 12-month-old SAMP8.
In the cerebral cortex of SAMR1,AMFR protein increased after 2-months of age while in SAMP8,it showed an apparent decrease tendency after 6-months of age. Compared with SAMR1,AMFR protein decreased significantly after 6-months of age in SAMP8.In the hippocampus of SAMR1,AMFR protein increased after 2-months of age while it decreased with aging in SAMP8.Compared with SAMR1,protein expression of AMFR increased significantly at the 2-month old SAMP8,but decreased significantly at 6-and 15-month old SAMP8.
In the cerebral cortex of SAMR1,AMF protein increased significantly at 10-and 15-month-old SAMR1 than its expression level at 2-and 6-month-old SAMR1 while in samples from SAMP8,its expression decreased after 6-month-old with aging. Compared with SAMR1,AMF protein decreased significantly after 6-months of age in SAMP8.In the hippocampus of SAMR1,AMF protein maintained at a certain level with aging process while its expression showed an increase before 10-month-old and a decrease since 10-month-old.Compared with SAMR1.AMF protein decreased at all of age.
1.1.2 Expression of AMFR in the cerebral cortex and hippocampus of SAMP8 treated with anti-AD drugs
Compared with the normal control group,the mRNA and protein expression in the cerebral cortex and hippocampus of SAMP8 was decreased significantly.After SAMP8 was treated with the six anti-AD drugs,the abnormal low expression of AMFR was up-regulated by LW and HupA for mRNA and LW,BW and HupA for protein in the cerebral cortex of SAMP8.In the hippocampus of SAMP8,the abnormal low expression of AMFR was up-regulated by BW,HL,DSS,TXF and HupA for mRNA and LW,BW and HupA for protein.
1.2.STUB1
The STIP1 homology and U-box-containing proteinl(STUB1),a cytoplasmic protein also named C terminus of Hsc70-interacting protein(CHIP),is a Hsp70 co-chaperone and an E3 ubiquitin ligase that protects cells from proteotoxic stress,so STUB1 plays the pivotal role in the protein QC system.By its U-box domain,STUB1 acts as an E3 ligase to facilitate the transfer of a polyubiquitin chain to misfolded substrates,while by its TPR domain,STUB1 binds heat shock cognate 70(HSC70)or HSP70,attenuates the HSP40-stimulated ATPase and refolding activities of HSP70, which contributes to the delivery of protein substrates to lysosomes-mediated autophagy. So STUB1 is one of the most important members of UPS and acts as a molecular switch between proteasomal and lysosomal degradation pathways,mediates crosstalk between molecular chaperones and the UPS.This study showed that,with aging process,both mRNA and protein expression of STUB1 in cerebral cortex and hippocampus from SAMR1 were increased after 2-month-old while decreased in the above brain tissues from SAMP8 after 6-month-old;compared with SAMR1,mRNA and protein expression of STUB1 were decreased after 10-month-old SAMP8 respectively and could be up-regulated by the six anti-AD drugs.
2.NF-L
Neurofilament proteins are specific intermediate filament for neurons and NF-L is the backbone for NF-M and NF-H to polymerize.NF-L plays an important role in maintenance and regulation of the dynamic cystoskeleton of neurons by regulating the growth of neural cells,axis caliber and axonal transport.Consequently,NF-L plays a key role in CNS of the mammal.This study showed that,with aging process,both mRNA and protein expression of NF-L were decreased significantly in cerebral cortex and hippocampus after 10 months of age and which could be up-regulated by HL,DSS, TXF and HupA in cerebral cortex at both mRNA and protein levels while in the hippocampus,all these drugs showed up-regulative effects on both mRNA and protein expression of NF-L.
3.NDUFS2
NDUFS2 is an important subunit of ironsulfur protein in Complex I for respiratory chain.NDUFS2 has the activity of oxidoreduction and involves in the energy metabolism,which could reflect the level of oxidative phosphorylation.This study showed that mRNA of NDUFS was abnormal expressed in SAMP8 with aging compared with SAMR1,which could be down-regulated by LW,BW,HL,DSS,TXF and HupA in the cerebral cortex of SAMP8 and could be up-regulated by BW,HL,DSS and TXF in the hippocampus of SAMP8.
4.Signal transduction related genes CAMK2A,EphB6 and CSTN1
4.1 CAMK2A
CAMK2A is one most abundant subunit of CaMKII in cerebral cortex and hippocampus.The autophosphorylation of the threonine at 286 of CAMK2A is essential for hippocampus long term potentiation(LTP)and spatial learning.CAMK2A may involve in not only phosphorylation at multiple sites of Tau in AD patients,but also phosphorylation of APP in vitro.This study showed that that the levels of both CaMKIIαmRNA and protein decreased significantly in the cerebral cortex of SAMR1 with aging,but increased significantly in the cerebral cortex of SAMP8.Compared with age-matched SAMR1,the expression of mRNA and protein of CaMKIIαsignificantly increased in the cerebral cortex and hippocampus of SAMP8 after 10-months of age. After SAMP8 was treated with the previously mentioned drugs,the abnormally high expression of CaMKIIαwas relatively down-regulated for mRNA in both cerebral cortex and hippocampus,for protein in hippocampus by all the six anti-AD drugs used in this study and was relatively down-regulated for protein in the cerebral cortex by all the six anti-AD drugs with the exception for LW.
4.2 EphB6
EphB6 is a transmembrane receptor and belongs to protein tyrosine kinase(PTK), which an unique protein that have two-way signal transduction system based on its specific structure.This study showed that EphB6 mRNA has different expression patterns in the hippocampus and cerenral cortex,that is,its expression was increased with aging while showed a highest expression at 6-month-old in hippocampus in SAMR1.While in SAMP8,its expression showed an increase tendency in both the hippocampus and the cerebral cortex.For protein expression of EphB6,it showed an increase at 10-month-old but an decrease at 15-month-old SAMR1 in both the hippocampus and cerebral cortex.However,the protein level was increased after 10-month of age in SAMP8.Compared with SAMR1,the EphB6 protein increased significantly after 10-months of age in SAMP8 which could be down-regulated by LW, HL,TXF and DSS in the cerebral cortex and by LW,BW.TXF and DSS in the hippocampus.
4.3 CSTN1
CLSTN1 is a specific molecule which could link the proteolysis outside of cytes and the signal transduction of Ca~(2+)within the cytes CLSTN1 locates at the postsynaptic membrane and binds with Ca~(2+)to regulate the signal transduction and cell communication.Study showed that this specific protein could cooperate with APP to increase the secretion of Aβ,thus,this protein may accelerate the AD process.Our study showed that CSTN1 mRNA was highest expressed at 6-month-old SAMR1 in both the cerebral cortex and hippocampus while its expression pattern showed an increase in SAMP8 in both of the above-mentioned brain regions.Compared with SAMR1,this gene increased significantly in SAMP8 and which could be down-regulated by LW,BW,HL and DSS in the cerebral cortex and by BW.HL,DSS and HupA in the hippocampus.
5.NRXN1 and NGRN
NeurexinⅠcould bind withα-latrotoxin as a postsynaptic receptor,thus further activate the release of neurotransmitter(NT).In addition,NRXN1 could play its roles as a cellular adhesion molecule(CAM)and closely linked to synaptogenesis.NGRN has been presumed to play an important role in cell growth,maintenance and neuron differentiation.This paper showed that both NRXN1 and NGRN mRNA were abnormally lower expressed in SAMP8 compared with SAMR1.The lower expression of NRXN1 could be up-regulated by BW,DSS,TXF and HupA in the cerebral cortex in SAMP8 and by all anti-AD drugs with the exception for LW in the hippocampus.And the lower expression of NGRN could be up-regulated by LW,HL,DSS and HupA in the cerebral cortex and by all drugs with the exception for TXF.
Part three.Relationship between candidate gene targets for anti-AD drugs and the key pathological proteins in AD
1.The distribution of key pathological proteins in AD
Results from the study by immunohistochemistry showed that with aging process, APP,Tan and P-Tau showed an apparent increase tendency in the subregions CA3 and dentate gyrus(DG)of hippocampus and the subregion temporal lobe(TL)of cerebral cortex.
2.The distribution of AMFR protein in SAM with aging
Results from the study by immunohistochemistry showed that with aging process, the positive neural cells decreased apparently in the subregions CA3 and dentate gyrus (DG)of hippocampus and the subregion temporal lobe(TL)of cerebral cortex.While in SAMR1,this phenomenen was not apparent or just showed an increase tendency in the same subregions.
3.Co-localization of AMFR,STUB1 and APP in subregion frontal lobe of the cerebral cortex
Results from the study by immunofluorescence showed a definite co-localization of AMFR and APP,STUB1 and APP in subregion frontal lobe of the cerebral cortex in 6-month-old SAMP8.
Part four.Interaction of candidate gene targets for anti-AD drugs and their roles in AD process
First.by the molecules interaction analysis software Osprey,the molecules that directly interact with the key pathological protein APP,Tau and the nine candidate molecules were analyzed.The results showed that there were total 49 and 16 molecules directly interacting with APP and Tau,respectively.The result indicated that there are many molecules directly interacting with APP and Tau,which were related to multiple functions.To further study these molecules will be no doubt benefiting the further understanding of the etiopathogenisis for AD and which will help to prevent and cure this disease.There were total 3 and 7 molecules directly interacting with AMFR and STUB1,respectively.The analysis result indicated that involvement in protein metabolism was the major function of AMFR,while for STUB1,besides this function, there were also other important functions,The results showed that there were total 4 molecules directly interacting with NF-L and which indicated that the main function of NF-L is cell organization and cytogenesis.However,the software analysis showed that there were no direct interaction molecules to NDUFS2 and NGRN till now.The direct interaction molecules to CAMK2A,EphB6 and CSTN1 were 14,4 and 3,respectively. Phosphorylation of substrates was the basic function for CAMK2A and CAMK2A is a muir-functional enzyme.EphB6 could involve in phosphorylation of substrates and cell organization and cytogenesis.To further study the functions of CSTN1 will benefit both the understanding and the prevention and cure of AD.The NRXN1 could interaction directly to 20 molecules and the major functions of NRXN1 were transport and cell organization and cytogenesis.There were no direct interacting molecules for NGRN. Then,the possible interaction within the 9 molecules as well as APP,Tau,PS1.PS2 and ApoE were analyzed by osprey and the results are as follows:There were no direct interaction within the 9 molecules and only CSTN1 could directly interact with APP and PS1.Finally,the possible interaction of the 9 molecules and their direct interaction molecules with the key pathological protein of AD was also analyzed by Osprey.The results predicted that nearly 80%of these candidate 9 molecules could indirectly interact with APP and Tan by several pathways.Among of which,AMFR could interact indirectly with APP and Tau through the linkage of EphB6;STUB1 could interact indirectly with APP and Tau;NF-L was much closer to Tau but it could also interact with APP;CaMK2A was much closer to APP but it could also interact with Tau by the linkage of APP;EphB6 could indirectly interact with both APP and Tau;NRXN1 was much closer to APP but it could also interact with Tau.These results indicated that these 9 molecules played their roles in AD onset by the interaction network within them and their interacting molecules instead of their respective sole action.The result supports the hypothesis that the interaction network instead of respective sole action of candidates from "combination targets" for anti-AD drugs involves in the pathology of AD.
In sum,in order to preliminary verification of the candidate "combination targets" for anti-AD drugs,from which,this paper selected totally 9 representative genes belonging to five types and used SAMP8 as the animal model for AD.By the methods such as real time Quantitative fluorescence RT-PCR,Western blot, Immunohistochemistry and Immunofluorescence,the auther firstly investigated the age-related expression changes of APP,Tau and these 9 molecules in SAMP8 and SAMR1 with aging.Then,the expression changes of these 9 molecules in 12-month SAMP8 treated with anti-AD drugs LW,BW,HL,DSS,TXF and natural product HupA were also investigated.Finally,the respective networks of these 9 molecules,the interaction of these 9 molecules and their possible roles in AD process had also been studied by bioinformatics.The results showed that these 9 molecules not only closely linked to degeneration in learning and memory ability of SAMP8,but also actively responded to the multiple anti-AD drugs used in this paper.The results also indicated that on the one hand,the same molecule expressed differently at its gene and protein levels,which may be due to the post-transcriptional processing,translation and post-translational modification.The same molecule showed differently distributed patterns in part in different brain regions,which may be due to the distinct functions of its own and different brain regions.On the other hand,the same molecule showed differently responses to the same drug in the different brain regions,which may be caused by the specificities of drugs and specific action region of the molecule.However, the same molecule showed the similar responses to different anti-AD drugs,which indicated that this molecule may be the common target for multiple drugs.And also,the different molecules showed the similar responses to the same drug,which may be resulted from the multiple mechanisms of the same drug.In addition,the 80%of the candidate molecules were predicted to interact with the key pathological protein APP and Tau,and they may involve in the pathology of AD by the interaction network instead of respective sole action of themselves.The results obtained by this paper settled the foundation for designing,screening and further evaluating new drugs for AD, which were based on the bio-regulative network of "combination targets" for anti-AD drugs.
In conclusion,this paper supported the hypothesis that "combination targets", based on the bio-regulative network of targets for anti-AD drugs,are of great value to designing,screening and further evaluating new drugs for AD.And the "combination targets" are composed by one group or several groups of molecules,which are closely related to cognitive defects in AD and show complementation and cooperation within them."Combination targets" based on the bio-regulative network of targets for anti-AD drugs,will be of great value not only for designing,screening and further evaluating new drugs for AD,but also for studying and elucidating the pathophysiological processes of AD from the view of pharmacology,and also,will give important cues for discoveries of biomarkers used to predict the risk for the onset of AD,diagnose AD and assess the therapeutic effect by anti-AD drugs.
创新药物研究和开发中最重要的环节之一就是药物靶标的发现。AD是典型的多基因复杂疾病,其发生发展过程涉及多系统、多环节结构和功能的异常,因此,针对单一靶点的防治策略和措施难以取得满意疗效,多靶点综合干预才是提高AD防治水平的重要策略。因此,寻找并确立一组或几组与AD脑内病理改变及认知功能障碍密切相关的、相互之间具有补充与协同关系的“组合靶标”,将为AD防治药物的筛选和发现提供新的筛选靶标体系,将大大提高AD防治药物的研究和开发水平。
中药复方具有通过多途径、多环节和多靶点发挥作用的特点,因此其在防治AD这类多基因复杂疾病方面,与作用靶点相对单一的化学药物相比,具有明显的优势和特色。我国和日本等国家在防治AD中广泛使用的中药复方如六味地黄汤(Liu-Wei-Di-Huang decoction,LW)、八味地黄汤(Ba-Wei-Di-Huang decoction,BW)、黄连解毒汤(Huang-Lian-Jie-Du decoction,HL)、当归芍药散(Dang-Gui-Shao-Yao-San,DSS)、调心方(Tiao-Xin-Fang decoction,TXF)等,不但在治疗AD中取得了良好的临床疗效,而且在实验药理学研究中也被证明对于AD模型动物或学习记忆功能障碍模型动物具有良好治疗作用。从中医药分类看,这些中药复方的功能主治不尽相同,如补肾、活血化瘀、清热解毒等,提示它们发挥药效的靶点和作用机理可能有所不同,但对AD病人或模型动物具有良好疗效是其共同特点。因此,从这些中药中选择几种疗效明确的代表方剂做为工具,研究和探索它们发挥益智作用的环节或靶点有望为防治AD药物筛选与研究新靶标的发现提供线索,同时还可能为从药理学角度研究和阐述AD的病理生理学过程和发病机制提供新的线索。
基于上述原因,本实验室在国家重点基础研究发展规划(973)项目的资助下,选择包括六味地黄汤、八味地黄汤、当归芍药散、调心方及黄连解毒汤在内的几种功能主治不同的中药复方作为工具药物,以与AD具有相似病理特点的快速老化模型小鼠(senescence-accelerated mouse,SAM)P8亚系(SAMP8)为模型,通过实验治疗并结合抑制消减杂交、基因芯片和蛋白质组学等技术,从与学习记忆或认知功能密切相关的脑区,即海马和大脑皮层,寻找药物反应基因和蛋白,并通过系统的分析与比较,初步提出了一组可能用于AD防治药物筛选和评价的候选“组合基因靶标”。本课题在上述研究的基础上进行,重点在于对上述所提出的“组合基因靶标”进行初步验证。
本研究主要从所提出的“组合基因靶标”中选择了2个与AD病理变化密切相关的分子APP和Tau,和5类9个具有其它功能代表性的基因进行研究,其中包括与异常蛋白降解相关的分子AMFR和STUB1、细胞骨架相关分子NF-L、线粒体功能相关分子NDUFS2、信号转导相关分子CAMK2A、EphB6、CSTN1以及神经发生生长相关分子NRXN1、NGRN基因等。主要开展了以下几方面工作:首先观察了所选基因及其蛋白在SAMP8及其正常对照SAM R1亚系(SAMR1)海马和皮层中的增龄性表达和分布变化,进而观察了防治AD有效的5种中药复方LW、BW、HL、DSS、TXF以及1种天然产物石杉碱甲(Hupzine A,HupA)对这些基因及其蛋白表达的影响,最后,采用生物信息学方法对所选基因之间的相互关系及其与已知的几种AD关键病理分子之间的可能关系等进行了分析。
一、AD关键病理分子APP和tau蛋白表达的动态变化及其对防治AD药物的反应
β淀粉样蛋白(βamyloid protein,Aβ)沉积形成老年斑(senile plaques,SP)、tau蛋白异常聚集形成神经原纤维缠结(neurofibrillary tangles,NFT)和神经元丢失是AD的主要病理学特征,其中,Aβ和tau蛋白被认为是AD形成和发展的关键因素。因此本研究首先对实验所采用的与AD具有相似病理学特征的模型动物SAMP8海马和皮层中β-淀粉样前体蛋白(β-amyloid precursor protein,APP)和tau蛋白的表达情况进行了探讨。
1.快速老化模型小鼠增龄过程中APP表达的动态变化及其对防治AD药物的反应
采用实时定量PCR技术,观察了SAMP8和SAMR1增龄过程中海马和皮层APP mRNA表达的变化。结果表明,增龄过程中,SAMR1海马和皮层中APP mRNA的表达维持在相对恒定水平,而SAMP8中的表达随增龄逐渐增加,其中,12月龄时的表达水平显著高于2月龄和6月龄。与同龄SAMR1相比,6和12月龄SAMP8皮层中、12月龄SAMP8海马中APP表达水平均显著升高。上述结果提示SAMP8海马和皮层中APP mRNA表达水平随增龄明显增高,这可能是其学习记忆功能随增龄进行性衰退的重要原因。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,除BW外,其余药物均能不同程度地纠正或逆转SAMP8皮层中APP mRNA表达的升高;BW和DSS能逆转海马中其表达的升高。表明所选中药复方和天然药物对SAMP8APP基因的异常表达具有不同程度的改善或纠正作用,提示APP基因对所选的防治AD药物具有明显反应性。
2.快速老化模型小鼠增龄过程中tau蛋白表达的动态变化及其对防治AD药物的反应
观察SAMP8和SAMR1增龄过程中海马和皮层中tau蛋白mRNA表达变化的结果表明,增龄过程中,SAMR1皮层中tau蛋白mRNA的表达无明显变化,而SAMP8皮层中其表达随增龄增加,12月龄时表达水平最高,且显著高于同龄SAMR1。在海马中,tau蛋白mRNA的表达虽然在二亚系动物中均随增龄进行性增高,但在各月龄SAMP8中,其表达水平均明显高于同龄SAMR1。提示tau蛋白基因表达水平的增高可能也是SAMP8学习记忆功能衰退的重要原因之一。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,均明显下调SAMP8皮层tau蛋白基因表达的升高;而在海马中,只有HL未表现出对该基因表达的下调作用,其余药物均表现出对其的下调作用。表明所选药物对SAMP8tau蛋白mRNA的异常表达具有不同程度的改善作用,提示tau蛋白基因对所选防治AD药物亦具有明显反应性。
结合文献和我们以往的研究结果,可以认为,APP与tau的表达随增龄在SAMP8海马和皮层中的进行性增高可能是SAMP8学习记忆功能进行性衰退的重要原因,药物对其表达异常的改善或纠正正是药物改善其认知功能的重要原因。上述研究结果进一步说明SAMP8是一个可用于AD发生机理和药物筛选的良好动物模型,从而为我们选择此模型开展防治AD药物作用候选“组合基因靶标”的验证提供了新的实验依据。
二、AMFR等9个候选基因表达的动态变化及其对防治AD药物的反应
(一)与异常蛋白降解相关的分子AMFR和STUB1
1.AMFR
AMFR(Autocrine motility factor receptor,自分泌运动因子受体)是一个G-蛋白偶联受体,具有E3泛素-蛋白连接酶活性,参与滑面型内质网介导的异常蛋白的降解,AMFR与其配体AMF结合后,能够产生刺激细胞运动、促进神经元的生长与存活等作用。为了研究AMFR是否参与AD的发病,本研究观察了随增龄,AMFR及其配体AMF在SAM脑中的表达以及AMFR对防治AD药物的反应。
1.1快速老化模型小鼠增龄过程中AMFR及其配体表达的动态变化
采用实时定量PCR技术,观察了SAMP8和SAMR1增龄过程中海马和皮层AMFR mRNA表达的变化。结果表明,增龄过程中,在SAMR1的皮层和海马中,AMFR表达水平在2月龄最低,在6月龄最高;而在SAMP8的上述脑区,其表达于12月龄时达最低水平,且显著低于同龄SAMR1。
进而,采用Western blot技术,观察了SAMP8和SAMR1增龄过程中海马和皮层中AMFR蛋白表达的变化。结果表明,SAMR1皮层和海马中AMFR蛋白的表达随增龄显著增加,6月龄时已基本达表达高峰,此后基本无显著变化;而在SAMP8中,其表达则随增龄显著下降,至15月龄降至最低。与同龄SAMR1比较,2月龄,即幼龄时,SAMP8海马和皮层中AMFR的表达均显著增高,而15月龄,即老龄时,SAMP8中其表达则显著降低。由于AMFR具有参与内质网介导的异常蛋白降解的功能,因此,我们推测,老龄SAMP8脑中包括Aβ在内的异常蛋白沉积的增多可能与AMFR表达水平的降低有关。
AMF(Autocrine motility factor,自分泌运动因子)是AMFR的配体,具有神经营养作用。接下来,又观察了SAMP8和SAMR1增龄过程中海马和皮层该蛋白表达的变化。结果表明,在SAMR1皮层中,AMF蛋白在10、15月龄的表达较2、6月龄显著增加;而在SAMP8皮层中,其表达在6月龄最高,其后随增龄逐渐降低,至15月龄降至最低。两亚系之间比较的结果表明,除6月龄外,各月龄SAMP8皮层中AMF的表达均显著低于同龄SAMR1。在海马中,该蛋白的表达在各月龄SAMR1间无显著差别,而在SAMP8中,其表达于10月龄时最高,于2月龄时最低。两亚系之间进行比较发现,该蛋白在各月龄SAMP8海马中的表达也显著低于同龄SAMR1。结果提示,AMF水平的显著低下可能也是SAMP8表现有增龄性脑神经病理损害和认知功能障碍的重要原因。
1.2防治AD药物对AMFR表达的影响
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,LW和HupA显著上调SAMP8皮层中AMFR mRNA异常低表达;而在海马中,该基因表达的降低可被除LW之外的所有受试药物显著上调。进一步观察上述药物对AMFR蛋白表达影响的结果表明,无论在皮层还是海马中,LW、BW、HupA均对SAMP8AMFR蛋白表达的降低显示出了显著的上调作用。上述结果表明所选药物对SAMP8 AMFR基因和蛋白的异常表达具有不同程度的改善作用,提示AMFR对所选防治AD药物具有明显反应性。
2.STUB1
STUB1(STIP1 homology and U-Box containing protein 1,STIP1同源和U-Box域包含蛋白)兼有辅助分子伴侣功能和E3泛素-蛋白连接酶活性,是异常蛋白降解的泛素-蛋白酶系统和溶酶体系统二者间转换的分子开关,能够协调细胞内蛋白质量控制系统的平衡和稳定,在蛋白质量控制中起关键作用。
2.1快速老化模型小鼠增龄过程中STUB1表达的动态变化
分别采用实时定量PCR和Western blot技术,观察了SAMP8和SAMR1增龄过程中海马和皮层STUB1 mRNA和蛋白表达的变化。结果表明,增龄过程中,SAMR1皮层和海马中STUB1 mRNA表达均随增龄进行性增加,12月龄时表达最高;而在SAMP8中,其在皮层和海马中的表达规律有所不同,其中,在皮层中的表达为2月龄时最高,在海马中,则为6月龄时表达最高。两亚系之间比较的结果表明,2月龄SAMP8皮层中STUB1表达水平明显高于同龄SAMR1,12月龄SAMP8皮层和海马中STUB1 mRNA表达水平均明显低于同龄SAMR1。STUB1蛋白表达的增龄性变化的趋势与mRNA基本一致。上述结果提示老龄SAMP8STUB1表达水平的低下可能造成该蛋白参与的异常蛋白降解功能降低,从而使其蛋白质量控制作用下降,这可能与文献报道的异常蛋白在老龄SAMP8中的累积有关。
2.2防治AD药物对STUB1表达的影响
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,观察药物对STUB1基因表达的影响,结果表明,皮层中除TXF外,海马中除DSS外的其余5个药物均对SAMP8 STUB1 mRNA表达水平的低下表现出了明显的上调作用。观察药物对STUB1蛋白表达的影响,结果表明,皮层中除LW外,海马中除LW和HupA外,其它药物均显著上调了SAMP8中STUB1蛋白的异常低表达。结果表明所选药物对SAMP8 STUB1基因和蛋白的异常表达具有不同程度的改善作用,说明STUB1对所选防治AD药物具有明显反应性。
(二)细胞骨架相关分子NF-L
神经微丝是神经元特有的微丝蛋白,NF-L(Neurofilament protein,light chain,神经丝蛋白-轻链)是神经微丝三联体蛋白的聚合骨架,可通过调节神经生长、轴突管径以及轴突转运,来维持和调节着神经元细胞骨架的可塑性,在神经系统正常发育和正常发挥其功能中具有重要作用。
1.快速老化模型小鼠增龄过程中NF-L表达的动态变化
采用实时定量PCR和Western blot技术,观察了SAMP8和SAMR1增龄过程中海马和皮层NF-L mRNA和蛋白表达的变化。结果表明,增龄过程中,SAMR1皮层中,NF-L mRNA和蛋白表达均维持在相对恒定的水平:而海马中,其表达均在6月龄最高;与SAMR1相比较,10月龄后SAMP8皮层和海马中NF-L基因和蛋白表达均显著减少。上述结果提示NF-L的异常低表达可能造成神经元细胞骨架受损,而诱发神经元丢失。
2.防治AD药物对NF-L表达的影响
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,除LW和BW外,其余药物均能够显著上调12月龄SAMP8皮层中NF-L基因和蛋白的异常低表达;而海马中,各给药组对SAMP8中NF-L基因和蛋白的异常低表达均有上调效应。表明所选药物对SAMP8 NF-L的异常表达具有不同程度的改善作用,提示调节NF-L表达,改善由于其下降导致的神经元骨架受损而保护神经元可能是上述防治AD药物发挥AD治疗作用的共有机制之一。
(三)线粒体相关分子NDUFS2
NDUFS2(NADH dehydrogenase(ubiquinone)Fe-S protein 2,还原型烟酰胺二核苷酸脱氢酶铁硫蛋白2)是线粒体复合体Ⅰ铁硫蛋白的一个亚基,具有氧化还原酶活性,参与能量代谢通路,可以反映出机体的氧化磷酸化水平。
采用实时定量PCR技术,观察了SAMP8和SAMR1增龄过程中海马和皮层NDUFS2 mRNA表达的变化。结果表明,SAMR1中,NDUFS2 mRNA的表达均为6月龄最高,而2月龄最低;而SAMP8中,该基因的表达均在6月龄最低;与SAMR1相比较,SAMP8中,NDUFS2表达均在2月龄显著增加,而6月龄则显著减少。表明SAMP8早期就存在氧化磷酸化功能的异常。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,所有药物均能显著下调12月龄SAMP8皮层中NDUFS2 mRNA的异常高表达;而海马中,除LW和HupA外,其余四种药物对SAMP8中海马中NDUFS2 mRNA的异常低表达均有上调作用。提示上述防治AD有效的药物能够调节NDUFS2的异常表达从而改善SAMP8中的氧化磷酸化异常,这可能是这些防治AD药物发挥AD防治作用的共有途径之一。
(四)信号转导相关分子CAMK2,EphB6和CSTN1
1.CAMK2A
CaMKⅡ(Calcium/calmodulin-dependent protein kinase typeⅡ,钙/钙调蛋白依赖性蛋白激酶Ⅱ型α亚基)在神经递质释放、神经突触可塑性、学习和记忆等方面发挥着重要作用。其α亚基即CAMK2A(CaMKⅡalpha chain,CAMK2α)是CaMKⅡ在大脑皮层和海马中表达最为丰富的一个亚基,它不仅参与了tau蛋白的磷酸化,而且也可能参与了APP的磷酸化,在AD的发病过程中发挥重要作用。观察SAMP8和SAMR1增龄过程中海马和皮层CAMK2A表达变化的结果表明,增龄过程中,在SAMR1皮层和海马中,CAMK2A基因和蛋白均呈现出递减的趋势;而SAMP8中,其表达则均呈现出递增的趋势;SAMP8与SAMR1相比,10月龄后SAMP8中CAMK2A的表达均显著增加。结果提示CAMK2A表达的增龄性降低可能是导致SAMR1正常老化过程中学习记忆功能衰退的原因;而其表达的异常增加则可能导致SAMP8加速老化过程中学习记忆功能衰退。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,所有药物均能够显著下调12月龄SAMP8皮层和海马中CAMK2A mRNA的异常高表达。除LW外,上述药物均能显著下调12月龄SAMP8皮层中该蛋白的异常高表达;而在海马中,除HL和DSS外,上述药物对SAMP8中该蛋白的异常高表达也具有显著的下调作用。提示上述防治AD药物能够调节CAMK2A的异常表达从而改善SAMP8中的认知功能障碍,这也可能是六种药物改善SAMP8学习记忆功能的共同机制之一。
2.EphB6
EphB6(erythropoietin-producing hepatocyte receptor,促红细胞生成素源肝细胞受体B6)是一种跨膜受体蛋白,归属于酪氨酸激酶,具有独特的双向信号转导系统,能通过调节细胞骨架而发挥重要的生物学功能,在某些细胞株上其家族蛋白可调节Tau蛋白的磷酸化。
观察SAMP8和SAMR1增龄过程中海马和皮层EphB6表达的变化,结果表明,增龄过程中,SAMR1 EphB6 mRNA和蛋白表达均在6-10月间最高;而SAMP8中,其表达则在10月龄后最高;SAMP8与SAMR1相比较,10月龄后其表达均显著升高。结果提示EphB6早期的异常低表达可能造成了SAMP8的细胞免疫功能的缺陷,而后期其表达的异常增加则可能是加速了SAMP8的异常老化。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,LW、HL、TXF和DSS显著下调12月龄SAMP8皮层中该基因的异常高表达;而LW、BW、TXF和DSS则能够下调12月龄SAMP8海马中该基因的异常高表达。提示上述防治AD药物能够通过调节EphB6的异常表达而起到一定防治AD作用,但如早期给药,可能会收到更好效果。
3.CSTN1
CSTN1(Calsyntenin-1,钙同线蛋白-1)是一个能够将胞外蛋白水解活性与胞内Ca2+信号转导相关联的独特的突触后膜蛋白,能与钙离子结合,参与信号转导和细胞交流,能通过胞外蛋白水解来调节突触后钙的动力,同时,APP和CSTN1共同作用可增加Aβ的分泌,在AD的发生过程中发挥作用。
观察SAMP8和SAMR1增龄过程中海马和皮层CSTN1 mRNA表达变化的结果表明,增龄过程中,SAMR1 CSTN1 mRNA表达均在6月龄最高,2月龄最低;而在SAMP8中,其表达则有增龄性增加的趋势,其中以皮层的变化较为显著;与SAMR1相比,其表达在12月龄SAMP8中均显著增加。结果提示CSTN1的异常高表达可能造成SAMP8中Aβ释放的增加,从而加速AD的进程。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和HupA一个月,LW、BW、HL和DSS显著下调12月龄SAMP8皮层中该基因的异常高表达;而BW、HL、DSS和HupA也能够显著下调12月龄SAMP8海马中该基因的异常高表达。提示上述防治AD药物可能通过调节CSTN1的异常表达而减少Aβ的释放,起到一定的防治AD作用。
(五)神经发生生长相关分子Neurexin1和NGRN
1.Neurexin 1
NeurexinⅠ(Neurexin蛋白-1)可作为突触后受体与黑寡妇蜘蛛毒素α-latrotoxin结合,激发神经递质的释放,可作为细胞黏附分子和受体而发挥其作用,并与突触发生紧密相关。
本研究还观察了SAMP8和SAMR1增龄过程中海马和皮层NRXN1的mRNA表达的变化。结果表明,增龄过程中,SAMR1中,NRXN1 mRNA的表达在皮层中在6月龄最低,而海马中则在6月龄最高;在SAMP8中,其在皮层中增龄性递减,而在海马的表达则维持在不变的低水平;与SAMR1相比,其表达均在12月龄SAMP8中显著减少。结果提示NRXN1的异常表达可能造成SAMP8中细胞的营养不良环境,从而加速AD的进程。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,除LW、HL外的其它药物均显著上调12月龄SAMP8皮层中该基因的异常低表达;同样,除LW外的其它药物均显著上调12月龄SAMP8海马该基因的异常低表达。提示上述防治AD药物能够通过上调NRXN1的异常低表达而部分改善SAMP8神经细胞的营养环境而发挥一定AD防治作用。
2.NGRN
NGRN(Neurite outgrowth associated protein,神经突生长相关蛋白)可能与细胞生长和维持有关,并可能在神经元分化过程中起重要作用。
观察SAMP8和SAMR1增龄过程中海马和皮层NGRN的mRNA表达变化的结果表明,增龄过程中,SAMR1中,NGRN mRNA的表达在皮层随增龄递减,在海马中则相反;在SAMP8中,其在皮层和海马的表达均为6月龄最低;与SAMR1相比,其在6月龄SAMP8的皮层和12月龄的海马中均显著减少。结果提示NGRN的异常表达可能造成SAMP8中细胞的营养不良环境,从而加速AD的进程。
分别灌胃给予SAMP8 LW、BW、HL、DSS、TXF和Hup A一个月,LW、HL、DSS和HupA显著上调SAMP8中该基因的异常低水平,而海马中,除TXF外,其它药物对SAMP8中该基因的异常低水平表达也均有上调效应。提示上述防治AD药物能够通过上调NRXN1的异常低表达而部分改善SAMP8神经细胞的营养环境而发挥一定的抗AD作用。
三、候选分子AMFR等与AD关键病理蛋白的关系研究
1.AD关键病理蛋白在快速老化模型小鼠脑中的组织分布变化
用免疫组织化学的方法研究了AD关键病理蛋白APP、Tau和P-Tau在SAM鼠脑中的组织分布变化,结果表明,随SAMP8增龄,这三种AD关键病理蛋白在海马齿状回、海马CA3区以及颞叶皮层中均呈现出表达增加的趋势。
2.AMFR在快速老化模型小鼠脑中的组织分布变化
用免疫组织化学的方法研究了AMFR蛋白在SAM鼠脑中的组织分布变化,结果表明,随SAMP8增龄,AMFR蛋白在海马齿状回、海马CA3区以及颞叶皮层中均呈现出表达减少的趋势。
3.AMFR、STUB1与APP在快速老化模型小鼠额叶皮层的共定位
免疫荧光共定位研究结果显示:AMFR、STUB1与APP能够共表达于SAMP8的额叶皮层,在6月龄的SAMP8小鼠大脑的额叶皮层,AMFR与APP、STUB1与APP存在共定位关系。
四、候选基因靶标间的相互关系及其与AD关键病理分子关系的生物信息学分析
采用Osprey软件,首先考察了能够与AD关键病理蛋白及能够与所选9个分子直接相互作用的分子情况,结果表明,能够与APP发生直接相互作用的分子共49个;能够与Tau发生直接相互作用的分子共16个;能够与AMFR和STUB1发生直接相互作用的分别有3个和8个分子;能够与NF-L发生直接相互作用的分子共4个;未发现与NDUFS2和NGRN直接作用的分子;能够与CAMK2A、EphB6和CSTN1发生直接相互作用的分子分别为14个、4个和3个。
其次,分析了所选9个分子间及他们与APP、Tau、PS1、PS2和ApoE的直接相互作用。结果表明,所选9个分子间不存在直接的相互作用;只有CSTN1可以和APP、PS1发生直接相互作用,而其余分子间则不发生直接作用。
最后,分析了所选9个分子及与之相互作用的分子和AD关键病理分子的可能关系,预测结果表明它们中的80%能够与AD关键病理分子APP、Tau发生间接相互作用,而且是通过多条通路。其中,AMFR可以通过EphB6与APP、Tau发生联系;STUB1、EphB6、NRXN1均能与APP、Tau发生联系;NF-L与Tau联系密切,但同时也能够与APP发生联系;CaMK2A与APP关系较近,但也可以通过APP与Tau发生联系。结果提示从候选“组合基因靶标”中所选的各分子不是孤立在发挥作用,而是与AD关键病理蛋白形成相互作用网络来参与AD的病理生理过程。这一结果支持我们的假说,即它们不是单个基因的作用,而是形成一个作用网络,来参与AD的病理。
上述结果表明,从候选“组合基因靶标”中选择的5类9个分别代表不同功能的分子在SAMP8海马和皮层中的表达水平随增龄出现不同程度的异常,尤其在老龄SAMP8中表达异常最为明显,抗AD有效药物对其异常表达具有改善或纠正作用。同时,研究还表明,AMFR和STUB1在海马和皮层中与AD关键病理分子之一APP存在共定位关系,提示其可能对APP代谢生成Aβ产生影响。生物信息学分析提示所选的候选基因及其蛋白可通过多种途径与AD关键病理分子发生相互作用,从而发挥干预AD病理生理过程及改善认知功能的作用。上述研究结果提示,所选9个候选基因及其蛋白表达的异常可能是SAMP8随增龄脑内出现多种神经病理损害,并导致其学习记忆功能进行性衰退的重要原因,改善这些基因和蛋白的异常表达可能是降低AD相关神经病理损害、改善认知功能障碍的重要途径。因此,上述这9个分子有成为抗AD药物筛选潜在靶标的可能,有待进一步深入研究。
Alzheimer's disease(AD),one of the most prevalent neurodegenerative diseases, geatly threatens the health of the elderly and has been the fourth killing disease.AD holds about 60-70%of the totally 32 kinds of dementia.Mainly due to the uncertain aetiology of AD,the complexity of this disease,limited therapeutic drugs,it is hard to make great progress on the prevention and cure of AD in the near future.Cholinesterase inhibitors are most widely used drugs for the treatment of AD now,but their effects are only to ameliorate symptoms but do not achieve any permanent improvement.Therefore, to develop more effective AD therapeutic drugs with lower toxicity and lower side-effect remains an urgent task.The definite targets for anti-AD drugs restrict the development of new promising therapeutic drugs for AD and the breakthrough in this field is urgently calling for discovery and verification of the new targets for anti-AD drugs.
However,for any of the compounds,aiming at a sole target for AD,it is indeed a difficulty to obtain the satisfied therapeutic effects since AD is with such a complex pathogenesis which is still unclear till now,and also partly due to the serious consequences of AD,which are linked to abnormal construction and anormal functions of multi-systems.This fact suggests that intervention of multiple targets for AD is more important and which may be a feasible strategy to make great progress in prevention and cure of this stubborn disease.Thus,we propose a hypothesis that "combination targets" may be of great value to design,screen and further evaluate new drugs for AD.
Traditional Chinese medicine(TCM)are composed of several herbs and have many ingredients,their effects have the characteristics of multi-mechanisms, multi-pathway and multi-targets,likewise AD is a complex,multi-factorial and genetically heterogeneous syndrome.So the character of the whole traditional Chinese medicinal prescription is consistent with AD,the complicated disease,It is well known that many widely used traditional Chinese medicinal(TCM)prescriptions,including Liu-Wei-Di-Huang decoction(LW),Ba-Wei-Di-Huang decoction(BW), Huang-Lian-Jie-Du decoction(HL),Dang-Gui-Shao-Yao decoction(DSS), Tiao-Xin-Fang decoction(TXF)and Huperzine A(HupA),play important roles in the clinical therapy of AD patients,and also have beneficial effects on cognitive abilities in animal models.Senescence-accelerated mouse(SAM)prone/8(SAMP8)is a good animal model to investigate the fundamental mechanisms of age-related learning and memory deficits such as Alzheimer's disease(AD)at the gene and protein levels,and SAM resistant/1(SAMR1)is its normal control.It has been proved that all of the above-mentioned five TCM,including LW,BW,HL,DSS and TXF,can improve the functional impairments of learning and memory in SAMP8 in our previous studies. Although these five TCM are initially designed for different diseases,they do show the common therapeutic effects on AD patients and animal models.However,they may indeed have the different characters and through the different action mechanisms on the cognitive functions.
Having influences on gene expression is one of the mechanisms on drug action. Cerebral cortex and hippocampus are the key regions which are closely linked to learning and memory.Since these five TCM can improve the ability of learning and memory in AD cases,gene expression pattern of SAMP8 should be changed after SAMP8 were given to these TCM.To study these different expressed genes in SAMP8, drug reaction genes could be picked out.Then,to further compare the difference and identity of these drug reaction genes,the common genes responding to all these drugs and also the special genes responding to certain drug could be discovered.The common responding genes may reflect the similar action mechanism of all these drugs while the specific responding genes may represent the distinct action mechanism of the special drug.Thus,it will be very helpful to investigate the changed genes during the degenerative process of cognitive ability in SAMP8,which will not only give cues to find new targets for anti-AD drags but also be an important way to study and search the molecular targets for AD.
Consequently,the distinct anti-AD drugs such as TCM LW,BW,HL,DSS,TXF and natural product Hupzine A(HupA)were used as tools and experimental therapy had been conducted to investigate their effects on cognitive ability of SAMP8.Meantime,by Suppression subtractive hybridization(SSH)and cDNA microarray technique,our team bad successfully screened out the candidate "combination targets" for anti-AD drugs, which are composed by various molecules,such as protein degradation related genes AMFR and STUB1,neural cystoskeleton related gene NF-L,mitochondria function related gene NDUFS,signal transduction related genes CAMK2A,EphB6 and CSTN1, and neurogenesis related genes NRXN1 and NGRN.
However,these results are mainly based on the cDNA technique,which has a relatively higher false positive rate(FPR).So the necessary verification procedure is needed for the further validation of these targets for anti-AD drugs.
In order to preliminary verification of the candidate "combination targets" for anti-AD drugs,from which,this paper selected totally 9 representative genes belonging to five types and used SAMP8 as an animal model for AD.By the methods such as real time Quantitative fluorescence RT-PCR,Western blot,Immunohistochemistry and Immunofluorescence.the auther firstly investigated the age-related expression changes of these 9 molecules in SAMP8 and SAMR1 with aging.Then,the expression changes of these 9 molecules in 12-month SAMP8 treated with anti-AD drugs LW,BW,HL, DSS,TXF and natural product HupA were also investigated.Finally,the respective networks of these 9 molecules,the interaction of these 9 molecules and their possible roles in AD process had also been studied by bioinformatics.This paper have provided the evidences for supporting the hypothesis that "'combination targets",based on the bio-regulative network of targets for anti-AD drugs,may be of great value to design, screen and further evaluate new drugs for AD.
Part one.Expression changes of APP and Tau in the cerebral cortex and hippocampus of SAMR1 and SAMP8 and their responses to anti-AD drugs
The main pathological feature of AD is neuron loss,senile plaques(SP)and neurofibrillary tangles(NFT)in the key brain regions.SP and NFT are formed by the intercellular and intracellular deposition and aggregation byβamyloid protein(Aβ)and hyperphosphorylation form of Tau(HP Tau),respectively.Thus,Aβand Tau has been considered as the key factors that lead to the onset and progress of AD.Aβis produced byβ-amyloid precursor protein(APP).Consequently,this paper firstly investigated the expression changes of APP and Tau in the key brain regions related to cognitive functions in SAM.
1.Age-related expression changes of APP and its responses to anti-AD drugs With aging process,the expression of APP mRNA is maintained at certain level in the hippocampus and cerebral cortex from SAMR1,while it was increased after 2-month-old in the hippocampus and cerebral cortex from SAMP8.Compared with SAMR1,the expression of APP is increased significantly in SAMP8;The results from immunohistochemistry(IHC)showed that the number of positive cells is increased with aging in the cortex and hippocampus in SAMP8.
After SAMP8 was given anti-AD drugs LW,BW,HL,DSS,TXF and HupA for a month,the abnormal high mRNA expression of APP was significantly down-regulated in cerebral cortex of 12-month-old SAMP8 treated with LW,HL,DSS,TXF and HupA. In hippocampus of SAMP8,only BW and DSS treated groups showed down-regulative effects on mRNA expression of APP.The results indicated that these anti-AD drugs could regulate the abnormal mRNA expression of APP and meantime they indicated that APP could actively respond to these anti-AD drugs.
2.Age-related expression changes of Tau and its responses to anti-AD drugs
With aging process,the expression of Tau mRNA is maintained at certain level in the cerebral cortex from SAMR1,while it was significantly increased in cerebral cortex in 12-month-old SAMP8.Compared with SAMR1,the expression of Tau is increased significantly in the cerebral cortex in 12-month-old SAMP8.In the hippocampus from both SAMP8 and SAMR1,Tau mRNA was increased with aging.Compared with SAMR1,the expression of Tau is significantly increased in hippocampus in every month of SAMP8.The different expression pattern for Tau in SAMP8 indicated that the abnormal higher expression of Tau may result in the degeneration ability in learning and memory.
After SAMP8 was given to anti-AD drugs LW,BW,HL,DSS,TXF and HupA for a month,the abnormal high mRNA expression of Tau was significantly down-regulated in cerebral cortex of 12-month-old SAMP8 treated with all these anti-AD drugs.In hippocampus of SAMP8,only HL-treated group didn't show down-regulative effects on mRNA expression of Tau.The results indicated that these anti-AD drugs could regulate the abnormal mRNA expression of Tau and meantime they indicated that Tau could actively respond to these anti-AD drugs.
Many studies have indicated that,there were age-related defects in cognitive abilities in SAMP8.Our studies showed that in SAMP8,both APP and Tau were increased with aging process both in the cerebral cortex and hippocampus,the key brain regions closely linked to learning and memory.At the same time,they showed the apparent responses to these anti-AD drugs.These results further indicated that SAMP8 is an excellent animal model and which could be used to preliminarily verify the candidate obtained "combination targets" for anti-AD drugs.
Part two.Expression changes of the 9 molecules from the candidate "combination targets" for anti-AD drugs and their responses to anti-AD drugs
1.Abnormal protein degradation related molecules AMFR and STUB1
1.1 AMFR
AMFR,a G-protein-coupled receptor,shows an ubiquitin protein ligase(E3) activity and participates in degradation of abnormal proteins from the smooth endoplasmic reticulumits-associated degradation(ERAD).Studies showed that after its binding with its endogenous ligand Autocrine motility factor(AMF),AMFR played various roles such as stimulating cell migration,facilitating the cell survival.To study whether AMFR involves in the AD process or not,this paper had studied the age-related mRNA and protein expression of AMFR and its distribution in SAM with aging.
1.1.1 Age-related expression of AMFR and its ligand AMF in the cerebral cortex and hippocampus of SAM with aging
In both cerebral cortex and hippocampus,AMFR mRNA increased significantly after 2-month-old in SAMR1 while it decreased significantly in both cerebral cortex and hippocampus from SAMP8.Compared with SAMR1,the expression of AMFR mRNA decreased significantly in both the cerebral cortex and hippocampus in 12-month-old SAMP8.
In the cerebral cortex of SAMR1,AMFR protein increased after 2-months of age while in SAMP8,it showed an apparent decrease tendency after 6-months of age. Compared with SAMR1,AMFR protein decreased significantly after 6-months of age in SAMP8.In the hippocampus of SAMR1,AMFR protein increased after 2-months of age while it decreased with aging in SAMP8.Compared with SAMR1,protein expression of AMFR increased significantly at the 2-month old SAMP8,but decreased significantly at 6-and 15-month old SAMP8.
In the cerebral cortex of SAMR1,AMF protein increased significantly at 10-and 15-month-old SAMR1 than its expression level at 2-and 6-month-old SAMR1 while in samples from SAMP8,its expression decreased after 6-month-old with aging. Compared with SAMR1,AMF protein decreased significantly after 6-months of age in SAMP8.In the hippocampus of SAMR1,AMF protein maintained at a certain level with aging process while its expression showed an increase before 10-month-old and a decrease since 10-month-old.Compared with SAMR1.AMF protein decreased at all of age.
1.1.2 Expression of AMFR in the cerebral cortex and hippocampus of SAMP8 treated with anti-AD drugs
Compared with the normal control group,the mRNA and protein expression in the cerebral cortex and hippocampus of SAMP8 was decreased significantly.After SAMP8 was treated with the six anti-AD drugs,the abnormal low expression of AMFR was up-regulated by LW and HupA for mRNA and LW,BW and HupA for protein in the cerebral cortex of SAMP8.In the hippocampus of SAMP8,the abnormal low expression of AMFR was up-regulated by BW,HL,DSS,TXF and HupA for mRNA and LW,BW and HupA for protein.
1.2.STUB1
The STIP1 homology and U-box-containing proteinl(STUB1),a cytoplasmic protein also named C terminus of Hsc70-interacting protein(CHIP),is a Hsp70 co-chaperone and an E3 ubiquitin ligase that protects cells from proteotoxic stress,so STUB1 plays the pivotal role in the protein QC system.By its U-box domain,STUB1 acts as an E3 ligase to facilitate the transfer of a polyubiquitin chain to misfolded substrates,while by its TPR domain,STUB1 binds heat shock cognate 70(HSC70)or HSP70,attenuates the HSP40-stimulated ATPase and refolding activities of HSP70, which contributes to the delivery of protein substrates to lysosomes-mediated autophagy. So STUB1 is one of the most important members of UPS and acts as a molecular switch between proteasomal and lysosomal degradation pathways,mediates crosstalk between molecular chaperones and the UPS.This study showed that,with aging process,both mRNA and protein expression of STUB1 in cerebral cortex and hippocampus from SAMR1 were increased after 2-month-old while decreased in the above brain tissues from SAMP8 after 6-month-old;compared with SAMR1,mRNA and protein expression of STUB1 were decreased after 10-month-old SAMP8 respectively and could be up-regulated by the six anti-AD drugs.
2.NF-L
Neurofilament proteins are specific intermediate filament for neurons and NF-L is the backbone for NF-M and NF-H to polymerize.NF-L plays an important role in maintenance and regulation of the dynamic cystoskeleton of neurons by regulating the growth of neural cells,axis caliber and axonal transport.Consequently,NF-L plays a key role in CNS of the mammal.This study showed that,with aging process,both mRNA and protein expression of NF-L were decreased significantly in cerebral cortex and hippocampus after 10 months of age and which could be up-regulated by HL,DSS, TXF and HupA in cerebral cortex at both mRNA and protein levels while in the hippocampus,all these drugs showed up-regulative effects on both mRNA and protein expression of NF-L.
3.NDUFS2
NDUFS2 is an important subunit of ironsulfur protein in Complex I for respiratory chain.NDUFS2 has the activity of oxidoreduction and involves in the energy metabolism,which could reflect the level of oxidative phosphorylation.This study showed that mRNA of NDUFS was abnormal expressed in SAMP8 with aging compared with SAMR1,which could be down-regulated by LW,BW,HL,DSS,TXF and HupA in the cerebral cortex of SAMP8 and could be up-regulated by BW,HL,DSS and TXF in the hippocampus of SAMP8.
4.Signal transduction related genes CAMK2A,EphB6 and CSTN1
4.1 CAMK2A
CAMK2A is one most abundant subunit of CaMKII in cerebral cortex and hippocampus.The autophosphorylation of the threonine at 286 of CAMK2A is essential for hippocampus long term potentiation(LTP)and spatial learning.CAMK2A may involve in not only phosphorylation at multiple sites of Tau in AD patients,but also phosphorylation of APP in vitro.This study showed that that the levels of both CaMKIIαmRNA and protein decreased significantly in the cerebral cortex of SAMR1 with aging,but increased significantly in the cerebral cortex of SAMP8.Compared with age-matched SAMR1,the expression of mRNA and protein of CaMKIIαsignificantly increased in the cerebral cortex and hippocampus of SAMP8 after 10-months of age. After SAMP8 was treated with the previously mentioned drugs,the abnormally high expression of CaMKIIαwas relatively down-regulated for mRNA in both cerebral cortex and hippocampus,for protein in hippocampus by all the six anti-AD drugs used in this study and was relatively down-regulated for protein in the cerebral cortex by all the six anti-AD drugs with the exception for LW.
4.2 EphB6
EphB6 is a transmembrane receptor and belongs to protein tyrosine kinase(PTK), which an unique protein that have two-way signal transduction system based on its specific structure.This study showed that EphB6 mRNA has different expression patterns in the hippocampus and cerenral cortex,that is,its expression was increased with aging while showed a highest expression at 6-month-old in hippocampus in SAMR1.While in SAMP8,its expression showed an increase tendency in both the hippocampus and the cerebral cortex.For protein expression of EphB6,it showed an increase at 10-month-old but an decrease at 15-month-old SAMR1 in both the hippocampus and cerebral cortex.However,the protein level was increased after 10-month of age in SAMP8.Compared with SAMR1,the EphB6 protein increased significantly after 10-months of age in SAMP8 which could be down-regulated by LW, HL,TXF and DSS in the cerebral cortex and by LW,BW.TXF and DSS in the hippocampus.
4.3 CSTN1
CLSTN1 is a specific molecule which could link the proteolysis outside of cytes and the signal transduction of Ca~(2+)within the cytes CLSTN1 locates at the postsynaptic membrane and binds with Ca~(2+)to regulate the signal transduction and cell communication.Study showed that this specific protein could cooperate with APP to increase the secretion of Aβ,thus,this protein may accelerate the AD process.Our study showed that CSTN1 mRNA was highest expressed at 6-month-old SAMR1 in both the cerebral cortex and hippocampus while its expression pattern showed an increase in SAMP8 in both of the above-mentioned brain regions.Compared with SAMR1,this gene increased significantly in SAMP8 and which could be down-regulated by LW,BW,HL and DSS in the cerebral cortex and by BW.HL,DSS and HupA in the hippocampus.
5.NRXN1 and NGRN
NeurexinⅠcould bind withα-latrotoxin as a postsynaptic receptor,thus further activate the release of neurotransmitter(NT).In addition,NRXN1 could play its roles as a cellular adhesion molecule(CAM)and closely linked to synaptogenesis.NGRN has been presumed to play an important role in cell growth,maintenance and neuron differentiation.This paper showed that both NRXN1 and NGRN mRNA were abnormally lower expressed in SAMP8 compared with SAMR1.The lower expression of NRXN1 could be up-regulated by BW,DSS,TXF and HupA in the cerebral cortex in SAMP8 and by all anti-AD drugs with the exception for LW in the hippocampus.And the lower expression of NGRN could be up-regulated by LW,HL,DSS and HupA in the cerebral cortex and by all drugs with the exception for TXF.
Part three.Relationship between candidate gene targets for anti-AD drugs and the key pathological proteins in AD
1.The distribution of key pathological proteins in AD
Results from the study by immunohistochemistry showed that with aging process, APP,Tan and P-Tau showed an apparent increase tendency in the subregions CA3 and dentate gyrus(DG)of hippocampus and the subregion temporal lobe(TL)of cerebral cortex.
2.The distribution of AMFR protein in SAM with aging
Results from the study by immunohistochemistry showed that with aging process, the positive neural cells decreased apparently in the subregions CA3 and dentate gyrus (DG)of hippocampus and the subregion temporal lobe(TL)of cerebral cortex.While in SAMR1,this phenomenen was not apparent or just showed an increase tendency in the same subregions.
3.Co-localization of AMFR,STUB1 and APP in subregion frontal lobe of the cerebral cortex
Results from the study by immunofluorescence showed a definite co-localization of AMFR and APP,STUB1 and APP in subregion frontal lobe of the cerebral cortex in 6-month-old SAMP8.
Part four.Interaction of candidate gene targets for anti-AD drugs and their roles in AD process
First.by the molecules interaction analysis software Osprey,the molecules that directly interact with the key pathological protein APP,Tau and the nine candidate molecules were analyzed.The results showed that there were total 49 and 16 molecules directly interacting with APP and Tau,respectively.The result indicated that there are many molecules directly interacting with APP and Tau,which were related to multiple functions.To further study these molecules will be no doubt benefiting the further understanding of the etiopathogenisis for AD and which will help to prevent and cure this disease.There were total 3 and 7 molecules directly interacting with AMFR and STUB1,respectively.The analysis result indicated that involvement in protein metabolism was the major function of AMFR,while for STUB1,besides this function, there were also other important functions,The results showed that there were total 4 molecules directly interacting with NF-L and which indicated that the main function of NF-L is cell organization and cytogenesis.However,the software analysis showed that there were no direct interaction molecules to NDUFS2 and NGRN till now.The direct interaction molecules to CAMK2A,EphB6 and CSTN1 were 14,4 and 3,respectively. Phosphorylation of substrates was the basic function for CAMK2A and CAMK2A is a muir-functional enzyme.EphB6 could involve in phosphorylation of substrates and cell organization and cytogenesis.To further study the functions of CSTN1 will benefit both the understanding and the prevention and cure of AD.The NRXN1 could interaction directly to 20 molecules and the major functions of NRXN1 were transport and cell organization and cytogenesis.There were no direct interacting molecules for NGRN. Then,the possible interaction within the 9 molecules as well as APP,Tau,PS1.PS2 and ApoE were analyzed by osprey and the results are as follows:There were no direct interaction within the 9 molecules and only CSTN1 could directly interact with APP and PS1.Finally,the possible interaction of the 9 molecules and their direct interaction molecules with the key pathological protein of AD was also analyzed by Osprey.The results predicted that nearly 80%of these candidate 9 molecules could indirectly interact with APP and Tan by several pathways.Among of which,AMFR could interact indirectly with APP and Tau through the linkage of EphB6;STUB1 could interact indirectly with APP and Tau;NF-L was much closer to Tau but it could also interact with APP;CaMK2A was much closer to APP but it could also interact with Tau by the linkage of APP;EphB6 could indirectly interact with both APP and Tau;NRXN1 was much closer to APP but it could also interact with Tau.These results indicated that these 9 molecules played their roles in AD onset by the interaction network within them and their interacting molecules instead of their respective sole action.The result supports the hypothesis that the interaction network instead of respective sole action of candidates from "combination targets" for anti-AD drugs involves in the pathology of AD.
In sum,in order to preliminary verification of the candidate "combination targets" for anti-AD drugs,from which,this paper selected totally 9 representative genes belonging to five types and used SAMP8 as the animal model for AD.By the methods such as real time Quantitative fluorescence RT-PCR,Western blot, Immunohistochemistry and Immunofluorescence,the auther firstly investigated the age-related expression changes of APP,Tau and these 9 molecules in SAMP8 and SAMR1 with aging.Then,the expression changes of these 9 molecules in 12-month SAMP8 treated with anti-AD drugs LW,BW,HL,DSS,TXF and natural product HupA were also investigated.Finally,the respective networks of these 9 molecules,the interaction of these 9 molecules and their possible roles in AD process had also been studied by bioinformatics.The results showed that these 9 molecules not only closely linked to degeneration in learning and memory ability of SAMP8,but also actively responded to the multiple anti-AD drugs used in this paper.The results also indicated that on the one hand,the same molecule expressed differently at its gene and protein levels,which may be due to the post-transcriptional processing,translation and post-translational modification.The same molecule showed differently distributed patterns in part in different brain regions,which may be due to the distinct functions of its own and different brain regions.On the other hand,the same molecule showed differently responses to the same drug in the different brain regions,which may be caused by the specificities of drugs and specific action region of the molecule.However, the same molecule showed the similar responses to different anti-AD drugs,which indicated that this molecule may be the common target for multiple drugs.And also,the different molecules showed the similar responses to the same drug,which may be resulted from the multiple mechanisms of the same drug.In addition,the 80%of the candidate molecules were predicted to interact with the key pathological protein APP and Tau,and they may involve in the pathology of AD by the interaction network instead of respective sole action of themselves.The results obtained by this paper settled the foundation for designing,screening and further evaluating new drugs for AD, which were based on the bio-regulative network of "combination targets" for anti-AD drugs.
In conclusion,this paper supported the hypothesis that "combination targets", based on the bio-regulative network of targets for anti-AD drugs,are of great value to designing,screening and further evaluating new drugs for AD.And the "combination targets" are composed by one group or several groups of molecules,which are closely related to cognitive defects in AD and show complementation and cooperation within them."Combination targets" based on the bio-regulative network of targets for anti-AD drugs,will be of great value not only for designing,screening and further evaluating new drugs for AD,but also for studying and elucidating the pathophysiological processes of AD from the view of pharmacology,and also,will give important cues for discoveries of biomarkers used to predict the risk for the onset of AD,diagnose AD and assess the therapeutic effect by anti-AD drugs.
引文
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