防治阿尔茨海默病药物筛选组合基因靶标的初步研究
|
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。其发病机制复杂，迄今为止尚不十分清楚，更无理想的防治药物问世。目前临床用于治疗AD的主要药物胆碱酯酶抑制剂只能减轻部分患者症状，对病情的发展尚不能有效控制。因此，研究开发疗效好、毒副作用小的防治AD的新型药物是当前创新药物研究开发领域的热点之一。
创新药物发现过程中最重要的环节之一是药物靶标的发现和确证。AD是典型的多因素复杂疾病，其发生发展过程涉及多系统、多环节结构和功能的异常，针对单一靶点的防治措施难以取得满意疗效，多靶点综合干预是提高AD防治水平的重要策略。因此，寻找、研究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、且具有相互补充与协同关系的“组合靶标”，将为防治AD创新药物的研究提供新的筛选靶标，并将大大提高AD防治药物的研究与开发水平。结合当今飞速发展的基因组及生物信息学技术，从基因入手研究上述组合药物靶标，是一条药物靶标研究的新路子，具有广阔的研究前景。
AD在临床表现为认知功能的进行性衰退。已有大量研究表明，基因表达或转录异常是AD发生、发展的重要因素之一，同时多种基因及其表达产物在中枢学习记忆功能衰退的过程中发挥了重要作用，如APO-E、APP、PS-1、PS-2、cystatin-3、LRP1等。海马和皮层是与学习和记忆功能关系最为密切的两个脑区，它们在AD脑内也呈现严重的病理改变和基因表达异常。因此，以这两个特定区域差异表达基因为线索，通过深入研究这些基因与AD发生、发展及病理变化的关系，有望发现用于防治AD的药物基因靶标。
快速老化模型小鼠(senescence-accelerated mouse，SAM)是一种自然发生的、全身性快速老化的模型动物。其快速老化亚系之一SAMP8的生物学特征是中枢学习记忆功能随增龄进行性衰退，同时脑内出现神经元丢失、Aβ沉积等类似AD的病理改变，是在基因和蛋白水平研究AD的良好模型。因此，通过研究SAMP8在增龄过程中海马和皮层基因表达的变化，是发现中枢学习记忆功能相关基因、开展防治AD药物靶标研究的一条重要途径。
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，就此而言，其在防治AD这类多因素复杂疾病方面，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。事实上，我国和日本等国家在防治AD中广泛使用的中药复方如当归芍药散、开心散、六味地黄汤、八味地黄汤、黄连解毒汤、调心方、钩藤散等，对AD病人或模型动物均具有良好的疗效。而我室前期考察六味地黄汤(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)等多种中药复方对学习记忆功能作用的结果也表明，上述几种中药方剂对SAMP8学习记忆功能障碍均具有改善作用。按照中医理论，这些中药复方的功能主治不尽相同，如补肾、清热解毒、活血化瘀等，因此它们对中枢认知功能的作用特点和作用机理亦可能不同，它们在药理作用和作用机理的方面的差异可能反应在用药后中枢神经系统的基因表达有所不同。因此，通过研究SAMP8应用这些中药复方后海马和皮层的反应基因，比较各药物反应基因群的异同，可从中发现这些中药复方所共有的药物反应基因和各自所特有的个性反应基因。药物反应基因或用药后基因表达的变化，是药物发挥药理作用的重要环节之一。共性反应基因可能代表了不同药物在药理作用、作用机理等方面的相似性，个性反应基因则可能代表了药物所特有的药效或作用机理等。因此，通过研究模型动物学习记忆功能衰退过程中表达变化的基因和对防治AD药物的反应基因，可为防治AD药物基因靶标的研究提供重要线索，是发现和研究防治AD药物基因靶标的一条重要途径。
本研究共分为两部分，一是采用cDNA芯片筛选SAMP8增龄过程中海马、皮层的差异表达基因以及SAMP8应用LW、BW和HL后其海马、皮层基因表达的变化，旨在获得一批增龄过程中SAMP8海马和皮层中表达明显变化的基因及一批应用具有改善学习记忆功能的三个中药复方后SAMP8海马和皮层中表达明显变化的基因，从而为防治AD药物候选组合基因靶标的提出奠定基础。二是通过对上述增龄性差异表达基因和药物反应基因进行生物信息学分析，初步提出一组防治AD药物筛选的候选组合基因靶标，并以SAMP8为模型，以防治AD的天然产物石杉碱甲(Hup A)和中药复方LW、BW、HL、DSS、TXF为工具，采用实时定量PCR方法，考察所提出的候选基因靶标在增龄性SAMP8海马、皮层中的表达变化以及对防治AD药物的反应性，以对这些候选基因靶标进行初步的验证。
一、防治AD药物作用基因靶标的筛选
为了寻找防治AD药物作用的基因靶标，本研究以快速老化模型小鼠(SAMP8)为动物模型，给予LW、BW和HL，采用我们自行研制的cDNA芯片，筛选和分析了SAMP8增龄过程中基因表达的变化以及给予LW、BW和HL对SAMP8海马和皮层基因表达的影响。
(一)不同月龄SAMP8海马和皮层差异表达基因的筛选
本研究采用cDNA芯片技术，比较了SAMP8和SAMR1增龄过程中海马和皮层基因表达的变化，并采用实时定量PCR方法对芯片筛选的结果进行了验证。
结果表明，与同龄SAMR1相比，2月龄SAMP8海马中差异表达克隆有124个，皮层中未见差异表达克隆。将这124个差异表达克隆进行测序，共成功测序100个，经过生物信息学分析，它们共对应45个基因。而与同龄SAMR1相比，6月龄SAMP8海马中的差异表达克隆仅有20个，共成功测序9个，对应5个基因；皮层中差异表达克隆为31个，共成功测序了21个克隆，其对应15个基因。12月龄SAMP8与同龄SAMR1比较，海马中的差异表达克隆有14个，共成功测序7个，对应4个基因；而在皮层中，表达差异的克隆有146个，成功测序了120个，对应36个基因。上述基因的功能涉及核酸代谢、核糖体装配合成、信号转导、转运、转录调节、蛋白质代谢、神经系统发育、细胞生长、能量代谢、免疫相关等，同时还有大量功能未知的基因。
随着增龄，SAMP8学习记忆功能进行性衰退。研究表明，SAMP8在4月龄时已出现AD相关的病理变化与学习记忆功能衰退。本研究发现，与SAMR1相比，SAMP8在增龄过程中其海马与皮层的基因表达发生了明显变化，其中包括与信号转导、蛋白质代谢、能量代谢等密切相关的基因。基因Ttc3、Uqcr的表达在2、6、12月龄中皆存在差异，提示这些基因可能与SAMP8脑老化以及学习记忆功能障碍的病理过程具有密切关系。这些随增龄而表达变化的基因不仅为深入研究SAMP8脑老化和学习记忆障碍的发生机理提供了有意义的线索，同时为防治AD药物研究的基因靶标筛选和研究提供了重要依据。
(二)六味地黄汤对6月龄SAMP8海马和皮层基因表达的影响
六味地黄汤(LW)是中医滋补肾阴的经典代表名方，近年来在临床上也被应用于AD的辅助治疗，取得了较好的效果。近年来我室从神经内分泌免疫调节(NIM)网络的角度对其药理作用及其机理进行了系统研究，结果表明，改善SAMP8学习记忆功能是LW的重要作用之一。为了探讨LW改善SAMP8学习记忆功能的作用途径或基因靶点，本研究选用6月龄雄性SAMP8，分别灌胃给予5、10、15g／kg剂量LW，连续1个月，然后取海马及皮层组织，提取总RNA，进而采用cDNA芯片技术观察了给药后SAMP8海马及皮层基因表达的变化。
结果表明，给予低剂量LW(5g／kg)后，SAMP8海马中差异表达的克隆有11个，皮层中有67个，将这些克隆进行测序，共成功测序53个，其对应基因28个；给予中剂量LW(10g／kg)后，SAMP8海马中差异表达的克隆有43个，皮层中有222个，共成功测序137个，其对应基因60个；给予高剂量LW(15g/kg)后，SAMP8海马中差异表达的克隆有80个，皮层中有233个，共成功测序197个，其对应基因55个。生物信息学分析的结果表明，不同剂量LW作用下所影响的基因种类大体相同，包括核酸代谢基因、递质转运基因、细胞生长、蛋白质代谢、核糖体生物合成装配、转录调节、神经系统发育、电子传递、免疫、能量代谢等，还有一些功能未知基因。
以上结果表明，给予不同剂量LW后，其海马和皮层反应基因的种类大致相同，但中、高剂量组发生变化的基因数量明显多于低剂量组。与SAMP8对照组相比，给予中、高剂量LW后，基因Ttc3、Oxr1、Slc17a7、Rps6ka1、C1qb、Nsf、Mast2、Rock1、Dusp12、Tac2、Def8、Prr6、Mink1、Acrbp、300002F03、AL845475.12等表达均具有显著差异。提示LW对SAMP8学习记忆功能障碍的改善作用可能与其影响上述基因的表达有关。
(三)八味地黄汤和黄连解毒汤对6月龄SAMP8海马和皮层基因表达的影响
BW和HL分别是中医温补肾阳和清热解毒的经典名方，近年临床和实验药理学研究资料表明，二者在AD治疗方面具有明显疗效，我室前期研究结果表明，它们对SAMP8学习记忆功能也具有明显改善作用。在本实验中我们选择BW和HL做为工具药物，采用cDNA芯片技术，观察了它们对6月龄SAMP8海马和皮层基因表达的影响，并与LW进行了比较，分析比较了这几个中药复方对SAMP8基因表达影响的异同，然后采用实时定量PCR方法对芯片筛选的结果进行了验证。
结果表明，给予BW(10.2g/kg)后，SAMP8海马中有42个差异表达克隆，其中表达上调的有21个，下调的有21个；皮层中有14个差异表达克隆，其中表达上调的有9个，下调的有5个。将这56个克隆进行测序，共成功测序39个，其对应19个基因，其中14个为已知功能基因，包括核酸代谢基因、核糖体合成装配基因、递质转运基因、信号转导基因、细胞生长相关基因、蛋白质代谢相关基因、电子传递相关基因等，另有5个为未知功能基因。
给予HL(5g/kg)后，SAMP8海马中有143个差异表达克隆，其中表达上调的有29个，下调的有114个；皮层中有93个差异表达克隆，其中表达上调的有9个，下调的有84个。将这些克隆进行测序，共成功测序168个，其对应61个基因，其中包括已知功能基因36个，按功能可分为核酸代谢、信号转导、转录调节、蛋白质代谢、能量代谢、神经系统发育等，此外还有25个未知功能基因。
为了便于分析比较，我们还以LW做为对照，与BW和HL一起进行了同步实验，观察了给予中等剂量LW(10g/kg)后，SAMP8海马和皮层海马中基因表达的变化，结果表明，LW作用后SAMP8海马中有44个差异表达克隆，其中表达上调的有40个，下调的有4个；皮层中有27个差异表达克隆，其中表达上调的有7个，下调的有20个。将上述克隆进行测序，共成功测序54个，其对应20个基因。经生物信息学分析，这些基因包括核酸代谢基因、递质转运基因、信号转导相关基因、细胞生长相关基因、转录调节基因等，另有未知功能基因8个。
以上研究结果表明，3个中药复方均对SAMP8海马和皮层的基因表达具有明显调节作用，不同的药物既有其共同的反应基因，又有其各自不同的反应基因，这些基因涉及多个功能及途径。三个复方共同的反应基因有8个，包括核酸代谢基因Fhit、Itm2c；蛋白质代谢基因Ube2d2；递质转运基因Slc17a7；信号转导基因Rps6ka1；核糖体生物合成装配基因Rn18s、18SrRNA；功能未知基因Rps19bp1。共同反应基因提示3个中药复方在药理作用或作用机理方面可能具有共同之处。
二、防治AD药物筛选候选组合基因靶标的提出及初步研究
(一)候选组合基因靶标的提出
对SAMP8增龄过程中海马和皮层基因表达变化规律及防治AD的中药复方LW、BW和HL对6月龄SAMP8海马和皮层基因表达的影响进行综合分析比较，结果表明，在差异表达基因中，有60个是有功能线索的基因，其中信号转导、核酸代谢、蛋白质代谢、细胞生长与维持、能量代谢等功能基因所占比例最高，提示这些基因可能与AD的发生发展过程密切相关，可能也与AD防治药物的作用途径或靶点有密切关系，值得进一步深入研究。此外在上述差异表达基因中，还有45个为没有检索到任何功能线索的基因，它们同样也可能与AD发生发展密切相关，而且可能蕴含着更多尚不被人们认识的神经生物学意义，因此为防治AD靶标的研究提供了更大的探索空间，具有更广阔的探索前景。
通过比较分析SAMP8和SAMR1在增龄过程中持续存在的差异表达基因、三种中药复方LW、BW和HL两两之间、三者之间的共同反应基因，以及仅出现于某些月龄SAMP8海马或皮层中，但对一个或几个中药复方有反应的药物反应基因，再结合公共数据库对这些基因功能、其与AD发病和治疗相关性的研究结果，初步提出了包括32个基因的拟用于防治AD药物筛选的候选基因群，我们称之为“候选组合基因靶标”，其中包括(1)核酸代谢相关基因2个，分别是Itm2c、Fhit；(2)信号转导相关基因12个，分别是Rps6ka1、Rock1、Dusp12、Mink1、Rab26、Mast2、Camk2α、Ephb6、S100a11、Tac2、Clstn1、Strn4；(3)蛋白质代谢相关基因4个，分别是Amfr、Ube2d2、Ttc3、Prr6；(4)递质转运相关基因1个，即Slc17a7；(5)转录调节相关基因2个，分别是D1ertd161e、Ssu72；(6)能量代谢相关基因4个，分别是Stub1、Uqcr、Ranbp5、Nsf；免疫相关基因1个，即C1qb；(7)细胞生长相关基因1个，即Ngrn；(8)神经系统发育相关基因1个，即Trim3；(9)电子传递相关基因1个，即Ndufs2；(10)神经胶质细胞分化相关基因1个，即Tspan2；(11)功能未知基因2个，分别是Rps19bp1、300002F03。
(二)候选基因群的初步验证
为了对上述初步提出的组成“组合基因靶标”的32个候选基因进行进一步的筛选和验证，我们采用实时定量PCR方法，进一步考察了这些基因随增龄在SAMP8海马和皮层中表达的变化，以及它们对LW、BW、HL的反应，此外，我们还考察了它们对其它一些对AD也具有良好疗效的中药复方和天然产物，如DSS、TXF和Hup A等的反应性。
结果表明，在增龄过程中，与SAMR1相比，这32个候选基因靶标在SAMP8海马中的表达均发生变化，而且其表达变化趋势有14种类型。这32个候选基因靶标除Ndufs2以外的31个在SAMP8皮层中表达均发生变化，而且其表达变化趋势有13种类型。分别给予SAMP8 Hup A、LW、BW、HL、DSS、TXF后，在海马和皮层中这32个候选基因靶标皆对这些药物呈现一定的反应性；在海马中这32个候选基因靶标的表达变化趋势分别有不同的6—7种类型，在皮层中则分别有不同的5—7种类型。其中，海马中基因Trim3、Tspan2、Ttc3、Rock1、Ube2d2、Mast2、Clstn1及皮层中基因Trim3、Rps19bp1、Dlertd161e、Rock1、Mast2、Stub1、Uqcr、Camk2α、Strn4对上述五种中药复方和一种天然产物皆有药物反应性；基因Amfr、C1qb、Dusp12、Ephb6、Itm2c、Mink1、Tac2、Ndufs2、Ngrn、Nsf、Ranbp5、S100a11、Fhit、Prr6、Rab26、300002F03、Slc17a7在海马或皮层中对这些药物中的一种或几种具有反应性；而基因Ssu72、Rps19bp1、Rps6ka1在海马中对上述五种中药复方和一种天然产物的治疗无反应。
综上所述，通过本研究筛选得到防治AD药物筛选的候选基因共计32个，经初步验证这32个候选基因在SAMP8中皆出现规律性的表达变化，提示这些基因可能和学习记忆功能障碍或AD发生发展过程有关。几种对AD治疗有效的中药复方和天然产物对这32个基因的表达既有相同的作用，又有不同的作用。相同作用的基因可能是防治AD药物作用的共同靶标；而不同作用的基因可能与所选药物的功能主治和作用机制及其发挥药效的生理途径不同有关。
我们充分认识到，寻找和确证药物靶标是一项异常艰巨的任务，并非通过一个学位论文或一个研究项目所能完成的，但我们相信，从“组合靶标”的角度开展药物靶标的筛选和研究是一个崭新的尝试，对于寻找AD这类多因素复杂疾病防治药物筛选靶标更是一个有益的尝试。本研究获得的一些结果仅仅为我们要建立的“组合靶标”提供了一些线索，更复杂更艰巨的任务是靶标验证工作，这将是我们下一步的研究重点。
Alzheimer's disease (AD) is the most common form of progressive dementia in the elderly. The aetiology of AD remains unknown and there is lack of effective cure. Cholinesterase inhibitors are most widely used for the treatment of AD. However, current drugs do not significantly ameliorate the corruption of neurodegeneration, and only provide limited or transient benefit to many patients. The development of drugs with more effective and less adverse drug reaction is imminent.
The discovery and validation of drug target are very important procedure of developing original drug. In the past time, drug therapy based on single-target-directed strategy seems inappropriate for the treatment of complex diseases, AD, that have multiple pathogenic factors and multiple dysfunctions. So the comprehensive treatment by means of multi-target is feasible to ameliorate therapy for AD. And the discovery, study and validation of one or one more groups of combinatorial targets associated with cognitive dysfunction and pathological abnormalities tightly will provide a new strategy for effective therapy for AD. Combining the techniques of genomics, proteomics, microarray and bioinformatics in drug discovery has been proposed as one of rapid and efficient approaches to search and find new potential drug targets.
The main clinical symptoms of AD are primarily memory loss and cognitive impairments. The expression and transcription of many genes and their encoding protein are associated with occurrence and development of AD. At present, genes of amyloid precursor protein (APP), APOE4, presenilin-1 (PSEN1), PSEN2, transcription factor CP2 (TCFP2), Alpha-T-catenin (CTNNA3) and LRP1 are known as some of the reasons of AD, but for this multi-factorial disease, there may be many unknown genes that play important roles in AD. Hippocampus and cerebral cortex are important brain regains for learning and memory. The study on expression levels of gene in the brain may provide clues of some function of CNS and potential gene targets.
The senescence-accelerated mouse (SAM) is a model of accelerated senescence that was established through phenotypic selection. The SAMP8 strain, a substrain of SAM, has been proposed as a good model for the study of aging of the brain and of deficits in learning and memory at gene and protein levels with the features of pathological abnormalities in the brain, learning and memory deficits. The study of gene expression change in hippocampus and cortex of SAMP8 will provide clues for discovery of genes about learning and memory in CNS and gene targets for AD.
The traditional Chinese medicine and their effective components have properly their own inimitable predominance with their multi-factorial, multi-target and multi-functional action than chemical compound with a single-target. In fact, Dang-Gui-Shao-Yao-San (DSS), Kai-Xin-San, Liu-Wei-Di-Huang decoction (LW), Ba-Wei-Di-Huang decoction (BW) and Huang-lian-Jie-Du Decoction (HL) etc, traditional Chinese medicinal prescriptions, have many clinical pharmacological actions including enhancing the cognitive function of CNS and benefits to AD patients in China and Japan. In the past, the study in our lab showed the above Chinese medicine ameliorated learning and memory. According to described in the theory of traditional Chinese medicine, the effect and indication of above Chinese medicine are different, including purging the fire and detoxifying, invigorating the kidney, promoting blood flow etc. These diversified effects indicated the different pharmacological actions and mechanisms maybe result in the difference of gene expression change in CNS. The difference and similarity of differential expression genes treated with diversified Chinese medicines were analyzed to discover the specific and common differential expression genes. The common differential expression represented the similarity of pharmacological actions and mechanisms. But the specific differential expression genes to single Chinese medicine represented drug action or mechanism. In this paper, the gene expression patterns of hippocampus and cortex of SAMP8 treatment with LW, BW and HL were investigated to provide insight into the study of the gene targets for AD.
This research consisted of two sections. One was the screening of differential expression genes in the period of aging and patterns of gene expression in hippocampus and cortex of SAMP8 through the technology of cDNA microarray. This aimed to discover a group of expression change genes associated with aging and drug action treatment with Chinese medicine. Meanwhile this aimed to establish the foundation of candidate gene targets discovery. The other was proposed a group of combinatorial candidate genes and primary validation. With the animal model of SAMP8, natural product HupA and Chinese medicine LW, BW, HL, DSS, TXF, the expression change of candidate genes in hippocampus and cortex of SAMP8 was carried out through the technology of real time quantitative RT-PCR.
1. The screening of gene targets for AD therapy
In order to investigate the potential gene targets, the differential month-old animal model SAMP8 and LW, BW and HL were applied in this study. The screening and analysis of differential gene expression in hippocampus and cortex were performed employing the cDNA microarray that designed of one's own.
1.1 The differential gene expression in hippocampus and cortex of differential ageSAMP8
The differential gene expression patterns in hippocampus and cortex of 2,6,12 month-old SAM were performed employing cDNA microarray and validation of real-time RT-PCR. The results showed there were 124 differential expression clones in hippocampus of 2-month SAMP8 and none in cortex of 2-month SAMP8 comparing with 2-month SAMR1. These clones were sequenced and 100 clones were sequenced successfully. The analysis of 100 differential expression clones by bioinformatics indicated these clones represented 45 genes. The results showed there were 20 differential expression clones in hippocampus of 6-month-old SAMP8 comparing with 6-month-old SAMR1. These clones were sequenced and 9 clones were sequenced successfully. These clones represented 5 genes. The results showed there were 31 differential expression clones in cortex of 6-month SAMP8 comparing with 6-month SAMR1. These clones were sequenced and 21 clones were sequenced successfully. The analysis of 21 differential expression clones by bioinformatics indicated that these clones represented 15 genes. The results showed there were 14 differential expression clones in hippocampus of 12-month-old SAMP8 comparing with 12-month-old SAMR1. These clones were sequenced and 7 clones were sequenced successfully. These represented 4 genes with distinct function. The results showed there were 146 differential expression clones in cortex of 12-month SAMP8 comparing with 12-month SAMR1. These clones were sequenced and 120 clones were sequenced successfully. These clones represented 36 genes. After the above results of differential expression genes were analyzed by bioinformatics, these represented distinct function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, regulation of transcription, protein metabolism, nervous system development, energy metabolism, cell growth and maintenance, immune response and many genes whose biological function and process were still unknown.
SAMP8 is a good animal model of age-related cognitive decline with relevance to alterations of the gene expression and protein abnormalities in AD. In this study, the results of differential gene expression in 2, 6, 12-month-old SAMP8 with SAMR1 showed more and more differential expression genes in the period of ageing and hint these genes were associated with cognitive decline and accelerated senescence, especially genes of signal transduction, protein and energy metabolism. These genes maybe provide significant clues for aging and cognitive impairment, meanwhile provide important base for study of potential targets for AD therapy.
1.2 Gene expression patterns of hippocampus and cortex of SAMP8 treated with LW
Liu-Wei-Di-Huang decoction (LW), a traditional Chinese medicinal prescription, hasmany clinical pharmacological actions including enhancing the cognitive function of CNS. Recently, pharmacological studies of our lab demonstrate that the major effect of LW was improvement of cognition and memory of SAMP8, according to research of neuroendocrine immunological regulation (NIM). In order to get the message of mechanism of LW enhancing the cognitive function and discover target for therapy, cDNA microarray was adopted.
The results showed there were 11 differential expression clones in hippocampus and 67 differential expression clones in cortex of SAMP8, after treated with LW (5g/kg). These clones were sequenced and 53 clones were sequenced successfully. They represented 28 genes with diversified biological function. The results showed there were 43 differential expression clones in hippocampus and 222 differential expression clones in cortex of SAMP8, after treated with LW (10g/kg). These clones were sequenced and 137 clones were sequenced successfully. They represented 60 genes with diversified biological function. The results showed there were 80 differential expression clones in hippocampus and 233 differential expression clones in cortex of SAMP8, after treated with LW (15g/kg). These clones were sequenced and 197 clones were sequenced successfully. They represented 55 genes with diversified biological function. After the above results of differential expression genes were analyzed by bioinformatics these genes treated with different dosage of LW represented similar biological function, including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, regulation of transcription, protein metabolism, nervous system development, energy metabolism, cell growth and maintenance, immune response, electron transport and many genes whose biological function and process were still unknown.
These results demonstrated there was similar functional category of differential expression genes. But there were more expression change genes in group medium and high dosage than group of low dosage. Gene Ttc3 Oxr1 Slc17a7 Rps6ka1 C1qb Nsf Mast2 Rock1 Dusp12 Tac2 Def8 Mink1. Prr6, Acrbp 300002F03 AL845475.12 expression changed significantly. It hints the effect of LW about learning and memory maybe is related to above genes.
1.3 Gene expression patterns of hippocampus and cortex of SAMP8 treated with BW and HL
BW and HL, traditional Chinese medicinal prescription related to invigorating kidney-YANG and purging the fire and detoxifying respectively, have many clinical pharmacological actions including enhancing the cognitive function of CNS. Recently, pharmacological studies demonstrate that the effect of improvement of cognition and memory was one of their diversified effects. In order to get the message of mechanism of BW and HL enhancing the cognitive function and discover target for therapy, cDNA microarray and 6-month-old SAMP8 were adopted and real time PCR was performed to validate the results from microarray. We analyzed and compared with the different expression patterns of hippocampus and cortex treated with LW, BW and HL.
The results showed there were 42 differential expression clones including 21 up-regulated clones and 21 down-regulated clones in hippocampus and 14 differential expression clones including 9 up-regulated and 5 down-regulated in cortex of SAMP8, after treated with BW (10.2g/kg). These clones were sequenced and 39 clones were sequenced successfully. They represented 14 genes with diversified biological function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, protein metabolism, cell growth and maintenance, and 5 genes whose biological function and process were still unknown.
The results showed there were 143 differential expression clones including 29 up-regulated clones and 114 down-regulated clones in hippocampus and 93 differential expression clones including 9 up-regulated and 84 down-regulated in cortex of SAMP8, after treated with HL (5g/kg). These clones were sequenced and 168 clones were sequenced successfully. They represented 36 genes with diversified biological function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, protein metabolism, energy metabolism, cell growth and maintenance, regulation of transcription, and nerve system development and 25 genes whose biological function and process were still unknown.
In order to analyze the patterns of gene expression treated with different Chinese medicine prescription, we investigated the differential gene expression treatment with LW meanwhile. The results showed there were 44 differential expression clones including 40 up-regulated clones and 4 down-regulated clones in hippocampus and 27 differential expression clones including 7 up-regulated and 20 down-regulated in cortex of SAMP8, after treated with LW (10g/kg). These clones were sequenced and 54 clones were sequenced successfully. They represented 12 genes with diversified biological function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, protein metabolism, energy metabolism, cell growth and maintenance, regulation of transcription, and nerve system development and 8 genes whose biological function and process were still unknown.
Comprehending the above screening results of HL, BW and LW, we detected that the three Chinese medicine prescriptions possessed significant ability of regulation of gene expression, including common effective genes to all medicine and specific effective genes to specific medicine. The common effective genes were eight, including nucleic acid metabolism gene Fhit and Itm2c, protein metabolism gene Ube2d2, transport gene Slc17a7, signal transduction gene Rps6kal, ribosome biogenesis and assembly gene Rn18s and 18srRNA, functional unknown gene Rps19bp1. These common genes hint there is some similarity with the three Chinese medicine prescriptions.
2. The propose and primary study of combinatorial candidate gene targets for AD therapy
2.1 The propose of combinatorial candidate gene targets for AD therapy
From the aggregate analysis the patterns of gene expression change in hippocampus and cortex in the period of aging and gene expression change in hippocampus and cortex treated with Chinese medicine prescription LW, BW and HL, there were 60 differential expression genes with clear biological function. Among the 60 genes with different function, signal transduction, nucleic acid metabolism, protein metabolism, energy metabolism was the major portion in ratio. This demonstrated these categories of biological function were mutually related to the occurrence and development of AD, pathway of drug action and drug targets. This is worth to further study. In addition, 45 genes with function unknown were interrelated to the occurrence and development of AD and possessed more significance of neurobiology. These results provided extensive prospect of research.
After comparing and analyzing the differential expression genes that always in the period of aging, that treated with two or three Chinese medicine prescription of LW, BW, HL or that in a specific month-old and treated with one Chinese medicine prescription simultaneously, and combining the bioinformatics and references, we posed the combinatorial candidate gene targets for AD therapy, namely (1) nucleic acid metabolism gene, including Itm2c, Fhit; (2) signal transduction gene, including Rps6ka1, Rock1, Dusp12, Mink1, Rab26, Mast2, Camk2α, Ephb6, S100a11, Tac2, Clstn1, Strn4; (3) protein metabolism gene, including Amfr, Ube2d2, Ttc3, Prr6; (4) transporter gene, including Slcl7a7; (5) regulation of transcript gene, including D1Ertd161e, Ssu72; (6) energy metabolism gene, including Stub1, Uqcr, Ranbp5, Nsf; (7) cell growth and maintenance gene, including Ngrn; (8) nerve system development gene, including Trim3; (9) electron transport gene, including Ndufs2; (10) Neurogila cell differentiation gene, including Tspan2; (11) function unknown gene, including Rps19bp1, 300002F03.
2.2 Primary study of combinatorial candidate gene targets
To validate the above combinatorial candidate gene targets, we applied the real time PCR to assess the patterns of gene expression in the period of aging and treated with LW, BW, HL, DSS, TXF, HupA that benefited to AD therapy.
The results showed there were 14 types of change of the 32 genes expression in the hippocampus of SAMP8 in the period of aging. And there were 13 types of change of the expression 31 genes in the cortex of SAMP8 in the period of aging, except for Ndufs2. After treated with Hup A, LW, BW, HL, DSS, TXF, there were all change of 32 genes expression in the hippocampus and cortex of SAMP8. In hippocampus, these changes of gene expression were defined 6-7 types. In cortex, these changes of gene expression were defined 5-7 types. After treated with 5 Chinese medicine prescriptions and HupA, gene Trim3, Tspan2, Ttc3, Rock1, Ube2d2, Mast2, Clstn1 in hippocampus all presented gene expression change, but gene Amfr, C1qb, Dusp12, Ephb6, Itm2c, Mink1, Tac2, Ndufs2, Ngrn, Nsf, Ranbp5, S100all, Fhit, Prr6, Rab26, 300002F03, Slc17a7 in hippocampus or cortex presented expression change for one or one more Chinese medicine prescriptions. Only gene Ssu72, Rpsl9bpl, Rps6kal in hippocampus presented no gene expression change.
Through the primary validation of 32 genes, the expression of them presented regularity. This hint these genes were interrelated to the occurrence and development of AD. The similarity and difference of 32 genes expression demonstrated the characters of effects of Chinese medicine and HupA.
The discovery and validation of drug targets is an arduous work, by no means achieve the goal through one paper or one research. But we believe the "combinatorial targets" is a helpful attempt. The results from this paper only provided clues and more complicated and hard works need our endeavor.
创新药物发现过程中最重要的环节之一是药物靶标的发现和确证。AD是典型的多因素复杂疾病，其发生发展过程涉及多系统、多环节结构和功能的异常，针对单一靶点的防治措施难以取得满意疗效，多靶点综合干预是提高AD防治水平的重要策略。因此，寻找、研究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、且具有相互补充与协同关系的“组合靶标”，将为防治AD创新药物的研究提供新的筛选靶标，并将大大提高AD防治药物的研究与开发水平。结合当今飞速发展的基因组及生物信息学技术，从基因入手研究上述组合药物靶标，是一条药物靶标研究的新路子，具有广阔的研究前景。
AD在临床表现为认知功能的进行性衰退。已有大量研究表明，基因表达或转录异常是AD发生、发展的重要因素之一，同时多种基因及其表达产物在中枢学习记忆功能衰退的过程中发挥了重要作用，如APO-E、APP、PS-1、PS-2、cystatin-3、LRP1等。海马和皮层是与学习和记忆功能关系最为密切的两个脑区，它们在AD脑内也呈现严重的病理改变和基因表达异常。因此，以这两个特定区域差异表达基因为线索，通过深入研究这些基因与AD发生、发展及病理变化的关系，有望发现用于防治AD的药物基因靶标。
快速老化模型小鼠(senescence-accelerated mouse，SAM)是一种自然发生的、全身性快速老化的模型动物。其快速老化亚系之一SAMP8的生物学特征是中枢学习记忆功能随增龄进行性衰退，同时脑内出现神经元丢失、Aβ沉积等类似AD的病理改变，是在基因和蛋白水平研究AD的良好模型。因此，通过研究SAMP8在增龄过程中海马和皮层基因表达的变化，是发现中枢学习记忆功能相关基因、开展防治AD药物靶标研究的一条重要途径。
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，就此而言，其在防治AD这类多因素复杂疾病方面，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。事实上，我国和日本等国家在防治AD中广泛使用的中药复方如当归芍药散、开心散、六味地黄汤、八味地黄汤、黄连解毒汤、调心方、钩藤散等，对AD病人或模型动物均具有良好的疗效。而我室前期考察六味地黄汤(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)等多种中药复方对学习记忆功能作用的结果也表明，上述几种中药方剂对SAMP8学习记忆功能障碍均具有改善作用。按照中医理论，这些中药复方的功能主治不尽相同，如补肾、清热解毒、活血化瘀等，因此它们对中枢认知功能的作用特点和作用机理亦可能不同，它们在药理作用和作用机理的方面的差异可能反应在用药后中枢神经系统的基因表达有所不同。因此，通过研究SAMP8应用这些中药复方后海马和皮层的反应基因，比较各药物反应基因群的异同，可从中发现这些中药复方所共有的药物反应基因和各自所特有的个性反应基因。药物反应基因或用药后基因表达的变化，是药物发挥药理作用的重要环节之一。共性反应基因可能代表了不同药物在药理作用、作用机理等方面的相似性，个性反应基因则可能代表了药物所特有的药效或作用机理等。因此，通过研究模型动物学习记忆功能衰退过程中表达变化的基因和对防治AD药物的反应基因，可为防治AD药物基因靶标的研究提供重要线索，是发现和研究防治AD药物基因靶标的一条重要途径。
本研究共分为两部分，一是采用cDNA芯片筛选SAMP8增龄过程中海马、皮层的差异表达基因以及SAMP8应用LW、BW和HL后其海马、皮层基因表达的变化，旨在获得一批增龄过程中SAMP8海马和皮层中表达明显变化的基因及一批应用具有改善学习记忆功能的三个中药复方后SAMP8海马和皮层中表达明显变化的基因，从而为防治AD药物候选组合基因靶标的提出奠定基础。二是通过对上述增龄性差异表达基因和药物反应基因进行生物信息学分析，初步提出一组防治AD药物筛选的候选组合基因靶标，并以SAMP8为模型，以防治AD的天然产物石杉碱甲(Hup A)和中药复方LW、BW、HL、DSS、TXF为工具，采用实时定量PCR方法，考察所提出的候选基因靶标在增龄性SAMP8海马、皮层中的表达变化以及对防治AD药物的反应性，以对这些候选基因靶标进行初步的验证。
一、防治AD药物作用基因靶标的筛选
为了寻找防治AD药物作用的基因靶标，本研究以快速老化模型小鼠(SAMP8)为动物模型，给予LW、BW和HL，采用我们自行研制的cDNA芯片，筛选和分析了SAMP8增龄过程中基因表达的变化以及给予LW、BW和HL对SAMP8海马和皮层基因表达的影响。
(一)不同月龄SAMP8海马和皮层差异表达基因的筛选
本研究采用cDNA芯片技术，比较了SAMP8和SAMR1增龄过程中海马和皮层基因表达的变化，并采用实时定量PCR方法对芯片筛选的结果进行了验证。
结果表明，与同龄SAMR1相比，2月龄SAMP8海马中差异表达克隆有124个，皮层中未见差异表达克隆。将这124个差异表达克隆进行测序，共成功测序100个，经过生物信息学分析，它们共对应45个基因。而与同龄SAMR1相比，6月龄SAMP8海马中的差异表达克隆仅有20个，共成功测序9个，对应5个基因；皮层中差异表达克隆为31个，共成功测序了21个克隆，其对应15个基因。12月龄SAMP8与同龄SAMR1比较，海马中的差异表达克隆有14个，共成功测序7个，对应4个基因；而在皮层中，表达差异的克隆有146个，成功测序了120个，对应36个基因。上述基因的功能涉及核酸代谢、核糖体装配合成、信号转导、转运、转录调节、蛋白质代谢、神经系统发育、细胞生长、能量代谢、免疫相关等，同时还有大量功能未知的基因。
随着增龄，SAMP8学习记忆功能进行性衰退。研究表明，SAMP8在4月龄时已出现AD相关的病理变化与学习记忆功能衰退。本研究发现，与SAMR1相比，SAMP8在增龄过程中其海马与皮层的基因表达发生了明显变化，其中包括与信号转导、蛋白质代谢、能量代谢等密切相关的基因。基因Ttc3、Uqcr的表达在2、6、12月龄中皆存在差异，提示这些基因可能与SAMP8脑老化以及学习记忆功能障碍的病理过程具有密切关系。这些随增龄而表达变化的基因不仅为深入研究SAMP8脑老化和学习记忆障碍的发生机理提供了有意义的线索，同时为防治AD药物研究的基因靶标筛选和研究提供了重要依据。
(二)六味地黄汤对6月龄SAMP8海马和皮层基因表达的影响
六味地黄汤(LW)是中医滋补肾阴的经典代表名方，近年来在临床上也被应用于AD的辅助治疗，取得了较好的效果。近年来我室从神经内分泌免疫调节(NIM)网络的角度对其药理作用及其机理进行了系统研究，结果表明，改善SAMP8学习记忆功能是LW的重要作用之一。为了探讨LW改善SAMP8学习记忆功能的作用途径或基因靶点，本研究选用6月龄雄性SAMP8，分别灌胃给予5、10、15g／kg剂量LW，连续1个月，然后取海马及皮层组织，提取总RNA，进而采用cDNA芯片技术观察了给药后SAMP8海马及皮层基因表达的变化。
结果表明，给予低剂量LW(5g／kg)后，SAMP8海马中差异表达的克隆有11个，皮层中有67个，将这些克隆进行测序，共成功测序53个，其对应基因28个；给予中剂量LW(10g／kg)后，SAMP8海马中差异表达的克隆有43个，皮层中有222个，共成功测序137个，其对应基因60个；给予高剂量LW(15g/kg)后，SAMP8海马中差异表达的克隆有80个，皮层中有233个，共成功测序197个，其对应基因55个。生物信息学分析的结果表明，不同剂量LW作用下所影响的基因种类大体相同，包括核酸代谢基因、递质转运基因、细胞生长、蛋白质代谢、核糖体生物合成装配、转录调节、神经系统发育、电子传递、免疫、能量代谢等，还有一些功能未知基因。
以上结果表明，给予不同剂量LW后，其海马和皮层反应基因的种类大致相同，但中、高剂量组发生变化的基因数量明显多于低剂量组。与SAMP8对照组相比，给予中、高剂量LW后，基因Ttc3、Oxr1、Slc17a7、Rps6ka1、C1qb、Nsf、Mast2、Rock1、Dusp12、Tac2、Def8、Prr6、Mink1、Acrbp、300002F03、AL845475.12等表达均具有显著差异。提示LW对SAMP8学习记忆功能障碍的改善作用可能与其影响上述基因的表达有关。
(三)八味地黄汤和黄连解毒汤对6月龄SAMP8海马和皮层基因表达的影响
BW和HL分别是中医温补肾阳和清热解毒的经典名方，近年临床和实验药理学研究资料表明，二者在AD治疗方面具有明显疗效，我室前期研究结果表明，它们对SAMP8学习记忆功能也具有明显改善作用。在本实验中我们选择BW和HL做为工具药物，采用cDNA芯片技术，观察了它们对6月龄SAMP8海马和皮层基因表达的影响，并与LW进行了比较，分析比较了这几个中药复方对SAMP8基因表达影响的异同，然后采用实时定量PCR方法对芯片筛选的结果进行了验证。
结果表明，给予BW(10.2g/kg)后，SAMP8海马中有42个差异表达克隆，其中表达上调的有21个，下调的有21个；皮层中有14个差异表达克隆，其中表达上调的有9个，下调的有5个。将这56个克隆进行测序，共成功测序39个，其对应19个基因，其中14个为已知功能基因，包括核酸代谢基因、核糖体合成装配基因、递质转运基因、信号转导基因、细胞生长相关基因、蛋白质代谢相关基因、电子传递相关基因等，另有5个为未知功能基因。
给予HL(5g/kg)后，SAMP8海马中有143个差异表达克隆，其中表达上调的有29个，下调的有114个；皮层中有93个差异表达克隆，其中表达上调的有9个，下调的有84个。将这些克隆进行测序，共成功测序168个，其对应61个基因，其中包括已知功能基因36个，按功能可分为核酸代谢、信号转导、转录调节、蛋白质代谢、能量代谢、神经系统发育等，此外还有25个未知功能基因。
为了便于分析比较，我们还以LW做为对照，与BW和HL一起进行了同步实验，观察了给予中等剂量LW(10g/kg)后，SAMP8海马和皮层海马中基因表达的变化，结果表明，LW作用后SAMP8海马中有44个差异表达克隆，其中表达上调的有40个，下调的有4个；皮层中有27个差异表达克隆，其中表达上调的有7个，下调的有20个。将上述克隆进行测序，共成功测序54个，其对应20个基因。经生物信息学分析，这些基因包括核酸代谢基因、递质转运基因、信号转导相关基因、细胞生长相关基因、转录调节基因等，另有未知功能基因8个。
以上研究结果表明，3个中药复方均对SAMP8海马和皮层的基因表达具有明显调节作用，不同的药物既有其共同的反应基因，又有其各自不同的反应基因，这些基因涉及多个功能及途径。三个复方共同的反应基因有8个，包括核酸代谢基因Fhit、Itm2c；蛋白质代谢基因Ube2d2；递质转运基因Slc17a7；信号转导基因Rps6ka1；核糖体生物合成装配基因Rn18s、18SrRNA；功能未知基因Rps19bp1。共同反应基因提示3个中药复方在药理作用或作用机理方面可能具有共同之处。
二、防治AD药物筛选候选组合基因靶标的提出及初步研究
(一)候选组合基因靶标的提出
对SAMP8增龄过程中海马和皮层基因表达变化规律及防治AD的中药复方LW、BW和HL对6月龄SAMP8海马和皮层基因表达的影响进行综合分析比较，结果表明，在差异表达基因中，有60个是有功能线索的基因，其中信号转导、核酸代谢、蛋白质代谢、细胞生长与维持、能量代谢等功能基因所占比例最高，提示这些基因可能与AD的发生发展过程密切相关，可能也与AD防治药物的作用途径或靶点有密切关系，值得进一步深入研究。此外在上述差异表达基因中，还有45个为没有检索到任何功能线索的基因，它们同样也可能与AD发生发展密切相关，而且可能蕴含着更多尚不被人们认识的神经生物学意义，因此为防治AD靶标的研究提供了更大的探索空间，具有更广阔的探索前景。
通过比较分析SAMP8和SAMR1在增龄过程中持续存在的差异表达基因、三种中药复方LW、BW和HL两两之间、三者之间的共同反应基因，以及仅出现于某些月龄SAMP8海马或皮层中，但对一个或几个中药复方有反应的药物反应基因，再结合公共数据库对这些基因功能、其与AD发病和治疗相关性的研究结果，初步提出了包括32个基因的拟用于防治AD药物筛选的候选基因群，我们称之为“候选组合基因靶标”，其中包括(1)核酸代谢相关基因2个，分别是Itm2c、Fhit；(2)信号转导相关基因12个，分别是Rps6ka1、Rock1、Dusp12、Mink1、Rab26、Mast2、Camk2α、Ephb6、S100a11、Tac2、Clstn1、Strn4；(3)蛋白质代谢相关基因4个，分别是Amfr、Ube2d2、Ttc3、Prr6；(4)递质转运相关基因1个，即Slc17a7；(5)转录调节相关基因2个，分别是D1ertd161e、Ssu72；(6)能量代谢相关基因4个，分别是Stub1、Uqcr、Ranbp5、Nsf；免疫相关基因1个，即C1qb；(7)细胞生长相关基因1个，即Ngrn；(8)神经系统发育相关基因1个，即Trim3；(9)电子传递相关基因1个，即Ndufs2；(10)神经胶质细胞分化相关基因1个，即Tspan2；(11)功能未知基因2个，分别是Rps19bp1、300002F03。
(二)候选基因群的初步验证
为了对上述初步提出的组成“组合基因靶标”的32个候选基因进行进一步的筛选和验证，我们采用实时定量PCR方法，进一步考察了这些基因随增龄在SAMP8海马和皮层中表达的变化，以及它们对LW、BW、HL的反应，此外，我们还考察了它们对其它一些对AD也具有良好疗效的中药复方和天然产物，如DSS、TXF和Hup A等的反应性。
结果表明，在增龄过程中，与SAMR1相比，这32个候选基因靶标在SAMP8海马中的表达均发生变化，而且其表达变化趋势有14种类型。这32个候选基因靶标除Ndufs2以外的31个在SAMP8皮层中表达均发生变化，而且其表达变化趋势有13种类型。分别给予SAMP8 Hup A、LW、BW、HL、DSS、TXF后，在海马和皮层中这32个候选基因靶标皆对这些药物呈现一定的反应性；在海马中这32个候选基因靶标的表达变化趋势分别有不同的6—7种类型，在皮层中则分别有不同的5—7种类型。其中，海马中基因Trim3、Tspan2、Ttc3、Rock1、Ube2d2、Mast2、Clstn1及皮层中基因Trim3、Rps19bp1、Dlertd161e、Rock1、Mast2、Stub1、Uqcr、Camk2α、Strn4对上述五种中药复方和一种天然产物皆有药物反应性；基因Amfr、C1qb、Dusp12、Ephb6、Itm2c、Mink1、Tac2、Ndufs2、Ngrn、Nsf、Ranbp5、S100a11、Fhit、Prr6、Rab26、300002F03、Slc17a7在海马或皮层中对这些药物中的一种或几种具有反应性；而基因Ssu72、Rps19bp1、Rps6ka1在海马中对上述五种中药复方和一种天然产物的治疗无反应。
综上所述，通过本研究筛选得到防治AD药物筛选的候选基因共计32个，经初步验证这32个候选基因在SAMP8中皆出现规律性的表达变化，提示这些基因可能和学习记忆功能障碍或AD发生发展过程有关。几种对AD治疗有效的中药复方和天然产物对这32个基因的表达既有相同的作用，又有不同的作用。相同作用的基因可能是防治AD药物作用的共同靶标；而不同作用的基因可能与所选药物的功能主治和作用机制及其发挥药效的生理途径不同有关。
我们充分认识到，寻找和确证药物靶标是一项异常艰巨的任务，并非通过一个学位论文或一个研究项目所能完成的，但我们相信，从“组合靶标”的角度开展药物靶标的筛选和研究是一个崭新的尝试，对于寻找AD这类多因素复杂疾病防治药物筛选靶标更是一个有益的尝试。本研究获得的一些结果仅仅为我们要建立的“组合靶标”提供了一些线索，更复杂更艰巨的任务是靶标验证工作，这将是我们下一步的研究重点。
Alzheimer's disease (AD) is the most common form of progressive dementia in the elderly. The aetiology of AD remains unknown and there is lack of effective cure. Cholinesterase inhibitors are most widely used for the treatment of AD. However, current drugs do not significantly ameliorate the corruption of neurodegeneration, and only provide limited or transient benefit to many patients. The development of drugs with more effective and less adverse drug reaction is imminent.
The discovery and validation of drug target are very important procedure of developing original drug. In the past time, drug therapy based on single-target-directed strategy seems inappropriate for the treatment of complex diseases, AD, that have multiple pathogenic factors and multiple dysfunctions. So the comprehensive treatment by means of multi-target is feasible to ameliorate therapy for AD. And the discovery, study and validation of one or one more groups of combinatorial targets associated with cognitive dysfunction and pathological abnormalities tightly will provide a new strategy for effective therapy for AD. Combining the techniques of genomics, proteomics, microarray and bioinformatics in drug discovery has been proposed as one of rapid and efficient approaches to search and find new potential drug targets.
The main clinical symptoms of AD are primarily memory loss and cognitive impairments. The expression and transcription of many genes and their encoding protein are associated with occurrence and development of AD. At present, genes of amyloid precursor protein (APP), APOE4, presenilin-1 (PSEN1), PSEN2, transcription factor CP2 (TCFP2), Alpha-T-catenin (CTNNA3) and LRP1 are known as some of the reasons of AD, but for this multi-factorial disease, there may be many unknown genes that play important roles in AD. Hippocampus and cerebral cortex are important brain regains for learning and memory. The study on expression levels of gene in the brain may provide clues of some function of CNS and potential gene targets.
The senescence-accelerated mouse (SAM) is a model of accelerated senescence that was established through phenotypic selection. The SAMP8 strain, a substrain of SAM, has been proposed as a good model for the study of aging of the brain and of deficits in learning and memory at gene and protein levels with the features of pathological abnormalities in the brain, learning and memory deficits. The study of gene expression change in hippocampus and cortex of SAMP8 will provide clues for discovery of genes about learning and memory in CNS and gene targets for AD.
The traditional Chinese medicine and their effective components have properly their own inimitable predominance with their multi-factorial, multi-target and multi-functional action than chemical compound with a single-target. In fact, Dang-Gui-Shao-Yao-San (DSS), Kai-Xin-San, Liu-Wei-Di-Huang decoction (LW), Ba-Wei-Di-Huang decoction (BW) and Huang-lian-Jie-Du Decoction (HL) etc, traditional Chinese medicinal prescriptions, have many clinical pharmacological actions including enhancing the cognitive function of CNS and benefits to AD patients in China and Japan. In the past, the study in our lab showed the above Chinese medicine ameliorated learning and memory. According to described in the theory of traditional Chinese medicine, the effect and indication of above Chinese medicine are different, including purging the fire and detoxifying, invigorating the kidney, promoting blood flow etc. These diversified effects indicated the different pharmacological actions and mechanisms maybe result in the difference of gene expression change in CNS. The difference and similarity of differential expression genes treated with diversified Chinese medicines were analyzed to discover the specific and common differential expression genes. The common differential expression represented the similarity of pharmacological actions and mechanisms. But the specific differential expression genes to single Chinese medicine represented drug action or mechanism. In this paper, the gene expression patterns of hippocampus and cortex of SAMP8 treatment with LW, BW and HL were investigated to provide insight into the study of the gene targets for AD.
This research consisted of two sections. One was the screening of differential expression genes in the period of aging and patterns of gene expression in hippocampus and cortex of SAMP8 through the technology of cDNA microarray. This aimed to discover a group of expression change genes associated with aging and drug action treatment with Chinese medicine. Meanwhile this aimed to establish the foundation of candidate gene targets discovery. The other was proposed a group of combinatorial candidate genes and primary validation. With the animal model of SAMP8, natural product HupA and Chinese medicine LW, BW, HL, DSS, TXF, the expression change of candidate genes in hippocampus and cortex of SAMP8 was carried out through the technology of real time quantitative RT-PCR.
1. The screening of gene targets for AD therapy
In order to investigate the potential gene targets, the differential month-old animal model SAMP8 and LW, BW and HL were applied in this study. The screening and analysis of differential gene expression in hippocampus and cortex were performed employing the cDNA microarray that designed of one's own.
1.1 The differential gene expression in hippocampus and cortex of differential ageSAMP8
The differential gene expression patterns in hippocampus and cortex of 2,6,12 month-old SAM were performed employing cDNA microarray and validation of real-time RT-PCR. The results showed there were 124 differential expression clones in hippocampus of 2-month SAMP8 and none in cortex of 2-month SAMP8 comparing with 2-month SAMR1. These clones were sequenced and 100 clones were sequenced successfully. The analysis of 100 differential expression clones by bioinformatics indicated these clones represented 45 genes. The results showed there were 20 differential expression clones in hippocampus of 6-month-old SAMP8 comparing with 6-month-old SAMR1. These clones were sequenced and 9 clones were sequenced successfully. These clones represented 5 genes. The results showed there were 31 differential expression clones in cortex of 6-month SAMP8 comparing with 6-month SAMR1. These clones were sequenced and 21 clones were sequenced successfully. The analysis of 21 differential expression clones by bioinformatics indicated that these clones represented 15 genes. The results showed there were 14 differential expression clones in hippocampus of 12-month-old SAMP8 comparing with 12-month-old SAMR1. These clones were sequenced and 7 clones were sequenced successfully. These represented 4 genes with distinct function. The results showed there were 146 differential expression clones in cortex of 12-month SAMP8 comparing with 12-month SAMR1. These clones were sequenced and 120 clones were sequenced successfully. These clones represented 36 genes. After the above results of differential expression genes were analyzed by bioinformatics, these represented distinct function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, regulation of transcription, protein metabolism, nervous system development, energy metabolism, cell growth and maintenance, immune response and many genes whose biological function and process were still unknown.
SAMP8 is a good animal model of age-related cognitive decline with relevance to alterations of the gene expression and protein abnormalities in AD. In this study, the results of differential gene expression in 2, 6, 12-month-old SAMP8 with SAMR1 showed more and more differential expression genes in the period of ageing and hint these genes were associated with cognitive decline and accelerated senescence, especially genes of signal transduction, protein and energy metabolism. These genes maybe provide significant clues for aging and cognitive impairment, meanwhile provide important base for study of potential targets for AD therapy.
1.2 Gene expression patterns of hippocampus and cortex of SAMP8 treated with LW
Liu-Wei-Di-Huang decoction (LW), a traditional Chinese medicinal prescription, hasmany clinical pharmacological actions including enhancing the cognitive function of CNS. Recently, pharmacological studies of our lab demonstrate that the major effect of LW was improvement of cognition and memory of SAMP8, according to research of neuroendocrine immunological regulation (NIM). In order to get the message of mechanism of LW enhancing the cognitive function and discover target for therapy, cDNA microarray was adopted.
The results showed there were 11 differential expression clones in hippocampus and 67 differential expression clones in cortex of SAMP8, after treated with LW (5g/kg). These clones were sequenced and 53 clones were sequenced successfully. They represented 28 genes with diversified biological function. The results showed there were 43 differential expression clones in hippocampus and 222 differential expression clones in cortex of SAMP8, after treated with LW (10g/kg). These clones were sequenced and 137 clones were sequenced successfully. They represented 60 genes with diversified biological function. The results showed there were 80 differential expression clones in hippocampus and 233 differential expression clones in cortex of SAMP8, after treated with LW (15g/kg). These clones were sequenced and 197 clones were sequenced successfully. They represented 55 genes with diversified biological function. After the above results of differential expression genes were analyzed by bioinformatics these genes treated with different dosage of LW represented similar biological function, including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, regulation of transcription, protein metabolism, nervous system development, energy metabolism, cell growth and maintenance, immune response, electron transport and many genes whose biological function and process were still unknown.
These results demonstrated there was similar functional category of differential expression genes. But there were more expression change genes in group medium and high dosage than group of low dosage. Gene Ttc3 Oxr1 Slc17a7 Rps6ka1 C1qb Nsf Mast2 Rock1 Dusp12 Tac2 Def8 Mink1. Prr6, Acrbp 300002F03 AL845475.12 expression changed significantly. It hints the effect of LW about learning and memory maybe is related to above genes.
1.3 Gene expression patterns of hippocampus and cortex of SAMP8 treated with BW and HL
BW and HL, traditional Chinese medicinal prescription related to invigorating kidney-YANG and purging the fire and detoxifying respectively, have many clinical pharmacological actions including enhancing the cognitive function of CNS. Recently, pharmacological studies demonstrate that the effect of improvement of cognition and memory was one of their diversified effects. In order to get the message of mechanism of BW and HL enhancing the cognitive function and discover target for therapy, cDNA microarray and 6-month-old SAMP8 were adopted and real time PCR was performed to validate the results from microarray. We analyzed and compared with the different expression patterns of hippocampus and cortex treated with LW, BW and HL.
The results showed there were 42 differential expression clones including 21 up-regulated clones and 21 down-regulated clones in hippocampus and 14 differential expression clones including 9 up-regulated and 5 down-regulated in cortex of SAMP8, after treated with BW (10.2g/kg). These clones were sequenced and 39 clones were sequenced successfully. They represented 14 genes with diversified biological function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, protein metabolism, cell growth and maintenance, and 5 genes whose biological function and process were still unknown.
The results showed there were 143 differential expression clones including 29 up-regulated clones and 114 down-regulated clones in hippocampus and 93 differential expression clones including 9 up-regulated and 84 down-regulated in cortex of SAMP8, after treated with HL (5g/kg). These clones were sequenced and 168 clones were sequenced successfully. They represented 36 genes with diversified biological function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, protein metabolism, energy metabolism, cell growth and maintenance, regulation of transcription, and nerve system development and 25 genes whose biological function and process were still unknown.
In order to analyze the patterns of gene expression treated with different Chinese medicine prescription, we investigated the differential gene expression treatment with LW meanwhile. The results showed there were 44 differential expression clones including 40 up-regulated clones and 4 down-regulated clones in hippocampus and 27 differential expression clones including 7 up-regulated and 20 down-regulated in cortex of SAMP8, after treated with LW (10g/kg). These clones were sequenced and 54 clones were sequenced successfully. They represented 12 genes with diversified biological function including signal transduction, nucleic acid metabolism, ribosome biogenesis and assembly, transport, protein metabolism, energy metabolism, cell growth and maintenance, regulation of transcription, and nerve system development and 8 genes whose biological function and process were still unknown.
Comprehending the above screening results of HL, BW and LW, we detected that the three Chinese medicine prescriptions possessed significant ability of regulation of gene expression, including common effective genes to all medicine and specific effective genes to specific medicine. The common effective genes were eight, including nucleic acid metabolism gene Fhit and Itm2c, protein metabolism gene Ube2d2, transport gene Slc17a7, signal transduction gene Rps6kal, ribosome biogenesis and assembly gene Rn18s and 18srRNA, functional unknown gene Rps19bp1. These common genes hint there is some similarity with the three Chinese medicine prescriptions.
2. The propose and primary study of combinatorial candidate gene targets for AD therapy
2.1 The propose of combinatorial candidate gene targets for AD therapy
From the aggregate analysis the patterns of gene expression change in hippocampus and cortex in the period of aging and gene expression change in hippocampus and cortex treated with Chinese medicine prescription LW, BW and HL, there were 60 differential expression genes with clear biological function. Among the 60 genes with different function, signal transduction, nucleic acid metabolism, protein metabolism, energy metabolism was the major portion in ratio. This demonstrated these categories of biological function were mutually related to the occurrence and development of AD, pathway of drug action and drug targets. This is worth to further study. In addition, 45 genes with function unknown were interrelated to the occurrence and development of AD and possessed more significance of neurobiology. These results provided extensive prospect of research.
After comparing and analyzing the differential expression genes that always in the period of aging, that treated with two or three Chinese medicine prescription of LW, BW, HL or that in a specific month-old and treated with one Chinese medicine prescription simultaneously, and combining the bioinformatics and references, we posed the combinatorial candidate gene targets for AD therapy, namely (1) nucleic acid metabolism gene, including Itm2c, Fhit; (2) signal transduction gene, including Rps6ka1, Rock1, Dusp12, Mink1, Rab26, Mast2, Camk2α, Ephb6, S100a11, Tac2, Clstn1, Strn4; (3) protein metabolism gene, including Amfr, Ube2d2, Ttc3, Prr6; (4) transporter gene, including Slcl7a7; (5) regulation of transcript gene, including D1Ertd161e, Ssu72; (6) energy metabolism gene, including Stub1, Uqcr, Ranbp5, Nsf; (7) cell growth and maintenance gene, including Ngrn; (8) nerve system development gene, including Trim3; (9) electron transport gene, including Ndufs2; (10) Neurogila cell differentiation gene, including Tspan2; (11) function unknown gene, including Rps19bp1, 300002F03.
2.2 Primary study of combinatorial candidate gene targets
To validate the above combinatorial candidate gene targets, we applied the real time PCR to assess the patterns of gene expression in the period of aging and treated with LW, BW, HL, DSS, TXF, HupA that benefited to AD therapy.
The results showed there were 14 types of change of the 32 genes expression in the hippocampus of SAMP8 in the period of aging. And there were 13 types of change of the expression 31 genes in the cortex of SAMP8 in the period of aging, except for Ndufs2. After treated with Hup A, LW, BW, HL, DSS, TXF, there were all change of 32 genes expression in the hippocampus and cortex of SAMP8. In hippocampus, these changes of gene expression were defined 6-7 types. In cortex, these changes of gene expression were defined 5-7 types. After treated with 5 Chinese medicine prescriptions and HupA, gene Trim3, Tspan2, Ttc3, Rock1, Ube2d2, Mast2, Clstn1 in hippocampus all presented gene expression change, but gene Amfr, C1qb, Dusp12, Ephb6, Itm2c, Mink1, Tac2, Ndufs2, Ngrn, Nsf, Ranbp5, S100all, Fhit, Prr6, Rab26, 300002F03, Slc17a7 in hippocampus or cortex presented expression change for one or one more Chinese medicine prescriptions. Only gene Ssu72, Rpsl9bpl, Rps6kal in hippocampus presented no gene expression change.
Through the primary validation of 32 genes, the expression of them presented regularity. This hint these genes were interrelated to the occurrence and development of AD. The similarity and difference of 32 genes expression demonstrated the characters of effects of Chinese medicine and HupA.
The discovery and validation of drug targets is an arduous work, by no means achieve the goal through one paper or one research. But we believe the "combinatorial targets" is a helpful attempt. The results from this paper only provided clues and more complicated and hard works need our endeavor.
引文
[1] Roberson ED, Mucke L. 100 years and counting: prospects for defeating Alzheimer's disease. Science 2006; 314 (5800): 781-4.
[2] Clark R, Goate A. Molecular genetics of Alzheimer's disease. Arch. Neurol. 1993; 50: 1164-72.
[3] Goedert M, Spillantini MG. A century of Alzheimer's disease. Science 2006; 314(5800): 777-81.
[4] Palop JJ, Chin J, Mucke L. A network dysfunction perspective on neurodegenerative diseases. Nature 2006; 443(7113): 768-73.
[5] Pericak-Vance MA, Haines JL. Genetic susceptibility to Alzheimer-disease. Trends Genet 1995; 11: 504-8.
[6] Martinez A, Castro A. Novel cholinesterase inhibitors as future effective drugs for the treatment of Alzheimer's disease. Expert Opin Investig Drugs 2006; 15(1): 1-12.
[7] Zoladz PR, Campbell AM, Park CR, Schaefer D, Danysz W, Diamond DM. Enhancement of long-term spatial memory in adult rats by the noncompetitive NMDA receptor antagonists, memantine and neramexane. Pharmacol Biochem Behav 2006; 85(2): 298-306.
[8] Tucker S, Ah1 M, Cho HH, Bandyopadhyay S, Cuny GD, Bush AI, Goldstein LE, Westaway D, Huang X, Rogers JT. RNA therapeutics directed to the non coding regions of APP mRNA, in vivo anti-amyloid efficacy of paroxetine, erythromycin, and N-acetyl cysteine. Curr Alzheimer Res 2006; 3(3): 221-7.
[9] van Muiswinkel FL, Kuiperij HB. The Nrf2-ARE Signalling pathway: promising drug target to combat oxidative stress in neurodegenerative disorders. Curr Drug Targets CNS Neurol Disord 2005; 4(3): 267-81.
[10] Kuipers SD, Bramham CR. Brain-derived neurotrophic factor mechanisms and function in adult synaptic plasticity: new insights and implications for therapy. Curr Opin Drug Discov Devel 2006; 9(5): 580-6.
[11] Youdim MB, Buccafusco JJ. CNS Targets for multi-functional drugs in the treatment of Alzheimer's and Parkinson's diseases. J Neural Transm 2005; 112(4): 519-37.
[12] Buccafusco JJ, Powers JC, Hernandez MA, Prendergast MA, Terry AV, Jonnala RR. MHP-133, a Drug with Multiple CNS Targets: Potential for Neuroprotection and Enhanced Cognition. Neurochem Res 2007. in press
[13] Anekonda TS, Reddy PH. Can herbs provide a new generation of drugs for treating Alzheimer's disease? Brain Res Brain Res Rev 2005; 50(2): 361-76.
[14] Yang S, Zhou W, Zhang Y, Yan C, Zhao Y. Effects of Liuwei Dihuang decoction on ion channels and synaptic transmission in cultured hippocampal neuron of rat. J Ethnopharmacol 2006; 106(2): 166-72.
[15] Kawashima K, Nagasawa H, Kogure K. Effect of Oren-gedoku-to on cerebral vascular accident. Pharma Medica 1998; 6(Suppl. 2): 33-7.
[16] Xu J, Murakami Y, Matsumoto K, Tohda M, Watanabe H, Zhang S, Yu Q, Shen J. Protective effect of Oren-gedoku-to(Huang-Lian-Jie-Du-Tang) against impairment of learning and memory induced by transient cerebral ischemia in mice. J Ethnopharmacol 2000; 73(3): 405-13.
[17] Kou J, Zhu D, Yan Y. Neuroprotective effects of the aqueous extract of the Chinese medicine Danggui-Shaoyao-san on aged mice. J Ethnopharmacol 2005; 97(2): 313-8.
[18] Shang WW, Qiao SY. [Advances on the study of Danggui Shaoyao powder]. Zhongguo Zhong Yao Za Zhi 2006;31 (8):630-3.
[19] Qiao HF, Yang S, Zhou WX, Zhang YX. NT-1, an active constituent extracted from Tiaoxin Recipe, enhances long-term potentiation of CAI subfield in rat hippocampal slices. Life Sci 2006;79(1):8-15.
[20] Morley J.E. BWA, Kumar V.B., Farr S.A.. The SAMP8 mouse as a model for Alzeimer disease: studies from Saint Louis University., 2004; 1260:23-38
[21] Butterfield DA, Poon HF. The senescence-accelerated prone mouse (SAMP8): a model of age-related cognitive decline with relevance to alterations of the gene expression and protein abnormalities in Alzheimer's disease. Exp Gerontol 2005;40 (10):774-83.
[22] Petursdottir AL, Farr SA, Morley JE, Banks WA, Skuladottir GV. Lipid peroxidation in brain during aging in the senescence-accelerated mouse (SAM). Neurobiol Aging 2006;27:330-8
[23] Kumar VB, Farr SA, Flood JF, Kamlesh V, Franko M, Banks WA, Morley JE. Site-directed antisense oligonucleotide decreases the expression of amyloid precursor protein and reverses deficits in learning and memory in aged SAMP8 mice. Peptides 2000;21 (12): 1769-75.
[24] Wu Y, Zhang AQ, Yew DT. Age related changes of various markers of astrocytes in senescence-accelerated mice hippocampus. Neurochem Int 2005 ;46 (7):565-74.
[25] Poon HF, Farr SA, Thongboonkerd V, Lynn BC, Banks WA, Morley JE, Klein JB, Butterfield DA. Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders. Neurochem Int 2005 ;46 (2): 159-68.
[26] Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D. Light-directed, spatially addressable parallel chemical synthesis. Science 1991;251 (4995):767-73.
[27] Grunblatt E. The benefits of microarrays as tools for studying neuropsychiatric disorders. Drugs Today (Barc) 2004;40 (2): 147-56.
[28] Glazier AM, Nadeau JH, Aitman TJ. Finding genes that underlie complex traits. Science 2002;298 (5602):2345-9.
[29] Ho L, Sharma N, Blackman L, Festa E, Reddy G, Pasinetti GM. From proteomics to biomarker discovery in Alzheimer's disease. Brain Res Brain Res Rev 2005 ;48 (2):360-9.
[30] Holbach LM, von Moller A, Decker C, Junemann AGM, Rummelt-Hofmann C, Ballhausen WG Loss of fragile histidine triad (FHIT) expression and microsatellite instability in periocular sebaceous gland carcinoma in patients with Muir-Torre syndrome. Am. J. Ophthal 2002; 134:147-8.
[31] Pekarsky Y, Garrison PN, Palamarchuk A, Zanesi N, Aqeilan RI, Huebner K, Barnes LD, Croce CM. Fhit is a physiological target of the protein kinase Src. Proc Natl Acad Sci U S A 2004;101 (11):3775-9.
[32] Cleaver JE, Thompson LH, Richardson AS, States JC. A summary of mutations in the UV-sensitive disorders: Xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. Hum. Mutat 1999;14:9-22.
[33] Kresse SH, Berner JM, Meza-Zepeda LA, Gregory SG, Kuo WL, Gray JW, Forus A, Myklebost O. Mapping and characterization of the amplicon near APOA2 in 1q23 in human sarcomas by FISH and array CGH. Mol Cancer 2005;4:39.
[34] Salojin K, Oravecz T. Regulation of innate immunity by MAPK dual-specificity phosphatases: knockout models reveal new tricks of old genes. J Leukoc Biol 2007;81 (4):860-9.
[35] Seki N, Yoshikawa T, Hattori A, Miyajima N, Muramatsu M, Saito T. cDNA cloning of a human RAB26-related gene encoding a Ras-like GTP-binding protein on chromosome 16p13.3 region. J. Hum. Genet 2000;45:309-14.
[36] McCarty N, Paust S, Ikizawa K, Dan I, Li X, Cantor H. Signaling by the kinase MINK is essential in the negative selection of autoreactive thymocytes. Nature Immun 2005;6:65-72.
[37] Chen B, Wang JF, Sun X, Young LT. Regulation of GAP-43 expression by chronic desipramine treatment in rat cultured hippocampal cells. Biol Psychiatry 2003;53 (6):530-7.
[38] Wang JY, Zhen DK, Bianchi DW, Androphy EJ, Chen JJ. Assignment of the gene for ERC-55 (RCN2) to human chromosome band 15q22.33→q24.1 by in situ hybridization. Cytogenet Cell Genet 1997;79(1-2):60-1.
[39] Romagnoli A, Oliverio S, Evangelisti C, Iannicola C, Ippolito G, Piacentini M. Neuroleukin inhibition sensitises neuronal cells to caspase-dependent apoptosis. Biochem Biophys Res Commun 2003;302 (3):448-53.
[40] Tsukahara F, Hattori M, Muraki T. Identification and cloning of a novel cDNA belonging to tetratricopeptide repeat gene family from Down syndrome-critical region 21q22.2. J. Biochem 1996; 120:820-7.
[41] Mirnics K, Middleton F, Marquez A, Lewis D, Levitt P. Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex. Neuron 2000;28:53-67.
[42] Jun W, Hemmings G. The KPNB3 locus is associated with schizophrenia. Neurosci Lett 2004;368:323-6.
[43] Imai Y, Soda M, Hatakeyama S, Akagi T, Hashikawa T, Nakayama K, Takahashi R. CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity. Molec. Cell 2002; 10:55-67.
[44] Chrast R, Verheijen MH, Lemke G. Complement factors in adult peripheral nerve: a potential role in energy metabolism. Neurochem Int 2004;45 (2-3):353-9.
[45] Zeniou M, Ding T, Trivier E, Hanauer A. Expression analysis of RSK gene family members: the RSK2 gene, mutated in Coffin-Lowry syndrome, is prominently expressed in brain structures essential for cognitive function and learning. Hum. Molec. Genet 2002; 11:2929-40.
[46] Cohen M, Zhang C, Shokat K, Taunton J. Structural bioinformatics-based design of selective, irreversible kinase inhibitors. Science 2005;308:1318-21.
[47] Hintsch G, Zurlinden A, Meskenaite V. The Calsyntenins-A Family of Postsynaptic Membrane Proteins with Distinct Neuronal Expression Patterns. Mol and Cell Neurosci 2002;21:393-409.
[48] Malenka R, Lancaster B, Zucker R S. Temporal limits on the rise in postsynaptic calcium required for the induction of long-term potentiation. Neuron 1992;9:121-8.
[49] Louise W, Suzanne B, Edwige M, Michel C, Nabil G. beta-Amyloid protein converting enzyme 1 and brain-specific type II membrane protein BRI3: binding partners processed by furin. Journal of Neurochemistry 2005;92:93-102.
[50] Kirvell SL, Esiri M, Francis PT. Down-regulation of vesicular glutamate transporters precedes cell loss and pathology in Alzheimer's disease. J Neurochem 2006;98 (3):939-50.
[51 ] Fremeau RT, Jr., Kam K, Qureshi T, Johnson J, Copenhagen DR, Storm-Mathisen J, Chaudhry FA, Nicoll RA, Edwards RH. Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites. Science 2004;304 (5678): 1815-9.
[52] Chen B, Mariano J, Tsai YC, Chan AH, Cohen M, Weissman AM. The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. Proc Natl Acad Sci U S A 2006; 103 (2):341-6.
[53] Grunwald IC, Korte M, Wolfer D, Wilkinson GA, Unsicker K, Lipp HP, Bonhoeffer T, Klein R. Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity. Neuron 2001;32 (6): 1027-40.
[54] Boyd AW, Lackmann M. Signals from Eph and ephrin proteins: a developmental tool kit. Sci STKE 2001;2001 (112):RE20.
[55] Keith B, Adelman D, Simon M. Targeted mutation of the murine arylhydrocarbon receptor nuclear translocator 2 (Arnt2) gene reveals partial redundancy with Arnt. Proc. Nat. Acad. Sci. 2001;98:6692-97.
[56] Loeffen J, Elpeleg O, Smeitink J, Smeets R, Stockler-Ipsiroglu S, Mandel H, Sengers R, Trijbels F, van den Heuvel L. Mutations in the complex I NDUFS2 gene of patients with cardiomyopathy and encephalomyopathy. Ann. Neurol 2001;49:195-201.
[57] Berrendero F, Mendizabal V, Robledo P, Galeote L, Bilkei-Gorzo A, Zimmer A, Maldonado R. Nicotine-induced antinociception, rewarding effects, and physical dependence are decreased in mice lacking the preproenkephalin gene. J Neurosci 2005;25:1103-12.
[58] Fukushima N, Sato N, Ueki T, Rosty C, Walter K, Wilentz R, Yeo C, Ralph H, Goggins M. Aberrant Methylation of Preproenkephalin and pl6 Genes in Pancreatic Intraepithelial Neoplasia and Pancreatic Ductal Adenocarcinoma. Am J Pathology 2002; 160:1573-81.
[59] Kolonin M, Saha P, Chan L, Pasqualini R, Arap W. Reversal of obesity by targeted ablation of adipose tissue. Nat Med 2004; 10:625-32.
[60] Otsuka M, Yoshioka K. Neurotransmitter functions of mammalian tachykinins. Physiol Rev 1993;73 (2):229-308.
[61] Drews J. Drug discovery: a historical perspective. Science 2000;287 (5460): 1960-4.
[62] Kanamori T, Takakura K, Mandai M, Kariya M, Fukuhara K, Sakaguchi M, Huh NH, Saito K, Sakurai T, Fujita J, Fujii S. Increased expression of calcium-binding protein S100 in human uterine smooth muscle tumours. Mol Hum Reprod 2004;10 (10):735-42.
[63] Duarte CR, Schutz B, Zimmer A. Incongruent pattern of neurokinin B expression in rat and mouse brains. Cell Tissue Res 2006;323 (1):43-51.
[64] Seki N, Yoshikawa T, Hattori A, Miyajima N, Muramatsu M, Saito T. cDNA cloning of a human RAB26-related gene encoding a Ras-like GTP-binding protein on chromosome 16p13.3 region. J Hum Genet 2000;45 (5):309-14.
[65] Ninan I, Arancio O. Presynaptic CaMKII is necessary for synaptic plasticity in cultured hippocampal neurons. Neuron 2004;42:129-41.
[66] Fukunaga K, Miyamoto E. Current studies on a working model of CaM kinase II in hippocampal long-term potentiation and memory. Jpn J Pharmacol 1999;79 (1):7-15.
[67] Chang BH, Mukherji S, Soderling TR. Calcium/calmodulin-dependent protein kinase II inhibitor protein: localization of isoforms in rat brain. Neuroscience 2001; 102 (4):767-77.
[68] Suenaga T, Morinobu S, Kawano K. Influence of immobilization stress on the levels of CaMKII and phospho-CaMKII in the rat hippocampus. Int J Neuropsychopharmacol 2004;26:1-11.
[69] Lin K, Chang R, Suen K. Modulation of calcium/calmodulin kinase-II provides partial neuroprotection against beta-amyloid peptide toxicity. Eur J Neurosci 2004; 19:2047-55.
[70] Tawara S, Shimokawa H. Progress of the study of rho-kinase and future perspective of the inhibitor. Yakugaku Zasshi 2007; 127 (3):501-14.
[71] Heffron D, Mandell JW. Differential localization of MAPK-activated protein kinases RSK1 and MSK1 in mouse brain. Brain Res Mol Brain Res 2005;136 (1-2):134-41.
[72] Shimamura A, Ballif BA, Richards SA, Blenis J. Rskl mediates a MEK-MAP kinase cell survival signal. Curr Biol 2000;10 (3): 127-35.
[73] Dufresne SD, Bjorbaek C, El-Haschimi K, Zhao Y, Aschenbach WG, Moller DE, Goodyear LJ. Altered extracellular signal-regulated kinase signaling and glycogen metabolism in skeletal muscle from p90 ribosomal S6 kinase 2 knockout mice. Mol Cell Biol 2001 ;21 (1):81-7.
[74] Shimura H, Schwartz D, Gygi SP, Kosik KS. CHIP-Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival. J Biol Chem 2004;279 (6):4869-76.
[75] Gray L, Scarr E, Dean B. N-Ethylmaleimide sensitive factor in the cortex of subjects with schizophrenia and bipolar I disorder. Neurosci Lett 2006;391 (3):112-5.
[76] Park H, Suzuki T, Lennarz WJ. Identification of proteins that interact with mammalian peptide:N-glycanase and implicate this hydrolase in the proteasome-dependent pathway for protein degradation. Proc Natl Acad Sci U S A 2001 ;98 (20): 11163-8.
[77] Shinsuke I, N. J-i, Y. T, Norimasa. Two novel genes, human neugrin and mouse m-neugrin, are upregulated with neuronal differentiation in neuroblastoma cells. Biochem and Biophy Research Comm2000;279:526-33.
[78] Carine G, Chaouki M, Celine F, Jean-Gabriel V, Samia B, Carl M, Gerard F. Kinase Cakl functionally interacts with the PAFl complex and Phosphatase Ssu72 via kinases Ctkl and Burl. Mol Gen Genomics 2006;275:136-47.
[2] Clark R, Goate A. Molecular genetics of Alzheimer's disease. Arch. Neurol. 1993; 50: 1164-72.
[3] Goedert M, Spillantini MG. A century of Alzheimer's disease. Science 2006; 314(5800): 777-81.
[4] Palop JJ, Chin J, Mucke L. A network dysfunction perspective on neurodegenerative diseases. Nature 2006; 443(7113): 768-73.
[5] Pericak-Vance MA, Haines JL. Genetic susceptibility to Alzheimer-disease. Trends Genet 1995; 11: 504-8.
[6] Martinez A, Castro A. Novel cholinesterase inhibitors as future effective drugs for the treatment of Alzheimer's disease. Expert Opin Investig Drugs 2006; 15(1): 1-12.
[7] Zoladz PR, Campbell AM, Park CR, Schaefer D, Danysz W, Diamond DM. Enhancement of long-term spatial memory in adult rats by the noncompetitive NMDA receptor antagonists, memantine and neramexane. Pharmacol Biochem Behav 2006; 85(2): 298-306.
[8] Tucker S, Ah1 M, Cho HH, Bandyopadhyay S, Cuny GD, Bush AI, Goldstein LE, Westaway D, Huang X, Rogers JT. RNA therapeutics directed to the non coding regions of APP mRNA, in vivo anti-amyloid efficacy of paroxetine, erythromycin, and N-acetyl cysteine. Curr Alzheimer Res 2006; 3(3): 221-7.
[9] van Muiswinkel FL, Kuiperij HB. The Nrf2-ARE Signalling pathway: promising drug target to combat oxidative stress in neurodegenerative disorders. Curr Drug Targets CNS Neurol Disord 2005; 4(3): 267-81.
[10] Kuipers SD, Bramham CR. Brain-derived neurotrophic factor mechanisms and function in adult synaptic plasticity: new insights and implications for therapy. Curr Opin Drug Discov Devel 2006; 9(5): 580-6.
[11] Youdim MB, Buccafusco JJ. CNS Targets for multi-functional drugs in the treatment of Alzheimer's and Parkinson's diseases. J Neural Transm 2005; 112(4): 519-37.
[12] Buccafusco JJ, Powers JC, Hernandez MA, Prendergast MA, Terry AV, Jonnala RR. MHP-133, a Drug with Multiple CNS Targets: Potential for Neuroprotection and Enhanced Cognition. Neurochem Res 2007. in press
[13] Anekonda TS, Reddy PH. Can herbs provide a new generation of drugs for treating Alzheimer's disease? Brain Res Brain Res Rev 2005; 50(2): 361-76.
[14] Yang S, Zhou W, Zhang Y, Yan C, Zhao Y. Effects of Liuwei Dihuang decoction on ion channels and synaptic transmission in cultured hippocampal neuron of rat. J Ethnopharmacol 2006; 106(2): 166-72.
[15] Kawashima K, Nagasawa H, Kogure K. Effect of Oren-gedoku-to on cerebral vascular accident. Pharma Medica 1998; 6(Suppl. 2): 33-7.
[16] Xu J, Murakami Y, Matsumoto K, Tohda M, Watanabe H, Zhang S, Yu Q, Shen J. Protective effect of Oren-gedoku-to(Huang-Lian-Jie-Du-Tang) against impairment of learning and memory induced by transient cerebral ischemia in mice. J Ethnopharmacol 2000; 73(3): 405-13.
[17] Kou J, Zhu D, Yan Y. Neuroprotective effects of the aqueous extract of the Chinese medicine Danggui-Shaoyao-san on aged mice. J Ethnopharmacol 2005; 97(2): 313-8.
[18] Shang WW, Qiao SY. [Advances on the study of Danggui Shaoyao powder]. Zhongguo Zhong Yao Za Zhi 2006;31 (8):630-3.
[19] Qiao HF, Yang S, Zhou WX, Zhang YX. NT-1, an active constituent extracted from Tiaoxin Recipe, enhances long-term potentiation of CAI subfield in rat hippocampal slices. Life Sci 2006;79(1):8-15.
[20] Morley J.E. BWA, Kumar V.B., Farr S.A.. The SAMP8 mouse as a model for Alzeimer disease: studies from Saint Louis University., 2004; 1260:23-38
[21] Butterfield DA, Poon HF. The senescence-accelerated prone mouse (SAMP8): a model of age-related cognitive decline with relevance to alterations of the gene expression and protein abnormalities in Alzheimer's disease. Exp Gerontol 2005;40 (10):774-83.
[22] Petursdottir AL, Farr SA, Morley JE, Banks WA, Skuladottir GV. Lipid peroxidation in brain during aging in the senescence-accelerated mouse (SAM). Neurobiol Aging 2006;27:330-8
[23] Kumar VB, Farr SA, Flood JF, Kamlesh V, Franko M, Banks WA, Morley JE. Site-directed antisense oligonucleotide decreases the expression of amyloid precursor protein and reverses deficits in learning and memory in aged SAMP8 mice. Peptides 2000;21 (12): 1769-75.
[24] Wu Y, Zhang AQ, Yew DT. Age related changes of various markers of astrocytes in senescence-accelerated mice hippocampus. Neurochem Int 2005 ;46 (7):565-74.
[25] Poon HF, Farr SA, Thongboonkerd V, Lynn BC, Banks WA, Morley JE, Klein JB, Butterfield DA. Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders. Neurochem Int 2005 ;46 (2): 159-68.
[26] Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D. Light-directed, spatially addressable parallel chemical synthesis. Science 1991;251 (4995):767-73.
[27] Grunblatt E. The benefits of microarrays as tools for studying neuropsychiatric disorders. Drugs Today (Barc) 2004;40 (2): 147-56.
[28] Glazier AM, Nadeau JH, Aitman TJ. Finding genes that underlie complex traits. Science 2002;298 (5602):2345-9.
[29] Ho L, Sharma N, Blackman L, Festa E, Reddy G, Pasinetti GM. From proteomics to biomarker discovery in Alzheimer's disease. Brain Res Brain Res Rev 2005 ;48 (2):360-9.
[30] Holbach LM, von Moller A, Decker C, Junemann AGM, Rummelt-Hofmann C, Ballhausen WG Loss of fragile histidine triad (FHIT) expression and microsatellite instability in periocular sebaceous gland carcinoma in patients with Muir-Torre syndrome. Am. J. Ophthal 2002; 134:147-8.
[31] Pekarsky Y, Garrison PN, Palamarchuk A, Zanesi N, Aqeilan RI, Huebner K, Barnes LD, Croce CM. Fhit is a physiological target of the protein kinase Src. Proc Natl Acad Sci U S A 2004;101 (11):3775-9.
[32] Cleaver JE, Thompson LH, Richardson AS, States JC. A summary of mutations in the UV-sensitive disorders: Xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. Hum. Mutat 1999;14:9-22.
[33] Kresse SH, Berner JM, Meza-Zepeda LA, Gregory SG, Kuo WL, Gray JW, Forus A, Myklebost O. Mapping and characterization of the amplicon near APOA2 in 1q23 in human sarcomas by FISH and array CGH. Mol Cancer 2005;4:39.
[34] Salojin K, Oravecz T. Regulation of innate immunity by MAPK dual-specificity phosphatases: knockout models reveal new tricks of old genes. J Leukoc Biol 2007;81 (4):860-9.
[35] Seki N, Yoshikawa T, Hattori A, Miyajima N, Muramatsu M, Saito T. cDNA cloning of a human RAB26-related gene encoding a Ras-like GTP-binding protein on chromosome 16p13.3 region. J. Hum. Genet 2000;45:309-14.
[36] McCarty N, Paust S, Ikizawa K, Dan I, Li X, Cantor H. Signaling by the kinase MINK is essential in the negative selection of autoreactive thymocytes. Nature Immun 2005;6:65-72.
[37] Chen B, Wang JF, Sun X, Young LT. Regulation of GAP-43 expression by chronic desipramine treatment in rat cultured hippocampal cells. Biol Psychiatry 2003;53 (6):530-7.
[38] Wang JY, Zhen DK, Bianchi DW, Androphy EJ, Chen JJ. Assignment of the gene for ERC-55 (RCN2) to human chromosome band 15q22.33→q24.1 by in situ hybridization. Cytogenet Cell Genet 1997;79(1-2):60-1.
[39] Romagnoli A, Oliverio S, Evangelisti C, Iannicola C, Ippolito G, Piacentini M. Neuroleukin inhibition sensitises neuronal cells to caspase-dependent apoptosis. Biochem Biophys Res Commun 2003;302 (3):448-53.
[40] Tsukahara F, Hattori M, Muraki T. Identification and cloning of a novel cDNA belonging to tetratricopeptide repeat gene family from Down syndrome-critical region 21q22.2. J. Biochem 1996; 120:820-7.
[41] Mirnics K, Middleton F, Marquez A, Lewis D, Levitt P. Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex. Neuron 2000;28:53-67.
[42] Jun W, Hemmings G. The KPNB3 locus is associated with schizophrenia. Neurosci Lett 2004;368:323-6.
[43] Imai Y, Soda M, Hatakeyama S, Akagi T, Hashikawa T, Nakayama K, Takahashi R. CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity. Molec. Cell 2002; 10:55-67.
[44] Chrast R, Verheijen MH, Lemke G. Complement factors in adult peripheral nerve: a potential role in energy metabolism. Neurochem Int 2004;45 (2-3):353-9.
[45] Zeniou M, Ding T, Trivier E, Hanauer A. Expression analysis of RSK gene family members: the RSK2 gene, mutated in Coffin-Lowry syndrome, is prominently expressed in brain structures essential for cognitive function and learning. Hum. Molec. Genet 2002; 11:2929-40.
[46] Cohen M, Zhang C, Shokat K, Taunton J. Structural bioinformatics-based design of selective, irreversible kinase inhibitors. Science 2005;308:1318-21.
[47] Hintsch G, Zurlinden A, Meskenaite V. The Calsyntenins-A Family of Postsynaptic Membrane Proteins with Distinct Neuronal Expression Patterns. Mol and Cell Neurosci 2002;21:393-409.
[48] Malenka R, Lancaster B, Zucker R S. Temporal limits on the rise in postsynaptic calcium required for the induction of long-term potentiation. Neuron 1992;9:121-8.
[49] Louise W, Suzanne B, Edwige M, Michel C, Nabil G. beta-Amyloid protein converting enzyme 1 and brain-specific type II membrane protein BRI3: binding partners processed by furin. Journal of Neurochemistry 2005;92:93-102.
[50] Kirvell SL, Esiri M, Francis PT. Down-regulation of vesicular glutamate transporters precedes cell loss and pathology in Alzheimer's disease. J Neurochem 2006;98 (3):939-50.
[51 ] Fremeau RT, Jr., Kam K, Qureshi T, Johnson J, Copenhagen DR, Storm-Mathisen J, Chaudhry FA, Nicoll RA, Edwards RH. Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites. Science 2004;304 (5678): 1815-9.
[52] Chen B, Mariano J, Tsai YC, Chan AH, Cohen M, Weissman AM. The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. Proc Natl Acad Sci U S A 2006; 103 (2):341-6.
[53] Grunwald IC, Korte M, Wolfer D, Wilkinson GA, Unsicker K, Lipp HP, Bonhoeffer T, Klein R. Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity. Neuron 2001;32 (6): 1027-40.
[54] Boyd AW, Lackmann M. Signals from Eph and ephrin proteins: a developmental tool kit. Sci STKE 2001;2001 (112):RE20.
[55] Keith B, Adelman D, Simon M. Targeted mutation of the murine arylhydrocarbon receptor nuclear translocator 2 (Arnt2) gene reveals partial redundancy with Arnt. Proc. Nat. Acad. Sci. 2001;98:6692-97.
[56] Loeffen J, Elpeleg O, Smeitink J, Smeets R, Stockler-Ipsiroglu S, Mandel H, Sengers R, Trijbels F, van den Heuvel L. Mutations in the complex I NDUFS2 gene of patients with cardiomyopathy and encephalomyopathy. Ann. Neurol 2001;49:195-201.
[57] Berrendero F, Mendizabal V, Robledo P, Galeote L, Bilkei-Gorzo A, Zimmer A, Maldonado R. Nicotine-induced antinociception, rewarding effects, and physical dependence are decreased in mice lacking the preproenkephalin gene. J Neurosci 2005;25:1103-12.
[58] Fukushima N, Sato N, Ueki T, Rosty C, Walter K, Wilentz R, Yeo C, Ralph H, Goggins M. Aberrant Methylation of Preproenkephalin and pl6 Genes in Pancreatic Intraepithelial Neoplasia and Pancreatic Ductal Adenocarcinoma. Am J Pathology 2002; 160:1573-81.
[59] Kolonin M, Saha P, Chan L, Pasqualini R, Arap W. Reversal of obesity by targeted ablation of adipose tissue. Nat Med 2004; 10:625-32.
[60] Otsuka M, Yoshioka K. Neurotransmitter functions of mammalian tachykinins. Physiol Rev 1993;73 (2):229-308.
[61] Drews J. Drug discovery: a historical perspective. Science 2000;287 (5460): 1960-4.
[62] Kanamori T, Takakura K, Mandai M, Kariya M, Fukuhara K, Sakaguchi M, Huh NH, Saito K, Sakurai T, Fujita J, Fujii S. Increased expression of calcium-binding protein S100 in human uterine smooth muscle tumours. Mol Hum Reprod 2004;10 (10):735-42.
[63] Duarte CR, Schutz B, Zimmer A. Incongruent pattern of neurokinin B expression in rat and mouse brains. Cell Tissue Res 2006;323 (1):43-51.
[64] Seki N, Yoshikawa T, Hattori A, Miyajima N, Muramatsu M, Saito T. cDNA cloning of a human RAB26-related gene encoding a Ras-like GTP-binding protein on chromosome 16p13.3 region. J Hum Genet 2000;45 (5):309-14.
[65] Ninan I, Arancio O. Presynaptic CaMKII is necessary for synaptic plasticity in cultured hippocampal neurons. Neuron 2004;42:129-41.
[66] Fukunaga K, Miyamoto E. Current studies on a working model of CaM kinase II in hippocampal long-term potentiation and memory. Jpn J Pharmacol 1999;79 (1):7-15.
[67] Chang BH, Mukherji S, Soderling TR. Calcium/calmodulin-dependent protein kinase II inhibitor protein: localization of isoforms in rat brain. Neuroscience 2001; 102 (4):767-77.
[68] Suenaga T, Morinobu S, Kawano K. Influence of immobilization stress on the levels of CaMKII and phospho-CaMKII in the rat hippocampus. Int J Neuropsychopharmacol 2004;26:1-11.
[69] Lin K, Chang R, Suen K. Modulation of calcium/calmodulin kinase-II provides partial neuroprotection against beta-amyloid peptide toxicity. Eur J Neurosci 2004; 19:2047-55.
[70] Tawara S, Shimokawa H. Progress of the study of rho-kinase and future perspective of the inhibitor. Yakugaku Zasshi 2007; 127 (3):501-14.
[71] Heffron D, Mandell JW. Differential localization of MAPK-activated protein kinases RSK1 and MSK1 in mouse brain. Brain Res Mol Brain Res 2005;136 (1-2):134-41.
[72] Shimamura A, Ballif BA, Richards SA, Blenis J. Rskl mediates a MEK-MAP kinase cell survival signal. Curr Biol 2000;10 (3): 127-35.
[73] Dufresne SD, Bjorbaek C, El-Haschimi K, Zhao Y, Aschenbach WG, Moller DE, Goodyear LJ. Altered extracellular signal-regulated kinase signaling and glycogen metabolism in skeletal muscle from p90 ribosomal S6 kinase 2 knockout mice. Mol Cell Biol 2001 ;21 (1):81-7.
[74] Shimura H, Schwartz D, Gygi SP, Kosik KS. CHIP-Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival. J Biol Chem 2004;279 (6):4869-76.
[75] Gray L, Scarr E, Dean B. N-Ethylmaleimide sensitive factor in the cortex of subjects with schizophrenia and bipolar I disorder. Neurosci Lett 2006;391 (3):112-5.
[76] Park H, Suzuki T, Lennarz WJ. Identification of proteins that interact with mammalian peptide:N-glycanase and implicate this hydrolase in the proteasome-dependent pathway for protein degradation. Proc Natl Acad Sci U S A 2001 ;98 (20): 11163-8.
[77] Shinsuke I, N. J-i, Y. T, Norimasa. Two novel genes, human neugrin and mouse m-neugrin, are upregulated with neuronal differentiation in neuroblastoma cells. Biochem and Biophy Research Comm2000;279:526-33.
[78] Carine G, Chaouki M, Celine F, Jean-Gabriel V, Samia B, Carl M, Gerard F. Kinase Cakl functionally interacts with the PAFl complex and Phosphatase Ssu72 via kinases Ctkl and Burl. Mol Gen Genomics 2006;275:136-47.