Functional characterization of long non-coding RNAs in Alzheimer´s disease

该项目将确定长链ncRNAs （lncRNAs）在阿尔茨海默病（AD）中的具体致病作用，并明确其与神经退行性变的联系。为此，我们将鉴定和表征lncrna -靶标相互作用的功能相关，lncrna -靶标相互作用先前在全基因组方法中鉴定为AD和人类对照脑之间的差异表达。在我们的前期工作中，我们将平铺阵列和定制阵列平台（humBrainChip）结合应用于40名患者和对照组的队列中，建立了AD和对照脑之间lncRNA表达差异的全基因组模式。利用细胞周期依赖的lncrna表达数据集进行富集分析和调整，发现了20个与染色质关联和细胞周期调节相关的lncrna，这支持了我们关于神经元分化控制在AD病理中关键参与的概念。在这里，我们将通过以下实验管道重点研究这20个已鉴定的与AD、细胞周期控制和染色质关联相关的lncRNA的功能特征，其结构分为五个目标：(i)将采用以下技术鉴定lncRNA的功能相关DNA和/或RNA靶序列：染色质分离RNA纯化- chirp (Chu et al. 2012; J Vis. Exp.； 3月25日；（61）。（ii） lncRNA调控的DNA位点将结合DNA测序、局部甲基化和表达分析以及对先前获得的ad特异性转录变化数据集的调整（humBrainChip）进行鉴定。（iii） rna结合蛋白（rbp）将通过两个步骤进行鉴定和进一步表征。首先，染色质复合物中附着在lncRNAs上的特定染色质蛋白组分（即polycomb、HDAC、组蛋白、甲基转移酶）的存在将被特异性抗体识别。在第二步，这将是第二个资助期的一部分，一个更系统的蛋白质组学方法将被应用于识别未知的rbp。（iv）涉及lncRNA、DNA和潜在rbp的多组分复合物将进一步通过特异性结合测定（例如EMSA）进行表征。(v) lncrna -靶标相互作用将通过esiRNA技术在功能上表征它们在AD中具有既定病理生理作用的细胞程序中的作用，例如细胞周期和分化控制、突触发生、凋亡、细胞死亡、淀粉样前体蛋白和tau蛋白的表达、加工和翻译后修饰。以下研究问题将得到解答：(1)中枢神经系统中的lncRNA是否专门参与AD病理生理程序的调节，并具有既定的功能，或者lncRNA的AD相关变化是现象性的？(2) lncrna与该功能相关的分子靶点是什么？(3) ad相关lncrna的作用模式是什么，与细胞死亡有何关系？

The project will establish the specific pathogenetic role of long ncRNAs (lncRNAs) in Alzheimer´s di¬sease (AD) and specify the link to neurodegeneration. To this end, we will identify and characterize functionally relevant lncRNA-target interactions of lncRNAs, previously identified in a genome-wide approach to be differentially expressed between AD and human control brain. In our preliminary work, applying a combination of tiling array and custom array platform (humBrainChip) to a cohort of 40 patients and controls we established the genome-wide pattern of lncRNA expression differences between AD and control brain. Enrichement analyses and adjustment with datasets on cell-cycle dependent expression of lncRNAs revelead 20 lncRNAs related to chromatin-association and cell cycle regulation which supports our concept on the critical involvement of neuronal differentiation control in AD pathology. Here, we will focus on functional characterization of these 20 identified lncRNA with established link to AD, cell cycle control and chromatin-association by the following experimental pipeline, structured into five aims: (i) Functionally relevant DNA and/or RNA target-sequences of lncRNAs will be identified, adapting the technique of: Chromatin Isolation by RNA Purification-ChIRP (Chu et al. 2012; J Vis. Exp.; Mar. 25;(61). (ii) DNA loci, regulated by lncRNA will be identified combining DNA-sequencing with local methylation and expression analyses and the adjustment to previously obtained datasets on AD-specific transcriptom changes (humBrainChip). (iii) RNA-binding proteins (RBPs) will be identified and further characterized by two steps. First, the presence of defined chromatin protein components (i.e polycomb, HDAC, histones, methyltransferases) attached to lncRNAs in the chromatin-complex will be identified by specific antibodies. In a second step, which will be part of the second funding period, a more systematic proteomic approach will be applied to identify unknown RBPs. (iv) Multicomponent complexes, involving lncRNA, DNA and potentially RBPs will further be characterized by specific binding assays (e.g. EMSA). (v) lncRNA-target interactions will functionally be characterized by esiRNA technology with respect to their role in cellular programmes with an established pathophysiological role in AD, such as cell-cycle and differentiation control, synaptogenesis, apoptose, cell death, expression, processing and posttranslational modification of amyloid precursor protein and tau protein. The following research questions will be answered: (1) Are lncRNA in the CNS specifically involved in the regulation of pathophysiological programmes with an established function in AD or is the AD-associated change of lncRNAs epiphenomenal? (2) What are the molecular targets of lncRNAs relevant to this function? (3) What is the mode of action of AD-related lncRNAs and how relates this to cell death?