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

该项目将确定长ncRNAs(LncRNAs)在阿尔茨海默病S病(AD)中的具体致病作用，并明确与神经退行性变的联系。为此，我们将识别和表征功能相关的lncRNA-靶相互作用的lncRNAs，以前在全基因组方法中确定在AD和人类对照脑之间差异表达。在我们的初步工作中，应用平铺阵列和定制阵列平台(HumBrainChip)的组合，对40名患者和对照组进行了研究，我们建立了阿尔茨海默病患者和对照组大脑中lncRNA表达差异的全基因组模式。对依赖细胞周期的lncRNAs表达的丰富分析和数据集揭示了20个与染色质结合和细胞周期调控相关的lncRNAs，这支持了我们关于神经元分化控制在AD病理中的关键参与的概念。在这里，我们将重点研究这20个已鉴定的与AD、细胞周期控制和染色质结合有关的lncRNA的功能特征，这些实验流程分为五个目标：(I)将鉴定功能相关的lncRNAs的DNA和/或RNA靶序列，采用以下技术：通过RNA纯化-Chirp分离染色质(Chu等人。2012年；J Vis.实验；3月25日；(61)。(Ii)将结合DNA测序与局部甲基化和表达分析以及对先前获得的AD特定转录变化数据集的调整来确定由lncRNA调控的DNA基因座(人脑芯片)。(3)将通过两个步骤鉴定和进一步鉴定RNA结合蛋白(RBPs)。首先，在染色质复合体中，结合到lncRNA上的明确的染色质蛋白质组分(即多梳、HDAC、组蛋白、甲基转移酶)的存在将被特定的抗体识别。第二步将是第二个资助期的一部分，将采用更系统的蛋白质组学方法来确定未知的限制性商业惯例。(4)涉及LncRNA、DNA和潜在限制性商业惯例的多组分复合体将进一步通过特定的结合分析(例如EMSA)进行表征。(V)esiRNA技术将从功能上表征LncRNA与靶分子的相互作用，以确定它们在细胞程序中的作用，并在AD中发挥既定的病理生理学作用，如细胞周期和分化控制、突触发生、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中具有既定功能的病理生理程序的调节，或者是AD相关的lncRNAs的变化是偶然的？(2)与这一功能相关的lncRNAs的分子靶点是什么？(3)与AD相关的lncRNAs的作用方式是什么，这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?