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

点击翻译按钮获取中文摘要

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?