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he project will establish the specific pathogenetic role of long ncRNAs (lncRNAs) in AlzheimerÂ´s diÂsease (AD). To this end, we will identify and characterize functionally relevant target interactions of lncRNAs, previously identified in a genome-wide approach. In our preliminary work, applying a combination of tiling array and custom array platform (â€œhumBrainChipâ€) to a cohort of 40 AD 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.
The experimental pipeline is structured into five aims: (i) Functionally relevant DNA and/or RNA target-sequences of lncRNAs will be identified, adapting the technique of â€œChromatin Isolation by RNA Purification-ChIRPâ€. (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 (â€œhumBrainChipâ€). (iii) RNA-binding proteins (RBPs) will be identified by a proteomic. (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.
The following questions will be answered: (1) Are lncRNA specifically involved in the regulation of pathophysiological programmes with an established role 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?
- Human brain bank
- Morphometry and Stereology (NeurolucidaTM, NeuroExplorerTM, Stereo InvestigatorTM)
- Multi-dye fluorescence microscopy, confocal microscopy, two-photon microscopy
- time-lap-microscopy for cell- and tissue cultures
- In situ hybridization FISH/CISH
- Laser scanning cytometry
- Laser microdissection
- quantitative RT-PCR
- Electromobility shift assays (EMSA)
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