Funktionelle Charakterisierung langer nicht-Protein-kodierender RNAs bei der Alzheimerschen Erkrankung
Zusammenfassung der Projektergebnisse
The project was developed to contribute information to the specific pathogenetic role of long ncRNAs (lncRNAs) in Alzheimer´s disease (AD) and to specify the causal link to neurodegeneration. Based on a custom made array platform (‘humBrainChip’) a genome-wide pattern of lncRNA expression differences was established between AD and control brains. HOTAIR which was amongst those loci that showed a robust association with AD was selected for further studies. Using ChIRP (Chromatin isolation by RNA purification) method, which required laborious methodical optimization, we isolated DNA domains which were associated with HOTAIR in human controls and AD brains and determined their sequence by next generation sequencing (Illumina). Most hits were detected on chromosome eight. Here we identified several homologs of REXO1 gene known as REXO1-like gene loci and comprising REXO1L1, REXO1L2P, REXO1L8, REXO1L9, REXO1L10 and REXO1L11. By PCR we could validate several REXO1L1 sequences in independent ChIRP isolates indicating regular transcription of REXO1L loci in human brain and confirming previous data (accessible on Sestan Lab Human Brain Atlas Microarrays) on translated REXO1L1 proteins in human brains. REXO1L2 and REXO1L8 were also validated in our brain samples. REXO1L1 gene produces a 3′-5 exonuclease (exo) which belongs to the DEDD family, composed of RNase T, RNase D and oligoribonuclease and involved in a wide range of activities including RNA maturation, nuclear mRNA surveillance and decay, and control of HBV and RNA virus infections. We focused our next steps on REXO1L1 because defects in this gene pointed to genomic instability (DSBs) and increased apoptosis, with both processes assumed to participate in neuronal degeneration in AD brains. To further examine interaction of HOTAIR with REXO1L1 sequences we started electrophoretic mobility shift assays (EMSA). Five different HOTAIR RNA fragments covering a broad range of HOTAIR total sequence were selected and several appropriate REXO1L1-DNA targets were chosen from the sequenced data list. While HOTAIR did not show a simple binding to REXO1L1 oligonucleotides in EMSA approaches the addition of proteins which were initially also isolated by ChIRP experiments (using biotinylated anti- HOTAIR-oligonucleotides) resulted in a pronounced shift of the newly built complex in the EMSA gel. The specificity of the binding was proved by administration of excessive non-biotinylated REXO1L1 oligonucleotides which (as expected) reduced the signal intensity of the detected complex. A second REXO1L1 sequence also showed this behavior, while all other REXO1L1 oligonucleotides tested did not. To quantitatively assess the binding strength we used a two channel surface plasmon resonance (SPR) system and reversibly immobilized HOTAIR RNA onto the gold chip surface using a specific short linker sequence. Applying a wide concentration range of different REXO1L1 oligonucleotides we obtained association and dissociation constants and rate constants, which showed a high binding affinity of REXO1L1 domain F4, which was about 100 fold higher (KD =2,32*10^-8 M) than the binding of TP73 (KD =2,58*10^-7 M), another DNA sequence which also was identified as a relevant binding target by sequencing of enriched HOTAIR-ChIRP derived DNA fragments. According to the initial working program we also studied the proteins bound to HOTAIR and isolated by ChIRP approach. To this end a TMT–based mass spectroscopy was performed (nULPC-ESI-MS/MS method) in collaboration with Martin von Bergen’s group at Helmholtz Centre at Leipzig. Data analysis of five controls and five AD cases resulted in a list of significantly enriched proteins (p<0.05) in AD brains which contained cytoskeletal, cell cycle related and synaptic proteins. VSNL1 (Visinin-line protein 1) had already been proposed as a promising marker for AD detection in blood samples and was therefore selected for further examinations. Using chromogenic and Fluorescence-based in situ hybridization we could detect a neuronal HOTAIR expression in temporal cortex of human brain. In AD a slightly more pronounced staining suggest a higher level of HOTAIR supporting our initial data from the custom array approach. Additionally, HOTAIR is also detected in ß-amyloid plaques and seems to mark tangle-like structures in AD neurons. Finally we also observed a co-localization of VSNL1 protein with HOTAIR expression in pyramidal cells allowing a direct interaction between both molecules.
Projektbezogene Publikationen (Auswahl)
- Functional characterization of long non-coding RNAs in Alzheimer's disease. In: Arendt Th, Heiker JT; Magin T, Schaefer M, Schulz-Siegmund M, Thiery J (eds.) 14th Leipzig Research Festival for Life Sciences 2018, p. 155
Krawetzke J, Bernhardt S, Stadler P, Arendt T, Ueberham U
- Amyloid precursor protein is regulated by ncRNAs: implications for Alzheimer´s Disease. In: Thiery J, Arendt Th. Beck-Sickinger AG (eds) 13 th Leipzig Research Festival for Life Sciences 2014, p. 255
Riekena B, Ueberham U, Arendt T
- The Antisense Transcriptome and the Human Brain. J Mol Neurosci. 2016 Jan;58(1):1-15
Mills JD, Chen BJ, Ueberham U, Arendt T, Janitz M.
(Siehe online unter https://doi.org/10.1007/s12031-015-0694-3) - Non-coding transcriptome in brain aging. Aging (Albany NY). 2017 Sep 12;9(9):1943-1944
Arendt T, Ueberham U, Janitz M
(Siehe online unter https://doi.org/10.18632/aging.101290) - RNA sequencing reveals pronounced changes in the noncoding transcriptome of aging synaptosomes. Neurobiol Aging. 2017 Aug;56:67-77
Chen BJ, Ueberham U, Mills JD, Kirazov L, Kirazov E, Knobloch M, Bochmann J, Jendrek R, Takenaka K, Bliim N, Arendt T, Janitz M
(Siehe online unter https://doi.org/10.1016/j.neurobiolaging.2017.04.005) - The emerging role of circular RNAs in transcriptome regulation. Genomics. 2017 Oct;109(5-6):401-407
Huang S, Yang B, Chen BJ, Bliim N, Ueberham U, Arendt T, Janitz M
(Siehe online unter https://doi.org/10.1016/j.ygeno.2017.06.005) - Multiple System Atrophy: Many Lessons from the Transcriptome. Neuroscientist. 2018 Jun;24(3):294-307
Curry-Hyde A, Chen BJ, Ueberham U, Arendt T, Janitz M
(Siehe online unter https://doi.org/10.1177/1073858417723915)
