Regulation krankheitsassoziierter Signalwege durch differenziell exprimierte miRNAs im Parkinson-Zellkulturmodell

Morbus Parkinson stellt eine schwerwiegende neurodegenerative Erkrankung dar, deren Kom-plexität sowohl die Diagnosestellung als auch die Therapie bis heute erschwert. Im letzten Jahrzehnt wurden unter anderem deregulierte microRNAs (miRNAs, miRs) als vielversprechende Kandidaten für neue Diagnose- und Therapiemöglichkeiten identifiziert (Condrat et al., 2020; Hanna et al., 2019). Ziel dieser Arbeit war es daher die miRNA-Signatur in erkrankten dopaminergen Neuronen anhand eines etablierten Parkinson-Zellkulturmodells genauer zu untersuchen sowie regulatorische Zielgennetzwerke differenziell exprimierter miRNAs zu identifizieren, die zur Progression der Erkrankung beitragen könnten. Zunächst erfolgte eine Charakterisierung des zellulären Parkinson-ähnlichen Phänotyps anhand einer Transkriptomanalyse sowie anschließender Signalweganalyse, in der die Deregulation der zentralen molekularen Pathomechanismen von Morbus Parkinson im Parkinson-Zellkulturmodell auf molekularer Ebene abgebildet werden konnte. Die nachfolgende miRnom-Analyse identifizierte dreizehn miRNAs mit signifikanter Expressionsveränderung nach Induktion des Parkinson-ähnlichen Phänotyps, darunter die induzierte miR-34a-5p und reprimierte miR-7-5p. Aufgrund dieser Ergebnisse sowie weiterer Studien, die einen signifikanten Einfluss der miR-34a-5p und miR-7-5p auf die Viabilität von dopaminergen Neuronen nachweisen, wurde im weiteren Verlauf dieser Arbeit der Fokus auf diese zentralen deregulierten miRNAs gelegt, um deren Zielgennetzwerke, die zur Progression von Morbus Parkinson beitragen, umfassend zu entschlüsseln. Durch eine kombinierte in silico Zielgenvorhersage mit anschließender Anreicherungsanalyse wurden insgesamt 112 Zielgensequenzen mit miR-34a-5p-Bindestellen und 160 Zielgensequenzen mit miR-7-5p-Bindestellen aus 14 Parkinson- und Dopamin-assoziierten Signalwegen zur experimentellen Validierung ausgewählt. Mittels Hochdurchsatz-miRNA-Interaktions-Reporterassay konnte eine miRNA-Zielgeninteraktion für 73,2 % der 112 Zielgensequenzen der miR-34a-5p und 51,9 % der 160 Zielgensequenzen der miR-7-5p verifiziert werden. In der nachfolgenden Analyse des Effektes der Länge und Anzahl von miRNA-Bindestellen auf die miRNA-bedingte Regulation konnte eine verstärkte Regulation mit einer erhöhten Anzahl potenziell bindender Nukleotide erfasst werden. Die Validierung der Ergebnisse des Hochdurchsatz-miRNA-Interaktions-Reporterassay mit mutierten Reporterkonstrukten konnte 90 % der zuvor erfassten Zielgeninteraktionen der miR-34a-5p und 60 % der Zielgeninteraktionen der miR 7 5p bestätigen. Zudem konnte für ausgewählte Zielgene die zuvor mittels Hochdurchsatz-miRNA-Interaktions-Reporterassay erfasste miRNA-bedingte Regulation ebenso auf Proteinebene verifiziert werden. Die in dieser Arbeit erfassten miRNA-Zielgeninteraktionen bilden die Grundlage zur Entschlüsselung der zentralen miRNA-regulierten Pathomechanismen von Morbus Parkinson und könnten in Zukunft zur spezifischen Modulation krankheitsassoziierter Signalwege genutzt werden.Parkinson’s Disease is a severe neurodegenerative disorder whose complexity impedes diagnosis as well as therapeutic approaches until now. In the last decade, microRNAs (miRNAs, miRs) were identified as promising candidates for new diagnostic and therapeutic approaches (Condrat et al., 2020; Hanna et al., 2019). Hence, the aim of this thesis was to examine the miRNA signature in diseased dopaminergic neurons based on a well-established Parkinson’s disease cell culture model as well as to identify gene regulatory networks that could contribute to the progression of the disease. First, transcriptome analysis and subsequent pathway analysis was performed to characterize the cellular Parkinson’s disease like phenotype. In this analysis, deregulation of the central pathogenic mechanisms of Parkinson’s disease was shown on molecular level. The following miRnome analysis identified thirteen miRNAs that showed a significant expression change upon induction of Parkinson’s disease like phenotype including the upregulated miR-34a-5p and the downregulated miR 7 5p. Based on these results as well as other studies that showed a significant impact of miR-34a-5p and miR-7-5p on the viability of dopaminergic neurons, these central miRNAs were focused in the following to decipher the gene regulatory networks that contribute to the progression of Parkinson’s disease. The combination of in silico target gene prediction with subsequent enrichment analysis led to the identification of 112 target sequences with miR 34a 5p binding sites and 160 target sequences with miR-7-5p binding sites in 14 signaling pathways associated with Parkinson’s disease and dopamine. High throughput miRNA interaction reporter assay verified miRNA targeting for 73,2 % of the 112 target sequences for miR-34-5p as well as for 51,9 % of the 160 target sequences for miR-7-5p. Further examination on the effects of length and number of miRNA binding sites on miRNA-mediated regulation revealed an augmented regulation with increasing number of potentially binding nucleotides. Validation of the results obtained from the high throughput miRNA interaction reporter assay with mutated reporter constructs verified 90 % of the miRNA-target interactions for miR-34a 5p and 60 % for miR-7-5p. The miRNA-mediated regulation was also verified for selected target genes on protein level. The miRNA-target-interactions detected in this study provide a basis for deciphering the central miRNA-regulated pathogenic mechanism of Parkinson’s disease and could be used in the future for specific modulation of disease-associated signaling pathways

miR-34a-5p as molecular hub of pathomechanisms in Huntington's disease

Background Although a pivotal role of microRNA (miRNA, miR) in the pathogenesis of Huntington’s disease (HD) is increasingly recognized, the molecular functions of miRNAs in the pathomechanisms of HD await further elucidation. One of the miRNAs that have been associated with HD is miR-34a-5p, which was deregulated in the mouse R6/2 model and in human HD brain tissues. Methods The aim of our study was to demonstrate interactions between miR-34a-5p and HD associated genes. By computational means we predicted 12 801 potential target genes of miR-34a-5p. An in-silico pathway analysis revealed 22 potential miR-34a-5p target genes in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway “Huntington’s disease”. Results Using our high-throughput miRNA interaction reporter assay (HiTmIR) we identifed NDUFA9, TAF4B, NRF1, POLR2J2, DNALI1, HIP1, TGM2 and POLR2G as direct miR-34a-5p target genes. Direct binding of miR-34a-5p to target sites in the 3’UTRs of TAF4B, NDUFA9, HIP1 and NRF1 was verifed by a mutagenesis HiTmIR assay and by determining endogenous protein levels for HIP1 and NDUFA9. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis identifed protein–protein interaction networks associated with HD like “Glutamine Receptor Signaling Pathway” and “Calcium Ion Transmembrane Import Into Cytosol”. Conclusion Our study demonstrates multiple interactions between miR-34a-5p and HD associated target genes and thereby lays the ground for future therapeutic interventions using this miRNA

Induction of the Endoplasmic-Reticulum-Stress Response: MicroRNA-34a Targeting of the IRE1α-Branch

Neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and the unfolded protein response (UPR). Modulating the UPR is one of the major challenges to counteract the development of neurodegenerative disorders and other diseases with affected UPR. Here, we show that miR-34a-5p directly targets the IRE1α branch of the UPR, including the genes BIP, IRE1α, and XBP1. Upon induction of ER stress in neuronal cells, miR-34a-5p overexpression impacts the resulting UPR via a significant reduction in IRE1α and XBP1s that in turn leads to decreased viability, increased cytotoxicity and caspase activity. The possibility to modify the UPR signaling pathway by a single miRNA that targets central genes of the IRE1α branch offers new perspectives for future therapeutic approaches against neurodegeneration

miR-34a as hub of T cell regulation networks

Background: Micro(mi)RNAs are increasingly recognized as central regulators of immune cell function. While it has been predicted that miRNAs have multiple targets, the majority of these predictions still await experimental confirmation. Here, miR-34a, a well-known tumor suppressor, is analyzed for targeting genes involved in immune system processes of leucocytes. Methods: Using an in-silico approach, we combined miRNA target prediction with GeneTrail2, a web tool for Multi-omics enrichment analysis, to identify miR-34a target genes, which are involved in the immune system process subcategory of Gene Ontology. Results: Out of the 193 predicted target genes in this subcategory we experimentally tested 22 target genes and confirmed binding of miR-34a to 14 target genes including VAMP2, IKBKE, MYH9, MARCH8, KLRK1, CD11A, TRAFD1, CCR1, PYDC1, PRF1, PIK3R2, PIK3CD, AP1B1, and ADAM10 by dual luciferase assays. By transfecting Jurkat, primary CD4+ and CD8+ T cells with miR-34a, we demonstrated that ectopic expression of miR-34a leads to reduced levels of endogenous VAMP2 and CD11A, which are central to the analyzed subcategories. Functional downstream analysis of miR-34a over-expression in activated CD8+ T cells exhibits a distinct decrease of PRF1 secretion. Conclusions: By simultaneous targeting of 14 mRNAs miR-34a acts as major hub of T cell regulatory networks suggesting to utilize miR-34a as target of intervention towards a modulation of the immune responsiveness of T-cells in a broad tumor context

Wrinkle in the plan: miR-34a-5p impacts chemokine signaling by modulating CXCL10/CXCL11/CXCR3-axis in CD4+, CD8+ T cells, and M1 macrophages

Background In 2016 the first-in-human phase I study of a miRNA-based cancer therapy with a liposomal mimic of microRNA-34a-5p (miR-34a-5p) was closed due to five immune related serious adverse events (SAEs) resulting in four patient deaths. For future applications of miRNA mimics in cancer therapy it is mandatory to unravel the miRNA effects both on the tumor tissue and on immune cells. Here, we set out to analyze the impact of miR-34a-5p over-expression on the CXCL10/CXCL11/CXCR3 axis, which is central for the development of an effective cancer control. Methods We performed a whole genome expression analysis of miR-34a-5p transfected M1 macrophages followed by an over-representation and a protein–protein network analysis. In-silico miRNA target prediction and dual luciferase assays were used for target identification and verification. Target genes involved in chemokine signaling were functionally analyzed in M1 macrophages, CD4+ and CD8+ T cells. Results A whole genome expression analysis of M1 macrophages with induced miR-34a-5p over-expression revealed an interaction network of downregulated target mRNAs including CXCL10 and CXCL11. In-silico target prediction in combination with dual luciferase assays identified direct binding of miR-34a-5p to the 3′UTRs of CXCL10 and CXCL11. Decreased CXCL10 and CXCL11 secretion was shown on the endogenous protein level and in the supernatant of miR-34a-5p transfected and activated M1 macrophages. To complete the analysis of the CXCL10/CXCL11/CXCR3 axis, we activated miR-34a-5p transfected CD4+ and CD8+ T cells by PMA/Ionomycin and found reduced levels of endogenous CXCR3 and CXCR3 on the cell surface. Conclusions MiR-34a-5p mimic administered by intravenous administration will likely not only be up-taken by the tumor cells but also by the immune cells. Our results indicate that miR-34a-5p over-expression leads in M1 macrophages to a reduced secretion of CXCL10 and CXCL11 chemokines and in CD4+ and CD8+ T cells to a reduced expression of CXCR3. As a result, less immune cells will be attracted to the tumor site. Furthermore, high levels of miR-34a-5p in naive CD4+ T cells can in turn hinder Th1 cell polarization through the downregulation of CXCR3 leading to a less pronounced activation of cytotoxic T lymphocytes, natural killer, and natural killer T cells and possibly contributing to lymphocytopenia

Quantitative and time-resolved miRNA pattern of early human T cell activation

T cells are central to the immune response against various pathogens and cancer cells. Complex networks of transcriptional and post-transcriptional regulators, including microRNAs (miRNAs), coordinate the T cell activation process. Available miRNA datasets, however, do not sufficiently dissolve the dynamic changes of miRNA controlled networks upon T cell activation. Here, we established a quantitative and time-resolved expression pattern for the entire miRNome over a period of 24 h upon human Tcell activation. Based on our time-resolved datasets, we identified central miRNAs and specified common miRNA expression profiles. We found the most prominent quantitative expression changes for miR155-5p with a range from initially 40 molecules/cell to 1600 molecules/cell upon T-cell activation. We established a comprehensive dynamic regulatory network of both the up- and downstream regulation of miR155. Upstream, we highlight IRF4 and its complexes with SPI1 and BATF as central for the transcriptional regulation of miR-155. Downstream of miR-155-5p, we verified 17 of its target genes by the time-resolved data recorded after T cell activation. Our data provide comprehensive insights into the range of stimulus induced miRNA abundance changes and lay the ground to identify efficient points of intervention for modifying the T cell response

miR-34a-5p as molecular hub of pathomechanisms in Huntington’s disease

Abstract Background Although a pivotal role of microRNA (miRNA, miR) in the pathogenesis of Huntington’s disease (HD) is increasingly recognized, the molecular functions of miRNAs in the pathomechanisms of HD await further elucidation. One of the miRNAs that have been associated with HD is miR-34a-5p, which was deregulated in the mouse R6/2 model and in human HD brain tissues. Methods The aim of our study was to demonstrate interactions between miR-34a-5p and HD associated genes. By computational means we predicted 12 801 potential target genes of miR-34a-5p. An in-silico pathway analysis revealed 22 potential miR-34a-5p target genes in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway “Huntington’s disease”. Results Using our high-throughput miRNA interaction reporter assay (HiTmIR) we identified NDUFA9, TAF4B, NRF1, POLR2J2, DNALI1, HIP1, TGM2 and POLR2G as direct miR-34a-5p target genes. Direct binding of miR-34a-5p to target sites in the 3’UTRs of TAF4B, NDUFA9, HIP1 and NRF1 was verified by a mutagenesis HiTmIR assay and by determining endogenous protein levels for HIP1 and NDUFA9. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis identified protein–protein interaction networks associated with HD like “Glutamine Receptor Signaling Pathway” and “Calcium Ion Transmembrane Import Into Cytosol”. Conclusion Our study demonstrates multiple interactions between miR-34a-5p and HD associated target genes and thereby lays the ground for future therapeutic interventions using this miRNA

Wrinkle in the plan: miR-34a-5p impacts chemokine signaling by modulating CXCL10/CXCL11/CXCR3-axis in CD4+, CD8+ T cells, and M1 macrophages

Background In 2016 the first-in-human phase I study of a miRNA-based cancer therapy with a liposomal mimic of microRNA-34a-5p (miR-34a-5p) was closed due to five immune related serious adverse events (SAEs) resulting in four patient deaths. For future applications of miRNA mimics in cancer therapy it is mandatory to unravel the miRNA effects both on the tumor tissue and on immune cells. Here, we set out to analyze the impact of miR-34a-5p over-expression on the CXCL10/CXCL11/CXCR3 axis, which is central for the development of an effective cancer control.Methods We performed a whole genome expression analysis of miR-34a-5p transfected M1 macrophages followed by an over-representation and a protein–protein network analysis. In-silico miRNA target prediction and dual luciferase assays were used for target identification and verification. Target genes involved in chemokine signaling were functionally analyzed in M1 macrophages, CD4+ and CD8+ T cells.Results A whole genome expression analysis of M1 macrophages with induced miR-34a-5p over-expression revealed an interaction network of downregulated target mRNAs including CXCL10 and CXCL11. In-silico target prediction in combination with dual luciferase assays identified direct binding of miR-34a-5p to the 3′UTRs of CXCL10 and CXCL11. Decreased CXCL10 and CXCL11 secretion was shown on the endogenous protein level and in the supernatant of miR-34a-5p transfected and activated M1 macrophages. To complete the analysis of the CXCL10/CXCL11/CXCR3 axis, we activated miR-34a-5p transfected CD4+ and CD8+ T cells by PMA/Ionomycin and found reduced levels of endogenous CXCR3 and CXCR3 on the cell surface.Conclusions MiR-34a-5p mimic administered by intravenous administration will likely not only be up-taken by the tumor cells but also by the immune cells. Our results indicate that miR-34a-5p over-expression leads in M1 macrophages to a reduced secretion of CXCL10 and CXCL11 chemokines and in CD4+ and CD8+ T cells to a reduced expression of CXCR3. As a result, less immune cells will be attracted to the tumor site. Furthermore, high levels of miR-34a-5p in naive CD4+ T cells can in turn hinder Th1 cell polarization through the downregulation of CXCR3 leading to a less pronounced activation of cytotoxic T lymphocytes, natural killer, and natural killer T cells and possibly contributing to lymphocytopenia

Time-resolved RNA signatures of CD4+ T cells in Parkinson’s disease

Abstract Parkinson’s disease (PD) emerges as a complex, multifactorial disease. While there is increasing evidence that dysregulated T cells play a central role in PD pathogenesis, elucidation of the pathomechanical changes in related signaling is still in its beginnings. We employed time-resolved RNA expression upon the activation of peripheral CD4+ T cells to track and functionally relate changes on cellular signaling in representative cases of patients at different stages of PD. While only few miRNAs showed time-course related expression changes in PD, we identified groups of genes with significantly altered expression for each different time window. Towards a further understanding of the functional consequences, we highlighted pathways with decreased or increased activity in PD, including the most prominent altered IL-17 pathway. Flow cytometric analyses showed not only an increased prevalence of Th17 cells but also a specific subtype of IL-17 producing γδ-T cells, indicating a previously unknown role in PD pathogenesis

Validation of human microRNA target pathways enables evaluation of target prediction tools.

MicroRNAs are regulators of gene expression. A wide-spread, yet not validated, assumption is that the targetome of miRNAs is non-randomly distributed across the transcriptome and that targets share functional pathways. We developed a computational and experimental strategy termed high-throughput miRNA interaction reporter assay (HiTmIR) to facilitate the validation of target pathways. First, targets and target pathways are predicted and prioritized by computational means to increase the specificity and positive predictive value. Second, the novel webtool miRTaH facilitates guided designs of reporter assay constructs at scale. Third, automated and standardized reporter assays are performed. We evaluated HiTmIR using miR-34a-5p, for which TNF- and TGFB-signaling, and Parkinson's Disease (PD)-related categories were identified and repeated the pipeline for miR-7-5p. HiTmIR validated 58.9% of the target genes for miR-34a-5p and 46.7% for miR-7-5p. We confirmed the targeting by measuring the endogenous protein levels of targets in a neuronal cell model. The standardized positive and negative targets are collected in the new miRATBase database, representing a resource for training, or benchmarking new target predictors. Applied to 88 target predictors with different confidence scores, TargetScan 7.2 and miRanda outperformed other tools. Our experiments demonstrate the efficiency of HiTmIR and provide evidence for an orchestrated miRNA-gene targeting