Browsing by Subject "microRNA"
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(2023)Progressive Multifocal Leukoencephalopathy (PML) is a rare but often fatal central nervous system demyelination disease caused by the reactivation of persistent JC polyomavirus (JCPyV) in immunosuppressed individuals. JCPyV infects oligodendrocytes in the brain, causing lysis of the glial cells, which leads to progressive demyelination and destruction of neurons seen as lesions in the white matter. The cause of JCPyV reactivation and how it reaches the brain are not well understood. MicroRNAs (miRNAs) are short non-coding RNAs which negatively regulate gene expression by marking mRNAs for destruction or by preventing translation. A Single miRNA can have multiple mRNA targets and multiple miRNAs can target the same mRNA, making the miRNA induced gene regulation a complex process affecting multiple different signaling pathways and cellular processes. The focus of the thesis is to study miRNA differential expression of PML patients compared to healthy individuals to find miRNAs and their target genes affected by JCPyV, while showing expertise in the data handling and data analysis of a miRNA sequencing experiment. The study was conducted by collecting miRNA samples from 8 PML patients and two controls and using Next-gen sequencing and the QuickMIRSeq analysis tool to collect miRNA counts for differential expression analysis. The analysis identified twelve miRNAs upregulated in the PML brain and multiple target genes interacting with two or more of the found miRNAs. The miRNAs were found to have connections to JCPyV replication, PML and important cellular processes such as neuroinflammation and BBB integrity.
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(2018)MicroRNAs are ~22 nucleotide long RNA strands which regulate gene expression by binding to the 3’UTRs of messenger RNAs. MicroRNAs are predicted to regulate about a half of all protein-coding genes in the human genome thus affecting many cellular processes. One crucial part of microRNA biogenesis is the cleaving of pre-miRNA strands into mature microRNAs by the type III RNase enzyme, Dicer. Dicer has been shown to be downregulated due to aging and in many disease states. Particularly central nervous system disorders are linked to dysregulated microRNA processing. According to the latest studies, Dicer is crucial to the survival of dopaminergic neurons and conditional Dicer knockout mice show severe nigrostriatal dopaminergic cell loss, which is a hallmark of Parkinson’s disease. By activating Dicer with a small-molecule drug, enoxacin, the survival of dopaminergic cells exposed to stress is significantly improved. However, enoxacin, which is a fluoroquinolone antibiotic, activates Dicer only at high concentrations (10-100 μM) and is polypharmacological, which may cause detrimental side effects. Therefore, enoxacin is not a suitable drug candidate for Dicer deficiencies and better Dicer-activating drug candidates are needed. The aim of this work was to develop a cell-based fluorescent assay to screen for Dicer-activating compounds. Assays which measure Dicer activity have already been developed, but they have some pitfalls which don’t make them optimal to use for high-throughput screening of Dicer-activating compounds. Some are cell-free enzyme-based assays and thus neglect Dicer in its native context. The RNA to be processed by Dicer does not represent a common mammalian RNA type. Most assays do not have internal normalizing factors, such as a second reporter protein to account for e.g. cell death, or the analysis method is not feasible for high-throughput screening data. Considering these disadvantages, the study started by designing a reporter plasmid in silico. The plasmid expresses two fluorescent proteins, mCherry (red) and EGFP (green), and a mCherry transcripttargeting siRNA implemented into a pre-miR155 backbone which is processed by Dicer. Thus, measuring the ratios of red and green fluorescence intensities will give an indication on Dicer activity. The plasmid also has additional regulatory elements for stabilizing expression levels. The plasmid was then produced by molecular cloning methods and its functionality was tested with Dicer-modulating compounds. The assay was optimised by testing it in different cell lines and varying assay parameters, and stable cell lines were created to make large-scale screening more convenient. Finally, a small-scale screen was done with ten pharmacologically active compounds. Transiently transfected, in Chinese hamster ovarian cells, mCherry silencing was too efficient for reliable detection of improvement in silencing efficiency due to floor effect. With an inducible, Tet-On, system in FLP-IN 293 T-Rex cells, the expression could be controlled by administering doxycycline and the improvement in silencing was quantifiable. The assay seemed to be functional after 72 hours and 120 hours of incubation using enoxacin (100 μM) as a positive control. However, the screening found no compounds to significantly reduce mCherry/EGFP fluorescence ratio and, additionally, the effect of enoxacin was abolished. Therefore, a more thorough analysis on the effects of enoxacin was done and, although statistically significant, enoxacin was only marginally effective in reducing mCherry/EGFP fluorescence ratio after 72 hours of treatment. It should be noted from the small-scale screening that metformin and BDNF, compounds previously shown to elevate Dicer levels, showed similar effects to enoxacin. The quality of the assay in terms of high-throughput screening was determined by calculating Zfactors and coefficients of variations for the experiments, which showed that the variability of the assay was acceptable, but the differences between controls was not large enough for reliable screening. In conclusion, the effects of metformin and BDNF should be further studied and regarding the assay, more optimisation is needed for large-scale, high-throughput, screening to be done with minimal resources.
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(2020)Selective degeneration and dysregulation of specific neuronal populations is a common hallmark shared by neurodegenerative diseases affecting the aging population. Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases with debilitating clinical manifestations that follow a chronic and progressive course. Pathological hallmarks of PD involve gradual and specific loss of DA (DA) neurons and widespread presence of Lewy body (LB) inclusions that consist of aggregated presynaptic protein, α-Synuclein (αSyn). Treatment of PD remains to be at symptomatic management as the underlying mechanisms that trigger neurodegeneration are still not fully elucidated. Over the past two decades, microRNAs (miRNAs) have become a major area of interest within biomedical fields and gained increasing momentum in the context of neurodegenerative diseases. In recent developments, changes in mature miRNA profiles have been reported in aging tissue and many age-related diseases, including PD. More recently, a number of studies have found that the most essential enzyme in the miRNA biogenesis pathway, Dicer, exhibits reduced expression with aging. To these ends, a genetic mouse model based on heterozygous knockout of Dicer (DicerHET) was introduced to simulate Dicer downregulation. Initial investigations identified the DicerHET model as a promising tool for studying the relationship between disrupted miRNA biogenesis and neurodegeneration associated with PD. To facilitate future investigations and speed up screening of potential therapeutic compounds using this genetic model, in the current work, we aimed to produce a DicerHET in vitro model with a practical and convenient genetic engineering approach. The main focus of this work was to validate the model and establish a standardized reproducible approach suitable for research that addresses the role of miRNA biogenesis in PD. The desired DicerHET genotype was generated in vitro by employing traditional Cre/loxP system in conjunction with a virally mediated Cre expression. More specifically, primary cortical cultures, derived from Dicer flox/+ mice embryos, were transduced with Cre expressing lentiviral vectors (lenti-hSYN-T2A-Cre) to delete the “floxed” Dicer allele. To establish optimal parameters for the procedure, we analysed recombination efficiency under different transduction conditions. The most efficient recombination was achieved after 5 days of induction in cultures. However, we observed that DicerHET genotype did not attenuate survival of the cells, as assessed by immunohistochemistry. Further, as a proof of concept, we exposed the DicerHET cultures to pre-formed fibrils (PFFs) - a PD related stressor that causes αSyn aggregation. pSer129-αSyn-positive LB-like aggregates were detected in all the PFF-treated cultures, however, with a greater accumulation in the DicerHET cultures. Interestingly, increased aggregation was not accompanied by increased cell death, suggesting that DicerHET genotype does not increase vulnerability of cortical neurons to pSer129-αSyn aggregation. Based on our earlier studies we presume that DA neurons may bear a specific vulnerability towards the age-related Dicer depletion. More conclusive evidence on this intriguing relationship could be provided in future research using the DicerHET model that can be readily applied to primary DA cultures.
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(2020)Selective degeneration and dysregulation of specific neuronal populations is a common hallmark shared by neurodegenerative diseases affecting the aging population. Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases with debilitating clinical manifestations that follow a chronic and progressive course. Pathological hallmarks of PD involve gradual and specific loss of DA (DA) neurons and widespread presence of Lewy body (LB) inclusions that consist of aggregated presynaptic protein, α-Synuclein (αSyn). Treatment of PD remains to be at symptomatic management as the underlying mechanisms that trigger neurodegeneration are still not fully elucidated. Over the past two decades, microRNAs (miRNAs) have become a major area of interest within biomedical fields and gained increasing momentum in the context of neurodegenerative diseases. In recent developments, changes in mature miRNA profiles have been reported in aging tissue and many age-related diseases, including PD. More recently, a number of studies have found that the most essential enzyme in the miRNA biogenesis pathway, Dicer, exhibits reduced expression with aging. To these ends, a genetic mouse model based on heterozygous knockout of Dicer (DicerHET) was introduced to simulate Dicer downregulation. Initial investigations identified the DicerHET model as a promising tool for studying the relationship between disrupted miRNA biogenesis and neurodegeneration associated with PD. To facilitate future investigations and speed up screening of potential therapeutic compounds using this genetic model, in the current work, we aimed to produce a DicerHET in vitro model with a practical and convenient genetic engineering approach. The main focus of this work was to validate the model and establish a standardized reproducible approach suitable for research that addresses the role of miRNA biogenesis in PD. The desired DicerHET genotype was generated in vitro by employing traditional Cre/loxP system in conjunction with a virally mediated Cre expression. More specifically, primary cortical cultures, derived from Dicer flox/+ mice embryos, were transduced with Cre expressing lentiviral vectors (lenti-hSYN-T2A-Cre) to delete the “floxed” Dicer allele. To establish optimal parameters for the procedure, we analysed recombination efficiency under different transduction conditions. The most efficient recombination was achieved after 5 days of induction in cultures. However, we observed that DicerHET genotype did not attenuate survival of the cells, as assessed by immunohistochemistry. Further, as a proof of concept, we exposed the DicerHET cultures to pre-formed fibrils (PFFs) - a PD related stressor that causes αSyn aggregation. pSer129-αSyn-positive LB-like aggregates were detected in all the PFF-treated cultures, however, with a greater accumulation in the DicerHET cultures. Interestingly, increased aggregation was not accompanied by increased cell death, suggesting that DicerHET genotype does not increase vulnerability of cortical neurons to pSer129-αSyn aggregation. Based on our earlier studies we presume that DA neurons may bear a specific vulnerability towards the age-related Dicer depletion. More conclusive evidence on this intriguing relationship could be provided in future research using the DicerHET model that can be readily applied to primary DA cultures.
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(2017)Cardiovascular disease is one of the leading causes of mortality worldwide. Upon myocardial infarction, billions of cardiomyocytes are lost, a fibrotic scar forms, and the heart's contractile function is compromised. Mammalian cardiomyocytes lose most of their proliferative capacity shortly after birth. This decline in proliferative capacity is associated with a switch from glycolysis to oxidative phosphorylation, yielding more ATP, but also inevitably forming reactive oxygen species (ROS). Therefore, finding a way to extend the proliferative window seems crucial to cardiac repair. microRNAs (miRNAs) are short, single-stranded noncoding RNAs that repress gene expression after transcription by binding to their target mRNAs. SIRT1-7, mammalian homologs of the Sirt2 protein in yeast, have been implicated in the regulation of metabolic homeostasis, cell proliferation, cardiac hypertrophy, and aging. The objective of our research was to investigate the differential expression of SIRT1-7 between day 1 and day 7 neonatal mice. Since cells continue to divide until day 7, we wanted to compare the differences in sirtuin expression during the two time points. By doing so, we hoped to gain insight into ways we could regulate sirtuin protein expression by manipulating miRNA and sirtuin gene expression in diseased hearts, thereby promoting the fetal gene program and inducing cells to reenter the cell cycle. Proteins were isolated from whole cell lysates of cardiac tissue of day 1 and day 7 neonatal mice, and western blotting technique was used to analyze SIRT1-7 expression. Expression of SIRT3 and 7 was significantly higher in day 7 as opposed to day 1 in at least two of the three runs, with SIRT7 levels being higher in day 7 in all three runs. Our study provides a basis for carrying out more quantitative analysis to validate gene and protein expression and protein activity, since expression is different at the gene and protein levels and does not necessarily translate into activity.
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(2016)The main purpose of this study was to participate in setting up the In situ hybridization for microRNA detection at the Department of Pharmacology. In situ hybridization (ISH) is an effective method for detection of molecules like DNA and RNA from paraffin fixed tissue sections. ISH provides information of expression and location of selected target molecules. Challenges of ISH for miRNA are the small size of the miRNAs and requirement of RNase free environment to prevent contamination. Micro-RNAs are single-stranded, noncoding, 19 to 25 nucleotides long RNAs, involved in post-transcriptional gene silencing. MiRNA-1 is known to be upregulated in ischemic heart muscle, which increases arrhythmias and apoptosis in heart. Adminstration of isoprenaline to Wistar rats induces similar conditions as acute myocardial infarction, which leads to rise in mir-1 levels. By in situ hybridization we were able to detect mir-1 from heart tissue and adjust suitable conditions for ISH.
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