| 1. |
- Kutsche, Lisa K., et al.
(författare)
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Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis
- 2018
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Ingår i: Cell systems. - : Elsevier BV. - 2405-4712. ; 7:4, s. 438-452
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Tidskriftsartikel (refereegranskat)abstract
- Non-coding RNAs regulate many biological processes including neurogenesis. The brain-enriched miR-124 has been assigned as a key player of neuronal differentiation via its complex but little understood regulation of thousands of annotated targets. To systematically chart its regulatory functions, we used CRISPR/Cas9 gene editing to disrupt all six miR-124 alleles in human induced pluripotent stem cells. Upon neuronal induction, miR-124-deleted cells underwent neurogenesis and became functional neurons, albeit with altered morphology and neurotransmitter specification. Using RNA-induced-silencing-complex precipitation, we identified 98 high-confidence miR-124 targets, of which some directly led to decreased viability. By performing advanced transcription-factor-network analysis, we identified indirect miR-124 effects on apoptosis, neuronal subtype differentiation, and the regulation of previously uncharacterized zinc finger transcription factors. Our data emphasize the need for combined experimental- and system-level analyses to comprehensively disentangle and reveal miRNA functions, including their involvement in the neurogenesis of diverse neuronal cell types found in the human brain.
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| 2. |
- Mallinson, Jay, et al.
(författare)
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ACouWash : A standalone instrument for the washing, separation and enrichment of cells.
- 2018
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Ingår i: 22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018. - 9781510897571 ; 1, s. 279-281
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Konferensbidrag (refereegranskat)abstract
- We developed the AcouWash (Figures 1 and 2), a benchtop instrument for flow-through cell and particle separation by acoustophoresis. Building on previous research [1-4] AcouWash can re-suspend cells in new medium, separate cells based on size or acoustic properties, or enrich cells, Figure 3. We present our initial evaluation of AcouWash regarding size-based separation of particles, cell washing, cell viability, and time savings compared to centrifugation. We repeatedly recovered 90-100% of target cells and particles, washed away 99.9% of a small fluorescent tracer molecule, viability of cells was unchanged, and reduced the time for standard cell labelling by 50%.
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| 3. |
- Pircs, Karolina, et al.
(författare)
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Crosstalk between MicroRNAs and Autophagy in Adult Neurogenesis : Implications for Neurodegenerative Disorders
- 2018
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Ingår i: Brain Plasticity. - 2213-6304. ; 3:2, s. 195-203
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Forskningsöversikt (refereegranskat)abstract
- Adult neurogenesis in the mammalian brain, including in humans, occurs throughout life in distinct brain regions. Alterations in adult neurogenesis is a common phenomenon in several different neurodegenerative disorders, which is likely to contribute to the pathophysiology of these disorders. This review summarizes novel concepts related to the interplay between autophagy and microRNAs in control of adult neurogenesis, with a specific focus on its relevance to neurodegenerative diseases.
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| 4. |
- Pircs, Karolina, et al.
(författare)
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Huntingtin Aggregation Impairs Autophagy, Leading to Argonaute-2 Accumulation and Global MicroRNA Dysregulation
- 2018
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 24:6, s. 1397-1406
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Tidskriftsartikel (refereegranskat)abstract
- Many neurodegenerative diseases are characterized by the presence of intracellular protein aggregates, resulting in alterations in autophagy. However, the consequences of impaired autophagy for neuronal function remain poorly understood. In this study, we used cell culture and mouse models of huntingtin protein aggregation as well as post-mortem material from patients with Huntington's disease to demonstrate that Argonaute-2 (AGO2) accumulates in the presence of neuronal protein aggregates and that this is due to impaired autophagy. Accumulation of AGO2, a key factor of the RNA-induced silencing complex that executes microRNA functions, results in global alterations of microRNA levels and activity. Together, these results demonstrate that impaired autophagy found in neurodegenerative diseases not only influences protein aggregation but also directly contributes to global alterations of intracellular post-transcriptional networks. Pircs et al. report that aggregation of the mutant huntingtin protein, a hallmark of Huntington's disease proteinopathy, impairs macroautophagy, leading to Argonaute-2 accumulation and global dysregulation of microRNAs. These results indicate that autophagy not only influences protein aggregation but also directly contributes to the global alterations of post-transcriptional networks in Huntington's disease.
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| 5. |
- Shrigley, Shelby, et al.
(författare)
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Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts
- 2018
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; 132
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Tidskriftsartikel (refereegranskat)abstract
- Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue thatis easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward andrapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each stepof the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cellsfor reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslipsfor electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examplesof the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skinbiopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a widearray of biomedical applications, including disease modeling, drug screening, and target validation.
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