MicroRNAs Engage in Complex Circuits Regulating Adult Neurogenesis

The finding that the adult mammalian brain is still capable of producing neurons has ignited a new field of research aiming to identify the molecular mechanisms regulating adult neurogenesis. An improved understanding of these mechanisms could lead to the development of novel approaches to delay cognitive decline and facilitate neuroregeneration in the adult human brain. Accumulating evidence suggest microRNAs (miRNAs), which represent a class of post-transcriptional gene expression regulators, as crucial part of the gene regulatory networks governing adult neurogenesis. This review attempts to illustrate how miRNAs modulate key processes in the adult neurogenic niche by interacting with each other and with transcriptional regulators. We discuss the function of miRNAs in adult neurogenesis following the life-journey of an adult-born neuron from the adult neural stem cell (NSCs) compartment to its final target site. We first survey how miRNAs control the initial step of adult neurogenesis, that is the transition of quiescent to activated proliferative adult NSCs, and then go on to discuss the role of miRNAs to regulate neuronal differentiation, survival, and functional integration of the newborn neurons. In this context, we highlight miRNAs that converge on functionally related targets or act within cross talking gene regulatory networks. The cooperative manner of miRNA action and the broad target repertoire of each individual miRNA could make the miRNA system a promising tool to gain control on adult NSCs in the context of therapeutic approaches

Kulturen des Entscheidens

Der Band thematisiert Entscheiden als eine soziale Praxis, die keineswegs selbstverständlich sondern in hohem Maße voraussetzungsvoll ist und die mit unterschiedlichen Zumutungen einhergeht. Entscheiden nimmt je nach sozialen Umständen ganz unterschiedliche Formen an und unterliegt demnach dem historischen Wandel. Die Beiträge des Bandes gehen anhand ausgewählter Fallbeispiele, die vom mittelalterlichen Europa bis hin zum gegenwärtigen Indien reichen, unterschiedlichen Aspekten von Kulturen des Entscheidens nach. Sie nehmen Narrative und Praktiken des Entscheidens ebenso in den Blick wie den Einsatz von Ressourcen in Prozessen des Entscheidens und diskutieren Ansätze, Entscheiden in einer geistes- und kulturwissenschaftlichen Perspektive zu analysieren. Der Band zeigt so die vielfältigen Möglichkeiten auf, wie Entscheiden untersucht werden kann, wenn dieses als eine historisch wandelbare soziale Praxis und als kulturell diverses Phänomen begriffen wird

MicroRNAs and orphan nuclear receptor GCNF as novel regulators of human neural stem cell differentiation and neuronal subtype specification

MicroRNAs (miRNAs) are currently recognized as important regulators of neural development. However, given the large number of miRNA species in existence, our understanding of miRNA-based regulation during neurogenesis remains incomplete, in particular with regard to human neural development. Human pluripotent stem cell (hPSC)-based neural stem cells (NSCs) now offer the possibility to study the function of miRNAs in association with early human neuronal differentiation. Thus, the aim of this study was to analyze the impact of miRNAs and their downstream effectors on human neuronal differentiation and subtype specification using a specific population of long-term 
self-renewing neuroepithelial-like stem (lt-NES) cells. First, a miRNA profiling analysis was performed covering the progression from human embryonic stem cells to neurons with lt-NES cells as a stable intermediate. Subsequent functional analyses demonstrated that miR-153, miR-181a/a* and miR-324-5p/3p are able to promote neuronal differentiation of lt-NES cells, similar to the impact of the neuronal-associated miR-124 and miR-125b. In addition, miR-124, miR-125b and miR-181a/a* were found to modulate the neuronal subtype composition of differentiating lt-NES cells, and transfection with respective miRNA oligonucleotides induced differentiation towards a dopaminergic phenotype. Further experiments using hPSC-derived floor plate progenitor cells, a more authentic source for midbrain dopaminergic neurons, confirmed the positive function of miR-181a and the negative function of miR-124 during dopaminergic differentiation. The last part of the thesis focused on deciphering the targets and down-stream effectors of miR-181a. With regard to its role as promoter of neuronal differentiation, miR-181a down-regulates several factors involved in NSC maintenance, including the orphan nuclear receptor GCNF. GCNF is a known transcriptional repressor of pluripotency genes, however, evidence collected in this work points to a yet unrecognized role for GCNF in human NSCs. Specifically, direct targeting of GCNF by miR-181a resulted in an increased rate of neuronal differentiation. Conversely, GCNF overexpression interfered with neuronal differentiation, while preserving the characteristic neural rosette morphology of undifferentiated lt-NES cells. On a mechanistic level, GCNF might act as a suppressor of pro-neural bHLH gene expression, similar to the role of Notch signaling. Indeed, ectopic expression of GCNF partially compensated for Notch inhibition by the gamma-secretase inhibitor DAPT. In addition to this general effect on neuronal differentiation, GCNF has a specific negative effect on the dopaminergic lineage. Thus, the action of miR-181a on GCNF might also account for the miR-181a-induced dopaminergic differentiation. Overexpression of miR-181a in lt-NES cells also increased Wnt activity, which might further contribute to the generation of dopaminergic neurons. Taken together, this work describes a comprehensive analysis from miRNA profiling to the functional study of specific miRNAs in the context of human neuronal differentiation. Based on these analyses, a mechanistic interaction between miR-181a and GCNF in regulating human NSC fate was discovered. Furthermore, this study is the first to describe a miRNA – miR-181a – that promotes the generation of dopaminergic neurons. These findings could be exploited to develop novel approaches for human neural stem cell maintenance as well as the in vitro differentiation of dopaminergic neurons

MicroRNA-based promotion of human neuronal differentiation and subtype specification.

MicroRNAs are key regulators of neural cell proliferation, differentiation and fate choice. Due to the limited access to human primary neural tissue, the role of microRNAs in human neuronal differentiation remains largely unknown. Here, we use a population of long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from human embryonic stem cells to study the expression and function of microRNAs at early stages of human neural stem cell differentiation and neuronal lineage decision. Based on microRNA expression profiling followed by gain- and loss-of-function analyses in lt-NES cells and their neuronal progeny, we demonstrate that miR-153, miR-324-5p/3p and miR-181a/a contribute to the shift of lt-NES cells from self-renewal to neuronal differentiation. We further show that miR-125b and miR-181a specifically promote the generation of neurons of dopaminergic fate, whereas miR-181a inhibits the development of this neurotransmitter subtype. Our data demonstrate that time-controlled modulation of specific microRNA activities not only regulates human neural stem cell self-renewal and differentiation but also contributes to the development of defined neuronal subtypes

Reciprocal Regulation between Bifunctional miR-9/9∗ and its Transcriptional Modulator Notch in Human Neural Stem Cell Self-Renewal and Differentiation

Tight regulation of the balance between self-renewal and differentiation of neural stem cells is crucial to assure proper neural development. In this context, Notch signaling is a well-known promoter of stemness. In contrast, the bifunctional brain-enriched microRNA miR-9/9∗ has been implicated in promoting neuronal differentiation. Therefore, we set out to explore the role of both regulators in human neural stem cells. We found that miR-9/9∗ decreases Notch activity by targeting NOTCH2 and HES1, resulting in an enhanced differentiation. Vice versa, expression levels of miR-9/9∗ depend on the activation status of Notch signaling. While Notch inhibits differentiation of neural stem cells, it also induces miR-9/9∗ via recruitment of the Notch intracellular domain (NICD)/RBPj transcriptional complex to the miR-9/9∗_2 genomic locus. Thus, our data reveal a mutual interaction between bifunctional miR-9/9∗ and the Notch signaling cascade, calibrating the delicate balance between self-renewal and differentiation of human neural stem cells

Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling

MiR-124, miR-125b and miR-181a/a*affect subspecification of lt-NES cell-derived neurons.

<p>(<b>A</b>, <b>B</b>) Immunostainings for β-III tubulin plus TH <b>(A)</b> or GAD65/67 (<b>B</b>) in lt-NES cells (I3 cell line) transduced with LVTHM-ctr or LVTHM-miR-124, -miR-125b and -miR-181a/a*, respectively, and differentiated for 15 days. Scale bars  = 100 µm. (<b>C</b>, <b>D</b>) Histograms showing the fold change in the number of TH-positive neurons (<b>C</b>) and GAD65/67-positive neurons (<b>D</b>) relative to the number of β-III tubulin-positive neurons in lt-NES cells transduced with LVTHM-ctr or LVTHM-miR-124, -miR-125b, -miR-153, -miR-181a/a* and -miR-324-5p/3p, compared to untransduced cells (equal to 1, dashed line). Data are presented as mean + SEM (n = 3; **, p≤0.01). (<b>E</b>) qRT-PCR analyses of NURR1, DAT, TH and GAD1 expression in 15 days differentiated lt-NES cell cultures overexpressing LVTHM-ctr, -miR-124, -miR-125b, and -miR-181a/a*, respectively. Data are normalized to 18S rRNA reference levels and presented as average changes + SEM relative to expression levels in LVTHM-ctr transduced lt-NES cells (equal to 1, n≥3; *, p≤0.05; **, p≤0.01; ***, p≤0.0001). Abbreviations: ctr, control; DAT, dopamine transporter; GAD, glutamic acid decarboxylase; lt-NES, long-term self-renewing neuroepithelial-like stem cells; NURR1, Nuclear receptor related 1 protein; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; rRNA, ribosomal RNA; TH, tyrosine-hydroxylase.</p

Analysis of microRNA expression in human ES cells, lt-NES cells and derived neural progeny.

<p>(<b>A</b>) Phase contrast image of a human ES cell colony (I3 line). (<b>B</b>–<b>D</b>) Immunofluorescent images of, respectively, self-renewing lt-NES cells stained for the neural precursor marker Nestin (<b>B</b>), and lt-NES cell cultures differentiated for 15 days (ND15; <b>C</b>), and 30 days (ND30; <b>D</b>), stained for the pan-neuronal marker β-III tubulin. DAPI labels nuclei. Scale bars = 100 µm. (<b>E</b>) Heat-map showing a hierarchical clustering of miRNA expression profiles in lt-NES cells (NES) and ND15 and ND30 differentiated neuronal cultures, compared to human ES cells (I3 ES, used as baseline). Relative expression levels in NES, ND15 and ND30 are displayed as log2 ratios compared to hES cells (yellow, expression increases; blue, expression decreases; “ns” stays for no significant expression). Representative miRNAs for the different expression groups identified are shown enlarged. In bold are indicated the newly identified neural-associated miRNAs further studied in this work. Abbreviations: DAPI, 4′,6-diamidino-2-phenylidole; ES, embryonic stem cells; lt-NES, long-term self-renewing neuroepithelial-like stem cells.</p

Validation of the identified microRNA expression patterns in two different cell lines.

<p>(<b>A</b>–<b>D</b>) Northern blot analyses showing expression of miRNAs in human ES cells (ES), lt-NES cells (NES) and lt-NES cells differentiated for 15 days (ND15) and 30 days (ND30) from the I3 and H9.2 cell lines. Representative miRNAs for the different expression groups identified are shown (Group 1, <b>A</b>; Group 2, <b>B</b>; Group 3, <b>C</b>). (<b>D</b>) Northern blot analyses showing expression of miR-181a and miR-181a* in the samples described above. Putative miRNA precursors are indicated by “pre”; mature miRNAs are indicated by “miR”. U6 snRNA was used as loading control. (<b>E, F</b>) qRT-PCR analyses monitoring expression of miR-153, miR-324-5p, miR-324-3p, miR-181a and miR-181a* in the samples described above from the I3 (<b>E</b>) and H9.2 (<b>F</b>) cell lines. Data are normalized to RNU5A snRNA reference levels and presented as average changes + SEM relative to expression in NES (baseline, set to 1; n = 3; *, p≤0.05; **, p≤0.005; ***, p≤0.0001). Abbreviations: ES, embryonic stem cells; lt-NES, long-term self-renewing neuroepithelial-like stem cells; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; snRNA, small nuclear RNA.</p