Protection against oxidative stress through SUA7/TFIIB regulation in Saccharomyces cerevisiae

The general transcription factor TFIIB, encoded by SUA7 in Saccharomyces cerevisiae, is required for transcription activation but apparently of a specific subset of genes, for example, linked with mitochondrial activity and hence with oxidative environments. Therefore, studying SUA7/TFIIB as a potential target of oxidative stress is fundamental. We found that controlled SUA7 expression under oxidative conditions occurs at transcriptional and mRNA stability levels. Both regulatory events are associated with the transcription activator Yap1 in distinct ways: Yap1 affects SUA7 transcription up regulation in exponentially growing cells facing oxidative signals; the absence of this activator per se contributes to increase SUA7 mRNA stability. However, unlike SUA7 mRNA, TFIIB abundance is not altered on oxidative signals. The biological impact of this preferential regulation of SUA7 mRNA pool is revealed by the partial suppression of cellular oxidative sensitivity by SUA7 overexpression, and supported by the insights on the existence of a novel RNA-binding factor, acting as an oxidative sensor, which regulates mRNA stability. Taken together the results point out a primarily cellular commitment to guarantee SUA7 mRNA levels under oxidative environments

Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

Extracellular matrix (ECM) proteins play a key role during oligodendrogenesis. While fibronectin (FN) is involved in the maintenance and proliferation of oligodendrocyte progenitor cells (OPCs), merosin (MN) promotes differentiation into oligodendrocytes (OLs). Mechanical properties of the ECM also seem to affect OL differentiation, hence this study aimed to clarify the impact of combined biophysical and biochemical elements during oligodendrocyte differentiation and maturation using synthetic elastic polymeric ECM-like substrates. CG-4 cells presented OPC- or OL-like morphology in response to brain-compliant substrates functionalised with FN or MN, respectively. The expression of the differentiation and maturation markers myelin basic protein — MBP — and proteolipid protein — PLP — (respectively) by primary rat oligodendrocytes was enhanced in presence of MN, but only on brain-compliant conditions, considering the distribution (MBP) or amount (PLP) of the protein. It was also observed that maturation of OLs was attained earlier (by assessing PLP expression) by cells differentiated on MN-functionalised brain-compliant substrates than on standard culture conditions. Moreover, the combination of MN and substrate compliance enhanced the maturation and morphological complexity of OLs. Considering the distinct degrees of stiffness tested ranging within those of the central nervous system, our results indicate that 6.5 kPa is the most suitable rigidity for oligodendrocyte differentiation

Profilin 1 is required for peripheral nervous system myelination

Myelination allows rapid saltatory propagation of action potentials along the axon and is an essential prerequisite for the normal functioning of the nervous system. During peripheral nervous system (PNS) development, myelin-forming Schwann cells (SCs) generate radial lamellipodia to sort and ensheath axons. This process requires controlled cytoskeletal remodeling, and we show that SC lamellipodia formation depends on the function of profilin 1 (Pfn1), an actinbinding protein involved in microfilament polymerization. Pfn1 is inhibited upon phosphorylation by ROCK, a downstream effector of the integrin linked kinase pathway. Thus, a dramatic reduction of radial lamellipodia formation is observed in SCs lacking integrinlinked kinase or treated with the Rho/ROCK activator lysophosphatidic acid. Knocking down Pfn1 expression by lentiviralmediated shRNA delivery impairs SC lamellipodia formation in vitro, suggesting a direct role for this protein in PNS myelination. Indeed,SC-specific gene ablation of Pfn1 in mice led to profound radial sorting and myelination defects, confirming a central role for this protein in PNS development. Our data identify Pfn1 as a key effector of the integrin linked kinase/Rho/ROCK pathway. This pathway, acting in parallel with integrin β1/LCK/Rac1 and their effectors critically regulates SC lamellipodia formation, radial sorting and myelination during peripheral nervous system maturation

Differentiation of Human Umbilical Cord Matrix Mesenchymal Stem Cells into Neural-Like Progenitor Cells and Maturation into an Oligodendroglial-Like Lineage

<div><p>Mesenchymal stem cells (MSCs) are viewed as safe, readily available and promising adult stem cells, which are currently used in several clinical trials. Additionally, their soluble-factor secretion and multi-lineage differentiation capacities place MSCs in the forefront of stem cell types with expected near-future clinical applications. In the present work MSCs were isolated from the umbilical cord matrix (Wharton's jelly) of human umbilical cord samples. The cells were thoroughly characterized and confirmed as <i>bona-fide</i> MSCs, presenting <i>in vitro</i> low generation time, high proliferative and colony-forming unit-fibroblast (CFU-F) capacity, typical MSC immunophenotype and osteogenic, chondrogenic and adipogenic differentiation capacity. The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity. The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of <i>bona-fide</i> oligodendrocyte precursor cells and differentiated oligodendrocytes.</p></div

Immunofluorescence microscopy images of OL-like cells derived from hUCM-MSCs in co-culture with mouse dorsal root ganglion (DRG) neurons.

<p>The co-cultures were stained using an anti-human Golgi antibody (green) to specifically identify the human cells (OL-like cells derived form MSCs) and an anti-beta-III-tubulin antibody (red) to label the mouse DRG neurons. The images acquired in the green channel (anti-human Golgi antibody) were deliberately slightly overexposed to allow for a better understanding of the cellular morphology, which did not affect the identification of human versus mouse cells, as evidenced by the lack of green signal in the mouse DRG neurons (in red). Counterstaining of the nuclei was performed using DAPI (blue). It was apparent that the OL-like cells and DRG neurons tend to cluster together, as illustrated in a lower magnification image (<i>A</i>). Higher magnification images (<i>B-D</i>) suggest the existence of contact points (arrows) between branches of OL-like cells displaying an immature oligodendrocyte-like morphology (asterisks) and neurites. Scale bars correspond to 50 µm (<i>A</i>) or 20 µm (<i>B-D</i>).</p

Cell morphology and expression of nestin in hUCM-MSC and during the first steps of differentiation.

<p>Phase contrast images of undifferentiated hUCM-MSCs (<i>A</i>), neuroectodermal-like induced MSCs – niMSCs (<i>B</i>) and NSC-like cells after 3 (<i>C</i>) and 18 days (<i>D</i>) in culture in NSC induction medium. Immunofluorescence microscopy images for the neural precursor marker nestin (in green) in undifferentiated MSCs (<i>E</i>) and NSC-like cells (<i>F</i>). Counterstaining of nuclei was performed with DAPI (in blue). In (<i>A-D</i>) the scale bar represents 200 µm and in (<i>E-F</i>) represents 50 µm. Images are representative of at least 3 independent experiments.</p

Quantification of mean fluorescence intensity (MFI) of immunofluorescence images and mRNA expression of differentiation stage markers.

<p>MFI values for nestin (<i>A</i>), O4 (<i>B</i>), GalC (<i>C</i>) and MBP (<i>D</i>) of immnunofluorescence images (representative images on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111059#pone-0111059-g004" target="_blank">Figures 4</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111059#pone-0111059-g006" target="_blank">6</a>) were quantified using Image J software. The graphics represent results regarding cells cultured during the OPC-like differentiation stage on fibronectin-coated TC polystyrene. OL-like cells were differentiated for 10 days on laminin 2 (merosin)-coated wells, cultured in the presence of T3, F3, T3F3 or 7dT3+3dT3F3, as indicated. Bars represent mean ± SEM of at least 3 independent experiments. Statistical analysis was performed by repeated measures one-way ANOVA followed by Tukey's Multiple Comparison Test. Statistically significant differences for each marker between MSCs and cells at other differentiation stages were indicated on top of the corresponding graphic bars, while differences between other conditions were represented using connectors (*<i>P</i><0,05; **<i>P</i><0,01 and ***<i>P</i><0,001). Real time RT-PCR analysis (<i>E</i>) was performed for genes representative of neural progenitors (<i>sox2</i> and <i>nestin</i>), neurons (<i>neurofilament</i>), astrocytes (<i>gfap</i>) and oligodendrocytes (<i>mbp</i>), while <i>fibronectin</i> was used as a mesenchymal marker. Bars represent mean ± SEM of at least 3 independent experiments and are expressed as fold change of 2^−ΔΔCt using <i>actin</i> as a reference gene and undifferentiated MSCs as the control condition. Statistical analysis was performed by Kruskal-Wallis one-way ANOVA followed by Dunn's multiple comparison test (*<i>P</i><0,05, **<i>P</i><0,01 and ***<i>P</i><0,001).</p