Roles of db-cAMP, IBMX and RA in Aspects of Neural Differentiation of Cord Blood Derived Mesenchymal-Like Stem Cells

Mesenchymal stem cells (MSCs) have multilineage differentiation potential which includes cell lineages of the central nervous system; hence MSCs might be useful in the treatment of neurodegenerative diseases such as Parkinson's disease. Although mesenchymal stem cells have been shown to differentiate into the neural lineage, there is still little knowledge about the underlying mechanisms of differentiation particularly towards specialized neurons such as dopaminergic neurons. Here, we show that MSCs derived from human umbilical cord blood (MSChUCBs) are capable of expressing tyrosine hydroxylase (TH) and Nurr1, markers typically associated with DA neurons. We also found differential phosphorylation of TH isoforms indicating the presence of post-translational mechanisms possibly activating and modifying TH in MSChUCB. Furthermore, functional dissection of components in the differentiation medium revealed that dibutyryl-cAMP (db-cAMP), 3-isobutyl-1-methylxanthine (IBMX) and retinoic acid (RA) are involved in the regulation of Nurr1 and Neurofilament-L expression as well as in the differential phosphorylation of TH. We also demonstrate a possible inhibitory role of the protein kinase A signaling pathway in the phosphorylation of specific TH isoforms

Asymmetric cell division and Notch signaling specify dopaminergic neurons in Drosophila.

In Drosophila, dopaminergic (DA) neurons can be found from mid embryonic stages of development till adulthood. Despite their functional involvement in learning and memory, not much is known about the developmental as well as molecular mechanisms involved in the events of DA neuronal specification, differentiation and maturation. In this report we demonstrate that most larval DA neurons are generated during embryonic development. Furthermore, we show that loss of function (l-o-f) mutations of genes of the apical complex proteins in the asymmetric cell division (ACD) machinery, such as inscuteable and bazooka result in supernumerary DA neurons, whereas l-o-f mutations of genes of the basal complex proteins such as numb result in loss or reduction of DA neurons. In addition, when Notch signaling is reduced or abolished, additional DA neurons are formed and conversely, when Notch signaling is activated, less DA neurons are generated. Our data demonstrate that both ACD and Notch signaling are crucial mechanisms for DA neuronal specification. We propose a model in which ACD results in differential Notch activation in direct siblings and in this context Notch acts as a repressor for DA neuronal specification in the sibling that receives active Notch signaling. Our study provides the first link of ACD and Notch signaling in the specification of a neurotransmitter phenotype in Drosophila. Given the high degree of conservation between Drosophila and vertebrate systems, this study could be of significance to mechanisms of DA neuronal differentiation not limited to flies

IBMX and db-cAMP upregulate <i>Nurr1</i> expression in a redundant manner and RA and IBMX synergistically regulate <i>NF-L</i> expression.

<p>(A) RT-PCR analysis showing mRNA expression of <i>Nurr1</i> and <i>NF-L</i> following different treatments. Note that IBMX and db-cAMP are sufficient for upregulating <i>Nurr1</i> expression. IBMX and db-cAMP have redundant roles as reflected by the reduction of <i>Nurr1</i> expression only in the absence of both components from the medium. RA and IBMX act synergistically on <i>NF-L</i> expression. (B) Quantification of RT-PCR results of (A). Experiments were performed in duplicates and data is expressed as mean ± SEM.</p

Analysis of neurite-like extensions of MSC^hUCB1 in different media composition.

<p>(A) Control. (B) Cytokine induction medium (CIM). (C, D, E, F and G) Substraction of IBMX, db-cAMP, RA, bFGF and NGF from CIM, respectively. (H, I, J, K and L) MSC^hUCB1 incubated with IBMX, db-cAMP, RA, bFGF and NGF, respectively. Note that IBMX and db-cAMP are necessary and sufficient for neurite-like outgrowth as their removal (C, D) resulted in less cells with neurite-like extensions and individually, (H, I) they induced neurite-like extensions. Removal of RA resulted in a higher frequency of cells with neurite-like extensions possibly due to a toxic effect of RA. If applied individually, RA resulted in small but statistically significant increase in cells with neurite-like extensions. bFGF and NGF did not have significant effects on neurite extension. (M) Quantification of cells with neurite-like outgrowth when individual components were removed from CIM. (N) Quantification of neurite-like extensions when MSC^hUCB1 were incubated with single components. Data in (M) and (N) were analysed using Student's t-test. Experiments were performed in triplicates and data is expressed as mean ± SEM.</p

MSC^hUCBs upregulate neural specific markers.

<p>(A) Band quantification readouts of RT-PCR results of the three MSC^hUCBs after incubation in differentiation medium for 24 h, normalized to β-actin and the respective controls. Note that <i>Nurr1</i> upregulation is statistically significant in all the three MSC^hUCBs. <i>NF-M</i> levels are also elevated, although not significantly. In contrast to MSC^hUCB3, MSC^hUCB1 and MSC^hUCB6 could considerably upregulate other neuronal specific marker gene expression such as <i>NF-H</i> and <i>NF-L</i>, with MSC^hUCB1 also showing upregulation of <i>TH</i> expression. Data are expressed as mean ± SEM (n = 3). (B) Gene expression profiling of MSC^hUCB1 with RT-PCR. Representative RT-PCR products for each marker are shown. Neural markers such as <i>NSE</i>, <i>NF-H</i>, <i>NF-M</i>, <i>NF-L</i>, <i>GFAP</i> as well as the dopaminergic markers <i>Nurr1</i> and <i>TH</i> are upregulated following differentiation. Experiments have been performed in triplicates and data are expressed as mean ± SEM. Note the down-regulation of <i>dopamine-β-hydroxylase</i> (<i>DβH</i>) which encodes for the key enzyme that converts dopamine into nor-adrenaline.</p

MSC^hUCB1 express neural specific markers suggesting neural differentiation.

<p>(A) Protein expression profile. Expression of general neural markers (Tau, NSE and GFAP) and dopaminergic markers (TH and P-TH) are upregulated. The four P-TH isoforms are of sizes between 50.7 kDa and 90 kDa. Note that the different P- TH isoforms (isoforms 1, 3 and isoforms 2, 3) are differentially regulated (day 3 and day 5 post-induction, respectively). A small molecular weight band (∼28.6 kDa), possibly representing an unknown P-TH isoform is marked by ##. The MAPK pathway is down-regulated as a consequence of differentiation as shown by decreasing levels of P-ERK1/2 correlated to increasing length of differentiation. (B) The small molecular weight band (∼28.6 kDa) is strongly elevated in the dopaminergic SY5Y neuroblastoma cells in response to the differentiation medium. β-actin is used as loading controls (A, B). (C) Summary of the FACS analysis data showing the average geometric mean fluorescence intensity (GMFI) of CD133 expression of the experimental and the respective isotype control. CD133 is expressed more strongly in a small population of undifferentiated MSCs and weakly in the rest of the population. However, CD133 is completely down-regulated within 24 h of differentiation. Analysis was done in triplicates and data is expressed as mean ± SEM. (D–G) Immunocytochemical analysis of controls (D, F) versus differentiated MSC^hUCB1 (E, G), showing cells stained with α-synaptophysin (Syp) (D and E) and α-P-TH (F and G). Cells were fixed after 3 days of incubation in differentiation medium. Note that Syp is strongly expressed at the projections (arrowheads in E). Although P-TH expression is already present at basal levels in the control, it becomes upregulated during differentiation. Arrowheads in G point to some cells with neurite-like extensions.</p

Additional file 1 of Genetic and pharmacologic p32-inhibition rescue CHCHD2-linked Parkinson’s disease phenotypes in vivo and in cell models

Additional file 1: Figure S1. Toxicity testing and validation of the p32-inhibitor. A A range of p32-inhibitor concentrations from 0 to 250 µM were tested on the Hela cells expressing CHCHD2-WT-MYC, CHCHD2-Thr61Ile-MYC and the Control cells expressing MYC alone. Note that 2.5 µM, 5 µM and 7.5 µM of p32-I are more toxic to the Hela cells expressing CHCHD2-WT-MYC as compared to the Control (n = 3, One-way ANOVA with Bonferroni post hoc test). Hence, 1 µM p32-I is chosen for cell treatment. B Representative Western blots showing effectiveness of 1 µM p32-I. Data are presented as mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01