Reelin induces a radial glial phenotype in human neural progenitor cells by activation of Notch-I

Background: Reelin and Notch-I signaling pathways have been recently found to be necessary to induce the expression of brain lipid binding protein ( BLBP) and to promote the process extension and the maturation of the neuronal progenitors, the radial glial cells. In this study, we report the cross talk between these two pathways. Results: Both in vitro Reelin treatment and overexpression of Notch-I intracellular domain (NICD) induced BLBP expression and a radial glial phenotype in an immortalized human neural progenitor (HNP) cell line, isolated from the cortex of 14 weeks old fetus. Reelin treatment increased the level of NICD, indicating that Reelin signaling directly activates Notch-I. In addition, reducing NICD release, by inhibiting gamma-secretase activity, inhibited the Reelin-induced radial glial phenotype in human neural progenitor cells. Furthermore, we found that Dab-I, an adaptor protein downstream of Reelin, was co-immunoprecipitated with Notch-I and NICD. Conclusion: These data indicate that Reelin signaling induces BLBP expression and a radial glial phenotype in human neural progenitor cells via the activation of Notch-I. This study suggest that Reelin signaling may act to fine tune Notch-I activation to favor the induction of a radial glial phenotype prenataly and would thus offer an insight into how Notch-I signaling leads to different cellular fates at different developmental stages

Reelin Signaling Promotes Radial Glia Maturation and Neurogenesis

The end of neurogenesis in the human brain is marked by the transformation of the neural progenitors, the radial glial cells, into astrocytes. This event coincides with the reduction of Reelin expression, a glycoprotein that regulates neuronal migration in the cerebral cortex and cerebellum. A recent study showed that the dentate gyrus of the adult reeler mice, with homozygous mutation in the RELIN gene, have reduced neurogenesis relative to the wild type. Based on the above findings, our first hypothesis states that Reelin expression is important for the formation of radial glia and the generation of neurons from the neural progenitors. In order to investigate the role of Reelin in the process of cortical neurogenesis during development, we used human neural progenitor cells (hNPCs) that were isolated from a fetal cortex. These cells do not express Reelin. In this study, we show that Reelin addition to these hNPCs in vitro induced the formation of radial glia and increased neurogenesis significantly. Next, we investigated the mechanism by which Reelin increases the formation of radial glia and the generation of neurons. The formation of radial glia is under the control of two pathways, these are the Reelin and the Notch-1 signaling pathways. Since the level of Notch-1 activation determines if a cell would become a radial glia or an astrocyte, and since the absence of Reelin allows the transformation of a radial glia into astrocyte, we hypothesized that Reelin induces the formation of radial glia via activating Notch-1 signaling. To test this hypothesis, we investigated the effect of Reelin addition on Notch-1 activation in hNPCs. We found that Reelin addition in vitro activated Notch-1 signaling by increasing the level of Notch-1 intracellular domain (NICD). On the other hand, reducing NICD release, by inhibiting γ -secretase activity, inhibited the Reelin-induced radial glia, confirming that Reelin\u27s effect on the formation of radial glia is dependent on Notch-1 activation. Furthermore, we found that the Reelin-induced tyrosine phosphorylation of Disabled-1 (Dab-1), an adaptor protein downstream of Reelin, and the subsequent activation of Src family kinases, are essential steps for Notch-1 activation by Reelin. Finally, we found that Reelin addition increased the binding of Dab-1, recently identified as a nucleoshuttling protein, to NICD and enhanced NICD translocation to the nucleus. This resulted in the induction of BLBP expression and the subsequent formation of radial glia. Taken together, these data show that Reelin signaling, mediated by Dab-1 and Src kinase, activates Notch-1 signaling in hNPCs resulting in the induction of BLBP expression, the formation of radial glia and the generation of neurons. This work is novel because it provides that first evidence that Reelin expression is an important signal for the neuronal differentiation of the hNPCs. It also shows the crosstalk between Reelin and Notch-1 signaling, two major pathways in development and cell fate determination. The work is significant because it improves our understanding of the role of Reelin signaling in cell fate determination, differentiation and neurogenesis for the future manipulation of these processes to restore adult brain functions after brain injury or in neurodegenerative diseases

Egr-1 induces DARPP-32 expression in striatal medium spiny neurons via a conserved intragenic element.

DARPP-32 (dopamine and adenosine 3\u27, 5\u27-cyclic monophosphate cAMP-regulated phosphoprotein, 32 kDa) is a striatal-enriched protein that mediates signaling by dopamine and other first messengers in the medium spiny neurons. The transcriptional mechanisms that regulate striatal DARPP-32 expression remain enigmatic and are a subject of much interest in the efforts to induce a striatal phenotype in stem cells. We report the identification and characterization of a conserved region, also known as H10, in intron IV of the gene that codes for DARPP-32 (Ppp1r1b). This DNA sequence forms multiunit complexes with nuclear proteins from adult and embryonic striata of mice and rats. Purification of proteins from these complexes identified early growth response-1 (Egr-1). The interaction between Egr-1 and H10 was confirmed in vitro and in vivo by super-shift and chromatin immunoprecipitation assays, respectively. Importantly, brain-derived neurotrophic factor (BDNF), a known inducer of DARPP-32 and Egr-1 expression, enhanced Egr-1 binding to H10 in vitro. Moreover, overexpression of Egr-1 in primary striatal neurons induced the expression of DARPP-32, whereas a dominant-negative Egr-1 blocked DARPP-32 induction by BDNF. Together, this study identifies Egr-1 as a transcriptional activator of the Ppp1r1b gene and provides insight into the molecular mechanisms that regulate medium spiny neuron maturation

Reelin Regulates Differentiation Of Neural Stem Cells By Activation Of Notch Signaling Through Disabled-1 Tyrosine Phosphorylation

Silicon nanowires were created via the electroless etching technique using silver nitrate (AgNO 3 )/hydrofluoric acid (HF) solution. The prepared raw samples were oxidized for various intervals, so as to have an end result of various nanowire thicknesses. Scanning electron microscope (SEM) images were taken of the original nanowires, the oxidized nanowires and then the oxidized and etched (in HF solution) nanowires. When silicon nanowires are made, the area of exposed silicon undergoes amplification, a formula for which is provided herein. When silicon nanowires are oxidized, the growth rate of the oxide layer varies according to the crystalline alignment. A formula for a polar plot is provided for illustrating the shape of a silicon nanowire after oxidation for various intervals, based on the Deal-Grove and Massoud models of oxidation. © 2011 Elsevier B.V. All rights reserved

Trichostatin A, valproic acid and HDACi 4b up-regulate markers of the differentiated MSN but do not increase survival of striatal neurons <i>in vitro</i>.

<p>(<b>a</b>) Over 2,000 neurons were counted for each condition, derived from 3 separate platings. Untreated control, BDNF (10 ng/ml), HDACi 4b (5 µM), trichostatin A (10 nM) and valproic acid (3 mM), p = 0.94, one-way ANOVA. (<b>b–d</b>) Primary striatal neurons were treated with (b) TSA (10 nM), (c) VPA (3 mM), or (d) HDACi 4b (5 µM) for 24 h, each of which increased the level of DARPP-32 protein. Representative of 3 platings, each performed in duplicate. (<b>e</b>) Calbindin is increased after 24 h of treatment with VPA, (<b>f</b>) Transcript levels of DARPP-32, ARPP-21, D1 type receptors (D1R), D2 type receptors (D2R), encephalin (ENK) and dynorphin (DYN) were measured by RT-qPCR after 24 h treatment with BDNF or TSA. N = 5–8; +/− SEM (*p<0.05, **p<0.01, ***p<0.001).</p

Bcl11b protein level is down-regulated in the presence of HDAC inhibitors.

<p>(<b>a</b>) Ontogeny of Bcl11b in striatum in Swiss-Webster mice. Bcl11b levels decrease markedly post-natally, particularly between the first and second week of life. (<b>b</b>) Primary striatal neurons were treated with VPA (3 mM) or TSA (10 nM) with or without BDNF (10 ng/ml) for 24 h. Similar results were observed after treatment with compound 4 b (not shown). N = 3–5; +/− SEM (*p<0.05, **p<0.01, ***p<0.001).</p

VPA and TSA do not inhibit induction of Egr-1 by BDNF, but do inhibit induction of Nab2 by BDNF.

<p>(<b>a</b>) Primary striatal neurons were treated with BDNF (10 ng/ml), VPA (3 mM), or BDNF (10 ng/ml) plus VPA (3 mM) for 15, 30, and 60 min. The level of Egr-1 was determined by western blotting. Results representative of at least 2 separate platings, performed in triplicate. Error bars indicate SEM (*p<0.05, **p<0.01, ***p<0.001). (<b>b</b>) Primary striatal neurons were treated with BDNF (10 ng/ml), VPA (3 mM), or BDNF (10 ng/ml) plus VPA (3 mM) for 3 hours and the level of Nab2 was determined by western blotting. Results representative of at least 2 separate platings, performed in triplicate. Error bars indicate SEM (*p<0.05). (<b>c</b>) Primary striatal neurons were treated with BDNF (10 ng/ml), TSA (10 nM), or BDNF (10 ng/ml) plus TSA (10 nM) for 3 hours. Levels of Egr-1 and Nab2 were determined by Western blotting. Results representative of 3 separate platings performed in duplicate. Error bars indicate SEM (*p<0.05, **p<0.01, ***p<0.001). (<b>d</b>) Primary striatal cultures were treated as in (b) followed by gel shift analysis. Anti-Nab2, 2 µg, was added before addition of DNA except in Lane 5, where it was added after the labeled oligonucleotide. Representative of 2 experiments performed in duplicate. The band representing binding of Egr-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076842#pone.0076842-Keilani1" target="_blank">[7]</a> is represented by the filled arrow, and the super-shift band generated by addition of α-Nab2 is indicated by the unfilled arrow. (<b>e</b>) Primary striatal cultures were treated with vehicle (NI:non-infected), Ad-GFP (Adenovirus – GFP), or Ad-Nab2 (Adenovirus – Nab2) with and without BDNF, as described in Methods. Representative of 3 platings, performed in duplicate. Error bars indicate SEM (*p<0.05, **p<0.01, ***p<0.001).</p

AcH3 is increased in striatal neurons by HDAC inhibitors and is enriched downstream of the transcription start site in <i>Ppp1r1b</i> striatal chromatin.

<p>(<b>a</b>) Cells were treated with BDNF (10 ng/mL), (a) trichostatin A (10 nM), (b) valproic acid (3 mM) or (c) HDACi 4b (5 µM) for 3 h. N = 3−5, +/− SEM (*p<0.05, ***p<0.001). (<b>b</b>) Chromatin was purified from adult mouse striatal tissue (STR) (DARPP-32-positive) and NIH-3T3 cells (DARPP-32-negative). Chromatin immunoprecipitation coupled with real-time PCR showed an increased association of acetylated histone H3 within a 1 kb region downstream of the DARPP-32 transcriptional start site (TSS) in striata relative to NIH 3T3 cells. Results are representative of two separate experiments performed in triplicates. Values for IgG are set at 100%. Error bars indicate SEM (*p<0.05).</p