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First published online January 25, 2006
doi: 10.1242/10.1242/dev.02233

Development 133, 675-684 (2006)
Published by The Company of Biologists 2006
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Extracellular nucleotide signaling in adult neural stem cells: synergism with growth factor-mediated cellular proliferation

Santosh K. Mishra1, Norbert Braun1, Varsha Shukla1, Marc Füllgrabe1, Christof Schomerus2, Horst-Werner Korf2, Christian Gachet3, Yukio Ikehara4, Jean Sévigny5, Simon C. Robson6 and Herbert Zimmermann1,*

1 Biocenter, J.W. Goethe-University, AK Neurochemistry, Frankfurt am Main, Germany.
2 Institute of Anatomy II, Medical School, J.W. Goethe-University, Frankfurt am Main, Germany.
3 INSERM U.311, EFS-Alsace, Strasbourg, France.
4 Department of Cell Biology, Fukuoka University School of Medicine, Japan.
5 Centre de Recherche en Rhumatologie et Immunologie, Sainte-Foy, (Québec), Canada.
6 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.


Figure 1
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  		Fig. 1. Characterization of neurospheres. (A-C) Immunocytochemistry identified nestin (A), GFAP (B), S-100ß (C), ßIII tubulin (D), and the ecto-nucleotidases NTPDase2 (E) and TNAP (F) in all neurospheres investigated (representative images). Scale bars: 50 µm. (G) Analysis of ecto-phosphatase activity. Viable neurospheres were incubated in the presence 1 mM ATP, ADP, AMP, PNPP or PNP-TMP. To determine the contribution of TNAP activity, ATP hydrolysis by neurospheres was analyzed in the presence of 1 mM levamisole. No phosphodiesterase activity (PNP-TMP hydrolysis) was observed. Phosphatase activities were normalized to the activity obtained with ATP as a substrate. The 100% value corresponds to 3.2±1.1 nmoles Pi/min/100 neurospheres (mean±s.d., n=9-12, *P<0.05, **P<0.01). (H) RT-PCR products revealing the presence of mRNAs in neurosphere extracts encoding NTPDase2 and TNAP. (I) Immunoblot using total neurosphere protein (5 µg/lane) detecting protein bands of 70 and 80 kDa (arrowheads) corresponding to NTPDase2 and TNAP, respectively.

 

Figure 2
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  		Fig. 2. Neurospheres express functional P2 receptors. (A) Neurospheres were loaded with the Ca2+ indicator fura-2 AM and stimulated with the P2 receptor agonists ATP, ADP, UTP and UDP (50 µM each). Representative plot of rapid and transient cytosolic Ca2+ elevations recorded from a single neurosphere, evoked by successive application (horizontal bars) of nucleotides. (B) Responses to ATP, ADP, UTP and UDP evoked in identical neurospheres, normalized to the ATP response (100%) (mean±s.d., n=14, **P<0.01). (C) Dose dependence of the neurosphere response. Ca2+ responses to increasing concentrations of either ATP (black), ADP (hatched) or UTP (white) were successively recorded from single neurospheres. The response to 50 µM nucleotide was set to 100% (mean±s.d., n=3-9).

 

Figure 3
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  		Fig. 3. Profiling of receptors responsible for cytosolic Ca2+ responses. (A) Ca2+ signals recorded from fura 2-loaded neurospheres evoked by a variety of P2-receptors agonists. Whereas all P2Y1-receptor agonists (ATP, 2-MeSATP, ADPßS, 2-ClATP, 2-MeSADP) induced a comparable response, the P2X receptor agonists {alpha},ß-meATP, ß,{gamma}-meATP and BzATP, the P2Y14 receptor agonist UDP-glucose, and the P1-receptor agonist adenosine were ineffective (50 µM each). Ca2+ signals were normalized to the ATP response (100%) obtained in the identical neurosphere. (B) Independence of the cytosolic Ca2+ response on extracellular Ca2+ (e-Ca2+) following application of ATP (50 µM). (C) Dependence of the cytosolic Ca2+ response on intracellular Ca2+. Pre-application for 10 minutes of the Ca2+ store blocker thapsigargin (Th, 5 µM) strongly diminished the Ca2+ elevations evoked by ATP or ADP (50 µM each). Responses were normalized to the ATP (or ADP) response (100%) obtained in the identical neurosphere (mean±s.d., n=5-19 (A), 27 (B) or 7-9 (C); *P<0.05, **P<0.01).

 

Figure 4
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  		Fig. 4. P2-receptor antagonists reduce the cytosolic Ca2+ elevations. Inhibitors (100 µM each) were applied 8 minutes prior to agonist application. The P2Y1-receptor-specific antagonist MRS2179 reduced the Ca2+ signal evoked by ATP or ADP (50 µM each). The non-selective P2-receptor antagonists suramin or PPADS reduced the Ca2+ signals evoked by either ATP, ADP or UTP (mean±s.d., n=5-8, *P<0.05, **P<0.01).

 

Figure 5
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  		Fig. 5. P2-receptor-evoked cytosolic Ca2+ signals in neurospheres derived from P2Y1-receptor knockout mice (C57BL/6J). The relative agonist potency in neurospheres from C57BL/6J wild-type mice corresponds to that of C57BL/6N mice (compare with Fig. 2B). In neurospheres derived from P2Y1-knockout mice, the ATP and UTP responses were equal and ADP and UDP were essentially inactive (mean±s.d., n=20-38).

 

Figure 6
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  		Fig. 6. P2 receptor expression. (A) RT-PCR. Neurospheres (NS) express mRNA for P2Y1, P2Y2 and P2Y6 receptors, but not for the P2Y4 receptor. A P2Y4 PCR product could, however, be amplified from a mouse brain genomic DNA preparation (gDNA). To exclude contamination of the mRNA preparation with genomic DNA, a primer pair for the immunoglobulin heavy chain binding protein (BiP) was used. Using the mRNA preparation from the neurospheres (NS), a 560 bp sequence was amplified. With mouse brain genomic DNA (gDNA), a 876 bp PCR product was amplified that contained a 316 bp long intron sequence. (B) In situ hybridization. Left: Using a 319 bp fragment of the P2Y1 receptor as an antisense probe, mRNA was detected in large cell bodies of the striatum and in select clusters of subependymal cells (arrows) of the lateral wall of the lateral ventricle (lv), as well as in the dorsolateral triangular area of the SVZ (arrowheads), where neuroblasts enter the RMS. A cluster of labeled subependymal cells boxed in the left image is enlarged in the middle panel. Right: Lack of staining for the corresponding sense probe. Scale bars: 50 µm in the left and right panels; 10 µm in the middle panel.

 

Figure 7
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  		Fig. 7. Modulation of cell proliferation by agonists of P2Y and P1 receptors. (A,B) Immunocytochemistry (representative images). Neurospheres from C57BL/6N mice were dissociated and expanded in the presence of either 20 ng/ml EGF and 10 ng/ml FGF2 (A) or 5 ng/ml EGF and 2.5 ng/ml FGF2 (B). Equal expression of nestin was observed in 4-day-old neurospheres whether cultured at high (A, H-nestin) or reduced (B, R-nestin) growth factor concentrations. (C-F) The daily addition to reseeded cells of adenosine (C), the P2Y1-receptor agonist ADPßS (D), the P2Y2 receptor agonist UTP (E) or of ADPßS and UTP (F) (50 µM each) had no effect on nestin expression. Scale bars: 10 µm. (G) Agonist-dependent effect on cell proliferation after daily application of ADPßS+UTP, ADPßS, UTP, UDP, {alpha},ßmeATP, BzATP (50 µM each) or adenosine (ADO) (1 µM to 50 µM) to cells dissociated from neurospheres and expanded in the presence of reduced growth factor concentrations. The cell number was determined after 4 days. (H) Cell proliferation in neurospheres derived from wild-type (+/+) and P2Y1-receptor knockout (-/-) mice. First, the cell number obtained after 4 days was compared between wild-type (control, 100%) and P2Y1-knockout mice (-/-) (both C57BL/6J). In addition, the effect of the P2Y1-receptor antagonist MRS2179 (100 µM) on neurospheres derived from wild-type mice was analyzed under two different experimental conditions. In one case [MRS2179(d4)], the antagonist was added immediately after reseeding and then daily for 4 days. In the other case [MRS2179(d7)], neurospheres were first cultured for 4 days in the presence of high growth factor concentrations, washed and reseeded at reduced growth factor concentrations. A single dose of MRS2179 (100 µM) was then applied and the cell number was determined at 7 days. (I) Comparison of the proliferative effect of P2-receptor agonists on neurosphere cells from wild-type (+/+) and P2Y1-receptor knockout (-/-) mice. Processing of cells was as for G. Values in G-I represent mean±s.d., n=3-7, *P<0.05, **P<0.01. The average cell number derived from a single well in the control experiments (100%) amounted to 4.0±1.2x106 (s.d.) for G, to 2.8±1.3x106 (s.d.) for H, and to 3.3±1.3x106 (s.d.) for I.

 

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© The Company of Biologists Ltd 2006