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P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord.

Luo J, Lee S, Wu D, Yeh J, Ellamushi H, Wheeler AP, Warnes G, Zhang Y, Bo X - Cell Death Dis (2013)

Bottom Line: Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice.All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro.These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary University of London, London E1 2AT, UK [2] Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT
The potential to use Schwann cells (SCs) in neural repair for patients suffering from neurotrauma and neurodegenerative diseases is well recognized. However, significant cell death after transplantation hinders the clinical translation of SC-based therapies. Various factors may contribute to the death of transplanted cells. It is known that prolonged activation of P2X7 purinoceptors (P2X7R) can lead to death of certain types of cells. In this study, we show that rat SCs express P2X7R and exposure of cultured SCs to high concentrations of ATP (3-5 mM) or a P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced significant cell death rapidly. High concentrations of ATP and BzATP increased ethidium uptake by SCs, indicating increased membrane permeability to large molecules, a typical feature of prolonged P2X7R activation. SC death, as well as ethidium uptake, induced by ATP was blocked by an irreversible P2X7R antagonist oxidized ATP (oxATP) or a reversible P2X7R antagonist A438079. oxATP also significantly inhibits the increase of intracellular free calcium induced by minimolar ATP concentrations. Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice. All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro. When rat SCs were treated with oxATP before transplantation into uninjured rat spinal cord, 35% more SCs survived than untreated SCs 1 week after transplantation. Moreover, 58% more SCs isolated from P2X7R-knockout mice survived after being transplanted into rat spinal cord than SCs from wild-type mice. This further confirms that P2X7R is involved in the death of transplanted SCs. These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells. As many other types of cells, including neural stem cells, also express P2X7R, deactivating P2X7R may improve the survival of other types of transplanted cells.

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P2X7R is expressed in isolated SCs and sciatic nerves from rat and mouse. (a) Photomicrograph of cultured rat SCs double-immunostained for the SC marker S100 and P2X7R. (b) Detection of P2X7R mRNA in cultured rat SCs using PCR. (c) Photomicrographs of longitudinal sections through the rat sciatic nerve double-immunostained for S100 and P2X7R or NF200 and P2X7R. Scale bar, 50 μm. (d) Photomicrographs of longitudinal sections through the sciatic nerves from C57Bl/6J wild-type (WT) and P2X7R-knockout (KO) mice double-immunostained for S100 and P2X7R. Scale bar, 100 μm
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fig1: P2X7R is expressed in isolated SCs and sciatic nerves from rat and mouse. (a) Photomicrograph of cultured rat SCs double-immunostained for the SC marker S100 and P2X7R. (b) Detection of P2X7R mRNA in cultured rat SCs using PCR. (c) Photomicrographs of longitudinal sections through the rat sciatic nerve double-immunostained for S100 and P2X7R or NF200 and P2X7R. Scale bar, 50 μm. (d) Photomicrographs of longitudinal sections through the sciatic nerves from C57Bl/6J wild-type (WT) and P2X7R-knockout (KO) mice double-immunostained for S100 and P2X7R. Scale bar, 100 μm

Mentions: Cultured rat SCs were double-immunostained for P2X7R and the SC marker S100. P2X7R immunoreactivity was distributed all over the cells, whereas S100 immunoreactivity was much stronger in the nuclei (Figure 1a). PCR using rat SC cDNAs and a pair of P2X7R-specific primers produced a DNA band of the same size as that using P2X7R cDNA as template, demonstrating that the P2X7R mRNA is expressed in SCs (Figure 1b). Immunostaining of rat sciatic nerves showed the colocalization of P2X7R and S100 immunoreactivity in SCs (Figure 1c). The P2X7R immunoreactivity was stronger in Schmidt-Lanterman incisures, the tubular cytoplasm structures inside the myelin sheath. P2X7R immunoreactivity was absent or very weak on axons labeled with N52 antibody for neurofilament 200 (Figure 1c). A similar pattern of immunostaining of P2X7R and S100 was seen in the sciatic nerve of wild-type C57Bl/6J mice (Figure 1d). However, no immunoreactivity for P2X7R was detected in the sciatic nerve from the P2X7R-knockout mice from GlaxoSmithKline (Figure 1d). This result confirms the specificity of the P2X7R antibody.


P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord.

Luo J, Lee S, Wu D, Yeh J, Ellamushi H, Wheeler AP, Warnes G, Zhang Y, Bo X - Cell Death Dis (2013)

P2X7R is expressed in isolated SCs and sciatic nerves from rat and mouse. (a) Photomicrograph of cultured rat SCs double-immunostained for the SC marker S100 and P2X7R. (b) Detection of P2X7R mRNA in cultured rat SCs using PCR. (c) Photomicrographs of longitudinal sections through the rat sciatic nerve double-immunostained for S100 and P2X7R or NF200 and P2X7R. Scale bar, 50 μm. (d) Photomicrographs of longitudinal sections through the sciatic nerves from C57Bl/6J wild-type (WT) and P2X7R-knockout (KO) mice double-immunostained for S100 and P2X7R. Scale bar, 100 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824653&req=5

fig1: P2X7R is expressed in isolated SCs and sciatic nerves from rat and mouse. (a) Photomicrograph of cultured rat SCs double-immunostained for the SC marker S100 and P2X7R. (b) Detection of P2X7R mRNA in cultured rat SCs using PCR. (c) Photomicrographs of longitudinal sections through the rat sciatic nerve double-immunostained for S100 and P2X7R or NF200 and P2X7R. Scale bar, 50 μm. (d) Photomicrographs of longitudinal sections through the sciatic nerves from C57Bl/6J wild-type (WT) and P2X7R-knockout (KO) mice double-immunostained for S100 and P2X7R. Scale bar, 100 μm
Mentions: Cultured rat SCs were double-immunostained for P2X7R and the SC marker S100. P2X7R immunoreactivity was distributed all over the cells, whereas S100 immunoreactivity was much stronger in the nuclei (Figure 1a). PCR using rat SC cDNAs and a pair of P2X7R-specific primers produced a DNA band of the same size as that using P2X7R cDNA as template, demonstrating that the P2X7R mRNA is expressed in SCs (Figure 1b). Immunostaining of rat sciatic nerves showed the colocalization of P2X7R and S100 immunoreactivity in SCs (Figure 1c). The P2X7R immunoreactivity was stronger in Schmidt-Lanterman incisures, the tubular cytoplasm structures inside the myelin sheath. P2X7R immunoreactivity was absent or very weak on axons labeled with N52 antibody for neurofilament 200 (Figure 1c). A similar pattern of immunostaining of P2X7R and S100 was seen in the sciatic nerve of wild-type C57Bl/6J mice (Figure 1d). However, no immunoreactivity for P2X7R was detected in the sciatic nerve from the P2X7R-knockout mice from GlaxoSmithKline (Figure 1d). This result confirms the specificity of the P2X7R antibody.

Bottom Line: Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice.All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro.These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary University of London, London E1 2AT, UK [2] Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT
The potential to use Schwann cells (SCs) in neural repair for patients suffering from neurotrauma and neurodegenerative diseases is well recognized. However, significant cell death after transplantation hinders the clinical translation of SC-based therapies. Various factors may contribute to the death of transplanted cells. It is known that prolonged activation of P2X7 purinoceptors (P2X7R) can lead to death of certain types of cells. In this study, we show that rat SCs express P2X7R and exposure of cultured SCs to high concentrations of ATP (3-5 mM) or a P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced significant cell death rapidly. High concentrations of ATP and BzATP increased ethidium uptake by SCs, indicating increased membrane permeability to large molecules, a typical feature of prolonged P2X7R activation. SC death, as well as ethidium uptake, induced by ATP was blocked by an irreversible P2X7R antagonist oxidized ATP (oxATP) or a reversible P2X7R antagonist A438079. oxATP also significantly inhibits the increase of intracellular free calcium induced by minimolar ATP concentrations. Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice. All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro. When rat SCs were treated with oxATP before transplantation into uninjured rat spinal cord, 35% more SCs survived than untreated SCs 1 week after transplantation. Moreover, 58% more SCs isolated from P2X7R-knockout mice survived after being transplanted into rat spinal cord than SCs from wild-type mice. This further confirms that P2X7R is involved in the death of transplanted SCs. These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells. As many other types of cells, including neural stem cells, also express P2X7R, deactivating P2X7R may improve the survival of other types of transplanted cells.

Show MeSH
Related in: MedlinePlus