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Nogo-66 promotes the differentiation of neural progenitors into astroglial lineage cells through mTOR-STAT3 pathway.

Wang B, Xiao Z, Chen B, Han J, Gao Y, Zhang J, Zhao W, Wang X, Dai J - PLoS ONE (2008)

Bottom Line: In the present study, we have found that myelin protein and Nogo-66 promoted the differentiation of NPCs into glial lineage.These results revealed a novel function of Nogo-66 in the fate decision of NPCs.This discovery could have profound impact on the understanding of CNS development and could improve the therapy of CNS injuries.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT

Background: Neural stem/progenitor cells (NPCs) can differentiate into neurons, astrocytes and oligodendrocytes. NPCs are considered valuable for the cell therapy of injuries in the central nervous system (CNS). However, when NPCs are transplanted into the adult mammalian spinal cord, they mostly differentiate into glial lineage. The same results have been observed for endogenous NPCs during spinal cord injury. However, little is known about the mechanism of such fate decision of NPCs.

Methodology/principal findings: In the present study, we have found that myelin protein and Nogo-66 promoted the differentiation of NPCs into glial lineage. NgR and mTOR-Stat3 pathway were involved in this process. Releasing NgR from cell membranes or blocking mTOR-STAT3 could rescue the enhanced glial differentiation by Nogo-66.

Conclusions/significance: These results revealed a novel function of Nogo-66 in the fate decision of NPCs. This discovery could have profound impact on the understanding of CNS development and could improve the therapy of CNS injuries.

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Related in: MedlinePlus

Myelin preparation and GST-Nogo-66 expression.(A) Immunofluorescence analysis of Nestin (green) in the primary culture NPCs. Nuclei were stained by PI (shown in red). Nearly all of the cells were immunostaining positive for Nestin. (B) Western blot analysis detecting Nogo-A and MBP proteins in myelin preparation with a Nogo-A antibody and a MBP antibody. In myelin preparation from adult rat spinal cord not only MBP, a major myelin component, was detected, but Nogo-A was also detected. (C) GST-Nogo-66 SDS-PAGE image. Soluble GST-Nogo-66 purified by glutathione-resin was broken and contained about 30% full-length GST-Nogo-66. Full-length GST-Nogo-66 was about 95% in the inclusion bodies. (D) In vitro analysis of renatured GST-Nogo-66 biological acitivity. Postnatal day 8 rat cerebellar granule neurons were dissociated and placed in culture on slides coated with poly-L-lysine and later supplemented with GST or GST-Nogo-66. After culturing for 48 hours, cells were fixed, permeabalized and stained with a β III tubulin antibody (green) and Nuclei were stained by Hoechst 33342 (blue). Micrographs of the treated cultures showed the inhibitory effects of GST-Nogo-66. (E) Dose-response curve of CGCs treated as in (D) of GST-Nogo-66 protein against neurite length. Results represent the mean neurite length per cell from two independent experiments, with more than 50 cells being measured in three separate wells for each treatment. GST-Nogo-66 significantly inhibited neurite outgrowth at 50 nM concentration (2-tailed t test, P<0.01).
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pone-0001856-g001: Myelin preparation and GST-Nogo-66 expression.(A) Immunofluorescence analysis of Nestin (green) in the primary culture NPCs. Nuclei were stained by PI (shown in red). Nearly all of the cells were immunostaining positive for Nestin. (B) Western blot analysis detecting Nogo-A and MBP proteins in myelin preparation with a Nogo-A antibody and a MBP antibody. In myelin preparation from adult rat spinal cord not only MBP, a major myelin component, was detected, but Nogo-A was also detected. (C) GST-Nogo-66 SDS-PAGE image. Soluble GST-Nogo-66 purified by glutathione-resin was broken and contained about 30% full-length GST-Nogo-66. Full-length GST-Nogo-66 was about 95% in the inclusion bodies. (D) In vitro analysis of renatured GST-Nogo-66 biological acitivity. Postnatal day 8 rat cerebellar granule neurons were dissociated and placed in culture on slides coated with poly-L-lysine and later supplemented with GST or GST-Nogo-66. After culturing for 48 hours, cells were fixed, permeabalized and stained with a β III tubulin antibody (green) and Nuclei were stained by Hoechst 33342 (blue). Micrographs of the treated cultures showed the inhibitory effects of GST-Nogo-66. (E) Dose-response curve of CGCs treated as in (D) of GST-Nogo-66 protein against neurite length. Results represent the mean neurite length per cell from two independent experiments, with more than 50 cells being measured in three separate wells for each treatment. GST-Nogo-66 significantly inhibited neurite outgrowth at 50 nM concentration (2-tailed t test, P<0.01).

Mentions: We have prepared myelin from adult rat spinal cord and assessed its effect on the differentiation of NPCs (see Fig 1B). After treated with 1.0 or 5.0 ug/ml myelin, NPCs were inhibited to differentiate into neuronal cells (β III tubulin positive cells) and promoted to differentiate into astrocytes (GFAP positive cells) (See Fig 2B and C). There are many components in myelin. We wonder if the major myelin components have the similar effects. Myelin basic protein (MBP) is one of the major myelin proteins, making up 40% of total myelin protein in CNS. We found that MBP protein did not regulate the glial and neuronal differentiation of NPCs (See Fig 2D and E). Some myelin proteins such as Nogo-A, MAG, and OMgp are axon outgrowth inhibitors in CNS. We have hypothesized that these axon inhibitory myelin proteins could mediate the NPCs fate decision. Central to the current understanding of axon inhibitors is the membrane protein Nogo-A. In the myelin preparation, Nogo-A protein was successfully detected by western blot analysis with a Nogo-A antibody (See Fig 1B).


Nogo-66 promotes the differentiation of neural progenitors into astroglial lineage cells through mTOR-STAT3 pathway.

Wang B, Xiao Z, Chen B, Han J, Gao Y, Zhang J, Zhao W, Wang X, Dai J - PLoS ONE (2008)

Myelin preparation and GST-Nogo-66 expression.(A) Immunofluorescence analysis of Nestin (green) in the primary culture NPCs. Nuclei were stained by PI (shown in red). Nearly all of the cells were immunostaining positive for Nestin. (B) Western blot analysis detecting Nogo-A and MBP proteins in myelin preparation with a Nogo-A antibody and a MBP antibody. In myelin preparation from adult rat spinal cord not only MBP, a major myelin component, was detected, but Nogo-A was also detected. (C) GST-Nogo-66 SDS-PAGE image. Soluble GST-Nogo-66 purified by glutathione-resin was broken and contained about 30% full-length GST-Nogo-66. Full-length GST-Nogo-66 was about 95% in the inclusion bodies. (D) In vitro analysis of renatured GST-Nogo-66 biological acitivity. Postnatal day 8 rat cerebellar granule neurons were dissociated and placed in culture on slides coated with poly-L-lysine and later supplemented with GST or GST-Nogo-66. After culturing for 48 hours, cells were fixed, permeabalized and stained with a β III tubulin antibody (green) and Nuclei were stained by Hoechst 33342 (blue). Micrographs of the treated cultures showed the inhibitory effects of GST-Nogo-66. (E) Dose-response curve of CGCs treated as in (D) of GST-Nogo-66 protein against neurite length. Results represent the mean neurite length per cell from two independent experiments, with more than 50 cells being measured in three separate wells for each treatment. GST-Nogo-66 significantly inhibited neurite outgrowth at 50 nM concentration (2-tailed t test, P<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2266802&req=5

pone-0001856-g001: Myelin preparation and GST-Nogo-66 expression.(A) Immunofluorescence analysis of Nestin (green) in the primary culture NPCs. Nuclei were stained by PI (shown in red). Nearly all of the cells were immunostaining positive for Nestin. (B) Western blot analysis detecting Nogo-A and MBP proteins in myelin preparation with a Nogo-A antibody and a MBP antibody. In myelin preparation from adult rat spinal cord not only MBP, a major myelin component, was detected, but Nogo-A was also detected. (C) GST-Nogo-66 SDS-PAGE image. Soluble GST-Nogo-66 purified by glutathione-resin was broken and contained about 30% full-length GST-Nogo-66. Full-length GST-Nogo-66 was about 95% in the inclusion bodies. (D) In vitro analysis of renatured GST-Nogo-66 biological acitivity. Postnatal day 8 rat cerebellar granule neurons were dissociated and placed in culture on slides coated with poly-L-lysine and later supplemented with GST or GST-Nogo-66. After culturing for 48 hours, cells were fixed, permeabalized and stained with a β III tubulin antibody (green) and Nuclei were stained by Hoechst 33342 (blue). Micrographs of the treated cultures showed the inhibitory effects of GST-Nogo-66. (E) Dose-response curve of CGCs treated as in (D) of GST-Nogo-66 protein against neurite length. Results represent the mean neurite length per cell from two independent experiments, with more than 50 cells being measured in three separate wells for each treatment. GST-Nogo-66 significantly inhibited neurite outgrowth at 50 nM concentration (2-tailed t test, P<0.01).
Mentions: We have prepared myelin from adult rat spinal cord and assessed its effect on the differentiation of NPCs (see Fig 1B). After treated with 1.0 or 5.0 ug/ml myelin, NPCs were inhibited to differentiate into neuronal cells (β III tubulin positive cells) and promoted to differentiate into astrocytes (GFAP positive cells) (See Fig 2B and C). There are many components in myelin. We wonder if the major myelin components have the similar effects. Myelin basic protein (MBP) is one of the major myelin proteins, making up 40% of total myelin protein in CNS. We found that MBP protein did not regulate the glial and neuronal differentiation of NPCs (See Fig 2D and E). Some myelin proteins such as Nogo-A, MAG, and OMgp are axon outgrowth inhibitors in CNS. We have hypothesized that these axon inhibitory myelin proteins could mediate the NPCs fate decision. Central to the current understanding of axon inhibitors is the membrane protein Nogo-A. In the myelin preparation, Nogo-A protein was successfully detected by western blot analysis with a Nogo-A antibody (See Fig 1B).

Bottom Line: In the present study, we have found that myelin protein and Nogo-66 promoted the differentiation of NPCs into glial lineage.These results revealed a novel function of Nogo-66 in the fate decision of NPCs.This discovery could have profound impact on the understanding of CNS development and could improve the therapy of CNS injuries.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT

Background: Neural stem/progenitor cells (NPCs) can differentiate into neurons, astrocytes and oligodendrocytes. NPCs are considered valuable for the cell therapy of injuries in the central nervous system (CNS). However, when NPCs are transplanted into the adult mammalian spinal cord, they mostly differentiate into glial lineage. The same results have been observed for endogenous NPCs during spinal cord injury. However, little is known about the mechanism of such fate decision of NPCs.

Methodology/principal findings: In the present study, we have found that myelin protein and Nogo-66 promoted the differentiation of NPCs into glial lineage. NgR and mTOR-Stat3 pathway were involved in this process. Releasing NgR from cell membranes or blocking mTOR-STAT3 could rescue the enhanced glial differentiation by Nogo-66.

Conclusions/significance: These results revealed a novel function of Nogo-66 in the fate decision of NPCs. This discovery could have profound impact on the understanding of CNS development and could improve the therapy of CNS injuries.

Show MeSH
Related in: MedlinePlus