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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

Astroglial induction of NPCs by Nogo-66 for 8 days.(A) Images of immunocytochemistry. (B) In western blot analysis, both NeuN and β III tubulin expressions were decreased and GFAP was upregulated in NPCs induced by Nogo-66. (C, D) The quantitative results of anti-NueN, and β III tubulin antibodies immunocytocheminstry. Nogo-66 significantly inhibited neuronal differentiation in NPCs in dose-dependent manner. (E, F) The quantitative results of anti-GFAP and S-100βantibodies immunocytocheminstry. Nogo-66 significantly induced NPCs differentiation into astroglial cells in dose-dependent manner. (G) Apoptosis assay with Annexin V-Fitc and PI staining by FACS. Nogo-66 did not induce significantly apoptosis compared to GST at day 2, 5, and 8. Data are mean±S.E. Error bars indicate SE. *p<0.05 **p<0.01(n = 3). Bar scale = 100 um.
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pone-0001856-g003: Astroglial induction of NPCs by Nogo-66 for 8 days.(A) Images of immunocytochemistry. (B) In western blot analysis, both NeuN and β III tubulin expressions were decreased and GFAP was upregulated in NPCs induced by Nogo-66. (C, D) The quantitative results of anti-NueN, and β III tubulin antibodies immunocytocheminstry. Nogo-66 significantly inhibited neuronal differentiation in NPCs in dose-dependent manner. (E, F) The quantitative results of anti-GFAP and S-100βantibodies immunocytocheminstry. Nogo-66 significantly induced NPCs differentiation into astroglial cells in dose-dependent manner. (G) Apoptosis assay with Annexin V-Fitc and PI staining by FACS. Nogo-66 did not induce significantly apoptosis compared to GST at day 2, 5, and 8. Data are mean±S.E. Error bars indicate SE. *p<0.05 **p<0.01(n = 3). Bar scale = 100 um.

Mentions: Nogo-A is predominantly expressed in oligodentrocytes of CNS and its axon growth inhibiting domain of 66 amino acids (Nogo-66) is expressed at the extracellular surface[11] and Nogo-66 receptor (NgR) mediates its regeneration inhibition[12]. The soluble, native GST-Nogo-66 protein purified with a glutathione-resin was broken and only contained about 30% full-length GST-Nogo-66. Thus we have renatured the GST-Nogo-66 from inclusion bodies (see Fig 1C). The renatured GST-Nogo-66 had biological activity and significantly inhibited neurite outgrowth of rat cerebellar granule neurons (See Fig 1D, E). We thus used the renatured Nogo-66 to assess its effect on NPCs differentiation in our study. We observed the differentiation of NPCs treated by Nogo-66 for 8 days in vitro (See Fig 3). Nogo-66 promoted NPCs to differentiate into astrogalial cells (GFAP and S-100β positive cells). We found that 50 nM and 100 nM Nogo-66 could significantly increase the proportion of GFAP or S-100β immunostaining positive cells compared to the corresponding dose of GST treatment. It indicated that Nogo-66 could promote astroglial differentiation of NPCs, with the similarity of astrocyte differentiation promotion in vivo in previous reports. Meanwhile, both the NeuN and β III tubulin antibodies were used to identify the differentiated neurons in immunostaining analysis and Nogo-66 suppressed the neuronal differentiation of NPCs in a dose-dependent manner. Comparing to β III tubulin expression in neuron cytoplasm and its axons, NeuN was mostly expressed in the nuclei. From the two consistent results, we concluded that Nogo-66 could inhibit the differentiation of NPCs into neurons in vitro. And the result was further confirmed by western blot analysis (See Fig 3B). After Nogo-66 treatment, NeuN and β III tubulin expressions were significantly decreased and GFAP expression was significantly increased. In addition, no difference of cell apoptosis percentages between Nogo-66 and GST treatment group was detected by FACS using Annexin V-FITC and propidine iodide (PI) staining (see Fig 3G). The results suggested that the promotion of astroglial differentiation was not the result of the possible enhanced neuronal cell apoptosis induced by Nogo-66. Due to the consistent results with the astrocytic marker, GFAP and S-100βimmunostaining analysis, we considered that Nogo-66 indeed promoted the glial differentiation of NPCs and GFAP was used as the marker of glial cells in our experiments.


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)

Astroglial induction of NPCs by Nogo-66 for 8 days.(A) Images of immunocytochemistry. (B) In western blot analysis, both NeuN and β III tubulin expressions were decreased and GFAP was upregulated in NPCs induced by Nogo-66. (C, D) The quantitative results of anti-NueN, and β III tubulin antibodies immunocytocheminstry. Nogo-66 significantly inhibited neuronal differentiation in NPCs in dose-dependent manner. (E, F) The quantitative results of anti-GFAP and S-100βantibodies immunocytocheminstry. Nogo-66 significantly induced NPCs differentiation into astroglial cells in dose-dependent manner. (G) Apoptosis assay with Annexin V-Fitc and PI staining by FACS. Nogo-66 did not induce significantly apoptosis compared to GST at day 2, 5, and 8. Data are mean±S.E. Error bars indicate SE. *p<0.05 **p<0.01(n = 3). Bar scale = 100 um.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2266802&req=5

pone-0001856-g003: Astroglial induction of NPCs by Nogo-66 for 8 days.(A) Images of immunocytochemistry. (B) In western blot analysis, both NeuN and β III tubulin expressions were decreased and GFAP was upregulated in NPCs induced by Nogo-66. (C, D) The quantitative results of anti-NueN, and β III tubulin antibodies immunocytocheminstry. Nogo-66 significantly inhibited neuronal differentiation in NPCs in dose-dependent manner. (E, F) The quantitative results of anti-GFAP and S-100βantibodies immunocytocheminstry. Nogo-66 significantly induced NPCs differentiation into astroglial cells in dose-dependent manner. (G) Apoptosis assay with Annexin V-Fitc and PI staining by FACS. Nogo-66 did not induce significantly apoptosis compared to GST at day 2, 5, and 8. Data are mean±S.E. Error bars indicate SE. *p<0.05 **p<0.01(n = 3). Bar scale = 100 um.
Mentions: Nogo-A is predominantly expressed in oligodentrocytes of CNS and its axon growth inhibiting domain of 66 amino acids (Nogo-66) is expressed at the extracellular surface[11] and Nogo-66 receptor (NgR) mediates its regeneration inhibition[12]. The soluble, native GST-Nogo-66 protein purified with a glutathione-resin was broken and only contained about 30% full-length GST-Nogo-66. Thus we have renatured the GST-Nogo-66 from inclusion bodies (see Fig 1C). The renatured GST-Nogo-66 had biological activity and significantly inhibited neurite outgrowth of rat cerebellar granule neurons (See Fig 1D, E). We thus used the renatured Nogo-66 to assess its effect on NPCs differentiation in our study. We observed the differentiation of NPCs treated by Nogo-66 for 8 days in vitro (See Fig 3). Nogo-66 promoted NPCs to differentiate into astrogalial cells (GFAP and S-100β positive cells). We found that 50 nM and 100 nM Nogo-66 could significantly increase the proportion of GFAP or S-100β immunostaining positive cells compared to the corresponding dose of GST treatment. It indicated that Nogo-66 could promote astroglial differentiation of NPCs, with the similarity of astrocyte differentiation promotion in vivo in previous reports. Meanwhile, both the NeuN and β III tubulin antibodies were used to identify the differentiated neurons in immunostaining analysis and Nogo-66 suppressed the neuronal differentiation of NPCs in a dose-dependent manner. Comparing to β III tubulin expression in neuron cytoplasm and its axons, NeuN was mostly expressed in the nuclei. From the two consistent results, we concluded that Nogo-66 could inhibit the differentiation of NPCs into neurons in vitro. And the result was further confirmed by western blot analysis (See Fig 3B). After Nogo-66 treatment, NeuN and β III tubulin expressions were significantly decreased and GFAP expression was significantly increased. In addition, no difference of cell apoptosis percentages between Nogo-66 and GST treatment group was detected by FACS using Annexin V-FITC and propidine iodide (PI) staining (see Fig 3G). The results suggested that the promotion of astroglial differentiation was not the result of the possible enhanced neuronal cell apoptosis induced by Nogo-66. Due to the consistent results with the astrocytic marker, GFAP and S-100βimmunostaining analysis, we considered that Nogo-66 indeed promoted the glial differentiation of NPCs and GFAP was used as the marker of glial cells in our experiments.

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