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Porf-2 Inhibits Neural Stem Cell Proliferation Through Wnt/β-Catenin Pathway by Its GAP Domain.

Huang GH, Yang XT, Chen K, Xing J, Guo L, Zhu L, Li HJ, Li XC, Zhang SY, Feng DF - Front Cell Neurosci (2016)

Bottom Line: Previous studies showed porf-2 functions as a modulator in central nerve system development.We here show that porf-2, a conserved family of RhoGAPs, is highly and specifically expressed in NSCs.By using neurosphere formation and Edu assay we confirmed the GAP domain is necessary for its function.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine Shanghai, China.

ABSTRACT
Neural stem cell (NSC) proliferation and differentiation play a pivotal role in the development of brain, the plasticity of the brain network, and the repair for brain function in CNS diseases. The mechanisms regulating NSC behavior are not well elucidated. Previous studies showed porf-2 functions as a modulator in central nerve system development. We here show that porf-2, a conserved family of RhoGAPs, is highly and specifically expressed in NSCs. We also demonstrate that porf-2 inhibits the proliferation of NSCs in vivo and in vitro, but has no effect on NSC differentiation. We investigated which domain is required for the role of porf-2 on NSC proliferation. By using neurosphere formation and Edu assay we confirmed the GAP domain is necessary for its function. In addition, we surveyed a few classical pathways on NSC proliferation and found that porf-2 inhibits NSC proliferation by suppressing the β-catenin nuclear translocation. Taken together, we show for the first time that porf-2 inhibits NSC proliferation through Wnt/β-catenin pathway by its GAP domain.

No MeSH data available.


Related in: MedlinePlus

Knockdown of porf-2 increased NSC proliferation but had no effect on NSC differentiation. (A) Knockdown of porf-2 by lenti-shRNA infection was confirmed by immunoblot analysis in NSCs. ***P < 0.001 vs. shCtrl. (B) Representative image of neurosphere after 4 days culture in lenti-shCtrl and lenti-shporf-2 group. Scale bar: 200 μm. (C) Comparison and Quantification of the diameter of NSC spheres between shCtrl and shporf-2 group after 4 days lentivirus infection. The percentage of different neurosphere size was quantified (right panel). Data are mean ± SEM (n = 4). *p < 0.05. (D) Representative image of Edu positive cells in shCtrl and shporf-2 group. Quantification of the percentage of Edu positive cell number in each group. The number of Edu positive cells is normalized to the total cell number. Scale bar: 100 μm. Data are mean ± SEM (n = 4). **p < 0.01. (E) Confocal images showing the Tuj1 and GFAP positvie cell after 7 days differentiation of NSCs in each group. Quantification of the percentage of Tuj1 and GFAP positive cell number in shCtrl and shporf-2 group. The number of Tuj1 or GFAP positive cells is normalized to the total cell number. Scale bar: 100 μm. Data are mean ± SEM (n = 4). NS: no significant difference P > 0.05.
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Figure 2: Knockdown of porf-2 increased NSC proliferation but had no effect on NSC differentiation. (A) Knockdown of porf-2 by lenti-shRNA infection was confirmed by immunoblot analysis in NSCs. ***P < 0.001 vs. shCtrl. (B) Representative image of neurosphere after 4 days culture in lenti-shCtrl and lenti-shporf-2 group. Scale bar: 200 μm. (C) Comparison and Quantification of the diameter of NSC spheres between shCtrl and shporf-2 group after 4 days lentivirus infection. The percentage of different neurosphere size was quantified (right panel). Data are mean ± SEM (n = 4). *p < 0.05. (D) Representative image of Edu positive cells in shCtrl and shporf-2 group. Quantification of the percentage of Edu positive cell number in each group. The number of Edu positive cells is normalized to the total cell number. Scale bar: 100 μm. Data are mean ± SEM (n = 4). **p < 0.01. (E) Confocal images showing the Tuj1 and GFAP positvie cell after 7 days differentiation of NSCs in each group. Quantification of the percentage of Tuj1 and GFAP positive cell number in shCtrl and shporf-2 group. The number of Tuj1 or GFAP positive cells is normalized to the total cell number. Scale bar: 100 μm. Data are mean ± SEM (n = 4). NS: no significant difference P > 0.05.

Mentions: We have described porf-2 expression pattern. Next, we explored its function on NSC behavior. Firstly, we performed knockdown of porf-2 in mouse hippocampal NSCs by using lentiviral-mediated shRNA delivery. As shown in Figure 2A and Figure S2, all the shRNAs have a high knockdown efficiency, especially the shRNA3, about 61% knockdown in protein level (Figure 2A). Thus we use shRNA3 to investigate the effect of porf-2 on NSCs for all the subsequent testings.


Porf-2 Inhibits Neural Stem Cell Proliferation Through Wnt/β-Catenin Pathway by Its GAP Domain.

Huang GH, Yang XT, Chen K, Xing J, Guo L, Zhu L, Li HJ, Li XC, Zhang SY, Feng DF - Front Cell Neurosci (2016)

Knockdown of porf-2 increased NSC proliferation but had no effect on NSC differentiation. (A) Knockdown of porf-2 by lenti-shRNA infection was confirmed by immunoblot analysis in NSCs. ***P < 0.001 vs. shCtrl. (B) Representative image of neurosphere after 4 days culture in lenti-shCtrl and lenti-shporf-2 group. Scale bar: 200 μm. (C) Comparison and Quantification of the diameter of NSC spheres between shCtrl and shporf-2 group after 4 days lentivirus infection. The percentage of different neurosphere size was quantified (right panel). Data are mean ± SEM (n = 4). *p < 0.05. (D) Representative image of Edu positive cells in shCtrl and shporf-2 group. Quantification of the percentage of Edu positive cell number in each group. The number of Edu positive cells is normalized to the total cell number. Scale bar: 100 μm. Data are mean ± SEM (n = 4). **p < 0.01. (E) Confocal images showing the Tuj1 and GFAP positvie cell after 7 days differentiation of NSCs in each group. Quantification of the percentage of Tuj1 and GFAP positive cell number in shCtrl and shporf-2 group. The number of Tuj1 or GFAP positive cells is normalized to the total cell number. Scale bar: 100 μm. Data are mean ± SEM (n = 4). NS: no significant difference P > 0.05.
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Related In: Results  -  Collection

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Figure 2: Knockdown of porf-2 increased NSC proliferation but had no effect on NSC differentiation. (A) Knockdown of porf-2 by lenti-shRNA infection was confirmed by immunoblot analysis in NSCs. ***P < 0.001 vs. shCtrl. (B) Representative image of neurosphere after 4 days culture in lenti-shCtrl and lenti-shporf-2 group. Scale bar: 200 μm. (C) Comparison and Quantification of the diameter of NSC spheres between shCtrl and shporf-2 group after 4 days lentivirus infection. The percentage of different neurosphere size was quantified (right panel). Data are mean ± SEM (n = 4). *p < 0.05. (D) Representative image of Edu positive cells in shCtrl and shporf-2 group. Quantification of the percentage of Edu positive cell number in each group. The number of Edu positive cells is normalized to the total cell number. Scale bar: 100 μm. Data are mean ± SEM (n = 4). **p < 0.01. (E) Confocal images showing the Tuj1 and GFAP positvie cell after 7 days differentiation of NSCs in each group. Quantification of the percentage of Tuj1 and GFAP positive cell number in shCtrl and shporf-2 group. The number of Tuj1 or GFAP positive cells is normalized to the total cell number. Scale bar: 100 μm. Data are mean ± SEM (n = 4). NS: no significant difference P > 0.05.
Mentions: We have described porf-2 expression pattern. Next, we explored its function on NSC behavior. Firstly, we performed knockdown of porf-2 in mouse hippocampal NSCs by using lentiviral-mediated shRNA delivery. As shown in Figure 2A and Figure S2, all the shRNAs have a high knockdown efficiency, especially the shRNA3, about 61% knockdown in protein level (Figure 2A). Thus we use shRNA3 to investigate the effect of porf-2 on NSCs for all the subsequent testings.

Bottom Line: Previous studies showed porf-2 functions as a modulator in central nerve system development.We here show that porf-2, a conserved family of RhoGAPs, is highly and specifically expressed in NSCs.By using neurosphere formation and Edu assay we confirmed the GAP domain is necessary for its function.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine Shanghai, China.

ABSTRACT
Neural stem cell (NSC) proliferation and differentiation play a pivotal role in the development of brain, the plasticity of the brain network, and the repair for brain function in CNS diseases. The mechanisms regulating NSC behavior are not well elucidated. Previous studies showed porf-2 functions as a modulator in central nerve system development. We here show that porf-2, a conserved family of RhoGAPs, is highly and specifically expressed in NSCs. We also demonstrate that porf-2 inhibits the proliferation of NSCs in vivo and in vitro, but has no effect on NSC differentiation. We investigated which domain is required for the role of porf-2 on NSC proliferation. By using neurosphere formation and Edu assay we confirmed the GAP domain is necessary for its function. In addition, we surveyed a few classical pathways on NSC proliferation and found that porf-2 inhibits NSC proliferation by suppressing the β-catenin nuclear translocation. Taken together, we show for the first time that porf-2 inhibits NSC proliferation through Wnt/β-catenin pathway by its GAP domain.

No MeSH data available.


Related in: MedlinePlus