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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

The GAP domain is required for the porf-2's function on NSC proliferation. (A) The construct pattern of the domain deleted plasmid. Verification of domain deleted plasmid by immunoblot. The same blot was probed with β-actin antibody as a loading control. (B) The morphology of neurosphere in different groups. Scale bar: 50 μm. (C) Quantification of neurosphere size in each group. *p < 0.05, **p < 0.01. (D) Representative images of Edu positive cells in indicated group. Quantification of the percentage of Edu positive cell number in each group. Scale bar: 50 μm. Data are mean ± SEM (n = 5). **P < 0.01.
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Figure 4: The GAP domain is required for the porf-2's function on NSC proliferation. (A) The construct pattern of the domain deleted plasmid. Verification of domain deleted plasmid by immunoblot. The same blot was probed with β-actin antibody as a loading control. (B) The morphology of neurosphere in different groups. Scale bar: 50 μm. (C) Quantification of neurosphere size in each group. *p < 0.05, **p < 0.01. (D) Representative images of Edu positive cells in indicated group. Quantification of the percentage of Edu positive cell number in each group. Scale bar: 50 μm. Data are mean ± SEM (n = 5). **P < 0.01.

Mentions: As porf-2 has three domains: WW, Myth4, and GAP domain, we tried to find out which domain is required in the NSC proliferation. Next, we created three domain deleted plasmids of porf-2. The construct pattern was shown in Figure 4A. Our western blot result suggested that the three plasmids were successfully constructed (Figure 4A). In agreement with our result above, overexpression of porf-2 showed smaller neurosphere phenotype compared to control (Figures 4B,C). Similarly, overexpression of Δww-porf-2 and ΔMyth4-porf-2 showed smaller neurosphere phenotype compared to control, which is consistant with porf-2 overexpression. While interestingly, the diameter of neurosphere in ΔGAP-porf-2 group was back to control level, which indicated that the GAP domain is the functional domain of porf-2 in NSC proliferation (Figures 4B,C).


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)

The GAP domain is required for the porf-2's function on NSC proliferation. (A) The construct pattern of the domain deleted plasmid. Verification of domain deleted plasmid by immunoblot. The same blot was probed with β-actin antibody as a loading control. (B) The morphology of neurosphere in different groups. Scale bar: 50 μm. (C) Quantification of neurosphere size in each group. *p < 0.05, **p < 0.01. (D) Representative images of Edu positive cells in indicated group. Quantification of the percentage of Edu positive cell number in each group. Scale bar: 50 μm. Data are mean ± SEM (n = 5). **P < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: The GAP domain is required for the porf-2's function on NSC proliferation. (A) The construct pattern of the domain deleted plasmid. Verification of domain deleted plasmid by immunoblot. The same blot was probed with β-actin antibody as a loading control. (B) The morphology of neurosphere in different groups. Scale bar: 50 μm. (C) Quantification of neurosphere size in each group. *p < 0.05, **p < 0.01. (D) Representative images of Edu positive cells in indicated group. Quantification of the percentage of Edu positive cell number in each group. Scale bar: 50 μm. Data are mean ± SEM (n = 5). **P < 0.01.
Mentions: As porf-2 has three domains: WW, Myth4, and GAP domain, we tried to find out which domain is required in the NSC proliferation. Next, we created three domain deleted plasmids of porf-2. The construct pattern was shown in Figure 4A. Our western blot result suggested that the three plasmids were successfully constructed (Figure 4A). In agreement with our result above, overexpression of porf-2 showed smaller neurosphere phenotype compared to control (Figures 4B,C). Similarly, overexpression of Δww-porf-2 and ΔMyth4-porf-2 showed smaller neurosphere phenotype compared to control, which is consistant with porf-2 overexpression. While interestingly, the diameter of neurosphere in ΔGAP-porf-2 group was back to control level, which indicated that the GAP domain is the functional domain of porf-2 in NSC proliferation (Figures 4B,C).

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