The protein phosphatase activity of PTEN is essential for regulating neural stem cell differentiation

Abstract

Background: The tumor suppressor gene Phosphatase and tensin homolog (PTEN) is highly expressed in neural progenitor cells (NPCs) and plays an important role in development of the central nervous system. As a dual-specificity phosphatase, the loss of PTEN phosphatase activity has been linked to various diseases.

Results: Here we report that the protein phosphatase activity of Pten is critical for regulating differentiation of neural progenitor cells. First we found that deletion of Pten promotes neuronal differentiation. To determine whether the protein or lipid phosphatase activity is required for regulating neuronal differentiation, we generated phosphatase domain-specific Pten mutations. Interestingly, only expression of protein phosphatase-deficient mutant Y138L could mimic the effect of knocking down Pten, suggesting the protein phosphatase of Pten is critical for regulating NPC differentiation. Importantly, we showed that the wild-type and lipid phosphatase mutant (G129E) forms of Pten are able to rescue neuronal differentiation in Pten knockout NPCs, but mutants containing protein phosphatase mutant cannot. We further found that Pten-dependent dephosphorylation of CREB is critical for neuronal differentiation.

Conclusion: Our data indicate that the protein phosphatase activity of PTEN is critical for regulating differentiation of NSCs during cortical development.

Figure 1

Pten deletion promotes differentiation of NSCs into neurons and enlarges neurosphere. (A) Neurospheres generated from Pten ^loxp/loxp NSCs, were transfected with lentivirus expressing GFP (control) or Cre-GFP (for Pten deletion), and induced to differentiate by growth factor withdrawal. PTEN protein expression was abolished by Cre-GFP-mediated Pten removal, as detected by western blotting. GAPDH was used as a loading control. (B) The fate change of differentiated NSCs was ascertained by labeling with an antibody against the neuronal marker Tuj1. GFP⁺ cells represent the cells transfected with lentivirus. Nuclei were stained with DAPI. Scale bar = 40 μm. (C) A greater number of Tuj1⁺GFP⁺ cells was observed upon Pten deletion compared to Pten ^loxP/loxP controls. Data are expressed as mean ± SD. **P < 0.01 (n > 800 cells per group from each experiment). (D) Neurospheres cultured 3 days after harvesting through flow cytometry in proliferation condition culture. Scale bar = 100 μm. (E) Values are expressed as the neurospheres diameter. Data are expressed as mean ± SD. *P < 0.05 (The average sizes with standard deviations determined for all neurospheres in one well).

Figure 2

PTEN protein phosphatase activity maintains NSCs in a progenitor state. (A) Cells were transduced with WT or mutant PTEN (Y138L, G129E, or C124S: mutations in either protein or lipid phosphatase domain, or both, respectively) or PTEN shRNA. Protein expression was absent in PTEN RNAi cells. (B) Lentiviral transduction of WT, Y138L, G129E, and C124S resulted in the expression of a 55 kDa band corresponding to the PTEN protein, as seen by western blotting. GAPDH was used as a loading control. (C) NSCs transduced with GFP, PTEN shRNA, or WT, Y138L, G129E, or C124S constructs were labeled with an antibody against the neuronal marker Tuj1. Scale bar = 40 μm. (D) Increased neuronal differentiation was observed upon loss of PTEN protein phosphatase activity (Y138L). Data are expressed as mean ± SD. *P < 0.05, ***P < 0.001 (n > 800 cells per group from each experiment).

Figure 3

RNAi-resistant PTEN rescues the effect of PTEN knockdown. (A) Schematic illustration of lentiviral-based PTEN wild type, G129E, Y138L rescue constructs. (B) 293 T cells were transfected with vector, HA-PTEN-WT, HA-PTEN-WT-rescue, HA-PTEN-G129E-rescue, HA-PTEN-Y138L-rescue resulted in the expression of a 55 kDa band corresponding to the PTEN protein, as seen by western blotting. (C) The data show the Tuj1 positive cells in control, PTEN RNAi, PTEN RNAi + HA-PTEN-(WT, G129E, Y138L)-rescue conditions respectively. (D) NSCs transduced with GFP, PTEN RNAi, or PTEN RNAi + HA-PTEN-(WT, G129E, Y138L)-rescue constructs were labeled with an antibody against the neuronal marker Tuj1. Scale bar = 40 μm. Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001 (n > 800 cells per group from each experiment).

Figure 4

Expression of wild-type, but not the protein phosphatase mutant form of PTEN, rescues the accelerated differentiation induced by the genetic deletion of PTEN. (A) Pten ^loxP/loxP NSCs were co-transduced with Cre-GFP and either GFP, or HA-tagged WT PTEN, Y138L, G129E, or C124S constructs; co-expression of the two constructs was visualized by simultaneously labeling cells with antibodies against GFP and HA. The mutations are described in the Figure 2 legend. Scale bar = 40 μm. (B) Overexpression of WT PTEN abolished the increase in the number of Tuj1⁺ cells induced by the loss of PTEN (Cre-GFP + HA PTEN-WT). G129E, which retains protein phosphatase activity, also rescued the precocious differentiation. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 (n > 200 cells per group from each experiment).

Figure 5

PTEN-dependent CREB dephosphorylation is critical for regulating neural stem cell differentiation. (A) NSCs transduced with GFP, PTEN RNAi, or PTEN RNAi + CREB (WT), PTEN RNAi + CREB (S133A) constructs were labeled with an antibody against the neuronal marker Tuj1. Scale bar = 40 μm. (B) Western analysis of CREB phosphorylation in control and PTEN RNAi primary cultured neurons. (C) Quantification of experiments in (A). Data are expressed as mean ± SD. *P < 0.05 **P < 0.01, ***P < 0.001 (n > 800 cells per group from each experiment).