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. 2010 Nov 10:5:49.
doi: 10.1186/1750-1326-5-49.

NO signaling and S-nitrosylation regulate PTEN inhibition in neurodegeneration

Young-Don Kwak  1 , Tao Ma, Shiyong Diao, Xue Zhang, Yaomin Chen, Janet Hsu, Stuart A Lipton, Eliezer Masliah, Huaxi Xu, Francesca-Fang Liao
Affiliations

NO signaling and S-nitrosylation regulate PTEN inhibition in neurodegeneration

Young-Don Kwak et al. Mol Neurodegener. .

Abstract

Background: The phosphatase PTEN governs the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway which is arguably the most important pro-survival pathway in neurons. Recently, PTEN has also been implicated in multiple important CNS functions such as neuronal differentiation, plasticity, injury and drug addiction. It has been reported that loss of PTEN protein, accompanied by Akt activation, occurs under excitotoxic conditions (stroke) as well as in Alzheimer's (AD) brains. However the molecular signals and mechanism underlying PTEN loss are unknown.

Results: In this study, we investigated redox regulation of PTEN, namely S-nitrosylation, a covalent modification of cysteine residues by nitric oxide (NO), and H2O2-mediated oxidation. We found that S-nitrosylation of PTEN was markedly elevated in brains in the early stages of AD (MCI). Surprisingly, there was no increase in the H2O2-mediated oxidation of PTEN, a modification common in cancer cell types, in the MCI/AD brains as compared to normal aged control. Using several cultured neuronal models, we further demonstrate that S-nitrosylation, in conjunction with NO-mediated enhanced ubiquitination, regulates both the lipid phosphatase activity and protein stability of PTEN. S-nitrosylation and oxidation occur on overlapping and distinct Cys residues of PTEN. The NO signal induces PTEN protein degradation via the ubiquitin-proteasome system (UPS) through NEDD4-1-mediated ubiquitination.

Conclusion: This study demonstrates for the first time that NO-mediated redox regulation is the mechanism of PTEN protein degradation, which is distinguished from the H2O2-mediated PTEN oxidation, known to only inactivate the enzyme. This novel regulatory mechanism likely accounts for the PTEN loss observed in neurodegeneration such as in AD, in which NO plays a critical pathophysiological role.

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Figures

Figure 1
Figure 1
Quantitative analysis of PTEN and P-Akt levels in relation to SNO-PTEN in human brains. (A) SNO-PTEN levels were detected by immunoblot analysis following biotin-switch assays. Other proteins were detected by Western blot analysis using 15 μg of brain lysate. (B) Quantification of the Western blots using densitometry analysis reveals a statistically significant elevation of SNO-PTEN levels in MCI and AD brains compared to NC brains *** indicates P < 0.001. (C) An inverse correlation between PTEN and p-Akt levels. * indicates P < 0.05 in MCI and AD compared to NC brains for total PTEN levels. ** indicates P < 0.005 between AD and NC groups for P-Akt. (D) Examination of H2O2-type oxidation of PTEN by band-shift assays: neuronal cells or brains were lysed in buffer containing 2% SDS and 40 mM N-ethylmaleimide and 20 μg of proteins was subjected to 10% SDS-PAGE under non-reducing condition as described [22]. "Unt" stands for untreated neurons. H2O2 was used at 100 μM for 2 h as a positive control for band-shift of the oxidized PTEN.
Figure 2
Figure 2
PTEN is S-nitrosylated by various chemical and biological NO donors in cultured neurons. (A) PTEN nitrosylation by SNOC in a dose-dependent manner as detected by biotin-switch assays: to detect S-nitrosylated cystein residues, the cysteine residues of PTEN was first masked by methylthiolation with MMTS. Nitrosothiols were then selectively reduced by ascorbate to reform free thiol group, which reacted with biotin-HPDH. In this experiment MMTS was added to serve as a positive control since all the cysteine residues in PTEN can react with biotin-HPDP. On the contrary, ascorbte in the untreated samples were used as negative control due to no reactive cysteine residues to biotin-HPDH. (B) Specificity of PTEN S-nitrosylation by DAN assay. (C) PTEN can be S-nitrosylated in cultured neurons by SNOC (200 μM, 30 min), glutamate (200 μM, 30 min), Aβ peptides (10 μM, 4 h) but not by staurosporine (STS, 200 nM, 30 min). (D) H2O2-induced oxidation in primary neurons with the same treatment conditions as in (C). H2O2 was used at 100 μM for 2 h. (E) P-Akt was detected by Western blot analysis 30 min after treatments with SNOC (200 μM), glutamate (200 μM), Aβ peptides (10 μM) in neurons. For the right panel, 10 mM DTT was added during the 30 min treatments.
Figure 3
Figure 3
PTEN S-nitrosylation correlates with protein degradation. (A) Effects of various neurotoxins on the steady-state protein level of PTEN 4 h after treatments. N = 6 experiments. (B) Cycloheximide-chase experiment on PTEN stability. PRCN cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times in the presence or absence of glutamate (200 μM). Cells were simultaneously treated with glutamate and cycloheximide. Two groups of neurons were also co-treated with glutamate and 25 μM MG132. Cells lysates were then prepared for Western blot analysis of steady-state levels of PTEN. (C) Glutamate-induced reduction of PTEN can be rescued by l-NMMA (1 mM) and MG132 (25 μM) pretreatment for 5 and 1 h, respectively, as examined by three independent experiments.
Figure 4
Figure 4
NO signals induce enhanced ubiquitination of PTEN, leading to protein degradation. (A) Treatments with SNOC, glutamate or Aβ peptides increase PTEN ubiquitination, as determined by IP-Western analysis. (B) Enhanced physical interaction between PTEN and NEDD4-1 upon various treatments at the conditions used in other experiments, as determined by co-immunoprecipitation/Western blot analysis. Figure is chosen as the representative of three independent experiments. (C) Downregulation of NEDD4-1 by siRNA ( 4 μg) prevents PTEN protein degradation upon SNOC treatment.
Figure 5
Figure 5
SNOC treatment inactivates PTEN's lipid phosphatase activity. (A) Dose-dependent effect of SNOC on lipid phosphatase activity as determined by Malachite Green assay. Data presented are means based on five independent experiments. ** indicates p < 0.05 and *** indicates p < 0.005. (B) DTT treatment (10 mM) completely restores the PTEN's lipid phosphatase activity abolished by SNOC (300 μM).
Figure 6
Figure 6
Mapping the SNO-PTEN sites by site-directed mutagenesis. (A) (Top scheme) Structural domains of PTEN: phosphatase domain, Ca-independent C2 and a PDZ binding domain at the C-terminus. (B) SNO-PTEN levels of Cys to Ala mutants as measured by biotin switch assays. Transfected N2a cells were treated with 200 μM SNOC for 30 min before cells were harvested for biochemical analysis. Data presented here is representative of 7 experiments. (C) PTEN ubiquitination as measured by IP-Western. Transfected WT or triple mutant PTEN were IPed with anti-HA antibody and immunoprobed with an antibody against ubiquitin. (D) Protein structure of the lipid phosphatase domain with three putative Cys residues labeled based on published PTEN crystal data [32].
Figure.7
Figure.7
Down regulation of PTEN is neuroprotective in acute experimental models. (A) and (B) Downregulation of PTEN with specific siRNA confers neuroprotection 24 h after Aβ exposure (25 μM): PTEN IHC staining (red, A) and MAP/NeuN staining for neuron morphology (green, B). (C) Reduced PTEN protein level in PTEN heterozygous mouse brain (frontal region), accompanied by elevated P-Akt level (2-month-old mice, n = 2). (D) Primary cultured cortical neurons from PTEN +/- pups manifest less apoptotic cell death than PTEN+/+ neurons 24 h after Aβ exposure. Data presented as means ± SD from 5 independent experiments.
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