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NO signaling and S-nitrosylation regulate PTEN inhibition in neurodegeneration.

Kwak YD, Ma T, Diao S, Zhang X, Chen Y, Hsu J, Lipton SA, Masliah E, Xu H, Liao FF - Mol Neurodegener (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Tennessee Health Science Center, College of Medicine, 874 Union Avenue, Memphis TN, 38163, USA. fliao@uthsc.edu.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Using cultured primary rat cortical neurons and biotin-switch assays, we found that SNO-PTEN can be rapidly induced in primary cultured cortical neurons treated with the physiological NO donor S-nitrosocysteine (SNOC) in a dose-dependent manner with detectable nitrosylation achieved by as little as 10 μM SNOC and a plateau with 300 μM (Figure 2A). This was also confirmed by a more quantitative fluorescent assay (DAN assay, Ref. [25]) using purified recombinant PTEN (Figure 2B). Additional neurotoxic compounds that induce NO generation, such as glutamate or β-amyloid peptides (Aβ25-35) also induced robust SNO-PTEN within minutes (Figure 2C) and lasted for more than 10 hours, even after the NO donors were removed from the cultured media. Interestingly, the classic apoptotic stimuli, staurosporine/STS, did not induce SNO-PTEN. Since STS is known to activate Ca2+ influx through non-NMDAR type ion channels, our results indicate that the source of NO induced by glutamate/Aβ is most likely via the activated nNOS in response to NMDAR-mediated Ca2+ influx. Two other mitochondrial ROS agents (rotenone and MPP+) also induced SNO-PTEN (data not shown); this is consistent with SNO-PTEN in PD brains and suggests that SNO-PTEN may play a common role in chronic degenerative diseases such as AD and PD. In parallel, we found that after treatment of SNOC/glutamate/Aβ, the majority of PTEN in neuronal cells (>85%) remained unmodified by H2O2-type oxidation, indicating that H2O2 may not be the dominant oxidizing species induced by these treatments (Figure 2D). This is consistent with one recent report [26]. Furthermore, we found that the same experimental conditions that induced PTEN nitrosylation led to Akt activation as assessed by increased P-Akt; both were diminished by DTT treatment (Figure 2E).


NO signaling and S-nitrosylation regulate PTEN inhibition in neurodegeneration.

Kwak YD, Ma T, Diao S, Zhang X, Chen Y, Hsu J, Lipton SA, Masliah E, Xu H, Liao FF - Mol Neurodegener (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2992530&req=5

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.
Mentions: Using cultured primary rat cortical neurons and biotin-switch assays, we found that SNO-PTEN can be rapidly induced in primary cultured cortical neurons treated with the physiological NO donor S-nitrosocysteine (SNOC) in a dose-dependent manner with detectable nitrosylation achieved by as little as 10 μM SNOC and a plateau with 300 μM (Figure 2A). This was also confirmed by a more quantitative fluorescent assay (DAN assay, Ref. [25]) using purified recombinant PTEN (Figure 2B). Additional neurotoxic compounds that induce NO generation, such as glutamate or β-amyloid peptides (Aβ25-35) also induced robust SNO-PTEN within minutes (Figure 2C) and lasted for more than 10 hours, even after the NO donors were removed from the cultured media. Interestingly, the classic apoptotic stimuli, staurosporine/STS, did not induce SNO-PTEN. Since STS is known to activate Ca2+ influx through non-NMDAR type ion channels, our results indicate that the source of NO induced by glutamate/Aβ is most likely via the activated nNOS in response to NMDAR-mediated Ca2+ influx. Two other mitochondrial ROS agents (rotenone and MPP+) also induced SNO-PTEN (data not shown); this is consistent with SNO-PTEN in PD brains and suggests that SNO-PTEN may play a common role in chronic degenerative diseases such as AD and PD. In parallel, we found that after treatment of SNOC/glutamate/Aβ, the majority of PTEN in neuronal cells (>85%) remained unmodified by H2O2-type oxidation, indicating that H2O2 may not be the dominant oxidizing species induced by these treatments (Figure 2D). This is consistent with one recent report [26]. Furthermore, we found that the same experimental conditions that induced PTEN nitrosylation led to Akt activation as assessed by increased P-Akt; both were diminished by DTT treatment (Figure 2E).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Tennessee Health Science Center, College of Medicine, 874 Union Avenue, Memphis TN, 38163, USA. fliao@uthsc.edu.

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.

No MeSH data available.


Related in: MedlinePlus