Enhancement of endogenous neurogenesis by nitric oxide: identification of s-nitrosylation targets

Santos, Ana Isabel Reis dos

http://hdl.handle.net/10400.1/9001

Use this identifier to reference this record.

Name:	Description:	Size:	Format:
PhD thesis_Ana_final.pdf		14.55 MB	Adobe PDF	Download

Send Feedback

Authors

Santos, Ana Isabel Reis dos

Advisor(s)

Araújo, Inês

Ruiz, Antonio Martínez

Abstract(s)

Nitric oxide (NO) is a well-established regulator of neurogenesis. NO enhances proliferation of neural stem cells (NSC) via activation of the ERK/MAPK pathway, and is essential for injury-induced hippocampal neurogenesis following seizures. In the ERK pathway, p21Ras (Ras) is a likely first target for NO to enhance NSC proliferation. S-nitrosylation, a post-translational modification that consists in the formation of a nitrosothiol group (R-SNO) in cysteine residues, may have a substantial role in the activation and/or inhibition of several proteins involved in the neurogenic process, including Ras. The aims of this work were to identify Ras as a first target of NO in NSC and to assess Ras activation through S-nitrosylation, and to identify proteins modified by S-nitrosylation in neurogenic conditions. We show an increase in S-nitrosylation of Ras in NSC after treatment with NO. NO stimulated cell proliferation and increased ERK phosphorylation in overexpressing WT Ras but not its C118S mutant (NO-insensitive), suggesting that NO-sensitive Ras mediates the effect of NO on NSC proliferation. In a seizure mouse model showing NO-dependent neurogenesis, there was a transient increase in cysteine S-nitrosylation of Ras at 2 days after seizures, suggesting that Ras activation precedes cell proliferation in the dentate gyrus. We demonstrate that treatment with S-nitroso-L-cysteine (CysSNO), a permeable nitrosothiol, increased cysteine oxidation and S-nitrosylation in several proteins in NSC. Separation by two-dimensional electrophoresis and analysis by mass spectrometry resulted in the identification of several proteins that presented modified cysteines. We validated the modification of proteins that can be relevant in neurogenesis, observing a clear increase in S-nitrosylation of PEBP-1, PCNA, 14-3-3 and hnRNP K in NSC treated with CysSNO. Overall, the present work highlights Ras as a target of NO-induced modification in the proliferation of NSC, and also identifies several proteins as targets of S-nitrosylation in NSC, suggesting new candidates for NO-induced regulation of neurogenesis.