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Identification of new targets of S-nitrosylation in neural stem cells by thiol redox proteomics
Publication . Santos, Ana Isabel; Lourenco, Ana S.; Simão, Sónia; Marques-da-Silva, Dorinda; Santos, Daniela F.; Carvalho, Ana Paula Onofre de; Pereira, Ana Catarina; Izquierdo-Álvarez, Alicia; Ramos, Elena; Morato, Esperanza; Marina, Anabel; Martínez-Ruiz, Antonio; Araújo, Inês
Nitric oxide (NO) is well established as a regulator of neurogenesis. NO increases the proliferation of neural stem cells (NSC), and is essential for hippocampal injury-induced neurogenesis following an excitotoxic lesion. One of the mechanisms underlying non-classical NO cell signaling is protein S-nitrosylation. This post-translational modification consists in the formation of a nitrosothiol group (R-SNO) in cysteine residues, which can promote formation of other oxidative modifications in those cysteine residues. S-nitrosylation can regulate many physiological processes, including neuronal plasticity and neurogenesis. In this work, we aimed to identify S-nitrosylation targets of NO that could participate in neurogenesis. In NSC, we identified a group of proteins oxidatively modified using complementary techniques of thiol redox proteomics. S-nitrosylation of some of these proteins was confirmed and validated in a seizure mouse model of hippocampal injury and in cultured hippocampal stem cells. The identified S-nitrosylated proteins are involved in the ERK/MAPK pathway and may be important targets of NO to enhance the proliferation of NSC.
Nitric oxide from inflammatory origin impairs neural stem cell proliferation by inhibiting epidermal growth factor receptor signaling
Publication . Carreira, Bruno P.; Morte, Maria I.; Santos, Ana I.; Lourenco, Ana S.; Ambrosio, Antonio F.; Carvalho, Caetana M.; Araújo, Inês
Neuroinflammation is characterized by activation of microglial cells, followed by production of nitric oxide (NO), which may have different outcomes on neurogenesis, favoring or inhibiting this process. In the present study, we investigated how the inflammatory mediator NO can affect proliferation of neural stem cells (NSCs), and explored possible mechanisms underlying this effect. We investigated which mechanisms are involved in the regulation of NSC proliferation following treatment with an inflammatory stimulus (lipopolysaccharide plus IFN-gamma), using a culture system of subventricular zone (SVZ)-derived NSCs mixed with microglia cells obtained from wild-type mice (iNOS(+/+)) or from iNOS knockout mice (iNOS(-/-)). We found an impairment of NSC cell proliferation in iNOS(+/+) mixed cultures, which was not observed in iNOS(-/-) mixed cultures. Furthermore, the increased release of NO by activated iNOS(+/+) microglial cells decreased the activation of the ERK/MAPK signaling pathway, which was concomitant with an enhanced nitration of the EGF receptor. Preventing nitrogen reactive species formation with MnTBAP, a scavenger of peroxynitrite (ONOO-), or using the ONOO- degradation catalyst FeTMPyP cell proliferation and ERK signaling were restored to basal levels in iNOS(+/+) mixed cultures. Moreover, exposure to the NO donor NOC-18 (100 mu M), for 48 h, inhibited SVZ-derived NSC proliferation. Regarding the antiproliferative effect of NO, we found that NOC-18 caused the impairment of signaling through the ERK/MAPK pathway, which may be related to increased nitration of the EGF receptor in NSC. Using MnTBAP nitration was prevented, maintaining ERK signaling, rescuing NSC proliferation. We show that NO from inflammatory origin leads to a decreased function of the EGF receptor, which compromised proliferation of NSC. We also demonstrated that NO-mediated nitration of the EGF receptor caused a decrease in its phosphorylation, thus preventing regular proliferation signaling through the ERK/MAPK pathway.
Nitric Oxide Regulates Neurogenesis in the Hippocampus following Seizures
Publication . Carreira, Bruno P.; Santos, Daniela F.; Santos, Ana Isabel; Carvalho, Caetana M.; Araújo, Inês
Hippocampal neurogenesis is changed by brain injury. When neuroinflammation accompanies injury, activation of resident microglial cells promotes the release of inflammatory cytokines and reactive oxygen/nitrogen species like nitric oxide (NO). In these conditions, NO promotes proliferation of neural stem cells (NSC) in the hippocampus. However, little is known about the role of NO in the survival and differentiation of newborn cells in the injured dentate gyrus. Here we investigated the role of NO following seizures in the regulation of proliferation, migration, differentiation, and survival of NSC in the hippocampus using the kainic acid (KA) induced seizuremouse model. We show that NO increased the proliferation of NSC and the number of neuroblasts following seizures but was detrimental to the survival of newborn neurons. NO was also required for the maintenance of long-term neuroinflammation. Taken together, our data show that NO positively contributes to the initial stages of neurogenesis following seizures but compromises survival of newborn neurons.
S-nitrosation and neuronal plasticity
Publication . Santos, Ana Isabel; Martinez-Ruiz, A.; Araújo, Inês
Nitric oxide (NO) has long been recognized as a multifaceted participant in brain physiology. Despite the knowledge that was gathered over many years regarding the contribution of NO to neuronal plasticity, for example the ability of the brain to change in response to new stimuli, only in recent years have we begun to understand how NO acts on the molecular and cellular level to orchestrate such important phenomena as synaptic plasticity (modification of the strength of existing synapses) or the formation of new synapses (synaptogenesis) and new neurons (neurogenesis). Post-translational modification of proteins by NO derivatives or reactive nitrogen species is a non-classical mechanism for signalling by NO. S-nitrosation is a reversible post-translational modification of thiol groups (mainly on cysteines) that may result in a change of function of the modified protein. S-nitrosation of key target proteins has emerged as a main regulatory mechanism by which NO can influence several levels of brain plasticity, which are reviewed in this work. Understanding how S-nitrosation contributes to neural plasticity can help us to better understand the physiology of these processes, and to better address pathological changes in plasticity that are involved in the pathophysiology of several neurological diseases. Linked ArticlesThis article is part of a themed section on Pharmacology of the Gasotransmitters. To view the other articles in this section visit
Enhancement of endogenous neurogenesis by nitric oxide: identification of s-nitrosylation targets
Publication . Santos, Ana Isabel Reis dos; Araújo, Inês; Ruiz, Antonio Martínez
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.
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
SFRH
Funding Award Number
SFRH/BD/77903/2011