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Advisor(s)
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.
Description
Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016
Keywords
Neurogenesis Neural stem cells Nitric oxide signaling S-nitrosylation Brain injury