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- Specificity in S-Nitrosylation: a short-range mechanism for NO signaling?Publication . Martinez-Ruiz, Antonio; Araújo, Inês; Izquierdo-Alvarez, Alicia; Hernansanz-Agustin, Pablo; Lamas, Santiago; Serrador, Juan M.Significance: Nitric oxide (NO) classical and less classical signaling mechanisms (through interaction with soluble guanylate cyclase and cytochrome c oxidase, respectively) operate through direct binding of NO to protein metal centers, and rely on diffusibility of the NO molecule. S-Nitrosylation, a covalent post-translational modification of protein cysteines, has emerged as a paradigm of nonclassical NO signaling. Recent Advances: Several nonenzymatic mechanisms for S-nitrosylation formation and destruction have been described. Enzymatic mechanisms for transnitrosylation and denitrosylation have been also studied as regulators of the modification of specific subsets of proteins. The advancement of modification-specific proteomic methodologies has allowed progress in the study of diverse S-nitrosoproteomes, raising clues and questions about the parameters for determining the protein specificity of the modification. Critical Issues: We propose that S-nitrosylation is mainly a short-range mechanism of NO signaling, exerted in a relatively limited range of action around the NO sources, and tightly related to the very controlled regulation of subcellular localization of nitric oxide synthases. We review the nonenzymatic and enzymatic mechanisms that support this concept, as well as physiological examples of mammalian systems that illustrate well the precise compartmentalization of S-nitrosylation. Future Directions: Individual and proteomic studies of protein S-nitrosylation-based signaling should take into account the subcellular localization in order to gain further insight into the functional role of this modification in (patho)physiological settings. Antioxid. Redox Signal. 19, 1220-1235.
- Ultrasensitive gold micro-structured electrodes enabling the detection of extra-cellular long-lasting potentials in astrocytes populationsPublication . Mestre, Ana L. G.; Cerquido, Monica; INÁCIO, PEDRO; Asgarifar, Sanaz; Lourenco, Ana S.; Lurdes S. Cristiano, M.; Aguiar, Paulo; Medeiros, Maria C. R.; Araújo, Inês; Ventura, Joao; Gomes, Henrique L.Ultra-sensitive electrodes for extracellular recordings were fabricated and electrically characterized. A signal detection limit defined by a noise level of 0.3-0.4 mu V for a bandwidth of 12.5 Hz was achieved. To obtain this high sensitivity, large area (4 mm(2)) electrodes were used. The electrode surface is also micro-structured with an array of gold mushroom-like shapes to further enhance the active area. In comparison with a flat gold surface, the micro-structured surface increases the capacitance of the electrode/electrolyte interface by 54%. The electrode low impedance and low noise enable the detection of weak and low frequency quasi-periodic signals produced by astrocytes populations that thus far had remained inaccessible using conventional extracellular electrodes. Signals with 5 mu V in amplitude and lasting for 5-10 s were measured, with a peak-to-peak signal-to-noise ratio of 16. The electrodes and the methodology developed here can be used as an ultrasensitive electrophysiological tool to reveal the synchronization dynamics of ultra-slow ionic signalling between non-electrogenic cells.
- Regulation of injury-induced neurogenesis by Nitric OxidePublication . Carreira, Bruno P.; Carvalho, Caetana M.; Araújo, InêsThe finding that neural stem cells (NSCs) are able to divide, migrate, and differentiate into several cellular types in the adult brain raised a new hope for restorative neurology. Nitric oxide (NO), a pleiotropic signaling molecule in the central nervous system (CNS), has been described to be able to modulate neurogenesis, acting as a pro-or antineurogenic agent. Some authors suggest that NO is a physiological inhibitor of neurogenesis, while others described NO to favor neurogenesis, particularly under inflammatory conditions. Thus, targeting the NO system may be a powerful strategy to control the formation of new neurons. However, the exact mechanisms by which NO regulates neural proliferation and differentiation are not yet completely clarified. In this paper we will discuss the potential interest of the modulation of the NO system for the treatment of neurodegenerative diseases or other pathological conditions that may affect the CNS.
- Identification of new targets of S-nitrosylation in neural stem cells by thiol redox proteomicsPublication . 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, InêsNitric 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.
- New fluorescent probes based on gallium(III) corrole complexes for the recognition of hydrogen sulfide: a journey from solution to intracellular sitePublication . Santos, Carla I.M.; Santiago, Ana M.; Araújo, Ana R.L.; Pinto, Sandra; Agostinho, Rafaela; Simão, Sónia; Azevedo, Tomás Pais de; Antunes, Catarina; Faustino, M. Amparo F.; Araujo, Ines; Neves, M. Graça P.M.S.; Martinho, José M.G.; Maçôas, Ermelinda M.S.In this work, three fluorescent probes for detection of hydrogen sulfide (H2S) where prepared based on gallium (III) corrole complexes bearing nitro groups at beta-pyrrolic positions. Two of the compounds selected, the 3-nitro5,10,15- tris(pentafluorophenyl)corrolatogallium(III)(pyridine) (CGa-NO2) and the 3,17-dinitro-5,10,15-tris (pentafluorophenyl)corrolatogallium(III)(pyridine) (CGa-2NO2) present one and two nitro groups directly linked to the beta-pyrrolic position. The third compound, the (E)-3-(2-nitroprop-1-en-1-yl)-5,10,15-tris(pentafluorophenyl)corrolatogallium(III)(pyridine) (CGa-EtNO2), has a carbon-carbon double bond spacer between the corrole unit and the nitro group. All these derivatives were obtained from 5,10,15-tris(pentafluorophenyl)corrolatogallium(III)(pyridine) (CGa). The precursor CGa and the derivative CGa-EtNO2 behaved as turn-OFF probes, while compound CGa-NO2 responded as a turn-ON probe in the presence of H2S in the pH range of 5-9. Mechanistic studies show that the interaction of H2S with the probes involves its coordination with gallium(III) and in some cases the reduction of the nitro group to a new aminated corrole. While the formation of the coordination complex with H2S is almost immediate, the kinetics of the reduction is slow. Interestingly, for CGaNO2 the two processes can be explored in a ratiometric sensing of H2S in a non-aqueous solution showing a good linearity over an extended concentration range (5-200 mu M). The response of the corroles to H2S in intracellular medium was studied in 2D cultured cells (HeLa).
- Ear wound regeneration in the African spiny mouse Acomys cahirinusPublication . Santos Matias, Dino; Rita, Ana Martins; Casanellas, Ignasi; Ova, Adelia Brito; Araújo, Inês; Power, Deborah; Tiscornia, GustavoWhile regeneration occurs in a number of taxonomic groups across the Metazoa, there are very few reports of regeneration in mammals, which generally respond to wounding with fibrotic scarring rather than regeneration. A recent report described skin shedding, skin regeneration and extensive ear punch closure in two rodent species, Acomys kempi and Acomys percivali. We examined these striking results by testing the capacity for regeneration of a third species, Acomys cahirinus, and found a remarkable capacity to repair full thickness circular punches in the ear pinna. Four-millimeter-diameter wounds closed completely in 2 months in 100% of ear punches tested. Histology showed extensive formation of elastic cartilage, adipose tissue, dermis, epidermis and abundant hair follicles in the repaired region. Furthermore, we demonstrated abundant angiogenesis and unequivocal presence of both muscle and nerve fibers in the reconstituted region; in contrast, similar wounds in C57BL/6 mice simply healed the borders of the cut by fibrotic scarring. Our results confirm the regenerative capabilities of Acomys, and suggest this model merits further attention.
- Differential contribution of the guanylyl cyclase-cyclic GMP-protein kinase g pathway to the proliferation of neural stem cells stimulated by nitric oxidePublication . Carreira, Bruno P.; Morte, Maria Inêss; Lourenço, Ana Sofia; Santos, Ana Isabel; Inácio, Ângela; Ambrósio, António F.; Carvalho, Caetana M.; Araújo, InêsNitric oxide (NO) is an important inflammatory mediator involved in the initial boost in the proliferation of neural stem cells following brain injury. However, the mechanisms underlying the proliferative effect of NO are still unclear. The aim of this work was to investigate whether cyclic GMP (cGMP) and the cGMP-dependent kinase (PKG) are involved in the proliferative effect triggered by NO in neural stem cells. For this purpose, cultures of neural stem cells isolated from the mouse subventricular zone (SVZ) were used. We observed that long-term exposure to the NO donor (24 h), NOC-18, increased the proliferation of SVZ cells in a cGMP-dependent manner, since the guanylate cyclase inhibitor, ODQ, prevented cell proliferation. Similarly to NOC-18, the cGMP analogue, 8-Br-cGMP, also increased cell proliferation. Interestingly, shorter exposures to NO (6 h) increased cell proliferation in a cGMP-independent manner via the ERK/MAP kinase pathway. The selective inhibitor of PKG, KT5823, prevented the proliferative effect induced by NO at 24 h but not at 6 h. In conclusion, the proliferative effect of NO is initially mediated by the ERK/MAPK pathway, and at later stages by the GC/cGMP/PKG pathway. Thus, our work shows that NO induces neural stem cell proliferation by targeting these two pathways in a biphasic manner. Copyright (C) 2012 S. Karger AG, Basel
- Deregulation of the retinal renin-angiotensin system precedes the onset of diabetic retinopathyPublication . S, Simão; Bitoque, D. B.; Santos, D. F.; Araújo, Inês; Silva, G. A.The renin-angiotensin system (RAS) is a set of complex pathways with a well-defined function in the regulation of blood pressure and body fluid homeostasis.
- Stimulation of neural stem cell proliferation by inhibition of phosphodiesterase 5Publication . Santos, Ana Isabel; Carreira, Bruno P.; Nobre, Rui Jorge; Carvalho, Caetana M.; Araújo, InêsThe involvement of nitric oxide (NO) and cyclic GMP (cGMP) in neurogenesis has been progressively unmasked over the last decade. Phosphodiesterase 5 (PDE5) specifically degrades cGMP and is highly abundant in the mammalian brain. Inhibition of cGMP hydrolysis by blocking PDE5 is a possible strategy to enhance the first step of neurogenesis, proliferation of neural stem cells (NSC). In this work, we have studied the effect on cell proliferation of 3 inhibitors with different selectivity and potency for PDE5, T0156, sildenafil, and zaprinast, using subventricular zone-(SVZ-) derived NSC cultures. We observed that a short-(6 h) or a long-term (24 h) treatment with PDE5 inhibitors increased SVZ-derived NSC proliferation. Cell proliferation induced by PDE5 inhibitors was dependent on the activation of the mitogen-activated protein kinase (MAPK) and was abolished by inhibitors of MAPK signaling, soluble guanylyl cyclase, and protein kinase G. Moreover, sildenafil neither activated ERK1/2 nor altered p27(Kip1) levels, suggesting the involvement of pathways different from those activated by T0156 or zaprinast. In agreement with the present results, PDE5 inhibitors may be an interesting therapeutic approach for enhancing the proliferation stage of adult neurogenesis.
- Rewired glycosylation activity promotes scarless regeneration and functional recovery in spiny mice after complete spinal cord transectionPublication . Nogueira-Rodrigues, Joana; Leite, Sérgio C.; Pinto-Costa, Rita; Sousa, Sara C.; Luz, Liliana L.; Sintra, Maria A.; Oliveira, Raquel; Monteiro, Ana C.; Pinheiro, Gonçalo; Vitorino, Marta; Silva, Joana A.; S, Simão; Vitor Fernandes, Dr; Provazník, Jan; Benes, Vladimir; Cruz, Célia D.; Safronov, Boris V.; Magalhães, Ana; Reis, Celso A.; Vieira, Jorge; Vieira, Cristina P.; Tiscórnia, Gustavo; Araujo, Ines; Sousa, Mónica M.Regeneration of adult mammalian central nervous system (CNS) axons is abortive, resulting in inability to recover function after CNS lesion, including spinal cord injury (SCI). Here, we show that the spiny mouse (Acomys) is an exception to other mammals, being capable of spontaneous and fast restoration of function after severe SCI, re-establishing hind limb coordination. Remarkably, Acomys assembles a scarless pro-regenerative tissue at the injury site, providing a unique structural continuity of the initial spinal cord geometry. The Acomys SCI site shows robust axon regeneration of multiple tracts, synapse formation, and electrophysiological signal propagation. Transcriptomic analysis of the spinal cord following transcriptome reconstruction revealed that Acomys rewires glycosylation biosynthetic pathways, culminating in a specific pro-regenerative proteoglycan signature at SCI site. Our work uncovers that a glycosylation switch is critical for axon regeneration after SCI and identifies beta 3gnt7, a crucial enzyme of keratan sulfate biosynthesis, as an enhancer of axon growth.
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