Papers by Roberto Paes de Carvalho
Sinalização purinérgica: implicações fisiopatológicas
All the contents of this work, except where otherwise noted, is licensed under a Creative Commons... more All the contents of this work, except where otherwise noted, is licensed under a Creative Commons Attribution 4.0 International license. Todo o conteúdo deste trabalho, exceto quando houver ressalva, é publicado sob a licença Creative Commons Atribição 4.0. Todo el contenido de esta obra, excepto donde se indique lo contrario, está bajo licencia de la licencia Creative Commons Reconocimento 4.0.
Neural plasticity, 2018
The regenerative capacity of CNS tracts has ever been a great hurdle to regenerative medicine. Al... more The regenerative capacity of CNS tracts has ever been a great hurdle to regenerative medicine. Although recent studies have described strategies to stimulate retinal ganglion cells (RGCs) to regenerate axons through the optic nerve, it still remains to be elucidated how these therapies modulate the inhibitory environment of CNS. Thus, the present work investigated the environmental content of the repulsive axon guidance cues, such as Sema3D and its receptors, myelin debris, and astrogliosis, within the regenerating optic nerve of mice submitted to intraocular inflammation + cAMP combined to conditional deletion of PTEN in RGC after optic nerve crush. We show here that treatment was able to promote axonal regeneration through the optic nerve and reach visual targets at twelve weeks after injury. The Regenerating group presented reduced MBP levels, increased microglia/macrophage number, and reduced astrocyte reactivity and CSPG content following optic nerve injury. In addition, Sema3D...
International Journal of Developmental Neuroscience, 2006
Nitric oxide (NO) is an intercellular messenger involved in many physiological and pathological p... more Nitric oxide (NO) is an intercellular messenger involved in many physiological and pathological processes of vertebrate and invertebrate animal tissues. In the embryonic chick retina, nitric oxide synthase (NOS) activity and a system for l‐arginine transport between neurons and glial cells were described, supporting the idea that nitric oxide is a critical molecule during retinal development. In the present work we show that nitric oxide is a modulator of cell proliferation in chick embryo retina. Mixed cultures of retinal neurons and glial cells were submitted to [3H]‐thymidine incorporation after drug treatment. Incubation for 24 h with the NO donors S‐nitroso‐N‐acetyl‐penicillamine (SNAP) or Spermine nitric oxide (SpNO) complex promoted a decrease of approximately 70% of [3H]‐thymidine incorporation in a dose‐dependent manner. SNAP did not increase Lactate dehydrogenase release and its effect was not mimicked by 8‐bromo cyclic GMP, or blocked by the guanylate cyclase inhibitor 1H...
Neuroscience, 2009
Adenosine is a neuromodulator implicated in nervous system development and plasticity and its eff... more Adenosine is a neuromodulator implicated in nervous system development and plasticity and its effects are mediated by inhibitory (A 1 , A 3) and excitatory (A 2a , A 2b) receptors. The role of adenosine in the synaptic activity depends mainly on a balanced activation of A 1 and A 2a receptors which are activated by various ranges of adenosine concentrations. Herein, we investigated the expression of A 1 and A 2a receptors and also the accumulation of cAMP in the superior colliculus at different stages of development. Furthermore, we examined the effects of an acute in vivo blockade of adenosine deaminase during the critical period when the elimination of misplaced axons/terminals takes place with a simultaneous fine tuning of terminal arbors into appropriate terminal zones. Lister Hooded rats ranging from postnatal days (PND) 0-70 were used for ontogeny studies. Our results indicate that A 1 expression in the visual layers of the superior colliculus is higher until PND 28, while A 2a expression increases after PND 28 in a complementary developmental pattern. Accordingly, the incubation of collicular slices with 5=-N-ethylcarboxamido-adenosine, a non-specific adenosine receptor agonist, showed a significant reduction in cAMP accumulation at PND 14 and an increase in adults. For the anatomical studies, the uncrossed retinotectal projections were traced after the intraocular injection of horseradish peroxidase. One group received daily injections of an adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl adenine), 10 mg/kg i.p.) between PND 10 and 13, while control groups were treated with vehicle injections (NaCl 0.9%, i.p.). We found that a short-term blockade of adenosine deaminase during the second postnatal week induced an expansion of retinotectal terminal fields in the rostrocaudal axis of the tectum. Taken together, the results suggest that a balance of purinergic A 1 and A 2a receptors through cAMP signaling plays a pivotal role during the development of topographic order in the retinotectal pathway.
Journal of Neurochemistry, 2007
Nitric oxide (NO) is an important signaling molecule in the CNS, regulating neuronal survival, pr... more Nitric oxide (NO) is an important signaling molecule in the CNS, regulating neuronal survival, proliferation and differentiation. Here, we explored the mechanism by which NO, produced from the NO donor S‐nitroso‐acetyl‐d‐l‐penicillamine (SNAP), exerts its neuroprotective effect in purified cultures of chick retinal neurons. Cultures prepared from 8‐day‐old chick embryo retinas and incubated for 24 h (1 day in culture, C1) were treated or not with SNAP, incubated for a further 72 h (up to 4 days in culture, C4), fixed, and the number of cells estimated, or processed for cell death estimation, by measuring the reduction of the metabolic dye 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). Experimental cultures were run in parallel but were re‐fed with fresh medium in the absence or presence of SNAP at culture day 3 (C3), incubated for a further 24 h up to C4, then fixed or processed for the MTT assay. Previous studies showed that the re‐feeding procedure promotes ex...
Journal of Neurochemistry, 2006
Journal of Neurochemistry, 2008
Cell Death & Differentiation, 2014
During early neurogenesis, retinal neuronal cells display a conserved differentiation program in ... more During early neurogenesis, retinal neuronal cells display a conserved differentiation program in vertebrates. Previous studies established that nitric oxide (NO) and cGMP accumulation regulate essential events in retinal physiology. Here we used pharmacological and genetic loss-of-function to investigate the effects of NO and its downstream signaling pathway in the survival of developing avian retinal neurons in vitro and in vivo. Six-day-old (E6) chick retinal cells displayed increased calcium influx and produced higher amounts of NO when compared with E8 cells. L-arginine (substrate for NO biosynthesis) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP; a nitrosothiol NO donor) promoted extensive cell death in E6 retinas, whereas in E8 both substances decreased apoptosis. The effect of NO at both periods was mediated by soluble guanylyl cyclase (sGC) and cGMP-dependent kinase (cGK) activation. In addition, shRNA-mediated cGKII knockdown prevented NO-induced cell death (E6) and cell survival (E8). This, NO-induced cell death or cell survival was not correlated with an early inhibition of retinal cell proliferation. E6 cells also responded differentially from E8 neurons regarding cyclic AMP-responsive element-binding protein (CREB) activation in the retina in vivo. NO strongly decreased nuclear phospho-CREB staining in E6 but it robustly enhanced CREB phosphorylation in the nuclei of E8 neurons, an effect that was completely abrogated by cGKII shRNAs at both embryonic stages. The ability of NO in regulating CREB differentially during retinal development relied on the capacity of cGKII in decreasing (E6) or increasing (E8) nuclear AKT (V-Akt murine thymoma viral oncogene) activation. Accordingly, inhibiting AKT prevented both cGKII shRNA-mediated CREB upregulation in E6 and SNAP-induced CREB activation in E8. Furthermore, shRNAmediated in vivo cGKII or in vitro CREB1 knockdown confirmed that NO/cGKII dualistically regulated the downstream CREB1 pathway and caspase activation in the chick retina to modulate neuronal viability. These data demonstrate that NO-mediated cGKII signaling may function to control the viability of neuronal cells during early retinal development via AKT/CREB1 activity.
The Open Nitric Oxide Journal, 2013
The retina is a highly organized structure responsible for transducing light stimulation into ele... more The retina is a highly organized structure responsible for transducing light stimulation into electrical responses. Retinal organization is conserved in all vertebrate species and the generation of retinal cell types is chronologically determined and fairly documented from amphibians to humans. Furthermore, the chick retina is a well-established experimental paradigm for neurochemical and developmental studies regarding the nervous system. Among many signaling molecules regulating retinal physiology, nitric oxide (NO) is likely to play a prominent role within the retina. NO is a gaseous signaling transmitter, which regulates a plethora of physiological functions within an organism, including high-order signaling events both in the developing and mature nervous system. In this review we focus on different aspects of NO signaling in regulating retinal cell neurochemistry, focusing mainly on developing chick retina as a prevalent experimental model. Based on literature and data gathered from our group we conclude that NO is a major atypical neurotransmitter in the retina, regulating signaling events associated with the development of embryonic retinal neurons and glial cells.
Free Radical Biology and Medicine
Adenosine is an important neuromodulator in the CNS, regulating neuronal survival and synaptic tr... more Adenosine is an important neuromodulator in the CNS, regulating neuronal survival and synaptic transmission. The antioxidant ascorbate (the reduced form of vitamin C) is concentrated in CNS neurons through a sodium-dependent transporter named SVCT2 and participates in several CNS processes, for instance, the regulation of glutamate receptors functioning and the synthesis of neuromodulators. Here we studied the interplay between the adenosinergic system and ascorbate transport in neurons. We found that selective activation of A3, but not of A1 or A2a, adenosine receptors modulated ascorbate transport, decreasing intracellular ascorbate content. Förster resonance energy transfer (FRET) analyses showed that A3 receptors associate with the ascorbate transporter SVCT2, suggesting tight signaling compartmentalization between A3 receptors and SVCT2. The activation of A3 receptors increased ascorbate release in an SVCT2-dependent manner, which largely altered the neuronal redox status without interfering with cell death, glycolytic metabolism, and bioenergetics. Overall, by regulating vitamin C transport, the adenosinergic system (via activation of A3 receptors) can regulate ascorbate bioavailability and control the redox balance in neurons.
Free Radical Biology and Medicine, 2018
Microglia are resident myeloid cells of the central nervous system that are critical for brain fu... more Microglia are resident myeloid cells of the central nervous system that are critical for brain functioning in health and disease. Using tissue-specific conditional gene targeting in mice, together with other methodologies, we have revealed essential functions for the of the Rho family of GTPases in regulating microglia homeostasis and brain physiology. In particular, we showed that microglia-specific ablation of RhoA in adult mice profoundly disrupted the homeostasis of brain microglia, resulting in the production of several mediators of inflammation, including reactive oxygen species, which impaired long-term synaptic plasticity and led to cognitive deficits. We found that RhoA exerted its microglial homeostatic functions by sustaining C-terminal Src kinase (Csk) negative regulation of c-Src tyrosine kinase activity. Accordingly, inhibition of Src with a clinically-relevant inhibitor almost completely restored microglia homeostasis and normal cognitive performance. Overall, our wor...
Neuroscience, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Frontiers in Neuroscience, 2019
Ascorbate, the reduced form of Vitamin C, is one of the most abundant and important low-molecular... more Ascorbate, the reduced form of Vitamin C, is one of the most abundant and important low-molecular weight antioxidants in living tissues. Most animals synthesize vitamin C, but some primates, including humans, have lost this capacity due to disruption in L-gulono-gamma-lactone oxidase gene. Because of this incapacity, those animals must obtain Vitamin C from the diet. Ascorbate is highly concentrated in the central nervous system (CNS), including the retina, and plays essential roles in neuronal physiology. Ascorbate transport into cells is controlled by Sodium Vitamin C Co-Transporters (SVCTs). There are four SVCT isoforms and SVCT2 is the major isoform controlling ascorbate transport in the CNS. Regarding ascorbate release from retinal neurons, Glutamate, by activating its ionotropic receptors leads to ascorbate release via the reversion of SVCT2. Moreover, dopamine, via activation of D 1 receptor/cyclic AMP/EPAC2 pathway, also induces ascorbate release via SVCT2 reversion. Because the dopaminergic and glutamatergic systems are interconnected in the CNS, we hypothesized that dopamine could regulate ascorbate release indirectly, via the glutamatergic system. Here we reveal that dopamine increases the release of D-Aspartate from retinal neurons in a way independent on calcium ions and dependent on excitatory amino acid transporters. In addition, dopamine-dependent SVCT2 reversion leading to ascorbate release occurs by activation of AMPA/Kainate receptors and downstream ERK/AKT pathways. Overall, our data reveal a dopamine-to-glutamate signaling that regulates the bioavailability of ascorbate in neuronal cells.
Biochemical pharmacology, Jan 19, 2018
Chlorogenic acids (CGAs) are a group of phenolic compounds found in worldwide consumed beverages ... more Chlorogenic acids (CGAs) are a group of phenolic compounds found in worldwide consumed beverages such as coffee and green tea. They are synthesized from an esterification reaction between cinnamic acids, including caffeic (CFA), ferulic and p-coumaric acids with quinic acid (QA), forming several mono- and di-esterified isomers. The most prevalent and studied compounds are 3-O-caffeoylquinic acid (3-CQA), 4-Ocaffeoylquinic acid (4-CQA) and 5-O-caffeoylquinic acid (5-CQA), widely described as having antioxidant and cell protection effects. CGAs can also modulate glutamate release from microglia by a mechanism involving a decrease of reactive oxygen species (ROS). Increased energy metabolism is highly associated with enhancement of ROS production and cellular damage. Glutamate can also be used as an energy source by glutamate dehydrogenase (GDH) enzyme, providing α-ketoglutarate to the tricarboxylic acid (TCA) cycle for ATP synthesis. High GDH activity is associated with some disorders...
Journal of neurochemistry, Jan 21, 2017
Vitamin C (in the reduced form ascorbate or in the oxidized form dehydroascorbate) is implicated ... more Vitamin C (in the reduced form ascorbate or in the oxidized form dehydroascorbate) is implicated in signaling events throughout the central nervous system (CNS). In the retina, a high-affinity transport system for ascorbate has been described and glutamatergic signaling has been reported to control ascorbate release. Here, we investigated the modulatory role played by vitamin C upon glutamate uptake and N-methyl-d-aspartate (NMDA) receptor activation in cultured retinal cells or in intact retinal tissue using biochemical and imaging techniques. We show that both forms of vitamin C, ascorbate or dehydroascorbate, promote an accumulation of extracellular glutamate by a mechanism involving the inhibition of glutamate uptake. This inhibition correlates with the finding that ascorbate promotes a decrease in cell surface levels of the neuronal glutamate transporter excitatory amino acid transporter 3 in retinal neuronal cultures. Interestingly, vitamin C is prone to increase the activity ...
Science signaling, Jan 28, 2017
Vitamin C is essential for the development and function of the central nervous system (CNS). The ... more Vitamin C is essential for the development and function of the central nervous system (CNS). The plasma membrane sodium-vitamin C cotransporter 2 (SVCT2) is the primary mediator of vitamin C uptake in neurons. SVCT2 specifically transports ascorbate, the reduced form of vitamin C, which acts as a reducing agent. We demonstrated that ascorbate uptake through SVCT2 was critical for the homeostasis of microglia, the resident myeloid cells of the CNS that are essential for proper functioning of the nervous tissue. We found that depletion of SVCT2 from the plasma membrane triggered a proinflammatory phenotype in microglia and resulted in microglia activation. Src-mediated phosphorylation of caveolin-1 on Tyr(14) in microglia induced the internalization of SVCT2. Ascorbate treatment, SVCT2 overexpression, or blocking SVCT2 internalization prevented the activation of microglia. Overall, our work demonstrates the importance of the ascorbate transport system for microglial homeostasis and hi...
Scientific reports, Jan 18, 2017
Dopamine and glutamate are critical neurotransmitters involved in light-induced synaptic activity... more Dopamine and glutamate are critical neurotransmitters involved in light-induced synaptic activity in the retina. In brain neurons, dopamine D1 receptors (D1Rs) and the cytosolic protein tyrosine kinase Src can, independently, modulate the behavior of NMDA-type glutamate receptors (NMDARs). Here we studied the interplay between D1Rs, Src and NMDARs in retinal neurons. We reveal that dopamine-mediated D1R stimulation provoked NMDAR hypofunction in retinal neurons by attenuating NMDA-gated currents, by preventing NMDA-elicited calcium mobilization and by decreasing the phosphorylation of NMDAR subunit GluN2B. This dopamine effect was dependent on upregulation of the canonical D1R/adenylyl cyclase/cAMP/PKA pathway, of PKA-induced activation of C-terminal Src kinase (Csk) and of Src inhibition. Accordingly, knocking down Csk or overexpressing a Csk phosphoresistant Src mutant abrogated the dopamine-induced NMDAR hypofunction. Overall, the interplay between dopamine and NMDAR hypofunction...
Analytical Methods, 2016
A HPLC-UV method has been developed and validated for the determination of ascorbic acid in chick... more A HPLC-UV method has been developed and validated for the determination of ascorbic acid in chicken embryo retina.
Neurochemical Research, 2003
Adenosine modulates the survival of chick embryo retinal neurons in culture. When cultures were i... more Adenosine modulates the survival of chick embryo retinal neurons in culture. When cultures were incubated for 3 days and refed with fresh medium, a large proportion of neurons died in the subsequent 3 days of culture. This cell death was prevented by preincubation of cultures for at least 24?h with adenosine plus the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an
Neurochemistry International, 1998
The regulation of adenylate cyclase by neurotransmitters is observed in early development of the ... more The regulation of adenylate cyclase by neurotransmitters is observed in early development of the chick retina[ In the present work we show that L!1!amine!3!phosphonobutyric acid "L!AP3#\ the major agonist of group III metabotropic glutamate receptors "mGluRs#\ inhibits the accumulation of cyclic AMP induced by forskolin in the chick retina[ This e}ect is observed after 7 days of development "E7#\ is maximal from E01ÐE06 and decreases at the post!hatching period "PH#[ The inhibition is also observed in cultures of retinal cells incubated for 1Ð7 days[ We have also investigated the interaction between group III mGluRs and other receptors coupled to adenylate cyclase in the developing retina[ The inhibition by L!AP3 is partially additive with that induced by the A0 adenosine agonist Cyclohexyladenosine and is not observed when cyclic AMP levels are increased with 1!chloroadenosine or dopamine[ The group II mGluR agonist trans!"0S\2R#!0!amino!cyclopentanedicarboxylic acid has an inhibitory e}ect only on PH retinas\ indicating that group II and group III mGluRs have a di}erential ontogenesis in this tissue[ The results show that Group III mGluRs are expressed early during chick retina development and do not interact with other receptors known to be coupled to adenylate cyclase in the developing retina[ Þ 0887 Elsevier Science Ltd[ All rights reserved[
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Papers by Roberto Paes de Carvalho