Alvaro Estevez | University of Central Florida (original) (raw)

Papers by Alvaro Estevez

The Journal of Neuroscience, Jul 25, 2007

Vascular endothelial growth factor (VEGF) plays a neuroprotective role in mice harboring mutation... more Vascular endothelial growth factor (VEGF) plays a neuroprotective role in mice harboring mutations of copper-zinc superoxide dismutase 1 (SOD1) in familial amyotrophic lateral sclerosis (ALS). Conversely, the loss of VEGF expression through genetic depletion can give rise to a phenotype resembling ALS independent of SOD1 mutations. Here, we observe a profound downregulation of VEGF mRNA expression in spinal cords of G93A SOD1 mice that occurred early in the course of the disease. Using an in vitro culture model of glial cells expressing mutant SOD1, we demonstrate destabilization and downregulation of VEGF RNA with concomitant loss of protein expression that correlates with level of transgene expression. Using a luciferase reporter assay, we show that this molecular effect is mediated through a portion of the VEGF 3Ј-untranslated region (UTR) that harbors a class II adenylate/uridylate-rich element. Other mutant forms of SOD1 produced a similar negative effect on luciferase RNA and protein expression. Mobility shift assay with a VEGF 3Ј-UTR probe reveals an aberrantly migrating complex that contains mutant SOD1. We further show that the RNA stabilizing protein, HuR (human antigen R), is translocated from nucleus to cytoplasm in mutant SOD1 cells in vitro and mouse motor neurons in vivo. In summary, our data suggest that mutant SOD1 gains a novel function, possibly by altering the ribonucleoprotein complex with the VEGF 3Ј-UTR. We postulate that the resultant dysregulation of VEGF posttranscriptional processing critically reduces the level of this neuroprotective growth factor and accelerates the neurodegenerative process in ALS.

Free Radical Biology and Medicine

In vivo (Athens, Greece)

Background: Overproduction of nitric oxide (NO) has been recognized as a major mechanism of neuro... more Background: Overproduction of nitric oxide (NO) has been recognized as a major mechanism of neurotoxicity. NO reacts with superoxide to generate peroxynitrite, a strong oxidizing and nitrating species. Peroxynitrite is formed in glial cells and degenerating neurons in neuropathological conditions, including amyotrophic lateral sclerosis (ALS). In ALS, motor neurons re-express the p75 neurotrophin receptor (p75 NTR ) and might become vulnerable to NGF. In the present study, we investigated whether peroxynitrite stimulated nerve growth factor (NGF) expression in spinal cord astrocytes. Materials and Methods: Astrocyte monolayers were exposed to peroxynitrite and nitrotyrosine formation was determined by immunofluorescence. mRNA levels for NGF, brain derived neutrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) were measured by semi-quantitative RT-PCR and NGF release was determined by ELISA. Results and Discussion: A single exposure to peroxynitrite specifically induced NGF expression and secretion in astrocytes coincident with reactive morphological changes and increased nitrotyrosine immunoreactivity. These results suggest that NGF expression in reactive astrocytes is under the control of oxidative stress.

Journal of Biological Chemistry

Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correla... more Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.

Journal of Neuroscience, 2007

Journal of Neuroscience, 2012

Mutant SOD1 has pleiotropic toxic effects on motor neurons, among which mitochondrial dysfunction... more Mutant SOD1 has pleiotropic toxic effects on motor neurons, among which mitochondrial dysfunction has been proposed as one of the contributing factors in motor neuron demise. Mitochondria are highly dynamic in neurons; they are constantly reshaped by fusion and move along neurites to localize at sites of high-energy utilization, such as synapses. The finding of abnormal mitochondria accumulation in neuromuscular junctions, where the SOD1-FALS degenerative process is though to initiate, suggests that impaired mitochondrial dynamics in motor neurons may be involved in pathogenesis. We addressed this hypothesis by live imaging microscopy of photo-switchable fluorescent mitoDendra in transgenic rat motor neurons expressing mutant or wild-type human SOD1. We demonstrate that mutant SOD1 motor neurons have impaired mitochondrial fusion in axons and cell bodies. Mitochondria also display selective impairment of retrograde axonal transport, with reduced frequency and velocity of movements. Fusion and transport defects are associated with smaller mitochondrial size, decreased mitochondrial density, and defective mitochondrial membrane potential. Furthermore, mislocalization of mitochondria at synapses among motor neurons, in vitro, correlates with abnormal synaptic number, structure, and function. Dynamics abnormalities are specific to mutant SOD1 motor neuron mitochondria, since they are absent in wild-type SOD1 motor neurons, they do not involve other organelles, and they are not found in cortical neurons. Together, these results suggest that impaired mitochondrial dynamics may contribute to the selective degeneration of motor neurons in SOD1-FALS.

Seminars in Perinatology, 2000

Many of the actions of nitric oxide are not due to nitric oxide itself, but rather by the seconda... more Many of the actions of nitric oxide are not due to nitric oxide itself, but rather by the secondary formation of oxidants like peroxynitrite. Peroxynitrite leaves a footprint in the nitration of tyrosine, which helps track the formation of reactive nitric oxide-derived species in diseases and even normal development.

Proceedings of the National Academy of Sciences, 2003

There has been a longstanding debate regarding the role of proteolysis in Huntington's disease. T... more There has been a longstanding debate regarding the role of proteolysis in Huntington's disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntington's disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is ''downstream'' of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival.

Proceedings of the National Academy of Sciences, 1999

NO 2 Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitric oxide-derived specie... more NO 2 Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitric oxide-derived species and has been implicated in the pathology of diverse human diseases. Nitration of active-site tyrosine residues is known to compromise protein structure and function. Although free NO 2 Tyr is produced in abundant concentrations under pathological conditions, its capacity to alter protein structure and function at the translational or posttranslational level is unknown. Here, we report that free NO 2 Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl terminus of ␣-tubulin via a posttranslational mechanism catalyzed by the enzyme tubulin-tyrosine ligase. In contrast to the enzymatically regulated carboxylterminal tyrosination͞detyrosination cycle of ␣-tubulin, incorporation of NO 2 Tyr shows apparent irreversibility. Nitrotyrosination of ␣-tubulin induces alterations in cell morphology, changes in microtubule organization, loss of epithelialbarrier function, and intracellular redistribution of the motor protein cytoplasmic dynein. These observations imply that posttranslational nitrotyrosination of ␣-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface-exposed carboxyl terminus of ␣-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of motorand microtubule-associated proteins. Collectively, these observations illustrate a mechanism whereby free NO 2 Tyr can impact deleteriously on cell function under pathological conditions encompassing reactive nitrogen species production. The data also yield further insight into the role that the ␣-tubulin tyrosination͞detyrosination cycle plays in microtubule function.

Neurotoxicity Research, 2012

Oxidative damage is a common and early feature of Alzheimer&amp;amp;amp;amp;amp;amp;amp;a... more Oxidative damage is a common and early feature of Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease, Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease, amyotrophic lateral sclerosis (ALS), and other neurodegenerative disorders. Dr. Mark Smith and his colleagues have built the case for oxidative stress being a primary progenitor rather than a secondary end-stage epiphenomenon of neurodegeneration. They proposed that reactive oxygen species contribute to the &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;age-related cascade of neurodegeneration,&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; whereby accumulative oxidative damage with age promotes other characteristic pathological changes in afflicted brain regions, including protein aggregation, metabolic deficiencies, and inflammation. Nitric oxide (NO) likely plays a critical role in this age-related cascade. NO is a major signaling molecule produced in the central nervous system to modulate neurological activity through stimulating cyclic GMP synthesis. However, the same physiological concentrations of NO, relevant in cellular signaling, may also initiate and amplify oxidative damage by diffusion-limited reactions with superoxide (O(2)(•-)) to produce peroxynitrite (ONOO(-)). This is perhaps best illustrated in ALS where physiological levels of NO promote survival of motor neurons, but the same concentrations can stimulate motor neuron apoptosis and glial cell activation under pathological conditions. While these changes represent a complex mechanism involving multiple cell types in the pathogenesis of ALS, they also reveal general processes underlying neurodegeneration.

Neuroscience Letters, 1997

In the present study we have assessed whether riluzole stimulates the production of trophic activ... more In the present study we have assessed whether riluzole stimulates the production of trophic activities for motoneurons by spinal astrocyte cultures. Astrocyte monolayers prepared from new-born rats were exposed to vehicle or riluzole (1-10 mM) for 30-36 h, then washed and further incubated without riluzole for 24 h in L15 medium to obtain the astrocyte conditioned media (ACM). Motoneuron-enriched cultures were used to test the ability of the ACM to support motoneuron viability. Astrocyte monolayers exposed to 1 mM riluzole did not show changes in morphology or in DNA or protein synthesis. However, the conditioned medium obtained from astrocyte monolayers after this treatment increased motoneuron survival compared to that from vehicle-treated cultures. A similar effect was found when astrocytes were exposed to a higher riluzole concentration (10 mM) but with greater dilutions of the conditioned medium. This trophic activity was abolished by boiling or after treatment with trypsin. These findings strongly suggest the existence of a new trophic mechanism, through which riluzole may exert motoneuron protection.

Journal of Neurochemistry, 2004

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic ani... more Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75 NTR ) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75 NTR was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75 NTR +/+ mouse embryos but had no effect in p75 NTR -/knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75 NTR blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.

Journal of Neurochemistry, 2002

Nerve growth factor (NGF) prevents apoptosis induced by the oxidant peroxynitrite in undifferenti... more Nerve growth factor (NGF) prevents apoptosis induced by the oxidant peroxynitrite in undifferentiated PC12 rat pheochromocytoma cells. Previous studies have shown that activation of phosphatidylinositol 3-kinase (P1 3-kinase) by NGF via the TrkA receptor tyrosine kinase protects PC12 cells from serum deprivation-induced apoptosis. We found that two P1 3-kinase inhibitors, wortmannin and LY294002, eliminated the protection NGF provided against peroxynitrite-induced apoptosis at concentrations consistent with their effectiveness as P1 3-kinase inhibitors. When the activity of Fl 3-kinase was assayed in phosphotyrosine immunoprecipitates after treatment of P012 cells with peroxynitrite, P1 3-kinase activity was reduced by 50% of that detected in control cells, whereas P1 3-kinase activity in NGFtreated cells was unaffected by peroxynitrite. If an antibody against P1 3-kinase was used to immunoprecipitate the enzyme, treatment with peroxynitrite had no effect on activity. Therefore, peroxynitrite appeared to disrupt interactions between P1 3-kinase and phosphotyrosine proteins, rather than directly inhibiting the enzyme. NGF also activates p21 Ras..dependent pathways, but this did not appear to be required for NGF to exert its protective effect against peroxynitrite. P012 cells expressing a dominant inhibitory mutant of p21 Ras were equally susceptible to peroxynitrite-induced apoptosis, which was prevented by NGF. Wortmannin was also able to block the protective effect of NGF in the p21 Ras mutant cell line. Although many signaling pathways are activated by NGF, these results suggest that a P1 3-kinase-dependent pathway is important for inhibiting peroxynitrite-induced apoptosis.

Journal of Biological Chemistry, 1999

Concentrations of up to 1.5 milliunits/ml xanthine oxidase (XO) (1.1 g/ml) are found circulating ... more Concentrations of up to 1.5 milliunits/ml xanthine oxidase (XO) (1.1 g/ml) are found circulating in plasma during diverse inflammatory events. The saturable, high affinity binding of extracellular XO to vascular endothelium and the effects of cell binding on both XO catalytic activity and differentiated vascular cell function are reported herein. Xanthine oxidase purified from bovine cream bound specifically and with high affinity (K d ‫؍‬ 6 nM) at 4°C to bovine aortic endothelial cells, increasing cell XO specific activity up to 10-fold. Xanthine oxidase-cell binding was not inhibited by serum or albumin and was partially inhibited by the addition of heparin. Pretreatment of endothelial cells with chondroitinase, but not heparinase or heparitinase, diminished endothelial binding by ϳ50%, suggesting association with chondroitin sulfate proteoglycans. Analysis of rates of superoxide production by soluble and cell-bound XO revealed that endothelial binding did not alter the percentage of univalent reduction of oxygen to superoxide. Comparison of the extent of CuZn-SOD inhibition of native and succinoylated cytochrome c reduction by cell-bound XO indicated that XO-dependent superoxide production was occurring in a cell compartment inaccessible to CuZn-SOD. This was further supported by the observation of a shift of exogenously added XO from extracellular binding sites to intracellular compartments, as indicated by both proteasereversible cell binding and immunocytochemical localization studies. Endothelium-bound XO also inhibited nitric oxide-dependent cGMP production by smooth muscle cell co-cultures in an SOD-resistant manner. This data supports the concept that circulating XO can bind to vascular cells, impairing cell function via oxidative mechanisms, and explains how vascular XO activity diminishes vasodilatory responses to acetylcholine in hypercholesterolemic rabbits and atherosclerotic humans. The ubiquity of cell-XO binding and endocytosis as a fundamental mechanism of oxidative tissue injury is also affirmed by the significant extent of XO binding to human vascular endothelial cells, rat lung type 2 alveolar epthelial cells, and fibroblasts.

European Journal of Pharmacology, 1995

Excitatory amino acid-mediated neurotoxicity was investigated in motoneuron-enriched cultures fro... more Excitatory amino acid-mediated neurotoxicity was investigated in motoneuron-enriched cultures from fetal rats at 12-14 days of gestation. The cultures were mainly composed of differentiated motoneurons identified by choline acetyl transferase and calcitonin gene-related peptide (CGRP) immunoreactivity. Addition of glutamate (600 microM) to the conditioned medium induced no acute neuronal swelling. However, it was followed by a widespread neuronal degeneration over the next 24 h, accounting for 77% of the total cell number. Glutamate toxicity was dose dependent, with an EC50 around 300 microM. Treatment for 24 h with the agonists, N-methyl-D-aspartate (NMDA, 100 microM), kainate (500 microM) or RS-alpha-amino-3-hydroxy-5-methyl-4-isoxalopropionate (AMPA, 10 microM), also induced a significant cell loss. Riluzole (2 amino 6-trifluoromethoxybenzothiazole), a compound known to interfere with glutamatergic transmission pre- and postsynaptically, significantly reduced glutamate and NMDA neurotoxicity in a dose-dependent manner. These results suggest that a prolonged activation of one or more subtypes of ionotropic excitatory amino acid receptors can lead to motoneuron degeneration in vitro, and provide direct experimental evidence supporting the neuroprotective effect of riluzole in cultured motoneurons.

Brain Research Reviews, 2004

A strong glial reaction typically surrounds the affected upper and lower motor neurons and degene... more A strong glial reaction typically surrounds the affected upper and lower motor neurons and degenerating descending tracts of ALS patients. Reactive astrocytes in ALS contain protein inclusions, express inflammatory makers such as the inducible forms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2), display nitrotyrosine immunoreactivity and downregulate the glutamate transporter EAAT2. In this review, we discuss the evidence sustaining an active role for astrocytes in the induction and propagation of motor neuron loss in ALS. Available evidence supports the view that glial activation could be initiated by proinflammatory mediators secreted by motor neurons in response to injury, axotomy or muscular pathology. In turn, reactive astrocytes produce nitric oxide and peroxynitrite, which cause mitochondrial damage in cultured neurons and trigger apoptosis in motor neurons. Astrocytes may also contribute to the excitotoxic damage of motor neurons by decreasing glutamate transport or actively releasing the excitotoxic amino acid. In addition, reactive astrocytes secrete pro-apoptotic mediators, such as nerve growth factor (NGF) or Fas-ligand, a mechanism that may serve to eliminate vulnerable motor neurons. The comprehensive understanding of the interactions between motor neurons and glia in ALS may lead to a more accurate theory of the pathogenesis of the disease. D

Biochemical and Biophysical Research Communications, 1988

Plasma membrane isolated by two-phase partition from rat liver showed rates of ascorbate free rad... more Plasma membrane isolated by two-phase partition from rat liver showed rates of ascorbate free radical reduction by NADH of 4-5 nmoles of oxidized NADH/min/mg protein. This activity was inhibited 80% by ConA and up to 97% by WGA and LFA lectins. NADH-ascorbate free radical reductase was also inhibited in rat liver plasma membranes preincubated with neuraminidase or trypsin, but no additional inhibition was observed in the presence of LFA after enzyme digestion. It appears that the integrity of glucan moieities of the cell surface glycoconjugates are necessary for the optimal function of this activity that could be considered as part of the transplasma membrane electron transport system.