Célia A. Aveleira

TNF-α signals through PKCζ/NF-κB to alter the tight junction complex and increase retinal endothelial cell permeability

OBJECTIVE Tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) are elevated in the vitreous of diabetic patients and in retinas of diabetic rats associated with increased retinal vascular permeability. However, the molecular mechanisms underlying retinal vascular permeability induced by these cytokines are poorly understood. In this study, the effects of IL-1β and TNF-α on retinal endothelial cell permeability were compared and the molecular mechanisms by which TNF-α increases cell permeability were elucidated. RESEARCH DESIGN AND METHODS Cytokine-induced retinal vascular permeability was measured in bovine retinal endothelial cells (BRECs) and rat retinas. Western blotting, quantitative real-time PCR, and immunocytochemistry were performed to determine tight junction protein expression and localization. RESULTS IL-1β and TNF-α increased BREC permeability, and TNF-α was more potent. TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins. Dexamethasone prevented TNF-α–induced cell permeability through glucocorticoid receptor transactivation and nuclear factor-kappaB (NF-κB) transrepression. Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α–stimulated permeability. Finally, inhibiting protein kinase C zeta (PKCζ) using both a peptide and a novel chemical inhibitor reduced NF-κB activation and completely prevented the alterations in the tight junction complex and cell permeability induced by TNF-α in cell culture and rat retinas. CONCLUSIONS These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy.

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H2O2). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H2O2 and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H2O2 and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.

Beclin 1 mitigates motor and neuropathological deficits in genetic mouse models of Machado–Joseph disease

Machado-Joseph disease or spinocerebellar ataxia type 3, the most common dominantly-inherited spinocerebellar ataxia, results from translation of the polyglutamine-expanded and aggregation prone ataxin 3 protein. Clinical manifestations include cerebellar ataxia and pyramidal signs and there is no therapy to delay disease progression. Beclin 1, an autophagy-related protein and essential gene for cell survival, is decreased in several neurodegenerative disorders. This study aimed at evaluating if lentiviral-mediated beclin 1 overexpression would rescue motor and neuropathological impairments when administered to pre- and post-symptomatic lentiviral-based and transgenic mouse models of Machado-Joseph disease. Beclin 1-mediated significant improvements in motor coordination, balance and gait with beclin 1-treated mice equilibrating longer periods in the Rotarod and presenting longer and narrower footprints. Furthermore, in agreement with the improvements observed in motor function beclin 1 overexpression prevented neuronal dysfunction and neurodegeneration, decreasing formation of polyglutamine-expanded aggregates, preserving Purkinje cell arborization and immunoreactivity for neuronal markers. These data show that overexpression of beclin 1 in the mouse cerebellum is able to rescue and hinder the progression of motor deficits when administered to pre- and post-symptomatic stages of the disease.

High glucose and oxidative/nitrosative stress conditions induce apoptosis in retinal endothelial cells by a caspase-independent pathway.

Diabetic retinopathy (DR) is a leading cause of vision loss among working-age adults. Retinal endothelial cell apoptosis is an early event in DR, and oxidative stress is known to play an important role in this pathology. Recently, we found that high glucose induces apoptosis in retinal neural cells by a caspase-independent mechanism. Here, we investigated the mechanisms underlying retinal endothelial cell apoptosis induced by high glucose and oxidative/nitrosative stress conditions. Endothelial cells (TR-iBRB2 rat retinal endothelial cell line) were exposed to high glucose (long-term exposure, 7 days), or to NOC-18 (nitric oxide donor; 250microM) or H(2)O(2) (100microM) for 24h. Cell viability was assessed by the MTT assay and cell proliferation by [methyl-(3)H]-thymidine incorporation into DNA. Apoptotic cells were detected with Hoechst or Annexin V staining. Active caspases were detected by an apoptosis detection kit. Active caspase-3 and apoptosis-inducing factor (AIF) protein levels were assessed by Western blot or immunohistochemistry. High glucose, NOC-18 and H(2)O(2) increased apoptosis in retinal endothelial cells. High glucose and mannitol decreased cell proliferation, but mannitol did not induce apoptosis. Caspase activation did not increase in high glucose- or NOC-18-treated cells, but it increased in cells exposed to H(2)O(2). However, the protein levels of AIF decreased in mitochondrial fractions and increased in nuclear fractions, in all conditions. These results are the first demonstrating that retinal endothelial cell apoptosis induced by high glucose is independent of caspase activation, and is correlated with AIF translocation to the nucleus. NOC-18 and H(2)O(2) also activate a caspase-independent apoptotic pathway, although H(2)O(2) can also induce caspase-mediated apoptosis.

Neuropeptide Y stimulates autophagy in hypothalamic neurons

Significance Autophagy impairment is a major hallmark of aging, and any intervention that enhances autophagy is of potential interest to delay aging. However, it was described that the hypothalamus is a brain area with a key role on whole-body aging. In the present study, we show that an endogenous molecule produced by the hypothalamus, the neuropeptide Y (NPY), stimulates autophagy in rodent hypothalamus. Because both hypothalamic autophagy and NPY levels decrease with age, a better understanding of hypothalamic neuronal autophagy regulation by NPY may provide new putative therapeutic strategies to ameliorate age-related deteriorations and delay aging. Aging is characterized by autophagy impairment that contributes to age-related disease aggravation. Moreover, it was described that the hypothalamus is a critical brain area for whole-body aging development and has impact on lifespan. Neuropeptide Y (NPY) is one of the major neuropeptides present in the hypothalamus, and it has been shown that, in aged animals, the hypothalamic NPY levels decrease. Because caloric restriction (CR) delays aging, at least in part, by stimulating autophagy, and also increases hypothalamic NPY levels, we hypothesized that NPY could have a relevant role on autophagy modulation in the hypothalamus. Therefore, the aim of this study was to investigate the role of NPY on autophagy in the hypothalamus. Using both hypothalamic neuronal in vitro models and mice overexpressing NPY in the hypothalamus, we observed that NPY stimulates autophagy in the hypothalamus. Mechanistically, in rodent hypothalamic neurons, NPY increases autophagy through the activation of NPY Y1 and Y5 receptors, and this effect is tightly associated with the concerted activation of PI3K, MEK/ERK, and PKA signaling pathways. Modulation of hypothalamic NPY levels may be considered a potential strategy to produce protective effects against hypothalamic impairments associated with age and to delay aging.

Alterations in phospholipidomic profile in the brain of mouse model of depression induced by chronic unpredictable stress

Depression is a worldwide disability disease associated with high morbidity and has increased dramatically in the last few years. The differential diagnosis and the definition of an individualized therapy for depression are hampered by the absence of specific biomarkers. The aim of this study was to evaluate the phospholipidomic profile of the brain and myocardium in a mouse model of depression induced by chronic unpredictable stress (CUS). The lipidomic profile was evaluated by thin layer and liquid chromatography and mass spectrometry and lipid oxidation was estimated by FOX II assay. Antioxidant enzyme activity and the oxidized/reduced glutathione (GSH/GSSG) ratio were also evaluated. Results showed that chronic stress affects primarily the lipid profile of the brain, inducing an increase in lipid hydroperoxides, which was not detected in the myocardium. A significant decrease in phosphatidylinositol (PI) and in cardiolipin (CL) relative contents and also oxidation of CL and a significant increase of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were observed in the brain of mice after unpredictable chronic stress conditions. In the myocardium only an increase in PC content was observed. Nevertheless, both organs present a decreased GSH/GSSG ratio when compared to control groups, corroborating the occurrence of oxidative stress. The enzyme activities of catalase (CAT) and superoxide dismutase (SOD) were found to be decreased in the myocardium and increased in the brain, while glutathione reductase (GR) was decreased in the brain. Our results indicate that in a mouse model for studying depression induced by CUS, the modification of the expression of oxidative stress-related enzymes did not prevent lipid oxidation in organs, particularly in the brain. These observations suggest that depression has an impact on the brain lipidome and that further studies are needed to better understand lipids role in depression and to evaluate their potential as future biomarkers.

Proliferative Hypothalamic Neurospheres Express NPY, AGRP, POMC, CART and Orexin-A and Differentiate to Functional Neurons

Some pathological conditions with feeding pattern alterations, including obesity and Huntington disease (HD) are associated with hypothalamic dysfunction and neuronal cell death. Additionally, the hypothalamus is a neurogenic region with the constitutive capacity to generate new cells of neuronal lineage, in adult rodents. The aim of the present work was to evaluate the expression of feeding-related neuropeptides in hypothalamic progenitor cells and their capacity to differentiate to functional neurons which have been described to be affected by hypothalamic dysfunction. Our study shows that hypothalamic progenitor cells from rat embryos grow as floating neurospheres and express the feeding-related neuropeptides Neuropeptide Y (NPY), Agouti-related Protein (AGRP), Pro-OpioMelanocortin (POMC), Cocaine-and-Amphetamine Responsive Transcript (CART) and Orexin-A/Hypocretin-1. Moreover the relative mRNA expression of NPY and POMC increases during the expansion of hypothalamic neurospheres in proliferative conditions. Mature neurons were obtained from the differentiation of hypothalamic progenitor cells including NPY, AGRP, POMC, CART and Orexin-A positive neurons. Furthermore the relative mRNA expression of NPY, CART and Orexin-A increases after the differentiation of hypothalamic neurospheres. Similarly to the adult hypothalamic neurons the neurospheres-derived neurons express the glutamate transporter EAAT3. The orexigenic and anorexigenic phenotype of these neurons was identified by functional response to ghrelin and leptin hormones, respectively. This work demonstrates the presence of appetite-related neuropeptides in hypothalamic progenitor cells and neurons obtained from the differentiation of hypothalamic neurospheres, including the neuronal phenotypes that have been described by others as being affected by hypothalamic neurodegeneration. These in vitro models can be used to study hypothalamic progenitor cells aiming a therapeutic intervention to mitigate feeding dysfunction that are associated with hypothalamic neurodegeneration.

NPY in rat retina is present in neurons, in endothelial cells and also in microglial and Muller cells

NPY is present in the retina of different species but its role is not elucidated yet. In this work, using different rat retina in vitro models (whole retina, retinal cells in culture, microglial cell cultures, rat Müller cell line and retina endothelial cell line), we demonstrated that NPY staining is present in the retina in different cell types: neurons, macroglial, microglial and endothelial cells. Retinal cells in culture express NPY Y(1), Y(2), Y(4) and Y(5) receptors. Retina endothelial cells express all NPY receptors except NPY Y(5) receptor. Moreover, NPY is released from retinal cells in culture upon depolarization. In this study we showed for the first time that NPY is present in rat retina microglial cells and also in rat Müller cells. These in vitro models may open new perspectives to study the physiology and the potential pathophysiological role of NPY in the retina.

Caloric restriction stimulates autophagy in rat cortical neurons through neuropeptide Y and ghrelin receptors activation

Caloric restriction is an anti-aging intervention known to extend lifespan in several experimental models, at least in part, by stimulating autophagy. Caloric restriction increases neuropeptide Y (NPY) in the hypothalamus and plasma ghrelin, a peripheral gut hormone that acts in hypothalamus to modulate energy homeostasis. NPY and ghrelin have been shown to be neuroprotective in different brain areas and to induce several physiological modifications similar to those induced by caloric restriction. However, the effect of NPY and ghrelin in autophagy in cortical neurons is currently not known. Using a cell culture of rat cortical neurons we investigate the involvement of NPY and ghrelin in caloric restriction-induced autophagy. We observed that a caloric restriction mimetic cell culture medium stimulates autophagy in rat cortical neurons and NPY or ghrelin receptor antagonists blocked this effect. On the other hand, exogenous NPY or ghrelin stimulate autophagy in rat cortical neurons. Moreover, NPY mediates the stimulatory effect of ghrelin on autophagy in rat cortical neurons. Since autophagy impairment occurs in aging and age-related neurodegenerative diseases, NPY and ghrelin synergistic effect on autophagy stimulation may suggest a new strategy to delay aging process.

Neuropeptide Y Receptors Y1 and Y2 are Present in Neurons and Glial Cells in Rat Retinal Cells in Culture

PURPOSE Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system (CNS), including the retina. This peptide activates various different G-coupled receptors (NPY Y(1), Y(2), Y(4), and Y(5)) that are also present in the retina. However, the localization of NPY receptors in the several types of retinal cells is not completely known. In this study, we have looked at the distribution of NPY Y(1) and Y(2) receptors in rat retinal cells to reveal new perspectives on the role of NPY receptors in retina physiology. METHODS Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and pure rat Müller cell culture was obtained after treatment of these cells with ascorbic acid. The presence of NPY Y(1) and Y(2) in retinal cell types was studied by immunocytochemistry. RESULTS We show that NPY Y(1) and Y(2) receptors are present on every cell type of rat retinal cell cultures. Neurons, as photoreceptors, bipolar, horizontal, amacrine, and ganglion cells, express these two types of NPY receptors. NPY Y(1) and Y(2) receptors are also located in macroglial cells (Müller cells and astrocytes) and microglial cells. CONCLUSIONS We have clarified the presence of the NPY Y(1) and Y(2) receptors in all different cell types that constitute the retina, which we believe will help open new perspectives for studying the physiology and the potential pathophysiologic function of NPY and its receptors in the retina.