Neuropeptide Y Enhances Progerin Clearance and Ameliorates the Senescent Phenotype of Human Hutchinson-Gilford Progeria Syndrome Cells

Hutchinson-Gilford progeria syndrome (HGPS, or classical progeria) is a rare genetic disorder, characterized by premature aging, and caused by a de novo point mutation (C608G) within the lamin A/C gene (LMNA), producing an abnormal lamin A protein, termed progerin. Accumulation of progerin causes nuclear abnormalities and cell cycle arrest ultimately leading to cellular senescence. Autophagy impairment is a hallmark of cellular aging, and the rescue of this proteostasis mechanism delays aging progression in HGPS cells. We have previously shown that the endogenous Neuropeptide Y (NPY) increases autophagy in hypothalamus, a brain area already identified as a central regulator of whole-body aging. We also showed that NPY mediates caloric restriction-induced autophagy. These results are in accordance with other studies suggesting that NPY may act as a caloric restriction mimetic and plays a role as a lifespan and aging regulator. The aim of the present study was, therefore, to investigate if NPY could delay HGPS premature aging phenotype. Herein, we report that NPY increases autophagic flux and progerin clearance in primary cultures of human dermal fibroblasts from HGPS patients. NPY also rescues nuclear morphology and decreases the number of dysmorphic nuclei, a hallmark of HGPS cells. In addition, NPY decreases other hallmarks of aging as DNA damage and cellular senescence. Altogether, these results show that NPY rescues several hallmarks of cellular aging in HGPS cells, suggesting that NPY can be considered a promising strategy to delay or block the premature aging of HGPS

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 diseas

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

O envolvimento de mediadores inflamatórios na disfunção microvascular da retina: implicações para a ruptura da barreira hemato-retiniana na retinopatia diabética

Tese de doutoramento em Ciências Biomédicas apresentada à Fac. de Medicina da Univ. de CoimbraA retinopatia diabética é uma das complicações mais comuns da diabetes mellitus, e uma das principais causas de perda de visão e cegueira em adultos em idade activa nos países desenvolvidos. A ruptura da barreira hemato-retiniana (BHR) é a principal característica desta doença, sendo uma das alterações mais precoces que ocorrem na retina de doentes diabéticos. Evidências recentes sugerem que processos inflamatórios crónicos desempenham um papel importante na patogénese da retinopatia diabética. Os níveis de citocinas como a interleucina-1β (IL-1β) e o factor de necrose tumoral-α (TNF-α) estão aumentados no vítreo de doentes diabéticos e nas retinas de animais diabéticos, e estes aumentos correlacionam-se com um aumento da permeabilidade da BHR. No entanto, os mecanismos moleculares envolvidos na disfunção endotelial e alteração da permeabilidade da vasculatura da retina induzidas por estas citocinas não estão esclarecidos. Assim, este estudo teve como principal objectivo avaliar o efeito da IL-1β e do TNF-α na permeabilidade de células endoteliais dos vasos da retina, prestando particular atenção aos mecanismos moleculares subjacentes ao aumento da permeabilidade celular induzida pelo TNF-α. Além disso, avaliou-se também o efeito da glucose elevada e da IL-1β na regulação do receptor da IL-1 tipo I (IL-1RI). As citocinas IL-1β e TNF-α induziram um aumento da permeabilidade das células endoteliais da retina de bovino (BREC), dependente da concentração e do tempo de exposição, sendo o TNF-α mais eficaz. O TNF-α induziu uma diminuição, a nível proteico e de RNA mensageiro, da zonula occludens -1 (ZO-1) e claudina-5. Contrariamente, a expressão de ocludina aumentou significativamente. Além disso, a localização celular destas proteínas alterou-se após exposição a TNF-α. Devido à natureza inflamatória da retinopatia diabética, avaliou-se o possível efeito protector do glucocorticóide dexametasona na permeabilidade celular induzida por TNF-α. A dexametasona preveniu completamente as alterações nas junções oclusivas e o aumento da permeabilidade celular induzido por TNF-α. O efeito protector da dexametasona foi dependente da transactivação do receptor dos glucocorticóides, uma vez que o antagonista deste receptor, RU486, reduziu o seu efeito protector. Porém, o efeito do RU486 foi apenas parcial, sugerindo que outros mecanismos estão envolvidos. O factor de transcrição nuclear κB (NF-κB) constitui um alvo central na via de sinalização do TNF-α, e foi demonstrado que a sua activação pode ser inibida pelo tratamento com glucocorticóides. A inibição da activação do NF-κB, através da inibição da cinase da proteína inibitória do NF-κB (IKK) e da sobre-expressão da proteína inibitória do NF-κB α (IκBα) mediada por adenovírus, preveniu, parcialmente, o aumento da permeabilidade induzida por TNF-α, sugerindo um novo papel do NF-κB na regulação da permeabilidade vascular. Mais ainda, a inibição da proteína cinase C ζ (PKCζ) reduziu a activação do NF-κB e preveniu completamente o efeito do TNF-α nas proteínas das junções oclusivas, bem como o aumento da permeabilidade em células endoteliais da retina e também dos vasos da retina de rato induzido por TNF-α. Este conjunto de observações mostra que o TNF-α altera a estrutura das junções oclusivas e aumenta a permeabilidade das células endoteliais de retina. Mais ainda, a inibição da PKCζ previne completamente o aumento de permeabilidade induzida por TNF-α, em parte, pela redução da activação do NF-κB. Estes resultados sugerem que a PKCζ pode ser considerada como um novo alvo terapêutico para a prevenção da permeabilidade vascular induzida por citocinas. Apesar das células endoteliais da retina constituírem um alvo preferencial da IL-1β e TNF-α, pouco se sabe sobre a regulação dos seus respectivos receptores, como por exemplo o IL-1RI, neste tipo de células. Como a actividade biológica da IL-1β é mediada principalmente pelo IL- 1RI, avaliou-se o efeito da glucose elevada e da IL-1β na regulação da expressão do IL-1RI numa linha celular de células endoteliais da retina de rato (TR-iBRB2). A exposição das células endoteliais a glicose elevada, manitol (controlo osmótico) ou IL-1β, durante períodos relativamente curtos (1-24 h), causou uma diminuição significativa, dependente do tempo de exposição, do receptor IL-1RI. A exposição longa (7 dias) a glicose elevada ou manitol causou também uma diminuição significativa do receptor nas células endoteliais da retina. A diminuição dos níveis proteicos de IL-1RI foi inibida pela presença de anticorpos contra IL-1β e IL-1RI, sugerindo que a activação do IL-1RI pela IL-1β é um evento necessário neste processo. A diminuição do IL-1RI induzida pela glucose elevada ou IL-1β foi prevenida por inibidores do lisossoma, mas não por inibidores do proteossoma. Além disso, observou-se também que o receptor da IL-1β transloca-se para o núcleo, onde se acumula, após a exposição a glucose elevada ou IL-1β. Os resultados obtidos mostram que a glicose elevada, provavelmente devido ao stress osmótico, e a IL-1β regulam o conteúdo do IL-1RI nas células endoteliais da retina. A diminuição de IL-1RI é induzida pela sua activação e depende, em parte, da sua degradação através do lisossoma e da sua translocação e acumulação no núcleo. Uma vez que a regulação do IL-1RI tem consequências importantes na via de sinalização da IL-1β, o IL-1RI pode ser considerado um possível alvo terapêutico para a tratamento da retinopatia diabética. Em conclusão, os resultados apresentados neste estudo oferecem uma nova perspectiva sobre o efeito de citocinas na regulação da permeabilidade celular das células endoteliais de retina. Além disso, os receptores de citocinas ou alvos específicos a jusante, tais como a PKCζ, podem ser considerados como potenciais novos alvos terapêuticos para o tratamento da permeabilidade vascular em doenças oculares caracterizadas por níveis elevados de citocinas, tais como a retinopatia diabética.Diabetic retinopathy is one of the most common complications of diabetes mellitus and remains a leading cause of vision loss and blindness in working-age adults in developed countries. Retinal microvascular dysfunction is an early feature of diabetic retinopathy, and the bloodretinal barrier (BRB) breakdown is the hallmark of the disease. Growing evidence indicates that low-grade and chronic inflammatory processes play an important role in the pathogenesis of diabetic retinopathy. Elevated levels of the cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the vitreous of diabetic patients and in diabetic rat retinas correlate with increased retinal vascular permeability. However, the mechanisms by which cytokines induce endothelial cell dysfunction and alter retinal vascular permeability remain unclear. Therefore, the main goal of the present study was to investigate the effect of IL-1β and TNF-α on retinal endothelial cell permeability, giving particular attention to the molecular mechanisms underlying the increase in cell permeability induced by the more potent permeabilizing factor, TNF-α. Additionally, the regulation of the interleukin-1 type I receptor (IL-1RI) content in retinal endothelial cells exposed to elevated glucose or IL-1β was also investigated. Both IL-1β and TNF-α increased bovine retinal endothelial cell (BREC) permeability in a concentration- and time-dependent manner, being TNF-α more effective. TNF-α induced a marked downregulation of zonula occludens-1 (ZO-1) and claudin-5 protein content, concomitant with a decrease in the respective transcripts expression. Conversely, TNF-α increased occludin expression. In addition, the junctional localization of these tight junction proteins was altered by TNF-α. Given the inflammatory nature of diabetic retinopathy, the ability of the glucocorticoid dexamethasone to prevent TNF-α-induced cell permeability was evaluated. Dexamethasone completely prevented the disruption of the tight junction complex and the increase in cell permeability induced by TNF-α. Moreover, the protective effect of dexamethasone was dependent on transcriptional activation of the glucocorticoid receptor, since RU486, a glucocorticoid receptor antagonist, reduced the protective effect of dexamethasone on TNF-α- induced cell permeability. However, RU486 effect was only partial, suggesting that additional mechanisms are involved. The transcription factor nuclear factor-κB (NF-κB) is a critical target of the TNF-α signaling pathway, and its activation can be inhibited by glucocorticoid treatment. Preventing NF-κBactivation with a chemical inhibitor of the inhibitor NF-κB protein kinase (IKK) or with adenovirus-mediated overexpression of the inhibitor NF-κB protein α (IκBα) partially prevented the increase in cell permeability induced by TNF-α, implying a new role for NF-κB in cell permeability regulation. Furthermore, protein kinase C ζ (PKCζ) inhibition reduced NF-κB activation and completely prevented the alterations in the tight junction complex and cell permeability induced by TNF-α, both in cell cultures and in the retinal vasculature of rats. Taken together, these results demonstrate that TNF-α alters the tight junction complex and increases retinal endothelial cell permeability. Further, inhibition of PKCζ completely prevents TNF-α-induced cell permeability, in part, by reducing NF-κB activation. These results suggest that PKCζ may be considered as a specific therapeutic target for the prevention of cytokineinduced vascular permeability. Although retinal endothelial cells are primarily affected by IL-1β and TNF-α, little attention has been given to the regulation of their cognate receptors, such as IL-1RI, in these cells. As IL-1β activity is regulated primarily by IL-1RI, the effect of high glucose and IL-1β on IL-1RI regulation was also investigated in a rat retinal capillary endothelial cell line (TR-iBRB2). A time-dependent downregulation of IL-1RI occurred in cells exposed to high glucose, mannitol (osmotic control) or IL-1β (1-24 h). Long-term exposure (7 days) of retinal endothelial cells to high glucose or mannitol also decreased IL-1RI protein content. The downregulation of IL-1RI was completely prevented by anti-IL-1RI or anti-IL-1β antibodies, indicating that IL-1RI activation by IL-1β is a necessary event in this process. IL-1RI downregulation was prevented by lysosome inhibitors but not by proteasome inhibitors. It was also found that IL-1RI translocates to the nucleus after high glucose, mannitol or IL-1β treatment. These results indicate that high glucose, probably due to osmotic stress, and IL-1β downregulate IL-1RI in retinal endothelial cells. The downregulation of IL-1RI is triggered by its activation and is due, at least partially, to lysosomal degradation and translocation and accumulation in the nucleus. Given the potential important role of IL-1β on the pathogenesis of diabetic retinopathy, the inhibition of IL-1β action by targeting IL-1RI may be considered another potential therapeutic strategy for the treatment of diabetic retinopathy. In summary, the results presented herein provide new insight into the role of pro-inflammatory cytokines on retinal endothelial cell barrier function. Moreover, cytokine receptors or specific downstream targets, such as PKCζ, may be considered potential novel therapeutic targets for the treatment of vascular permeability in ocular diseases characterized by elevated levels of cytokines, such as diabetic retinopathy

PI3K/AKT/MTOR and ERK1/2-MAPK signaling pathways are involved in autophagy stimulation induced by caloric restriction or caloric restriction mimetics in cortical neurons

Caloric restriction has been shown to robustly ameliorate age-related diseases and to prolong lifespan in several model organisms, and these beneficial effects are dependent on the stimulation of autophagy. Autophagy dysfunction contributes to the accumulation of altered macromolecules, and is a key mechanism of promoting aging and age-related disorders, as neurodegenerative ones. We have previously shown that caloric restriction (CR), and CR mimetics Neuropeptide Y (NPY) and ghrelin, stimulate autophagy in rat cortical neurons, however by unknown molecular mechanisms. Overall, we show that CR, NPY, and ghrelin stimulate autophagy through PI3K/AKT/MTOR inhibition and ERK1/2-MAPK activation. The knowledge of these kinases in autophagy regulation and the contribution to the understanding of molecular mechanism facilitates the discovery of more targeted therapeutic strategies to stimulate autophagy, which is relevant in the context of age-related disorders

Elevated Glucose and Interleukin-1β Differentially Affect Retinal Microglial Cell Proliferation

Diabetic retinopathy is considered a neurovascular disorder, hyperglycemia being considered the main risk factor for this pathology. Diabetic retinopathy also presents features of a low-grade chronic inflammatory disease, including increased levels of cytokines in the retina, such as interleukin-1 beta (IL-1β). However, how high glucose and IL-1β affect the different retinal cell types remains to be clarified. In retinal neural cell cultures, we found that IL-1β and IL-1RI are present in microglia, macroglia, and neurons. Exposure of retinal neural cell cultures to high glucose upregulated both mRNA and protein levels of IL-1β. High glucose decreased microglial and macroglial cell proliferation, whereas IL-1β increased their proliferation. Interestingly, under high glucose condition, although the number of microglial cells decreased, they showed a less ramified morphology, suggesting a more activated state, as supported by the upregulation of the levels of ED-1, a marker of microglia activation. In conclusion, IL-1β might play a key role in diabetic retinopathy, affecting microglial and macroglial cells and ultimately contributing to neural changes observed in diabetic patients. Particularly, since IL-1β has an important role in retinal microglia activation and proliferation under diabetes, limiting IL-1β-triggered inflammatory processes may provide a new therapeutic strategy to prevent the progression of diabetic retinopathy

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

http://www.sciencedirect.com/science/article/B6WFD-4V94X19-2/2/49680e499c2d31bd74eb7ad7fc99a8c

Fluoxetine Induces Proliferation and Inhibits Differentiation of Hypothalamic Neuroprogenitor Cells In Vitro

A significant number of children undergo maternal exposure to antidepressants and they often present low birth weight. Therefore, it is important to understand how selective serotonin reuptake inhibitors (SSRIs) affect the development of the hypothalamus, the key center for metabolism regulation. In this study we investigated the proliferative actions of fluoxetine in fetal hypothalamic neuroprogenitor cells and demonstrate that fluoxetine induces the proliferation of these cells, as shown by increased neurospheres size and number of proliferative cells (Ki-67+ cells). Moreover, fluoxetine inhibits the differentiation of hypothalamic neuroprogenitor cells, as demonstrated by decreased number of mature neurons (Neu-N+ cells) and increased number of undifferentiated cells (SOX-2+ cells). Additionally, fluoxetine-induced proliferation and maintenance of hypothalamic neuroprogenitor cells leads to changes in the mRNA levels of appetite regulator neuropeptides, including Neuropeptide Y (NPY) and Cocaine-and-Amphetamine-Regulated-Transcript (CART). This study provides the first evidence that SSRIs affect the development of hypothalamic neuroprogenitor cells in vitro with consequent alterations on appetite neuropeptides