Ghrelin delays premature aging in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal genetic condition that arises from a single nucleotide alteration in the LMNA gene, leading to the production of a defective lamin A protein known as progerin. The accumulation of progerin accelerates the onset of a dramatic premature aging phenotype in children with HGPS, characterized by low body weight, lipodystrophy, metabolic dysfunction, skin, and musculoskeletal age-related dysfunctions. In most cases, these children die of age-related cardiovascular dysfunction by their early teenage years. The absence of effective treatments for HGPS underscores the critical need to explore novel safe therapeutic strategies. In this study, we show that treatment with the hormone ghrelin increases autophagy, decreases progerin levels, and alleviates other cellular hallmarks of premature aging in human HGPS fibroblasts. Additionally, using a HGPS mouse model (LmnaG609G/G609G mice), we demonstrate that ghrelin administration effectively rescues molecular and histopathological progeroid features, prevents progressive weight loss in later stages, reverses the lipodystrophic phenotype, and extends lifespan of these short-lived mice. Therefore, our findings uncover the potential of modulating ghrelin signaling offers new treatment targets and translational approaches that may improve outcomes and enhance the quality of life for patients with HGPS and other age-related pathologies.

Ataxin-2 in the hypothalamus at the crossroads between metabolism and clock genes.

ATXN2 gene, encoding for ataxin-2, is located in a trait locus for obesity. Atxn2 knockout (KO) mice are obese and insulin resistant; however, the cause for this phenotype is still unknown. Moreover, several findings suggest ataxin-2 as a metabolic regulator, but the role of this protein in the hypothalamus was never studied before. The aim of this work was to understand if ataxin-2 modulation in the hypothalamus could play a role in metabolic regulation. Ataxin-2 was overexpressed/re-established in the hypothalamus of C57Bl6/Atxn2 KO mice fed either a chow or a high-fat diet (HFD). This delivery was achieved through stereotaxic injection of lentiviral vectors encoding for ataxin-2. We show, for the first time, that HFD decreases ataxin-2 levels in mouse hypothalamus and liver. Specific hypothalamic ataxin-2 overexpression prevents HFD-induced obesity and insulin resistance. Ataxin-2 re-establishment in Atxn2 KO mice improved metabolic dysfunction without changing body weight. Furthermore, we observed altered clock gene expression in Atxn2 KO that might be causative of metabolic dysfunction. Interestingly, ataxin-2 hypothalamic re-establishment rescued these circadian alterations. Thus, ataxin-2 in the hypothalamus is a determinant for weight, insulin sensitivity and clock gene expression. Ataxin-2's potential role in the circadian clock, through the regulation of clock genes, might be a relevant mechanism to regulate metabolism. Overall, this work shows hypothalamic ataxin-2 as a new player in metabolism regulation, which might contribute to the development of new strategies for metabolic disorders.

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.

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.

Elevated Glucose and Interleukin-1ß 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-1ß). However, how high glucose and IL-1ß affect the different retinal cell types remains to be clarified. In retinal neural cell cultures, we found that IL-1ß 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-1ß. High glucose decreased microglial and macroglial cell proliferation, whereas IL-1ß 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-1ß 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-1ß has an important role in retinal microglia activation and proliferation under diabetes, limiting IL-1ß-triggered inflammatory processes may provide a new therapeutic strategy to prevent the progression of diabetic retinopathy.

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.

The pathophysiology of defective proteostasis in the hypothalamus - from obesity to ageing.

Hypothalamic dysfunction has emerged as an important mechanism involved in the development of obesity and its comorbidities, as well as in the process of ageing and age-related diseases, such as type 2 diabetes mellitus, hypertension and Alzheimer disease. In both obesity and ageing, inflammatory signalling is thought to coordinate many of the cellular events that lead to hypothalamic neuronal dysfunction. This process is triggered by the activation of signalling via the toll-like receptor 4 pathway and endoplasmic reticulum stress, which in turn results in intracellular inflammatory signalling. However, the process that connects inflammation with neuronal dysfunction is complex and includes several regulatory mechanisms that ultimately control the homeostasis of intracellular proteins and organelles (also known as 'proteostasis'). This Review discusses the evidence for the key role of proteostasis in the control of hypothalamic neurons and the involvement of this process in regulating whole-body energy homeostasis and lifespan.

Long-term exposure to high glucose increases the content of several exocytotic proteins and of vesicular GABA transporter in cultured retinal neural cells.

Diabetic retinopathy is a leading cause of vision loss and blindness. Increasing evidence has shown that the neuronal components of the retina are affected even before the detection of vascular lesions. Hyperglycemia is considered the main pathogenic factor for the development of diabetic complications. Nevertheless, other factors like neuroinflammation, might also contribute for neural changes. To clarify whether hyperglycemia can be the main trigger of synaptic changes, we evaluated whether prolonged elevated glucose per se, mimicking chronic hyperglycemia, is able to change the content and distribution of several exocytotic proteins and vesicular glutamate and GABA transporters in retinal neurons. Moreover, we also tested the hypothesis that an inflammatory stimulus (interleukin-1ß) could exacerbate the effects induced by exposure to elevated glucose, contributing for changes in synaptic proteins in retinal neurons. Rat retinal neural cells were cultured for 9 days. Cells were exposed to elevated D-glucose (30 mM) or D-mannitol (osmotic control), for 7 days, or were exposed to interleukin-1ß (10 ng/ml) or LPS (1 µg/ml) for 24 h. The protein content and distribution of SNARE proteins (SNAP-25, syntaxin-1, VAMP-2), synapsin-1, synaptotagmin-1, rabphilin 3a, VGluT-1 and VGAT, were evaluated by western blotting and immunocytochemistry. The protein content and immunoreactivity of syntaxin-1, synapsin-1, rabphilin 3a and VGAT increased in retinal neural cells exposed to high glucose. No changes were detected when cells were exposed to interleukin-1ß, LPS or mannitol per se. Particularly, exposure to interleukin-1ß for 24 h did not exacerbate the effect of high glucose on the content and immunoreactivity of exocytotic proteins, suggesting the primordial role of hyperglycemia for neuronal changes. In summary, prolonged exposure to elevated glucose alters the total content of several proteins involved in exocytosis, suggesting that hyperglycemia per se is a fundamental factor for neuronal changes caused by diabetes.

Impaired adrenal medullary function in a mouse model of depression induced by unpredictable chronic stress.

Stress has been considered determinant in the etiology of depression. The adrenal medulla plays a key role in response to stress by releasing catecholamines, which are important to maintain homeostasis. We aimed to study the adrenal medulla in a mouse model of depression induced by 21 days of unpredictable chronic stress (UCS). We observed that UCS induced a differential and time-dependent change in adrenal medulla. After 7 days of UCS, mice did not show depressive-like behavior, but the adrenal medullae show increased protein and/or mRNA levels of catecholamine biosynthetic enzymes (TH, DßH and PNMT), Neuropeptide Y, the SNARE protein SNAP-25, the catecholamine transporter VMAT2 and the chromaffin progenitor cell markers, Mash1 and Phox2b. Moreover, 7 days of UCS induced a decrease in the chromaffin progenitor cell markers, Sox9 and Notch1. This suggests an increased capacity of chromaffin cells to synthesize, store and release catecholamines. In agreement, after 7 days, UCS mice had higher NE and EP levels in adrenal medulla. Opposite, when mice were submitted to 21 days of UCS, and showed a depressive like behavior, adrenal medullae had lower protein and/or mRNA levels of catecholamine biosynthetic enzymes (TH, DßH, PNMT), catecholamine transporters (NET, VMAT1), SNARE proteins (synthaxin1A, SNAP25, VAMP2), catecholamine content (EP, NE), and lower EP serum levels, indicating a reduction in catecholamine synthesis, re-uptake, storage and release. In conclusion, this study suggests that mice exposed to UCS for a period of 21 days develop a depressive-like behavior accompanied by an impairment of adrenal medullary function.

NPY/neuropeptide Y enhances autophagy in the hypothalamus: a mechanism to delay aging?

Aging was recently described as a life event programmed by the hypothalamus, a key brain region that is crucial for the neuroendocrine interaction between the central nervous system and the periphery. Autophagy impairment is a hallmark of aging, contributing to the aging phenotype and to the aggravation of age-related diseases. Since hypothalamic autophagy decreases with age, strategies to promote autophagy in the hypothalamus may be relevant for control of the aging process. NPY (neuropeptide Y) is an endogenous neuropeptide mainly produced by the hypothalamus. We recently reported, for the first time, that NPY stimulates autophagy in rodent hypothalamus and mediates caloric restriction-induced autophagy in hypothalamic neurons. Moreover, we observed that NPY acts through NPY1R (neuropeptide Y receptor Y1) or NPY5R activation involving a concerted action of different signaling pathways. Since both hypothalamic autophagy and NPY levels decrease with age, modulation of NPY levels could provide new putative therapeutic tools to ameliorate age-related deteriorations and extend longevity.

Neuropeptide Y stimulates autophagy in hypothalamic neurons.

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.

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.

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 (H(2)O(2)). 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 H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.

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-1ß) 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-1ß 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-1ß 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.

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.

Müller cells do not influence leukocyte adhesion to retinal endothelial cells.

PURPOSE: Diabetic retinopathy is associated with inflammation. The authors investigated the influence of Müller cells on leukocyte adhesion to retinal endothelial cells. METHODS: ICAM-1 levels were assessed by Western blotting and immunocytochemistry. Leukocyte adhesion was quantified using a fluorescence assay. RESULTS: High glucose and oxidative/nitrosative stress conditions increased ICAM-1 levels in endothelial cells and leukocyte adhesion. Under the influence of Müller cells (co-cultures/conditioned medium), the effects were comparable to those found when endothelial cells were exposed, alone, to similar conditions. CONCLUSIONS: These results show that Müller cells do not influence leukocyte adhesion under the in vitro conditions used in this study.