Cellular Senescence: From Mechanisms to Current Biomarkers and Senotherapies.

An increase in life expectancy in developed countries has led to a surge of chronic aging-related diseases. In the last few decades, several studies have provided evidence of the prominent role of cellular senescence in many of these pathologies. Key traits of senescent cells include cell cycle arrest, apoptosis resistance, and secretome shift to senescence-associated secretory phenotype resulting in increased secretion of various intermediate bioactive factors important for senescence pathophysiology. However, cellular senescence is a highly phenotypically heterogeneous process, hindering the discovery of totally specific and accurate biomarkers. Also, strategies to prevent the pathologic effect of senescent cell accumulation during aging by impairing senescence onset or promoting senescent cell clearance have shown great potential during in vivo studies, and some are already in early stages of clinical translation. The adaptability of these senotherapeutic approaches to human application has been questioned due to the lack of proper senescence targeting and senescence involvement in important physiologic functions. In this review, we explore the heterogeneous phenotype of senescent cells and its influence on the expression of biomarkers currently used for senescence detection. We also discuss the current evidence regarding the efficacy, reliability, development stage, and potential for human applicability of the main existing senotherapeutic strategies. SIGNIFICANCE STATEMENT: This paper is an extensive review of what is currently known about the complex process of cellular senescence and explores its most defining features. The main body of the discussion focuses on how the multifeature fluctuation of the senescence phenotype and the physiological role of cellular senescence have both caused a limitation in the search for truly reliable senescence biomarkers and the progression in the development of senotherapies.

The impact of insomnia on frailty and the hallmarks of aging.

Throughout the course of life, there are age-related changes in sleep. Despite these normal changes, there is a high percentage of older adults that report sleep dissatisfaction with a high pervasiveness of chronic insomnia, the most common sleep disorder worldwide, with its prevalence being expected to continuously increase due to the growing rates of aging and obesity. This can have different adverse health outcomes, especially by promoting both physical and cognitive decline, which ultimately may aggravate frailty in older adults. Moreover, age-related frailty and sleep dysfunction may have a common mechanism related to the hallmarks of cellular aging. Cellular aging was categorized into nine hallmarks, such as DNA damage, telomere attrition and epigenetic changes. In the context of geriatric and chronic insomnia research, this review aims at discussing the current evidence from both animal models and human cohorts addressing the link between chronic insomnia, the hallmarks of aging and their impact on frailty. Moreover, the most recent research about the putative effect of insomnia therapeutic approaches on hallmarks of aging will be also highlighted.

SIRT1 activation and its circadian clock control: a promising approach against (frailty in) neurodegenerative disorders.

With the increase in life expectancy, the incidence of neurodegenerative disorders and their impact worldwide has been increasing in recent years. Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, have complex and varied mechanisms of pathogenesis. Importantly, they share the common feature of disrupted circadian rhythms. This hallmark is believed to underlie the symptoms of such diseases and even potentially contribute to their onset. In addition, the association of physical frailty with dementia and neurodegenerative disorders has been demonstrated. In fact, frail persons are 8 times more likely to have some form of dementia and population studies report a significant prevalence for frailty in older patients with AD and PD. SIRT1 regulates the acetylation status of clock components and controls circadian amplitude of clock genes. However, the mechanisms responsible for this circadian clock control have been the subject of contradictory findings. Importantly, the activation of SIRT1 has been shown to have very relevant therapeutic potential against neurodegeneration. Nevertheless, few studies have attempted to connect the therapeutic reestablishing of SIRT1 as an approach against circadian disruption in neurodegenerative diseases. In this review, we address: circadian rhythms as an important early biomarker of neurodegenerative disorders; mechanisms for SIRT1 activation and the novel sirtuin-activating compounds (STACs); SIRT1 circadian paradox and subsequent studies in an unprecedented way in the literature; the beneficial role of SIRT1 activation in neurodegeneration; innovative proposals of how circadian-based interventions (e.g., SIRT1 activators) may become an important therapeutic approach against neurodegenerative disorders and how non-pharmacologic interventions (e.g., Mediterranean-style diet) might help in the prevention and/or treatment of these high-burden disorders, while tackling frailty and enhancing robustness.

SIRT2 Deficiency Exacerbates Hepatic Steatosis via a Putative Role of the ER Stress Pathway.

Nonalcoholic fatty liver disease (NAFLD), a condition strongly associated with obesity and insulin resistance, is characterized by hepatic lipid accumulation and activation of the endoplasmic reticulum (ER) stress response. The sirtuin 2 (SIRT2) protein deacetylase is emerging as a new player in metabolic homeostasis, but its role in the development of hepatic steatosis and its link with ER stress activation remains unknown. SIRT2-knockout (SIRT2-KO) and wild-type mice were fed either a control or a high-fat diet (HFD) for 4 weeks. Genetic manipulation of SIRT2 levels was performed in human hepatic cells. Although apparently normal under a control diet, SIRT2-KO mice showed accelerated body weight gain and adiposity on a HFD, accompanied by severe insulin resistance. Importantly, SIRT2-KO mice exhibited worsened hepatic steatosis independently from diet, consistent with upregulated gene expression of lipogenic enzymes and increased expression of ER stress markers. Exposure of hepatic cells to palmitate induced lipid accumulation, increased ER stress, and decreased SIRT2 expression. Moreover, SIRT2-silenced cells showed enhanced lipid accumulation and ER stress activation under basal conditions, whereas SIRT2 overexpression abrogated palmitate-induced lipid deposition and ER stress activation. Our findings reveal a role for SIRT2 in the regulation of hepatic lipid homeostasis, potentially through the ER stress response, suggesting that SIRT2 activation might constitute a therapeutic strategy against obesity and its metabolic complications.

PET Imaging of the Neuropeptide Y System: A Systematic Review.

Neuropeptide Y (NPY) is a vastly studied biological peptide with numerous physiological functions that activate the NPY receptor family (Y1, Y2, Y4 and Y5). Moreover, these receptors are correlated with the pathophysiology of several diseases such as feeding disorders, anxiety, metabolic diseases, neurodegenerative diseases, some types of cancers and others. In order to deepen the knowledge of NPY receptors' functions and molecular mechanisms, neuroimaging techniques such as positron emission tomography (PET) have been used. The development of new radiotracers for the different NPY receptors and their subsequent PET studies have led to significant insights into molecular mechanisms involving NPY receptors. This article provides a systematic review of the imaging biomarkers that have been developed as PET tracers in order to study the NPY receptor family.

Common risk factors and therapeutic targets in obstructive sleep apnea and osteoarthritis: An unexpectable link?

Osteoarthritis (OA) and Obstructive Sleep Apnea (OSA) are two highly prevalent chronic diseases for which effective therapies are urgently needed. Recent epidemiologic studies, although scarce, suggest that the concomitant occurrence of OA and OSA is associated with more severe manifestations of both diseases. Moreover, OA and OSA share risk factors, such as aging and metabolic disturbances, and co-morbidities, including cardiovascular and metabolic diseases, sleep deprivation and depression. Whether this coincidental occurrence is fortuitous or involves cause-effect relationships is unknown. This review aims at collating and integrating present knowledge on both diseases by providing a brief overview of their epidemiology and pathophysiology, analyzing current evidences relating OA and OSA and discussing potential common mechanisms by which they can aggravate each other. Such mechanisms constitute potential therapeutic targets whose pharmacological modulation may provide more efficient ways of reducing the consequences of OA and OSA and, thus, lessen the huge individual and social burden that they impose.

Trehalose alleviates the phenotype of Machado-Joseph disease mouse models.

BACKGROUND: Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is the most common of the dominantly inherited ataxias worldwide and is characterized by mutant ataxin-3 aggregation and neuronal degeneration. There is no treatment available to block or delay disease progression. In this work we investigated whether trehalose, a natural occurring disaccharide widely used in food and cosmetic industry, would rescue biochemical, behavioral and neuropathological features of an in vitro and of a severe MJD transgenic mouse model. METHODS: Two MJD animal models, a lentiviral based and a transgenic model, were orally treated with 2% trehalose solution for a period of 4 and 30 weeks, respectively. Motor behavior (rotarod, grip strength and footprint patterns) was evaluated at different time points and neuropathological features were evaluated upon in-life phase termination. RESULTS: Trehalose-treated MJD mice equilibrated for a longer time in the rotarod apparatus and exhibited an improvement of ataxic gait in footprint analysis. Trehalose-mediated improvements in motor behaviour were associated with a reduction of the MJD-associated neuropathology, as MJD transgenic mice treated with trehalose presented preservation of cerebellar layers thickness and a decrease in the size of ataxin-3 aggregates in Purkinje cells. In agreement, an improvement of neuropathological features was also observed in the full length lentiviral-based mouse model of MJD submitted to 2% trehalose treatment. CONCLUSIONS: The present study suggests trehalose as a safety pharmacological strategy to counteract MJD-associated behavioural and neuropathological impairments.

The NAD+-dependent deacetylase SIRT2 attenuates oxidative stress and mitochondrial dysfunction and improves insulin sensitivity in hepatocytes.

Insulin resistance is a major predictor of the development of metabolic disorders. Sirtuins (SIRTs) have emerged as potential targets that can be manipulated to counteract age-related diseases, including type 2 diabetes. SIRT2 has been recently shown to exert important metabolic effects, but whether SIRT2 regulates insulin sensitivity in hepatocytes is currently unknown. The aim of this study is to investigate this possibility and to elucidate underlying molecular mechanisms. Here, we show that SIRT2 is downregulated in insulin-resistant hepatocytes and livers, and this was accompanied by increased generation of reactive oxygen species, activation of stress-sensitive ERK1/2 kinase, and mitochondrial dysfunction. Conversely, SIRT2 overexpression in insulin-resistant hepatocytes improved insulin sensitivity, mitigated reactive oxygen species production and ameliorated mitochondrial dysfunction. Further analysis revealed a reestablishment of mitochondrial morphology, with a higher number of elongated mitochondria rather than fragmented mitochondria instigated by insulin resistance. Mechanistically, SIRT2 was able to increase fusion-related protein Mfn2 and decrease mitochondrial-associated Drp1. SIRT2 also attenuated the downregulation of TFAM, a key mtDNA-associated protein, contributing to the increase in mitochondrial mass. Importantly, we found that SIRT2 expression in PBMCs of human subjects was negatively correlated with obesity and insulin resistance. These results suggest a novel function for hepatic SIRT2 in the regulation of insulin sensitivity and raise the possibility that SIRT2 activators may offer novel opportunities for preventing or treating insulin resistance and type 2 diabetes.

Role of Beta-adrenergic Receptors and Sirtuin Signaling in the Heart During Aging, Heart Failure, and Adaptation to Stress.

In the heart, catecholamine effects occur by activation of beta-adrenergic receptors (ß-ARs), mainly the beta 1 (ß1-AR) and beta 2 (ß2-AR) subtypes, both of which couple to the Gs protein that activates the adenylyl cyclase signaling pathway. The ß2-ARs can also couple to the Gi protein that counterbalances the effect of the Gs protein on cyclic adenosine monophosphate production and activates the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. In several cardiovascular disorders, including heart failure, as well as in aging and in animal models of environmental stress, a reduction in the ß1/ß2-AR ratio and activation of the ß2-AR-Gi-PI3K-Akt signaling pathway have been observed. Recent studies have shown that sirtuins modulate certain organic processes, including the cellular stress response, through activation of the PI3K-Akt signaling pathway and of downstream molecules such as p53, Akt, HIF1-α, and nuclear factor-kappa B. In the heart, SIRT1, SIRT3, and ß2-ARs are crucial to the regulation of the cardiomyocyte energy metabolism, oxidative stress, reactive oxygen species production, and autophagy. SIRT1 and the ß2-AR-Gi complex also control signaling pathways of cell survival and death. Here, we review the role played by ß2-ARs and sirtuins during aging, heart failure, and adaptation to stress, focusing on the putative interplay between the two. That relationship, if proven, merits further investigation in the context of cardiac function and dysfunction.

Dipeptidyl peptidase IV (DPP-IV) inhibition prevents fibrosis in adipose tissue of obese mice.

BACKGROUND: During the development of obesity the expansion of white adipose tissue (WAT) leads to a dysregulation and an excessive remodeling of extracellular matrix (ECM), leading to fibrosis formation. These ECM changes have high impact on WAT physiology and may change obesity progression. Blocking WAT fibrosis may have beneficial effects on the efficacy of diet regimen or therapeutical approaches in obesity. Since dipeptidyl peptidase IV (DPP-IV) inhibitors prevent fibrosis in tissues, such as heart, liver and kidney, the objective of this study was to assess whether vildagliptin, a DPP-IV inhibitor, prevents fibrosis in WAT in a mouse model of obesity, and to investigate the mechanisms underlying this effect. METHODS: We evaluated the inhibitory effect of vildagliptin on fibrosis markers on WAT of high-fat diet (HFD)-induced obese mice and on 3T3-L1 cell line of mouse adipocytes treated with a fibrosis inducer, transforming growth factor beta 1 (TGFß1). RESULTS: Vildagliptin prevents the increase of fibrosis markers in WAT of HFD-fed mice and reduces blood glucose, serum triglycerides, total cholesterol and leptin levels. In the in vitro study, the inhibition of DPP-IV with vildagliptin, neuropeptide Y (NPY) treatment and NPY Y1 receptor activation prevents ECM deposition and fibrosis markers increase induced by TGFß1 treatment. CONCLUSIONS: Vildagliptin prevents fibrosis formation in adipose tissue in obese mice, at least partially through NPY and NPY Y1 receptor activation. GENERAL SIGNIFICANCE: This study highlights the importance of vildagliptin in the treatment of fibrosis that occur in obesity.

In vitro susceptibility of Trypanosoma brucei brucei to selected essential oils and their major components.

Aiming for discovering effective and harmless antitrypanosomal agents, 17 essential oils and nine major components were screened for their effects on T. b. brucei. The essential oils were obtained by hydrodistillation from fresh plant material and analyzed by GC and GC-MS. The trypanocidal activity was assessed using blood stream trypomastigotes cultures of T. b. brucei and the colorimetric resazurin method. The MTT test was used to assess the cytotoxicity of essential oils on macrophage cells and Selectivity Indexes were calculated. Of the 17 essential oils screened three showed high trypanocidal activity (IC50 < 10 µg/mL): Juniperus oxycedrus (IC50 of 0.9 µg/mL), Cymbopogon citratus (IC50 of 3.2 µg/mL) and Lavandula luisieri (IC50 of 5.7 µg/mL). These oils had no cytotoxic effects on macrophage cells showing the highest values of Selectivity Index (63.4, 9.0 and 11.8, respectively). The oils of Distichoselinum tenuifolium, Lavandula viridis, Origanum virens, Seseli tortuosom, Syzygium aromaticum, and Thymbra capitata also exhibited activity (IC50 of 10-25 µg/mL) but showed cytotoxicity on macrophages. Of the nine compounds tested, α-pinene (IC50 of 2.9 µg/mL) and citral (IC50 of 18.9 µg/mL) exhibited the highest anti-trypanosomal activities. Citral is likely the active component of C. citratus and α-pinene is responsible for the antitrypanosomal effects of J. oxycedrus. The present work leads us to propose the J. oxycedrus, C. citratus and L. luisieri oils as valuable sources of new molecules for African Sleeping Sickness treatment.

Molecular Mechanisms and Cellular Pathways Implicated in Machado-Joseph Disease Pathogenesis.

Machado-Joseph disease (MJD) is a dominantly inherited disorder originally described in people of Portuguese descent, and associated with the expansion of a CAG tract in the coding region of the causative gene MJD1/ATX3. The CAG repeats range from 10 to 51 in the normal population and from 55 to 87 in SCA3/MJD patients. MJD1 encodes ataxin-3, a protein whose physiological function has been linked to ubiquitin-mediated proteolysis. Despite the identification of the causative mutation, the pathogenic process leading to the neurodegeneration observed in the disease is not yet completely understood. In the past years, several studies identified different molecular mechanisms and cellular pathways as being impaired or deregulated in MJD. Autophagy, proteolysis or post-translational modifications, among other processes, were implicated in MJD pathogenesis. From these studies it was possible to identify new targets for therapeutic intervention, which in some cases proved successful in models of disease.

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 mitigates neuropathology and motor deficits in mouse models of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal, dominantly inherited neurodegenerative disorder associated with an expanded polyglutamine tract within the ataxin-3 protein, and characterized by progressive impairment of motor coordination, associated with neurodegeneration of specific brain regions, including cerebellum and striatum. The currently available therapies do not allow modification of disease progression. Neuropeptide Y (NPY) has been shown to exert potent neuroprotective effects by multiple pathways associated with the MJD mechanisms of disease. Thus, we evaluated NPY levels in MJD and investigated whether raising NPY by gene transfer would alleviate neuropathological and behavioural deficits in cerebellar and striatal mouse models of the disease. For that, a cerebellar transgenic and a striatal lentiviral-based models of MJD were used. NPY overexpression in the affected brain regions in these two mouse models was obtained by stereotaxic injection of adeno-associated viral vectors encoding NPY. Up to 8 weeks after viral injection, balance and motor coordination behaviour and neuropathology were analysed. We observed that NPY levels were decreased in two MJD patients' cerebella and in striata and cerebella of disease mouse models. Furthermore, overexpression of NPY alleviated the motor coordination impairments and attenuated the related neuropathological parameters, preserving cerebellar volume and granular layer thickness, reducing striatal lesion and decreasing mutant ataxin-3 aggregation. Additionally, NPY mediated increase of brain-derived neurotrophic factor levels and decreased neuroinflammation markers. Our data suggest that NPY is a potential therapeutic strategy for MJD.

Hypoxia mimetic induces lipid accumulation through mitochondrial dysfunction and stimulates autophagy in murine preadipocyte cell line.

BACKGROUND: Hypoxia occurs within adipose tissue of obese human and mice. However, its role in adipose tissue regulation is still controversial. METHODS: We used murine preadipocyte 3T3-L1 cells and hypoxia was induced by using hypoxia mimetic agents, as CoCl2. To study adipocyte differentiation, we evaluated the adipocyte markers (PPARγ, C/EBPα and aP2), and a preadipocyte marker (pref-1) by qPCR, western blotting and immunofluorescence. Lipid accumulation was evaluated by Oil red-O assay and perilipin levels by western blotting and immunofluorescence. The effect of CoCl2 in microRNA, miR-27a and miR-27b, levels was evaluated by qPCR. We also assessed the mitochondrial membrane potential and reactive oxygen species (ROS), superoxide and ATP production. The effect of hypoxia mimetic in autophagy was determined by LC3B and p62 level evaluation by western blotting. RESULTS: Our results show that the hypoxia mimetic cobalt chloride increases lipid accumulation with no expression of PPARγ2. Furthermore, using qPCR we observed that the hypoxia mimetic increases microRNAs miR-27a and miR-27b, which are known to block PPARγ2 expression. In contrast, cobalt chloride induces mitochondrial dysfunction, and increases ROS production and autophagy. Moreover, an antioxidant agent, glutathione, prevents lipid accumulation induced by hypoxia mimetic indicating that ROS are responsible for hypoxia-induced lipid accumulation. CONCLUSIONS: All these results taken together suggest that hypoxia mimetic blocks differentiation and induces autophagy. Hypoxia mimetic also induces lipid accumulation through mitochondrial dysfunction and ROS accumulation. GENERAL SIGNIFICANCE: This study highlights the importance of adipocyte response to hypoxia, which might impair adipocyte metabolism and compromise adipose tissue function.

Neuropeptide Y (NPY) as a therapeutic target for neurodegenerative diseases.

Neuropeptide Y (NPY) and NPY receptors are widely expressed in the mammalian central nervous system. Studies in both humans and rodent models revealed that brain NPY levels are altered in some neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and Machado-Joseph disease. In this review, we will focus on the roles of NPY in the pathological mechanisms of these disorders, highlighting NPY as a neuroprotective agent, as a neural stem cell proliferative agent, as an agent that increases trophic support, as a stimulator of autophagy and as an inhibitor of excitotoxicity and neuroinflammation. Moreover, the effect of NPY in some clinical manifestations commonly observed in Alzheimer's disease, Parkinson's disease, Huntington's disease and Machado-Joseph disease, such as depressive symptoms and body weight loss, are also discussed. In conclusion, this review highlights NPY system as a potential therapeutic target in neurodegenerative diseases.

Antileishmanial activity of antiretroviral drugs combined with miltefosine.

Co-infection of Leishmaniasis, a neglected tropical disease, with human immunodeficiency virus (HIV) has hindered treatment efficacy. In this study, we aim to evaluate the antileishmanial activity of two protease inhibitors (darunavir and atazanavir) and four reverse transcriptase inhibitors (tenofovir, efavirenz, neviraprine, and delavirdine mesylate) on Leishmania infantum. The activity of different antiretrovirals combinations and of antiretroviral with miltefosine, a drug used on leishmaniasis treatment, was also evaluated. Only two non-nucleoside reverse transcriptase inhibitors (NNRTIs) were active on L. infantum. Efavirenz showed the best antileishmanial activity on promastigotes cells with IC50 value of 26.1 µM followed by delavirdine mesylate with an IC50 value of 136.2 µM. Neviraprine, tenofovir, atazanavir, and darunavir were not active at the concentrations tested (IC50 > 200 µM). The efavirenz also showed high antileishmanial activity on intramacrophage amastigotes with IC50 of 12.59 µM. The interaction of efavirenz with miltefosine improved antileishmanial activity on promastigotes and intracellular amastigotes (IC50 values of 11. 8 µM and 8.89 µM, respectively). These results suggest that combined-therapy including efavirenz and miltefosine could be alternative options for treating Leishmaniasis and Leishmania/HIV co-infections.

Lipodystrophy as a target to delay premature aging.

Lipodystrophy syndromes are rare diseases characterized by low levels and an abnormal distribution of adipose tissue, caused by diverse genetic or acquired causes. These conditions commonly exhibit metabolic complications, including insulin resistance, diabetes, hypertriglyceridemia, nonalcoholic fatty liver disease, and adipose tissue dysfunction. Moreover, genetic lipodystrophic laminopathies exhibit a premature aging phenotype, emphasizing the importance of restoring adipose tissue distribution and function. In this opinion, we discuss the relevance of adipose tissue reestablishment as a potential approach to alleviate premature aging and age-related complications in genetic lipodystrophy syndromes.

Modeling hypothalamic pathophysiology in vitro for metabolic, circadian, and sleep disorders.

The hypothalamus, a small and intricate brain structure, orchestrates numerous neuroendocrine functions through specialized neurons and nuclei. Disruption of this complex circuitry can result in various diseases, including metabolic, circadian, and sleep disorders. Advances in in vitro models and their integration with new technologies have significantly benefited research on hypothalamic function and pathophysiology. We explore existing in vitro hypothalamic models and address their challenges and limitations as well as translational findings. We also highlight how collaborative efforts among multidisciplinary teams are essential to develop relevant and translational experimental models capable of replicating intricate neural circuits and neuroendocrine pathways, thereby advancing our understanding of therapeutic targets and drug discovery in hypothalamus-related disorders.

Improved Chemical and Radiochemical Synthesis of Neuropeptide Y Y2 Receptor Antagonist N-Methyl-JNJ-31020028 and Preclinical Positron Emission Tomography Studies.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals and is involved in several physiological processes through NPY Y1, Y2, Y4 and Y5 receptors. Of those, the Y2 receptor has particular relevance for its autoreceptor role in inhibiting the release of NPY and other neurotransmitters and for its involvement in relevant mechanisms such as feeding behaviour, cognitive processes, emotion regulation, circadian rhythms and disorders such as epilepsy and cancer. PET imaging of the Y2 receptor can provide a valuable platform to understand this receptor's functional role and evaluate its potential as a therapeutic target. In this work, we set out to refine the chemical and radiochemical synthesis of the Y2 receptor antagonist N-[11C]Me-JNJ31020028 for in vivo PET imaging studies. The non-radioactive reference compound, N-Me-JNJ-31020028, was synthesised through batch synthesis and continuous flow methodology, with 43% and 92% yields, respectively. N-[11C]Me-JNJ-31020028 was obtained with a radiochemical purity > 99%, RCY of 31% and molar activity of 156 GBq/µmol. PET imaging clearly showed the tracer's biodistribution in several areas of the mouse brain and gut where Y2 receptors are known to be expressed.