Ion-Pairing Chromatography and Amine Derivatization Provide Complementary Approaches for the Targeted LC-MS Analysis of the Polar Metabolome.

Liquid chromatography coupled to mass spectrometry is a key metabolomics/metabonomics technology. Reversed-phase liquid chromatography (RPLC) is very widely used as a separation step, but typically has poor retention of highly polar metabolites. Here, we evaluated the combination of two alternative methods for improving retention of polar metabolites based on 6-aminoquinoloyl-N-hydroxysuccinidimyl carbamate derivatization for amine groups, and ion-pairing chromatography (IPC) using tributylamine as an ion-pairing agent to retain acids. We compared both of these methods to RPLC and also to each other, for targeted analysis using a triple-quadrupole mass spectrometer, applied to a library of ca. 500 polar metabolites. IPC and derivatization were complementary in terms of their coverage: combined, they improved the proportion of metabolites with good retention to 91%, compared to just 39% for RPLC alone. The combined method was assessed by analyzing a set of liver extracts from aged male and female mice that had been treated with the polyphenol compound ampelopsin. Not only were a number of significantly changed metabolites detected, but also it could be shown that there was a clear interaction between ampelopsin treatment and sex, in that the direction of metabolite change was opposite for males and females.

High-fat diet during adulthood interacts with prenatal stress, affecting both brain inflammatory and neuroendocrine markers in male rats.

Prenatal stress (PNS) affects foetal programming and, through an interaction with subsequent challenges, can increase vulnerability to mood and metabolic disorders. We have previously shown that, following PNS, adult male rats are characterized by increased vulnerability to a metabolic stressor experienced at adulthood (8-week-high-fat diet-HFD). In this study, we specifically assessed whether PNS might interact with an adult metabolic challenge to induce an inflammatory phenotype. Changes in the expression levels of inflammatory (Il-1ß, Tnf-α, Il-6) and of stress response mediators (Nr3c1, Fkbp5) as well as of mood and metabolic regulators (Bdnf, Ghs-R) were investigated in the hippocampus, prefrontal cortex and hypothalamus, brain regions involved in the pathogenesis of depression and prone to inflammation in response to stress. Overall, PNS reduced the expression of Bdnf and Tnf-α, while HFD administered at adulthood counteracted this effect suggesting that PNS impinges upon the same pathways regulating responses to a metabolic challenge at adulthood. Furthermore, HFD and PNS affected the expression of both Nr3c1 and Fkbp5, two neuroendocrine mediators involved in the response to stress, metabolic challenges and in the modulation of the emotional profile (as shown by the correlation between Fkbp5 and the time spent in the open arms of the elevated plus-maze). Overall, these results indicate that the same metabolic and neuroendocrine effectors engaged by PNS are affected by metabolic challenges at adulthood, providing some mechanistic insight into the well-known comorbidity between mood and metabolic disorders.

Health and longevity studies in C. elegans: the "healthy worm database" reveals strengths, weaknesses and gaps of test compound-based studies.

Several biogerontology databases exist that focus on genetic or gene expression data linked to health as well as survival, subsequent to compound treatments or genetic manipulations in animal models. However, none of these has yet collected experimental results of compound-related health changes. Since quality of life is often regarded as more valuable than length of life, we aim to fill this gap with the "Healthy Worm Database" ( http://healthy-worm-database.eu ). Literature describing health-related compound studies in the aging model Caenorhabditis elegans was screened, and data for 440 compounds collected. The database considers 189 publications describing 89 different phenotypes measured in 2995 different conditions. Besides enabling a targeted search for promising compounds for further investigations, this database also offers insights into the research field of studies on healthy aging based on a frequently used model organism. Some weaknesses of C. elegans-based aging studies, like underrepresented phenotypes, especially concerning cognitive functions, as well as the convenience-based use of young worms as the starting point for compound treatment or phenotype measurement are discussed. In conclusion, the database provides an anchor for the search for compounds affecting health, with a link to public databases, and it further highlights some potential shortcomings in current aging research.

Molecular mechanisms underlying metabolic syndrome: the expanding role of the adipocyte.

The metabolic syndrome (MetS) is defined as a cluster of 3 or more metabolic and cardiovascular risk factors and represents a serious problem for public health. Altered function of adipose tissue has a significant impact on whole-body metabolism and represents a key driver for the development of these metabolic derangements, collectively referred as to MetS. In particular, increased visceral and ectopic fat deposition play a major role in the development of insulin resistance and MetS. A large body of evidence demonstrates that aging and MetS share several metabolic alterations. Of importance, molecular pathways that regulate lifespan affect key processes of adipose tissue physiology, and transgenic mouse models with adipose-specific alterations in these pathways show derangements of adipose tissue and other metabolic features of MetS, which highlights a causal link between dysfunctional adipose tissue and deleterious effects on whole-body homeostasis. This review analyzes adipose tissue-specific dysfunctions, including metabolic alterations that are related to aging, that have a significant impact on the development of MetS.-Armani, A., Berry, A., Cirulli, F., Caprio, M. Molecular mechanisms underlying metabolic syndrome: the expanding role of the adipocyte.

Dynamic changes in p66Shc mRNA expression in peripheral blood mononuclear cells following resistance training intervention in old frail women born to obese mothers: a pilot study.

The p66Shc gerontogene may affect healthspan by promoting fat accumulation. We assessed changes of p66Shc-mRNA in peripheral tissues in relation to maternal obesity and the moderating effects of resistance-training (RT) exercise in elderly frail women. Thirty-seven women participated in a 4-month RT program. Twenty were offspring of lean/normal weight mothers and 17 were offspring of overweight/obese mothers (OOM). P66Shc was assessed in peripheral blood mononuclear cells (PBMC) and in subcutaneous adipose tissue (SAT) before and after RT. Overall, OOM showed elevated p66Shc mRNA levels in the PBMC. Independently from maternal obesity, following RT there was a decrease in p66Shc expression in PBMC but not in SAT, particularly in subjects with a high body mass index. Results suggest that maternal obesity has long-term effects on the expression of genes involved in mitochondrial function and fat deposition and that RT modifies p66Shc expression in PBMC with greater effects in obese subjects.ClinicalTrials.gov ID: NCT01931540.

Decreased Bdnf expression and reduced social behavior in periadolescent rats following prenatal stress.

Prenatal stress (PNS) is a risk factor for the development of neuropsychiatric disorders. This study was aimed at assessing, in a rodent model, changes in gene expression profiles and behavioral output as a result of PNS, during periadolescence, a critical developmental period for the onset of psychopathology. Social behavior was studied in a standardized social interaction paradigm and the expression of Brain-Derived Neurotrophic Factor (Bdnf), a marker of neuronal plasticity, and of inhibitory and excitatory mechanisms (Na(+)-K(+)-2Cl(-) and K(+)-Cl(-) cotransporters ratio, NKCC1/KCC2) was analyzed. Results indicate that PNS reduced Bdnf transcripts while increasing the NKCC1/KCC2 ratio, primarily in the hippocampus. In the prefrontal cortex, changes in Bdnf were found to be gender-dependent. These effects were accompanied by reduced levels of affiliative and investigative social behaviors. Interestingly, interaction with non-stressed subjects was able to improve sociality in PNS rats suggesting that the social environment could be exploited for therapeutic intervention.

Quality and timing of stressors differentially impact on brain plasticity and neuroendocrine-immune function in mice.

A growing body of evidence suggests that psychological stress is a major risk factor for psychiatric disorders. The basic mechanisms are still under investigation but involve changes in neuroendocrine-immune interactions, ultimately affecting brain plasticity. In this study we characterized central and peripheral effects of different stressors, applied for different time lengths, in adult male C57BL/6J mice. We compared the effects of repeated (7 versus 21 days) restraint stress (RS) and chronic disruption of social hierarchy (SS) on neuroendocrine (corticosterone) and immune function (cytokines and splenic apoptosis) and on a marker of brain plasticity (brain-derived neurotrophic factor, BDNF ). Neuroendocrine activation did not differ between SS and control subjects; by contrast, the RS group showed a strong neuroendocrine response characterized by a specific time-dependent profile. Immune function and hippocampal BDNF levels were inversely related to hypothalamic-pituitary-adrenal axis activation. These data show a fine modulation of the crosstalk between central and peripheral pathways of adaptation and plasticity and suggest that the length of stress exposure is crucial to determine its final outcome on health or disease.

Rosmarinic Acid Improves Cognitive Abilities and Glucose Metabolism in Aged C57Bl/6N Mice While Disrupting Lipid Profile in Young Adults in a Sex-Dependent Fashion.

A growing body of evidence suggests that regular consumption of natural products might promote healthy aging; however, their mechanisms of action are still unclear. Rosmarinic acid (RA) is a polyphenol holding anti-inflammatory, antioxidant and neuroprotective properties. The aim of this study was to characterise the efficacy of an oral administration of RA in promoting healthspan in a mouse model of physiological aging. Aged C57Bl/6 male and female (24-month-old) mice were either administered with RA (500 mg/Kg) or a vehicle in drinking bottles for 52 days while 3-month-old mice receiving the same treatment were used as controls. All subjects were assessed for cognitive abilities in the Morris water maze (MWM) and for emotionality in the elevated-plus maze test (EPM). Brain-derived Neurotrophic Factor (BDNF) protein levels were evaluated in the hippocampus. Since the interaction between metabolic signals and cerebral functions plays a pivotal role in the etiopathogenesis of cognitive decline, the glycaemic and lipid profiles of the mice were also assessed. RA enhanced learning and memory in 24-month-old mice, an effect that was associated to improved glucose homeostasis. By contrast, the lipid profile was disrupted in young adults. This effect was associated with worse glycaemic control in males and with reduced BDNF levels in females, suggesting powerful sex-dependent effects and raising a note of caution for RA administration in young healthy adult subjects.

The predictive role of proinflammatory and neuroendocrine factors in the acute phases of bipolar disorder: study protocol of a longitudinal controlled trial.

INTRODUCTION AND AIMS: Bipolar disorder (BD) is a severe and recurring mental illness associated with a significant personal and social burden. It has been recently hypothesized that increased levels of pro-inflammatory cytokines and cortisol, which is also associated with a reduced expression of the brain-derived neurotrophic factor (BDNF), may influence affective recurrences in BD. Our study aims to: 1) assess changes in the levels of peripheral cytokines, BDNF and salivary cortisol during acute and euthymic phases of bipolar disorder, compared to that of a sample oh healthy controls; 2) evaluate whether these changes represent a biosignature for the different phases of the illness. MATERIALS AND METHODS: Patients aged 18-65 years old, with a diagnosis of BD I or II types, will be enrolled during an acute episode, according to DSM-5 criteria, together with age- and gender-matched healthy controls. Blood and salivary samples will be collected at baseline and after 3 and 6 months. Validated assessment instruments will be administered to all participants for the evaluation of symptom severity, global functioning, suicidal risk, stress levels and physical comorbidities. EXPECTED RESULTS: We expect changes in inflammatory and neuroendocrine indices to be predictive of the onset of an acute phase of bipolar disorder and that overall levels of cytokines, cortisol and BDNF are overall significantly different between BD patients and healthy controls. CONCLUSIONS: The longitudinal design of the study will allow to assess whether the presence of acute affective symptoms in BD patients correlates with significantly higher levels of cytokines and salivary cortisol and with reduced BDNF levels compared to euthymic phases. Moreover, the comparison with healthy control subjects will allow to understand if inflammatory mediators as well as the hypothalamic-pituitary-adrenal (HPA) axis are chronically elevated in BD patients and are independent from mood swings.

Bdnf-Nrf-2 crosstalk and emotional behavior are disrupted in a sex-dependent fashion in adolescent mice exposed to maternal stress or maternal obesity.

Maternal obesity has been recognized as a stressor affecting the developing fetal brain, leading to long-term negative outcomes comparable to those resulting from maternal psychological stress, although the mechanisms have not been completely elucidated. In this study, we tested the hypothesis that adverse prenatal conditions as diverse as maternal stress and maternal obesity might affect emotional regulation and stress response in the offspring through common pathways, with a main focus on oxidative stress and neuroplasticity. We contrasted and compared adolescent male and female offspring in two mouse models of maternal psychophysical stress (restraint during pregnancy - PNS) and maternal obesity (high-fat diet before and during gestation - mHFD) by combining behavioral assays, evaluation of the hypothalamic-pituitary-adrenal (HPA) axis reactivity, immunohistochemistry and gene expression analysis of selected markers of neuronal function and neuroinflammation in the hippocampus, a key region involved in stress appraisal. Prenatal administration of the antioxidant N-acetyl-cysteine (NAC) was used as a strategy to protect fetal neurodevelopment from the negative effects of PNS and mHFD. Our findings show that these two stressors produce overlapping effects, reducing brain anti-oxidant defenses (Nrf-2) and leading to sex-dependent impairments of hippocampal Bdnf expression and alterations of the emotional behavior and HPA axis functionality. Prenatal NAC administration, by restoring the redox balance, was able to exert long-term protective effects on brain development, suggesting that the modulation of redox pathways might be an effective strategy to target common shared mechanisms between different adverse prenatal conditions.

Sustained hippocampal neurogenesis in females is amplified in P66(Shc-/-) mice: An animal model of healthy aging.

Aging is accompanied by poor learning and memory abilities and by decreased hippocampal neurogenesis, a process that is also modulated by oxidative stress (OS). P66(Shc) has recently emerged as a novel mammalian gerontogene able to affect healthspan during aging. Deletion of this gene in mice leads to reduced OS accompanied by decreased incidence of age-related pathologies and reduced signs of behavioral aging. We hypothesized that p66(Shc-/-) mutants might show increased neurogenesis in the hippocampus, a brain region involved in learning and memory processes. To this aim, granule cell number, proliferation, neuronal differentiation, and cell death were assessed in the hippocampus in senescent p66(Shc-/-) [knock out (KO)] and p66(Shc+/+) [wild type (WT)] male and female mice. Spatial learning abilities and spontaneous activity were also investigated in a multifunctional behavioral system-IntelliCages. The behavioral analysis revealed that females in general perform better in spatial learning tasks, with genotype effects being apparent in the activity pattern only. Likewise, all females showed increased neuronal differentiation, whereas increased proliferation was found only in those belonging to the p66(Shc-/-) genotype, indicating that they might be protected from precursor cell loss. The number of dying cells was not affected by genotype or sex; however, all KO mice showed less granule cells than WT. Overall, our data suggest that hippocampal function is protected in the female gender at older age, an effect amplified by reduced OS in the p66(Shc-/-) mutant.

NGF, brain and behavioral plasticity.

Nerve Growth Factor (NGF) was initially studied for its role as a key player in the regulation of peripheral innervations. However, the successive finding of its release in the bloodstream of male mice following aggressive encounters and its presence in the central nervous system led to the hypothesis that variations in brain NGF levels, caused by psychosocial stressor, and the related alterations in emotionality, could be functional to the development of proper strategies to cope with the stressor itself and thus to survive. Years later this vision is still relevant, and the body of evidence on the role of NGF has been strengthened and expanded from trophic factor playing a role in brain growth and differentiation to a much more complex messenger, involved in psychoneuroendocrine plasticity.

Developing effective animal-assisted intervention programs involving visiting dogs for institutionalized geriatric patients: a pilot study.

AIM: An ever increasing interest in the therapeutic aspects of the human-animal bond has led to a proliferation of animal-assisted interventions (AAI) involving dogs. However, most of these programs lack a solid methodological structure, and basic evaluative research is needed. The purpose of this study was to test the value of dog-assisted interventions as an innovative tool to increase quality of life in the geriatric population. METHODS: Nineteen patients (men and women) with a mean age of 85 years participated in the study. Interactions between patients and visiting dogs occurred either in a social situation (socialization sessions) or in a therapeutic context (physical therapy sessions). We derived and characterized a specific ethogram of elderly-dog interactions aimed at evaluating the effectiveness of visiting dogs in improving mood, catalyzing social interactions and reducing their everyday apathetic state. Cortisol levels were also measured in the saliva, and depressive state was evaluated. RESULTS: Overall, results show a time-dependent increase in social behaviour and spontaneous interactions with the dogs. Dog-mediated interactions affected the daily increase in cortisol levels, thus having an 'activational effect', in contrast to the apathetic state of institutionalized elderly. CONCLUSIONS: Dog-mediated intervention programs appear to be promising tools to improve the social skills and enrich the daily activities of the institutionalized elderly.

Prenatal psychological or metabolic stress increases the risk for psychiatric disorders: the "funnel effect" model.

Adverse stressful experiences in utero can redirect fetal brain development, ultimately leading to increased risk for psychiatric disorders. Obesity during pregnancy can have similar effects as maternal stress, affecting mental health in the offspring. In order to explain how similar outcomes may originate from different prenatal conditions, we propose a "funnel effect" model whereby maternal psychological or metabolic stress triggers the same evolutionarily conserved response pathways, increasing vulnerability for psychopathology. In this context, the placenta, which is the main mother-fetus interface, appears to facilitate such convergence, re-directing "stress" signals to the fetus. Characterizing converging pathways activated by different adverse environmental conditions is fundamental to assess the emergence of risk signatures of major psychiatric disorders, which might enable preventive measures in risk populations, and open up new diagnostics, and potentially therapeutic approaches for disease prevention and health promotion already during pregnancy.

Inflammatory Signatures of Maternal Obesity as Risk Factors for Neurodevelopmental Disorders: Role of Maternal Microbiota and Nutritional Intervention Strategies.

Obesity is a main risk factor for the onset and the precipitation of many non-communicable diseases. This condition, which is associated with low-grade chronic systemic inflammation, is of main concern during pregnancy leading to very serious consequences for the new generations. In addition to the prominent role played by the adipose tissue, dysbiosis of the maternal gut may also sustain the obesity-related inflammatory milieu contributing to create an overall suboptimal intrauterine environment. Such a condition here generically defined as "inflamed womb" may hold long-term detrimental effects on fetal brain development, increasing the vulnerability to mental disorders. In this review, we will examine the hypothesis that maternal obesity-related gut dysbiosis and the associated inflammation might specifically target fetal brain microglia, the resident brain immune macrophages, altering neurodevelopmental trajectories in a sex-dependent fashion. We will also review some of the most promising nutritional strategies capable to prevent or counteract the effects of maternal obesity through the modulation of inflammation and oxidative stress or by targeting the maternal microbiota.

Curcuma Longa, the "Golden Spice" to Counteract Neuroinflammaging and Cognitive Decline-What Have We Learned and What Needs to Be Done.

Due to the global increase in lifespan, the proportion of people showing cognitive impairment is expected to grow exponentially. As target-specific drugs capable of tackling dementia are lagging behind, the focus of preclinical and clinical research has recently shifted towards natural products. Curcumin, one of the best investigated botanical constituents in the biomedical literature, has been receiving increased interest due to its unique molecular structure, which targets inflammatory and antioxidant pathways. These pathways have been shown to be critical for neurodegenerative disorders such as Alzheimer's disease and more in general for cognitive decline. Despite the substantial preclinical literature on the potential biomedical effects of curcumin, its relatively low bioavailability, poor water solubility and rapid metabolism/excretion have hampered clinical trials, resulting in mixed and inconclusive findings. In this review, we highlight current knowledge on the potential effects of this natural compound on cognition. Furthermore, we focus on new strategies to overcome current limitations in its use and improve its efficacy, with attention also on gender-driven differences.

Natural products improve healthspan in aged mice and rats: A systematic review and meta-analysis.

Over the last decades a decrease in mortality has paved the way for late onset pathologies such as cardiovascular, metabolic or neurodegenerative diseases. This evidence has led many researchers to shift their focus from researching ways to extend lifespan to finding ways to increase the number of years spent in good health; "healthspan" is indeed the emerging concept of such quest for ageing without chronic or disabling diseases and dysfunctions. Regular consumption of natural products might improve healthspan, although the mechanisms of action are still poorly understood. Since preclinical studies aimed to assess the efficacy and safety of these compounds are growing, we performed a systematic review and meta-analysis on the effects of natural products on healthspan in mouse and rat models of physiological ageing. Results indicate that natural compounds show robust effects improving stress resistance and cognitive abilities. These promising data call for further studies investigating the underlying mechanisms in more depth.

Chronic Isolation Stress Affects Central Neuroendocrine Signaling Leading to a Metabolically Active Microenvironment in a Mouse Model of Breast Cancer.

Social isolation is a powerful stressor capable of affecting brain plasticity and function. In the case of breast cancer, previous data indicate that stressful experiences may contribute to a worse prognosis, activating neuroendocrine and metabolism pathways, although the mechanisms underlying these effects are still poorly understood. In this study, we tested the hypothesis that chronic isolation stress (IS) may boost hypothalamic-pituitary-adrenal (HPA) axis activity, leading to changes in the hypothalamic expression of genes modulating both mood and metabolism in an animal model of breast cancer. This centrally activated signaling cascade would, in turn, affect the mammary gland microenvironment specifically targeting fat metabolism, leading to accelerated tumor onset. MMTVNeuTg female mice (a model of breast cancer developing mammary hyperplasia at 5 months of age) were either group-housed (GH) or subjected to IS from weaning until 5 months of age. At this time, half of these subjects underwent acute restraint stress to assess corticosterone (CORT) levels, while the remaining subjects were characterized for their emotional profile in the forced swimming and saccharin preference tests. At the end of the procedures, all the mice were sacrificed to assess hypothalamic expression levels of Brain-derived neurotrophic factor (Bdnf), Neuropeptide Y (NpY), Agouti-Related Peptide (AgRP), and Serum/Glucocorticoid-Regulated Protein Kinase 1 (SgK1). Leptin and adiponectin expression levels, as well as the presence of brown adipose tissue (BAT), were assessed in mammary fat pads. The IS mice showed higher CORT levels following acute stress and decreased expression of NpY, AgRP, and SgK1, associated with greater behavioral despair in the forced swimming test. Furthermore, they were characterized by increased consumption of saccharin in a preference test, suggesting an enhanced hedonic profile. The IS mice also showed an earlier onset of breast lumps (assessed by palpation) accompanied by elevated levels of adipokines (leptin and adiponectin) and BAT in the mammary fat pads. Overall, these data point to IS as a pervasive stressor that is able to specifically target neuronal circuits, mastered by the hypothalamus, modulating mood, stress reactivity and energy homeostasis. The activation of such IS-driven machinery may hold main implications for the onset and maintenance of pro-tumorigenic environments.

Maternal Obesity as a Risk Factor for Brain Development and Mental Health in the Offspring.

Maternal obesity plays a key role in the health trajectory of the offspring. Although research on this topic has largely focused on the potential of this condition to increase the risk for child obesity, it is becoming more and more evident that it can also significantly impact cognitive function and mental health. The mechanisms underlying these effects are starting to be elucidated and point to the placenta as a critical organ that may mediate changes in the response to stress, immune function and oxidative stress. Long-term effects of maternal obesity may rely upon epigenetic changes in selected genes that are involved in metabolic and trophic regulations of the brain. More recent evidence also indicates the gut microbiota as a potential mediator of these effects. Overall, understanding cause-effect relationships can allow the development of preventive measures that could rely upon dietary changes in the mother and the offspring. Addressing diets appears more feasible than developing new pharmacological targets and has the potential to affect the multiple interconnected physiological pathways engaged by these complex regulations, allowing prevention of both metabolic and mental disorders.

Trehalose administration in C57BL/6N old mice affects healthspan improving motor learning and brain anti-oxidant defences in a sex-dependent fashion: a pilot study.

Aim of this study was to characterize the effects of oral trehalose administration (2%w/v) on healthspan in old mice. Trehalose was administered in drinking water for 1 month to male and female C57BL/6N mice aged 25-months. After behavioral phenotyping (grip strength, beam walking and rotarod tests), autophagy (LC3-II/actin) and oxidative stress were tested in the cerebral cortex and gastrocnemius muscle. The latter parameter was indirectly assessed by evaluating carbonyl groups added to proteins as a result of oxidative reactions, in addition to central levels of NRF2 protein, a transcription factor that regulates the expression of antioxidant enzymes. In comparison with sex-matched controls, trehalose-treated males performed better in motor planning and coordination tasks. This behavioral phenotype was associated with an activation of the ubiquitin-proteasome system, autophagy and antioxidant defences in cerebral cortex. Independently from trehalose administration, females were characterized by better motor performance and showed higher levels of ubiquitinated proteins and NRF2 in cerebral cortex, suggesting an up-regulation of basal antioxidant defences. In conclusion, trehalose was effective in counteracting some aspects of age-related decay, with specific effects in male and female subjects.