Shared mechanisms underlying the impact of maternal psychophysical stress and obesity on offspring neurodevelopment

Vulnerability to mental illness might find its roots very early during development, already during fetal life. In fact, prenatal adversities can affect brain development by shaping neuronal circuits involved in stress responses, resulting in embedded biological traces that persist throughout life. In this perspective, maternal environment plays a pivotal role in driving fetal neurodevelopment, even more important than purely heritable genetic background. Chapter 1 of this thesis introduces the concept that maternal obesity - a growing public health issue - can be considered as a stressor that, by contributing to establish a sub-optimal intrauterine environment, may derange fetal neurodevelopment. We reviewed in detail clinical and preclinical evidence showing an association between the prenatal exposure to an “obesogenic environment” and a higher risk for the occurrence of neurodevelopmental and psychiatric disorders. An ever increasing body of evidence shows that similar mental health outcomes in the offspring have been observed as a result of either maternal obesity or maternal distress during pregnancy. Thus, in Chapter 2 we propose a “funnel effect” model hypothesizing that prenatal stressors of different nature might trigger shared stress-responsive pathways affecting neuroendocrine system, immune-inflammatory processes and energy metabolism regulation, ultimately resulting in increased vulnerability to psychopathology. Chapter 3 and Chapter 4 (original studies) investigate the shared biological mechanisms underlying the above-mentioned stressors and their effects during specific time windows across neurodevelopment in two C57Bl6/N mouse models of maternal psychophysical stress (PNS) and maternal obesity (mHFD). We focused on oxidative stress as a central player driving fetal brain programming by adverse prenatal conditions. Also for this reason in our mouse models, we administered as preventive strategy the antioxidant N-acetyl-cysteine (NAC) to protect fetal neurodevelopment from stress-derived derangements. In particular, when we focused on the short-term effects, we found a widespread pro-inflammatory profile in fetal brains exposed either to PNS or mHFD - with females being more susceptible - to be associated to placental dysfunctions (Chapter 3). Moreover, investigation of the long-term effects of PNS and mHFD specifically during adolescence showed similar effects in the offspring, characterized by reduced brain anti-oxidant defenses and impairments in hippocampal Bdnf levels, overall leading to alterations in the emotional behavior and hypothalamic-pituitary-adrenal axis functionality, in a sex-dependent fashion. Maternal NAC administration, by restoring the redox balance, showed long-term protective effects on brain development (Chapter 4). Together, our findings contribute to support our original “funnel effect” model to explain the converging effects of different stressors on offspring brain development. Above all, a pivotal role of redox signaling was highlighted as the orchestrator of a synchronized response to early adversities by the neuroendocrine and the immune system, among others. In addition, we unveil clear sex-specific differences that drive the programming effects of prenatal stressors on neurodevelopment

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

Nutraceuticals have sex-dependent positive effects on healthy ageing in murine models

Introduction Human lifespan has been rising in developed countries however this phenomenon has not been paralleled by an increase in disease-free life expectancy (healthspan). In this context, although great efforts are constantly put through to develop novel target-specific drugs, aimed at alleviating age-related frailty, the occurrence of multiple side effects do not render them the best choice to improve overall healthspan. The use of nutraceutical compounds show many advantages in terms of biosafety, acceptance by the patients and economy. Thus, the interest of scientific community is growing in this field of research with the main aim to characterize the anti-ageing properties and to shed light to the mechanisms of action of natural compounds. Aim The present study was aimed at investigating the effects of a nutraceutical compound, the ACD-0503, on the health phenotype in a murine model of physiological ageing. Methods Two different doses of ACD-0503 (low dose - 50 mg/Kg - and high dose - 500 mg/Kg) were administered to 3- and 24-month-old mice through drinking water for 52 days. Following 2 weeks on the nutraceutical treatment a thorough characterization of motor functions and physical strength, metabolic as well as cognitive and emotional profile was carried out. Results ACD-0503 improved several aspects that are important for the promotion of healthy ageing, acting in an age-, sex- and dose-dependent fashion. More in detail, ACD-0503 high dose was effective in buffering the age-related decline in old male mice since it improved motor function (Beam Walking) and glucose homeostasis (Insulin Sensitivity Test) in addition to the HDL/LDL cholesterol ratio, an index of lower cardiovascular risk. By contrast, aged females treated with both doses of ACD-0503 showed a trend to spend more time in the target zone during the Morris Water Maze Test, suggesting improved memory retention compared to controls. By contrast, only ACD-0503 low dose improved muscle strength (Grip Strength Test) while worsening glucose homeostasis, as assessed both in the Insulin Sensitivity and in the Glucose Tolerance tests. Currently, metabolomic and transcriptomic analysis are in progress on central and peripheral tissues to assess potential underlying mechanisms. Conclusions Overall, these findings indicate that ACD-0503 exerts promising pro-healthspan effects, probably via its anti-oxidant activity, although further data are required to characterise the most important pathways affected by ACD-0503 and to better understand the age, dose and sex differences observed in this study. In particular, one important conclusion is that data obtained in males cannot be immediately extrapolated to female subjects, raising a note of caution for the use of nutraceutical compounds in the general population. - Support: H2020 AwE (grant N. 633589)

Antioxidant N-acetyl-cysteine administration in pregnant mice has long-term positive effects on metabolic and behavioural endpoints of male and female offspring prenatally exposed to a high-fat-diet

Consumption of a high-fat-diet (HFD) during pregnancy acts as powerful prenatal stressor leading to adult stress-related metabolic or behavioural disorders. We hypothesized that administration of antioxidants throughout gestation might counteract the negative effects of maternal HFD feeding during pregnancy on the developing foetus. Female C57BL/6J mice were fed HFD for 13 weeks and exposed to the N-acetyl-cysteine (NAC) antioxidant from 10-weeks of age until right before delivery. Body weight of the offspring was assessed from birth until adulthood. At this age, the metabolic, neuroendocrine and emotional profiles were also tested. Prenatal HFD increased mother and offspring body weight when administered in conjunction with NAC. In female offspring, NAC reduced the increase in leptin levels resulting from prenatal HFD. NAC administration resulted also in greater glucose tolerance, insulin sensitivity and increased adiponectin levels. Exploratory behaviour was increased in NAC offspring, an effect accompanied by reduced stress-induced plasma corticosterone levels. Moreover, prenatal HFD administration led to reduced glutathione levels in the hypothalamus of males while NAC was able to revert this effect both in the brain (males) as well as in the brown adipose tissue (both males and females). Overall, results from this study indicate that the body redox milieu should be tightly regulated during foetal life and that buffering oxidative stress during pregnancy can have important long-term consequences on metabolic and behavioural endpoints. Support: H2020 AwE (N. 633589)

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

Improving our understanding of sex-dependent mechanisms regulating healthspan through novel methodological approaches

Improving our understanding of sex-dependent mechanisms regulating healthspan through novel methodological approaches Alessandra Berry1, Carla Raggi1, Chiara Musillo1, Gaia De Cristofaro1, Igor Branchi1, Silvia Poggini1, Aurelia Viglione1, Francesca Cirulli1 1Center for Behavioral Sciences and Mental Health, Istituto Superiore di Sanità, Viale Regina Elena, Rome, Italy A well-described clinical phenomenon is that females live longer but tend to experience greater levels of co-morbidity and disability than males, a condition that has been regarded as the “frailty paradox”. Frailty is a common condition associated with ageing relying on an increased vulnerability including decline of physical conditions in addition to metabolic and cognitive impairment. Notwithstanding this evidence, standardized measures of frailty in the ageing population (and in animal models) are still lacking. In the framework of the European project “Ageing with Elegans”, we sought to device a comprehensive protocol to assess frailty in both male and female mice. A frailty index was adapted from previously published protocols. Motor coordination and balance were assessed by means of the rotarod and beam walking tests, while muscle strength was measured using the grip strength test. Specific protocols were developed to measure cognitive performance, emotionality and motivation in the automated IntelliCage (TSE, Germany). This apparatus allows testing mice in their home-cage avoiding social isolation and the experimenter’s manipulation, markedly reducing the stress imposed by testing, a condition to which old animals are highly sensitive. Using this testing battery, we found that, despite females appeared in worst physical condition than males, they showed better performance in most of the above-described tasks, in agreement with the gender differences described in the human population. In conclusion, this protocol may be applied to better characterize the age-related frailty condition and to devise specific interventions. Support: H2020 AwE (grant N. 633589)

Nutraceuticals counteract memory decline in murine models of aging

Cognitive decline is a major disability during aging and, due to the global increase in lifespan, the proportion of people affected is expected to grow. The lack of target-specific drugs has shifted the attention to natural products as an appealing alternative. Notwithstanding the interest of the scientific community, the mechanisms of action of these compounds are still poorly understood and sound evidence is needed to support their use. Animal models have been successfully used to assess safety and efficacy of natural compounds. Through a systematic search, we sought to investigate the effects of nutraceuticals on: a. “natural aging” (mice older than 15 months); b. chemically-induced aging (e.g.: D-Galactose or scopolamine) or c. genetically selected lines (e.g.: SAMP8). In particular, we focused on the specific aspect of memory retention as assessed by means of the Morris water maze, that is one of the most widely used and reliable tests for spatial memory in rodents. Results show that natural compounds are effective in improving cognitive abilities in the three types of mouse models selected. The observed positive effects were independent from the nature of the substance, the route of administration and treatment duration. This suggests that common mechanisms, impinging upon cellular mechanisms underlying resistance to stress, characterize the efficacy of natural compounds. Our group is now investigating key questions related to dose-dependency of effects and sex/gender differences in efficacy. Funding: H2020 AwE ‘‘Ageing with Elegans’’ [grant-agreement N. 633589]

Rosmarinic acid administration improves cognitive and emotional behaviour and brain anti-oxidant defences in a sex-dependent manner in C57BL/6N aged mice fed a Western diet

A growing body of research suggests that regular consumption of nutraceuticals might improve healthspan. However, a better understanding of their therapeutic efficacy, as well as their mechanism of action, is needed. We investigated the ability of the nutraceutical compound rosmarinic acid (RA) to counteract the negative effects of a Western Diet (WD, high in sugar and fat) in a murine model of aging. Male and female 22-month-old C57BL/6N mice received either RA (500 mg/kg daily) or vehicle (KH2PO4/K2HPO4) via drinking water. Two weeks later, all subjects received either WD or control diet for 28 days, and were tested for emotional (Elevated Plus Maze - EPM), cognitive (Morris Water Maze - MWM) and metabolic parameters. At sacrifice, brain areas were dissected and used for transcriptomic analyses. Results show that RA prevented the WD-induced increase in body weight in males, although it failed to counteract the deleterious effects observed on glucose metabolism. Moreover, RA improved learning abilities in the MWM and increased exploration in the EPM in females only. These behavioural effects were associated with increased hippocampal expression levels of NFE2L2/NRF2, a master transcription factor for regulating the expression of multiple antioxidant proteins involved in mitochondrial redox homeostasis. Overall, our data suggest that RA may promote healthy aging in a sex-dependent fashion, at least in part through its ability to boost the organisms’ anti-oxidant defences. A thorough analysis of the transcriptomic profile (in process) will help elucidating all the pathways affected by RA. Support: H2020 AwE (grant-N. 633589)

Prenatal N-acetyl-cysteine administration prevents social anxiety and modulates brain immune- and plasticity-related genes in adolescent offspring born from high-fat diet C57Bl6/N mouse dams

Aims Maternal obesity is associated to increased oxidative stress (OS) representing a risk factor for adult mental health however the mechanisms underlying the negative long-term effects are poorly understood. We investigated inflammation, OS and hypothalamic-pituitary-adrenal (HPA) axis function in a mouse model of maternal high-fat diet (HFD) as potential mechanisms affecting brain development and emotional behaviour in the offspring. We also tested the antioxidant N-acetyl-cysteine (NAC) in preventing the long-term effects of HFD consumption during pregnancy. Methods Female C57BL/6N mice were fed HFD before and during pregnancy (13 weeks); after 5 weeks, half of them received NAC (1g/kg) for 8 weeks. Emotionality and social behaviour of male and female adolescent offspring (35-45 days) were assessed through the elevated plus maze (EPM) and the social interaction test (SIT); plasma corticosterone levels were assessed under basal conditions and following an acute stress. Gene expression levels of CD68, Bdnf and Nrf2 were measured in hippocampus as markers of microglial activation, brain plasticity and antioxidant capacity respectively by RealTime PCR. We focused on adolescence, an age of vulnerability for the onset of psychopathology. Results HFD offspring showed reduced exploration in the EPM and sociability in the SIT. These effects were associated to decreased hippocampal Bdnf levels in females while males showed increased CD68 expression and reduced basal corticosterone levels. Prenatal NAC administration prevented social anxiety, restored HPA axis basal activity in males and Bdnf levels in females. These effects may be partly mediated by Nrf2, an important regulator of antioxidant defence, as indicated by its upregulation in the hippocampus of both sexes. Conclusions Prenatal HFD showed detrimental sex-dependent effects on brain, neuroendocrine function and emotional behaviour; these changes were buffered by prenatal NAC suggesting that immune and OS signalling may play an important role in foetal programming of adult diseases. Funding: ERANET-NEURON-JTC-2018 Project EMBE