Joanne Weinberg - US grants

The ability to respond to stress is an important basic adaptive mechanism. Hyper-responsiveness or a deficit in the capacity to adapt to adapt to or recover from stress could ultimately affect an organism's health, in that prolonged stress levels of glucocorticoids are associated with sustained alterations in metabolism and immunosuppression. The present research will systematically document changes in stress responsiveness induced by ethanol exposure, either in utero or in adulthood, and will investigate one possible mechanism which may mediate these changes. The following hypotheses will be tested: 1) sex differences in stress responsiveness consistently occur following prenatal ethanol exposure; 2) the impaired capacity of fetal ethanol- exposed animals to adapt to and recover from stress is a robust and generalized phenomenon; 3) chronic ethanol intake in adulthood increases stress responsiveness and produces deficits in adaptation to or recovery from stress; 4) chronic ethanol consumption may differentially affect adult males and females; 5) a decrease in hippocampal glucocorticoid receptor concentration, induced prenatally by maternal ethanol intake and in adulthood by chronic ethanol consumption, might mediate changes in stress responsiveness. Animals will be exposed to ethanol either in utero or for varying periods in adulthood, and will be tested to determine 1) pituitary- adrenal response to prolonged or repeated exposure to a stressor; 2) response to chronic unpredictable stress; and 3) recovery following acute or chronic stress. Hippocampal glucocorticoid receptor concentration will be determined in fetal ethanol exposed animals: 1) under basal conditions; 2) following repeated exposure to a stressor; and 3) during recovery from stress. Experiments will also determine whether early handling can attenuate adverse effects of prenatal ethanol exposure on both pituitary-adrenal activity and hippocampal receptor concentration. In adult animals chronically consuming ethanol, hippocampal receptor concentration will be determined at varying intervals following cessation of 3, 6 or 12 weeks of chronic intake. In addition, testing will determine whether subjecting adult "alcoholic" animals to repeated or chronic stress exacerbates ethanol's adverse effect on receptor concentration.

This research will systematically document changes in stress responsiveness induced by ethanol exposure, either in utero or in adulthood, and will investigate mechanisms that might mediate these changes. 1. Fetal alcohol exposure (FAE). Our working hypothesis is that pituitary-adrenal hyperresponsiveness and deficits in recovery following stress which have been observed in FAE animals result from ethanol-induced deficits in feedback control of the hypothalamo-pituitary-adrenal (HPA) axis. HPA function in FAE animals will be explored at multiple levels: 1) by both challenging and inhibiting the system, and 2) by examining hormonal responsiveness in parallel experiments both in vivo and in vitro. The role of hippocampal glucocorticoid receptors in mediating increased stress responsiveness of FAE animals will be studied by examining receptor occupancy under conditions of varying hormone levels, receptor plasticity (up-regulation following adrenalectomy [ADX]), and receptor down-regulation (during chronic stress or chronic corticoid administration). Possible deficits in feedback regulation at other levels of the HPA axis (hypothalamus, pituitary) will also be assessed. Sex differences in response will be examined in all studies. 2.Adult chronic alcoholism. Our working hypothesis is that chronic alcohol consumption, which consistently elevates basal corticoid levels in both animals and humans, will result in HPA hyperresponsiveness to stress. Chronic alcoholic males and females will be tested using acute and chronic stressors to determine HPA activation and recovery, and to examine HPA responsiveness both in vivo and in vitro. The role of hippocampal glucocorticoid receptors in mediating changes in stress responsiveness will be studied by examining receptor occupancy following acute and chronic stress and receptor plasticity (up-regulation following ADX). Possible deficits in feedback regulation at other levels of the HPA axis will also be examined. The ability to respond to stress is an important basic adaptive mechanism. Hyperresponsiveness or deficits in recovery following stress could have adverse physiological and behavioral consequences and thus impact negatively on health. The proposed research will have relevance to our understanding of sex differences in ethanol's effects, both in utero and in adulthood, on the adaptive function of the organism.

DESCRIPTION (provided by applicant): Children with Fetal Alcohol Spectrum Disorder (FASD) exhibit cognitive, neuropsychological and neurobehavioral problems that range from mild to severe. Moreover, secondary disabilities in a number of life domains are common, including a high incidence of depression and anxiety disorders. Hypothalamic- pituitary-adrenal (HPA) dysregulation is a common finding in major depression. Furthermore, there is a strong relationship between depression in adulthood and adverse early life events. Consistent with these findings, brain areas implicated in depression overlap with areas that mediate the stress response, with the HPA axis a key player in both. We have shown that prenatal alcohol exposure (PAE) reprograms the fetal HPA axis such that HPA tone is increased throughout life. PAE offspring are typically hyperresponsive to stressors, and show both increased HPA drive and deficits in feedback regulation. Furthermore, interactions between the HPA axis and both the gonadal and serotonergic systems are altered by PAE. This pattern of dysregulation parallels in many ways what is observed in depression. The present proposal will utilize our well-established animal model of PAE to examine the links among PAE, stress system abnormalities, the gonadal and serotonergic systems and depression. In the context of the stress-diathesis model, we will test the hypothesis that fetal programming of HPA activity by PAE permanently sensitizes neuroadaptive mechanisms that mediate responses to stress, resulting in hyper- reactivity to subsequent, even mild, stressful life events. Ultimately, repeated stress exposure results in a maladaptive cascade of events and increased vulnerability to depression and anxiety. Our Preliminary Studies demonstrate sex-dependent increases in depressive symptomatology in PAE animals following exposure to chronic mild stress (CMS) in adulthood. The proposed studies will extend these finding in important and novel directions. Our Specific Aims are to investigate: 1) the role of the maternal hormonal milieu in programming fetal HPA activity, sensitizing the offspring HPA axis, and mediating increased vulnerability to adverse effects of stress on offspring HPA, brain and behavioural outcomes;2) the role of HPA dysregulation in adult PAE males and females in mediating increased vulnerability to adverse effects of stress on HPA, brain and behavioural outcomes, and the role of the gonadal hormones in mediating the sexually dimorphic effects of PAE and stress that are observed;and 3) whether antidepressant treatment can reverse or alleviate the adverse effects of stress on HPA, brain and behavioural outcomes in PAE offspring. The proposed studies will elucidate mechanisms mediating the link between PAE and increased vulnerability to depression and anxiety disorders in children with FASD, and have important implications for the development of novel therapeutic interventions. PUBLIC HEALTH RELEVANCE Children with Fetal Alcohol Spectrum Disorder (FASD) exhibit cognitive, neuropsychological and behavioral problems, as well as numerous secondary disabilities, the most prominent being mental illnesses such as depression. The present research utilizes our well established animal model of prenatal alcohol exposure to examine the role of stress system abnormalities in mediating this increased incidence of depression. The data from these studies will significantly increase our understanding of the mechanisms underlying the long term and far reaching consequences of prenatal alcohol exposure on health and well-being, and will have important implications for the development of novel therapeutic interventions for depression in individuals with FASD.

[unreadable] DESCRIPTION (provided by applicant): Prenatal ethanol exposure programs the fetal hypothalamic-pituitary-adrenal (HPA) axis such that HPA tone is increased throughout life, resulting in HPA hyperresponsiveness to stressors. The developmental programming concept states that early environmental stimuli can program metabolic and physiologic processes and as such increase vulnerability to illnesses or disorders later in life. Thus a long-term increase in HPA function will increase exposure to glucocorticoids over the lifespan and have adverse consequences for health. The mechanism(s) underlying such programming are unknown, but probably involve an interaction between environmental stimuli and epigenetic regulation of gene expression, such as changes in DNA methylation and/or chromatin structure (e.g. histone methylation, acetylation). An organism is especially vulnerable to epigenetic changes during development and the epigenome is highly susceptible to environmental stimuli such as maternal diet (including drugs and other toxic agents) and maternal behavior. We propose that in utero ethanol exposure is an environmental stimulus that elicits epigenetic changes with long-term adverse consequences. Because of the metabolic link of ethanol to methyl group metabolism we speculate that the HPA programming observed in adult animals exposed in utero to ethanol is a consequence of alterations in methyl group metabolism, gene-specific changes in DNA methylation patterns and a gene expression profile that promotes hyperresponsiveness to stress. Utilizing our well established model of prenatal ethanol exposure, we will test the hypothesis that prenatal ethanol exposure alters the methionine cycle and programs the fetal HPA axis through gene-specific changes in DNA methylation patterns and expression of the genes crucial to normal HPA activity and regulation. Our Specific Aims are to determine if fetal ethanol exposure results in: 1) altered methyl status in offspring; and 2) changes in DNA methylation patterns and the expression profile in brain of the glucocorticoid receptor (Nr3c1) and the mineralocorticoid receptor (Nr3c2) genes. The proposed studies will begin to elucidate possible mechanisms underlying HPA programming and altered behavioral responses to stress in animals exposed to ethanol in utero. Our goal is to provide a framework for the future development of interventions that could be effective in rescuing these systems. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Among the physiological abnormalities reported in fetal alcohol spectrum disorder (FASD), both clinical studies and studies using animal models and have demonstrated marked impairments in immune competence, including delayed and deficient development of the immune system, depressed or overactive immune responses to challenge, and increased risk for infection and various forms of cancer, with many changes persisting into adolescence and adulthood. Impaired immune function in the alcohol-consuming mother, altered immunity at the fetal-placental interface, and disruption of the finely-tuned bidirectional communication between the neuroendocrine and neuroimmune systems appear to play a role in the deficits observed in prenatal alcohol-exposed (PAE) offspring. The present proposal investigates when and how PAE can impact immune function, and will elucidate possible mechanisms underlying PAE's adverse effects on the mother, the fetus, and the interaction between maternal and fetal systems. The role of early life experience in programming the set-point or tone for stress and immune responsiveness to later life challenge will also be investigated. Our Specific Aims are: 1) To investigate the effects of alcohol consumption on neuroimmune function of the pregnant dam and fetus, and their possible link, in mediating alcohol-induced immune impairments in dams and PAE offspring via cytokine analysis (Expt 1A) and fetal microglia responsivity (Expt 1B). 2) To elucidate a developmental neuroimmune profile of PAE compared to control offspring from birth to adulthood to gain insight into how PAE, in the presence or absence of stress during the adolescent period, may differentially alter resting neuroimmune function and set the stage for vulnerability to stress or immune challenge in later life; 3) To investigate the role of early life immune challenge, imposed on an already sensitized organism (PAE), in modulating differential immune and neural outcomes of PAE and control animals following immune challenge in adulthood; 4) To elucidate mechanisms underlying the differential course of inflammation in PAE and control offspring observed previously, and to investigate possible differential effects of stress during adolescence, a sensitive developmental period, in modulating the course of inflammation in adulthood. Our working hypothesis is that fetal programming of stress and immune systems by PAE results in a primed, vulnerable pro-inflammatory-biased organism that is predisposed to increased responsiveness to immune challenge in adulthood. Elucidation of mechanisms underlying neuroimmune deficits in PAE offspring is critical for understanding the nature of the immune abnormalities seen in children with FASD, which will improve clinical care of these children, and ultimately improve their long-term health and well-being.

Risk for adult diseases or disorders is known to be influenced by the prenatal/early life environment. Building on seminal studies by Barker and colleagues, who reported associations between low birth weight and biological risk for adult disease, support for the ?developmental origins of health and disease? (DOHaD) hypothesis has grown to include early life adversities beyond low birth weight and to extend to outcomes beyond metabolic syndrome. Of particular relevance, prenatal alcohol exposure (PAE), in addition to its teratogenic effects, can program developing neurobiological systems and thus increase risk for diseases/ disorders over the life course. Our CIFASD Developmental Project is the first to identify links among maternal alcohol consumption, inflammation, and child outcomes; unique immune signatures in pregnant women were identified in association with whether or not they consumed alcohol and with neurodevelopmental outcomes of their children. The proposed UO1 builds on these findings to examine immune profiles in pregnant women and children from birth through adulthood. Our working hypothesis is that prenatal alcohol-induced dysregulation of immune/ inflammatory systems will be associated with adverse health, functional and adaptive outcomes, providing insight into factors underlying risk and resilience. Specific Aims are to: 1) Use validation cohorts to confirm the utility of immune parameters as possible biomarkers and predictors of alcohol-related health and neurobehavioral outcomes, by analyzing: a) plasma samples and neurobehavioral outcomes from matched mother-infant or mother-child pairs from Ukraine (Chambers) and San Diego (Chambers, Jones, Mattson), respectively, and a child cohort from Minnesota (Wozniak). Assessment of individuals from different cultural/ethnic, SES, and environmental conditions will provide insight into factors modulating alcohol?s programming effects, and increase understanding of immune variables as biomarkers of alcohol intake and predictive factors for PAE-related outcomes. 2) Extend assessment of the immune system into adulthood with assessment of cohorts from Atlanta (Coles), Seattle (Grant), and British Columbia (Weinberg, Loock, Oberlander, Lutke). We will investigate whether physical/mental health and impaired cognitive and adaptive outcomes are associated with immune/inflammatory system dysregulation resulting from prenatal programming effects of alcohol. For both Aims, we will analyze cytokines/inflammatory markers in plasma, obtain past and current mental/physical health information, and measure neurobehavioral and adaptive outcomes, providing novel insight into links among immune function, cognitive and adaptive function, and health outcomes. Together, these studies bring a new dimension to CIFASD, a focus on DOHAD/health outcomes, a critically important but relatively understudied area. Moreover, as the immune system plays a key role in brain development, this work has broad implications for understanding the role of immune/inflammatory mechanisms in FASD-associated neurobehavioral and adaptive deficits.