Tracy Bale - US grants

DESCRIPTION (provided by applicant): The predisposition to stress-related mood disorders likely involves a genetic vulnerability to increased stress sensitivity and may be a direct result of a dysregulation in central corticotrophin releasing factor (CRF) pathways. The focus of this grant proposal is the examination of central CRF pathway dysregulation involvement with increased susceptibility for stress-related mood disorders via interactions with serotonin (5-HT) neurocircuitry. Our studies utilize a genetic mouse model in which CRF expression is elevated in the amygdala in addition to showing increased stress responsivity under conditions of homeostatic challenge. In order to influence synaptic 5-HT levels in our behavioral, biochemical, and molecular comparisons, we will treat animals with the SSRI fluoxetine. The following aims are proposed using an integrated view of how stress pathways impact emotional behavior, specifically the interactions of CRF and 5-HT neurocircuitry. Aim 1 will examine the possible role of CRF dysregulation in the development of stress-related mood disorders resulting from an impact on serotonin pathways by examining behavioral stress responses. Aim 2 will then examine the molecular and biochemical changes corresponding with behavioral outputs following fluoxetine treatment. We will study alterations in CRF and 5-HT receptor gene expression, protein levels and biochemical state, and receptor occupancy in specific brain regions. In Aim 3 we will create a conditional, inducible, site-specific mouse deficient for CRFR1 in 5-HT producing cells in order to more directly examine the specific interaction of elevated central CRF expression and 5-HT pathways impacting stress-sensitive behaviors. These mice will be crossed with our CRFR2-deficient mice to produce mice with elevated amygdalar CRF and deficient in CRFR1 in 5-HT cells. We hypothesize that results from these proposed studies will demonstrate an involvement of a dysregulation of CRF pathways in alterations in 5-HT neurotransmission leading to a predisposition to stress-related mood disorders.

DESCRIPTION (provided by applicant): The purpose of this conference on Early Life Programming of Neurodevelopmental Disorders is to bring together both basic and clinical scientists to facilitate collaborative efforts between these groups with overlapping interests in affective disorders, schizophrenia, and obesity. This meeting is designed to have brief talks by speakers on their most recent studies and include new directions that their work is heading. This will allow attendees to determine what resources could be shared, what information may benefit new studies, and will encourage a more rapid endpoint to translational outcomes. The meeting will have a format where talks are centered on disease focus. Each session will include basic, clinical, and non-human primate speakers. Each afternoon will have a breakout session focused on each disease. These sessions will include the speakers from that day as well as all attendees interested in that disease, and will be led by the session chair. Each participant will present one slide detailing their interest in the field and what their future plans and goals are. Discussion will then promote collaborative interactions such that materials, reagents and resources can be effectively utilized, new mechanistic findings can be applied to proposed studies, and funding opportunities focused on translational abilities will be emphasized. Discussion will focus on defining the direction the field needs to go and what resources would get us there faster and more effectively. This conference will allow experts in the field from all disease areas to come together and build networks and promote collaborations that will undoubtedly facilitate our understanding of disease treatment and prevention. Bale, Tracy L. Neurodevelopmental disorders currently impact 6-10% of Americans. As studies support a major contribution of early life programming in disease susceptibility, there is a critical need for a forum in which basic and clinical scientists can share their most recent findings and future directions in order to facilitate collaborations and the best utilization of materials, and for the promotion of cross-disease interest discussions to occur such that underlying mechanisms, genes, and strategies can be shared. The justification for this meeting is for the initial presentation of science to provide the stimulus for the critical discussions to occur in the afternoon Breakout Sessions by providing the time and forum to do so where scientists across these diseases can share information and build networks and collaborations that will promote faster and more effective outcomes in translational research.

DESCRIPTION (provided by applicant): The mechanism through which fetal antecedents contribute to disease development is not understood, though likely involves a complex interaction between maternal environment, placental changes, embryo sex and epigenetic programming. As most neurodevelopmental disorders exhibit a sex bias in presentation, elucidation of the mechanisms by which sex-specific susceptibility arises is likely to provide critical insight into disease etiology. We have recently identified a sensitive period of early gestation where stress has sex-specific long- term programming effects on offspring stress pathway neurodevelopment. Mechanistically, we have detected sex-specific effects of maternal stress on placental inflammatory cytokines, growth factors and epigenetic machinery. From these studies, we hypothesize that early pregnancy is a highly sensitive period for the long- term sex-specific consequences of maternal stress through effects on placental inflammation, epigenetic machinery and nutrient transport altering programming of the developing embryo. Therefore, our studies will examine: 1) how early prenatal stress (EPS) alters the long-term programming of stress pathway neurodevelopment through sex-specific placental and embryonic inflammation, nutrient transport and changes in epigenetic machinery during a highly sensitive period of early gestation, 2) the possible rescue of the sex- specific programming effects of EPS by maternal treatment with an anti-inflammatory or a specific inhibitor of NFkB activation, NBD, and 3) genomic and proteomic technology including ChIP-Sequencing and pathway focused PCR Arrays to analyze placental and embryonic brain tissues and proteomic analysis of amniotic fluid across gestation to identify potential translatable biomarkers and genes important in the programming of stress dysregulation predictive of neurodevelopmental disorders in EPS offspring.

DESCRIPTION (provided by applicant): Sex-biased neurodevelopmental disorders, including schizophrenia, have been associated with maternal stress experienced during pregnancy. We have recently identified a specific period of early pregnancy where male offspring were sensitive to the effects of maternal stress, displaying as adults behaviors and stress physiology suggestive of brain feminization. Our hypothesis to be examined in these proposed studies is that prenatal stress exerts programming effects on the developing male brain via changes in methylation patterns affecting testis development and testosterone production during the organizational period of sexually dimorphic brain development. Organizational and activational testosterone has been shown to be important in programming of male stress neurocircuitry. Stress pathway dysregulation and sensitivity is a hallmark of most neuropsychiatric disorders. Therefore, these studies are designed: 1) To determine the contribution of SRY gene methylation and expression in the programming of a feminized stress-sensitive phenotype of early prenatal stress male mice, 2) To examine the heritability of early prenatal stress effects in second generation offspring to identify possible gene targets of PNS that may be epigenetically modified in the germline, and 3) To utilize prenatal testosterone treatment or DNMT1 inhibition to ameliorate the effects of early prenatal stress on male offspring stress sensitivity. Determination of the fetal antecedents and mechanisms by which alterations in brain development of these circuits occur, and identification of potential heritable aspects of this phenotype may provide insight into novel therapeutic targets and disease prevention.

It has been well established that early life adversity exposure (prior to puberty) is one of the strongest predictors of adult affective disorders such depression, anxiety, PTSD, and bipolar disorder, especially in women. The most common underlying feature in affective disorders is that of stress dysregulation. The goal of this proposal is to determine how the prepubertal adversity experience reprograms the brain toward stress dysregulation, or stress resilience, and how this is influenced during dynamic hormonal changes across the female life span, including pregnancy and aging. We are proposing to examine similar outcome measures proposed in the human subject studies in Projects I & II in a mouse model. The mouse model allows greater control over manipulations in the environment and dissection of potential mechanisms involved in programming of endpoints than what is possible in humans, and thus is an invaluable model for this SCOR proposal. Aims for these studies include: 1) To examine the programming effects of prepubertal adversity on adult stress responsivity during the period of dynamic hormonal change, pregnancy, in a mouse model. Behavioral and physiological analyses during this period will include assessment of the HPA stress axis, and anxiety-like behaviors as well as stress responsivity in both male and female offspring; 2) To examine the programming effects of prepubertal adversity on adult stress responsivity during the period of dynamic hormonal change, aging, in a mouse model. Sex differences in behavioral and physiological analyses in aged mice will include assessment of the HPA stress axis, cognitive flexibility and performance, and anxiety- like behaviors; and 3) To examine the potential molecular mechanisms by which prepubertal adversity may program changes in stress pathway maturation and thus altering long-term adult stress responses during dynamic hormonal periods, including examination of the sex-specific brain miRNA environment and epigenetic histone marks in relevant brain regions during the prepubertal window.

DESCRIPTION (provided by applicant): Stress pathway dysregulation is the most pervasive symptom in neuropsychiatric disease, yet we understand little as to the developmental programming and maturation of this system and the sensitive periods during which perturbations may be disruptive. Stress during pregnancy has been strongly associated with an increased incidence of neurodevelopmental disorders, including depression, anxiety, schizophrenia, and autism. We have developed a mouse model of early prenatal stress in which male offspring present with increased stress sensitivity. Mechanisms for how stress during pregnancy contributes to reprogramming of stress pathways likely involve complex connections between the maternal and fetal environments. One such interaction that has not been explored is the effect of prenatal stress on the vaginal microbiome. As the neonatal gut is initially populated from the maternal vaginal microbiome, changes in the vaginal ecology produced by maternal stress will similarly affect this microbial population. Hence, such changes in neonatal gut microbial diversity could impact neurodevelopmental processes via changes in vital nutrient metabolism and absorption. Therefore, our proposal will utilize our mouse model of early prenatal stress to examine: 1) the effects of stress on the maternal vaginal and pup gut microbiome composition, including changes in relative Lactobacillus levels and beta diversity using MiSeq technology, 2) mechanisms by which the microbiome is involved in neurodevelopmental programming through direct manipulation of the microbiome to rescue and recapitulate aspects of the EPS phenotype and effects on hypothalamic development, and 3) epigenetic mechanisms by which the pup gut microbiome reprograms the hypothalamus, focusing on metabolomics outcomes linked with functional gene sets and examining their upstream epigenetic marks.

? DESCRIPTION (provided by applicant): Parental lifetime exposures to perturbations such as stress, infection, malnutrition, and to advanced age have been linked with an increased risk for neurodevelopmental disorders in their children. While maternal insults during pregnancy can directly impact fetal development, the mechanisms by which paternal lifelong experiences can alter germ cell programming and affect offspring neurodevelopment are not known. However, transmission of these epigenetic marks to the next generation can significantly elevate disease risk, and if programmed into the germline can affect future generations as well. Using male stress experience as a model in which we can examine mechanisms involved in offspring neurodevelopmental programming, we found that paternal stress significantly altered offspring HPA stress axis regulation and reprogrammed the hypothalamus. Mechanistically, we identified 9 specific miRNAs in the mature sperm from stressed males that contributed to the offspring phenotype. Further, we were able to completely recapitulate the offspring phenotype by microinjection of these 9 miRNAs into fertilized zygotes. Using single-cell amplification technology, we were identified a novel role for these miRNAs to significantly affect post-fertilization embryo development, providing substantial evidence that sperm miRNAs are programmable by the environment and are able to transmit this information allowing for paternally directed embryo development. Therefore, our proposal will utilize our mouse model of paternal stress to examine: 1) the mechanisms whereby stress alters paternal germ cell miRNA that affect neurodevelopment and give rise to the offspring phenotype, 2) the transgenerational transmission of stress dysregulation to a second generation programmed by paternal stress and sperm miRNAs, and 3) the mechanism within the offspring brain that promotes the paternal stress phenotype through increased BBB permeability and repressive histone epigenetic programming.

Maternal lifetime exposures to perturbations such as stress, infection, malnutrition, and advanced age have been linked with an increased risk for offspring disease, including a strong association with neuropsychiatric disorders. While maternal insults during pregnancy can directly impact fetal development, the mechanisms by which lifelong experiences can alter germ cell programming and affect offspring neurodevelopment are not known. Surprisingly few animal models have been developed to examine programming mechanisms of preconception perturbations. In this proposal, we utilize our mouse model of maternal preconception stress (MPS) in which female, but not male, offspring present with elevated stress sensitivity, to examine the sex- specific mechanisms contributing to neurodevelopmental programming. Based on our exciting published and preliminary data, we hypothesize that MPS imparts changes in oocyte stored maternal mRNAs that interact during blastocyst and placental development to program sex-specific changes in the developing hypothalamus. Our hypothesis will be tested in this proposal and examined at 3 distinct mechanistic levels: the oocyte, the placenta, and the hypothalamus. Sex differences in the placenta are likely to produce sex-specific trans- placental signals to the developing brain and begin with sex chromosomes. Our studies and others have identified X-linked genes that are expressed at higher levels in the female placenta. Through a genome-wide screen following maternal stress, we identified the X-linked gene, OGT, as causal in programming a sex- specific stress phenotype. Placental OGT is poised to respond to changes in the environment that are important to the developing embryo, ultimately serving as a sex-specific regulator of hypothalamic development. Therefore, our proposal will focus on defining mechanisms contributing to MPS programming examining the: 1) oocyte: To identify the lasting molecular changes that occur in response to preconception stress that are present at fertilization and their necessity for reproducing the female offspring phenotype, 2) placenta: To determine the maternal preconception stress sex-specific mechanisms involved in the female offspring phenotype focusing on the placental contribution, and 3) hypothalamus: To determine the sex-specific hypothalamic programming changes following maternal preconception stress and the role this plays as an underlying factor in female neuropsychiatric disease risk.

Defining the mechanisms by which stress in the environment during pregnancy promotes changes in development is critical in identifying factors predictive of disease risk or resilience. One major consistency across prenatal insults is the increased vulnerability of males. In this proposal, we utilize our mouse model of early prenatal stress (EPS) to examine sex-specific placental transcriptional regulation. In our EPS model, male, but not female, offspring present with increased stress sensitivity, including increased HPA stress axis activity, reduced post-weaning growth, and hypothalamic mitochondrial dysfunction. Sex differences in the placental function are likely to produce sex-specific transplacental signals to the developing fetal brain. Sex differences in the placenta begin with sex chromosomes. Through a genome-wide screen following maternal stress, we identified the X-linked gene, OGT, as causal in programming the male-specific stress phenotype via its regulation of the histone transcriptional repressive mark, H3K27me3. This proposal uses innovative approaches to determine the mechanisms by which the female placenta is able to restrict transcriptional responses to stress in the environment, where males are not, thus placing the male developing brain at greater risk prenatally. The transplacental signals received by the developing brain appear to be related to energy availability and impact metabolic and mitochondrial programming. Of key translational importance, we have also found the same biochemical and molecular outcomes are predicted by fetal sex in human placental tissue. Therefore, our proposal will focus on defining the causal importance of H3K27me3 in risk for developmental changes in response to stress, identify the sex-specific transplacental signals resulting from these changes in placental function using ex vivo perfusion, and determine the cellular compartment and mechanism by which these changes promote hypothalamic mitochondrial reprogramming and the EPS phenotype.