Rajesh C. Miranda - US grants

DESCRIPTION (Adapted from applicant's abstract): The overall purpose of this proposal is to examine the role of the estrogen receptor, a nuclear transcription factor, in the differentiation of cerebral cortical circuitry that subserves cognition and undergoes a period of naturally occurring cell death, with features characteristics of apoptosis. The proposed experiments examine the interactions of the estrogen receptor with a family of growth factors, the neurotrophins, in the regulation of this neuronal death within the developing rodent prefrontal/limbic cerebral cortex. We hypothesized that this transcription factor, activated by endogenous growth factors, may selectively alter the process of naturally occurring cell death, and under conditions of inappropriate activation, lead to altered patterns of cortical differentiation. The first specific aim will be to determine whether estrogen receptors mediate the neurotrophin regulation of apoptosis in the developing rodent prefrontal/limbic cerebral cortex. The experimental design will make use of an in vitro explant culture model of chemically induced apoptosis and a complementary in vivo model of naturally occurring apoptosis. The second specific aim will be to determine estrogen receptor-dependent components of the cellular signaling pathway that initiates apoptotic cell death in the cerebral cortex. Specifically, experiments will investigate the estrogen receptor-mediated regulation of specific cyto-protective (i.e., Bcl-2 and Bcl-x) and cytotoxic (TNFr, p75 and fas) components of the signaling pathway that mediate cell survival using in vitro and complementary in vivo models of naturally occurring apoptosis in the developing limbic/prefrontal cerebral cortex. The answers to these questions have clinical implications. Altered activation of transcription factors in the immature cerebral cortex may lead to inappropriate patterns of neuronal survival and differentiation and impairments in the development of cognitive and affective behaviors. Such perturbations may be an important etiologic feature of the impaired functioning characteristics of a variety of psychiatric disorders including schizophrenia and pediatric dementias. Elucidation of basic developmental processes is essential to understanding the pathogenesis of these important psychiatric disorders. Additionally, these experiments will provide a framework for future studies examining the role of the estrogen receptor in cortical plasticity and in the formation of cognitive circuits, integrating the cerebral cortex with its efferent targets.

[unreadable] DESCRIPTION (provided by applicant): Alcohol is an important teratogen. Much research has focused on the sensitivity of the third- trimester period of brain development, where ethanol has been shown to be neuro-toxic. However, we know very little about the vulnerability of the second trimester period. The second trimester is important, because during this trimester, fetal neural stem (NSCs) and progenitor (NPCs) cells give rise to most neurons of the adult brain. Previously, we showed that ethanol did not kill fetal cortical NSCs/NPCs. Rather, ethanol induced cell proliferation, but depleted NSCs and NPCs, suggesting that ethanol promoted aberrant NSC maturation. Importantly, ethanol suppressed four microRNAs; small non-coding RNA molecules that control large gene networks to determine cell fate. Our central hypothesis is that ethanol depletes NSCs by driving stem to blast maturation of NSCs. A secondary hypothesis is that microRNAs mediate teratogenic effects of ethanol. We will address three specific aims. (Aim#l) To determine the extent to which ethanol promotes premature cortical neuroepithelial maturation. Our hypotheses are that (a) in utero binge ethanol exposure, during the second trimester period will significantly deplete resident NSCsINPCs and (b) isolated NSCs/NPCs will exhibit aberrant maturation patterns following ethanol exposure. We will test the ethanol- sensitivity of NSC/NPC populations using flow cytometric approaches. (Aim#2) To identify ethanol- sensitive microRNAs and their biological mechanisms. Our hypothesis is that ethanol persistently suppresses microRNAs that promote NSC/NPC renewal. We will use microarray technologies to identify candidate microRNAs, and manipulate levels of these microRNAs, to identify their role in NSC/NPC maturation. (Aim#3) To determine the extent to which ethanol-sensitive microRNAs prevent or reverse the effects of ethanol on fetal NSCs/NPCs. Our hypothesis is that microRNA supplementation will prevent and reverse ethanol's effects on NSC/NPC renewal and maturation. Cell biological approaches and mRNA expression analyses and will be used to assess the capacity of ethanol-regulated microRNAs to reverse or prevent the effects of prior ethanol exposure. At the end of the project period, we expect to have identified sensitive NSC/NPC populations, critical second-trimester periods of vulnerability to ethanol, and specific microRNAs and microRNA- mediated mechanisms that are persistently altered by ethanol. These outcomes will be significant because they are expected to provide the foundations for developing therapeutic strategies to manage the persistent neural effects of a leading teratogen. PUBLIC HELTH RELEVANCE Alcohol is a potent teratogen. Alcohol consumption during pregnancy can lead to a stereotypic spectrum of disorders in children, called the `Fetal Alcohol Spectrum Disorders' or FASD. At the neuroanatomical level, birth defects can include microencephaly and brain malformations. Much research has focused on the sensitivity of the third-trimester period of brain development, where ethanol has been shown to be neurotoxic. However, we know very little about the vulnerability of the second trimester period. The second trimester is important, because during this trimester, neuroepithelial cells of the ventricular walls give birth to millions of new neurons, creating a cellular framework for the rest of neural development. Previously, we showed that fetal cerebral cortical neuroepithelial cells did not die when exposed to ethanol. Rather, ethanol induced cell proliferation, while depleting cells expressing neural stem (NSC) and progenitor cell (NPC) markers. This proposal will test two hypotheses, that ethanol depletes stem and progenitor cells by driving stem to blast maturation of NSCs, and that the effects of ethanol are mediated by, and consequently, can be reversed by microRNAs. At the end of the project period, we expect to have identified sensitive NSC/NPC populations, critical second-trimester periods of vulnerability to ethanol, and specific microRNAs and microRNA-mediated mechanisms that are persistently altered by ethanol. These outcomes will be significant because they are expected to provide the foundations for developing therapeutic strategies to manage the persistent neural effects of a leading teratogen. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Maternal alcohol consumption during early pregnancy is difficult to prevent due to the prevalence of both unplanned pregnancies and binge patterns of alcohol consumption in the US. Exposure is common during the 1st trimester, when neural stem cells (NSCs) begin producing neurons, increasing the risk for neurodevelopmental disability. There is a critical, un-met need for biomedical interventions to mitigate effects of alcohol exposure. A lack of such interventions means that we can do little to help women who subsequently seek prenatal care for fetal alcohol exposure. Our long-term goal is to find ways to mitigate brain damage due to teratogens like ethanol. Our approach to reversing ethanol's effects focuses on intervening prenatally to manipulate the growth potential of residual fetal NSCs. This approach is based on our key findings that ethanol does not kill NSCs, but promotes premature maturation. A class of small regulatory RNAs, miRNAs, mediates many of these ethanol effects. Ethanol deregulates miRNA (miR153, miR335) control of differentiation-promoting transcription factors (ndTFs) like Nfia and Nfib and NeuroD1, resulting in premature ndTF expression in NSCs. Moreover, nicotinic acetylcholine receptor (nAChR) agonists can prevent and even reverse effects of ethanol on miRNAs and their target ndTFs. Collectively, these data support two hypotheses: (1) ethanol depletes NSCs by interfering with miRNA-ndTF networks that prevent premature NSC maturation, and (2) both miRNAs and nAChR agonists prevent and perhaps even reverse effects of fetal ethanol exposure. Aim 1 will identify key ndTFs that facilitate ethanol effects on NSC self-renewal and maturation while Aim 2 will identify key miRNAs that block ethanol effects. Aim 3 will identify pharmacological interventions that control miRNA-ndTF networks and prevent ethanol- mediated loss of NSCs. We will assess direct nAChR effects (i.e., varenicline exposure), as well as ndTF- and miRNA-mediated effects of nAChR activation, on NSC renewal and maturation. In these aims, we will manipulate ndTFs and miRNAs with innovative viral-mediated strategies, and a novel murine inducible reporter model to track affected NSCs and their daughter progeny. This proposal is significant in that it is expected to lay the theoretical and experimental framework for a new approach to address the un-met need for reparative fetal therapy to prevent FASD. It is innovative because it advances a novel conceptual model that links a cellular target (miRNA-ndTF networks) to a therapeutic approach (varenicline and nAChR pharmacology), to reprogram neurogenesis in the aftermath of alcohol exposure. As an outcome of these studies we expect to identify core molecular and pharmacological approaches to repair fetal damage following exposure to a potent and common teratogen, alcohol.

Project Summary There is a fundamental knowledge gap in understanding the substantial variability in the incidence, onset, duration, and severity of neonatal abstinence syndrome (NAS) in infants born to women with opioid use disorder (OUD). Despite a growing body of literature in the NAS field, no single factor or combination of known factors have sufficiently explained this variability. Current clinical tools to identify infants needing pharmacological treatment for NAS are reactive rather than proactive since they focus on the severity of physical symptoms of withdrawal at onset. Thus, there is an unmet need for biomarkers in the neonate that proactively predict the severity of NAS, before the onset of physical withdrawal. The long-term goal of our program is to improve the short- and long-term outcomes of infants born to women with OUD. The objective in this particular application is to assess the extent to which intra- and extra-cellular miRNAs are sensitive to opioid withdrawal in the period preceding physiological manifestations of NAS. The central hypothesis is that umbilical cord-derived plasma and leukocyte miRNAs may be assessed as surrogate markers for brain health at birth, following opioid exposure, and predict severity of NAS before the emergence of physiological signs of withdrawal. Our preliminary data indicate that important brain miRNAs are secreted by developing neural cells and present in neonatal plasma, and can serve as a biomarker for brain effects of prenatal drug exposure. Guided by these preliminary data, the hypothesis will be tested by pursuing the specific aim to identify an infant miRNA signature that precedes and predicts NAS. This will be assessed in 70 well-characterized infants prenatally exposed to opioids by identifying 1A) intra-cellular (leukocytes) and extra-cellular (plasma) miRNAs that differentiate infants with NAS (cases) and without NAS (controls); 1B) the extent to which this miRNA composite signature of cell and tissue adaptation predict the severity of NAS. The rationale for the proposed research is that miRNAs can be used not only as diagnostic biomarkers for drug exposure and effects, but also can be manipulated to diminish effects of drug exposure and to provide neuroprotection. The approach is innovative, because it examines a unique set of biomarkers within the context of other perinatal predictors of infant health and will leverage resources of an established, well-characterized longitudinal cohort. The proposed research is significant because it is expected to lead to better understanding of maternal, newborn, and epigenetic factors contributing to NAS variability, and is expected to lead to identification of new therapeutic targets for preventing NAS. Better understanding of mechanisms involved in NAS pathophysiology has the potential to change clinical management of NAS, which now affects 6 per 1000 hospital births. Ultimately, such knowledge has the potential to improve short- and long-term outcomes of the growing number of infants prenatally exposed to opioids in the U.S.

Project(Summary( Fetal Alcohol Spectrum Disorders (FASD) result in life-long systemic disabilities that contribute to disease and premature mortality in FASD adults. We recently found that prenatal alcohol-exposure (PAE) led to long-term deficits in cranially-directed vascular function in aging mice. PAE also diminished neurological recovery in young adult mice following cerebrovascular ischemic stroke. Preliminary data indicate that middle-aged PAE animals experience larger stroke infarcts compared to age-matched controls or young PAE adults. Moreover, reduced levels of the peptide hormone, IGF1, and epigenetic re-programming of IGF pathways contribute to ischemia-induced brain damage and disability, while intracranial IGF1 delivery after stroke improves tissue survival and behavior. Therefore, we hypothesize that ?PAE accelerates the age-dependent increase in brain vulnerability to ischemic stroke by epigenetically programming IGF1 signaling pathways??. We plan to assess effects of PAE on brain adaptation to ischemia in aging male and female adults in rat models, and consistent with stroke research guidelines, use two models for ischemic stroke by intraluminal suture-occlusion and by endtothelin-1-mediated vasoconstriction of the middle cerebral artery. Aim 1 will determine the extent to which PAE influences brain damage, sensorimotor impairment, and blood brain barrier (BBB) permeability, in aging adults following ischemia. Our working hypothesis is that the middle- aged PAE brain will exhibit a larger infarct volume following ischemia, compared to age-matched controls, and comparable to the aged non-PAE adult brain. Middle-aged and aged PAE animals will also exhibit increased sensorimotor impairment, accompanied by prolonged BBB permeability following an ischemic episode compared to age-matched, non-PAE controls. Aim 2 will assess the contribution of PAE to aging-related epigenetic reprogramming of IGF1 pathways. Our working hypothesis is that PAE epigenetically reprograms liver and brain resulting in aging-related loss of IGF1 in adulthood. We expect that PAE will result in chromatin silencing or miRNA-mediated translation-repression of IGF1 signaling. Aim 3 will determine the impact of exogenous IGF1, or epigenetic stimulators of hepatic or brain IGF1, on ischemia outcomes in PAE adults. Our working hypothesis is that IGF1 supplementation after ischemia will ameliorate effects of PAE on the BBB, infarct volume, and sensorimotor function in aging animals. We will test the extent to which effects of PAE on stroke-induced impairment are ameliorated by post-stroke treatment with IGF1, or with agents that promote IGF function, like sodium butyrate, a histone deacetylase inhibitor, and an antagomir to the microRNA Let7. This proposal tests an innovative hypothesis that PAE increases risk for adverse outcomes due to adult-onset disease, in an experimentally rigorous way. It is significant because it addresses a critical knowledge gap about brain vulnerability in aging adults with FASD. The investigators have a history of collaboration, and bring complementary expertise to studies that will inform clinical care of adults with FASD.

Prenatal alcohol exposure (PAE) is an established cause of brain-based disability, though co- exposure to other drugs, i.e., poly-substance use, can exacerbate adverse PAE-associated infant outcomes. The practice of Simultaneous Alcohol and Cannabinoid (SAC) use, i.e., co- ingestion, is an emerging trend among adolescents and adults of child-bearing age. SAC is motivated and maintained because combined use of alcohol and cannabinoids amplifies each drug?s psychological effect. Cannabinoids are known contributors to teratogenicity. However, we know virtually nothing about potential consequences of SAC for birth outcomes. The premise of the proposed studies is informed by the published literature, which indicates that PAE effects are, in part, mediated by activation of cannabinoid receptor signaling pathways and that PAE and prenatal cannabinoids engender similar fetal developmental outcomes. Moreover, recently published data shows developmental synergy between sub-teratogenic doses of cannabinoids and ethanol in non-mammalian vertebrate models. Based on these, as well as our own preliminary data we plan to address two questions: Firstly, ?is SAC more damaging to fetal development than either alcohol or cannabinoids alone??; Secondly, and importantly, ?will cannabinoid antagonists protect against effects of PAE & SAC??. Our studies will focus on the effects of SAC on neurogenesis and vasculogenesis, the complementary growth of vasculature that supports fetal brain growth. We plan to use in vivo and ex vivo mouse PAE models in combination with molecular assays and behavioral assays for hyperactivity and conditioned place preference, as well as state-of-the-art optical imaging (optical coherence tomography and light-sheet microscopy) and high-resolution ultrasound imaging, to assess the effects of SAC on, (Aim #1) brain and behavior, and (Aim #2) on vasculogenesis and cerebrovascular blood flow. Our overarching goal, to identify and minimize the contribution of factors, including poly-drug use, that contribute to increased risk for brain disabilities due to PAE, is consistent with the goals of the Collaborative Research on Addiction at NIH (CRAN) initiative. The rapid spread of recreational cannabis use means that SAC is an important emerging mode of drug consumption, and a potential contributor to the severity of PAE effects. As an outcome of these studies, we will acquire evidence to guide human studies on SAC birth outcomes, and to assess the efficacy of novel pharmacological intervention strategies targeted to cannabinoid receptors as a means to prevent or reverse effects of PAE.