Scott E. Parnell - US grants

Among the most common, yet devastating, effects of prenatal ethanol exposure are those that involve the developing brain. While both structural and functional abnormalities of the brain have been described in individuals with Fetal Alcohol Syndrome (FAS)/Fetal Alcohol Spectrum Disorders (FASD), gaps remain in our understanding of the full range of these defects and of expected structural/functional correlates. Following up on the previous work of others, as well as the applicant's recent research, the experiments proposed herein are designed to examine the long-term effects of early gestational exposure on both brain structure and function and to provide correlative data. Overall, the proposed work will test the hypothesis that ethanol exposure at early gestational stages (gestational day [GD] 8 in mice; equivalent to the fourth week post fertilization in humans) results in a correlative pattern of brain dysmorphology and neurofunctional deficits that persists into adulthood. The proposed work will employ a mouse FASD model, state of the art high-resolution Magnetic Resonance Imaging (MRI), Diffusion Tensor Imaging (DTI), and a battery of cognitive, sensory, motor and other behavioral tests. In addition to furthering the applicant's training in MRI/DTI techniques, analyses and interpretation, the experiments and educational opportunities outlined in this proposal will greatly enhance the candidate's knowledge and understanding of methods designed to characterize neurofunctional phenotypes. Promise for the successful completion of this work is provided by the exceptional research environment of the University of North Carolina - Chapel Hill and of Duke University, mentorship by and collaboration with experts in the FASD field (Dr. K Sulik), behavioral analyses (Dr. S Moy), and imaging technologies (Drs A Johnson and M Styner), as well as the applicant's previous FASD research experience. Having illustrated the utility of high resolution MRI for discovery of ethanol-induced brain dysmorphology in fetal mice (Parnell et al, 2009), the proposed work will extend these analyses into postnatal stages. This work will be conducted by addressing 3 sub-hypotheses and the associated specific aims as follows: SPECIFIC AIM #1 will test the hypothesis that acute ethanol exposure on GD 8 will produce long-term morphological effects on specific regions of the mouse brain. The experiments for this aim will utilize high-resolution MRI and will entail analyses of the brains of postnatal day (PD) 12, 30, and 90 mice. SPECIFIC AIM #2 will test the hypothesis that this same ethanol exposure paradigm will alter the interconnecting neural pathways of the brain. Fiber tracts of the brains of PD 12, 30, and 90 mice will be assessed utilizing DTI. SPECIFIC AIM #3 will test the hypothesis that acute GD 8 ethanol exposure will result in neurofunctional abnormalities in adolescent and adult mice that are consistent with the observed dysmorphology. The results of these studies will provide important data regarding the long-term consequences of early gestational ethanol exposure and will, undoubtedly, promise to inform FASD diagnosis and prevention efforts. Additionally, the research and training described in this proposal will provide a solid foundation for both future studies regarding ethanol's teratogenesis, and the candidate's goal of pursuing a career as an academician.

DESCRIPTION (provided by applicant): The objective of the proposed basic research is to make clinically-relevant discoveries regarding prenatal alcohol (ethanol) exposure-induced pathology involving the brain and face. This proposal builds naturally on our CIFASD-supported basic research to date and continues to address the need for a more complete understanding of the spectrum and exposure stage-dependency of abnormalities caused by maternal alcohol use. Utilizing a well-established FASD mouse model, along with innovative technologies and approaches, and addressing 3 Specific Aims, we propose to test the overall hypothesis that alcohol induces structural abnormalities of the brain and face in mice that are consistent with and informative for those in human FASD. The Aim 1 studies compliment and extend the clinical research proposed by Drs. Foroud and Hammond. They employ Magnetic Resonance Imaging (MRI) and dense surface modeling (DSM) for experiments that are designed to identify exposure stage-dependent correlative abnormalities of the brain and face. The Aim 2 studies compliment and extend the Sowell group's neuroimaging-based clinical studies while following up on preliminary findings of cerebro-cortical thickness alterations in our mouse model. For this, MRI-based assessments of regional brain volumes and cerebro-cortical thickness changes, along with DTI-based investigations of fiber tract and structural connectivity alterations in adult animals are proposed. The Aim 3 studies are directed toward further defining the histopathology and genesis of early prenatal alcohol exposure- induced regional brain dysmorphology. They will employ routine histological methods, as well as immunohistochemistry, and stereology. Specimens selected for detailed histological analyses will include those postnatal brains that had previously been imaged and analyzed for Aim 2. Additional Aim 3 studies will focus on prenatal stages and will address the novel concept that early alcohol insult yields changes in Cajal-Retzius cell populations; changes that underlie subsequent cerebro-cortical lamination defects. The proposed work is consistent with the overall purpose/goals of the CIFASD in that it will facilitate diagnosis of the full range of birth defects associated with prenatal alcohol exposure, and it will aid in elucidating biological mechanisms that contribute to alcohol teratogenesis. The results of the proposed studies promise to fill a significant FASD research void, inform human clinical research, and continue to highlight the first trimester as a critical period for alcohol-induced defects of the face and brain.

Alcohol (ethanol) exposure during pregnancy is a well-recognized cause of birth defects and central nervous system disturbances that lead to cognitive and behavioral problems across the lifespan. Although clusters of physical features, in particular those involving the craniofacies, and neurobehavioral symptoms define fetal alcohol spectrum disorders (FASDs), there is considerable variation in the consequences of prenatal alcohol exposure. This variation impedes the accurate diagnosis of FASDs and confounds our complete understanding of the damage that can be caused by alcohol exposure. While some of the individual differences in the consequences of alcohol exposure are due to variations in the timing of exposure, genetic variability is a strong modifier of the effects of ethanol exposure. Elucidating the genetic factors that confer risk and resilience has been a slow process, usually accomplished by comparing ethanol?s effects among various strains of animals, or by candidate gene approaches. Here, we propose a cross-species genetic analysis, utilizing state-of-the-art whole transcriptomic sequencing (RNA-Seq), high-throughput CRISPR/Cas9 gene editing techniques, and genetic screening to drive the discovery of novel candidate genes that modify susceptibility to early gestational ethanol exposure. In Aim 1, RNA-Seq will be performed after ethanol or vehicle exposure in two closely related mouse strains that differ in their susceptibility to the teratogenic effects of ethanol. This experiment will reveal a number of genes that are differentially expressed in these ?at risk? and ?resilient? strains. Candidate genes are then refined and tested for significant associations with craniofacial and neuroanatomical dysmorphology, as well as neurobehavioral changes, using our zebrafish high-throughput screens, mouse MRI analysis (with Hammond) and mouse behavioral phenotyping. The dual species approach affords a highly conserved FASD model that is more relevant than studying either species alone. In Aim 2, we will perform an unbiased forward genetic screen in zebrafish to identify mutations that suppress the teratogenicity of ethanol. The roles of these genes will be tested in mice to identify conserved mechanisms of ethanol teratogenesis. These conserved genetic mechanisms of ethanol teratogenesis can then be tested by CIFASD members Foroud, Hammond, Mattson in human populations with prenatal ethanol exposure who vary in their craniofacial and neurobehavioral manifestations. Likewise, human whole-exome sequencing experiments proposed by Dr. Foroud will generate numerous candidate genes that will be tested and confirmed in our animal models for the purpose of identifying conserved teratogenic mechanisms. These highly translational studies will significantly contribute to our understanding of the genetic factors underlying the susceptibility to prenatal ethanol exposure, which may be used to improve diagnosis, treatment, and prevention of FASD, as well as provide insight into the teratogenic mechanisms of FASD.

Alcohol has long been recognized as a teratogen. Cannabinoids (CBs), however, have been popularly believed to be safe during pregnancy. Behavioral observations in children prenatally exposed to marijuana, and animal studies of THC and the more recently introduced, highly potent synthetic cannabinoids (SCBs), contradict this belief and indicate that cannabinoid exposure is, indeed, teratogenic. Importantly, in rodents, early prenatal exposure to SCBs causes birth defects similar to those in fetal alcohol syndrome. During early gestation, both alcohol and cannabinoids independently induce brain abnormalities involving ventral midline-derived structures, including the hypothalamus, septal area and striatum. We hypothesize that these two drugs, when administered together during the early periods of gastrulation and neurulation, will have a significant persistent teratogenic effect as evidenced by altered response to novelty/habituation processes, corticosterone responses and alcohol self-administration in adolescent offspring. Using a rat model of prenatal teratogen exposure during the key developmental events of gastrulation and neurulation, the proposed research will explore this premise, addressing the following Specific Aims: Aim 1 will examine the effect of prenatal alcohol+SCB exposure on locomotor activity and habituation to a novel environment, and changes in corticosterone in response to a mild stressor. Aim 2 will test the hypothesis that early gestational alcohol+SCB exposure will potentiate alcohol self- administration, motivation to self-administer alcohol and alcohol-seeking behavior. These data will be important information on the functional effects of prenatal co-exposure to two widely used drugs and how this may increase adolescent drinking behaviors.

Many of the structural and functional abnormalities associated with Fetal Alcohol Spectrum Disorders (FASD) have been uncovered, yet major gaps remain in our understanding of the associated pathogenesis and mechanisms. For example, it is well known that ethanol exposure during gastrulation results in the classic hypoteloric FAS face and midline forebrain dysgenesis; yet, exposure just slightly later, during neurulation, induces expanded midline brain structures and hypertelorism. Interestingly, these abnormalities resemble (phenocopy) those of many genetic ciliopathies, such as Joubert syndrome. The central pathogenic mechanism of ciliopathies is a perturbation of the structure and/or function of primary cilia, hair-like organelles found on most cells that integrate extra- and intra-cellular signals. The proposed research tests the overall novel hypothesis that neurulation-stage ethanol exposure induces a ?transient ciliopathy? (i.e., a temporary disruption of primary cilia function) that is the basic cellular mechanism for the expansion of midline brain structures and hyperteloric dysmorphologies. The proposed experiments are designed to meet the following integrated specific aims. Aim 1 will define the direct effects of early prenatal ethanol exposure on primary cilia structure and function. For this, confocal microscopy and immunohistochemistry will be used to examine the effects of ethanol on primary cilia number while gene expression assays will be used to assess cilia function. It is hypothesized that ethanol exposure causes abnormal ciliary number and/or function, reducing activation of the Shh signaling pathway. Aim 2 will characterize the secondary cellular pathogenic events in the neural tube resulting from an ethanol-induced transient ciliopathy. The experiments in this aim will test the hypothesis that the ethanol-induced transient ciliopathy and subsequent down-regulation of the Shh pathway will decrease downstream cell proliferation genes in the ventral neural tube and expand morphogen gradients that pattern the dorsal neural tube. Following ethanol exposure, genes with known roles in cell proliferation will be assessed using qRT-PCR and the gradients of ventral and dorsal morphogens will be assessed using in situ hybridization. These data will help to determine the precise mechanisms by which ethanol alters development. Aim 3 is to determine the primary cellular mechanistic events underlying an ethanol-induced transient ciliopathy. This final Aim will use RNA-seq to determine in an unbiased manner how ethanol disrupts normal ciliogenesis by examining the total transcriptomic profile at several time points immediately following ethanol exposure. We hypothesize that ethanol will alter key ciliogenesis genes; however, using this non-biased approach will aid in identifying other potential changes. Finally, we test the alternative/complementary hypothesis that ethanol alters tubulin post-translational modification, thereby disrupting normal cilia stability and function. Together, these novel experiments will provide fundamental insights into the pathogenic mechanisms underlying the effects of ethanol exposure during development, and propel alcohol research into new primary ciliary-related studies.

Title: In vivo evaluation of safety and pharmacology of a sustained release formulation of dolutegravir in pre- conception and early stages of pregnancy Abstract: Injectable long-acting (LA) formulations of antiretrovirals (ARVs) represent an important alternative to improve adherence to HIV/AIDS treatment and prevention. Dolutegravir (DTG) is a highly effective ARV drug with low toxicity, improved tolerability, better drug?drug interaction profile, low side-effects, and high genetic barrier to resistance. Due to its excellent properties, dolutegravir became widely used as part of ARV therapies for HIV. Recently, we used dolutegravir for development of an ultra-LA, removable system that delivers drug for up to 9 months and can be safely removed to stop drug delivery. Although this approach represents a potentially effective strategy for the ultra-LA drug delivery for HIV treatment and prevention, long-time exposure to ARV, especially during pregnancy, raises questions of safety. These concerns are exacerbated by the recent discovery that DTG-based treatment for women in early stages of pregnancy may be associated with several cases of severe neural tube defects (NTDs) in children whose mothers were being treated with DTG. It is thus vital to systematically assess the teratogenic potential of long-term exposures to dolutegravir using relevant in vivo models. Mice are an ideal animal model because they allow for rapid evaluation of drug effects, easy access to embryos, and analysis of drug levels that is not possible in humans. We will use inbred mouse strains with differential sensitivity to NTDs (BALB/cJ, C57BL/6J, and FVB/NJ) as tools for an accurate evaluation of the relative risks of long-term DTG exposure under conditions that are most relevant to the use of DTG in humans: (i) DTG exposure after a single injection of the long-acting DTG formulation designed to improve adherence to drug regimen in humans; (ii) long-term exposure to DTG after daily oral administration as all current ARV regimens are oral; (iii) exposure to DTG in preconception and during pregnancy. We will use acute exposure to DTG at critical stages of embryonic development equivalent to human pregnancy at weeks 3, 4, 5, or 6 to gain insight into the mechanism of potential DTG action during pregnancy. Specifically, this analysis will be able to identify and classify a wide spectrum of potential teratogenic effects observed in human populations in developmental stages of gastrulation and the beginning of neurulation, neural tube closure, the beginning of limb development, and stages following neural tube closure, including palate formation. In addition, we will evaluate the role of the folic acid, one of the most critical factors involved in NTDs. These data will be critical in evaluating and interpreting the human birth defects data that will likely emerge over the next several years. We will also provide a comprehensive analysis of DTG concentration in maternal plasma, placenta, amniotic fluid and embryonic tissues during chronic daily oral DTG administration, after a single dose of a long- acting formulation of DTG and after an acute oral dose of DTG at critical stages of embryonic development. This will allow us to correlate concentration of DTG in embryonic tissues with observed birth defects. Evaluation of teratogenic effect of long-term oral administration of DTG and a long-acting DTG formulation using mouse strains with differential sensitivities to NTD represents a novel and valuable approach to demonstrate the safety profile of DTG in pre-conception and during early stages of pregnancy.