Marieta B. Heaton - US grants

The proposed research is designed as analytical and manipulative investigations of mechanisms critical to cell migration and nerve fiber outgrowth in patterning of the developing nervous system. The studies will focus on growth factors influencing these events during early stages of neurogenesis, and will examine the potential of these factors to enhance and guide later regenerative growth. The developing trigeminal (V) motor nucleus in the chick embryo will be used as a model system. For these studies, tissue culture techniques will be used to evaluate the role of the trigeminal ganlion and normal target tissue (jaw muscle) in providing trophic or directing effects on fiber outgrowth and somal translocation. Our previous in vivo studies have indicated the ganglion is vital to normal development of V motor nucleus, and our in vivo and in vitro studies suggest that muscle tissue plays a trophic or directing role during later developmental stages. Basal plate explants of the trigeminal region, taken before the onset of cell migration and nerve fiber outgrowth, will be cultured alone, with disaggregated ganglion tissue, with control neural tissue, with ganlia conditioned media, with whole ganglia plated in varying positions, and with appropriate and inappropriate muscle tissue. Explants from embryos at later developmental stages will also be grown alone and with ontogenetically sequenced ganglionic tissue or target musculature in order to examine the potential of these tissues to provide their trophic influences during later stages, and the capacity of the V motoneurons to respond to these influences during their subsequent development. These studies will be significant in analyzing previously unknown growth factors, and the capacity of these factors to direct both initial neurogenesis and later regenerative growth. Such analyses will contribute to an improved understanding of normal nervous system development and may provide insights which will have significance in remediation of nervous system damage.

The proposed research will investigate the hypothesis that an important mechanism leading to certain of the CNS anomalies seen in the fetal alcohol syndrome (FAS) is a disruption of normal trophic interactions during neurogenesis. We propose that chronic prenatal ethanol treatment (CPET) results in a decrease in the synthesis, availability, delivery, or biological activity of normally occurring neurotrophic substances, such as nerve growth factor (NGF), or may alter the capacity of target neurons to respond appropriately to these factors. These developing relationships will be studied in the fetal and neonatal rat septo-hippocampal system. This system was chosen because its importance to normal cognitive functioning makes it likely to be involved in the severe intellectual impairment seen in FAS. Prior study has shown that the septal nuclei and their target hippocampus are selectively vulnerable to ethanol insult following adult chronic ethanol treatment (CET) in both humans and animal models. The hippocampus is similarly vulnerable to ethanol during development, and there is some limited evidence indicating that the basal forebrain nuclei are also damaged following CPET. The hippocampus synthesizes NGF, basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and certain uncharacterized substances which provide normal trophic support for basal forebrain neurons. Initial experiments will examine developing basal forebrain neurons (of medial septal/vertical diagonal band of Broca nuclei [MS/VDB]) in control and CPET animals. Total population numbers will be compared, as will the number of cholinergic, GABAergic, and NGF-- receptor-positive neurons. In addition, autoradiographic studies will compare neuronal generation in MS/VDB in control and CPET groups. We will also examine the influence of CPET on the development of trophic activity in the hippocampal formation. In our prior studies in adult animals, we found that CET reduces trophic activity produced in the hippocampus. We will measure trophic activity of hippocampal extract from control and ethanol-exposed animals using both bioassays and ELISA quantifications (assessing NGF and bFGF content). We will also examine the influence of CPET on the capacity of septal and hippocampal neurons to respond normally to trophic factors (e.g., NGF, bFGF, hippocampal extract), and we will assess direct ethanol effects on cultured septal and hippocampal neurons, and septalhippocampal co-cultured explants, and the capacity of neurotrophic factors to ameliorate these effects. We will also investigate the ontogeny of cholinergic expression within the basal forebrain populations, in control and CPET animals. ChAT activity, high affinity choline uptake and ACh synthesis will be examined in vivo and in vitro. These studies have the potential to elucidate important cellular mechanisms underlying the devastating CNS damage seen in FAS, and could open the eventual possibility of therapeutic intervention in FAS mental retardation via neurotrophic factor replacement procedures.

DESCRIPTION: This continuation proposal outlines a comprehensive, interdisciplinary program in the neurosciences leading to research, education and training in alcoholic neurodegenerative disease. This training program will continue to be performed under the auspices of the Center for Alcohol Research and the University of Florida Brain Institute within the Health Science Center of the University of Florida. Our training preceptors includes 16 faculty members spanning six Departments in three Colleges within the Health Center. Funding is requested to provide stipends for three predoctoral and three postdoctoral trainees. Our program takes advantage of the history and strength in interdisciplinary neuroscience research at the University of Florida to provide the necessary context, experience and environment conducive to the training of highly-qualified medical research professionals. Research areas in which trainees can participate includes studies of: The deleterious actions of ethanol in developing animals (Heaton, Walker) and humans (Behnke, Eyler); the role of neurotrophic factors in ethanol neurotoxicity (Walker, Heaton, King, MacLennan); the role of excitotoxicity and excitatory amino acids in alcohol degeneration (Anderson, Freund, Vickroy); the role of ethanol in disruption of signal transduction and synaptic plasticity (King, Meyer, Pen's, Papke); the role of dopaminergic and GABAergic receptor interactions in mediating the actions of ethanol and cocaine co-abuse (MacLennan, Pen's) as well as mechanisms for interaction of ethanol with other commonly abused drugs (Meyer, Papke, Vickroy); the role of ion currents and channels in ethanol neurotoxicity (Posner, Walker, King); and ethanol effects of gliogenesis and cytokine expression (Streit). Our training plan also includes an educational component encompassing limited coursework in medical neurosciences, molecular neurobiology, grant writing and scientific ethics, and the neurobiology and neuropharmacology of alcohol and alcoholism. This training program will be integrated with our strong, existing interdisciplinary training centers in the neurobiological sciences, the neurobiology of aging and the University of Florida Brain Institute to provide comprehensive and integrated neuroscience training in neurodegenerative disease.

The proposed research will investigate the relationship between developmental ethanol neurotoxicity and the expression of apoptosis effector and repressor molecules of the Bcl-2 family. Members of this family can inhibit apoptosis (e.g., Bcl-2, Bcl- xl) or promote it (e.g., Bax, Bad, Bcl-xs). These relationships will be explored in the developing cerebellum, which exhibits a differential temporal susceptibility to ethanol during the early postnatal period, with a brief period of sensitivity on postnatal days 45 (P45), which results in loss of Purkinje and granule cells, followed by a period during which this region is relatively refractory to ethanol effects (P7-8). We hypothesize that alterations in the expression of Bcl-2-related molecules contribute significantly to this population's relative temporal vulnerability to ethanol. Neonatal rats will be exposed to ethanol via artificial rearing, which we have found to produce increases in bax and bcl-xs mRNA on P4, but not on P7. In specific experiments we will use quantitative Western blot procedures to characterize the dynamics of expression of Bcl-2- related proteins following ethanol exposure at P4 and P7. We will also examine the influence of ethanol on certain post- translational modifications of Bcl-2-related proteins (e.g., dimerization, phosphorylation, and altered molecular integrity). Such processes affect the capacity of these molecules to implement or inhibit cell death. We will determine the regional distribution of Bcl-2-related proteins within the developing cerebellum following ethanol exposure at a vulnerable time (P4) and at a "protected" time (P7), using immunohistochemical procedures. Finally, we will establish whether a causal relationship exists between expression of Bcl-2-related proteins and ethanol-induced neurotoxicity. For this study we will use genetically engineered animals lacking the pro-apoptotic bax gene. Homozygous, heterozygous and wild-type animals will be exposed to ethanol on P45 and Purkinje and granule cell counts subsequently made. We hypothesize that loss of this apoptosis promoter will eliminate or significantly mitigate ethanol-induced cerebellar neuronal death. These studies will be important in producing the first characterization of the role of cell death effector and repressor molecules in developmental ethanol neurotoxicity, and will provide new information concerning a critical mechanism underlying the devastating central nervous system damage seen in the Fetal Alcohol Syndrome.

DESCRIPTION (provided by applicant): The objectives of the proposed research will be to define cellular mechanisms critical to the devastating consequences produced by exposure to ethanol during the development of the nervous system. Such exposure can lead to the fetal alcohol syndrome (FAS) or alcohol-related birth defects (ARBD). Improved understanding of these mechanisms will make it possible to eventually devise therapeutic strategies for preventing or mitigating ethanol neurotoxicity. Of particular interest in these studies is the effect of ethanol on proteins of the Bcl-2 survival-regulatory gene family. Members of this family can inhibit apoptosis (e.g., Bcl-2, Bcl-xl) or promote it (e.g., Bax, Bad, Bid). Proposed experiments will focus on the Bax protein, an apoptosis agonist strongly linked to ethanol-induced cell death. These relationships are being explored in developing cerebellum, which is highly susceptible to ethanol during the early postnatal period. This region is maximally vulnerable to ethanol on postnatal days 4-5 (P4-5), but is resistant to these effects by P7-9. For these studies, we will use a two-pronged in vivo <>in vitro approach. P4 and P7 neonatal rats will be exposed to ethanol via vapor inhalation, and cultured cerebellar granular cells will be used for parallel manipulative analyses. Techniques to be used include Western blot protein analyses for characterizing Bax activation and subcellular localization, and the ELISA procedure to assess protein-protein interactions. Cell survival assays will be made in the cultured cells, via the MTT assay. In addition, protein purification and circular dichroism (CD) methodologies will be used to examine protein structure. Specific experiments will define (1) ethanol influences on activation of the JNK kinase;JNK phosphorylation of the 14-3-3 Bax anchoring protein;subsequent Bax activation and insertion into the mitochondrial membrane;(2) ethanol effects on cleavage of the Bid protein, and subsequent tBid:Bax dimerization;and (3) the pathway of Bax disruption of the mitochondrial membrane, i.e., via the mitochondrial permeability transition pore or by Bax formation of membrane channels. In each of these series of studies, the granule cell model system will enable us to perform manipulative assessments, and to measure cell death in order to confirm the importance of the events of interest. In addition, purified P4 and P7 Bax will be subjected to CD analyses of protein conformation at the two ages, and ethanol effects on this conformation.

[unreadable] DESCRIPTION (provided by applicant): Animal fetal alcohol syndrome (FAS) models have demonstrated a temporal window during the brain growth spurt in which cerebellum is particularly vulnerable to the neurotoxic effects of ethanol (EtOH). In rodents, the brain growth spurt period occurs during postnatal day 4 (P4) through P10. In humans, this developmental stage occurs during the third trimester of pregnancy when neurobehavioral abnormalities associated with FAS are introduced. Rat cerebellum is particularly sensitive to EtOH during P4-6. At a slightly later neonatal period (P7 and later), the effects of comparable exposure are minimal. During the brain growth spurt, many neurons undergo apoptosis. This EtOH-mediated neuronal death is primarily Bax-dependent. There are two known general mechanisms by which Bax induces mitochondria to release apoptotic death effectors: 1) Bax can directly interact with the mitochondrial permeability transition pore (PTP) to prolong its opening [PTP-dependent]; or, 2) Bax molecules can homo-oligomerize to create a channel in the mitochondrial membrane [PTP-independent]. Under most apoptotic conditions, apoptotic activation is PTP-independent. Under physiological conditions (e.g., no interaction with Bax), regulatory proteins connect oxidative phosphorylation to glucose metabolism and maintain membrane integrity. The mechanism by which EtOH mediates mitochondrial-activated apoptosis at this pivotal point is unknown. However, based on previous studies and preliminary data, we hypothesize that during a period of maximum EtOH susceptibility (P4), EtOH-mediated apoptosis in the rat cerebellum is activated by the PTP-dependent Bax mechanism. At a slightly later more EtOH resistant period (P7), we hypothesize that EtOH effects on PTP regulatory proteins are minimal. This proposed investigation uniquely addresses the role of PTP regulatory proteins specific to alcohol neurotoxicity in developing neurons. Additionally, it tests a novel, potential regulatory mechanism of Bad-a Bax-supporting protein-which we speculate promotes apoptosis in cerebellum of P4 EtOH-exposed animals by binding and sequestering mitochondrial hexokinase, a glucose metabolizing enzyme. For the proposed research, Long-Evans rats are exposed to a single dose of EtOH on P4 and P7. Novel elements used for this in vivo investigation include: 1) isoelectric focusing to identify the mechanism of Bax-dependent apoptotic activation induced by EtOH; and, 2) an ELISA-based technique recently developed in our laboratory (Siler-Marsiglio et al., 2005a, 2006) that detects and quantifies potential competing native protein-protein interactions. Analyses of differential regulatory protein-protein interactions in cerebellum at EtOH-sensitive compared to EtOH-resistant ages are important for revealing mechanisms critical to developmental EtOH neurotoxicity, and will be particularly important in identifying possible sites for eventual therapeutic intervention. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Exposure to ethanol during the development of the central nervous system (CNS) can produce a wide array of neuroanatomical, behavioral and cognitive abnormalities, termed Fetal Alcohol Spectrum Disorder (FASD), or the more severe Fetal Alcohol Syndrome (FAS). It is estimated that nearly 1 in 100 live births worldwide exhibit at least some adverse effects of prenatal ethanol exposure. A goal of FAS/FASD research has been to elucidate the mechanisms contributing to developmental ethanol neurotoxicity, and to devise therapeutic strategies for mitigating the severity of the resultant deficits. One recent lin of research has focused on the nutrient, choline, as a therapeutic tool for ameliorating ethanol-mediated damage to the developing brain. This substance, which serves several vital biological functions, produces dramatic, long-lasting improvement in a number of behavioral and cognitive measures, following early ethanol exposure. The mechanisms underlying this amelioration have not been fully characterized, however. The proposed studies will investigate a number of possible mechanisms whereby choline may modulate ethanol neurotoxicity. The three categories of processes chosen for these analyses, i.e., apoptosis, neurotrophic factor (NTF) expression, and neurogenesis, have been shown to be critical aspects of ethanol-related neuropathology in the developing CNS, and each has been shown to be amenable to choline modulation, but their role has not been investigated in the ethanol + choline paradigm. These analyses will be made in developing prefrontal cortex and hippocampus, following prenatal or neonatal treatment, and will be related to analyses of neuron number and performance on behavioral tasks known to be sensitive to damage within these regions. These CNS regions were chosen because choline supplementation has been shown to attenuate ethanol effects on behaviors dependent on their functional integrity. We hypothesize that choline-mediated mitigation of ethanol neurotoxicity will be accompanied by: (1) alteration in the expression of apoptosis-related proteins (e.g., Bax, Bcl-2) in a manner favoring neuronal survival; (2) up-regulation of neurotrophic factors BDNF and/or NGF; and (3) attenuation of ethanol-related disruption of fetal and/or adult neurogenesis. In addition, a manipulative study using cultured cortical and hippocampal neurons will begin a determination of the role of BDNF in the protective or reparative effects of choline with respect to ethanol toxicity. These multi-faceted studies will generate important new insights concerning critical mechanisms contributing to choline's neuroprotective actions, and may suggest molecular mechanisms which should be considered in formulating future therapeutic strategies.

The psychotropic substance, cannabis (marijuana), is becoming increasingly accepted, for both medicinal and recreational purposes. Cannabis is used predominantly by young adults of childbearing ages and since warnings against its use during pregnancy have not been forthcoming, it is frequently used/abused during these critical periods. It has in fact been estimated that more than 10% of pregnancies in the United States and Europe are affected by maternal cannabis use. Although both human clinical observations and animal studies have demonstrated that prenatal and/or neonatal exposure to cannabis produces a range of neurocognitive and neurobehavioral deficits, relatively little is known of the molecular mechanisms involved, and the extent of the central nervous system (CNS) damage produced. The proposed studies will combine behavioral and neuroanatomical analyses of the neurobiological consequences of prenatal and early postnatal cannabis (CB) exposure, to test the global hypothesis that a critical mechanism underlying cannabis neurotoxicity to the developing CNS is the cannabis receptor-mediated triggering of the intrinsic apoptosis pathway, leading to cell death within CNS regions corresponding to many of the previously demonstrated cannabis-related behavioral/cognitive/motor deficits, i.e., prefrontal cortex, hippocampus, and cerebellum. For these studies, ?9-tetrahydrocannabinol (?9THC), the primary psychoactive component of marijuana, will be administered during gestation or in the early neonatal period, to wild-type mice, and to genetically engineered mice, lacking the pro-apoptotic bax gene. We will then conduct behavioral tests chosen to reveal functional deficits specific to these CNS regions, and will determine whether loss of Bax attenuates these deficits. We will then perform stereological counts of neurons in prefrontal cortex, hippocampus, and cerebellum, regions critical to the behavioral tasks chosen, to determine whether ?9THC-induced neuronal loss contributes to the behavioral deficits, and whether loss of Bax mitigates this loss. Individual identities will be retained so that behavioral and neuroanatomical data may be correlated. We hypothesize that loss of this primary apoptosis effector will significantly improve performance in behavioral/cognitive/motor tasks, accompanied by blocking or blunting ?9THC-mediated neuronal death. This project will be the first to investigate apoptotic neuronal loss as a consequence of developmental THC exposure, and as an antecedent to THC-mediated behavioral/cognitive deficits; and will also be the first to use gene-deletion technologies to define critical mechanisms underlying the harmful effects of ?9THC on the developing brain.