Lisa A. Opanashuk, Ph.D. - US grants

The objective of this research is to understand the role of adrenal steroids in mediating normal cerebellar development and to establish the mechanisms by which developmental events are perturbed in response to high concentrations of glucocorticoids. Postnatal administration of glucocorticoids disrupts cerebellar development and causes functional deficits involving locomotor activity in motor coordination. Granule neuron progenitors are potential targets for glucocorticoids since they are responsible for defining a critical period of postnatal development in the cerebellum. However, it is unclear whether glucocorticoids intrinsically effect cerebellar development. Furthermore, the direct effects or precise mechanisms of action on granule neuroblast cell division or cell survival have not been systematically explored. The specific aims of this proposal are 1) test the hypothesis that glucocorticoids attenuate granule neuroblast proliferation and 2) characterize the influence of glucocorticoids on EGL cell turnover by simultaneously evaluating both cell proliferation and survival. These studies will be performed in a noel EGL murine cell culture using biochemical, morphometric and flow cytometric techniques. Results from these experiments should provide clinically relevant insight regarding the neurodevelopmental consequences attributable to postnatal stress or administration of glucocorticoids in the neonate.

DESCRIPTION: The objective of this research is to understand the role of adrenal steroids in mediating normal cerebellar development and to establish the mechanisms by which developmental events are perturbed in response to high concentrations of glucocorticoids. Postnatal administration of glucocorticoids disrupts cerebellar development and causes functional deficits involving locomotor activity in motor coordination. Granual neuron progenitors are potential targets for glucocorticoids since they are responsible for defining critical period of postnatal development in the cerebellum. However, it is unclear whether glucocorticoids intrinsically effect cerebellar development. Furthermore, the direct effects of precise mechanisms of action on granule neuroblast cell division or cell survival have not been systematically explored. The specific aims of this proposal are to: 1) test the hypothesis that glucocorticoids attenuate granule neuroblast proliferation; and 2) characterized the influence of glucocorticoids on EGL cell turnover by simultaneously evaluation of both cell proliferation and survival. These studies will be performed in novel EGL murine cell cultures using biochemical, morphometric and flow cytometric techniques. Results form these experiments should provide clinically relevant insight regarding the neurodevelopmental consequence attributable to postnatal stress or administration or glucocorticoids in the neonate.

DESCRIPTION (Taken from the Investigator's Abstract) Polychlorinated biphenyls (PCBS) are widespread and persistent environmental contaminants. Perinatal PCB exposure is related to developmental cognitive and motor abnormalities in humans. Therefore, exposure to these toxicants is recognized as a significant human health-related concern. Corroborative animal studies have identified neurobehavioral changes in perinatal PCB exposed rats which include defective locomotor activity, altered eyeblink conditioning, and impaired delayed spatial alternation and reversal learning. The nature of these alterations likely reflects PCB-induced disruption of cerebellar output. Based on these considerations it is hypothesized that gestational PCB exposure disrupts neuronal maturation during a critical period of cerebellar development. Exposure hinders neuronal differentiation, thereby leading to abnormal formation of cytoarchitecture and dysregulation of neurochemical function. These structural and functional perturbations ultimately interfere with cerebellar output. This hypothesis will be addressed by the following specific aims: Specific aim 1: Does gestational exposure to Aroclor 1254 influence cell acquisition or synaptogenesis during rat cerebellar development? Specific aim 2: Is gestational exposure to Aroclor 1254 related to alterations in the molecular differentiation state of neurons in the developing cerebellum? Specific aim 3: Does gestational exposure to Aroclor 1254 alter cerebellar amino acid neurotransmitter levels, turnover, and neurochemical output? The actions of PCBs on the developing cerebellum have been largely unexplored despite clear indications of regional vulnerability. The proposed project will provide a foundation for identifying cellular sites of PCB action in cerebellum and corresponding molecular mechanisms. Such studies will lead to a better understanding of pathophysiology which can be used as a rational basis for reducing exposure risks and developing efficacious pharmacotherapies. Since there is evidence that PCBs induce a state of hypothyroidism in developing brain, future research will explore the role of thyroid hormone in PCB neurotoxicity and the possibility of thyroid replacement therapy.

[unreadable] DESCRIPTION (provided by applicant): Dioxin (2,3,7,8 -tetrachlorodibenzo-p-dioxin; TCDD) is a ubiquitous and persistent environmental contaminant that produces developmental neurotoxicity associated with deficits in cognitive function, locomotor development, and sexual behavior. The molecular mechanisms underlying these neurotoxic actions have not been determined. However, TCDD interacts with the aryl hydrocarbon receptor (AhR) to exert toxicity in other tissues. The AhR is a transcription factor that regulates the expression of regulatory molecules that play critical roles in cellular development. Thus, it is conceivable that TCDD disrupts normal brain development through binding to the AhR. Although the precise anatomical regions and cell types targeted by TCDD during brain development have not been identified, preliminary results indicate that AhR is expressed by pluripotent neuroepithelial stem cells (NSC) and cerebellar granule cell progenitors (CGNP). Therefore, these precursor cells, among others, might be important sites of action for AhR-mediated TCDD neurotoxicity. Based on these observations, it is hypothesized that TCDD binds to AhR and interferes with precursor cell development. These signaling events disrupt the normal genetic programs required for differentiation and thereby produce neurotoxicity. This exploratory proposal is designed to identify critical developmental periods in which CNS precursor cells are vulnerable to TCDD exposure. Accordingly, the studies proposed in this application will determine whether TCDD, through interaction with the AhR, interferes with the development of NSC, CGNP, and lineage-restricted precursor cells by modifying gene expression and precursor cell function. Results of the proposed research have significant implications for understanding the risks of TCDD exposure during pregnancy, and offer new approaches to the analysis of TCDD toxicity in the developing central nervous system. [unreadable] [unreadable]

The University of Rochester is offering a REU Site program in Cellular and Molecular Biology to promising undergraduates. The goal is to encourage students to pursue graduate education and careers in the Biological Sciences. Participants will experience the excitement of hands-on, hypothesis-driven research under the mentorship of a faculty. The Site will recruit underrepresented minority students and students from smaller, non-research-intensive, undergraduate colleges. Students will be selected from a large national pool of applicants, and will participate in a 10-week independent research project under the supervision of a faculty mentor. Students will attend research seminars as well as seminars on ethics, graduate school and career issues, and will also be offered the opportunity to participate in a GRE prep course and in discussion groups. Participants will present their research in poster form at a mini-symposium at the end of the summer. Finally, continuation of research work during the academic year will be strongly supported and used as a mechanism to support linkages between UR and other colleges. More information is available by contacting Dr. David A Pearce at David_Pearce@urmc.rochester.edu, or by visiting the program website at http://www.urmc.rochester.edu/GEBS/summer.htm.

DESCRIPTION (provided by applicant): The ubiquitous and persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been linked to developmental neurotoxicity in humans and experimental animals. TCDD, the most potent dioxin, mediates toxicity via binding to the aryl hydrocarbon receptor (AhR), a ligand-activated member of the bHLH/PAS transcription factor superfamily. These transcription factors serve as environmental sensors and transducers of physiological signals, particularly during development. The endogenous functions of AhR remain unknown. Certain bHLH/PAS proteins have been implicated in the regulation of cell fate determination, proliferation, and differentiation during neurogenesis. Therefore, it is conceivable that AhR plays similar roles. TCDD, through its high affinity binding and activation of the AhR, causes numerous biochemical and pathological abnormalities, particularly following developmental exposure. Deficits in cognitive function, locomotor development, and sexual behavior are some of the most sensitive endpoints associated with perinatal exposure to dioxin-like chemicals. However, the regional, cellular, and gene targets of AhR-mediated TCDD neurotoxicity remain unclear. Our laboratory has determined that cerebellar granule neuron precursors (GNPs) express high levels of transcriptionally active AhR during a critical period of cerebellar neurogenesis. The proposed studies are an extension of our previous work toward understanding the neurotoxic impact of AhR activation by TCDD in GNPs during cerebellar development. One approach will explore the manner in which inappropriate AhR activation by TCDD disrupts cerebellar neurogenesis. A second strategy will investigate the developmental role of AhR by defining the effects of AhR deletion on GNP maturation in mutant mice. Behavioral correlates of cerebellar function will be assessed in both of these models. Our preliminary studies demonstrate striking effects of TCDD on GNP proliferation, early differentiation, survival, and also on gene expression patterns associated with those processes. We have additional evidence that cerebellar growth is abnormal in AhR-/- mice. Together, these observations raise the intriguing hypothesis that TCDD disrupts cerebellar maturation by impeding AhR actions during GNP development. The primary hypothesis of the proposed studies is that AhR intrinsically controls the balance between GNP proliferation, differentiation, and apoptosis during cerebellar development. It is postulated that AhR alters cell cycle regulation, transcription factor activity, and programmed cell death by modulating gene expression in immature GNPs. Consequently, TCDD exposure disrupts endogenous AhR-mediated spatiotemporal gene profiles associated with the proper formation of cerebellar cytoarchitecture, which, in turn, adversely impacts behavioral function. The findings from this research will be important to further our understanding of transcriptional and cell cycle regulation during GNP differentiation, which may lead to the identification of factors that contribute to the anatomical and functional deficits following exposure to dioxins.

Neurological symptoms with methamphetamine (METH) toxicity of the nigrostriatal system, both in rodent models and in drug abusers, are associated with neuroinflammation, a fundamental reaction to brain injury characterized by activated microglia and astrocytes, local expression of inflammatory mediators, and possible infiltration of peripheral cells such as monocytes. This prominent and local tissue response most likely represents an adaptive and restorative repair process. Yet reminiscent of many inflammatory conditions in peripheral diseases, neuroinflammation can also contribute to the pathophysiology of CNS disorders. Thus observations of neuroinflammation in the setting of METH neurotoxicity raise questions about the contribution of glial directed inflammation to neuronal damage as well as the possible mechanisms underlying this association. One such mechanism is oxidative stress, which is known to occur in both METH toxicity and neuroinflammation. Among HIV infected individuals, METH abuse is associated with significant enhancement of HIV encephalitis, greater neuropathology, and increased expression of inflammation-associated genes. Thus neuroinflammation represents a factor common to both METH exposure and CNS HIV infection that may underlie the enhanced disease and neuropathology seen when these two risk factors coexist. One of the major driving forces in CNS inflammation is the proinflammatory cytokine interleukin (IL)-1[unreadable], which is produced by activated microglia. Among its many actions, IL-1[unreadable] promotes phenotypic activation of astrocytes leading to expression of other inflammatory mediators including chemokines, which facilitate CNS infiltration by monocytes and other blood-borne cells. Moreover, there is clear evidence that IL-1 is an important contributor to neuronal damage in several CNS injury and disease models. We have recently developed a transgenic mouse model that provides temporal and spatial control of sustained IL-1[unreadable] expression. We propose using this mouse to test the hypothesis that coexistent neuroinflammation enhances METH-induced damage in the ventral midbrain dopaminergic system. A corollary hypothesis is that attenuation of neuroinflammation will protect against METH neurotoxicity. Finally, we hypothesize that one mechanism underlying the association between neuroinflammation and METH toxicity is production of microglial/ macrophage derived reactive oxygen species (ROS). To explore these hypotheses, we will: 1) quantify striatal dopaminergic nerve terminal toxicity elicited by METH exposure in the presence of sustained neuroinflammation;2) determine whether abrogation of IL-1 signaling reduces neurotoxicity following METH exposure, and 3) characterize oxidant injury in METH and METH + neuroinflammation elicited neurotoxicity. By better understanding the role of neuroinflammation in METH-associated neurotoxicity, we may be able to develop strategies to curb the neurological side effects of METH abuse, particularly when associated with conditions like HIV where neuroinflammation is prominent.