John A. Watt, Ph.D. - US grants

DESCRIPTION (provided by applicant): Our working hypothesis is that interleukin-1-beta plays a fundamental role in mediating intercellular communication in the central nervous system. Interestingly, the cellular source of IL-1-beta often determines whether its activity is beneficial or deleterious to brain function. Glial-derived IL-1-beta is predominantly associated with pathophysiological conditions, including inflammatory and neurodegenerative disorders. In contrast, neuronal IL-1-beta appears to play a critical role in modulating specific brain functions by acting as either an autocrine or paracrine signaling molecule under normal physiological conditions. However, little is known regarding the mechanisms of its actions Recent findings from this and other laboratories have demonstrated that IL-1-beta is present in the oxytocin (OT) and vasopressin (VP) neurons of the rat magnocellular neurosecretory system (MNS) where it may act to modulate neurosecretion of OT and VP by influencing neuronal-glial interactions. Our long-term objectives are to utilize the MNS as a model system for elucidating the mechanisms by which neuronal IL-1-beta modulates neuronal-glial interactions and neurosecretory activity in the CNS. The specific objectives of the proposed studies are to: 1) investigate the physiological conditions under which neuronal IL-1-beta and IL-1-beta receptor type 1 gene expression may be altered; 2) confirm and extend our preliminary observations which provided strong evidence for activity-dependent release of neuronal IL-1-beta from neurosecretory terminals. Pursuit of our objectives will be accomplished using quantitative autoradiographic in situ hybridization analysis and quantitative enzyme-linked immunoassay (ELISA) following potassium-evoked release of IL-1-beta from individual neural lobe (NL) explants maintained in culture. The R03 mechanism will allow the PI to establish the in situ hybridization histochemistry, autoradiographic and ELISA techniques critical for pursuit of these and future objectives. Fulfillment of these specific aims will increase our knowledge of the mechanisms by which cytokines affect neuronal-glial interactions and neurosecretory activity in the CNS, insights that could in turn suggest innovative approaches for cytokine therapies in neuroimmune, neuroendocrine and neurodegenerative disorders throughout the nervous system.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): It has been recently recognized that chronic intermittent hypoxia (IH), as occurs in human obstructive sleep apnea (OSA), is associated with substantial cortico-hippocampal damage and leads to impairments in neurobehavioral functions. The objective of the current research application is to delineate the molecular mechanisms of reactive oxygen species (ROS) and antioxidant enzymes in the modulation and prevention of chronic IH-mediated neuronal cell vulnerability. The working hypothesis of the current application is that the cyclical oscillations of oxygen during IH mimics the ischemia (hypoxia)/re-oxygenation process and may increase cellular ROS production. The cumulative damage from oxidative propagation may result in neurological dysfunction. On the other hand, targeted increases in antioxidant enzymatic activity may reduce ROS-mediated propagation, decrease IH-mediated cortical neuronal cell death and prevent the neurobehavioral impairments. This proposal will therefore focus on the following specific aims: (1) To identify IH-mediated specific ROS production at specific cellular compartments of cortical neuronal cells and analyze the specific redox alterations in mitochondria, cytosol and membrane as they relate to neuronal cell vulnerability. (2) To analyze the molecular processes of ROS-mediated neuronal cell death induced by chronic IH, and delineate signal transduction pathways underlying neuronal cell death using cell culture and mouse models. (3) To define molecular relationships between chronic IH-mediated ROS production, protein aggregation, and neuronal cell vulnerability. Specifically, we will use primary cell culture and rodent models to elucidate the roles of protein oxidation and protein aggregation in the modulation of chronic IH-mediated neuronal cell death. (4). To study the protective roles of mitochondrial MnSOD and phospholipid glutathione peroxidase (GPX4) in primary neuronal cells in vitro and in vivo in response to chronic IH exposures. Specifically, we will use gene transfer, anti-sense, and transgenic mouse approaches to analyze the effects of increased/decreased mitochondrial anti-oxidant activity in preventing/facilitating chronic IH-mediated cortical neuronal cell death. We anticipate that increased understanding of the mechanisms underlying neuronal vulnerability to cyclical hypoxia will lead to delineation of effective interventional strategies aiming to reduce the substantial neurocognitive and behavioral morbidities associated with obstructive sleep apnea.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Neurotrophic factors in general and ciliary neurotrophic factor (CNTF) in particular continue to be considered attractive therapeutic agents for the treatment of a variety of acute and chronic neurodegenerative conditions including traumatic brain injury, Parkinson's and Huntington's disease and amytrophic lateral sclerosis. However, despite the potential clinical importance of CNTF, little is known regarding its expression in adult brain or the mechanisms of its actions in vivo. The long term goal of my laboratory is to gain a greater understanding of the mechanisms by which neurotrophins and related cytokines promote neuronal survival and process outgrowth in the damaged CNS. Our central hypothesis is that CNTF acts as both a neuronal survival factor and sprouting factor for magnocellular neurosecretory neurons in vivo through activation of specific signal transduction pathways. In support of this hypothesis, we have demonstrated that axotomy results in a significant increase in CNTF expression in both the axotomized supraoptic nucleus and in the contralateral sprouting supraoptic nucleus. Furthermore, we have preliminary data which shows activation of Signal Transducer and Activator of Transcription (STAT) proteins in response to axotomy and following direct infusion of rat recombinant CNTF (rrCNTF) in the supraoptic nucleus in vivo. In light of these observations, the objectives of the proposed studies are to: 1) Determine the specific signal transduction pathways involved in the magnocellular response to injury and to exogenous rrCNTF and their role in the promotion of neuronal survival and process outgrowth;2) Define the cellular responses induced by chronically administered rrCNTF in vivo;and 3) Determine if stimulation of magnocellular neurons with exogenous rrCNTF will overcome the maturational decline in neuronal sprouting efficacy and determine if reduced CNTF or STAT activity contributes to this decline. Pursuit of these objectives will be accomplished in part using chronic infusion of growth factors, immunocytochemistry, Western blot analysis, quantitative RT-PCR, in situ hybridization, electrophoretic mobility shift assays and organotypic cultures. Eludicating the mechanisms by which CNTF mediates neuronal survival and axonal sprouting either through direct interactions on neurons or indirectly through glial activation will provide a significant advancement in our understanding of how neurotrophin-mediated neurorestorative activities are controlled throughout the CNS.

____________________________________________________________________________________ Project Summary Summary: The objective of the Histology Core Facility at the University of North Dakota School of Medicine and Health Sciences, is to provide high quality instrumentation, sample preparation and training in a wide spectrum of fundamental and advanced histological techniques to COBRE research investigators. The Histology Core Facility will be located in the first level of the new UND SMHS Building adjacent to the established Imaging Core which consists of the multiphoton, confocal, intravital imaging, fluorescent, TIRF and electron microscopy suites. The Histology facility will be critical to the completion of the proposed scientific projects from the five junior investigators. Furthermore, the Histology core will allow junior investigators to access the equipment and technical expertise to prepare samples for a wide variety of high resolution imaging techniques. The Core Director and technical staff will provide all project investigators and their students/staff assistance in experimental design, sample preparations for specific types of morphological analyses, and selection of the appropriate levels of imaging resolution. Additionally, the core will provide investigators and their trainees with comprehensive training in a wide variety of sample preparation techniques and analysis across a spectrum of tissue-specific applications. To maximize the impact of the NIH investment in biomedical research within North Dakota, the Histology Core Facility will provide access and support to other investigators, within the UND SMHS UND researchers who are not part of the medical school, and researchers from other North Dakota universities such as North Dakota State University, tribal colleges through INBRE, and other research institutions such as USDA-Grand Forks Human Nutrition Research Center; however, a fee-for-service approach will be applied to investigators outside this COBRE, and these fees will help to sustain this center.

Epigenetics of regeneration in the aging brain PROJECT SUMMARY This project is designed to investigate the maturational decline in regenerative capability in the rat central nervous system. Our working hypothesis is that the intrinsic capacity for axon or dendritic regeneration results from an age-related alteration of epigenetic factors that regulate the organization of chromatin and accessibility of genes associated with neuronal survival and process outgrowth. This study will directly link altered transcriptome and acetylation and methylation enzymatic activity with axotomy and collateral axonal sprouting in vivo. Furthermore, we will provide the first evidence for the maturation-induced changes in the epigenetic landscape that lead to loss of neuronal plasticity in vivo. Our long term goal is to reverse age-induced alterations in the epigenetic landscape to promote neuronal survival and process outgrowth in the mature mammalian CNS. Reversal of maturation associated inhibition of regeneration will provide an important tool for promoting, regulating and directing a functionally relevant regeneration event in humans following traumatic brain injury, ischemia or neurodegenerative disease. The principle goals of this project are as follows: Aim 1: We will use an unbiased approach to compare the transcriptome and epigenomic profile in young regenerating vs aged non-regenerating hypothalamic neurons Aim 2: We will test how CNTF-induced JAK/STAT3 signaling triggers epigenetic and transcriptional events to mediate neuronal survival and axonal outgrowth. Aim 3: To determine how the PI3K-AKT pathway mediates CNTF-induced process outgrowth. In addition to applying a novel and highly relevant model system to the study of maturational changes in the SON neural and astrocyte epigenome, we propose to utilize new and innovative methods to address our specific objectives. We will take advantage of laser capture microdissection to directly assess the methylation and acetylation status of young versus mature and sprouting versus non sprouting neurons and astrocytes isolated from SON in situ. We will also interpret this data in conjunction with analysis of alterations in enzymatic activity of specific Dnmts, 5-mC hydroxylase TET activity, histone acetyltransferase, histone de-acetyltransferase and histone methyltransferase in isolated SON under similar experimental conditions.