Gerald Dienel - US grants

IBN 97-28171 DIENEL The functions of two major classes of brain cells, neurons and glia, are highly specialized. Neurons carry out the signaling processes, transferring information from one cell to another to control and coordinate the activities of the body. Glial cells surround neurons and have many important functions, including control of the extracellular environment which can modify neuronal activity thereby influencing behavior. Glial functions and glial- neuronal interactions in brain are poorly understood because it is very difficult to assay the activities of specific classes of cells in the living brain. Brain function is often evaluated by metabolic brain imaging procedures using radiolabeled glucose analogs because there is a close correlation between changes in functional activity and local rate of glucose utilization to provide energy for brain work. Functional metabolism is the breakdown of compounds to extract energy for the working cell, and metabolic imaging is based on local trapping of labeled products of the metabolic tracer in the cell where it is metabolized; the quantity of trapped label reflects the magnitude of functional activity. Because glucose is a fuel for all cells, it cannot distinguish between the functional metabolic changes in the different cell types in brain. In the present study, glial- specific "reporter molecules", i.e., labeled compounds known to be metabolized mainly in glial cells, will be used to measure stimulus-induced metabolic changes in glial cells and evaluate working relationships between glia and neurons. Experiments are designed to test the hypothesis that metabolic activity in glial cells increases in proportion to the activity of neurons when neurons are stimulated by graded changes in sensory input, and that compounds made in glial cells are transferred from glia to neurons in increased quantities. Neurons will be activated by changing the magnitude of a visual stimulus (flash rate or intensity of a strobe light) to cause progressive increases in the signaling activity of neurons in the eye as the visual information is processed in the brains of experimental rats; altered neuronal activity is predicted to cause parallel changes in the activity of glial cells in the visual pathway and rates of metabolism of glial reporter molecules. Some compounds made in glial cells are exported to neurons, and the quantity of labeled compounds transferred and incorporated into a neuron-specific marker (a neurotransmitter called GABA) will be measured, thereby permitting assessment of neuronal-glial metabolic interactions in working brain. The results of the proposed experiments will increase our understanding of how glia obtain their energy to carry out their work and how neurons and glia work together to process visual information. Knowledge of the cellular basis of metabolic brain images will also help to form the basis for future development of new brain imaging techniques to visualize and quantify glial cell activity in human brain.

DESCRIPTION (Based on Applicant Abstract): The focus of this application is on brain "aerobic glycosis", a term applied to the disproportionately greater increase in glucose than oxygen utilization in working brain. The general hypothesis to be tested is that glucose taken up in excess of oxygen is used to restore metabolic pools depleted at the onset of stimulation, rather than to generate lactate. Dr. Dienel proposes to develop a rat model of aerobic glycosis using generalized sensory stimulation, then assay changes in brain levels of major metabolites and flux of radiolabelled glucose into major pathways, some specific to neurons (GABA) or glia (glycogen and glutamine), using biochemical, autoradiographic, and microdialysis techniques. To determine the cellular basis of metabolism during aerobic glycosis, Dr. Dienel will use radiolabelled "glial reporter molecules" (butyrate, acetate) and autoradiography to examine glial contributions.

The major long-term objectives of this study are to elucidate glial function and neuronal-glial interactions in brain in vivo, and establish novel neuroimaging tracers for glial brain tumors. Glia, a major heterogeneous class of brain cells, are involved in critical aspects of brain structure and function and injury repair. In spite of numerous previous studies, the roles of glial cells in vivo in health and disease are not well understood. Autoradiographic studies are particularly useful for in vivo studies to examine glial function because they can take into account the complex architecture and cell-cell interactions in mature, fully-developed brain. Because acetate is preferentially transported into astrocytes compared to neurons, preliminary studies for this project used radiolabeled acetate to assess metabolic responses of glia to sensory stimulation of neurons and chemical disruption of ion homeostasis. Local acetate metabolism increased by 8-40 percent in response to brain activation, whereas a decrement (7 percent) was observed after acute denervation to eliminate neuronal input; estimates of the rate of acetate utilization suggest that acetate might be a significant fuel for brain. These results indicate that acetate is a useful glial reporter molecule (i.e., a compound metabolized mainly in glia at variable rates) that can be used to assess neuronal-glial interactions in brain in vivo, and test the overall hypothesis that the local rate of acetate utilization by glia varies in proportion to functional activity. The specific aims of this project are (1) determine the acetate utilization rate in rat brain in vivo by both direct biochemical assays of product formation and by autoradiography; (2) determine the rates of acetate transport into different brain cell types and relationships among acetate, glucose, and oxygen utilization in cultured brain cells; (3) establish glial responses to graded changes in neuronal activity induced by various sensory (e.g., visual, auditory, and whisker) stimuli to normal rats, and assess neuronal-glial interactions; and (4) determine responses of glia to pathophysiological conditions (e.g., puncture wound) that induce reactive astrocytes; (5) establish the use of labeled acetate to localize and monitor growth of glial tumors in rat brain and determine specificity of glial tumor labeling by acetate in human tumor pathological specimens. The proposed studies will lead to a better understanding of glial metabolism in brain, establish fundamental relationships between glial and neuronal activity in brain under normal and pathophysiological conditions, and the develop the use of labeled acetate for human positron emission tomographic (PET) studies of glial function and dysfunction, particularly neuroimaging of brain tumors.

DESCRIPTION (provided by applicant): The functional roles of gap junctional communication among astrocytes and other brain cells are believed to include trafficking of nutrients, energy metabolites, signaling molecules, and electrolytes. Although trafficking of biological molecules within the cellular syncytia in brain is not well understood due to the lack of sensitive assays for unlabeled or non-fluorescent molecules, hereditary mutations in connexin proteins that impair gap junctional signaling can cause deafness, underscoring the importance of trans-cellular communication among brain cells. Our initial findings demonstrate that gap junctional dye transfer is markedly reduced in cultured astrocytes after prolonged exposure to high levels of glucose and in brain slices from streptozotocin-treated diabetic rats. Disruption of syncytial trafficking and the slower onset of increased expression of endoplasmic reticulum chaperone proteins in experimental diabetes suggests that reduced communication among gap junction-coupled brain cells and endoplasmic reticulum stress may be unrecognized, 'subtle'complications of diabetes in the central nervous system. Gap junctional trafficking deficits in cultured astrocytes occurred after increased generation of reactive oxygen species (ROS) and could be prevented or restored by various pharmacological agents. These findings led to our overall hypothesis that diabetes-induced oxidative- nitrosative stress impairs gap junctional trafficking of biological molecules, gradually causes endoplasmic reticulum stress, and causes subtle impairment of brain function. The three specific aims are as follows, (1) establish roles of oxidative-nitrosative modification of cellular proteins in impairment of astrocytic gap junction communication and manifestation of endoplasmic reticulum stress, (2) establish impairment of gap junctional transfer of biological molecules during experimental diabetes, and (3) establish effects of experimental diabetes on auditory pathway functions. This proposal addresses some of the long-standing, basic issues related to connexin channel function - permeability of biological molecules, functions of syncytial communication in situ, and development of novel assays, tools, or probes for studies of connexin structure and function. It then assesses permeability of connexin channels to biological molecules in experimental diabetes. Our long-term goals are to understand the functions of the astrocytic syncytium in health and disease and improve knowledge of the cellular basis of metabolic brain images. Translational aspects of this study include potential treatment strategies for diabetes and improved interpretation of brain imaging and spectroscopic studies using technologies that detect signals arising from metabolic responses to changes in physiological activity. The anticipated results will lead to a better understanding of nutritional and signaling roles of gap junctions in astrocytes and other brain cells and how these functions are disrupted by hyperglycemia and experimental diabetes. PUBLIC HEALTH RELEVANCE: Complications of diabetes cause serious, progressive health problems and many diabetic patients acquire altered hearing characteristics and develop hearing loss. We found that experimental diabetes impairs the ability of brain cells to the shuttle nutrients and signaling compounds between different cells in the brain, and we test the hypothesis that disruption of cellular function by diabetes progressively interferes with communication between brain and inner ear cells, leading to hearing impairment. Therapeutic molecules will be tested for their ability to overcome these deficits with the goal of developing new treatment strategies to minimize diabetes-induced deficits in brain.

Astrocytes, a major brain cell type, have key roles in brain function including extracellular ion buffering, de novo synthesis of neurotransmitters, transmitter uptake from the synaptic cleft, communication between the vasculature and neurons to regulate blood flow, modulation of neuronal activities, and responsiveness to injury. Astrocytes are highly coupled by gap junction channels, but the functional roles of the syncytium are not well understood because dye transfer studies cannot predict transfer of biological molecules and radiolabeled tracers can be metabolically transformed in donor and recipient cells. Our two initial findings, (1) very restricted transjunctional transfer of glucose-6-phosphate compared to other phosphorylated compounds related to the glycolytic pathway, and (2) marked impairment of dye transfer via astrocytic gap junctions by chronic hyperglycemia in vitro and in rat brain slices led to our hypothesis that transfer of molecules with fluxgenerating regulatory roles through astrocytic gap junctions is restricted, and hyperglycemia impairs gap junction-mediated shuttling of energy-related metabolites and signaling molecules within the astrocytic syncytium. The hypothesis is tested in three specific aims, (1) identify cytoplasmic molecules with fluxgenerating regulatory roles, redox signaling, or energy-donating or sensing roles that have restricted transjunctional transfer, (2) establish the role of connexin proteins in the specificity of transjunctional transfer of these biological molecules, and (3) establish the impact of chronic hyperglycemia on gap junction channelmediated trafficking. Our long-term goals are to understand the functions of the astrocytic syncytium, contributions of astrocytes to the brain[unreadable]s energy budget in health and disease, and the cellular basis of metabolic brain imaging. Novel methods are developed to assess selective permeability of connexin channels to biological molecules in cultured astrocytes, N2A cells stably transfected with different connexins, and adult rat brain slices. This proposal addresses some of the long-standing, basic issues related to connexin channel function [unreadable] permeability of biological molecules, functions of syncytial communication in situ, and development of assays, tools, or probes for studies of connexin structure and function. The translational component of the study focuses on impairment of gap junction trafficking during chronic hyperglycemia and development of a treatment protocol to restore the high glucose-induced deficit in gap junctional trafficking. The anticipated results will lead to a better understanding of nutritional sensing and coordination of energy production in gap junction-coupled astrocytes in normal and experimentally-diabetic rat brain and improve interpretation of brain imaging studies using technologies that detect signals arising from metabolic responses to changes in physiological activity