Gary Banker - US grants

DESCRIPTION (Investigator's Abstract): The long-term goal of this research is to understand from a cell biological perspective how neurons establish and maintain distinct axonal and dendritic domains that differ in structure and function. This feature of nerve cells-referred to as neuronal polarity- is essential to normal neural function. Disruption of neuronal polarity is thought to contribute to the pathophysiology of many neurologic diseases. The aims for the coming award period are to elucidate the cellular mechanisms underlying the targeting and transport of membrane proteins and to assess the role that these processes play in the development of neuronal polarity. In the previous award period it was shown that basolateral targeting signals that contain a tyrosine motif target exogenous proteins to dendrites, where exogenous proteins carrying motifs in the cytoplasmic domain of endogenous dendritic protein mediate their targeting and will identify the sequence within an endogenous axonal protein that govern its polarization to the axon. The earliest event in the establishment of neuronal polarity occurs when one of several, initially identical processes undergoes a prolonged period of growth, becoming the cell's axon. In order to assess possible causal relationships between specification of the axon and the development of protein targeting, changes in the distribution of representative axonal and dendritic proteins labeled with Green Fluorescent Protein will be followed as cells begin axonal outgrowth. Further experiments will perturb the localization of an endogenous cell adhesion molecule involved in the signaling that governs the specification of polarity in order to assess its impact on axonal outgrowth and the development of polarity. After the initial sorting event, which is presumed to direct different proteins to distinct vesicle populations, transport vesicles must be targeted to appropriate domains within the cell. The expression of appropriately tagged axonal, dendritic, and uniformly distributed proteins will be used to define the populations of transport vesicles present in hippocampal neurons and to visualize their transport in living cells.

[unreadable] DESCRIPTION (provided by applicant): The goal of this training program is to provide first- and second-year predoctoral students with broadly based, multidisciplinary training in neuroscience. This will be accomplished by providing instruction in molecular, cellular, developmental, systems, and behavioral approaches to research on the nervous system through course work, seminars, and laboratory rotations. Each of the three OHSU graduate programs that train students in neuroscience--the Behavioral Neuroscience Program, the Neuroscience Graduate Program, and the Program in Molecular & Cellular Biology will participate. The core faculty consists of 57 scientists selected from nearly 150 neuroscientists within the OHSU community. All of the trainers are affiliated with the Neuroscience Graduate Program; most also participate in the Behavioral Neuroscience Program or the Program in Molecular and Cellular Biosciences. The trainers are drawn from the Schools of Medicine and Dentistry, the Vollum Institute, the Center for Research on Occupational and Environmental Toxicology, the Oregon Hearing Research Center, the Oregon National Primate Research Center, and the Neurological Sciences Institute. These scientists provide expertise in state-of-the art approaches to neuroscience research. The training program will be directed by Dr. Gary Banker, who has a long record of commitment to graduate education. Dr. Edwin McCleskey will direct our program for under-represented minorities. He has worked for more than 15 years to develop innovative strategies for recruiting students from diverse cultural backgrounds to careers in science. Drs. Banker and McCleskey will work in close cooperation with a Steering Committee, which includes representatives drawn from the various participating entities. The long history of collegiality within the neuroscience community at OHSU and the many programs that have been developed to enhance interaction among scientists and clinicians provide a unique opportunity for predoctoral students to develop a cross-disciplinary approach and to gain an appreciation of the health-relatedness of the basic science research that they undertake. [unreadable] [unreadable]

DESCRIPTION (Applicant's abstract): Over the last two decades, primary neural cell cultures have been widely used to study many aspects of cellular neurobiology, ranging from development to pathophysiology. Such cultures offer several experimental advantages: their cellular composition is much more homogeneous than neural tissue, the cells are readily accessible to analysis and experimental perturbation, and they permit living cells to be monitored over time. In the present application, we proposed to explore the application of emerging technologies for gene expression profiling to the three best-defined culture systems for studying the biology of CNS neurons, oligodendroglia, and astroglia. We will examine the changes in gene expression that occur during the cells normal development in culture. We will also examine rapid changes in gene expression in response to agents that enhance neuronal maturation or cause oxidative stress to developing glial cells. The database detailing developmental changes in gene expression in these three culture systems will be made available on the website of OHSUs genomics core facility. These data will provide quantitative information on the cell-type specific expression of the unique ESTs being prepared as part of the Brain Molecular Anatomy Project; this information will be of general use to all neuroscientists studying gene expression in the nervous system. In addition, our data will enable any of the hundreds of neuroscientists who use these cell culture systems to follow the pattern of expression of genes relevant to their research that are included among the ESTs available during the period of this project.

DESCRIPTION (provided by applicant): Nearly every aspect of neuronal function depends on the correct polarization of membrane proteins to axons or dendrites. Following exit from the Golgi complex, membrane proteins destined for different cellular domains are sorted into carrier vesicles and transported along microtubules to their destinations, where they are delivered to the plasma membrane by exocytosis. The long-term goal of our research is to uncover the mechanisms that govern the selectivity of these trafficking pathways.We have developed novel methods that allow protein trafficking to be visualized in living hippocampal neurons, permitting each step along the exocytic pathway -- sorting, transport, and delivery -- to be assessed independently. In addition, we have generated a catalog of GFP tagged protein markers designed to label a representative subset of the different carriers that deliver proteins to dendrites and axons. We propose to use these tools to address three specific aspects of the trafficking of polarized proteins in nerve cells. First, we will estimate the number of different carriers that convey dendritically polarized proteins and identify the motifs in dendritic proteins that govern their sorting into these carriers. Second, we will test the hypothesis that interaction of carriers with minus-end directed motors prevents the transport of dendritic proteins into axons, thereby ensuring their delivery only to the correct domain. Third, we will define the principal populations of carriers that convey axonally polarized proteins, identify the motifs that govern the sorting of axonal proteins into these carriers, and confirm that these carriers deliver axonal proteins exclusively to the axonal membrane.The complexity of neuronal protein trafficking combined with the extreme dimensions of nerve cells contribute to their selective vulnerability to injury and degenerative disease. Improved understanding of these aspects of neuronal biology may significantly advance our understanding of the etiology of these diseases and suggest potential avenues for therapy.

DESCRIPTION (provided by applicant): Nearly every aspect of neuronal function depends on the accurate polarization of membrane proteins to axons or dendrites. During the current award period we have identified the principal trafficking pathways that underlie the polarized localization of neuronal plasma membrane proteins in cultured hippocampal neurons and developed methods to image each of the different populations of the long-range carriers that transport proteins along these pathways. Our results show that the selectivity of anterograde, kinesin- mediated transport plays a central role in the targeting of polarized proteins. Carriers containing dendritic proteins are transported into dendrites but excluded from axons;carriers containing axonal proteins enter both dendrites and axons, but are transported preferentially into axons. We also developed an assay to assess the selective transport of constitutively active kinesin motor domains in the absence of cargo. We demonstrated that Kinesin-1 motor domains translocate preferentially into the axon, whereas a Kinesin-3 motor domain translocates with equal efficiency into both axons and dendrites. Recent evidence demonstrates that posttranslational modifications of tubulin (acetylation and glutamylation) regulate the efficiency of kinesin translocation and our preliminary data show that axonal and dendritic microtubules differ in both of these posttranslational modifications. Using the unique methods we have developed during the current award period, we now propose to identify the kinesins that transport each population of long-range carriers and to investigate the molecular features of kinesins and the molecular modifications of microtubules that determine the selectivity of carrier transport in hippocampal neurons. We will use two-color imaging to identify the carrier populations labeled by expressed kinesins. We will also use RNAi to inhibit the expression of individual kinesins and evaluate the populations of long-range carriers that are affected. We will compare the selectivity of motor domain translocation for all kinesin organelle motors expressed in hippocampal neurons and examine how posttranslational modifications of tubulin influence the selectivity of motor domain transport and the transport of long-range carriers. Defects in kinesin-mediated transport cause neuronal dysfunction in animal models and have been implicated in several human neurodegenerative diseases. Our results will identify novel targets for pharmacological manipulations that could compensate for transport defects and protect against neural degeneration. PUBLIC HEALTH RELEVANCE: Nearly every aspect of neuronal function depends on the accurate transport and trafficking of membrane proteins;defects in the long-range transport of membrane proteins are thought to underlie several neurodegenerative diseases. By elucidating the regulatory mechanisms that underlie the accuracy of kinesin-driven transport, our work may identify a novel set of targets for pharmacologic manipulations that could enhance long-range transport and perhaps ameliorate neural degeneration.