David A. Harris - US grants

The formation of chemical synapses is an essential step in development of the nervous system, and one that is likely to be involved in a wide range of neurological and psychiatric disorders. The process of synaptogenesis can be analyzed in particular detail at the vertebrate neuromuscular junction, because of its accessibility to electrophysiological, pharmacological, and biochemical manipulation. Our purpose is to explore how motor neurons regulate the expression of specific genes, and the assembly of their protein products, in muscle cells during development of the neuromuscular junction. In addition, the molecular action of a trophic factor that may mediate these effects will be investigated. This study will make use of a well-established nerve-muscle culture system that permits ready observation and experimental manipulation of nascent synapses. The principal objectives of the project are as follows: 1) To examine by cytochemical methods how the accumulation of synapse-associated proteins such as the acetylcholine receptor, and the mRNAs that encode them, is modulated in regions of the muscle cytoplasm underlying developing synapses. 2) To dissect pharmacologically the relationship between insertion of new AChRs into the subsynaptic membrane, and clustering there of preexisting receptors; both processes are operative during synaptogenesis. 3) To investigate the mechanism of actionof a polypeptide from chick brain that stimulates inserrtion and clustering of AChRs on the surface of muscle cells. Its effect on synthesis and assembly of receptor subunits, and on the amounts of the corresponding mRNAs will be tested. Whether it influences the distribution and synthesis of other synaptic molecules will also be examined. 4) To clone a cDNA encoding the trophic polypeptide using recombinant DNA technology. This clone will be essential for obtaining further structural information about the polypeptide, as well as for determining its sites of synthesis in the developing nervous system.

Prion proteins (PrPs) are cellular polypeptides of unknown function, modified forms of which have been implicated in the pathogenesis of several degenerative diseases of the central nervous system, including Creutzfeldt-Jakob disease and Gerstmann-Straussler syndrome in man, and scrapie in animals. We have recently identified a cDNA that encodes a chicken prion-like protein (ch-PLP), the first non-mammalian example to be described. The objective of the present application is to carry out further studies of ch-PLP using molecular and cell biological techniques. These studies are likely to provide important information about the structure, function and metabolism of PrPs that is relevant to understanding their role in neurological disease. Only a single PrP gene and protein have been described in each of several mammalian species examined, and this fact has played an prominent role in hypotheses about the pathogenesis of prion diseases. Several considerations suggest that ch-PLP may not be the only prion-related protein encoded in the chicken genome. We propose here to characterize the gene that encodes chPLP; and to search for cDNAs that encode other prion-like proteins in chicken, as well as mouse and man, by low-stringency screening of libraries, and by use of the polymerase chain reaction. Preliminary studies indicate that ch-PLP undergoes several kinds of posttranslational processing, including a novel proteolytic cleavage near its N-terminus that has not been previously observed for mammalian PrP. We propose to further characterize the posttranslational processing of ch-PLP using antibody mapping and pulse-chase labeling. We will also define critical processing sites in the molecule by expression in transfected cells of deleted and chimeric proteins, as well as proteins harboring mutations that have been linked to prion diseases in man. Ch-PLP was originally identified as the major sequenceable protein in purified preparations of an acetylcholine receptor-inducing activity (ARIA) from chick brain that is postulated to play a role in neuromuscular synaptogenesis. Indirect evidence suggests that ch-PLP and ARIA are identical or closely related molecules. We propose to determine which posttranslationally processed forms of ch-PLP are present in preparations of ARIA, and to raise antisera against these molecules that can be used to immunoprecipitate AChR-inducing activity. We will also create N- and C-terminally deleted forms of ch-PLP that mimic the molecules present in ARIA preparations, and assay the receptor-inducing activity of these recombinant proteins.

DESCRIPTION: (adapted from Applicant's Abstract) The scrapie prion protein (PrPsc) is the major component of the infectious particle (prion) responsible for a group of fatal neurodegenerative disorders, including Creutzfeldt-Jakob disease and kuru in human beings, and scrapie in animals. During prion infection, PrPsc is generated by posttranslational modification of a glycolipid-anchored membrane protein of the host called PrPc. While PrPsc has been the subject of intensive study, much less attention has been paid to PrPc, whose physiological function is unknown. The purpose of the present application is to explore several interrelated questions concerning the cellular properties of PrPc, with a view to understanding the normal function of this isoform, as well as to establishing possible therapeutic strategies for inhibiting its conversion to PrPsc.

DESCRIPTION: (Adapted from Applicant's Abstract) Prion diseases are fatal neurodegenerative disorders of humans and animals that are characterized by dementia, motor dysfunction, and cerebral amyloidosis. These disorders are unique because they can be either infectious, genetic or sporadic in origin. All three forms result from a posttranslational alteration in the conformation of a host-encoded membrane glycoprotein called PrPc. Infectious cases are caused by prion particles composed of a protease-resistant isoform of PrPc called PrPSc, and inherited cases are linked to mutations in the host gene that encodes PrPc. Although cell culture models for infectious forms of prion disease are available, there has been no analogous system for familial forms. Applicant and his colleagues have recently developed such a model, in which mutant prion proteins associated with each of the known inherited prion disorders undergo conversion to PrPSc in cultured cells. The purpose of this application is to use this system to carry out a detailed analysis of the cellular and molecular mechanisms responsible for familial prion diseases, and to elucidate general features that are shared by familial, infectious and sporadic forms of these disorders. First, we propose to define the molecular mechanisms responsible for attachment of mutant and infectious forms of PrPsc to cellular membranes, using differential extraction, lipid-soluble labeling reagents, mapping of antibody and protease accessibility, and characterization of the aggregation state of protein on the cell surface. Second, we plan to analyze the kinetics of PrPsc biosynthesis in cells expressing mutant PrPs using pulse-chase metabolic labeling, in conjunction with assays of membrane attachment, aggregation state, and association with molecular chaperons. Third, we will investigate the localization and cellular trafficking of mutant PrPs by light and electron microscopy, subcellular fractionation, pharmacological treatments, temperature block, and surface labeling to monitor endocytosis. Fourth, we propose to characterize "strain-specific" differences between mutant PrPs in their glycosylation patterns and proteinase K cleavage sites, and in the efficiency with which they are converted to PrPsc in cultured neurons from several brain regions. Fifth, we will develop in vitro systems for generation of PrPsc from mutant PrPs, using permeabilized cells, isolated fractions of microsomes and Golgi, and purified proteins.

Although the conformational conversion of PrPC into PrPSc is the central molecular event in prion diseases, the biological function of PrPC, a normal cell-surface glycoprotein, remains a mystery. Cell biological work from my laboratory has demonstrated that PrPC constitutively cycles between the plasma membrane and an early endosomal compartment, and that clathrin-coated pits mediate internalization of the protein. The significance of this recycling pathway has been obscure, although it suggested the possibility that the protein might serve as a receptor for uptake of an extracellular ligand. However, the identity of this putative ligand was unknown. Several intriguing new pieces of data from other laboratories now suggest a candidate ligand for PrPC: copper ions. Copper ions bind with low micromolar affinity to the histidine-rich peptide repeats that are found in the N-terminal half of PrPC, and membrane fractions from the brains of PrP0/0 mice that do not express PrPC show a markedly reduced content of ionic copper. This new information on the interaction of copper with PrPC can be melded with our own data on cellular trafficking of the protein to suggest a specific and testable hypothesis about the physiological function of PrPC: We propose that PrPC is a copper receptor that serves to increase the efficiency of copper uptake by cells in the CNS. Specifically, we suggest that, by virtue of the cellular recycling pathway that it follows, PrPC can continuously deliver copper ions from the extracellular milieu to an early endosomal compartment within which the bound ions dissociate and are moved into the cytosol by transmembranetransporters. To test this hypothesis, we propose in AIM number 1 to analyze the effect of copper ions on the endocytic trafficking of wild-type and N- terminally deleted forms of PrPC expressed in transfected neuroblastoma cells. In AIM number 2, we will characterize the uptake of radioactive 64Cu by transfected neuroblastoma cells expressing PrPC, and by cerebellar and cortical neurons cultured from the brains of normal mice, PrP0/0 mice, and transgenic mice over-expressing wild-type or N- terminally deleted PrP. The data to be obtained in the proposed experiments will serve as an important starting point for a number of future studies into the role of copper in the biology of PrP and it associated diseases.

DESCRIPTION: (Adapted from Applicant's Abstract) Prion diseases are fatal neurodegenerative disorders of humans and animals that are characterized by dementia, motor dysfunction, and cerebral amyloidosis. These disorders are unique because they can be either infectious, genetic or sporadic in origin. All three forms result from a posttranslational alteration in the conformation of a host-encoded membrane glycoprotein called PrPc. Infectious cases are caused by prion particles composed of a protease-resistant isoform of PrPc called PrPSc, and inherited cases are linked to mutations in the host gene that encodes PrPc. Although cell culture models for infectious forms of prion disease are available, there has been no analogous system for familial forms. Applicant and his colleagues have recently developed such a model, in which mutant prion proteins associated with each of the known inherited prion disorders undergo conversion to PrPSc in cultured cells. The purpose of this application is to use this system to carry out a detailed analysis of the cellular and molecular mechanisms responsible for familial prion diseases, and to elucidate general features that are shared by familial, infectious and sporadic forms of these disorders. First, we propose to define the molecular mechanisms responsible for attachment of mutant and infectious forms of PrPsc to cellular membranes, using differential extraction, lipid-soluble labeling reagents, mapping of antibody and protease accessibility, and characterization of the aggregation state of protein on the cell surface. Second, we plan to analyze the kinetics of PrPsc biosynthesis in cells expressing mutant PrPs using pulse-chase metabolic labeling, in conjunction with assays of membrane attachment, aggregation state, and association with molecular chaperons. Third, we will investigate the localization and cellular trafficking of mutant PrPs by light and electron microscopy, subcellular fractionation, pharmacological treatments, temperature block, and surface labeling to monitor endocytosis. Fourth, we propose to characterize "strain-specific" differences between mutant PrPs in their glycosylation patterns and proteinase K cleavage sites, and in the efficiency with which they are converted to PrPsc in cultured neurons from several brain regions. Fifth, we will develop in vitro systems for generation of PrPsc from mutant PrPs, using permeabilized cells, isolated fractions of microsomes and Golgi, and purified proteins.

DESCRIPTION (From the applicant's abstract): Prion diseases are neurodegenerative disorders that result from changes in the conformation of a single, highly unusual membrane glycoprotein called PrP (prion protein). This molecular transition converts a normal version of the protein (PrPc) into a pathogenic form (PrPsc) that constitutes the major component of an unprecedented type of infectious particle (prion) devoid of nucleic acid. Although a wealth of information is now available about the role of PrPsc in the disease process, relatively little is known about the normal, physiological function of PrPc. Aside from its intrinsic biological interest, identifying the function of PrPc is likely to be important in understanding the pathogenesis of prion disease, as it has been suggested that impairment of this function as a result of conversion to PrPsc may explain some features of the disease phenotype. Several lines of evidence have emerged recently suggesting that PrPc may play an important role in the cellular metabolism of the essential trace metal, copper. The most compelling results are that copper binds with low micromolar affinity to PrPc, that membrane fractions from the brains of PrP-null mice show 5 percent of the normal content of ionic copper, and that neuronal Cu-Zn superoxide dismutase from these mice is less enzymatically active and incorporates less radioactive copper than the enzyme from normal mice. In addition, my own laboratory has recently shown that copper ions rapidly and dramatically alter the cellular trafficking of PrPc in cultured neurons. Taken together, these findings constitute the most substantial clues to the normal function of PrPc to emerge in the 15 years since the protein was discovered. They suggest the hypothesis that PrPc may function in cellular pathways responsible for uptake delivery, or excretion of copper ions. The results also raise the possibility that copper metabolism may be altered during prion diseases, and that manipulation of copper levels may be useful in treatment of the disorders. In this application, we will investigate these ideas by (1) analyzing the interactions between PrPc and copper at the cellular and biochemical levels in mammalian cells, (2) by using the yeast S. cerevisiae to elucidate the role of PrPc in copper trafficking, (3) by characterizing the interplay between copper and the disease-specific isoform PrPsc, and (4) by using PET to image the distribution of radioactive copper in living mice.

DESCRIPTION (From the Applicant's Abstract): The overall goal of this project is to utilize transgenic (Tg) mice as models for human familial prion diseases, which are linked to point and insertional mutations in the gene encoding the prion protein (PrP) on chromosome 20. We have previously constructed lines of transgenic mice that express a PrP molecule with a nine-octapeptide insertional mutation (PG14) associated with a familial form of Creutzfeldt-Jakob disease in humans. These Tg(PG14) mice develop a progressive neurological disorder characterized by ataxia, apoptosis of cerebellar granule cells, punctate deposition of PrP, and astrocytic gliosis. In addition, beginning at birth the mice accumulate mutant PrP molecules in their brains that display the major biochemical hallmarks of PrPSc, the pathogenic isoform of PrP. Thus, Tg(PG14) mice recapitulate several of the essential clinical, neuropathological, and biochemical features of inherited human prion diseases. These mice offer a unique opportunity to study the molecular and cellular basis of familial prion diseases in an in vivo setting, and to establish a rational basis for the future development of more effective diagnostic and therapeutic modalities. The purpose of the present application is to carry out further studies of the PG14 mutation in transgenic mice, with a view toward understanding how mutant PrP molecules cause the pathology seen in familial prion diseases, and what role the PrPSc isoform plays in this process. We plan to: (1) create new lines of Tg in which expression of mutant PrP is controlled by neuron-specific and inducible promoters; (2) investigate whether degradation of mutant PrP by theubiquitin-proteasome plays a role in the neuropathology observed in Tg(PG14) mice; and (3) compare the molecular, pathogenic, and transmission properties of two forms of mutant PrP that differ significantly in their degree of protease resistance.

[unreadable] DESCRIPTION (provided by applicant): This renewal application requests continued support to train Ph.D. students in the Cell and Molecular Biology (CMB) Programs within the Division of Biology and Biomedical Sciences (the "Division") at Washington University. The four CMB Programs are Molecular Cell Biology, Molecular Genetics, Developmental Biology, and Molecular Microbiology and Microbial Pathogenesis. These Programs have a history of excellence in research and education that is nationally recognized. A unique feature of these Programs is their administration by the Division, which was established more than 30 years ago with its own endowment, in recognition of the increasingly interrelated nature of all aspects of biological and biomedical research. As a result of the Divisional structure, all predoctoral programs and degree-granting units in the biological sciences at Washington University are both interdisciplinary and interdepartmental. Continued support of this training grant is proposed to be used for two main purposes: 1) to promote the professional growth of students whose research interests lie in an interdisciplinary area of crucial importance in the post-genomic era: cell and molecular biology; and 2) to foster interactions among the faculty as a means of providing new research directions and training opportunities for students. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The candidate is an established scientist in the medical device field, who seeks to retool in fundamental auditory neurobiology and redirect his career back to hearing science. Multi-unit and field potential-derived current source density mapping will be used to study the spatial and temporal properties of the synaptic drive and post-synaptic activity of neuronal populations in chicken Nucleus Laminaris (NL). Localized activity in a tuned population of coincidence detectors is assumed to provide a "place code" for the location of a sound based on interaural timing differences (ITD). In vitro brain slices and in vivo recordings are used as preparations for spatiotemporal mapping experiments to detail the interaural timing difference ITD topography in NL. We propose to identify the contribution of GABAergic input to the spatiotemporal topographic patterns. Neuronal excitation patterns are assessed with spatial profiles (1-D) or contours (2-D) extracted from space-time maps of multi-unit activity and the timing and distribution of current sources and sinks are defined with maps of field potentials, processed with current source density (CSD) analysis. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Prion diseases are fatal neurodegenerative disorders of humans and animals. A wealth of evidence suggests that the central molecular event in these diseases is the conformational conversion of PrPC, a normal cell-surface glycoprotein, into PrPSc, an abnormal isoform that is infectious in the absence of nucleic acid. Although we now have a detailed picture of how PrPSc figures in the disease process, the normal biological function of PrPC has remained a mystery. The major objective of this grant is to investigate an exciting new hypothesis concerning the physiological function of PrPC. We propose that PrPC plays a key role in protection of cells from pro-apoptotic stresses, and that subversion of this cytoprotective activity causes neurodegeneration. First, we will utilize novel, genetically-based screens in yeast, in addition to proteomics technologies, to identify interacting proteins that play a role in the cytoprotective and cytotoxic actions of PrP. Next, we will investigate the mechanisms underlying the cytoprotective effects of PrPC using a variety of biochemical and cell-based approaches. Finally, we will utilize transgenic mice to analyze the role of Bax in the neurotoxic actions of PrPSc and other abnormal forms of PrP. In addition to elucidating the normal function of PrPC, our findings are likely to provide insights into the mechanisms by which prions cause neurodegeneration, and will yield clues as to how this process can be blocked as a therapeutic strategy. The PrP-interacting proteins and cellular pathways identified here may also be relevant to other neurodegenerative disorders, such as Alzheimer's, Parkinson's and Huntington's diseases, in which cellular stress plays a prominent role. [unreadable] [unreadable] [unreadable]