Eugene M. Johnson - US grants

Work carried out in the laboratory has characterized a new method of producing permanent sympathectomy in the rat by the administration of the antihypertensive agent, guanethidine. Our data show that this method is superior to previously used methods of producing sympathectomy, especially in the critical parameters of completeness (including vasculature) and specificity for peripheral neurons (CNS unaffected). Our objectives now are to exploit the permanent denervation of the cardiovascular system produced by guanethidine in order to define the role of the sympathetic nervous system in several forms of hypertension, to determine the mechanism by which guanethidine destroys sympathetic neurons, and to determine the reasons for the observed resistance of other species to guanethidine sympathectomy. Experiments will be carried out to determine whether animals sympathectomized by guanethidine treatment develop mineralocorticoid (DOCA-salt) hypertension or genetic hypertension (Japanese SHR rat). The hypothesis that guanethidine destroys sympathetic neurons by inhibiting utilization of nerve growth factor probably by blocking the retrograde transport of the trophic protein will be tested by monitoring accumulation of locally applied 125I-NGF in sympathetic neuron cell bodies. Experiments designed to explain the lack of effect of guanethidine on other species will involve measurement of guanethidine accumulation in ganglia of treated animals of these species (chemical assay) and by comparing the direct effects of guanethidine on the neurons (organ culture). Attempt will be made to sympathectomize these species (rabbit, cat) by combined treatment of guanethidine and the antibody to NGF. The studies will expand our knowledge of the pharmacology of guanethidine, especially as related to its cytotoxic effect on sympathetic neuron and demonstrate its utility as a tool in the understanding of the development and pathophysiology (e.g. hypertension, arrythmia) of the sympathetic nervous system.

Nerve growth factor (NGF) is essential for the survival of embryonic sensory neurons in dorsal root ganglia (DRG). Recent observations indicate that the role of NGF is more complex and probably more important throughout the life of a DRG neuron than previously thought. We have found: 1) I125-NGF is retrogradely transported from the spinal cord to the DRG; 2) cutting the central process of DRG in newborn rats results in massive neuronal death; 3) exogenous systemic NGF prevents the cell death produced by lesions of the central or peripheral process of the DRG; and 4) intraspinal tracts bears NGF receptors. These results show that the central process of sensory neurons is important in providing trophic support, at least during development and suggest a physiological role for NGF in that support. We will systematically characterize the effects of lesioning the central, the peripheral, or combined central/peripheral lesions on DRG neurons. We will examine cell survival, and morphological (chromtolysis, etc.) and biochemical changes (peptide levels, lysosomal markers) caused by these lesions in animals of various ages. We will thereby determine the relative role of the central vs. peripheral process in the trophic support of sensory neurons at different stages of development. We will examine these same parameters in animals deprived of NGF by anti-NGF to assess the physiological role of NGF in the response to these lesions in animals of various ages. We will examine the ability of exogenous, systemic or locally applied NGF to prevent cell death or modulate the injury response to the lesion and thereby assess the pharmacological potential of NGF to amelionate those injuries. We will quantify by immunoassay the amount of NGF being retrogradely transported via the central and peripheral process of DRG neurons in animals of different ages and correlate these values with the relative importance of the central vs. peripheral process at different stages of development. We will measure NGF levels in normal and denervated (dorsal rhizotomized) spinal cord. Lastly, we will examine and identify by autoradiographic and immunohistochemical methods the intraspinal tracts found to bear NGF receptors and look for retrograde transport of I125-NGF in specific CNS tracts. These experiments will increase our understanding of the role of the central process and of NGF in sensory trophic support and will assess possible roles of NGF in the CNS.

We have discovered that Schwann cells, distal to an injury of sciatic nerve express high densities of nerve growth factor (NGF) receptors. We propose that Schwann cells, when deprived of axonal contact, both express NGF receptors and produce NGF. This results in an "NGF-laden substratum" which provides tropic guidance and trophic support to facilitate regeneration of NGF responsive neurons. We propose to characterize the phenomenon of NGF receptor induction on Schwann cells after nerve injury, to assess the physiological significance of this phenomenon, and to determine the mechanism(s) involved in regulation of NGF receptors on Schwann cells. We shall describe by receptor quantitation and by microscopic localization the time course of receptor induction after transection or crush of sciatic nerve to determine effects of reinnervation and to provide a precise anatomical description of NGF receptor interactions during these processes. We shall determine whether a similar induction of NGF receptor occurs after various lesions within the CNS and whether NGF receptor induction only occurs on glial cells previously associated with axons of NGF responsive neurons. We have found that NGF receptors appear in high numbers when Schwann cells are cultured. We shall define the time course of receptor induction and the kinetic properties of the receptors. Once the culture system is well defined, it will be used to examine the mechanisms by which axonal interaction modulates NGF receptor density on Schwann cells. We shall examine the possibility that the NGF can directly exert effects on Schwann cells. In parallel with these experiments, NGF protein levels (two-site immunoassay) and NGF mRNA (quantitative Northern blot analysis) will be determined under similar conditions. Sites of NGF biosynthesis will be determined by in situ hybridization. We shall thus be able to correlate the phenomena and mechanisms controlling NGF receptor expression and NGF synthesis in cultured Schwann cells and in injured nerve. We shall test the hypothesis that the NGF- laden substratum is important in sympathetic axonal regeneration by assessing the effects of systemic or local NGF deprivation on regeneration after sciatic nerve crush. Lastly, we shall examine the possibility that, during development, Schwann cells bear NGF receptors by measuring NGF receptors in nerve during development and by localizing by EM immunohistochemistry NGF receptors in developing nerve. This project will provide an assessment of NGF receptors on Schwann cells and their possible role in Schwann cell/axon interactions.

Neurons deprived of access to neurotrophic factors undergo programmed cell death (PCD). We and others have hypothesized that PCD is caused by the expression of a specific genetic program whose function is to bring about physiologically appropriate death. We have recently provided the first evidence for the increased expression of a specific gene in neurons undergoing PCD. That gene is cyclin D1, a molecule whose only known function is in progression through the Go-G1/S transition of the cell cycle. Based on this and other similarities of PCD with mitosis, we shall test the general hypothesis that neuronal PCD involves an aborted attempt of the cell to enter the cell cycle and the specific hypothesis that an increased expression of cyclin D1 us a critical event in neuronal PCD. Toward this objective, we shall further analyze NGF-deprived sympathetic neurons in vitro for expression of other cell cycle-related genes and for biochemical events normally associated with mitosis. We shall examine the expression of cell cycle-related genes in other cell types, thymocytes and prostate epithelium, undergoing PCD. We shall prepare specific cyclin D1 antibodies to examine the expression, posttranslational modification, and subcellular localization, of cyclin D1 in dying neurons. We shall also look for evidence for association of cyclin D1 with other proteins in dying neurons. We shall examine the expression and localization of cyclin D1 in vivo in neurons undergoing naturally occurring or experimentally induced PCD. Last, we shall utilize Herpes Simplex vectors to assess directly the role of cyclin D1, and other cell cycle-related genes in neuronal PCD. These studies should either prove or disprove the working hypotheses that cyclin D1 specifically is involved in neuronal PCD and that PCD represents an aborted attempt of the neuron to enter the cell cycle. The work will further our long-term goals of elucidating the molecular mechanism of neuronal PCD, of developing the means to manipulate the process pharmacologically, and of determining whether the inappropriate expression of PCD after the period of naturally occurring, physiologically appropriate, neuronal death might underlie human neurodegenerative conditions.

Programmed cell death (PCD) is a well-recognized developmental phenomenon that occurs by a process of apoptosis. Recent evidence strongly suggests that PCD also occurs in pathological conditions. Several lines of evidence have indicated that PCD is the result of the expression of a specific genetic program leading to proteins that kill the cell. We have identified several genes whose expression is increased in sympathetic neurons undergoing PCD in response to deprivation of the neurotrophic factor, NGF. We have presented evidence that expression of member(s) of the Fos and Jun family of transcriptional activators are required for neuronal PCD, and we have identified novel genes expressed as the cells die. In addition we have identified a phenomenon wherein total cellular RNA, including the vast majority of mRNA species, are degraded well before the cell is committed to die. We propose a multi-faceted attack on this problem to elucidate the mechanism of, and ultimately exert pharmacological control over, neuronal PCD. We shall use a variety of molecular genetic approaches to assess further the role of the Fos and Jun family in neuronal PCD, and in doing so establish methods to examine other potential genes of interest We shall continue our use of the differential mRNA display approach to the identification of mRNAs expressed in dying neurons. We shall determine the role of already identified genes, as well as newly identified genes, in neuronal PCD in our model system. We shall determine whether the genes we have identified, and those we hope to identify, are expressed in dying neurons in vivo after death-producing axotomy and whether these genes are expressed in other cell types (prostate epithelium, thymocytes) undergoing PCD in response to hormonal signals. We shall characterize the phenomenon of marked, early, and apparently global degradation of RNA we have observed in sympathetic neurons undergoing PCD. We shall attempt to determine the mechanism of the activation of RNA degradation and its importance in PCD. Finally, we shall examine the biochemical and genetic events associated with PCD of cerebellar granule cells. The granule cell system will allow an assessment of the generality of the events we have identified in sympathetic neurons undergoing PCD, and, we hope, provide a system with many logistical advantages for future study of neuronal PCD. These studies will provide considerable insight into the mechanism of neuronal PCD. Given the increasing evidence for a role of neuronal PCD in pathological conditions of the nervous system (stroke, neurotoxicity, neurodegenerative disease), these insights may lead to strategies to intervene pharmacologically to prevent or retard death in these conditions.

In collaboration with the lab of Dr.Jeffrey Milbrandt, we have recently isolated and cloned a novel neurotrophic factor, neurturin, that bears homology to glial cell-derived neurotrophic factor (GDNF). Neurturin was purified based on its ability to block sympathetic neuron death after nerve growth factor (NGF) deprivation. Experiments are proposed that will examine the physiological functions and pharmacological actions of neurturin. We shall examine the expression of neurturin mRNA by Northern and RT-PCR analysis and by in situ hybridization, and neurturin protein by immunohistochemistry, in developing and adult rats. Particular attention will be paid to the adult CNS. Expression in response to injury to the nervous system will be examined. We shall examine the ability of neurturin exert survival-promoting or trophic effects on a variety of neuronal and hematopoietic cell types in culture. In addition we shall examine the retrograde transport of 125I- neurturin and endogenous neurturin in the adult PNS and CNS. We shall determine the physiological roles of neurturin by two complementary approaches. First, we shall analyze neurturin "knockout" mice generated in the Milbrandt lab. Secondly, we will examine the effects of neurturin neutralizing antibodies on fetal, neonatal, and adult rats, and in rats subjected to injury. We shall examine the pharmacological effects of neurturin when administered exogenously to neonatal rats. We shall examine the ability of neurturin to ameliorate the effects of immunological, mechanical, and chemical insults to sympathetic neurons in vivo and other neuronal types to be identified. We shall contrast and compare the pattern of trophic and survival promoting effects of neurturin with that of NGF and leukemia inhibitory factor (LW) on sympathetic neurons. We shall similarly compare potential signal transduction mechanisms mediating the survival promoting and growth promoting effects of these factors. These studies will define the physiological role and pharmacological actions of neurturin. Such results may indicate the potential of this factor as a therapeutic agent in neurodegenerative disease, stroke, or neuronal injury.

This ADRC is an integrated unit of Washington University that coordinates and supports the work of established investigators and teachers from diverse backgrounds. Mechanisms are provided for the support of programs and projects with a focus on both clinical and basic science research, and the development of improved training of professionals within the University and outreach programs for health care professionals and the public in the regional community. The clinical theme of the research component in this application is the identification and characterization of mild and very mild dementia of the Alzheimer type (DAT) in comparison with healthy aging.The basic science theme is the characterization of neurobiological principles and mechanisms relevant to aging of the nervous system and neuronal dysfunction and/or death in Alzheimer's disease (AD). Five cores (Biostatistics, Clinical, Psychometric, Neuropathology/Tissue Resource, Administration) will support five projects and five pilots. The topics of the projects include: 1) ApoE clearance receptor in CNS; 2) cellular prion proteins; 3) gene expression and neuronal vulnerability in neurodegenerative disease; 4) molecular genetics of dementia; and 5) processing speed, working memory and fluid cognitive abilities in DAT. The pilots study: 1) structure of the A-beta-protein; 2) AD and fibroblast bradykinin receptor modulation; 3) an AMPA receptor modulatory site; 4) protein fatty acylation in neuronal growth and remodeling; and 5) cognitive deficits and speech perception in DAT. A Training and Information-Transfer core supports our educational activities. Two Satellite components reach out to African Americans in our community and to the underserved rural areas of our region. This ADRC also interacts with and supports other investigators at this University, funded through other mechanisms, in order to focus attention on issues relevant to aging, AD and other dementias. Finally, the ADRC interacts with other Centers and the Alzheimer's Association to coordinate our efforts.

DESCRIPTION (Adapted from applicant's abstract): By chance, the applicant has produced a truncated mutant of Bax, which turned out to be a potent suppressor of neuronal apoptosis. Much of his application will focus on this finding. He will begin by investigating the mechanism by which sympathetic neurons mature to a non-NGF dependent state; he proposes 3 hypotheses to test here: (a) loss of dependence is due to an alteration in pattern of expression of members of the BCL-2 family; (b) a protein is expressed that sequesters BAX in cytoplasm, thus preventing its translocation to the mitochondria; or (c) there is an age-dependent post-translational modification of BAX. The second specific aim is to extend structure-function analysis of truncation mutants of Bax as suppressors of neuronal apoptosis. He will also determine if truncation mutants of other pro-apoptotic members of the BCL-2 family are anti-apoptotic. His third specific aim is see if truncated BAX also protects other cell types, including macroglia, fibroblasts, and lymphocytes, against apoptosis. Specific aim 4 will test whether truncated Bax blocks translocation of BAX to the mitochondria, or the loss of cytochrome c from the mitochondria. Finally, with an experienced collaborator, the investigator will generate tBAX transgenics.

The Washington University ADRC has been functioning since 1985. Emphasis on the work of the Center has focused on interdisciplinary longitudinal clinical analysis of dementia of the Alzheimer's type and the study of clinicopathologic correlates, with emphasis on distinguishing the earliest symptoms of AD. The ADRC performs and provides logistical and material support for research projects and this and many other institutes. It also provides for a range of informational and training activities aimed at both the professional and lay communities. To execute these activities the ADRC consists the ADRC consists of five Cores, Administrative, Clinical, Biostatistics, Neuropathology and Tissue Resource, and Training and Information Transfer (TITC). Two Satellites are supported by the Center. An African American Outreach Satellite of the Clinical Core designed to provide services to the African American Community in St. Louis and to increase the participation. of these subjects in the research activities of the Clinical Core. The Missouri/Iowa Satellite of TITC is designed to provide informational services to the rural populations of the states and the physicians and other health professionals that serve them Four research projects will be carried out using genetic, cell biological, biochemical, and neuroimaging, technologies to address fundamental mechanisms of the pathogenesis of Alzheimer's disease.

Description: (from the abstract) Apoptotic cell death occurs in the nervous system during normal and in pathological situations. The PI's preliminary data indicate that apoptosis has a major role in the ES cell graft after transplant into the injured rodent spinal cord. The extent of cell death underlie this process are currently unknown. The goal of this proposal is to apoptotic cell death of ES cell-derived neural lineage cells (ESNLCs). In relevant stages of development. In the second, he will characterize the apoptosis-inducing activity of the syrinx fluid which accumulates in the spinal apoptosis-inducing activity, and determine the changes in this activity as a function of time post-injury. Third, he will utilize the paradigms developed in to the molecular mechanisms of apoptosis and to develop cell clines that are resistant to apoptosis. These experiments should help to define the optimal molecules that are responsible for apoptosis induction seen after the success of ESNLC transplants.

DESCRIPTION (provided by applicant): In the previous funding period of this IRPG grant submitted with that of Jeffrey Milbrandt, we reported the discovery of neurotrophic factors neurturin, persephin, and artemin, which along with GDNF, constitute the GFL family of neurotrophic factors. We, and others, also discovered GPI-anchored co-receptors for neurturin and artemin. Among many other issues, we have addressed signal transduction of GFLs and provided evidence for a critical role for lipid rafts and the cytoplasmic tyrosine kinase C-src in mediating GFL action. In preliminary experiments we have discovered in sensory and sympathetic neurons a novel, non-GFL-mediated, maturation-dependent phosphorylation of the receptor kinase Ret. Surprisingly, this maturation-dependent phosphorylation, at least in sympathetic neurons, appears reliant upon nerve growth factor. We have also observed robust effects of GFLs on basal forebrain neurons in vitro and atrophic changes in basal forebrain neurons from aged neurturin-/- mice in vivo. We shall perform experiments (Aim 1) to understand the biochemical mechanism, cellular specificity, and generality of the ligand-independent, maturation-dependent phosphorylation of Ret. We shall do experiments (Aim 2) both in vitro and in vivo to assess potential biological significance of this phenomenon. We shall examine (Aim 3A) the structural/biochemical basis of the interaction of Ret with C-src and use cells from src-/- mice to assess critically the role C-src in mediating GFL actions and determine whether neuronal populations dependent on GFLs show neuronal loss and/or atrophy in src-/- mice (Aims 3B and 3C). We shall complete experiments (Aim 4) to define further the role and mechanisms of the interaction of the GFL-signaling apparatus and to determine the nature of the lipid-raft environment used by GPI-anchored co-receptors to activate Ret in response to GPL stimulation. We shall study (Aim 5) the role of the GFLs in basal forebrain neurons in vitro, examine their potential as neuroprotectants in vivo, and examine neurturin-/- mice for age-dependent neurodegeneration by anatomical, neurochemical, and behavioral criteria. Lastly, we shall continue to collaborate with the Milbrandt lab in the execution of the Aims in the companion IRPG grant, including analysis of the artemin- and persephin-knock-out animals.

The Washington University ADRC has been functioning since 1985. Emphasis on the work of the Center has focused on interdisciplinary longitudinal clinical analysis of dementia of the Alzheimer's type and the study of clinicopathologic correlates, with emphasis on distinguishing the earliest symptoms of AD. The ADRC performs and provides logistical and material support for research projects and this and many other institutes. It also provides for a range of informational and training activities aimed at both the professional and lay communities. To execute these activities the ADRC consists the ADRC consists of five Cores, Administrative, Clinical, Biostatistics, Neuropathology and Tissue Resource, and Training and Information Transfer (TITC). Two Satellites are supported by the Center. An African American Outreach Satellite of the Clinical Core designed to provide services to the African American Community in St. Louis and to increase the participation. of these subjects in the research activities of the Clinical Core. The Missouri/Iowa Satellite of TITC is designed to provide informational services to the rural populations of the states and the physicians and other health professionals that serve them Four research projects will be carried out using genetic, cell biological, biochemical, and neuroimaging, technologies to address fundamental mechanisms of the pathogenesis of Alzheimer's disease.

Grant funds are requested to support a new research Program Project aimed at initiating an exploration of the potential of embryonic stem (ES) cell transplantation for restoring lost functions after spinal cord injury. Members of the proposed Project team have already developed experience working with mouse ES cells differentiated down neural lineages, methods for reducing cell death in the nervous system, and rodent models of traumatic spinal cord injury. In the experiments that form the specific background for the present proposal, Project team members have obtained evidence that mouse ES-cell derived neural lineage cells (ESNLCs) can be successfully transplanted into the syrinx/cyst that forms nine days after impact injury to the rat spinal cord . These transplanted ESNLCs survived for at least many weeks, partially filling the syrinx and migrating into host tissue, and expressed markers for differentiated oligodendrocytes, astrocytes, and neurons. The requested Program Project funding would enable Project team members to build on these early experiments in a systematic and hypothesis-driven fashion. Based on direct observations that a majority of transplanted ESNLCs die over the first 48 hours following transplantation and that neurons in particular continue to die over subsequent days, the emphasis of the experiments proposed here will be on improving the survival of transplanted ESNLCs through pharmacological and genetic engineering approaches aimed at blocking apoptosis and excitotoxicity. It is anticipated that work performed in subsequent years may be directed at optimizing the ESNLC transplantation procedure in other ways.