Mary Bartlett Bunge - US grants

Our goal is to increase fundamental knowledge of factors that govern nerve fiber growth, formation of appropriate connections in target tissues, and modifications of these connections following experimental manipulation. The individual projects seek to investigate: a) if Schwann cells or fibroblasts from peripheral nerve provide trophic support for neurons (in tissue culture) and if these cells, aligned in "bridges", foster axonal regrowth in damaged spinal cord (in the animal), b) the success of axonal growth and connection formation of normal and modified "substrata" and in the absence of the usual targets in CNS tracts in fetal and neonatal animals, c) the correlation of developing synaptic activity with fine structural changes in growth cone and target cell during synapse formation, d) the signals that govern synthesis of specific proteins that may play a key role in axonal regeneratio, e) the development of a culture system to determine the role of glia in migration of nerve cells derived from normal and genetically abnormal animals, f) the pharmacological basis for plasticity in the visual cortex, g) physiological and anatomical differences between animals lesioned in infancy or adulthood with only the former exhibiting behavioral plasticity, and h) if learning can be detected at the cellular level in the cerebellum and thus serve as a model for studying adaptive control of behavior. The nine investigators involved bring expertise in diverse experimental paradigms and techniques. The purpose of this proposal is to seek funds to support shared facilities (computer, electron micro scopy, and animal support) and to stimulate interaction and collaboration to facilitate progress toward the goal of better understanding basic mechanisms of neuronal growth and interactions. We need new knowledge of mechanisms by which axonal regeneration and useful plasticity occur if we are to successfully intervene to prompt regeneration in the CNS of afflicted human patients.

This application has two goals. The first is to replace an outdated and unreliable electron microscope with a current, reliable model. The second goal is to extend the capabilities available to the Department of Anatomy and Neurobiology through the installation of a high performance electron microscope with special ancillary devices to which we currently do not have access. Together, this will give ongoing PHS supported biomedical research projects within the department: (1) enhanced quality of ultrastructural information, (2) increased efficiency for gaining this information, and (3) the ability to acquire new ultrastructural information not previously obtainable.

DESCRIPTION (Adapted from applicant's abstract) When transplanted into the central nervous system, Schwann cells (Scs) promote regeneration of axons and remyelination of demyelinated axons. Autologous transplantation of human SCs generated by growth in vitro from small nerve biopsies might thus be of significant clinical value. A procedure for growing adult human SCs and testing their function by transplantation into immune-deficient rodents was developed. SCs are stimulated to divide rapidly by the addition of a combination of the growth factor, heregulin, and an activator of adenylyl cyclase, forskolin, but human SCs appear to growth arrest after 12-14 population doublings and their ability to myelinate axons decreases after growth with mitogens. These observations suggest that the molecular and functional properties of human SCs are changed by growth with soluble mitogens in vitro. Experiments are proposed to test this hypothesis and to characterize changes in the properties of human SCs. First, tissue culture effects on expression of heregulin receptors and receptor-mediated signal transduction will be evaluated. Second, the mechanism by which forskolin potentiates the mitogenic effect of heregulin and whether prolonged exposure to combined mitogens alters the SCs response to forskolin will be examined. Third, experiments will test whether arrest of human SC growth reflects their entry into a state of replicative senescence. These studies will focus on the ability of heregulin/forskolin to activate senescence-related proteins, p53 and p21. Effect on phosphorylation of the growth-regulating protein, pRb, will be determined. The role of telomere shortening will be assessed and prevention of growth arrest by introduction of the catalytic subunit of telomerase into early passage human SCs will be tested. Fourth, changes in the human SC's ability to interact with axons by growth in vitro will be evaluated Effects on myelination capability in vivo will be examined and an in vitro model developed for study of the earliest SC-axon interactions: the initial contact, recognition, and association of the SC with the axonal surface. Experiments include an assessment of adhesion molecules, N-cadherin and L1, and on the ability of the SC to assemble an extracellular matrix. These studies explore the potential for use of human SCs in clinical applications, while expanding our knowledge of the biological and molecular properties of Schwann cells.

The ultimate goal of these studies is to develop effective strategies for improving the outcome of injuries to the spinal cord in humans. A moderate-severity contusion injury model in rats, characterize as are most injuries in humans by the interruption of axons that control lesion will not occur without treatment. This proposal first describes a series of three Aims comparing progressively more complex strategies to promote regeneration across a lesion site and into distal spinal cord. The strategy that maximizes regeneration will then be tested at delayed time points. A neuroprotective agent will be injected into all rats immediately following injury. In Aim 1, the lesion site will be modified at 1 week after injury by the transplantation, in medium containing fibrinogen and fibroblast growth factor-I, or Schwann cells or unsheathing glia (EG), or a 1:1 mixture of these regeneration-promoting cell types. A double-labeling retrograde tracing technique will be used to detect neurons in locomotion centers in the brain and brainstem which regenerate their axons into the lesion. At 12 weeks after injury the transplant type resulting in the regeneration of the highest number of axons into the lesion will be determined by counting the appropriately labeled neurons. In Aim 2, the most effective transplant from Aim 1 will be tested in the presence of increased levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the lesion. Axonal regeneration induced by 1) transplanted cells infected with adeno-associated viral (AAV) constructs coding for BDNF and NT-3, 2) uninfected cells transplanted with BDNF/NT-3 laden biodegradable microspheres, or 3) embryonic stem cells (naive or astrocyte-restricted) infected with a retroviral construct coding for a bi-functional neurotrophin with BDNF and NT-3 activity will be compared. In Aim 3, the most effective of these strategies will be combined with treatments to induce growth of the axons out of the lesion and into the distal cord. EG will be transplanted just distal to the lesion, alone or with the more effective of AAV-BDNF/AAV-NT-3 or BDNF/NT-3 laden microspheres injected mm distal to the lesion. The treatment resulting in the most axonal regeneration into the distal cord will be determined. In all studies, regeneration will also be assessed using anterograde tracing of axons from neurons in the hindlimb region of the cortex, in the lateral vestibular nucleus, and the reticular formation. Immunocytochemical methods will be used to classify the regenerating axons and to determine the expression of trkB and trkC receptors on responsive axons. Basso-Beattie-Bresnahan open field locomotion tests and electrophysiological analysis will also be performed. Importantly, in the fourth and final Aim, the response of severed locomotion-related axons to the most effective strategy defined in Aim 3 will be determined when cellular transplantation and neurotrophin treatments are delayed until 4 and 4 weeks after injury. An improved understanding of the change in potential of axons to regenerate with time after injury is critical for predicting the clinical merit of any regeneration-promoting strategy.

The Morphology Core is designed to function as a Core facility for the histopathology, immunocytochemistry, transmission electron microscopy, and image analysis requirements for the Program Project faculty. The Core facilities consist of personnel and equipment needed to perform the proposed studies in Projects 1-4. The Core will perform perfusion fixation and removal of spinal cords for light and electron microscopic examination. Core personnel will carry out immunocytochemical procedures, as well as develop new procedures for the visualization of additional antibodies. The Core will process tissues for plastic embedding for both light and electron microscopic analysis. High quality thin sections will be produced by personnel within the Core and stained for ultrastructural analysis. Established image analysis procedures will be utilized to quantitate spinal cord injury areas and volumes, as well as selective neuronal damage and white matter pathology. Finally, this Core will assist investigators in darkroom procedures and the generation of photographic slides are prints.