Jane E. Bottenstein - US grants

A fundamental and crucial aspect of normal development of the CNS is regulation of oligodendrocyte proliferation. It may also be involved in several neuropathological conditions and be a critical factor in failure of recovery in demyelinating disorders and CNS regeneration. We hypothesize that in addition to hormonal and other systemic regulation of oligodendrocyte proliferation, neuron-oligodendrocyte interactions play a critical role in the development of oligodendrocytes, that this interaction is mediated in part by soluble factors derived from neurons, and that the target cells for neuronal-derived growth factors are glial precursor cells. We have identified a factor produced by a CNS neuronal cell line that stimulates division of oligodendrocytes and inhibits division of Type I astrocytes in dissociated neonatal rat brain cultures. We will purify this factor using chromatographic procedures and a sensitive ELISA bioassay, characterize the chemical properties of the factor, determine whether the oligodendrocyte-stimulating and astrocyte-inhibiting factors are identical, define the target cells for the oligodendrocyte growth factor with the aid of differentiation stage-specific antibodies, and verify that developing CNS contains a factor with similar properties. In parallel we will develop monoclonal antibodies to the growth factor using in vitro immunization methods and screening neutralization of factor activity. The monoclonal antibody will be used for immunoaffinity chromatography in future purifications of the factor and for detecting the presence of antigen and its cellular localization in the developing rat nervous system. Our results should provide information about oligodendrocyte cell lineage in the brain and the capability of cells within this lineage to divide in response to a neuronal- derived growth factor. This data will be relevant to normal development processes and may provide insight into ways to accelerate recovery from demyelinating disorders.

This proposal is to purchase an image analysis system to be shared by a multidisciplinary group of PHS-funded investigators engaged in biomedical research. Dr. Bottenstein's project studies the proliferation of oligodendrocytes in vitro. A novel growth factor is being isolated and characterized that markedly stimulates the production of oligodendrocytes in neonatal rat brain cultures. 3H- thymidine autoradiographs will be correlated with differentiation- specific antigen profiles of cells to identify target of the growth factor. Cryostat sections of developing rat brain stained with monoclonal antibodies to the growth factor will detect the presence of immunoreactive endogenous factors. Finally, morphological development of mature oligodendrocytes from precursor cells will be followed with time-lapse analysis. Dr. Hulsebosch's project studies the effects of Nerve Growth Factor (NGF) deprivation on development of sprouting and synaptogenesis of primary afferent fibers in rat spinal cord using immunocytochemical and ultrastructural methods. The distribution of 125I-NGF, NGF receptors, and NGF mRNA will be analyzed in newborn, young, adult, and aged rat CNS and PNS. Dr. High's first project studies the immunocytochemical localization of monoamine oxidase A and B in normal human brain compared to several neurological syndromes, including Parkinson's and Alzheimer's disease, schizophrenia, and chronic alcohol abuse. The second project characterizes and quantitates catecholamine synapses on primate spinothalamic tract (STT) cells with light and EM methods. Also, mapping of brainstem neurons that project to the thalamus will be done with fluorescent tracers. Dr. Carlton's projectinvolves light and EM analysis of terminal inputs onto identified primate STT cells. After characterization of the normal terminal population, immuno-staining will identify and show the distribution of serotonin, substance P, and enkephalin terminals on STT cells. The sources of these terminals from descending, primary afferent, or intrinsic neurons will be evaluated. Dr. Chung's project studies the organization of primary afferent and propriospinal fibers in cat spinal cord. The number and termination sites of unmyelinated primary afferent fibers in dorsal funiculus will be determined. The number of propriospinal fibers in each funiculus will also be analyzed. The projects described in this application are relevant to various aspects of normal development, aging, demyelinating diseases, neuronal regeneration, mental and motor disorders, and pain mechanisms.

Nagle 9511773 Small protein molecules or peptides, play an important role in communication between cells in the brain. The synthesis of these peptides is a complex task involving the excision of the peptide from a larger precursor molecule by specific enzymes. Moreover, these peptides must be compartmentalized or packaged in the brain in such away that they can be released upon stimulation of the brain cell. To accomplish this release, the larger precursor molecules and the enzymes to process the precusor must be co-stored so the information transmitting peptide can be cut from the larger molecule and released between cells. However, how this packaging of material is accomplished remains unknown. Dr. Nagle will use a well-established system to investigate how the enzymes responsible for cleaving these peptides from their precursors is accomplished. It is anticipated that these studies will also identify specific domains in the enzymes responsible for targeting the peptide-containing precursors and enzymes to the appropriate secretory apparatus. These studies are of fundamental importance in understanding the transfer of information between cells. The findings from these studies are applicable to cells in the brain, as well as peripheral tissues. More importantly, however, these studies provide information on the biochemical basis by which information is transmitted between cells in the nervous system or the basis of the way the brain functions.