Lawrence F. Kromer, Ph.D. - US grants

Although functional regeneration occurs following peripheral nerve lesions, as yet, it has not been possible to promote clinically significant axonal regeneration in the adult mammalian central nervous system (CNS). Thus, the major emphasis of this research proposal is to analyze various cellular and molecular components of the peripheral nerve environment in order to identify factors that facilitate axonal growth for both PNS and CNS neurons. Since the immature CNS is capable of exhibiting considerable plasticity following injury, cellular and molecular components associated with immature CNS glia (astrocytes) also will be evaluated for their ability to foster axonal growth. Two basic experimental procedures will be used for these studies. First, in vitro septal explants will be grown in partially defined or conditioned media on a variety of substrates composed of glial cell layers (astrocytes and Schwann cells) or extracellular matrix molecules in order to identify culture conditions that facilitate neurite growth. Second, those in vitro environments that promote neurite growth from immature septal explant neurons will be adapted for transplantation in the CNS of adult rodents to further determine whether preparation that enhance neurite growth in culture also enhances regeneration from mature septal cholinergic neurons in vivo. These experiments will specifically evaluate whether: 1) CNS axonal growth is mediated by membrane associated molecules, 2) cell secreted diffusible factors enhance axon growth, and 3) components of the extracellular matrix (ECM) promote regeneration. Once individual cellular components have been identified as promoting axonal growth, then a final series of experiments will evaluate whether a combination of ECM components, diffusible factors and/or membrane associated molecules are necessary to promote optimal CNS regeneration. Immunocytochemical procedures will be used to identify specific cell phenotypes such as astrocytes, Schwann cells and cholinergic neurons and for localizing different molecular components in the culture and transplant substrates. A variety of neuroanatomical techniques will be employed for tracing axonal projections and for identifying the source of regenerating axons within the CNS. Results from the proposed in vitro explant studies and in vivo transplantation experiments will help characterize specific cellular components and extracellular matrix constituents that can stimulate regeneration following injury to the adult mammalian brain and spinal cord.

Degeneration of cholinergic and non-cholinergic neurons in the septal-basal forebrain region has been observed in a number of disease states such as Alzheimer's disease and Parkinsonism-dementia. Although it is uncertain what factors are responsible for this cell death, it has been proposed that neurodegenerative changes resulting from CNS injuries or associated with some aging disorders, such as Alzheimer's disease, result from the loss of target-derived neuronotrophic factors. This hypothesis is consistent with recent studies which indicate that neuronotrophic factors released in response to selective lesions of the adult mammalian CNS promote the survival of CNS and PNS neurons in culture and influence the survival of transplanted neurons. Thus, the long-term objectives of this proposed research are to develop in vivo experimental preparations for the identification of specific trophic substances and cellular environments that prevent age related cell death and influence neuronal survival following lesions in the mature mammalian CNS. The experimental model system to be employed in these studies is the septal projection to the hippocampal formation. The specific aims of the present proposal are to utilize intracephalic neural transplants in conjunction with a variety of lesion paradigms and the intraventricular or systemic administration of proposed trophic substances in order to identify experimental manipulations that: 1) prevent retrograde degeneration of cholinergic and non-cholinergic septal neurons following complete bilateral transections of the fornix/fimbria and 2) promote the survival of these neuronal populations in aged individuals. Specific neuronal populations within the septal region and their afferent projections will be identified using a variety of neuroanatomical techniques employing retrograde and anterograde axonal tracing procedures, acetylcholinesterase histochemistry, immunocytochemical identification of cholinergic and gabaergic neurons, and biochemical assay procedures. Data from the proposed research will provide insight into molecular factors and cellular environments that influence the survival of cholinergic and non-cholinergic septal neurons within the mature and aged mammalian CNS. These results should prove beneficial for evaluating whether the loss of target-derived trophic factors may be involved in the neuronal degeneration observed following neural injuries and during aging of the CNS. Moreover, these results will provide insight into possible treatment therapies that may promote cell survival in the adult mammalian CNS.

receptor; protein tyrosine kinase; developmental neurobiology; neurogenesis; basal ganglia; enzyme activity; receptor expression; ligands; synaptosomes; cell migration; receptor binding; phosphoproteins; chimeric proteins; in situ hybridization; immunoprecipitation; laboratory rat; western blottings;

DESCRIPTION (provided by applicant): This proposal will investigate the hypothesis that ephrins and their associated Eph receptor tyrosine kinases regulate inhibitory cell-cell interactions that mediated segregation of glial cells and meningeal fibroblasts, induce glial scar formation, and inhibit axonal regeneration following spinal cord lesions. To address these questions, we will perform over-hemisection lesions of the thoracic spinal cord in adult rats and mice with gene deletions or mutations of specific ephrins or Eph receptors. Additional in vitro studies will evaluate the effects of cytokines/growth factors and proteins that block or stimulate signaling between ephrins and Eph receptors on glial cell and meningeal fibroblast functions. A variety of neuroanatomical, biochemical and molecular techniques will be used to address three specific aims. Aim I will test the hypothesis that changes in the levels and activity state of Eph/ephrins after spinal cord injury regulate cell intermixing, glial scar formation, and the reformation of the glial limitans. Cell types expressing specific Eph/ephrins will be identified and their location correlated with cellular changes in the glial scar and the distribution of inhibitory CSPGs. Aim II will use in vitro preparations to test the hypotheses that cytokineslgrowth factors regulate expression of Eph/ephrins in CNS glia and meningeal fibroblasts and that signaling through specific ephrins and Eph receptors mediates astrocyte-meningeal cell segregation and boundary formation. In these studies cytokines/trophic factors, ephrin/Eph-Fc proteins and antisense oligonucleotides will be used to regulate the production of individual ephrins/Eph receptors or alter their activation to identify their functions. Cultures of primary cells from mice with deletions and/or mutations of specific Eph/ephrins also will be used to identify the functions of specific molecules. Aim III will test the hypothesis that interactions between Eph receptors expressed on corticospinal axons and ephrins present on reactive glia in the lesioned spinal cord inhibit axonal regeneration. In these studies bilateral dorsal funiculus lesions in mice with deletions and/or mutations in ephrin-B3 or EphA4 and rats with fetal spinal cord transplants and intraspinal infusions of ephrin/Eph-Fc proteins will be used to study axonal regeneration from lesioned corticospinal axons. These experiments will permit us to investigate whether disrupting Eph-ephrin signaling enhances regeneration through the lesion/transplant interface and into the distal spinal cord. Thus, the proposed studies will expand our knowledge of the function of Eph/ephrins beyond current assumptions that they primarily regulate compartment formation and axonal guidance during nervous system development. It is anticipated that these studies also will provide important information for developing strategies to treat human spinal cord injuries.