John A. Freeman - US grants

The overall objective of the proposed research is to identify the cellular functions and mechanisms of regulation of GAP43, the most prominent of a recently discovered class of growth associated proteins whose synthesis is selectively enhanced during periods of axonal growth and regeneration in retinal ganglion cells and other CNS neurons. We have obtained preliminary evidence suggesting that GAP43 expression is dually regulated by transcriptional as well as by post-transcriptional mechanisms, and postulate that retinal ganglion cells constitutively express the gene for GAP43, but fail to upregulate GAP43 levels following injury due to inhibitory factors, associated with contact with non-neuronal cells, conveyed by retrograde transport from the nerve terminal to the nucleus. We propose experiments designed to provide definitive information about the cellular function of this protein, to identify molecules that control its expression, and to identify the mechanisms by which its expression is regulated post-transcriptionally. Specific residues associated with known functional domains of GAP43 will be modified by oligonucleotide-directed site-specific mutagenesis. These include sequences associated with membrane attachment, phosphorylation by protein kinase C (pKC) and by casein kinase II (CKII), and GTP-G-O protein binding. Resulting functional effects will be measured in growth cones of rat retinal ganglion cells, including transport targeting, motility (using quantitative time-lapse video microscopy), intracellular calcium levels (using fura-2 imaging), and calcium channel conductance (using patch clamp analysis). Retrograde regulatory mechanisms will be investigated using blockers of axonal transport and quantitative 2D gel autoradiography. Experiments are designed to determine whether retrogradely transported molecules endocytosed from or modified by contact with mature oligodendrocytes in the optic nerve regulate the expression of GAP43, and if so, to isolate and test the regulatory molecules. The regulation of GAP43 message will be analyzed using quantitative RNA hybridization analysis and nuclear run-on assays to examine GAP43 message quantitatively. This will determine the extent to which GAP43 mRNA is regulated post-transcriptionally by alterations of message stability and translation efficiency during development and following optic nerve injury. Understanding the functions and molecular genetic control of this protein is likely to provide important insights more generally into the mechanisms of axonal growth, and in particular, might provide information critical to attempts to promote optic nerve regeneration and repair in the mammalian CNS. This information is of paramount importance in understanding the development of the visual system, and its response to injury.

Recent work has led to the discovery of a class of growth-associated proteins, called GAPs, whose expression is selectively enhanced in regenerating and developing neurons of some species. The purpose of this investigation is to study the cellular role of these growth-associated proteins, and in particular to determine whether tje abo;otu pf a meirpm tp regenerate depends on its ability to induce their synthesis. There are five related objectives. The first objective is to determine, by means of a limited phylogenetic survey, whether these proteins appear in CNS neurons lacking the capability to regenerate, or whether they occur only in growing or regenerating neurons. Two very sensitive and powerful techniques will be employed to detect and quantitate these proteins: computer-analyzed 2-dimensional gel electrophoresis, and immunological techniques. The second objective is to determine the cellular localization of GAPs, using EM immunocytochemical localization methods. The third objective is to characterize the major distinguishing characteristics of GAPs, and the fourth is to investigate the cellular functions of GAPs, by correlating the kinetics of GAP expression with different phases of axonal growth in developing and regenerating systems. The last objective is to determine how the expression of GAPs is regulated. We plan to study the effects of several likely regulatory molecules on GAP synthesis, including nerve growth factor (NGF) and cyclic AMP. The site of regulation (transcriptional, translational, of post- translational) will be determined with inhibitors of RNA and protein synthesis. Finally, specific changes in GAP messenger RNA during regeneration will be observed using a cell-free synthesis system. The significance of these studies lies in their promise to reveal some molecular mechanisms which control axonal growth. These are of paramount importance in understanding the development of the nervous system, and its response to injury.

Growth of organ primordia during post-gastrula morphogenesis, along with cell shape change and cell differentiation, leads to the form and shape of the developing tissue. Although cell replication occurs throughout the organ forming period in most tissues, its role in generating the force for forming the three dimensional shape of the tissue such as the evagination of a tube from an epithelial sheet, is not well understood. Factors that have hindered comprehension of the potential of cell division for shape generation include the previous inability to experimentally separate mitotic pressure from other shape generating processes and the lack of a system in which the process can be followed in situ. In the proposed study the role of cell replication in generating both the number and pattern of cells in the epithelium of a segment, as a stimulant for cell differentiation of specific cell types within the epithelium, and as a motive force in the shape change of the epithelium during limb bud formation will be investigated using larvae of the brine shrimp, Artemia. The simplicity of the tissue organization of these larvae makes this organism ideally suited for this study. The cells involved in the limb bud and segment forming regions are few in number, clearly defined, not surrounded by other cells, and have well timed growth periods. Analysis of the experiments will be carried out using both video-based image analysis and immunocytochemistry. The following hypotheses will be tested: 1) Cell replication occurs in a spatio-temporally directed manner that results in a set pattern of cells and regions of unequal cell density. 2) Attainment of this pattern leads to the differentiation of arthrodial membrane and tendonal cells in which microtubules are involved in cell shape change. 3) Deformation of the epithelium and evagination of the thoracopod limb bud result form unequal cell density, the presence of differentiated arthrodial membrane cells, and region-specific apolysis. 4) Maintenance of the form and shape of the limb bud involves continued patterned cell replication, new cuticle formation, and tendonal cell differentiation. The role of microtubules and microfilaments in the evagination process will also be determined. Some sections of the study will provide preliminary evidence necessary for future studies on regulation of cell replication during development of segmental structures.