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.
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