CELLULAR MECHANISMS OF AXONAL REPAIR AND DEGENERATION

University Of Texas Medical Br Galveston, Galveston TX

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Abstract

DESCRIPTION (From the Applicant's Abstract): The long-term goal of this project is to identify and characterize fundamental cellular/molecular mechanisms that help repair axolemmal damage or lead to axonal degeneration. In previous years of this project on axonal repair of membrane damage, we described several previously unreported cellular processes (formation, movement, accumulation, and interactions of Ca2+ induced vesicles) that we showed are necessary for successful repair (sealing) of axolemmal lesions (transections and punctures) to prevent degeneration of invertebrate giant axons (GAs) or mammalian axons. Given our descriptions of fundamental mechanisms of axolemmal repair, we now focus on two short term objectives: 1) To obtain cellular and molecular details of processes by which increased axoplasmic [Ca2+] enables vesicles to seal a damaged axolemma in our invertebrate GA preparations whose large size enables us to obtain detailed data more easily from fluorescently-labeled probes (antibodies to membrane proteins and endogenous and/or exogenous vesicles), time-lapse confocal imaging, fluorescence resonance energy transfer, and electron microscopy. Application of these techniques to these myelinated (earthworm) and unmyelinated (crayfish, squid) GA preparations enables us to identify proteins that are essential for successful axolemmal repair and to discriminate between several proposed models of vesicle-mediated sealing. 2) To determine whether the principles and cellular/molecular mechanisms that repair unmyelinated or myelinated invertebrate GAs are conserved in the repair of injured mammalian axons using neurites in nerve-model cultures of unmyelinated rat PNS pheochromocytoma (PC12), rat CNS (B104) cells, and in unmyelinated versus myelinated rat embryonic forebrain cultures at the same stage of development. We will also determine whether degeneration of the perikaryon after neurite transection closer to, or further from, the cell body in cell cultures is attributable to a failure of plasmalemmal repair. Such cellular/molecular data on vesicle mediated axolemmal sealing in invertebrate GAs and validation of their applicability to the plasmalemmal repair of mammalian neurons are essential for development of successful strategies of axolemmal repair and for restoration of axonal functions following injury to mammalian axons.