In humans, vision is the most important sense and damage to the retina or the optic nerve can cause irreversible vision loss. This is because the retina and the optic nerve are part of the central nervous system (CNS), which in adult mammals has lost its regenerative capacity. In rodents, several neuron intrinsic signaling pathways have now been identified that majorly boost axonal growth of injured retinal ganglion cells (RGC) axons, yet this has come with the realization that enhanced axonal regrowth frequently results in extensive misguidance, detrimental to function regeneration. Currently, the identity of extrinsic guidance cues, the mechanisms by which they direct regenerating RGC axons, and the identity of glia and other cell types along the optic nerve path that provide guidance are not well understood. Surprisingly, even the cellular behaviors of resident glial cells and immune cells summoned to the injury site, and how they interact with regenerating RGC axons is not well understood, mainly due to challenges of live cell imaging in mammals. In contrast to mammals, amphibians and fish, including zebrafish, have retained a remarkable capacity for optic nerve regeneration. We have established a powerful assay to transect the optic nerve in larval zebrafish, and monitor axonal and functional regeneration. RGC axons regenerate within a few days independently of neurogenesis, providing a unique opportunity to study the genes critical for spontaneous regeneration independently of the confound of neural survival and neurogenesis. From a genetic screen we identified mutants in two genes, the glycosyltransferase lh3 and one of its substrate col18a1 critical for the guidance of injured RGC axons. Our preliminary data support a hypothesis by which lh3 and col18a1 participate in a pathway to provide extrinsic guidance ?likely by surrounding glia- to guide regenerating RGC axons towards the CNS midline. The goal of this proposal are to define fundamental behaviors of regenerating axons, glia and immune cells in their native environment, and to determine the cellular and molecular mechanism by which lh3 and Col18a1 guide regenerating optic nerve axons. The experiments in this proposal will: (1) reveal and define for the first time in any vertebrate system the fundamental behaviors of regenerating optic nerve axons, glia and immune cells in their native environment; (2) determine the cellular and molecular mechanisms by lh3 and col18a1 direct optic nerve regeneration; and (3) determine how col18a1 function connects to axonal guidance of RGC axons. These studies are relevant to the study of human diseases that cause damage to the optic nerve, including hereditary optic neuropathies and glaucoma. Although spontaneous optic nerve regeneration is largely absent in mammals, boosting axonal regeneration via neuron intrinsic manipulation frequently results in misguidance, underscoring the importance to define the cellular interplay of injured RGC axons with surrounding glia and to decipher the molecular mechanism underlying regenerative guidance. Finally, the expected results will form a powerful foundation to formulate specific hypotheses of optic nerve regeneration across the board.
The mammalian CNS has minimal capacity for regeneration, and injury or disease induced damage to the human optic nerve leads to irreversible loss of vision. This proposal takes advantage of the remarkable CNS regenerative capacity, exquisite live cell imaging and genetic tools of the zebrafish model to quantify for the first time the cellular interactions between individual optic nerve axons and surrounding glia during the process of regeneration, and to determine the mechanisms by which two newly identified pro regenerative factors enable damaged optic nerve axons to regenerate. Thus, by characterizing fundamental cellular and molecular mechanisms that promote optic nerve regeneration, the proposal is highly relevant to restoring human vision.
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