Publication Date

2015-11-17

Availability

Embargoed

Embargo Period

2016-11-16

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Accounting (Business)

Date of Defense

2015-10-29

First Committee Member

Kevin K. Park

Second Committee Member

Sanjoy Bhattacharya

Third Committee Member

Rong Wen

Fourth Committee Member

Miguel Perez-Pinzon

Abstract

Neuroregeneration research seeks to address why mature neurons of the central nervous system (CNS) fail to re-extend a severed axon. However, equally important is understanding what happens after axons are induced to regenerate through the environment of the injured nervous system. In optic neuropathies like glaucoma, retinal ganglion cell (RGC) axon degeneration and cell death result in loss of visual function and blindness. Treatment strategies have been developed that promote long distance regeneration of RGC axons but are largely dependent on genetically modified knockout (KO) mice. Given the expanded use of RNA interference (RNAi)-based treatments in human patients, therapeutically relevant non-transgenic strategies for reinnervating targets have important implications in clinical and research settings and may also inspire treatments for brain and spinal cord injuries. In this dissertation I evaluate the efficacy of a novel treatment paradigm in an optic nerve crush injury model by combining viral, RNAi, and pharmacological approaches. These target both the phosphatase and tensin homolog (PTEN) and signal transducer and activator of transcription-3 (STAT3) pathways to allow axons to travel long distances in wild-type mice. In fact, I find regenerate RGC axons innervating a proximal visual target in the brain, the suprachiasmatic nucleus (SCN). Using this treatment strategy, I was able to explore several crucial challenges to visual restoration. Since the targeting of regenerate RGC axons must be sufficient to reconnect with proper areas in the brain, I provide a novel analysis of patterns of axon pathfinding through the visual pathway in unsectioned, cleared nervous tissue. Three-dimensional visualization of single regenerate axons allow us to document heterogeneous terminal patterns in the SCN, including examples of extensive arborization. Next, I find evidence of optic synapse formation in the SCN, although this is not accompanied by integration of photic input or restoration of SCN-mediated circadian function as measured by running-wheel activity. Lastly, I induce optic nerve regeneration in reporter mouse lines that label a melanopsin-expressing RGC subset that is intrinsically photosensitive (ipRGCs). I demonstrate these cells not only are able to regenerate after viral treatment but also comprise a disproportionately high percentage of regenerating axons. Collectively, I introduce a non-transgenic approach to induce long distance RGC axon regeneration, examine the cells of origin of regrown axons, and characterize RGC axons’ ability to target brain targets, reform synapses, and restore a visual function. Experimental findings that directly resulted from this approach contribute to this field’s understanding of both the behavior and the identity of adult regenerating RGC axons. Future research that reveals obstacles in these and other contexts will be critical for restoring vision.

Keywords

axon regeneration; retinal ganglion cells; circadian; melanopsin; gene therapy; viral vector

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