Defining the Local Landscape of Retinal Ganglion Cell Axons

The vertebrate visual system involves a series of complex steps which cover a number ofdiverse anatomical structures. Light first enters the eye and is projected onto the retina, a thin layer of tissue that lines the posterior of the eye and is comprised of millions of specialized neurons. Rods and cones initiate the process light signaling upon absorption of photons, with stimulation of retinal ganglion cells (RGCs) representing the final step of intraretinal signaling. RGC somas localize to the innermost layers of the retina, while their axons converge at the optic nerve head and bundle together to form the optic nerve carrying visual information to the brain. The RGC structure is unique in that the soma and dendrites are compartmentalized inside of the eye whereas the RGC axons exist primarily outside of the eye as they project via the optic nerve to synaptic targets in the CNS. In the proximal region of the optic nerve is a short, unmyelinated region of axons running between an astrocytic meshwork termed the glial lamina (GL). Seminal studies have shown that mitochondria are particularly abundant in the GL compared to other compartments of RGCs, potentially suggesting a particularly high demand for ATP production in this region. Of relevance, the GL is the first region of axonal degeneration in glaucoma, suggesting an inherent susceptibility of this axonal compartment to cellular stress. The focus of this dissertation centers on elucidating the local landscape and regulation of the GL in healthy mice and disease models. Our earliest work is a comprehensive analysis of the enrichment of mitochondria in the GL in wild type mice, improving upon the low-resolution histological studies on which this concept was based. Using complementary immunofluorescence and electron microscopy techniques, we confirm that mitochondria are more abundant in the GL compared to the retrolaminar (RL) optic nerve, but show that the overall mitochondrial accumulation arises only because of differential mitochondrial abundance in the largest diameter RGC axons. We also show that the mitochondrial accumulation is established by postnatal day 6- v 9, preceding the onset of axonal myelination. Therefore, the enrichment of mitochondria in the GL is not a direct consequence of the unique absence of myelin in this region. The remainder of our work is an exploration of differences between the GL and RL in mitochondrial and axonal morphology as well as in proteomic signatures. In preliminary studies, we have observed distinct mitochondrial morphologies between the two compartments, with small differences in mitochondrial length and cristae structure. We also have found preliminary evidence of unprecedented inter-axonal fusion events between RGC axons of the GL, a phenomenon we term short axonal merging sites (SAMS). SAMS are characterized as two individual axons that run parallel and exhibit focal breakdown of their plasma membranes with apparent fusion between the two. While additional experiments are required to eliminate the possibility that SAMS are non-physiological artifacts of tissue fixation, should we confirm their presence it would open a new direction exploring the functional significance of the fusion events in light signaling to the central nervous system and in the propagation of pathology in optic nerve disorders. Finally, we describe our early efforts proving the feasibility of characterizing the compartment-specific proteomes of optic nerve tissue and of RGC mitochondria specifically. We also describe preliminary studies designed to compare the GL proteomes of DBA/2J mice with early glaucoma to control non-glaucomatous littermates. Completion of this analysis may identify key biological differences between the GL and RL and highlight cellular processes that may subject the GL to early axonal degeneration in optic neuropathies like glaucoma. It is our hope that this work will help to identify pathways that may be targeted pharmacologically to combat RGC neurodegeneration in glaucoma and other blinding diseases.