Compartmental Differences in the Retinal Ganglion Cell Mitochondrial Proteome

Abstract

Retinal ganglion cells (RGCs) are projection neurons of the retina that are a nexus for integrating light signals originating from retinal photoreceptors and transmitting them to the visual processing centers of the brain. RGCs have a highly polarized morphology broadly divided into somatodendritic and axonal compartments. The drastically dissimilar structures and functions of these compartments implies that they face different bioenergetic and other physiological demands. As RGCs are known to be uniquely sensitive to dysfunction of mitochondria, it is believed that these organelles are key to maintaining RGC homeostasis and survival. Differences in mitochondrial biology are likely to be tailored to the specific physiological needs of each RGC compartment.This dissertation focuses on identifying fundamental differences between RGC mitochondria in the somatodendritic and axonal compartments. Compartment-specific functional disparities between mitochondria may not only highlight unique physiological demands inherent to these compartments but also play a pathophysiological role in the development of RGC dysfunction and death in various optic neuropathies. We hypothesized that compartmental differences in mitochondrial biology would be reflected by dissimilarities in mitochondrial protein composition and therefore be amenable to interrogation using proteomics. We describe an optimized protocol to isolate intact mitochondria separately from mouse RGC somatodendritic and axonal compartments by immunoprecipitating labeled mitochondria from novel RGC MitoTag mice. These genetically modified mice express a cytosol-facing GFP tag specifically on RGC mitochondria, allowing for GFP-based immunoprecipitation of mitochondria from the RGC somatodendritic compartment in the retina and from the axonal compartment in the optic nerve. Using liquid chromatography-mass spectrometry techniques, we identified several hundred proteins in the RGC somatodendritic and axonal mitochondrial immunoprecipitates, including a number of proteins highly enriched or exclusively identified in either compartment. To validate these findings, we further analyzed three mitochondrial proteins with distinct compartmental enrichment profiles: superoxide dismutase (SOD2), enriched in the RGC somatodendritic compartment; sideroflexin-3 (SFXN3), expressed equally in both compartments; and trifunctional enzyme subunit α (HADHA), enriched in the RGC axonal compartment. The expression and localization profiles of these proteins within RGCs were assessed using immunofluorescence techniques and compared to the protein abundance data obtained in the proteomics analysis. We subsequently explored RGC axon-specific metabolic programs after the identification of several enzymes involved in long-chain fatty acid catabolism as being enriched RGC axonal mitochondria. Metabolite profiling of mitochondria obtained in a compartment-specific manner from RGC MitoTag mice revealed an abundance of long-chain fatty acylcarnitine molecules primed for fatty acid oxidation in axonal mitochondria. Building from this finding, we performed RGC-specific genetic ablation of carnitine palmitoyltransferase 1, thereby depriving RGC mitochondria of the ability to import fatty acylcarnitine molecules to be used as metabolic substrates for the generation of cellular ATP. When assessing RGC abundance in the setting of chronic impairment of mitochondrial fatty acid import, there appeared to be a subtle trend towards decreased RGC survival in the mutant mice. Our findings provide clues to compartment-specific distinctions in the roles of RGC mitochondria. While compartmentalized energy gradients have been previously identified in other neuronal populations, our work represents the first to pursue metabolic differences within a type of retinal neuron. Exploring how several optic nerve disease states impact mitochondrial proteomic differences in RGCs is the subject of ongoing work in the lab, with preliminary results described here.