Investigating the Role of the Mitochondrial Protein Armcx1

Abstract

This dissertation describes my efforts to explore the functions of the poorly-characterized mitochondrial protein Armcx1. Armcx1 has previously been shown increase neurite outgrowth when overexpressed in cortical neurons, while this extension was diminished when Armcx1 was knocked down. Fascinatingly, adeno-associate virus-mediated overexpression of Armcx1 in retinal ganglion cells (RGCs) in live mice was shown to increase RGC survival and promote regeneration of their axons following optic nerve crush. This motivated me to pursue a project aimed at investigating the endogenous functions Armcx1, with a particular emphasis on RGCs.My initial investigations focused on examining the mitochondrial localization of Armcx1 in cultured cells using confocal microscopy. I observed a dual localization pattern for Armcx1: some cells exhibited uniform distribution along the outer mitochondrial membrane, while others demonstrated a punctate distribution on mitochondria. The punctate pattern was somewhat reminiscent of that of the mitochondrial fission protein Drp1, yet we observed incomplete co-localization between Armcx1 and Drp1 puncta. Live cell imaging also led to variable observations regarding whether Armcx1 puncta were present at the sites of active mitochondrial fission. The significance of the variable localization pattern of Armcx1 thus remains uncertain and will require additional investigation. To investigate the functions of endogenous Armcx1 in RGCs, I generated genetically modified mouse strains with either global deletion or the capacity for conditional deletion of Armcx1. Using a Cre-lox approach, I demonstrated the ability to achieve cell-specific deletion of Armcx1 from retinal neurons. I then assessed whether the complete loss of Armcx1 was detrimental to RGC survival by aging global Armcx1 knockout mice to 15 months. However, in the absence of any other cellular stressors, I found no evidence of reduced RGC survival or axonopathy in the aged mice lacking Armcx1. I also explored whether loss of Armcx1 might sensitize RGCs to pathology other than optic nerve crush by crossing the Armcx1 knockout line with a mouse model of severe mitochondrial complex I deficiency. However, I did not observe any hastening of RGC degeneration in this context. Overall, these results may indicate that any neuroprotective role of Armcx1 may be limited to the setting of axonal trauma, or that there exists functional redundancy among Armcx family proteins. Finally, I investigated the function of Armcx1 in the in vivo projection of RGC axons during embryonic development. By using a genetically modified mouse strain in which RGC axons are labeled with a membrane-bound GFP construct, I observed the progress of axon projection toward the central nervous system in mice at embryonic days 13 and 14. I tested the hypothesis that deleting Armcx1 would result in delay of the convergence of RGC axons from each eye at the optic chiasm. However, my analysis revealed that RGC axons from Armcx1 knockout mice achieved age-appropriate lengths at both of these developmental time points. This result again highlights the need to asses for functional redundancy with other Armcx proteins and for potential compensatory mechanisms involving other mitochondrial motility proteins. Overall, this research resulted in intriguing observations regarding variability in the sub-mitochondrial localization of Armcx1 and led to the development of a useful animal model for evaluating the functions of endogenous Armcx1 in mitochondrial biology. The absence of a pronounced phenotype following global Armcx1 deletion underscores the potential for functional redundancy, which will need to be considered in designing future studies.