ORCID
https://orcid.org/0000-0002-2181-923X
Date of Award
12-19-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Retinal Ganglion Cell (RGC) death is the final outcome in optic neuropathies like glaucoma and traumatic optic nerve injury, which represent the leading cause of irreversible blindness worldwide. There are currently no treatments for these diseases targeting RGCs directly, and mechanisms underlying RGC death are unclear. Calcium ion (Ca2+) dysregulation occurs in almost all neurodegenerative conditions, including glaucoma, which has been studied in rodents using a robust injury model of traumatic axon injury, optic nerve crush (ONC). However, the impact of homeostatic Ca2+ regulation in RGCs response to glaucoma or ONC is unknown. Furthermore, while some mechanisms that regulate Ca2+ within and between intracellular sources have been elucidated, their role in RGC degeneration has not been investigated. This dissertation aims to understand how intracellular Ca2+ levels within the cytoplasm (Chapter 1) and mitochondria (Chapter 2) influence RGC survival to ONC axon injury. We utilized an intersectional viral and genetic approach to express a homeostatic Ca2+ biosensor in mouse RGCs and observe many individual RGCs at single-cell resolution using in-vivo two-photon (2p) trans-pupillary imaging. We observed RGCs acutely after ONC to assess the role of Ca2+ as an acute injury signal for axon injury, and surprisingly did not identify any Ca2+ dynamics in RGC axons or cell bodies, neither at 1-2 minutes immediately after ONC nor up to two hours after. Rather, RGCs had differential homeostatic Ca2+ levels at baseline that were stable over time. We performed longitudinal 2p imaging for up to two weeks after ONC, imaging the same RGCs every two days, to track and determine Ca2+ related survival characteristics of individual RGCs. We compared baseline Ca2+ levels with RGC survivability and identified a strong association of high baseline intracellular Ca2+ levels with resilience. When comparing surviving and dying RGCs 14 days post ONC, surviving cells have significantly higher baseline Ca2+ levels than those that died. Additionally, cells that retained high baseline Ca2+ levels survived extremely well compared to always low Ca2+ cells or ‘dynamic’ cells, i.e. RGCs with large day to day fluctuations in Ca2+ levels. Additionally, well-surviving RGC subtypes had high baseline Ca2+ levels compared to the general population, and within those subtypes high Ca2+ RGCs survived best. We performed injections of a Ca2+ chelating compound and found a strong reduction of intracellular Ca2+ levels. Longitudinal injections after ONC reduced RGC survival, especially in previously well-surviving ‘high baseline calcium’ RGCs. Together these experiments revealed a previously unknown heterogeneity in homeostatic Ca2+ levels that is associated with RGC resilience to axon injury. Questions remained regarding the source of these Ca2+ differentials and the mechanisms underlying the survival phenotype. Mito-Ca2+ dysregulation occurs in neurodegenerative responses, yet mechanisms for mito-Ca2+ regulation and their influence in RGC degeneration is unknown. We investigated intra-mitochondrial calcium (mito-Ca2+) levels by expressing the same Ca2+ biosensor, Twitch2b, with a localization signal targeting mitochondria to assess mito-Ca2+ levels in-vivo. We identified a baseline Ca2+ differential across the RGC population of individual retinas, like cytosolic Ca2+. We also found a relationship to baseline mito-Ca2+ levels and survival, where high baseline mito-Ca2+ cells survived well, but not to the same extent as high cytosolic Ca2+ RGCs. We targeted a specific mechanism that regulates Ca2+ entry into the inner mitochondrial matrix, the Mitochondrial Calcium Uniporter (MCU) complex, to investigate how we could affect mito-Ca2+ levels and RGC degenerative responses to ONC. We used genetic and pharmacologic tools to alter MCU and assess RGC survival following ONC. When overexpressing the pore forming MCU subunit to increase mito-Ca2+ by allowing more passive transport of Ca2+ into the mitochondria, RGCs had decreased survival to ONC. Conversely, when either using shRNA knockdown of MCU or pharmacological injection of drugs to block the MCU complex, RGCs had increased survival following ONC. These results suggest that mito-Ca2+ is inversely related to cytoplasmic Ca2+ in their relationship to RGC survival to ONC axon injury; higher baseline mito-Ca2+ and less-regulated mito-Ca2+ entry is detrimental to RGC, whereas reduced mito-Ca2+ influx is protective. Together, these data provide a mechanistic relationship between intracellular Ca2+ regulation, especially within the mitochondria, and RGC survival to axon injury.
Language
English (en)
Chair and Committee
Philip Williams
Committee Members
Daniel Kerschensteiner; Edward Han; Ghazal Ashrafi; Terrance Kummer
Recommended Citation
McCracken, Sean, "Cytoplasmic and Mitochondrial Calcium Influences Retinal Ganglion Cell Survival to Axon Injury" (2024). Arts & Sciences Electronic Theses and Dissertations. 3364.
https://openscholarship.wustl.edu/art_sci_etds/3364