Peter Halcrow

Date of Award

January 2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Jonathan D. Geiger

Abstract

Endosomes and lysosomes (endolysosomes) are acidic organelles that are important both physiologically and pathologically. Implicated in the physiological and pathophysiological processes regulated by endolysosomes are readily releasable stores of cations including ferrous iron (Fe2+); an essential cofactor for the generation of reactive oxygen species (ROS). In determining the extent to which and mechanisms by which Fe2+ released from endolysosomes affects cellular functions it was important to determine levels of Fe2+ in endolysosomes. In some cells and by other researchers, FeRhoNox-1 was found to detect Fe2+ in acidic organelles known as Golgi. Here, using U87MG astrocytoma cells and primary cultures of rat neurons we report that FeRhoNox-1 is highly specific for Fe2+, that FeRhoNox-1 positive stores are largely localized in endolysosomes and not in Golgi, that control levels of Fe2+ were 36.3 ± 13.6 µM in endolysosomes, and that the stores of Fe2+ in endolysosomes increased to 75 ± 15.7 µM when cells were incubated with ferric ammonium citrate and decreased to 0.08 ± 0.05 µM when cells were incubated with the iron chelator deferoxamine. Furthermore, subpopulations of endolysosomes exist with extensive variability in Fe2+ content. Our findings demonstrate the utility of using FeRhoNox-1 to measure Fe2+ stores in endolysosomes and suggest that this probe will find important uses in better understanding cellular events downstream of released endolysosome Fe2+.

Mitochondria are subject to iron overload under a variety of conditions and disease states, but it is not clear what are the subcellular origins of this iron nor its consequences. Endolysosomes are storage sites of ferrous iron (Fe2+), and the degree to which and the precise mechanisms by which endolysosome Fe2+ contribute to iron-dependent changes to mitochondria and cell injury remains uncertain. Here, our studies were aimed to determine the role of inter-organellar signaling of Fe2+ from iron-rich endolysosomes to mitochondria under pharmacologically-induced conditions. We demonstrated, in U87MG astrocytoma cells, mouse primary hepatocytes, and primary cultures of rat cortical neurons, that Fe2+ within endolysosomes was translocated to the mitochondria resulting in mitochondrial dysfunction and cell death. The weak-base chloroquine and the vacuolar-ATPase inhibitor bafilomycin A1 both de-acidify endolysosomes and both induced the release of Fe2+ and this resulted in increased concentrations of Fe2+ in the cytoplasm and in mitochondria. Furthermore, the endocytosed iron chelator, deferoxamine, inhibited the release of bafilomycin A1- and chloroquine-induced release of endolysosome stores of Fe2+ and prevented the induced increases of ROS in cytoplasm and mitochondria. These findings demonstrate that redox-active Fe2+ in endolysosomes plays a key upstream role in mitochondrial iron accumulation and dysfunction, and deferoxamine might be potential adjunctive therapeutic strategies in preventing neurotoxicity and enhancing therapeutic outcomes of disease.

Drugs of abuse including the opioid morphine increase levels of reactive oxygen species (ROS) and predispose cells to insult-induced cell death. However, it remains uncertain as to the underlying mechanisms. Iron has long been known to be required for the generation of mitochondrial ROS and endolysosomes are major storage sites of ferrous iron (Fe2+). Yet, the degree to which and the precise mechanisms by which endolysosome iron plays a role in mitochondrial dysfunction remains uncertain. Here, our studies were aimed to determine the effects of morphine on inter-organellar signaling of Fe2+ from iron-rich endolysosomes to mitochondria. We demonstrated, in U87MG astrocytoma cells, that endolysosome Fe2+ is translocated to mitochondria and results in mitochondrial dysfunction. Morphine de-acidification of endolysosomes caused the release of Fe2+ from endolysosomes and increased levels of Fe2+ in cytosol and in. The morphine-induced effects on endolysosome Fe2+ appeared to be regulated through mu opioid receptors because naloxone blocked the de-acidification of endolysosomes by morphine and the release of endolysosome iron. Furthermore, the endocytosed iron chelator, deferoxamine, inhibited the release of redox-active Fe2+ into the cytosol and the morphine-induced increases in mitochondrial ROS. These findings demonstrate that redox-active Fe2+ in endolysosomes plays a key upstream role in mitochondrial dysfunction, and deferoxamine might be a potentially useful therapeutic strategy associated with opioid use disorders.

HIV-associated neurocognitive disorder (HAND) affects 50% of people living with HIV-1 despite viral suppression achieved by antiretroviral therapies. Pathologically, brain tissue from HAND patients has shown morphological changes to intracellular organelles including endolysosomes and mitochondria. Moreover, people living with HIV-1 show elevated iron serum levels and iron chelators have been suggested as an adjuvant therapy to antiretroviral therapeutics. Mechanistically, soluble factors including the HIV-1 coat protein gp120 have been implicated in HAND pathogenesis. Here, we tested the hypothesis that HIV-1 gp120-induced de-acidification of endolysosomes leads to an efflux of iron from endolysosomes and a subsequent increase in levels of cytosolic and mitochondrial reactive oxygen species (ROS). We used U87MG glioblastoma cells and time-lapse confocal microscopy to measure gp120-induced changes in endolysosome pH, endolysosome iron, cytosolic and mitochondrial iron, and ROS levels. HIV-1 gp120 de-acidified endolysosomes, reduced endolysosome iron levels, increased levels of cytosolic and mitochondrial iron, and increased levels of cytosolic and mitochondrial ROS. These effects were all attenuated significantly by the iron chelator deferoxamine that only enters cells via endocytosis. These results suggest that cellular and subcellular effects of HIV-1 gp120 can be downstream of its ability to de-acidify endolysosomes and increase the release of iron from endolysosomes. Thus, endolysosomes might represent an early and upstream target for therapeutic strategies against HAND.

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