Date of Award

January 2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Jonathan D. Geiger

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized clinically as progressive decline in cognition and dementia. Pathologically, AD is characterized by the extracellular presence of senile plaques composed of accumulated amyloid beta (Aβ) proteins and intraneuronal accumulation of neurofibrillary tangles composed of hyperphosphorylated tau proteins. In addition to these pathological hallmarks, AD pathogenesis is characterized by increases in intracellular iron and reactive oxygen species (ROS) and the link between the two have been well described by Fenton’s chemistry. Endolysosomes appear to be affected very early in the onset of AD. Endolysosomes contain labile stores of iron which can be readily released by endolysosome-targeting insults including HIV proteins and weak base drugs; events which lead to endolysosome damage and cell death. The role of endolysosome iron in AD-pathology is not well understood. Therefore, we hypothesized that endolysosome iron plays an important role in Aβ-induced endolysosome damage and cell death. In Chapter I, we reviewed literature about how endolysosomes are involved in Aβ production, Aβ degradation, cellular iron metabolism, autophagy, and their potential as therapeutic targets in the development of drugs against AD.

In Chapter II, we investigated the role of endolysosome iron in Aβ protein-induced increases in reactive oxygen species, mitochondrial depolarization, and regulated cell death. We found that Aβ is uptaken into endolysosomes where they deacidified endolysosomes, decreased endolysosome ferrous iron (Fe2+) levels, increased cytosolic iron and ROS levels, increased mitochondrial iron and ROS levels, and increased cell death. These effects were blocked by chelating the labile Fe2+ stores in endolysosomes with deferoxamine (DFO), suggesting that endolysosome iron may play a critical role in Aβ-induced endolysosome damage and cell death.

In Chapter III, we investigated mechanism(s) by which Aβ proteins cause ferroptosis as well as mechanisms by which DFO rescues Aβ-induced ferroptosis. In SH-SY5Y human neuroblastoma cells, we found that Aβ increased lipid peroxidation and caused decreases in levels of the anti-ferroptotic enzyme SLC7A11. These events increased cell death, which was blocked by a well-known inhibitor of ferroptosis, ferrostatin-1. We also found that DFO blocked Aβ-induced increases in lipid peroxidation as well as blocked Aβ-induced decreases in SLC7A11.

In Chapter IV we investigated the uptake of Aβ proteins into endolysosomes via low-density lipoprotein receptor-related protein-1 (LRP-1)-mediated endocytosis. In endolysosomes, Aβ degradation is catalyzed by Aβ-degrading enzymes such as cathepsins B and D. Previous studies have shown that in AD-like pathology, LRP-1 is negatively affected and the low levels of LRP-1 suggests that Aβ uptake and degradation is negatively affected. However, the mechanisms that drive Aβ-induced negative regulation of LRP-1 are not known. We investigated mechanisms by which Aβ proteins contributed to decreases in levels of LRP-1 expression in AD-like pathology and we found that Aβ-induced increases in levels of intracellular iron and ROS contributed to decreases in LRP-1 protein levels. Similarly, intracellular iron and ROS overload from ferrous ammonium citrate (FAC) and H2O2 respectively, also decreased levels of LRP-1 protein. Taken together, our findings show that Aβ-induced iron overload and ROS are major contributors to decreased LRP-1 protein levels in AD-like pathology. It remains to be determined how iron surplus specifically decrease LRP-1 protein levels.

In Chapter V we investigated the role of endolysosome iron in available options for treatment against AD. Our previous studies have shown that weak base drugs that get trapped in endolysosomes induce endolysosome damage and cause neurotoxicity. Also, we have previously shown that beta site Aβ precursor protein cleaving enzyme -1 (BACE-1) which is the rate-limiting enzyme that catalyzes the production of Aβ resides in endolysosomes. Though this class of drugs reduce Aβ burden, they have been discontinued due to neurotoxicity concerns. Therefore we sought to explain mechanism(s) by which BACE-1 inhibitor drugs cause neurotoxicity. We examined BACE-1 inhibitor drug-induced effects on endolysosome iron levels, intraorganellar iron and ROS levels, and mitochondrial membrane polarization. We found that BACE-1 inhibitor drugs but not non-BACE-1 anti-AD drugs decreased endolysosome Fe2+ levels, increased intracellular Fe2+ levels as well as ROS, and depolarized mitochondrial membrane potentials. All of these effects were blocked by chelating endolysosome iron with DFO. Thus, neurotoxicity associated with BACE-1 inhibitor drugs appears to be associated with perturbation of endolysosome iron, and the labile iron pool in endolysosomes contribute to cellular iron metabolism, Aβ uptake and degradation, and the development of therapeutic drugs against AD.

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