Date of Award
Doctor of Philosophy (PhD)
Jonathan D. Geiger
Endosomes and lysosomes (endolysosomes) are organelles that play key roles in cell survival and cell death. These acidic intracellular organelles are the principal sites for intracellular hydrolytic activity required for the maintenance of cellular homeostasis. Endolysosomes are involved in the degradation of plasma membrane components, extracellular macromolecules, as well as intracellular macromolecules and cellular fragments. Understanding the physiological significance and pathological relevance of endolysosomes is now complicated by relatively recent findings of physical and functional interactions between endolysosomes and other intracellular organelles including endoplasmic reticulum, mitochondria, plasma membranes, and peroxisomes. Indeed, evidence clearly indicates that endolysosome dysfunction and inter-organellar signaling occurs in different neurodegenerative diseases including HIV-1 associated neurocognitive disease (HAND), Parkinson’s disease (PD), and Alzheimer’s disease (AD), as well as various forms of brain cancer such as glioblastoma multiforme (GBM). These findings open new areas of cell biology research focusing on understanding the physiological actions and pathophysiological consequences of inter-organellar communication. Although others and we have studied the role of endolysosomes and inter-organellar signaling in different brain diseases including HAND, PD, AD, and GBM, here, we focused on AD disease which is the most common cause of dementia. Indeed, endolysosomes are the organelles suggested to participate in early and upstream pathological signaling events that occur in the development of AD pathology. In the two studies presented here we focused on the role played by aurora kinase A (AURKA) and Rab4a proteins in the development of AD-like pathology. Alzheimer’s disease is the leading cause of dementia worldwide in people older than 65 years of age. There is currently no cure for AD and mechanisms underlying the pathogenesis of the disease are still under investigation. The pathogenesis of AD has been linked with increased levels of Aβ, p-tau, and reactive oxygen species using cellular and animal models; subcellular organelles including endolysosomes, mitochondria, and endoplasmic reticulum play key roles in AD pathogenesis. Endolysosomes are single membrane acidic compartments that contain over 60 different types of acid hydrolase enzymes. The low luminal pH of endolysosomes is an important hallmark of these organelles, which is critical for the optimum activity of pH-sensitive hydrolytic enzymes in the lumen of endolysosomes. Endolysosome dysfunction has been associated with accelerated tauopathy, amyloidogenesis, and neuritic dystrophy in the development of AD, whereas endolysosome acidification decreases amyloidogenesis and levels of p-tau. Therefore, acidifying endolysosomes may be a potential therapeutic strategy against AD pathogenesis. Based on findings that AURKA affects the activity of v-ATPase, a proton pump that plays a key role in maintaining the acidic pH of endolysosomes, and findings on decreased AURKA activity in postmortem human brain tissues, here our studies were aimed to determine the extent to which and mechanisms by which AURKA regulates endolysosome pH and amyloidogenesis. We demonstrated the presence of AURKA in primary cultured rat cortical neurons, in brains of C57BL/6J mice, and in postmortem human AD brain. AURKA phosphorylation, which correlates directly with the activity of AURKA, was decreased in human AD brain and in hippocampus of 3xTg-AD mouse brain. Using dual-excitation ratiometric measurement of endolysosome pH in neurons, we found that activation of AURKA with anacardic acid acidified endolysosomes. In SH-SY5Y cells and using pH-sensitive LysoBriteTM Red we also demonstrated that AURKA activation increased endolysosome acidification, whereas AURKA inhibition with MLN8237 de-acidified endolysosomes. AURKA activation decreased levels of Aβ, decreased the activity of BACE-1, the rate-limiting enzyme for Aβ generation, and increased the activity of cathepsin D, a lysosomal protease involved in Aβ degradation. Conversely, AURKA inhibition increased levels of Aβ and decreased the activity of cathepsin D. These findings suggest that AURKA is involved in the development of AD pathology through endolysosome-mediated actions. Our findings provide new insight into the pathogenesis of AD and highlight endolysosomes as a potential upstream therapeutic target for Alzheimer’s disease. Besides endolysosome dysfunction, endolysosome trafficking is also impaired in AD. The Rab family of small GTPases plays a critical role in regulating intracellular membrane trafficking. Early endosome enlargement is associated with upregulation of Rab4 in AD brain. However, how Rab4a dysregulation contributes to the development of AD pathology has yet to be determined. Here, we tested the hypothesis that Rab4a through endolysosome-mediated actions contributed to the development of AD-like pathology. To study the role played by Rab4a in amyloidogenesis, we generated a stable cell of SH-SY5Y cells overexpressing wild-type AβPP in which Rab4a was knocked down using shRNA lentiviral particles. We demonstrated that knocking down Rab4a expression resulted in decreased protein levels of AβPP. Moreover, we found a decrease in intracellular and secreted levels of Aβ1-40 and Aβ1-42 in Rab4a KD SH-SY5Y cells. We also found that endolysosome acidification was increased in Rab4a KD SH-SY5Y cells compared to controls. Moreover, we demonstrated that knocking down Rab4a expression resulted in decreased protein levels and activity of BACE-1, the rate-limiting Aβ generating enzyme, and increased the activity of the Aβ degrading enzyme, cathepsin D. These findings suggest that Rab4a plays a key role in the development of AD-like pathology. These studies provide new insights into understanding the mechanisms involved in the development of AD pathology and may provide us with new therapeutic strategies against AD.
Afghah, Zahra, "Role Of Aurora Kinase A, Rab4a, And Endolysosomes In The Development Of Alzheimer’s Disease-Like Pathology" (2022). Theses and Dissertations. 4247.
Available for download on Friday, June 14, 2024