Master of Science
Pasternak, Stephen H.
Extracellular deposition of beta-Amyloid (Aβ) is an early event in Alzheimer’s disease development. However, it is not known how Aβ is secreted. Lysosomes readily undergo calcium-dependent exocytosis, a process that relies on small GTPase Rab27b. In addition, lysosomal enzymes have been found within extracellular amyloid plaques. We hypothesized that lysosomes mediate Rab27b-dependent exocytosis of Aβ. Neuro-2a cells were transfected with wild-type or mutant Rab27b constructs and/or a lysosomal marker. Cells were incubated with Aβ monomers and imaged using a confocal microscope before and after stimulation of calcium-dependent exocytosis. We observed a significant decrease in lysosome and Aβ co-localization post-treatment in comparison to pre-treatment in control samples. We also observed a significant increase in lysosome and Aβ co-localization post-treatment in Rab27b dominant-negative mutants in comparison to control. These results demonstrate that lysosomes can mediate Rab27b-dependent exocytosis of Aβ, thus elucidating a mechanism by which Aβ could be secreted in Alzheimer’s disease.
Summary for Lay Audience
Alzheimer’s disease is the leading form of dementia, in which patients experience progressive cognitive decline. In Alzheimer’s disease, a toxic protein named beta-Amyloid is produced within and subsequently released from cells, forming deposits within the brain. It has been suggested that the production of beta-Amyloid triggers a cascade of events that results in the development of Alzheimer’s disease. However, it is not known how beta-Amyloid is released from cells.
Previous research has implicated the involvement of lysosomes in Alzheimer’s disease progression. Lysosomes are classically viewed as waste disposal compartments within the cell. Recent studies have shown that lysosomes are also capable of discharging their contents to the outside of the cell. This process has been suggested to be dependent on protein Rab27b, which brings lysosomes closer to the membrane of a cell. When lysosomes are near the cell’s membrane, an increase in calcium within the cell enables lysosomes to release their contents. In this study, it was hypothesized that beta-Amyloid is discharged from lysosomes with the assistance of protein Rab27b.
To investigate this hypothesis, cells were manipulated to express inactive, active, or overactive protein Rab27b and/or a lysosome indicator. Cells were also loaded with beta-Amyloid. In this manner, lysosomes and beta-Amyloid present within cells that were or were not manipulated to express inactive, active, or overactive Rab27b were able to be observed under a microscope. Cells were then stimulated to discharge the contents of their lysosomes by increasing the amount of calcium within these cells. Cells were again observed under a microscope. Microscope images taken before and after stimulation of discharge were compared to determine differences in beta-Amyloid release. It was observed that lysosomes can release beta-Amyloid from cells with the assistance of protein Rab27b.
By the year 2050, it is predicted that more than 130 million new cases of Alzheimer’s disease will arise worldwide. Understanding how beta-Amyloid is released from cells would facilitate the development of targeted treatments for Alzheimer’s disease, which are essential to preventing future disease progression.
Lahan, Shany, "Lysosomes Mediate Rab27b-Dependent Secretion of Beta-Amyloid" (2019). Electronic Thesis and Dissertation Repository. 6603.
Available for download on Friday, October 15, 2021