EFFECT OF MONOMERIC AMYLOID-β ON CELL SECRETED EXOSOMES Discerning the pathology of Alzheimer’s disease
Typ
Examensarbete för masterexamen
Master Thesis
Master Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2019
Författare
Merchant, Nandan Amol
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Amyloid-β is a significant protein associated with the pathogenesis of Alzheimer’s disease. Dysregulation and overproduction of the protein after cleavage from the transmembrane Amyloid Precursor Protein (APP) by β and γ secretases, will lead to its aggregation into amyloid fibrils and subsequent accumulation into senile plaques. The development and progression of plaque pathology across the brain is gradual and Alzheimer’s patients will progressively show marked cognitive disabilities, decreased social abilities, dementia and reduced life expectancy. In healthy individuals, there are several clearance mechanisms that control the levels of Aβ42 in the body and prevent its aggregation. They include several intracellular processes that maintain cellular homeostasis via autophagy systems and microglial intervention. Exosomes are a class of cell secreted nanovesicles that are involved in many cell signaling processes as well as spread of neurological disorders such as Parkinson’s disease, ALS and Alzheimer’s disease. They are formed through exocytosis of intraluminal vesicles of multivesicular bodies, a trafficking organelle in the cell. Exosomes trap many proteins for cell signaling, transport, and intercellular communication. There is literature that describes the spread of Alzheimer’s disease through the formation of misfolded and aggregated forms of Aβ42, including their association with exosomes, which could mediate release into the extracellular environment as well as propagation to neighboring cells. But nothing is known about how Aβ42 peptides affect the exosome population that is secreted from a neuronal cell. Can Aβ accumulations in neurons alter exosome numbers, and alter exosome size distributions, which in turn could lead to release of more Aβ? How does Aβ peptides interact with the exosome? The aim of my thesis is to explore the effect of Aβ peptides on the exosome population secreted from a human neuroblastoma (SH-SY5Y) cell model. To achieve this, I developed small volume protocols to treat SHSY5Y cells with Aβ monomers and to isolate extracellular vesicles from harvested media. The extracellular vesicles were analysed for exosome markers and Aβ content using Western blot, their population size and numbers were determined by nanoparticle tracking analysis (NTA), and thioflavinT (ThT) aggregation assays were used to monitor the ability of exosomes to catalyze the in vitro aggregation of Aβ monomers. Further, using a modified SH-SY5Y cell line, expressing a GFP-tagged version of the exosome marker CD63, I used time lapse confocal microscopy to analyse the in-situ colocalization of Aβ with exosomes. Also, to scale down the experiments and analyze the exosome particles released in real time, we are developing a micro/nanofluidic device that will contain a live culture of SHSY5Y cells to analyze exosomes released by them through nanochannels built in it. Through these experiments, I found that exosomes secreted from cells that had been treated with monomeric Aβ42 had higher concentration and also increased mean size (diameter) compared to untreated control (mock). The exosomes slowed down the aggregation of Aβ42 into fibrils, primarily by extending the lag phase of the aggregation reaction. Altogether, these results suggest that exosomes are not only carriers of Aβ42 peptides, but that their physicochemical properties and concentrations can also be altered, which in turn can have both aggravating and protective effects on the accumulations of Aβ42 that occur during the development and progression of Alzheimer’s disease.
Beskrivning
Ämne/nyckelord
Neurovetenskaper , Cell- och molekylärbiologi , Medicinsk bioteknologi (med inriktning mot cellbiologi) , Livsvetenskaper , Neurosciences , Cell and Molecular Biology , Medical Biotechnology (with a focus on Cell Biology) , Life Science