Differentiated SH-SY5Y cells as a model to study amyloid-β pathology in Alzheimer’s disease

Abstract

Alzheimer’s disease (AD) is the most prevalent form of dementia and has been linked to misfolding and aggregation of amyloid-β (Aβ) peptides in and around neurons in the brain. Current understanding of these mechanisms and their relationship to neurodegeneration is incomplete and requires more research for ultimately developing novel therapeutic agents. This project has developed and optimized protocols for differentiating human neuroblastoma SH-SY5Y cells to exhibit neuron-like morphologies and functional presynapses, establishing a research tool to study Aβ pathology in AD. The differentiated cells were used to investigate the uptake and intracellular accumulation of Aβ, focusing on the pathological variant Aβ(1-42). Differentiation efficiency was assessed through morphological analysis. It was determined that the most vital parts of the differentiation procedure after RA pre-differentiation was BDNF induced differentiation, alone or in combination with NGF and/or VitD3 in serum-free conditions. Confocal imaging and flow cytometry were used to study the uptake of fluorescently labelled Aβ monomers and fibrils in both undifferentiated and differentiated cells, using FM 1-43 as a complementary membrane probe. Morphological characterization of Aβ(1-42) fibrils was done through SDS-page and atomic force microscopy. A higher uptake of Aβ(1-42) was observed in differentiated cells and Aβ(1-42) seemed to increase endocytic activity in differentiated cells. The uptake rate of Aβ(1-42) was higher in differentiated cells, however, Aβ(1-42) monomers and fibrils did not seem to colocalize with FM 1-43 stained vesicles. Undifferentiated cells seemed to internalize FM 1-43 stained vesicles to a higher extent, suggesting higher activity of endocytic pathways involving FM 1-43 stained vesicles. Differentiated cells showed minimal dextran uptake in comparison to undifferentiated cells, indicating alterations in endocytic pathways post-differentiation. Differentiated cells were also used to study Acetylcholine release using an enzyme-based amperometric biosensor. The established differentiation protocol showed improved Acetylcholine release compared to previously tested protocols. For future experiments, this cell model could be used to investigate how Aβ(1-42) affects Acetylcholine release. This project resulted in the establishment of a neuron-like cell model useful in research related to AD, hopefully contributing to new understanding of the pathological mechanisms linked to the disease.