The lack of a preventive therapy for neurodegenerative diseases, such as Alzheimer’s Disease (AD) has increased the urgency for gaining an understanding of the mechanisms that lead to cognitive decline and eventually death. The identification of several potential targets including the amyloid plaque, hyperphosphorylated tau tangles, microglial activation, reactive oxidative stress and neuroinflammation has led to the generation of various in vitro and in vivo models. In particular, the development of in vitro models of AD has been crucial to the understanding of heretofore poorly understood disease pathology. Such in vitro models have been able to partly recapitulate key pathological hallmarks of the disease using various neuronal cell lines including induced pluripotent stem cells, immortalized neural progenitor cells and neuroblastoma cells in 2D and 3D. While amyloid aggregation previously has been observed in 3D models, the use of a high throughput 3D platform as a screening tool to quantify changes in amyloid aggregation using high content imaging could have a wide range of applications. The primary focus of this research is to establish a stable cell line with mutations in the Amyloid Precursor Protein (APP) associated with Familial AD such as the Swedish double mutation (KM670/671NL) and the Indiana mutation (V717F) and use a high-throughput 384-well pillar plate system to understand the complexities of targeting aggregated amyloid beta in a 3D Familial AD model. To this end, we established a stable cell line by producing lentiviruses harboring the APP gene with the Swe/Ind mutation and transducing a HEK293T cell line to generate a stable cell population by the use of an antibiotic selection marker. A 50-fold increase in the level of Aβ42 was secreted by the cells in comparison to levels secreted by a wildtype APP-containing cell line, such as the BE (2)-M17 neuroblastoma cell line. Upon drug treatment with common gamma-secretase inhibitors such as gamma secretase inhibitor XXI and DAPT as well as other compounds inhibiting amyloid beta secretion, a dose dependent reduction in Aβ42 was observed in both 2D and 3D. This work provides a screening tool that can be used to mimic key pathological disease hallmarks that serve as drug targets for the applications of drug discovery.

Screening potential therapeutic targets with an in vitro 3D familial Alzheimer’s disease model using a high-throughput platform

ACS Fall 2021

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