Elucidating the causal mechanisms of Alzheimer's disease using cell-based models

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Narayanan, Vibha
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Electronic thesis
Chemical engineering
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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, and circadian rhythm disruption 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 in gaining a more complete understanding of AD 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 cell lines in 2D and 3D cultures. It is important to elucidate further the interplay among the various disease mechanisms using cell-based models that can aid in improving the drug discovery and development process. In this thesis research, various potential mechanisms of Alzheimer’s Disease are addressed using in vitro 2D and 3D cell-based models. First, a stable AD cell line capable of overexpressing mutations in the Amyloid Precursor Protein (APP) associated with Familial AD was established, which included the Swedish double mutation (KM670/671NL) and the Indiana mutation (V717F). This cell line was then used in a cell-based assay platform to determine the efficacy of three test compounds capable of inhibiting the generation of A by targeting the transmembrane domain of APP. Moreover, it was shown that these compounds targeted the APP specifically while possessing minimal off-target activity against the function of the -secretase. This sequential assay platform can be adapted for high throughput screening of various compounds capable of targeting A inhibition. Second, an immortalized microglial cell line was used to elucidate the neuroinflammatory facet of AD. The pro-inflammatory response of BV2 microglia was characterized upon exposure to soluble A oligomers and compared with the response of the microglial cells upon exposure to A monomers. Exposure of specific chemokines and cytokines detected in an AD environment to BV2 cells altered the pro-inflammatory response into an anti-inflammatory response. These findings may be used to target modulation of microglial phenotypes in early AD. Finally, to study the effects of circadian regulation in vitro, a 3D spheroid model was established using HepG2 cells to understand further the influence of circadian regulation on genes involved in processes associated with drug metabolism, a highly integrated and complex biological mechanism. Bulk RNA sequencing was used to compare the effects of circadian synchronization in a 3D spheroid cell culture system with that of a more conventional 2D cell culture system. Using transcriptomic analysis, significant differences in circadian and drug metabolism genes were observed between the 3D and 2D culture formats. Moving forward, this 3D spheroid model may be adapted to developing neurospheroids to study the influence of circadian regulation in AD.
August 2022
School of Engineering
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Rensselaer Polytechnic Institute, Troy, NY
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