Study of NAD metabolism in protein aggregation

• Autor(a) principal: Teixeira, Ana Andreia Mendes
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/25054
• topic: NAD biosynthesis; NAD; NAMPT; NAPRT; NMNAT-2; NAD precursors; Protein aggregation; Cell viability; Metabolic state; Gene expression; Aging

NAD (Nicotinamide Adenine Dinucleotide) is a pyridine involved in numerous biological processes, namely nutrient catabolism sustaining cellular energy metabolism. During aging, NAD levels decrease and a global proteostasis deregulation is observed. As many age-related diseases are neurodegenerative and characterized by the accumulation of protein aggregates, we hypothesized that NAD could prevent or ameliorate protein aggregation. To study the role of NAD metabolism in proteostasis, we used SH-SY5Y cells exposed to chemicals that modulate the levels of protein aggregation and NAD metabolism. Cells were stained with the ProteoStat® kit to detect protein aggregates and analysed by flow cytometry and fluorescence microscopy. Cell viability was measured with propidium iodide by flow cytometry and metabolic state was measured using the colorimetric resazurin assay. SH-SY5Y cells showed increased protein aggregation levels in the presence of the proteasome inhibitor MG132 over time. MG132 induced more aggregation than treatment of cells with a NAD metabolism inhibitor, although supplementation with NAD appeared to decrease protein aggregates. Fluorescence microscopy analysis corroborated the flow cytometry result. In all tested conditions cell viability was not altered, in contrast with metabolic state that was altered by chemical treatments. Supplementation with NAD appeared to decrease protein aggregates, and further studies are warranted to elucidate the role in proteostasis of the different NAD precursors and associated pathways.