Exploring cellular actions and interactions of SIRT1 that may counteract ageing

Abstract

The NAD⁺-dependent class III histone deacetylase SIRT1 appears to increase healthspan in some model organisms. Details of how SIRT1 is regulated and affects specific systems linked to ageing are limited. Here we aimed to gather a body of data to be used to generate a mathematical model of the interactions between SIRT1, resveratrol, NAD⁺, Poly-ADP ribose polymerase (PARP) and chaperone mediated autophagy (CMA). There is evidence that the dietary polyphenol resveratrol can increase healthspan and is believed to do this through activating SIRT1. PARP repairs DNA single-strand breaks and is also considered to be a modifier of ageing. Both SIRT1 and PARP consume NAD+, providing a point of interaction at the centre of cellular metabolism. Chaperone mediated autophagy (CMA) is reduced in ageing but evidence from our laboratory suggests the CMA regulator LAMP2 may be regulated by SIRT1, including by effects on DNA methylation at the LAMP2 promoter. Firstly, we observed that resveratrol increased SIRT1 mRNA, SIRT1 promoter activity and NAD⁺ in cultured cells (Caco-2). However, we also found that an increase in NAD⁺ reduced SIRT1 mRNA dramatically. Thus, interactions between SIRT1, resveratrol and NAD+ are complex. Secondly, SIRT1, PARP or NAD⁺ were each manipulated pharmaceutically or genetically and the response of the other two variables measured. Overall, the data suggested that SIRT1 and PARP have mutually inhibitory effects, which we hypothesise is driven by competition for NAD⁺. Next we developed and tested a functional assay based on a fluorescent substrate for CMA to measure activity directly. Preliminary findings indicated that reducing SIRT1 by siRNA decreased the activity of the pathway. Finally, LAMP2 mRNA expression was increased by the use of 5-aza-deoxycytidine to induce DNA hypomethylation, providing proof of principle that the gene is regulated by DNA methylation. Thus, the observed effect of SIRT1 on DNA methylation of the LAMP2 promoter may be the basis of its stimulatory effect on CMA. This body of work uncovers more information on the pleiotropic actions of SIRT1 relevant to modifying cellular ageing. The complexity of interactions with the other modifiers of ageing studied highlights the need for a system-level approach to data integration, and for further data to develop such a model, ultimately to identify target nodes for intervention to improve health-span.