Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity 1 – 3 . Changes to DNA methylation patterns over time form the basis of ageing clocks 4 , but whether older individuals retain the information needed to restore these patterns—and, if so, whether this could improve tissue function—is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity 5 – 7 . Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information—encoded in part by DNA methylation—that can be accessed to improve tissue function and promote regeneration in vivo.

SUMMARY A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD+ precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ precursor nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.

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Cells do not live in a vacuum, but in a milieu defined by cell-cell communication that can be measured via emerging high-resolution spatial transcriptomics approaches. However, analytical tools that fully leverage such data for kinetic modeling remain lacking. Here we present Spateo (http://spateo-release.readthedocs.io/), a general framework for quantitative spatiotemporal modeling of single-cell resolution spatial transcriptomics. Spateo delivers novel methods for digitizing spatial layers/columns to identify spatially-polar genes, and develops a comprehensive framework of cell-cell interaction to reveal spatial effects of niche factors and cell type-specific ligand-receptor interactions. Furthermore, Spateo reconstructs 3D models of whole embryos, and performs 3D morphometric analyses. Lastly, Spateo introduces the concept of "morphometric vector field" of cell migrations, and integrates spatial differential geometry to unveil regulatory programs underlying various organogenesis patterns of Drosophila. Thus, Spateo enables the study of the ecology of organs at a molecular level in 3D space, beyond isolated single cells.

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor and signaling molecule that controls the activity of enzymes involved in metabolism, DNA repair, and cellular survival, such as the PARPs, CD38, and the sirtuins. Here, we describe three methods for measuring the activity of these enzymes: the etheno-NAD assay measures NAD hydrolase activity using an NAD analog to produce a fluorescent product that is measured in real time; the PNC1 assay converts a native product of NAD hydrolysis, nicotinamide, into a quantitative fluorescent readout; and liquid chromatography tandem mass spectrometry (LC-MS/MS) is used to characterize the entire NAD metabolome in a sample. These methods will enable new insights into the roles that NAD and the enzymes that utilize it play in health and disease.

SIRT1 is an NAD+-dependent lysine deacetylase that promotes healthy aging and longevity in diverse organisms. Small molecule allosteric activators of SIRT1 such as resveratrol and SRT2104 directly bind to the N-terminus of SIRT1 and lower the Km for the protein substrate. In rodents, sirtuin-activating compounds (STACs) protect from age-related diseases and extend life span. In human clinical trials, STACs have a high safety profile and anti-inflammatory activities. Here, we describe methods for identifying and characterizing STACs, including production of recombinant protein, in vitro assays with recombinant protein, and cellular assays based on mitochondrial dynamics. The methods described in this chapter will facilitate this discovery of improved STACs, natural and synthetic, in the pursuit of interventions to treat age-related diseases.

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