Epigenetic drug valproic acid in vertebrate aging

Aging is arguably the most familiar yet least well-understood aspect of human biology. Aging has been defined as the progressive decline in physiological integrity leading to increased susceptibility to disease and ultimately to death. The quest for methods to halt or slow the aging process is probably as old as human history itself. While modern society has produced a plethora of improvements to increase life expectancy, there are still no pharmacological compounds that have been shown to delay human aging. Recently, there has also been increasing evidence that aging is not only a genetic process, but that epigenetic factors can also have an effect on the aging process. By definition, epigenetics represents the reversible and heritable mechanisms that occur without any alteration of the underlying DNA sequence. The aging process is associated with profound changes in the epigenome that lead to alterations in gene expression, the epigenetic landscape, and genome architecture. Unlike DNA mutations, epigenetic alterations are - at least theoretically - reversible, hence offering the potential to explore epigenetic compounds for the design of novel anti-aging treatments to rejuvenate the epigenome. The impact of epigenetic changes on aging was recently confirmed in a nematode experiment, as treatment with an epigenetic compound significantly extended both average and maximum lifespan. Whether this life-extending effect following administration of an epigenetic agent can also be reproduced in vertebrates is a central question of the present project. A major obstacle to studying the aging process directly in humans is their long lifespan and the associated slow process of aging. Therefore, animal models that are both short-lived and experimentally accessible are critical for aging research. The African killifish Nothobranchius furzeri is currently the shortest-lived vertebrate that can be bred in captivity and has been proposed as an ideally suited new model organism to study vertebrate aging and aging-related diseases. The present project focuses on the analysis of epigenetic compounds on the aging process in the model system Nothobranchius furzeri and investigates whether life-prolonging effects can also be observed in the vertebrate system. In the first specific aim of the project, we will use transcriptome sequencing to investigate whether treatment with an epigenetic agent affects gene expression. In the second part of the project, we will use histological analysis of fish tissues to determine whether delayed aging after treatment can also be visualized by imaging methods. Thus, the strategy of our project follows an emerging paradigm shift in medicine, whereby the aging process as such is treated preventively rather than a single disease.

The key question of this project was to determine whether the administration of an epigenetic drug exerts a geroprotective effect in the vertebrate system thereby extending life and healthspan. Here, we show that exposure to the epigenetic compound valproic acid (VPA) significantly extended both average and maximum lifespan of the vertebrate research model Nothobranchius furzeri. The African turquoise killifish Nothobranchius furzeri is currently the shortest-lived vertebrate that can be bred in captivity constituting an ideally suited new model organism to study vertebrate aging and aging-related diseases. First, to characterize the potentially effective range of VPA and to identify the optimal concentration inducing lifespan extension, we conducted a dose-response analysis in N. furzeri. We identified the concentration of 0.25mM VPA to be most effective in prolonging lifespan. An additional important point was the question at which initiation time point during the life cycle anti-aging VPA intervention could result in a longevity promoting effect. As the incidence of age-related pathologies strikingly increases after having passed midlife, we implemented a late-onset protocol starting VPA administration at week 10 of adult killifish life, roughly corresponding to 90% survivorship of our killifish colony. In this study, VPA treatment initiated at middle age produced a highly significant (**** p = < 0.0001) life-extending effect. Specifically, median and maximum survival was increased by 30% and 28,5%, respectively. With regard to in vivo reaction kinetics indicating epigenetic modifications, administration of the geroprotective concentration of 0.25mM VPA via the system water induced histone hyperacetylation within the first 24 hours indicative of a rapidly responding transcriptome. Next, to identify longevity-associated gene expression signatures and to distinguish immediate from long-term manifestation effects upon VPA treatment we performed an elaborate transcriptomic profiling experiment employing a late-onset VPA administration protocol of biological samples spanning the second half of the killifish lifespan including sampling time points week 10, 24 hours post drug administration, week 14, 17, and 21. In addition to identifying longevity-associated gene expression signatures, we collected histone and protein samples plus tissue samples for histopathological analyses of all the time points. The histopathological analyses will provide a link between VPA induced transcriptional deregulation and characteristic morphological changes in aging tissues. Taken together, the results of this project show that epigenetic interventions initiated passed midlife hold the potential to slow down the aging process by "rejuvenating" the epigenome thereby extending the disease free portion of life even at advanced age. Thus, the strategy of this project follows an emerging paradigm shift in medicine, whereby the aging process as such is treated preventively rather than a single disease.