An Introduction to DAF-16 and FOXO in the Context of Aging and Longevity

In the early 1990s Cynthia Kenyon and others produced the first C. elegans nematode worms to exhibit significantly extended longevity through a single gene mutation, in daf-2, the nematode version of the insulin-like growth factor 1 (IGF-1) receptor, and went on to map the relevant nearby biochemical landscape of these mutants. It is perhaps overly simplistic to mark this as the dividing line between a research mainstream whose members believed aging to be an intractably complex process, and a research mainstream increasingly interested in slowing aging through adjustment of metabolism, but that is the story as it is commonly told these days.

The mechanisms of longevity enhancement in daf-2 mutants depend on daf-16, a FOXO family transcription factor. The roles of these and other related proteins have been studied intensively in nematodes and other species since the first discoveries. Insulin metabolism - involving insulin, IGF-1, growth hormone, and their cell surface receptors - has emerged as one of the more influential means by which cellular mechanisms determine variations in longevity, both in response to circumstances for individuals within the same species, and to some degree between species. The record for mouse longevity is still held by growth hormone receptor loss of function mutants, for example. These proteins and their relationships are tied to cell growth, nutrient sensing, the calorie restriction response, temperature regulation, autophagy, and many other fundamental aspects of biochemistry.

From a historical perspective, to understand how the research community came to its present distribution of attitudes and focus, it helps to know something about this body of research and its central position in the modern study of aging. It has progressed and grown alongside the slow awakening to view aging as a treatable medical condition. If reliable changes of any sort can be achieved, so the thinking goes, then in principle something can be done to reduce the terrible toll of suffering, pain, and death that accompanies aging. The ability make even small changes means that aging is not intractable. Manipulating insulin metabolism and its surrounding mechanisms, such as through the development of calorie restriction mimetic drugs, is not the future of longevity science, however. It is not a road to rejuvenation, because rejuvenation can only occur when the causes of aging are reversed. All that can be done with the manipulation of insulin metabolism is to modestly slow down aging.

Thus the future of the field, for the treatment of aging at least, will involve a transition away from the study of processes that explain natural variations in longevity between individuals, or due to environmental factors such as calorie intake. A transition away from the work that awoke the possibility of influencing aging, and towards effective means of turning back aging. Since tinkering with insulin metabolism, or any similar approach, cannot produce rejuvenation, other methods must be adopted. This future is best represented by the SENS portfolio, the strategies for engineered negligible senescence, and similar programs focused on repairing the cell and tissue damage that causes aging. This is an entirely distinct focus, orthogonal to topics such as the way in which insulin metabolism functions to adjust the pace of aging. Metabolism generates various forms of damage even when operating normally, and that damage accumulates over time to cause age-related dysfunction, disease, and death. Removing this damage will turn back the state of aging, and thus be a form of rejuvenation.

DAF-16/FOXO Transcription Factor in Aging and Longevity

The genetic pathways and biochemical processes that modulate aging and longevity are well conserved from budding yeast to the nematode worm Caenorhabditis elegans and mammals. The forkhead transcription factor FOXO as the key downstream regulator that integrates different signals from these pathways plays a crucial role in aging and longevity. The roundworm C. elegans has been considered to be an excellent system for studying molecular mechanisms in regulating animal aging and longevity. Here we discuss the evidence for the role of DAF-16/FOXO in aging and longevity, especially the data in C. elegans, which could give clues to the further studies for human aging and longevity.

FOXOs belong to the class O of the Forkhead transcription factors, which is featured by a conserved DNA-binding domain that participates a wide range of important cellular processes such as cell cycle arrest, apoptosis, and metabolism besides its function in stress resistance and longevity. There are four FOXO genes in mammals: FOXO1 (FKHR), FOXO3 (FKHRL1), FOXO4 (AFX), and FOXO6 sharing high similarity in their structure and function as well as regulation with each other, while invertebrates have only one FOXO gene, named daf-16 in C. elegans.

"Deregulated nutrient sensing" as one of the aging hallmarks to be firstly described to influence longevity, is mainly regulated by the insulin and IGF-1 signaling (IIS) pathway. And this pathway is so highly conserved to modulate aging and longevity across a great evolutionary distance from invertebrates to mammals that the components in every step found in C. elegans could be corresponded to the homologs in mice or human. Any conditions that cause inner stress to block the IIS pathway, like in the presence of food restriction or signals failing to be transduced to DAF-16/FOXO, would increase the transcriptional activity of DAF-16/FOXO by inducing the translocation of DAF-16/FOXO to the cell nucleus, which could subsequently promote or repress the expression of downstream targets to trigger the resistance to different kinds of stress and prolong the lifespan of the organisms.

Another pathway correlated with nutrition affecting longevity is the TOR (target of rapamycin) pathway, which was firstly described in C. elegans and was proved evolutionarily conserved later in other organisms. Various dietary interventions such as caloric restriction may inactivate TOR pathway to promote lifespan extension. The TOR kinase exists in two distinct complexes, TORC1 and TORC2. TORC1-mediated longevity is dependent on DAF-16/FOXO.

AMPK pathway as an energy-sensing signaling pathway responses to stimuli of decreased energy as well as reduced glucose or leptin levels, and it is the theoretical basis of dietary restriction regimen that is considered to extend both the mean and maximal lifespan in a wide range of species. DAF-16 is necessary for AMPK function in oxidative stress resistance and longevity, as the increased longevity caused by overexpression of AMPK was reverted when DAF-16 was inhibited.

The JNK (Jun N-terminal kinase) family, a subgroup of MAPK (mitogen-activated protein kinase) superfamily, as a part of a signal transduction cascade that is activated by cytokines such as TNF and IL-1, serves as a molecular sensor for various stresses including UV irradiation, ROS (reactive oxygen species), DNA damage, heat, and inflammatory cytokines. In C. elegans, overexpression of JNK showed extension lifespan and resistance to heavy metal toxicity, which may function through phosphorylation of DAF-16.

A reproductive system that may integrate nutrient signaling and communicate with other tissues through germline to affect aging has been observed in C. elegans, flies, and mice, indicating a conserved regulation mechanism across different organisms. And it has been reported that lifespan could be extended by 40-60% if the germline precursor cells were removed or the germline stem cell division were prevented in C. elegans. A steroid hormone pathway that includes the key components DAF-36/NVD, DAF-9/CYP27 as well as DAF-12/NHR is required for lifespan extension in response to germline loss, and DAF-12/NHR and DAF-9/CYP27 probably form a complex with DAF-16/FOXO to function, although the detailed mechanisms remain to be further determined.

Thus multiple signaling pathways such as the insulin/IGF-1 signaling pathway, TOR signaling, AMPK pathway, JNK pathway, and germline signaling have been found to be involved in aging and longevity. DAF-16/FOXO, as a key transcription factor, could integrate different signals from these pathways to modulate aging, and longevity via shuttling from cytoplasm to nucleus. Hence, understanding how DAF-16/FOXO functions will be pivotal to illustrate the processes of aging and longevity.

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