FOXO transcription factors have long been associated with longevity control and tissue homeostasis. Although the transcriptional regulation of FOXO have been previously characterized (especially in long-lived insulin mutants and under stress conditions), how normal aging impacts the transcriptional activity of FOXO is poorly understood. Here, we conducted a chromatin immunoprecipitation sequencing (ChIP-Seq) analysis in both young (2-week-old) and aged (5-week-old) wild-type female fruit flies, Drosophila melanogaster , to evaluate the dynamics of FOXO gene targeting during aging. Intriguingly, the number of FOXO-bound genes dramatically decreases with age (from 2617 to 224). Consistent to the reduction of FOXO binding activity, many genes targeted by FOXO in young flies are transcriptionally altered with age, either up-regulated (FOXO-repressing genes) or down-regulated (FOXO-activating genes) in adult head tissue. In addition, we show that many FOXO-bound genes in wild-type flies are unique from those in insulin receptor substrate chico mutants. Distinct from chico mutants, FOXO targets specific cellular processes (e.g., actin cytoskeleton) and signaling pathways (e.g., Hippo, MAPK) in young wild-type female flies. FOXO targeting on these pathways decreases with age. Interestingly, FOXO targets in aged flies are enriched in cellular processes like chromatin organization and nucleosome assembly. Furthermore, FOXO binding to core histone genes is well maintained at aged flies. Together, our findings provide new insights into dynamic FOXO targeting under normal aging and highlight the diverse and understudied regulatory mechanisms for FOXO transcriptional activity.

Transcriptional coordination is a vital process contributing to metabolic homeostasis. As one of the key nodes in the metabolic network, the forkhead transcription factor FOXO has been shown to interact with diverse transcription co-factors and integrate signals from multiple pathways to control metabolism, oxidative stress response, and cell cycle. Recently, insulin/FOXO signaling has been implicated in the regulation of insect development via the interaction with insect hormones, such as ecdysone and juvenile hormone. In this study, we identified an interaction between Drosophila FOXO (dFOXO) and the zinc finger transcription factor Kruppel homolog 1 (Kr-h1), one of the key players in juvenile hormone signaling. We found that Kr-h1 mutants show delayed larval development and altered lipid metabolism, in particular induced lipolysis upon starvation. Notably, Kr-h1 physically and genetically interacts with dFOXO in vitro and in vivo to regulate the transcriptional activation of insulin receptor (InR) and adipose lipase brummer (bmm). The transcriptional co-regulation by Kr-h1 and dFOXO may represent a broad mechanism by which Kruppel-like factors integrate with insulin signaling to maintain metabolic homeostasis and coordinate organism growth.

The transcription factor foxo is a known regulator of lifespan extension and tissue homeostasis. It has been linked to the maintenance of neuronal processes across many species and has been shown to promote youthful characteristics by regulating cytoskeletal flexibility and synaptic plasticity at the neuromuscular junction (NMJ). However, the role of foxo in aging neuromuscular junction function has yet to be determined. We profiled adult Drosophila foxo- null mutant abdominal ventral longitudinal muscles and found that young mutants exhibited morphological profiles similar to those of aged wild-type flies, such as larger bouton areas and shorter terminal branches. We also observed changes to the axonal cytoskeleton and an accumulation of late endosomes in foxo null mutants and motor neuron-specific foxo knockdown flies, similar to those of aged wild-types. Motor neuron-specific overexpression of foxo can delay age-dependent changes to NMJ morphology, suggesting foxo is responsible for maintaining NMJ integrity during aging. Through genetic screening, we identify several downstream factors mediated through foxo-regulated NMJ homeostasis, including genes involved in the MAPK pathway. Interestingly, the phosphorylation of p38 was increased in the motor neuron-specific foxo knockdown flies, suggesting foxo acts as a suppressor of p38/MAPK activation. Our work reveals that foxo is a key regulator for NMJ homeostasis, and it may maintain NMJ integrity by repressing MAPK signaling.

Skeletal muscle has recently arisen as a novel regulators of Central Nervous System (CNS) function and aging, secreting bioactive molecules known as myokines with proteostasis and metabolism-modifying functions in targeted tissues, including the CNS. Here we report the generation of a novel transgenic mouse with enhanced muscle proteostasis via moderate overexpression of TFEB. We have discovered that the resulting enhanced skeletal muscle proteostasis function can significantly ameliorate proteotoxicity, reduce neuroinflammation and promotes transcriptional remodeling of the aging CNS, preserving cognition/memory in aging mice. Enhancing skeletal muscle proteostasis also reduced neuroinflammation and accumulation of tau-associated pathological hallmarks in a mouse model of tau pathology and was accompanied by increased secretion of CNS-targeting circulating factors from skeletal muscle. Our results implicate maintenance of skeletal muscle proteostasis throughout aging to direct regulations of the aging CNS metabolism and function, and suggest that skeletal-muscle originating factors may act as novel therapeutic targets against age-associated neurodegenerative diseases.