The transcription factor FOXO is a known regulator of tissue homeostasis and animal lifespan. It was first identified in its ability to promote longevity through the insulin signaling pathway, and has since been implicated in numerous cellular processes. Members of the FOXO protein family control a wide array of cellular functions including metabolism, cell cycle arrest, apoptosis, stress resistance, and aging. In response to various signaling cues, FOXO proteins can localize into the nucleus and interact with DNA to regulate transcription. FOXO is known as a longevity gene, however, how FOXO behavior changes during aging is not well understood. Normal aging involves a progressive decline in cell function and an accumulation of oxidative damage. Additionally, with age comes a reduction in FOXO gene expression, and FOXO protein activity can become dysregulated. To resolve how FOXO activity changes with normal aging, we began with chromatin immunoprecipitation sequencing (ChIP-seq) to compare differences in FOXO chromatin binding between young and old organisms, using Drosophila melanogaster as a model. In Drosophila, there is only one homolog representing the FOXO protein family, known as dFOXO. Through our investigation, we found that the number of dFOXO-bound DNA regions decreases with age, and see a number of these targeted genes undergo changes in expression with aging. Some pathways targeted by FOXO at a young age are Hippo, WNT, and MAPK signaling pathways.

FOXO is also known to mitigate oxidative stress, which is a contributing factor to age-related cellular degeneration. To understand FOXO dynamics in response to oxidative stress, we used Mass Spectrometry to evaluate dFOXO protein interacting networks under control and stress conditions. We observed a change in several dFOXO partners under paraquat-induced stress such as Stonewall, a chromatin modulator, and Hangover, a key transcription factor regulating neuromuscular junction (NMJ) morphology and neuronal activity. Both FOXO and Hangover show altered NMJ morphologies, and have potentially disrupted vesicle cycling.

Given that FOXO has been linked to homeostatic maintenance of neuronal processes across animal species, we performed genetic analysis to investigate how dFOXO regulate NMJ aging in adult flies. Adult NMJs are known to undergo loss of synaptic homeostasis with aging, which causes functional decline and can lead to neurodegeneration. We profiled adult Drosophila abdominal NMJs and found that loss of function FOXO mutants exhibited morphological profiles similar to those of middle aged wild-type flies. We also observed an abnormal accumulation of late endosomes associated with the NMJ both in aged flies and with knockdown of motor neuron FOXO. Overexpression of FOXO can delay this accumulation, suggesting FOXO is a positive regulator of neuronal homeostasis. We performed a genetic screen and identified pathways such as MAPK that act downstream of FOXO to control NMJ homeostasis during aging.

Collectively, this thesis provides evidence for global changes in FOXO chromatin binding activity under normal aging, highlights FOXO protein network dynamics that occur under oxidative stress, and displays homeostatic regulation of the adult Drosophila neuromuscular junction. These results illustrate how the transcription factor FOXO acts as a modulator of cellular homeostasis, and how FOXO activity is able to promote health and longevity.