Ashley E. Webb - US grants

PROJECT SUMMARY/ABSTRACT During aging, the ability of neural stem cells (NSCs) in the brain to form new neurons is reduced, but the molecular mechanisms underlying the deterioration of NSC function remain unclear. There is currently a critical need to understand the mechanisms by which NSCs are activated to form neurons, and why this process declines with age. The long term goal is to identify the mechanisms responsible for the loss of NSC function with age, and discover interventions that harness the regenerative capacity of these cells to increase cognitive function in aged and diseased individuals. The objective of this proposal is to identify the mechanisms by which the conserved ?pro-longevity? transcription factor, FOXO3, preserves NSC quiescence during aging. The central hypothesis is that FOXO3 directly regulates a network of target genes and pathways that are critical for preserving NSCs during aging. This hypothesis will be tested by pursuing the following specific aims: 1) Determine the specific pathways regulated by FOXO3 in NSCs that preserve the quiescent state; 2) Investigate how FOXO3 and ASCL1 govern the balance between stem cell preservation and neurogenesis, a process that is drastically altered with age; and 3) Determine the extrinsic inputs that control FOXO3 activity and function. The first aim will be accomplished by combining a model of primary adult mouse NSC quiescence with loss of function and overexpression approaches to test the hypothesis that FOXO3 directly promotes quiescence by regulating specific genes and pathways. The second aim will be performed using methods to reveal the dynamic and antagonistic interaction between FOXO3 and ASCL1, and test the extent to which levels or activity of these factors are responsible for reduced activation of NSCs with age. The third aim will be accomplished using a combination of mouse genetics and molecular methods to test the hypothesis that BMP signaling directly regulates FOXO3 expression in vivo to promote NSC quiescence during aging. The outcome of this project will be the identification of the mechanisms by which FOXO3 regulates NSC function, how these mechanisms deteriorate with age, and reveal a strategy to counter the loss of NSC function during aging. This work is significant because it will determine why NSC activation is reduced in the aged brain, and uncover strategies to reverse it. This proposed research is innovative because it will use a unique system to elucidate the direct, genome-wide mechanisms that promote adult NSC quiescence, and parlay these findings into the in vivo setting. This work will provide key mechanistic insight into how gene networks are coordinated in young and aging NSCs, and have the potential to reveal new mechanisms underlying cognitive decline during aging.

PROJECT SUMMARY This proposal is motivated by the scarcity of studies and approaches that directly investigate the mechanisms underlying aging and neurodegeneration in the context of physiologically aged neurons. This gap in our knowledge is significant because neurodegeneration is an age-related condition. This long-term goal of this work is to understand the mechanisms responsible for degeneration of the hypothalamus, a key region involved in neuroendocrine regulation. The objective of this proposal is to use cellular reprogramming methods to establish a new platform to investigate the mechanisms of degeneration of a particular hypothalamic neuronal subtype: POMC neurons. POMC neurons are a rare cell type in the brain with a critical neuroendocrine function. The hypothesis is that directly reprogrammed iPOMCs from aged animals and models of Alzheimer's disease will provide a useful system to uncover the mechanisms of POMC aging and degeneration. This hypothesis is supported by strong preliminary data and will be tested through two specific aims: 1) Establish and optimize an in vitro system to study how POMC neurons age and 2) Generate iPOMC cells with mutations associated with familial AD. The first aim will characterize the age-associated phenotypes in iPOMC cells in the context of physiological aging. The second aim will, for the first time, establish a system to study a particular hypothalamic cell type in the context of Alzheimer's Disease. This system is a highly innovative approach to studying rare but critical cell types in the context of aging and neurodegeneration. Moreover, it can be expanded in future studies to generate other hypothalamic cell types, perform mechanistic studies, and implement small molecule screens to study and treat neuroendocrine dysfunction. This work is significant because it will provide a system to study the mechanisms responsible for aging of particular hypothalamic neuronal subtypes, which currently is not feasible with available tools. Ultimately, this approach has the potential to transform our understanding of neurodegenerative disease and lead to new therapies to prevent and treat these conditions.

PROJECT SUMMARY A number of transcriptional regulators have been found to regulate the hallmarks of aging. In previous work, we found that the FOXO family of transcriptional regulators, which have been implicated in healthy aging across species, directly regulate a conserved network of target genes. In mice, FOXOs are central regulators of stem cell homeostasis during aging and are critical for tissue integrity. In humans, SNPs in the FOXO3 locus have been linked to longevity, and the upstream regulator of FOXO?s, insulin/IGF signaling must be tightly regulated to preserve healthy aging. Yet, we still lack an understanding of how FOXOs function at the chromatin level, and how their activity is altered during aging and in the context of neurodegeneration. In preliminary work, we identified for the first time the direct network of FOXO3 targets in human cells, and found that FOXOs function as pioneer factors to deploy a secondary network of transcriptional regulators to extend their target gene network. Here, we will address this critical question of the mechanisms underlying the pioneer activity, its heterogeneous nature, and how it changes in the context of aging and Alzheimer?s Disease. Completion of this work will reveal the chromatin-level changes that influence the activity of factors that counter aging and neurodegeneration, which may lead to strategies to improve associated pathologies.

Project Summary Adult Hippocampal Neurogenesis (AHN) is critical for normal learning and memory and reduced AHN is an early hallmark of Alzheimer?s Disease (AD). Thus, restoring AHN has emerged as an attractive target for AD therapy. The accumulation of negative signals that degrade the neurogenic niche contributes to the reduction in newborn neurons in AD and aging. BMPs are components of the niche that negatively regulate neurogenesis and their levels are increased in AD in humans and in mouse FAD models. We recently reported that full length MuSK harboring its Ig3 domain, which is necessary for high affinity BMP binding, is a BMP co-receptor that augments and shapes BMP signaling. In preliminary studies we established that MuSK is endogenously expressed in neural stem cells (NSCs). We generated knock-in mice engineered to constitutively express an alternatively spliced form of MuSK lacking the Ig3 domain (??Ig3- MuSK?). The animals are viable, fertile and have a normal life span. NSCs isolated from ?Ig3- MuSK mice show impaired BMP responsiveness. Remarkably, the ?Ig3-MuSK mice exhibit over a two-fold increase in AHN and improved hippocampal-dependent learning. These results suggest that reducing MuSK-BMP activity by modulating MuSK alternative splicing is a potential target for promoting AHN in AD. Importantly, such alternative splicing is expected to be amenable to manipulation by exon-skipping antisense oligonucleotides. The recent success of the ASO Spinraza for Spinal Muscular Atrophy has demonstrated that this class of drugs can be highly effective in the human CNS, with a favorable pharmacokinetic and safety profile. In the proposed experiments we will use mouse FAD models to test whether inhibition of the MuSK- BMP pathway can promote AHN in the plaque-rich and inflammatory ?AD environment?. To more closely model the pathological and therapeutic landscape in humans, we will also use conditional mutants to test whether manipulating the MuSK-BMP pathway after amyloid plaque formation can promote AHN and improve cognition. If successful, this work will form an important part of the rationale and impetus for a pursuing a MuSK-directed ASO therapy for AD.