Joseph Bedont - US grants

Project Summary / Abstract Sleep disruption is tightly associated with Alzheimer's disease (AD), and recent studies suggest it can be causal. While somnopathic paths to AD remain poorly understood, the variety of such disorders associated with chronic sleep loss suggest that these mechanisms are general. This proposal aims to leverage a Drosophila model of presenilin (psn) hypofunction, the leading cause of familial Alzheimer's disease in humans, to investigate these mechanisms. Psn+/- flies have sleep-dependent memory and synaptic defects reminiscent of AD, and a whole- fly metabolomic screen we conducted identified a possible mechanism for these phenotypes: urea cycle hyper- activity, present in both psn+/- and sleep-deprived wild-type flies. I hypothesize that urea cycle disruption couples sleep loss to Alzheimer's disease, perhaps via regulation of autophagy. Aim 1 will test whether urea cycle dysfunction links sleep disturbance to dementia-like psn+/- memory phenotypes, by examining how additional sleep manipulation modulate urea cycle throughput and testing the effects of urea cycle blockade and sleep deprivation on psn+/- memory defects. Aim 2 will test whether sleep disturbance and urea cycle dysfunction are the causes of reduced autophagy in psn+/- flies, by testing the effects of urea cycle blockade, enforced sleep, and sleep deprivation on deposition of insoluble ubiquitinated protein aggregates and live- imaged autophagy flux. Aim 2 will also test whether sleep deprivation is sufficient to disrupt autophagy in wild-type flies, and whether sleep deprivation in psn+/- flies can induce neuronal apoptosis as measured by TUNEL. Finally, Aim 3 will screen the brain metabolomes of psn+/- and sleep-deprived wild-type flies by mass spectroscopy for metabolic pathways our whole-fly screen may have missed, as well as determining whether urea cycle dysregulation by sleep disturbance occurs locally in the psn+/- brain. Through the innovative research strategy described in this proposal, Dr. Joseph Bedont will be trained in a new organism, to study fields in which he has room for growth, using several new techniques including mass spectroscopy. The proposed site, the University of Pennsylvania, provides all of the technical and intellectual resources necessary for success. The proposed sponsor, Dr. Amita Sehgal, is a world expert in Drosophila behavioral genetics and sleep, and a very successful mentor who will provide excellent scientific and career development support to the applicant. And the proposed training plan, especially activities planned with the sponsor and her long-standing collaborator Dr. Aalim Weljie, will provide all of the skills the applicant requires to fully leverage the training potential of the proposal. This proposal will give insight into whether urea cycle and autophagy dysregulation by sleep disturbance is an important contributor to somnopathic neurodegeneration in Alzheimer's disease, and identify possible additional paths to somnopathy for investigation in future studies.

Sleep is essential for life, and chronic sleep deprivation (SD) is associated with pathology including Alzheimer's disease in humans. Urea cycle abnormalities have been observed by others in animal SD paradigms and human sleep and fatigue disorders. I found that polyamines (PAs), coupled to urea cycle by the metabolite ornithine, are elevated in Drosophila sleep mutants, especially acetylated PAs and putrescine. I hypothesize that nitrogen diversion from urea cycle to PA synthesis drives sleep when SD is acute and neurodegeneration when SD is chronic. Mentored Aim 1 will test what mechanisms link SD to nitrogen metabolism by using sleep mutants for 13C-ornithine mass spectrometry to identify the metabolites that ornithine is channeled toward, and enzyme assays to assess what catalytic differences shape the nitrogen metabolome under chronic SD. Mass spectrometry will also be conducted under more acute SD to determine if this mirrors chronic SD in PA profile. Mentored Aim 2 will test how PA supplements, and broadly expressed RNAi that promote putrescine synthesis, both promote sleep in wild-type flies. Subpopulation RNAi sleep experiments will assess what neural circuits are involved in PA sleep responses. Broadly expressed RNAi sleep experiments will determine whether PAs generally are required for rebound sleep following SD, and whether production of putrescine specifically is required for PA supplement sleep increases. Independent Aim 3 will test whether PA increases contribute to the well-documented worsening of Alzheimer's pathology by SD. Mass spectrometry will test whether PA increases observed in mouse Alzheimer's models by others, which are very similar to my fly sleep mutants, carry over to multiple fly Alzheimer's models. I will also test whether broad PA synthesis RNAi that blunts production of acetyl-PAs and putrescine can block the worsening effects of SD in multiple fly Alzheimer's models. Measures of protein pathology, cell death, lethality, and memory will be used as metrics. Independent Aim 4 will test whether PA synthesis is sleep-regulated and sleep-promoting in mouse brain. Mass spectrometry under chronic environmental enrichment SD will test whether mice exhibit similar PA changes to what I observe in my fly sleep mutants. Motion-sensing and EEG sleep metrics will be used to assess whether both PA supplementation and pharmacological depletion of PAs alter sleep in mice. Any one of these four aims has the potential to launch a long-running research project if successful, enhancing my ability to launch an independent career from this proposal. My training plan builds on my core mouse and fly skillsets, adding important complementary skills in genetic engineering, fly memory behavior, specialized mouse sleep behavior protocols, and isotopic labeling metabolomics. My training plan will also enhance my theoretical knowledge of neurodegeneration and enhance my mentorship skills. This proposal will give me the data and skills I need to succeed as an independent investigator at a research institution, building out a lab of my own focused on biochemical regulation of homeostasis, and linkages to neural pathology.