Satchidananda Panda, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): In mammals, a master circadian clock in the suprachiasmatic nucleus of the hypothalamus synchronizes metabolism, cardiac output, alertness, and sleep state with the daily light/dark cycle. Chronic desynchronization of the circadian clock is a significant contributor to metabolic diseases, as well as sleep and mood disorders. Even temporary desynchronization of the clock with the natural day:night cycle, such as in jet lag or shift work syndrome, is a leading cause of productivity loss and accidental injury or death. Thus, a firm understanding of circadian clock function that leads to treatment of related disorders will lead to health and economic benefits of the society. [unreadable] [unreadable] Synchrony of the clock with the external environment is largely governed by exposure to light received by the eye. However, the precise mechanism by which light entrains the circadian oscillator is not known. An intriguing feature of circadian light entrainment is its resistance to several physiological conditions and mutations that usually causes blindness. This suggested novel photopigment, signaling property and chromophore use may be involved. Recently, a new opsin-like photopigment, melanopsin, was found in the inner retina and has been established as a dominant mediator of light input to the circadian clock. Experiments proposed in this project are to understand some key aspects of melanopsin's photosensitivity. Specifically, we want to understand the spectral and photochemical properties of melanopsin and how its interaction with arrestin modulates such function. We have developed heterologous expression systems for the measurement of melanopsin photosensitivity in living cells and for high level expression of the opsin for biochemical analysis. Using both cellular and biochemical assays we will (a) determine whether melanopsin photopigment exists in two distinct spectrally active and photoreversible states, (b) whether melanopsin has an intrinsic photoisomerase activity to regenerate its own chromophore and (c) how interaction with arrestin determines its photochemical properties. Successful completion of these aims will identify key functional features of melanopsin phototransduction, elucidate the underlying mechanism, and provide potential entry points for therapeutic intervention in circadian disorders. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Light received through the eye regulates an array of non-image forming photoresponses, including entrainment of the circadian clock to the ambient light dark cycle, light modulation of alertness/sleep, light suppression of pinel melatonin synthesis and release, and acute regulation of transcription in adrenal galnds. By classical genetics and cell biological studies we have determined an opsin class of GPCR, called melanopsin is the dominant photopigment that mediate these non-image forming responses. Melanopsin is expressed in a small subset of retinal ganglion cells. Unlike the classical vertebrate rod/cone photopigments, melanopsin uses a different mechanism for regeneration of its photopigment and a distinct signaling mechanism to transduce the light information. This has opened up potential to develop pharmacological agents to specifically modulate melanopsin function. Although photosensitive opsins are the founding members of GPCRs, no HTS compatible assay for this class currently exists. In this proposal we plan to generate cell lines stably expressing melanopsin and beta arrestin each bearing specific tags, such that photoactivation of melanopsin leads to a luminescent readout. We plan to use this cell line to develop a high through-put screening (HTS) compatible assay to monitor photoactivation of melanopsin. The assay will be optimized and miniaturized to be run in 384-well format, and the optimized assay will be used to screen a small library of compounds. Successful completion of the experiments will generate a novel HTS compatible assay for an opsin class of photopigment and a few potential modulators of an opsin class of photopigment. Ultimately, small molecule modulators of melanopsin will offer valuable tools to interrogate the role of melanopsin in non-image forming photoresponses in non-model organisms. PUBLIC HEALTH RELEVANCE: We have established a critical role of a novel light receptor melanopsin in adjusting our biological clock and sleep-wake rhythms to the day: night cycle. The goal of this research proposal is to develop a method to reliably measure melanopsin function. This method would then be used to screen a large number of potential drug compounds to find ones that can mimic light or darkness and can be ultimately used to treat diseases like depression, various sleep disorders and jet-lag.

DESCRIPTION (provided by applicant): The proposal is to obtain funds for an automated high throughput analysis system for the instruments core at the Salk Institute. The instrument system will comprise of an Eppendorf epMotion 5075 VAC-TMX high throughput DNA preparation workstation, an Eppendorf epMotion 5075 LH liquid handling station for liquid dispensing and assay set up, and a Molecular Devices FlexStation 3 multi- mode detector for various cell based and in vitro assays. The requested high throughput analyses system is needed to complement the strong chemical library screening core at the Salk Institute. We will use this system to generate and use libraries of cDNA and shRNA reagents for genomics screening. The high throughput genomic DNA purification system will allow preparation of libraries of cDNA or shDNA constructs for cell based assays. The liquid handling station will dispense the cDNA, shDNA and lentiviral particles into mammalian cells. The multimode detector plate reader will measure absorbance, fluorescence intensity, luminescence, fluorescence polarization and time-resolved fluorescence. The Flexstation 3 plate reader will enable researchers at the Salk Institute to use fluorescence based dyes to measure rapid changes in intracellular calcium level or of membrane potential in a high throughput manner. The combination of these instruments will offer a powerful yet flexible platform for use of genomics approaches to accelerate understanding of biological processes in multiple diverse laboratories. We have identified 10 user laboratories for immediate use of the system for various assay development, screening and post-screening follow-up. The proposed instrument system will be integrated to the existing Salk Institute instrument cores. A core instrument use committee will ensure that the instrument system is operated and maintained by trained personnel. PUBLIC HEALTH RELEVANCE: The proposal is to obtain funds for a robotic system at the Salk Institute. This instrument system will enable researchers to screen through thousands of biological samples with high degree of accuracy, reproducibility and speed to understand how genes and proteins function in healthy and diseased cells.

DESCRIPTION (provided by applicant): Feeding behavior in mammals is both episodic and circadian. Accordingly, mammals have developed mechanisms to temporally regulate liver gluconeogenesis to maintain glucose homeostasis. The CREB/CRTC2 pathway regulates transcription of gluconeogenic genes via cis-acting cAMP Response Elements (CREs) in response to fasting and feeding bouts. In parallel, the self sustaining hepatic circadian clock mediates circadian rhythm in expression of metabolic genes- a number of which are regulated by CREB and CRTC2- with peak levels aligned to appropriate time of the day. Although molecular interactions between the circadian clock and the CREB/CRTC2 pathway are thought to shape the overall adaptation to feeding regimen, these interactions are not well defined. The core circadian clock is based on a transcriptional feedback loop in which Clock/Bamal1 transcriptional activators bind to cis acting E-box in the promoters of Per and Cry genes, whose protein products in turn inhibit Clock/Bmal1 function, thus producing circadian rhythms in Per and Cry proteins. The current application tests the hypothesis that the circadian clock and CREB pathway promote metabolic adaptation to environmental changes through reciprocal regulatory interactions between the Per/CRY inhibitors and CREB/CRTC2 activators. Aims 1 to 3 address the role of Cry proteins in the circadian modulation of hepatic CREB and CRTC2 activities in response to episodic feeding. In particular, we will evaluate the proposed role of Crys in attenuating glucagon-dependent increases in cAMP production. Does cytoplasmic Cry interfere with G protein coupled receptor signaling? Aims 4-6 address counter-regulatory effects of the CREB/CRTC2 pathway on clock activity. In particular, we will examine the regulatory importance of CREB binding sites on Per genes, which offer a node for synchronizing Per transcription and consequently the circadian clock with the daily feeding rhythms. Finally, we will test how these interactions shape long term adaptation of the organism to feeding regimens.

Abstract Disruption in metabolic homeostasis is increasingly recognized as the root cause of obesity, insulin resistance, nonalcoholic steatohepatitis, hyperlipidemia, and cardiovascular diseases. Current efforts on prevention and therapies are focused on key components that mediate nutrient utilization, interconversion and storage. However, metabolic diseases are complex in nature with disruption in multiple pathways, often requiring progressively more complex combination therapies. Novel intervention that is effective both as a preventative method and to augment therapy is urgently needed. The circadian regulation of metabolism and physiology offer a novel promising avenue for the prevention and the treatment of these diseases. There is growing evidence that disruption of natural circadian rhythm in sleep and nutrition as occurs in people doing shiftwork is associated with increased incidence of metabolic diseases in humans. Furthermore, even among the general population, the daily rhythm of sleep and nutrition is significantly disrupted. Therefore, behavioral changes that sustain robust circadian function are considered to be beneficial against challenges that predispose to metabolic diseases. Time-restricted feeding (TRF), in which animals are fed within an 8-12 hour time interval during their natural circadian wakeful hours is both preventative and therapeutic against metabolic diseases in both mice and insects. There is growing precedence that the time of food intake has a profound impact on body weight regulation in humans. While these preliminary findings are encouraging, major questions remain to be answered before any potential human translation. Is TRF beneficial under a shiftwork paradigm that chronically disrupts circadian rhythm? And what are the potential mechanisms underlying TRF benefits? This proposal will test these questions in mice that are effectively used to model circadian rhythm disruption and metabolic diseases.

The circadian clock regulates physiologic processes by establishing cyclical rhythms. Circadian clock genes control key pathways altered during tumorigenesis including metabolism, inflammation, cell cycle, autophagy and DNA damage responses. Importantly, disruption of circadian rhythms increases cancer risk, and several physiologically oscillating pathways lose their rhythmic activity in cancer. These observations suggest that pharmacological modulation of the circadian clock machinery can be targeted for cancer treatment. However, the link between pharmacological intervention of the circadian clock and new therapeutic strategies for cancer prevention and treatment has yet to be demonstrated. This project provided a first-proof of concept using a pre-clinical animal model of glioblastoma by deploying the first-generation chemical tools SR9009 and SR9011 targeting nuclear receptor (NR) subfamily 1 group D member 1 (NR1D1) and NR1D2 (REV-ERSs). Although these small molecules were more effective than the standard of care drug for glioblastoma treatment, this first generation of NR ligands have relatively poor pharmacological characteristics limiting potency and general applicability for patient disease treatment. Therefore, the lab proposes to optimize REV-ERB agonists and validate their anticancer activity towards glioblastomas in vitro and pre-clinically in vivo alone or in combination with established clinical modalities to support translation of lead molecules for treating devastating diseases with limited therapeutic treatments.

Geroscience hypothesis posits that aging itself is the underlying major risk factor for age-related chronic diseases, including Alzheimer's Disease (AD); therefore, delaying aging delays disease, including AD. This proposal will examine the reciprocal relationship between circadian rhythm disruption (CRD) and AD pathology. Circadian rhythms, which are intimately interlinked with cellular metabolism, orchestrate the coordinated expression and function of multiple pathways that support normal cellular function and repair in both neural and peripheral tissues. Circadian rhythms deteriorate with aging, and Alzheimer's patients show disrupted circadian rhythm. However, the causal role of CRD for AD is not clear. The proposal will assess the relationship between circadian rhythm disruption and AD pathology, will test whether improvement in circadian rhythms delay the onset and progression of AD. and will attempt to identify underlying molecular mechanisms. Well-characterized Drosophila melanogaster (fruit fly) models of AD with Drosophila genetic tools will be used to test the impact of genetic or environmental CRD on the onset and severity of the multiorgan functional decline in AD. One feature of CRD is the lack of feeding consolidation to daylight hours in diurnal animals. To restore aspects of circadian rhythm in older flies, animals will be subject to time-restricted feeding (TRF) in which food is provided to flies only during the 12 h day time. TRF does not reduce daily caloric intake and imparts a molecular signature that is distinct from that under caloric restriction. Disease onset and severity will be assessed in TRF, and ad lib fed (ALF) flies to determine if TRF is an effective behavioral intervention for AD. To test the molecular pathways mediating the opposite effects of CRD and TRF on AD pathologies, time-series transcriptomes from these flies will be analyzed to find candidate pathways. The functional significance of these pathways will be tested by expressing genetic gain of function and loss of function alleles in AD flies. Our novel in vivo genetic-transgenic Drosophila disease model coupled with ultrastructural, functional, metabolic, and transcriptome techniques will generate unbiased insights into the mechanistic basis of accelerated aging in production of AD. Successful completion of this project will provide a deeper molecular understanding of the interaction between circadian rhythm and genetic risk of AD. Additionally, this research will assess the efficacy of a behavioral intervention that would have a high potential for adoption in humans.

Project Summary This application, in response to RFA-CA-004 ?Research Answers to National Cancer Institute's (NCI) Provocative Questions (R01 Clinical Trial Optional),? will address ?PQ2: How does intermittent fasting affect cancer incidence, treatment response, or outcome?? Obesity and age are two major risk factors for cancer development. Thus, therapeutic interventions that prevent or delay the development of excessive weight gain and/or age-associated physiological dysfunction hold great promise for reducing cancer risk in the increasingly obese and elderly global population. One such intervention is time-restricted eating (TRE), a pragmatic form of intermittent fasting in which daily caloric intake is constrained to a consistent window of 8?12 hours without explicitly reducing total caloric intake. In young male mice, time-restricted feeding (TRF) reduces cancer risk by preventing obesity and metabolic diseases. TRF has also been shown to reduce breast cancer xenograft progression in obese mice. In humans, short-term clinical studies of TRE have revealed metabolic improvements that predict reduced cancer risk, and epidemiological evidence suggests that prolonged nightly fasting can reduce the risk of cancer, independent of changes in body weight. This promising preliminary evidence suggests that TRE may be an effective intervention for reducing cancer risk. However, the effects of TRF in aged animals and in the context of an obesogenic Western diet have not yet been established, and the mechanisms by which TRF reduces cancer risk remain unknown. This application builds upon promising preliminary data and leverages the complementary skills of the research team to address these critical gaps in knowledge. Both obesity and aging are associated with mitochondrial dysfunction and the production of pro-tumorigenic mitochondrial metabolites. Proposed experiments test the hypothesis that TRF optimizes mitochondria function through both cell-autonomous and systemic mechanisms, thereby reducing cancer risk. In Aim 1, the impact of TRF on mitochondria function and related physiologies will be established in aged mice. Nutrient metabolism, energy consumption, and mitochondria function will be assessed in these mice. In Aim 2, an innovative combination of metabolomics and mitochondria respiration assays will be used to test the impact of TRF on mitochondria function in normal and cancer cells (assessing both cell-autonomous and non-cell-autonomous mechanisms). The effects of TRF on tumor incidence will be assessed by subjecting tumor-prone mice to TRF. In Aim 3, plasma collected from a recently concluded human TRE intervention study will be used to test the effect of TRE on mitochondria function and cancer risk in humans. The proposed comparative analysis of TRE in humans and mice will provide critical mechanistic insight into how one form of intermittent fasting can help prevent cancer onset and improve treatment outcomes.