Ravi Allada - US grants

DESCRIPTION (provided by applicant): Disturbed daily rhythms have been implicated in a variety of brain disorders. These rhythms are regulated by at least two intertwined pathways, one is the circadian clock and a second is the masking pathway that mediates activity in response to light. This proposal addresses these pathways using the fruit fly, Drosophila melanogaster. The genetic basis of circadian and masking function appears conserved, suggesting that findings in the fly will be widely applicable. A critical role for an enigmatic and highly conserved ion channel narrow abdomen (na) has been identified in both circadian output and masking regulation. These observations underlie the overall goal of this proposal, which is to provide an integrated view of na function in both masking and circadian rhythms at the behavioral, neuronal, and molecular levels. The specific aims of this proposal are: 1. To map the neural substrates of na function. NA may function in circadian pacemaker neurons to regulate masking behavior. Tissue-specific rescue and assessments of NA distribution will be performed to address NA function in circadian and photoreceptor neurons. The regulation of NA by the clock and/or light will also be examined. 2. To define the electrophysiological phenotype of na mutant neurons. A method has been developed to examine the electrical properties of Drosophila pacemaker neurons. Characterization of ionic currents in these key neurons, the effects of na on these currents, as well as the electrophysiological properties of the elusive NA channel itself will be examined, including regulation by the circadian clock and/or light. 3. To assess the consequences of altered NA channel properties. NA is a unique and conserved member of the voltage-gated cation channel family. NA reveals many signatures of ion channels including highly conserved pore selectivity and voltage sensor sequences. The function of NA channels with altered pore or voltage sensor sequences will be tested by behavioral rescue. In total, these studies will define the electrophysiological function of NA within distinct anatomic pathways relevant to circadian and masking regulation. Given the conservation of clocks and NA, this proposal should illuminate human daily rhythms and their contribution to disease.

DESCRIPTION (provided by applicant): Disturbed daily rhythms have been implicated in a variety of brain disorders. These rhythms are regulated by at least two intertwined pathways, one is the circadian clock and a second is the masking pathway that mediates activity in response to light. This proposal addresses these pathways using the fruit fly, Drosophila melanogaster. The genetic basis of circadian and masking function appears conserved, suggesting that findings in the fly will be widely applicable. A critical role for an enigmatic and highly conserved ion channel narrow abdomen (na) has been identified in both circadian output and masking regulation. These observations underlie the overall goal of this proposal, which is to provide an integrated view of na function in both masking and circadian rhythms at the behavioral, neuronal, and molecular levels. The specific aims of this proposal are: 1. To map the neural substrates of na function. NA may function in circadian pacemaker neurons to regulate masking behavior. Tissue-specific rescue and assessments of NA distribution will be performed to address NA function in circadian and photoreceptor neurons. The regulation of NA by the clock and/or light will also be examined. 2. To define the electrophysiological phenotype of na mutant neurons. A method has been developed to examine the electrical properties of Drosophila pacemaker neurons. Characterization of ionic currents in these key neurons, the effects of na on these currents, as well as the electrophysiological properties of the elusive NA channel itself will be examined, including regulation by the circadian clock and/or light. 3. To assess the consequences of altered NA channel properties. NA is a unique and conserved member of the voltage-gated cation channel family. NA reveals many signatures of ion channels including highly conserved pore selectivity and voltage sensor sequences. The function of NA channels with altered pore or voltage sensor sequences will be tested by behavioral rescue. In total, these studies will define the electrophysiological function of NA within distinct anatomic pathways relevant to circadian and masking regulation. Given the conservation of clocks and NA, this proposal should illuminate human daily rhythms and their contribution to disease.

DESCRIPTION (provided by applicant): Defects in circadian clocks have been implicated in a variety of clinical disorders. Emerging evidence implicates communication between neurons in synchronizing and sustaining circadian clocks. The fruit fly Drosophila has been a powerful model to elucidate the underlying mechanisms of clocks, many aspects of which are highly conserved with humans. In the fruit fly, the neuropeptide PIGMENT DISPERSING FACTOR (PDF) is central to synchronizing neural pacemakers and regulating neural outputs. We have recently identified the G-protein coupled receptor for PDF (PDFR). The identification of this receptor affords an opportunity to address central questions related to circadian pacemaker function. How do circadian clocks drive downstream neural circuits to control behavior, such as sleep and wake? What is the role of PDF in coupling of neural oscillators? What are the mechanisms by which PDF resets core oscillators and drives rhythmic behaviors? The specific aims of the proposal are: 1. To map the cellular substrates of PDF receptor function in behavioral and molecular circadian rhythms. Remarkably little is known about the neural substrates that mediate PDF receptor action in circadian behavior. To address this issue, we will use tissue-specific PDFR rescue and overexpression in circadian and potential downstream neural circuits. In addition, we will assess the distribution of PDFR in the brain. 2. To examine the role of PDF signaling in coupling of neural circadian pacemakers. To assay coupling, we will manipulate the speed of the clock in subsets of the circadian neural network and assay the consequences on interconnected oscillators in the presence or absence of PDF signalling. 3. To examine the molecular mechanisms by which PDF resets the core circadian clock and output pathways. We will examine the molecular consequences of loss of PDFR on the core clock as well as cAMP and MAPK signalling pathways. Using novel electrophysiological approaches, we will examine the effects of exogenous PDF on electrical properties of pacemaker and output neurons. We will analyze genetic interactions between PDF/PDFR, core clock, cAMP/MAPK, and membrane excitability mutants. These studies should elucidate the molecular and neural circuitry essential for PDFR action in circadian behavior. They also exploit the unique advantages of the Drosophila system, including the ease of tissue-specific rescue studies, the ability to manipulate the clock in identified subsets of pacemaker neurons, and the extensive genetic resources to examine signalling pathways and the core clock in the whole animal. Given the conservation with mammalian systems, this work should provide insights into the mechanisms by which neuropeptides mediate normal and disrupted circadian rhythms in human disease. Project Narrative Defects in circadian clocks have been implicated in a variety of clinical disorders. Communication between neural pacemakers and to their downstream targets is mediated by neuropeptides. We will elucidate the role of a circadian neuropeptide in synchronizing circadian clocks and communicating timing information in a simple animal model. Given the potential conservation with humans, this work should provide insight into the mechanisms by which neuropeptides mediate normal and disrupted circadian rhythms in human disease. Project Narrative Defects in circadian clocks have been implicated in a variety of clinical disorders. Communication between neural pacemakers and to their downstream targets is mediated by neuropeptides. We will elucidate the role of a circadian neuropeptide in synchronizing circadian clocks and communicating timing information in a simple animal model. Given the potential conservation with humans, this work should provide insight into the mechanisms by which neuropeptides mediate normal and disrupted circadian rhythms in human disease.

DESCRIPTION (provided by applicant): Inadequate sleep afflicts over 50 million Americans, contributing to reduced cognitive function and psychiatric disorders. How underlying sleep mechanisms go awry in sleep disorders and mental illness to disrupt cognitive function is not well understood at the molecular level. Studies suggest an intimate link between mental illness, sleep homeostasis, synaptic plasticity, and learning and memory. To address the cellular and molecular mechanisms linking these neural processes, we have been using a simple model organism, the fruit fly Drosophila. Remarkably, the fruit fly exhibits many of the core features of sleep. In fact, genetic analysis suggests that many genes involved in regulating sleep levels are conserved between Drosophila and mammals. As part of our studies to understand sleep regulation in Drosophila, we identified a role for specific neural loci, termed the mushroom bodies (MBs), in promoting restorative sleep. Of note, the MBs are also central to learning and memory. To further understand the neural and molecular basis linking sleep, arousal, and neuroplasticity, we propose to further refine MB neurons playing specific roles in sleep homeostasis. In addition, we will assess the effects of sleep deprivation on MB-dependent learning and memory and MB synapses. We will also examine the link between learning and memory genes and sleep homeostasis, including a focus on their role in mediating the effects of sleep loss on learning and memory. These studies take advantage of many aspects of the Drosophila system, including conserved mechanisms of sleep, arousal, and memory, the ability to manipulate in a targeted manner the function of neural circuits in vivo and to easily manipulate gene function to elucidate mechanisms linking sleep, memory and plasticity. Given the genetic conservation of sleep and memory pathways, these studies may shed light on underlying mechanisms of disordered sleep and its relationship to learning and memory as well as mental illness in humans.

Neurodegenerative diseases, such as Alzheimer's and Huntington's, commonly involve the accumulation and aggregation of neurotoxic proteins that impair and ultimately destroy specific neurons. Identifying processes that can slow neurodegeneration, especially before irreversible cell death, is a major challenge for the development of effective therapeutics. Accumulating evidence suggests that disrupted clocks are associated with, and even potentially alter, neurodegeneration at this early stage. To address the mechanistic relationship between circadian clocks and neurodegenerative diseases, we are using Huntington's disease (HD) as a model. HD is caused by a triplet repeat expansion resulting in an expansion of a polyglutamine repeat in the Huntingtin protein (mHtt). Accumulation of mHtt results in degeneration of striatal as well as cortical neurons, resulting in the characteristic motor and cognitive symptoms, and ultimately death. Considerable evidence from human and animal studies indicates that mHtt impairs circadian rhythms often before characteristic motor symptoms are even evident. In fact, master circadian pacemaker neurons are lost in HD patients. Yet little is known about the molecular mechanisms by which mHtt impairs circadian rhythmicity. In addition, it is unknown if circadian clocks, in turn, can modulate HD pathogenesis. Tostudy the interplay between clocks and HD, the fruit fly Drosophila, a well-established model organism in the study of neurodegenerative disease and circadian clocks, has been employed. Both environmental and genetic perturbations of the circadian clock were shown to alter mHtt-mediated neurodegeneration, revealing that circadian clocks are not only a target of mHtt but may also be an important player in mediating mHtt-mediated pathogenesis. To identify potential genetic pathways that mediate the effect of the clock on mHtt, a novel behavioral platform has been developed for screening HD modifiers that would allow the identification of those genes that can modify pre-degenerative/functional and/or cell death effects of mHtt. As part of this screen, several novel pathways that mediate mHtt effects on behavior have been discovered. Here this successful screen will be expanded to discover novel pathways. In addition, the molecular mechanisms by which novel modifiers function will be explored in terms of mHtt inclusions, molecular clocks, and cell death. These deep conservation with vertebrate studies exploit the advantages of the Drosophila system including the models of circadian clocks and mechanisms of pathogenicity. HighmHtt throughput fly genetics will be applied to reveal the elusive molecular and cellular pathways that bi-directionally link mHtt to clock disruption, a relatively understudied area of HD pathology and thus one ripe for the discovery of novel mechanisms.