Publications by authors named "Amita Sehgal"

135 Publications

Short and long sleeping mutants reveal links between sleep and macroautophagy.

Elife 2021 Jun 4;10. Epub 2021 Jun 4.

Chronobiology and Sleep Institute, Perelman Medical School of University of Pennsylvania, Philadelphia, United States.

Sleep is a conserved and essential behavior, but its mechanistic and functional underpinnings remain poorly defined. Through unbiased genetic screening in , we discovered a novel short-sleep mutant we named . Positional cloning and subsequent complementation, CRISPR/Cas9 knock-out, and RNAi studies identified Argus as a transmembrane protein that acts in adult peptidergic neurons to regulate sleep. mutants accumulate undigested Atg8a(+) autophagosomes, and genetic manipulations impeding autophagosome formation suppress sleep phenotypes, indicating that autophagosome accumulation drives short-sleep. Conversely, a neurodegenerative mutant that impairs autophagosome formation was identified independently as a gain-of-sleep mutant, and targeted RNAi screens identified additional genes involved in autophagosome formation whose knockdown increases sleep. Finally, autophagosomes normally accumulate during the daytime and nighttime sleep deprivation extends this accumulation into the following morning, while daytime gaboxadol feeding promotes sleep and reduces autophagosome accumulation at nightfall. In sum, our results paradoxically demonstrate that wakefulness increases and sleep decreases autophagosome levels under unperturbed conditions, yet strong and sustained upregulation of autophagosomes decreases sleep, whereas strong and sustained downregulation of autophagosomes increases sleep. The complex relationship between sleep and autophagy suggested by our findings may have implications for pathological states including chronic sleep disorders and neurodegeneration, as well as for integration of sleep need with other homeostats, such as under conditions of starvation.
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http://dx.doi.org/10.7554/eLife.64140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8177895PMC
June 2021

clock cells use multiple mechanisms to transmit time-of-day signals in the brain.

Proc Natl Acad Sci U S A 2021 Mar;118(10)

Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;

Regulation of circadian behavior and physiology by the brain clock requires communication from central clock neurons to downstream output regions, but the mechanism by which clock cells regulate downstream targets is not known. We show here that the pars intercerebralis (PI), previously identified as a target of the morning cells in the clock network, also receives input from evening cells. We determined that morning and evening clock neurons have time-of-day-dependent connectivity to the PI, which is regulated by specific peptides as well as by fast neurotransmitters. Interestingly, PI cells that secrete the peptide DH44, and control rest:activity rhythms, are inhibited by clock inputs while insulin-producing cells (IPCs) are activated, indicating that the same clock cells can use different mechanisms to drive cycling in output neurons. Inputs of morning cells to IPCs are relevant for the circadian rhythm of feeding, reinforcing the role of the PI as a circadian relay that controls multiple behavioral outputs. Our findings provide mechanisms by which clock neurons signal to nonclock cells to drive rhythms of behavior.
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http://dx.doi.org/10.1073/pnas.2019826118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958456PMC
March 2021

Loss of circadian protection against influenza infection in adult mice exposed to hyperoxia as neonates.

Elife 2021 Mar 2;10. Epub 2021 Mar 2.

The Children's Hospital of Philadelphia, Philadelphia, United States.

Adverse early-life exposures have a lasting negative impact on health. Neonatal hyperoxia that is a risk factor for bronchopulmonary dysplasia confers susceptibility to influenza A virus (IAV) infection later in life. Given our previous findings that the circadian clock protects against IAV, we asked if the long-term impact of neonatal hyperoxia vis-à-vis IAV infection includes circadian disruption. Here, we show that neonatal hyperoxia abolishes the clock-mediated time of day protection from IAV in mice, independent of viral burden through host tolerance pathways. We discovered that the lung intrinsic clock (and not the central or immune clocks) mediated this dysregulation. Loss of circadian protein, , in alveolar type 2 (AT2) cells recapitulates the increased mortality, loss of temporal gating, and other key features of hyperoxia-exposed animals. Our data suggest a novel role for the circadian clock in AT2 cells in mediating long-term effects of early-life exposures to the lungs.
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http://dx.doi.org/10.7554/eLife.61241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924938PMC
March 2021

Time-of-day specificity of anticancer drugs may be mediated by circadian regulation of the cell cycle.

Sci Adv 2021 Feb 12;7(7). Epub 2021 Feb 12.

Howard Hughes Medical Institute, Chronobiology and Sleep Institute (CSI), Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Circadian rhythms are an integral part of physiology, underscoring their relevance for the treatment of disease. We conducted cell-based high-throughput screening to investigate time-of-day influences on the activity of known antitumor agents and found that many compounds exhibit daily rhythms of cytotoxicity concomitant with previously reported oscillations of target genes. Rhythmic action of HSP90 inhibitors was mediated by specific isoforms of HSP90, genetic perturbation of which affected the cell cycle. Furthermore, clock mutants affected the cell cycle in parallel with abrogating rhythms of cytotoxicity, and pharmacological inhibition of the cell cycle also eliminated rhythmic drug effects. An HSP90 inhibitor reduced growth rate of a mouse melanoma in a time-of-day-specific manner, but efficacy was impaired in clock-deficient tumors. These results provide a powerful rationale for appropriate daily timing of anticancer drugs and suggest circadian regulation of the cell cycle within the tumor as an underlying mechanism.
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http://dx.doi.org/10.1126/sciadv.abd2645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880601PMC
February 2021

A circadian clock regulates efflux by the blood-brain barrier in mice and human cells.

Nat Commun 2021 01 27;12(1):617. Epub 2021 Jan 27.

Chronobiology and Sleep Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.

The blood-brain barrier (BBB) is critical for neural function. We report here circadian regulation of the BBB in mammals. Efflux of xenobiotics by the BBB oscillates in mice, with highest levels during the active phase and lowest during the resting phase. This oscillation is abrogated in circadian clock mutants. To elucidate mechanisms of circadian regulation, we profiled the transcriptome of brain endothelial cells; interestingly, we detected limited circadian regulation of transcription, with no evident oscillations in efflux transporters. We recapitulated the cycling of xenobiotic efflux using a human microvascular endothelial cell line to find that the molecular clock drives cycling of intracellular magnesium through transcriptional regulation of TRPM7, which appears to contribute to the rhythm in efflux. Our findings suggest that considering circadian regulation may be important when therapeutically targeting efflux transporter substrates to the CNS.
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http://dx.doi.org/10.1038/s41467-020-20795-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841146PMC
January 2021

The 2020 Pittendrigh/Aschoff Lecture: My Circadian Journey.

Authors:
Amita Sehgal

J Biol Rhythms 2021 02 11;36(1):84-96. Epub 2021 Jan 11.

Howard Hughes Medical Institute, Chronobiology and Sleep Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

The circadian field has come a long way since I started as a postdoctoral fellow ~30 years ago. At the time, the only known animal clock gene was , so I had the privilege of witnessing, and participating in, the molecular revolution that took us from the discovery of the circadian clock mechanism to the identification of pathways that link clocks to behavior and physiology. This lecture highlights my role and perspective in these developments, and also demonstrates how the successful use of Drosophila for studies of circadian rhythms inspired us to develop a fly model for sleep. I also touch upon my experiences as a non-white immigrant woman navigating my way through the US science and education system, and hope my story will be of interest to some.
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http://dx.doi.org/10.1177/0748730420982398DOI Listing
February 2021

Manipulations of the olfactory circuit highlight the role of sensory stimulation in regulating sleep amount.

Sleep 2021 05;44(5)

Howard Hughes Medical Institute, Chronobiology and Sleep Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

Study Objectives: While wake duration is a major sleep driver, an important question is if wake quality also contributes to controlling sleep. In particular, we sought to determine whether changes in sensory stimulation affect sleep in Drosophila. As Drosophila rely heavily on their sense of smell, we focused on manipulating olfactory input and the olfactory sensory pathway.

Methods: Sensory deprivation was first performed by removing antennae or applying glue to antennae. We then measured sleep in response to neural activation, via expression of the thermally gated cation channel TRPA1, or inhibition, via expression of the inward rectifying potassium channel KIR2.1, of subpopulations of neurons in the olfactory pathway. Genetically restricting manipulations to adult animals prevented developmental effects.

Results: We find that olfactory deprivation reduces sleep, largely independently of mushroom bodies that integrate olfactory signals for memory consolidation and have previously been implicated in sleep. However, specific neurons in the lateral horn, the other third-order target of olfactory input, affect sleep. Also, activation of inhibitory second-order projection neurons increases sleep. No single neuronal population in the olfactory processing pathway was found to bidirectionally regulate sleep, and reduced sleep in response to olfactory deprivation may be masked by temperature changes.

Conclusions: These findings demonstrate that Drosophila sleep is sensitive to sensory stimulation, and identify novel sleep-regulating neurons in the olfactory circuit. Scaling of signals across the circuit may explain the lack of bidirectional effects when neuronal activity is manipulated. We propose that olfactory inputs act through specific circuit components to modulate sleep in flies.
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http://dx.doi.org/10.1093/sleep/zsaa265DOI Listing
May 2021

Monitoring Electrical Activity in Drosophila Circadian Output Neurons.

Methods Mol Biol 2021 ;2130:221-232

Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA, USA.

Drosophila melanogaster is a powerful model organism used to study circadian rhythms, historically for elucidating the molecular basis of the clock and, more recently, for allowing for dissection of neural circuits underlying rhythmic behavior. The fly can be used to investigate the neuronal basis of complex behaviors at single-neuron resolution. Patch clamp electrophysiology permits single-neuron recording of resting membrane potential and action potential firing in response to genetic or environmental manipulations or application of drugs and neurotransmitters. Here we describe a protocol for dissecting Drosophila brains for electrophysiology, setting up and using a patch clamp system, and analyzing firing data around the circadian day and in stimulation-response experiments to test for functional neuronal connectivity in circadian circuits.
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http://dx.doi.org/10.1007/978-1-0716-0381-9_17DOI Listing
March 2021

Availability of food determines the need for sleep in memory consolidation.

Nature 2021 01 2;589(7843):582-585. Epub 2020 Dec 2.

Howard Hughes Medical Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.

Sleep remains a major mystery of biology, with little understood about its basic function. One of the most commonly proposed functions of sleep is the consolidation of memory. However, as conditions such as starvation require the organism to be awake and active, the ability to switch to a memory consolidation mechanism that is not contingent on sleep may confer an evolutionary advantage. Here we identify an adaptive circuit-based mechanism that enables Drosophila to form sleep-dependent and sleep-independent memory. Flies fed after appetitive conditioning needed increased sleep for memory consolidation, but flies starved after training did not require sleep to form memories. Memory in fed flies is mediated by the anterior-posterior α'/β' neurons of the mushroom body, while memory under starvation is mediated by medial α'/β' neurons. Sleep-dependent and sleep-independent memory rely on distinct dopaminergic neurons and corresponding mushroom body output neurons. However, sleep and memory are coupled such that mushroom body neurons required for sleep-dependent memory also promote sleep. Flies lacking Neuropeptide F display sleep-dependent memory even when starved, suggesting that circuit selection is determined by hunger. This plasticity in memory circuits enables flies to retain essential information in changing environments.
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http://dx.doi.org/10.1038/s41586-020-2997-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856038PMC
January 2021

The Lineage Before Time: Circadian and Nonclassical Clock Influences on Development.

Annu Rev Cell Dev Biol 2020 10;36:469-509

Chronobiology and Sleep Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; email:

Diverse factors including metabolism, chromatin remodeling, and mitotic kinetics influence development at the cellular level. These factors are well known to interact with the circadian transcriptional-translational feedback loop (TTFL) after its emergence. What is only recently becoming clear, however, is how metabolism, mitosis, and epigenetics may become organized in a coordinated cyclical precursor signaling module in pluripotent cells prior to the onset of TTFL cycling. We propose that both the precursor module and the TTFL module constrain cellular identity when they are active during development, and that the emergence of these modules themselves is a key lineage marker. Here we review the component pathways underlying these ideas; how proliferation, specification, and differentiation decisions in both developmental and adult stem cell populations are or are not regulated by the classical TTFL; and emerging evidence that we propose implies a primordial clock that precedes the classical TTFL and influences early developmental decisions.
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http://dx.doi.org/10.1146/annurev-cellbio-100818-125454DOI Listing
October 2020

AANAT1 functions in astrocytes to regulate sleep homeostasis.

Elife 2020 09 21;9. Epub 2020 Sep 21.

Centre for Research in Neuroscience, Department of Neurology and Neurosurgery, McGill University, Montreal, Canada.

How the brain controls the need and acquisition of recovery sleep after prolonged wakefulness is an important issue in sleep research. The monoamines serotonin and dopamine are key regulators of sleep in mammals and in . We found that the enzyme arylalkylamine N-acetyltransferase 1 (AANAT1) is expressed by astrocytes and specific subsets of neurons in the adult brain. AANAT1 acetylates monoamines and inactivates them, and we found that AANAT1 limited the accumulation of serotonin and dopamine in the brain upon sleep deprivation (SD). Loss of AANAT1 from astrocytes, but not from neurons, caused flies to increase their daytime recovery sleep following overnight SD. Together, these findings demonstrate a crucial role for AANAT1 and astrocytes in the regulation of monoamine bioavailability and homeostatic sleep.
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http://dx.doi.org/10.7554/eLife.53994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550187PMC
September 2020

Glial Metabolic Rewiring Promotes Axon Regeneration and Functional Recovery in the Central Nervous System.

Cell Metab 2020 11 16;32(5):767-785.e7. Epub 2020 Sep 16.

Raymond G. Perelman Center for Cellular and Molecular Therapeutics, the Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Axons in the mature central nervous system (CNS) fail to regenerate after axotomy, partly due to the inhibitory environment constituted by reactive glial cells producing astrocytic scars, chondroitin sulfate proteoglycans, and myelin debris. We investigated this inhibitory milieu, showing that it is reversible and depends on glial metabolic status. We show that glia can be reprogrammed to promote morphological and functional regeneration after CNS injury in Drosophila via increased glycolysis. This enhancement is mediated by the glia derived metabolites: L-lactate and L-2-hydroxyglutarate (L-2HG). Genetically/pharmacologically increasing or reducing their bioactivity promoted or impeded CNS axon regeneration. L-lactate and L-2HG from glia acted on neuronal metabotropic GABA receptors to boost cAMP signaling. Local application of L-lactate to injured spinal cord promoted corticospinal tract axon regeneration, leading to behavioral recovery in adult mice. Our findings revealed a metabolic switch to circumvent the inhibition of glia while amplifying their beneficial effects for treating CNS injuries.
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http://dx.doi.org/10.1016/j.cmet.2020.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642184PMC
November 2020

Anandamide Metabolites Protect against Seizures through the TRP Channel Water Witch in Drosophila melanogaster.

Cell Rep 2020 06;31(9):107710

Pharmacology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Chronobiology and Sleep Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Howard Hughes Medical Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Endocannabinoids protect against seizures, but their mechanism of action is still unclear, as they can have effects independent of known cannabinoid receptors. Using Drosophila melanogaster, which lacks canonical cannabinoid receptors, we report that the endocannabinoids anandamide and 2-arachidonoylglycerol protect against seizures in multiple fly seizure models. Surprisingly, inhibition of anandamide catabolism renders flies insensitive to protection by anandamide, indicating that anandamide metabolites are responsible for seizure protection. Consistent with this finding, arachidonic acid, a direct metabolite of anandamide, protects against seizures. To identify downstream effectors, we test for a role of transient receptor potential (TRP) channels and find that the TRPV1 antagonist capsazepine blocks the protective effect of anandamide. Also, a targeted genetic screen of TRP channels identifies water witch as a mediator of protection by anandamide. Using a Drosophila model, we reveal the role of arachidonic acid in seizure protection and identify a cannabinoid-receptor-1/2-independent mechanism of endocannabinoid seizure protection.
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http://dx.doi.org/10.1016/j.celrep.2020.107710DOI Listing
June 2020

Circadian and Sleep Metabolomics Across Species.

J Mol Biol 2020 05 3;432(12):3578-3610. Epub 2020 May 3.

Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Under normal circadian function, metabolic control is temporally coordinated across tissues and behaviors with a 24-h period. However, circadian disruption results in negative consequences for metabolic homeostasis including energy or redox imbalances. Yet, circadian disruption has become increasingly prevalent within today's society due to many factors including sleep loss. Metabolic consequences of both have been revealed by metabolomics analyses of circadian biology and sleep. Specifically, two primary analytical platforms, mass spectrometry and nuclear magnetic resonance spectroscopy, have been used to study molecular clock and sleep influences on overall metabolic rhythmicity. For example, human studies have demonstrated the prevalence of metabolic rhythms in human biology, as well as pan-metabolome consequences of sleep disruption. However, human studies are limited to peripheral metabolic readouts primarily through minimally invasive procedures. For further tissue- and organism-specific investigations, a number of model systems have been studied, based upon the conserved nature of both the molecular clock and sleep across species. Here we summarize human studies as well as key findings from metabolomics studies using mice, Drosophila, and zebrafish. While informative, a limitation in existing literature is a lack of interpretation regarding dynamic synthesis or catabolism within metabolite pools. To this extent, future work incorporating isotope tracers, specific metabolite reporters, and single-cell metabolomics may provide a means of exploring dynamic activity in pathways of interest.
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http://dx.doi.org/10.1016/j.jmb.2020.04.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781158PMC
May 2020

The Glial Perspective on Sleep and Circadian Rhythms.

Annu Rev Neurosci 2020 07 19;43:119-140. Epub 2020 Feb 19.

Chronobiology and Sleep Institute, Perelman School of Medicine, and Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; email:

While neurons and circuits are almost unequivocally considered to be the computational units and actuators of behavior, a complete understanding of the nervous system must incorporate glial cells. Far beyond a copious but passive substrate, glial influence is inextricable from neuronal physiology, whether during developmental guidance and synaptic shaping or through the trophic support, neurotransmitter and ion homeostasis, cytokine signaling and immune function, and debris engulfment contributions that this class provides throughout an organism's life. With such essential functions, among a growing literature of nuanced roles, it follows that glia are consequential to behavior in adult animals, with novel genetic tools allowing for the investigation of these phenomena in living organisms. We discuss here the relevance of glia for maintaining circadian rhythms and also for serving functions of sleep.
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http://dx.doi.org/10.1146/annurev-neuro-091819-094557DOI Listing
July 2020

RNA Splicing Factor Mutations That Cause Retinitis Pigmentosa Result in Circadian Dysregulation.

J Biol Rhythms 2020 02 15;35(1):72-83. Epub 2019 Nov 15.

Chronobiology and Sleep institute (CSI) and Howard Hughes Medical Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Circadian clocks regulate multiple physiological processes in the eye, but their requirement for retinal health remains unclear. We previously showed that Drosophila homologs of spliceosome proteins implicated in human retinitis pigmentosa (RP), the most common genetically inherited cause of blindness, have a role in the brain circadian clock. In this study, we report circadian phenotypes in murine models of RP. We found that mice carrying a homozygous H2309P mutation in () display a lengthened period of the circadian wheel-running activity rhythm. We show also that the daily cycling of circadian gene expression is dampened in the retina of H2309P mice. Surprisingly, molecular rhythms are intact in the eye cup, which includes the retinal pigment epithelium (RPE), even though the RPE is thought to be the primary tissue affected in this form of RP. Downregulation of , another RNA splicing factor implicated in RP, leads to period lengthening in a human cell culture model. The period of circadian bioluminescence in primary fibroblasts of human RP patients is not significantly altered. Together, these studies link a prominent retinal disorder to circadian deficits, which could contribute to disease pathology.
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http://dx.doi.org/10.1177/0748730419887876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7428848PMC
February 2020

Misregulation of Drosophila Myc Disrupts Circadian Behavior and Metabolism.

Cell Rep 2019 11;29(7):1778-1788.e4

Ludwig Institute for Cancer Research, New York, NY 10017, USA; The Wistar Institute, Philadelphia, PA 19104, USA. Electronic address:

Drosophila Myc (dMyc) is highly conserved and functions as a transcription factor similar to mammalian Myc. We previously found that oncogenic Myc disrupts the molecular clock in cancer cells. Here, we demonstrate that misregulation of dMyc expression affects Drosophila circadian behavior. dMyc overexpression results in a high percentage of arrhythmic flies, concomitant with increases in the expression of clock genes cyc, tim, cry, and cwo. Conversely, flies with hypomorphic mutations in dMyc exhibit considerable arrhythmia, which can be rescued by loss of dMnt, a suppressor of dMyc activity. Metabolic profiling of fly heads revealed that loss of dMyc and its overexpression alter steady-state metabolite levels and have opposing effects on histidine, the histamine precursor, which is rescued in dMyc mutants by ablation of dMnt and could contribute to effects of dMyc on locomotor behavior. Our results demonstrate a role of dMyc in modulating Drosophila circadian clock, behavior, and metabolism.
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http://dx.doi.org/10.1016/j.celrep.2019.10.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910219PMC
November 2019

The NRON complex controls circadian clock function through regulated PER and CRY nuclear translocation.

Sci Rep 2019 08 15;9(1):11883. Epub 2019 Aug 15.

Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.

Post-translational regulation plays a central role in the circadian clock mechanism. However, nucleocytoplasmic translocation of core clock proteins, a key step in circadian timekeeping, is not fully understood. Earlier we found that the NRON scaffolding complex regulates nuclear translocation of NFAT and its signaling. Here, we show that components of the NRON complex also regulate the circadian clock. In peripheral cell clock models, genetic perturbation of the NRON complex affects PER and CRY protein nuclear translocation, dampens amplitude, and alters period length. Further, we show small molecules targeting the NFAT pathway alter nuclear translocation of PER and CRY proteins and impact circadian rhythms in peripheral cells and tissue explants of the master clock in the suprachiasmatic nucleus. Taken together, these studies highlight a key role for the NRON complex in regulating PER/CRY subcellular localization and circadian timekeeping.
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http://dx.doi.org/10.1038/s41598-019-48341-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695496PMC
August 2019

Splicing the Clock to Maintain and Entrain Circadian Rhythms.

J Biol Rhythms 2019 12 7;34(6):584-595. Epub 2019 Aug 7.

Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, Pennsylvania.

Circadian clocks drive daily rhythms of physiology and behavior in multiple organisms and synchronize these rhythms to environmental cycles of light and temperature. The basic mechanism of the clock consists of a transcription-translation feedback loop, in which key clock proteins negatively regulate their own transcription. Although much of the focus with respect to clock mechanisms has been on the regulation of transcription and on the stability and activity of clock proteins, it is clear that other regulatory processes also have to be involved to explain aspects of clock function. Here, we review the role of alternative splicing in circadian clocks. Starting with a discussion of the clock and then extending to other major circadian model systems, we describe how the control of alternative splicing enables organisms to maintain their circadian clocks as well as to respond to environmental inputs, in particular to temperature changes.
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http://dx.doi.org/10.1177/0748730419868136DOI Listing
December 2019

Regulation of the Blood-Brain Barrier by Circadian Rhythms and Sleep.

Trends Neurosci 2019 07;42(7):500-510

Center for Sleep and Circadian Neurobiology, Chronobiology Program, and Howard Hughes Medical Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The blood-brain barrier (BBB) is an evolutionarily conserved, structural, and functional separation between circulating blood and the central nervous system (CNS). By controlling permeability into and out of the nervous system, the BBB has a critical role in the precise regulation of neural processes. Here, we review recent studies demonstrating that permeability at the BBB is dynamically controlled by circadian rhythms and sleep. An endogenous circadian rhythm in the BBB controls transporter function, regulating permeability across the BBB. In addition, sleep promotes the clearance of metabolites along the BBB, as well as endocytosis across the BBB. Finally, we highlight the implications of this regulation for diseases, including epilepsy.
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http://dx.doi.org/10.1016/j.tins.2019.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602072PMC
July 2019

Altered ocular parameters from circadian clock gene disruptions.

PLoS One 2019 18;14(6):e0217111. Epub 2019 Jun 18.

Center for Visual and Neurocognitive Rehabilitation, Atlanta VA Medical Center, Decatur, Georgia, United States of America.

The pathophysiology of refractive errors is poorly understood. Myopia (nearsightedness) in particular both blurs vision and predisposes the eye to many blinding diseases during adulthood. Based on past findings of diurnal variations in the dimensions of the eyes of humans and other vertebrates, altered diurnal rhythms of these ocular dimensions with experimentally induced myopia, and evolving evidence that ambient light exposures influence refractive development, we assessed whether disturbances in circadian signals might alter the refractive development of the eye. In mice, retinal-specific knockout of the clock gene Bmal1 induces myopia and elongates the vitreous chamber, the optical compartment separating the lens and the retina. These alterations simulate common ocular findings in clinical myopia. In Drosophila melanogaster, knockouts of the clock genes cycle or period lengthen the pseudocone, the optical component of the ommatidium that separates the facet lens from the photoreceptors. Disrupting circadian signaling thus alters optical development of the eye in widely separated species. We propose that mechanisms of myopia include circadian dysregulation, a frequent occurrence in modern societies where myopia also is both highly prevalent and increasing at alarming rates. Addressing circadian dysregulation may improve understanding of the pathogenesis of refractive errors and introduce novel therapeutic approaches to ameliorate myopia development in children.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217111PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581257PMC
February 2020

G1/S cell cycle regulators mediate effects of circadian dysregulation on tumor growth and provide targets for timed anticancer treatment.

PLoS Biol 2019 04 30;17(4):e3000228. Epub 2019 Apr 30.

Penn Chronobiology, Howard Hughes Medical Institute, Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

Circadian disruption has multiple pathological consequences, but the underlying mechanisms are largely unknown. To address such mechanisms, we subjected transformed cultured cells to chronic circadian desynchrony (CCD), mimicking a chronic jet-lag scheme, and assayed a range of cellular functions. The results indicated a specific circadian clock-dependent increase in cell proliferation. Transcriptome analysis revealed up-regulation of G1/S phase transition genes (myelocytomatosis oncogene cellular homolog [Myc], cyclin D1/3, chromatin licensing and DNA replication factor 1 [Cdt1]), concomitant with increased phosphorylation of the retinoblastoma (RB) protein by cyclin-dependent kinase (CDK) 4/6 and increased G1-S progression. Phospho-RB (Ser807/811) was found to oscillate in a circadian fashion and exhibit phase-shifted rhythms in circadian desynchronized cells. Consistent with circadian regulation, a CDK4/6 inhibitor approved for cancer treatment reduced growth of cultured cells and mouse tumors in a time-of-day-specific manner. Our study identifies a mechanism that underlies effects of circadian disruption on tumor growth and underscores the use of treatment timed to endogenous circadian rhythms.
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http://dx.doi.org/10.1371/journal.pbio.3000228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6490878PMC
April 2019

Genetic Mechanisms Underlying Sleep.

Cold Spring Harb Symp Quant Biol 2018 1;83:57-61. Epub 2019 Apr 1.

Howard Hughes Medical Institute, Perelman School of Medicine University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Sleep is important for cognitive ability, and perturbations of sleep are associated with a myriad of brain disorders. However, how sleep promotes health and function during wake is poorly understood. To address the cellular and molecular mechanisms underlying sleep, we use the fruit fly as a genetic model. Forward genetic approaches in flies were critical for deciphering molecular mechanisms of the circadian clock. Using similar approaches, we and others are gaining insights into the pathways that control sleep amount.
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http://dx.doi.org/10.1101/sqb.2018.83.037705DOI Listing
April 2019

A sleep-inducing gene, , links sleep and immune function in .

Science 2019 02;363(6426):509-515

Howard Hughes Medical Institute, Chronobiology Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Sleep remains a major mystery of biology. In particular, little is known about the mechanisms that account for the drive to sleep. In an unbiased screen of more than 12,000 lines, we identified a single gene, , that induces sleep. The NEMURI protein is an antimicrobial peptide that can be secreted ectopically to drive prolonged sleep (with resistance to arousal) and to promote survival after infection. Loss of increased arousability during daily sleep and attenuated the acute increase in sleep induced by sleep deprivation or bacterial infection. Conditions that increase sleep drive induced expression of in a small number of fly brain neurons and targeted it to the sleep-promoting, dorsal fan-shaped body. We propose that NEMURI is a bona fide sleep homeostasis factor that is particularly important under conditions of high sleep need; because these conditions include sickness, our findings provide a link between sleep and immune function.
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http://dx.doi.org/10.1126/science.aat1650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505470PMC
February 2019

Spliceosome factors target timeless () mRNA to control clock protein accumulation and circadian behavior in Drosophila.

Elife 2018 12 5;7. Epub 2018 Dec 5.

Chronobiology Program at Penn, Howard Hughes Medical Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States.

Transcription-translation feedback loops that comprise eukaryotic circadian clocks rely upon temporal delays that separate the phase of active transcription of clock genes, such as Drosophila and from negative feedback by the two proteins. However, our understanding of the mechanisms involved is incomplete. Through an RNA interference screen, we found that pre-mRNA processing 4 (PRP4) kinase, a component of the U4/U5.U6 triple small nuclear ribonucleoprotein (tri-snRNP) spliceosome, and other tri-snRNP components regulate cycling of the molecular clock as well as rest:activity rhythms. Unbiased RNA-Sequencing uncovered an alternatively spliced intron in whose increased retention upon downregulation leads to decreased TIM levels. We demonstrate that the splicing of is rhythmic with a phase that parallels delayed accumulation of the protein in a 24 hr cycle. We propose that alternative splicing constitutes an important clock mechanism for delaying the daily accumulation of clock proteins, and thereby negative feedback by them.

Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
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http://dx.doi.org/10.7554/eLife.39821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281371PMC
December 2018

Endocytosis at the blood-brain barrier as a function for sleep.

Elife 2018 11 26;7. Epub 2018 Nov 26.

Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States.

Glia are important modulators of neural activity, yet few studies link glia to sleep regulation. We find that blocking activity of the endocytosis protein, dynamin, in adult glia increases sleep and enhances sleep need, manifest as resistance to sleep deprivation. Surface glia comprising the fly equivalent of the blood-brain barrier (BBB) mediate the effect of dynamin on sleep. Blocking dynamin in the surface glia causes ultrastructural changes, albeit without compromising the integrity of the barrier. Supporting a role for endocytic trafficking in sleep, a screen of Rab GTPases identifies sleep-modulating effects of the recycling endosome Rab11 in surface glia. We also find that endocytosis is increased in BBB glia during sleep and reflects sleep need. We propose that endocytic trafficking through the BBB represents a function of sleep.
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http://dx.doi.org/10.7554/eLife.43326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6255390PMC
November 2018

Circadian and Sleep Circuits Ring Together.

Neuron 2018 11;100(3):514-516

Penn Chronobiology, Howard Hughes Medical Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Prolonged wakefulness stimulates the homeostatic need to sleep, but transition to sleep also depends on the circadian time of day. However, links between circadian and homeostatic influences are not well understood. Guo et al. (2018) identify a Drosophila circuit connecting circadian clock neurons to sleep-promoting ring neurons in the ellipsoid body.
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http://dx.doi.org/10.1016/j.neuron.2018.10.030DOI Listing
November 2018

Molecular and circuit mechanisms mediating circadian clock output in the Drosophila brain.

Eur J Neurosci 2020 01 16;51(1):268-281. Epub 2018 Aug 16.

Department of Neuroscience, Perelman School of Medicine, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, Pennsylvania.

A central question in the circadian biology field concerns the mechanisms that translate ~24-hr oscillations of the molecular clock into overt rhythms. Drosophila melanogaster is a powerful system that provided the first understanding of how molecular clocks are generated and is now illuminating the neural basis of circadian behavior. The identity of ~150 clock neurons in the Drosophila brain and their roles in shaping circadian rhythms of locomotor activity have been described before. This review summarizes mechanisms that transmit time-of-day signals from the clock, within the clock network as well as downstream of it. We also discuss the identification of functional multisynaptic circuits between clock neurons and output neurons that regulate locomotor activity.
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http://dx.doi.org/10.1111/ejn.14092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353709PMC
January 2020

Asymmetric vasopressin signaling spatially organizes the master circadian clock.

J Comp Neurol 2018 09 22;526(13):2048-2067. Epub 2018 Aug 22.

Department of Biomedical Sciences, Marquette University, Milwaukee, WI, 53233.

The suprachiasmatic nucleus (SCN) is the neural network that drives daily rhythms in behavior and physiology. The SCN encodes environmental changes through the phasing of cellular rhythms across its anteroposterior axis, but it remains unknown what signaling mechanisms regulate clock function along this axis. Here we demonstrate that arginine vasopressin (AVP) signaling organizes the SCN into distinct anteroposterior domains. Spatial mapping of SCN gene expression using in situ hybridization delineated anterior and posterior domains for AVP signaling components, including complementary patterns of V1a and V1b expression that suggest different roles for these two AVP receptors. Similarly, anteroposterior patterning of transcripts involved in Vasoactive Intestinal Polypeptide- and Prokineticin2 signaling was evident across the SCN. Using bioluminescence imaging, we then revealed that inhibiting V1A and V1B signaling alters period and phase differentially along the anteroposterior SCN. V1 antagonism lengthened period the most in the anterior SCN, whereas changes in phase were largest in the posterior SCN. Further, separately antagonizing V1A and V1B signaling modulated SCN function in a manner that mapped onto anteroposterior expression patterns. Lastly, V1 antagonism influenced SCN period and phase along the dorsoventral axis, complementing effects on the anteroposterior axis. Together, these results indicate that AVP signaling modulates SCN period and phase in a spatially specific manner, which is expected to influence how the master clock interacts with downstream tissues and responds to environmental changes. More generally, we reveal anteroposterior asymmetry in neuropeptide signaling as a recurrent organizational motif that likely influences neural computations in the SCN clock network.
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http://dx.doi.org/10.1002/cne.24478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158041PMC
September 2018

Cold Temperatures Fire up Circadian Neurons.

Cell Metab 2018 May;27(5):951-953

Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA, USA.

Circadian clocks monitor both light and temperature cycles to entrain behavior and physiology to the environment. Recently in Nature, Yadlapalli et al. (2018) identified a subgroup of Drosophila clock neurons that responds to temperature input with changes in intracellular calcium and mediates effects of temperature on circadian entrainment and sleep.
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http://dx.doi.org/10.1016/j.cmet.2018.04.016DOI Listing
May 2018