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1.
Cell Metab ; 35(10): 1704-1721.e6, 2023 10 03.
Article in English | MEDLINE | ID: mdl-37607543

ABSTRACT

Circadian disruptions impact nearly all people with Alzheimer's disease (AD), emphasizing both their potential role in pathology and the critical need to investigate the therapeutic potential of circadian-modulating interventions. Here, we show that time-restricted feeding (TRF) without caloric restriction improved key disease components including behavioral timing, disease pathology, hippocampal transcription, and memory in two transgenic (TG) mouse models of AD. We found that TRF had the remarkable capability of simultaneously reducing amyloid deposition, increasing Aß42 clearance, improving sleep and memory, and normalizing daily transcription patterns of multiple genes, including those associated with AD and neuroinflammation. Thus, our study unveils for the first time the pleiotropic nature of timed feeding on AD, which has far-reaching effects beyond metabolism, ameliorating neurodegeneration and the misalignment of circadian rhythmicity. Since TRF can substantially modify disease trajectory, this intervention has immediate translational potential, addressing the urgent demand for accessible approaches to reduce or halt AD progression.


Subject(s)
Alzheimer Disease , Mice , Animals , Humans , Alzheimer Disease/therapy , Alzheimer Disease/genetics , Mice, Transgenic , Disease Models, Animal , Circadian Rhythm , Brain/metabolism , Amyloid beta-Peptides
2.
Sci Adv ; 8(35): eabn9867, 2022 Sep 02.
Article in English | MEDLINE | ID: mdl-36054362

ABSTRACT

Seasonal changes in day length (photoperiod) affect numerous physiological functions. The suprachiasmatic nucleus (SCN)-paraventricular nucleus (PVN) axis plays a key role in processing photoperiod-related information. Seasonal variations in SCN and PVN neurotransmitter expression have been observed in humans and animal models. However, the molecular mechanisms by which the SCN-PVN network responds to altered photoperiod is unknown. Here, we show in mice that neuromedin S (NMS) and vasoactive intestinal polypeptide (VIP) neurons in the SCN display photoperiod-induced neurotransmitter plasticity. In vivo recording of calcium dynamics revealed that NMS neurons alter PVN network activity in response to winter-like photoperiod. Chronic manipulation of NMS neurons is sufficient to induce neurotransmitter switching in PVN neurons and affects locomotor activity. Our findings reveal previously unidentified molecular adaptations of the SCN-PVN network in response to seasonality and the role for NMS neurons in adjusting hypothalamic function to day length via a coordinated multisynaptic neurotransmitter switching affecting behavior.

3.
Mol Metab ; 57: 101431, 2022 03.
Article in English | MEDLINE | ID: mdl-34974160

ABSTRACT

OBJECTIVE: The increasing prevalence of obesity makes it important to increase the understanding of the maturation and function of the neuronal integrators and regulators of metabolic function. METHODS: Behavioral, molecular, and physiological analyses of transgenic mice with Sine oculis 3 (Six3) deleted in mature neurons using the Synapsincreallele. RESULTS: Conditional deletion of the homeodomain transcription factor Six3 in mature neurons causes dwarfism and weakens circadian wheel-running activity rhythms but increases general activity at night, and improves metabolic function, without impacting pubertal onset or fertility in males. The reduced growth in 6-week-old Six3fl/fl:Synapsincre (Six3syn) males correlates with increased somatostatin (SS) expression in the hypothalamus and reduced growth hormone (GH) in the pituitary. In contrast, 12-week-old Six3syn males have increased GH release, despite an increased number of the inhibitory SS neurons in the periventricular nucleus. GH is important in glucose metabolism, muscle function, and bone health. Interestingly, Six3syn males have improved glucose tolerance at 7, 12, and 18 weeks of age, which, in adulthood, is associated with increased % lean mass and increased metabolic rates. Further, 12-week-old Six3syn males have reduced bone mineralization and a lower bone mineral density, indicating that reduced GH levels during early life cause a long-term reduction in bone mineralization. CONCLUSION: Our study points to the novel role of Six3 in post-proliferative neurons to regulate metabolic function through SS neuron control of GH release.


Subject(s)
Dwarfism , Homeodomain Proteins , Animals , Dwarfism/genetics , Dwarfism/metabolism , Eye Proteins/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Male , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/metabolism
4.
J Neurosci Res ; 99(10): 2625-2645, 2021 10.
Article in English | MEDLINE | ID: mdl-34212416

ABSTRACT

The homeodomain transcription factors sine oculis homeobox 3 (Six3) and ventral anterior homeobox 1 (Vax1) are required for brain development. Their expression in specific brain areas is maintained in adulthood, where their functions are poorly understood. To identify the roles of Six3 and Vax1 in neurons, we conditionally deleted each gene using Synapsincre , a promoter targeting maturing neurons, and generated Six3syn and Vax1syn mice. Six3syn and Vax1syn females, but not males, had reduced fertility, due to impairment of the luteinizing hormone (LH) surge driving ovulation. In nocturnal rodents, the LH surge requires a precise timing signal from the brain's circadian pacemaker, the suprachiasmatic nucleus (SCN), near the time of activity onset. Indeed, both Six3syn and Vax1syn females had impaired rhythmic SCN output, which was associated with weakened Period 2 molecular clock function in both Six3syn and Vax1syn mice. These impairments were associated with a reduction of the SCN neuropeptide vasoactive intestinal peptide in Vax1syn mice and a modest weakening of SCN timekeeping function in both Six3syn and Vax1syn mice. Changes in SCN function were associated with mistimed peak PER2::LUC expression in the SCN and pituitary in both Six3syn and Vax1syn females. Interestingly, Six3syn ovaries presented reduced sensitivity to LH, causing reduced ovulation during superovulation. In conclusion, we have identified novel roles of the homeodomain transcription factors SIX3 and VAX1 in neurons, where they are required for proper molecular circadian clock function, SCN rhythmic output, and female fertility.


Subject(s)
Circadian Rhythm/physiology , Eye Proteins/metabolism , Fertility/physiology , Homeodomain Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neuropeptides/metabolism , Running/physiology , Suprachiasmatic Nucleus/metabolism , Animals , Eye Proteins/genetics , Female , Homeodomain Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , NIH 3T3 Cells , Nerve Tissue Proteins/genetics , Neuropeptides/genetics , Homeobox Protein SIX3
5.
Mol Psychiatry ; 26(7): 3383-3394, 2021 07.
Article in English | MEDLINE | ID: mdl-33674753

ABSTRACT

Bipolar disorder (BD) is a neuropsychiatric illness defined by recurrent episodes of mania/hypomania, depression and circadian rhythm abnormalities. Lithium is an effective drug for BD, but 30-40% of patients fail to respond adequately to treatment. Previous work has demonstrated that lithium affects the expression of "clock genes" and that lithium responders (Li-R) can be distinguished from non-responders (Li-NR) by differences in circadian rhythms. However, circadian rhythms have not been evaluated in BD patient neurons from Li-R and Li-NR. We used induced pluripotent stem cells (iPSCs) to culture neuronal precursor cells (NPC) and glutamatergic neurons from BD patients characterized for lithium responsiveness and matched controls. We identified strong circadian rhythms in Per2-luc expression in NPCs and neurons from controls and Li-R, but NPC rhythms in Li-R had a shorter circadian period. Li-NR rhythms were low amplitude and profoundly weakened. In NPCs and neurons, expression of PER2 was higher in both BD groups compared to controls. In neurons, PER2 protein levels were higher in BD than controls, especially in Li-NR samples. In single cells, NPC and neuron rhythms in both BD groups were desynchronized compared to controls. Lithium lengthened period in Li-R and control neurons but failed to alter rhythms in Li-NR. In contrast, temperature entrainment increased amplitude across all groups, and partly restored rhythms in Li-NR neurons. We conclude that neuronal circadian rhythm abnormalities are present in BD and most pronounced in Li-NR. Rhythm deficits in BD may be partly reversible through stimulation of entrainment pathways.


Subject(s)
Bipolar Disorder , Lithium , Bipolar Disorder/drug therapy , Circadian Rhythm , Humans , Lithium/pharmacology , Lithium Compounds/pharmacology , Neurons
6.
Methods Mol Biol ; 2130: 287-294, 2021.
Article in English | MEDLINE | ID: mdl-33284452

ABSTRACT

Circadian rhythms in cellular function can be monitored in real time with bioluminescence imaging. In this approach, bioluminescence is produced by an enzymatic reaction, which can be used to report dynamic changes in gene or protein expression in living cells. Bioluminescence imaging in circadian experiments typically uses an ex vivo slice preparation, with the most commonly studied structure being the master clock in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Here we describe procedures for dissecting and collecting SCN slices for bioluminescence imaging experiments.


Subject(s)
Circadian Clocks , Connectome/methods , Suprachiasmatic Nucleus/physiology , Animals , Genes, Reporter , Luciferases/genetics , Luciferases/metabolism , Mice , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolism , Suprachiasmatic Nucleus/metabolism
7.
Proc Natl Acad Sci U S A ; 117(24): 13771-13782, 2020 06 16.
Article in English | MEDLINE | ID: mdl-32487727

ABSTRACT

The nucleus accumbens (NAc), a central component of the midbrain dopamine reward circuit, exhibits disturbed circadian rhythms in the postmortem brains of depressed patients. We hypothesized that normal mood regulation requires proper circadian timing in the NAc, and that mood disorders are associated with dysfunctions of the NAc cellular circadian clock. In mice exhibiting stress-induced depression-like behavior (helplessness), we found altered circadian clock function and high nighttime expression of the core circadian clock component CRYPTOCHROME (CRY) in the NAc. In the NAc of helpless mice, we found that higher expression of CRY is associated with decreased activation of dopamine 1 receptor-expressing medium spiny neurons (D1R-MSNs). Furthermore, D1R-MSN-specific CRY-knockdown in the NAc reduced susceptibility to stress-induced helplessness and increased NAc neuronal activation at night. Finally, we show that CRY inhibits D1R-induced G protein activation, likely by interacting with the Gs protein. Altered circadian rhythms and CRY expression were also observed in human fibroblasts from major depressive disorder patients. Our data reveal a causal role for CRY in regulating the midbrain dopamine reward system, and provide a mechanistic link between the NAc circadian clock and vulnerability to depression.


Subject(s)
Circadian Clocks , Cryptochromes/metabolism , Depression/metabolism , Nucleus Accumbens/metabolism , Animals , Behavior, Animal , Depression/genetics , Depression/physiopathology , Depression/psychology , Dopamine/metabolism , Female , Helplessness, Learned , Humans , Male , Mice , Neurons/metabolism , Receptors, Dopamine/genetics , Receptors, Dopamine/metabolism
8.
Eur J Neurosci ; 51(12): 2329-2342, 2020 06.
Article in English | MEDLINE | ID: mdl-30044021

ABSTRACT

Shift-work and jet-lag-related disorders are caused by the limited flexibility of the suprachiasmatic nucleus (SCN), a master circadian clock in the hypothalamus, to adjust to new light-dark (LD) cycles. Recent findings confirmed here establish that behavioral jet lag after simulated time-zone travel is virtually eliminated following bifurcated circadian entrainment under a novel and atypical 24-h light:dark:light:dark (LDLD) cycle. To investigate the mechanisms of this fast resetting, we examined the oscillatory stability of the SCN and peripheral tissues in LDLD-bifurcated mice employing the dissection procedure as a perturbing resetting stimulus. SCN, lung, liver, and adrenal tissue were extracted at times throughout the day from female and male PER2::Luciferase knock-in mice entrained to either LDLD or a normal LD cycle. Except for adrenals, the phase of the cultured explants was more strongly set by dissection under LDLD than under normal LD. Acute bioluminescence levels of SCN explants indicate that the rhythm amplitude of PER2 is reduced and phase is altered in LDLD. Real-time quantitative PCR suggests that amplitude and rhythmicity of canonical clock genes in the lung, liver, and kidney are also significantly reduced in LDLD in vivo. Furthermore, spatiotemporal patterns of PER2 peak time in cultured SCN were altered in LDLD. These results suggest that altered gene expression patterns in the SCN caused by bifurcation likely result in fast resetting of behavior and cultured explants, consistent with previously reported mathematical models. Thus, non-invasive, simple light manipulations can make circadian rhythms more adaptable to abrupt shifts in the environmental LD cycle.


Subject(s)
Circadian Clocks , Circadian Rhythm , Animals , Female , Light , Male , Mice , Photoperiod , Suprachiasmatic Nucleus
9.
Yale J Biol Med ; 92(2): 251-258, 2019 06.
Article in English | MEDLINE | ID: mdl-31249486

ABSTRACT

Bmal1 is the only single circadian clock gene that is essential for rhythmic gene expression in the mammalian circadian timing system. Genetic approaches targeting Bmal1 expression have been used to further assess its role in the circadian clock and to test for behavioral effects of clock disruption. In particular, disruptions in circadian clock function have been implicated in human mood disorders, and clock gene manipulation in mice may provide valuable models for studying depression-like behavior. In this review, we explore various approaches to manipulating Bmal1 in mouse models and review their effects on the brain's master circadian pacemaker, on circadian rhythmicity in other brain regions, and on circadian and mood-related behavior.


Subject(s)
ARNTL Transcription Factors/metabolism , Brain/physiology , Circadian Clocks/physiology , Circadian Rhythm/physiology , ARNTL Transcription Factors/genetics , Animals , Brain/metabolism , Gene Expression Regulation , Humans , Mice, Knockout , Models, Biological , Time Factors
10.
Neuropsychopharmacology ; 44(3): 620-628, 2019 02.
Article in English | MEDLINE | ID: mdl-30487653

ABSTRACT

Bipolar disorder (BD) is a serious mood disorder associated with circadian rhythm abnormalities. Risk for BD is genetically encoded and overlaps with systems that maintain circadian rhythms. Lithium is an effective mood stabilizer treatment for BD, but only a minority of patients fully respond to monotherapy. Presently, we hypothesized that lithium-responsive BD patients (Li-R) would show characteristic differences in chronotype and cellular circadian rhythms compared to lithium non-responders (Li-NR). Selecting patients from a prospective, multi-center, clinical trial of lithium monotherapy, we examined morning vs. evening preference (chronotype) as a dimension of circadian rhythm function in 193 Li-R and Li-NR BD patients. From a subset of 59 patient donors, we measured circadian rhythms in skin fibroblasts longitudinally over 5 days using a bioluminescent reporter (Per2-luc). We then estimated circadian rhythm parameters (amplitude, period, phase) and the pharmacological effects of lithium on rhythms in cells from Li-R and Li-NR donors. Compared to Li-NRs, Li-Rs showed a difference in chronotype, with higher levels of morningness. Evening chronotype was associated with increased mood symptoms at baseline, including depression, mania, and insomnia. Cells from Li-Rs were more likely to exhibit a short circadian period, a linear relationship between period and phase, and period shortening effects of lithium. Common genetic variation in the IP3 signaling pathway may account for some of the individual differences in the effects of lithium on cellular rhythms. We conclude that circadian rhythms may influence response to lithium in maintenance treatment of BD.


Subject(s)
Antimanic Agents/pharmacology , Bipolar Disorder/drug therapy , Bipolar Disorder/physiopathology , Circadian Rhythm , Fibroblasts , Lithium Compounds/pharmacology , Adult , Animals , Bipolar Disorder/genetics , Cells, Cultured , Circadian Rhythm/drug effects , Circadian Rhythm/physiology , Fibroblasts/drug effects , Fibroblasts/physiology , Genotyping Techniques , Humans , Inositol 1,4,5-Trisphosphate Receptors/genetics , Luminescent Measurements , Mice , NIH 3T3 Cells , Period Circadian Proteins , Polymorphism, Single Nucleotide , Prospective Studies
11.
Ann Med ; 50(8): 637-654, 2018 12.
Article in English | MEDLINE | ID: mdl-30265156

ABSTRACT

Disruption of circadian clocks is strongly associated with mood disorders. Chronotherapies targeting circadian rhythms have been shown to be very effective treatments of mood disorders, but still are not widely used in clinical practice. The mechanisms by which circadian disruption leads to mood disorders are poorly characterized and, therefore, may not convince clinicians to apply chronotherapies. Hence, in this review, we describe specific potential mechanisms, in order to make this connection more credible to clinicians. We believe that four major features of disrupted clocks may contribute to the development of mood disorders: (1) loss of synchronization to environmental 24-h rhythms, (2) internal desynchronization among body clocks, (3) low rhythm amplitude, and (4) changes in sleep architecture. Discussing these attributes and giving plausible examples, we will discuss prospects for relatively simple chronotherapies addressing these features that are easy to implement in clinical practice. Key messages In this review, we describe specific potential mechanisms by which disrupted clocks may contribute to the development of mood disorders: (1) loss of synchronization to environmental 24-h rhythms, (2) internal desynchronization among body clocks, (3) low rhythm amplitude, and (4) changes in sleep architecture. We provide prospects for relatively simple chronotherapies addressing these features that are easy to implement in clinical practice.


Subject(s)
Chronotherapy/methods , Circadian Clocks/physiology , Circadian Rhythm/physiology , Mood Disorders/therapy , Diet, Healthy , Exercise/physiology , Humans , Light , Mood Disorders/physiopathology , Sleep/physiology , Wakefulness/physiology
12.
Cell ; 174(1): 72-87.e32, 2018 06 28.
Article in English | MEDLINE | ID: mdl-29861175

ABSTRACT

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.


Subject(s)
Cell Hypoxia , Circadian Clocks , Mechanistic Target of Rapamycin Complex 1/metabolism , Adaptor Proteins, Signal Transducing , Amino Acids, Dicarboxylic/pharmacology , Animals , CLOCK Proteins/metabolism , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins , Cells, Cultured , Circadian Clocks/drug effects , Culture Media/chemistry , Eukaryotic Initiation Factors , Hydrogen-Ion Concentration , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mice , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Ras Homolog Enriched in Brain Protein/metabolism , Signal Transduction/drug effects , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Transcriptome/drug effects , Tuberous Sclerosis Complex 2 Protein/deficiency , Tuberous Sclerosis Complex 2 Protein/genetics
13.
Proc Natl Acad Sci U S A ; 115(16): 4276-4281, 2018 04 17.
Article in English | MEDLINE | ID: mdl-29610316

ABSTRACT

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.


Subject(s)
Circadian Rhythm/physiology , Cryptochromes/biosynthesis , Fiber Optic Technology/methods , Fluorometry/methods , Neurons/metabolism , Period Circadian Proteins/biosynthesis , Suprachiasmatic Nucleus/metabolism , Animals , Bacterial Proteins/analysis , Bacterial Proteins/genetics , CA1 Region, Hippocampal/metabolism , CA2 Region, Hippocampal/metabolism , Cells, Cultured , Circadian Rhythm/genetics , Cryptochromes/deficiency , Cryptochromes/genetics , Dependovirus/genetics , Fiber Optic Technology/instrumentation , Fluorometry/instrumentation , Genes, Reporter , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Hypothalamus, Anterior/metabolism , Longitudinal Studies , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Mice , Mice, Inbred C57BL , Movement , Neurons/chemistry , Neurons/classification , Optical Fibers , Organ Specificity , Period Circadian Proteins/genetics , Photoperiod , Suprachiasmatic Nucleus/cytology , Transcription, Genetic , Vasoactive Intestinal Peptide/analysis
14.
Neural Plast ; 2018: 5147585, 2018.
Article in English | MEDLINE | ID: mdl-29681926

ABSTRACT

Seasonal changes in light exposure have profound effects on behavioral and physiological functions in many species, including effects on mood and cognitive function in humans. The mammalian brain's master circadian clock, the suprachiasmatic nucleus (SCN), transmits information about external light conditions to other brain regions, including some implicated in mood and cognition. Although the detailed mechanisms are not yet known, the SCN undergoes highly plastic changes at the cellular and network levels under different light conditions. We therefore propose that the SCN may be an essential mediator of the effects of seasonal changes of day length on mental health. In this review, we explore various forms of neuroplasticity that occur in the SCN and other brain regions to facilitate seasonal adaptation, particularly altered phase distribution of cellular circadian oscillators in the SCN and changes in hypothalamic neurotransmitter expression.


Subject(s)
Circadian Clocks/physiology , Circadian Rhythm/physiology , Neuronal Plasticity/physiology , Photoperiod , Suprachiasmatic Nucleus/physiology , Animals , Humans , Nerve Net/physiology , Seasons
15.
eNeuro ; 4(4)2017.
Article in English | MEDLINE | ID: mdl-28828400

ABSTRACT

Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca2+ ([Ca2+]i), and neuronal firing rate. [Ca2+]i oscillates in SCN neurons even in the absence of neuronal firing. To determine the causal relationship between circadian clock gene expression and [Ca2+]i rhythms in the SCN, as well as the SCN neuronal network dependence of [Ca2+]i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca2+ indicator, into SCN neurons from PER2::LUC knock-in reporter mice. Then, PER2 and [Ca2+]i were imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca2+]i both exhibited cell autonomous circadian rhythms, but [Ca2+]i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model in which clock gene rhythms drive [Ca2+]i rhythms. As predicted by the model, PER2 and [Ca2+]i rhythms were both stronger in SCN slices than in dispersed cells and were weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca2+]i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca2+]i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca2+]i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network.


Subject(s)
Calcium/metabolism , Circadian Rhythm/physiology , Nerve Net/physiology , Neurons/physiology , Suprachiasmatic Nucleus/cytology , Suprachiasmatic Nucleus/physiology , ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/metabolism , Animals , Circadian Rhythm/drug effects , Circadian Rhythm/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , In Vitro Techniques , Luciferases/genetics , Luciferases/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Theoretical , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolism , Sodium Channel Blockers/pharmacology , Tetrodotoxin/pharmacology , Transduction, Genetic , Zona Pellucida Glycoproteins/genetics , Zona Pellucida Glycoproteins/metabolism
16.
Neuroscience ; 357: 1-11, 2017 08 15.
Article in English | MEDLINE | ID: mdl-28576728

ABSTRACT

In mammals, the master circadian clock resides in the suprachiasmatic nucleus (SCN). The SCN is characterized by robust circadian oscillations of clock gene expression and neuronal firing. The synchronization of circadian oscillations among individual cells in the SCN is attributed to intercellular coupling. Previous studies have shown that gap junctions, specifically those composed of connexin-36 (Cx36) subunits, are required for coupling of electrical firing among SCN neurons at a time scale of milliseconds. However, it remains unknown whether Cx36 gap junctions also contribute to coupling of circadian (∼24h) rhythms of clock gene expression. Here, we investigated circadian expression patterns of the clock gene Period 2 (Per2) in the SCN of Cx36-deficient mice using luminometry and single-cell bioluminescence imaging. Surprisingly, we found that synchronization of circadian PER2 expression rhythms is maintained in SCN explants from Cx36-deficient mice. Since Cx36 expression levels change with age, we also tested circadian running-wheel behavior of juvenile (3-4weeks old) and adult (9-30weeks old) Cx36-deficient mice. We found that impact of connexin-36 expression on circadian behavior changes greatly during postnatal development. However, consistent with the intact synchrony among SCN cells in cultured explants, Cx36-deficient mice had intact locomotor circadian rhythms, although adults displayed a lengthened period in constant darkness. Our data indicate that even though Cx36 may be required for electrical coupling of SCN cells, it does not affect coupling of molecular clock gene rhythms. Thus, electrical coupling of neurons and coupling of circadian clock gene oscillations can be regulated independently in the SCN.


Subject(s)
Circadian Clocks/physiology , Circadian Rhythm/physiology , Connexins/deficiency , Period Circadian Proteins/metabolism , Suprachiasmatic Nucleus/metabolism , Adaptation, Physiological/physiology , Animals , Connexins/genetics , Female , Male , Mice, Transgenic , Suprachiasmatic Nucleus/growth & development , Tissue Culture Techniques , Gap Junction delta-2 Protein
17.
BMC Biol ; 15(1): 13, 2017 02 14.
Article in English | MEDLINE | ID: mdl-28196531

ABSTRACT

BACKGROUND: Circadian clocks control cell cycle factors, and circadian disruption promotes cancer. To address whether enhancing circadian rhythmicity in tumor cells affects cell cycle progression and reduces proliferation, we compared growth and cell cycle events of B16 melanoma cells and tumors with either a functional or dysfunctional clock. RESULTS: We found that clock genes were suppressed in B16 cells and tumors, but treatments inducing circadian rhythmicity, such as dexamethasone, forskolin and heat shock, triggered rhythmic clock and cell cycle gene expression, which resulted in fewer cells in S phase and more in G1 phase. Accordingly, B16 proliferation in vitro and tumor growth in vivo was slowed down. Similar effects were observed in human colon carcinoma HCT-116 cells. Notably, the effects of dexamethasone were not due to an increase in apoptosis nor to an enhancement of immune cell recruitment to the tumor. Knocking down the essential clock gene Bmal1 in B16 tumors prevented the effects of dexamethasone on tumor growth and cell cycle events. CONCLUSIONS: Here we demonstrated that the effects of dexamethasone on cell cycle and tumor growth are mediated by the tumor-intrinsic circadian clock. Thus, our work reveals that enhancing circadian clock function might represent a novel strategy to control cancer progression.


Subject(s)
Circadian Clocks , Melanoma, Experimental/genetics , Melanoma, Experimental/pathology , ARNTL Transcription Factors/metabolism , Animals , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Circadian Clocks/drug effects , Circadian Clocks/genetics , Circadian Rhythm/drug effects , Colforsin/pharmacology , Dexamethasone/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , HCT116 Cells , Heat-Shock Response/drug effects , Humans , Mice, Inbred C57BL , Models, Biological
18.
PLoS One ; 11(7): e0159618, 2016.
Article in English | MEDLINE | ID: mdl-27459195

ABSTRACT

Circadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in culture. In the present study, we examined the relationship between intrinsic myocardial function and circadian rhythms in cultures from mouse heart. We cultured ventricular explants or dispersed cardiomyocytes from neonatal mice expressing a PER2::LUC bioluminescent reporter of circadian clock gene expression. We found that isoproterenol, a ß-adrenoceptor agonist known to increase heart rate and contractility, also amplifies PER2 circadian rhythms in ventricular explants. We found robust, cell-autonomous PER2 circadian rhythms in dispersed cardiomyocytes. Single-cell rhythms were initially synchronized in ventricular explants but desynchronized in dispersed cells. In addition, we developed a method for long-term, simultaneous monitoring of clock gene expression, contraction rate, and basal intracellular Ca2+ level in cardiomyocytes using PER2::LUC in combination with GCaMP3, a genetically encoded fluorescent Ca2+ reporter. In contrast to robust PER2 circadian rhythms in cardiomyocytes, we detected no rhythms in contraction rate and only weak rhythms in basal Ca2+ level. In summary, we found that PER2 circadian rhythms of cardiomyocytes are cell-autonomous, amplified by adrenergic signaling, and synchronized by intercellular communication in ventricle explants, but we detected no robust circadian rhythms in contraction rate or basal Ca2+.


Subject(s)
Circadian Clocks/physiology , Heart Ventricles/metabolism , Myocytes, Cardiac/metabolism , Receptors, Adrenergic, beta/metabolism , Signal Transduction , Animals , Calcium/metabolism , Cell Communication , Cells, Cultured , Circadian Rhythm Signaling Peptides and Proteins/genetics , Circadian Rhythm Signaling Peptides and Proteins/metabolism , Gene Expression Regulation , Heart Ventricles/drug effects , Luminescent Measurements , Mice , Mice, Transgenic , Myocardial Contraction/genetics , Period Circadian Proteins/genetics , Ventricular Function/drug effects , Ventricular Function/genetics
19.
Eur Neuropsychopharmacol ; 26(8): 1310-9, 2016 08.
Article in English | MEDLINE | ID: mdl-27216486

ABSTRACT

UNLABELLED: Bipolar disorder (BD) is characterized by depression, mania, and circadian rhythm abnormalities. Lithium, a treatment for BD stabilizes mood and increases circadian rhythm amplitude. However, in fibroblasts grown from BD patients, lithium has weak effects on rhythm amplitude compared to healthy controls. To understand the mechanism by which lithium differentially affects rhythm amplitude in BD cells, we investigated the extracellular-signal-regulated kinase (ERK) and related signaling molecules linked to BD and circadian rhythms. In fibroblasts from BD patients, controls and mice, we assessed the contribution of the ERK pathway to lithium-induced circadian rhythm amplification. Protein analyses revealed low phospho-ERK1/2 (p-ERK) content in fibroblasts from BD patients vs. CONTROLS: Pharmacological inhibition of ERK1/2 by PD98059 attenuated the rhythm amplification effect of lithium, while inhibition of two related kinases, c-Jun N-terminal kinase (JNK), and P38 did not. Knockdown of the transcription factors CREB and EGR-1, downstream effectors of ERK1/2, reduced baseline rhythm amplitude, but did not alter rhythm amplification by lithium. In contrast, ELK-1 knockdown amplified rhythms, an effect that was not increased further by the addition of lithium, suggesting this transcription factor may regulate the effect of lithium on amplitude. Augmentation of ERK1/2 signaling through DUSP6 knockdown sensitized NIH3T3 cells to rhythm amplification by lithium. In BD fibroblasts, DUSP6 knockdown reversed the BD rhythm phenotype, restoring the ability of lithium to increase amplitude in these cells. We conclude that the inability of lithium to regulate circadian rhythms in BD may reflect reduced ERK activity, and signaling through ELK-1.


Subject(s)
Antimanic Agents/pharmacology , Bipolar Disorder/metabolism , Circadian Rhythm/drug effects , Lithium/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , ets-Domain Protein Elk-1/metabolism , Animals , Antimanic Agents/therapeutic use , Bipolar Disorder/drug therapy , Bipolar Disorder/pathology , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/antagonists & inhibitors , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Dual Specificity Phosphatase 6/antagonists & inhibitors , Dual Specificity Phosphatase 6/genetics , Dual Specificity Phosphatase 6/metabolism , Early Growth Response Protein 1/antagonists & inhibitors , Early Growth Response Protein 1/genetics , Early Growth Response Protein 1/metabolism , Genes, Reporter/drug effects , Humans , Lithium/therapeutic use , MAP Kinase Signaling System/drug effects , Mice , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/chemistry , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/chemistry , NIH 3T3 Cells , Period Circadian Proteins/agonists , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , RNA Interference , ets-Domain Protein Elk-1/antagonists & inhibitors , ets-Domain Protein Elk-1/genetics
20.
J Biol Rhythms ; 31(4): 337-51, 2016 08.
Article in English | MEDLINE | ID: mdl-27221103

ABSTRACT

Light is the primary signal that calibrates circadian neural circuits and thus coordinates daily physiological and behavioral rhythms with solar entrainment cues. Drosophila and mammalian circadian circuits consist of diverse populations of cellular oscillators that exhibit a wide range of dynamic light responses, periods, phases, and degrees of synchrony. How heterogeneous circadian circuits can generate robust physiological rhythms while remaining flexible enough to respond to synchronizing stimuli has long remained enigmatic. Cryptochrome is a short-wavelength photoreceptor that is endogenously expressed in approximately half of Drosophila circadian neurons. In a previous study, physiological light response was measured using real-time bioluminescence recordings in Drosophila whole-brain explants, which remain intrinsically light-sensitive. Here we apply analysis of real-time bioluminescence experimental data to show detailed dynamic ensemble representations of whole circadian circuit light entrainment at single neuron resolution. Organotypic whole-brain explants were either maintained in constant darkness (DD) for 6 days or exposed to a phase-advancing light pulse on the second day. We find that stronger circadian oscillators support robust overall circuit rhythmicity in DD, whereas weaker oscillators can be pushed toward transient desynchrony and damped amplitude to facilitate a new state of phase-shifted network synchrony. Additionally, we use mathematical modeling to examine how a network composed of distinct oscillator types can give rise to complex dynamic signatures in DD conditions and in response to simulated light pulses. Simulations suggest that complementary coupling mechanisms and a combination of strong and weak oscillators may enable a robust yet flexible circadian network that promotes both synchrony and entrainment. A more complete understanding of how the properties of oscillators and their signaling mechanisms facilitate their distinct roles in light entrainment may allow us to direct and augment the circadian system to speed recovery from jet lag, shift work, and seasonal affective disorder.


Subject(s)
Biological Clocks/physiology , Darkness , Light , Neurons/physiology , Animals , Brain/physiology , Circadian Rhythm/radiation effects , Computer Systems , Cryptochromes/physiology , Drosophila , Luminescent Measurements , Mammals , Models, Theoretical
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