bims-ciryme 2020-05-03 papers

Am J Physiol Regul Integr Comp Physiol. 2020 Apr 29.

Circadian clock genes and respiratory neuroplasticity genes oscillate in the phrenic motor system.

Mia N Kelly, Danelle N Smith, Michael D Sunshine, Ashley Ross, Xiping Zhang, Michelle L Gumz, Karyn A Esser, Gordon S Mitchell.

Circadian rhythms are endogenous and entrainable daily patterns of physiology and behavior. Molecular mechanisms underlie circadian rhythms, characterized by an ~24-hour pattern of gene expression of core clock genes. Although it has long been known that breathing exhibits circadian rhythms, little is known concerning clock gene expression in any element of the neuromuscular system controlling breathing. Further, we know little concerning gene expression necessary for specific respiratory functions, such as phrenic motor plasticity. Thus, we tested the hypotheses that transcripts for clock genes (Bmal1, Clock, Per1 & Per2) and molecules necessary for phrenic motor plasticity (Htr2a, Htr2b, Bdnf & Ntrk2) oscillate in regions critical for phrenic/diaphragm motor function via RT-PCR. Tissues were collected from male Sprague-Dawley rats entrained to a 12-hour light-dark cycle at 4 zeitgeber times (ZT; n=8/group): ZT5, ZT11, ZT17 and ZT23; ZT0 = lights on. Here, we demonstrate that 1) circadian clock genes (Bmal1, Clock, Per1, Per2) oscillate in regions critical for phrenic/diaphragm function, including the caudal medulla, ventral C3-C5 cervical spinal cord, and diaphragm, 2) the clock protein BMAL1 is localized within CtB-labelled phrenic motor neurons, 3) genes necessary for intermittent hypoxia-induced phrenic/diaphragm motor plasticity (Htr2b, Bdnf) oscillate in the caudal medulla and ventral C3-C5 spinal cord and 4) higher intensity of immunofluorescent BDNF protein within phrenic motor neurons at ZT23 compared to ZT11 (n=11/group). These results suggest local circadian clocks exist in the phrenic motor system and confirm the potential for local circadian regulation of neuroplasticity and other elements of the neural network controlling breathing.

Keywords: Circadian Rhythms; Control of breathing; motor neuron; plasticity; spinal cord

DOI: https://doi.org/10.1152/ajpregu.00010.2020

PLoS Genet. 2020 Apr 30. 16(4): e1008729

Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator.

Nan Yang, Nicola J Smyllie, Honor Morris, Cátia F Gonçalves, Michal Dudek, Dharshika Pathiranage, Johanna E Chesham, Antony Adamson, David Spiller, Egor Zindy, James Bagnall, Neil Humphreys, Judith Hoyland, Andrew S I Loudon, Michael H Hastings, Qing-Jun Meng.

Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.

DOI: https://doi.org/10.1371/journal.pgen.1008729

Am J Physiol Renal Physiol. 2020 Apr 27.

Differences in renal BMAL1 contribution to sodium homeostasis and blood pressure control in male and female mice.

G Ryan Crislip, Lauren Grace Douma, Sarah Howland Masten, Kit-Yan Cheng, I Jeanette Lynch, Jermaine G Johnston, Dominique Barral, Krystal B Glasford, Meaghan Rayann Holzworth, Jill W Verlander, Charles S Wingo, Michelle L Gumz.

The renal circadian clock has a major influence on the function of the kidney. Aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL; or BMAL1) is a core clock protein and transcription factor that regulates the expression of nearly half of all genes. Using male and female kidney-specific cadherin BMAL1 knockout mice (KS-BMAL1 KO), we examined the role of renal distal segment BMAL1 in blood pressure control and solute handling. We confirmed that this mouse model does not express BMAL1 in the thick ascending limb, distal convoluted tubule, and collecting duct cells which are the final locations for solute and fluid regulation. Male KS-BMAL1 KO displayed a substantially lower basal systolic blood pressure (SBP) compared to littermate control mice (CNTL), yet their circadian rhythm in pressure remained unchanged [Male: CNTL 127±0.7 (5) vs. KS-BMAL KO 119±2.3 mmHg (4); p<0.05]. Female mice, however, did not display a genotype difference in basal SBP [F: CNTL 120±1.6 (7) vs. KS-BMAL1 119±1.5 mmHg (5); p=0.4]. In addition, male KS-BMAL1 KO had less sodium retention compared to CNTL in response to a potassium-restricted diet (15% less following 5 days of treatment). However, there was not a genotype difference in sodium handling after a potassium-restricted diet in female mice. Furthermore, there is evidence indicating a sex-specific response to potassium restriction where females reabsorb less sodium in response to this dietary challenge compared to males. We propose that BMAL1 in the distal nephron and collecting duct contributes to blood pressure regulation and sodium handling in a sex-specific manner.

Keywords: clock genes; gene expression; potassium depletion; sex differences

DOI: https://doi.org/10.1152/ajprenal.00014.2020

Proc Natl Acad Sci U S A. 2020 May 01. pii: 201922388. [Epub ahead of print]

Noise-driven cellular heterogeneity in circadian periodicity.

Yan Li, Yongli Shan, Ravi V Desai, Kimberly H Cox, Leor S Weinberger, Joseph S Takahashi.

Nongenetic cellular heterogeneity is associated with aging and disease. However, the origins of cell-to-cell variability are complex and the individual contributions of different factors to total phenotypic variance are still unclear. Here, we took advantage of clear phenotypic heterogeneity of circadian oscillations in clonal cell populations to investigate the underlying mechanisms of cell-to-cell variability. Using a fully automated tracking and analysis pipeline, we examined circadian period length in thousands of single cells and hundreds of clonal cell lines and found that longer circadian period is associated with increased intercellular heterogeneity. Based on our experimental results, we then estimated the contributions of heritable and nonheritable factors to this variation in circadian period length using a variance partitioning model. We found that nonheritable noise predominantly drives intercellular circadian period variation in clonal cell lines, thereby revealing a previously unrecognized link between circadian oscillations and intercellular heterogeneity. Moreover, administration of a noise-enhancing drug reversibly increased both period length and variance. These findings suggest that circadian period may be used as an indicator of cellular noise and drug screening for noise control.

Keywords: circadian oscillation; heterogeneity/variance; period; single-cell imaging; transcriptional noise

DOI: https://doi.org/10.1073/pnas.1922388117