measuring-diurnal-rhythms-in-autophagic-and-proteasomal-flux

Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main ...

Washington University School of Medicine

Critical illness is associated with profound sleep disruption. Causality is diverse and includes physiologic, psychological, and environmental factors. There are limited pharmacologic interventions available to treat sleep disturbances in critical illness; however, multidisciplinary strategies that alter the intensive care unit (ICU) environment and cluster care delivery have shown promise in sleep and circadian promotion and delirium reduction. With the appropriate administrative support and involvement of diverse ICU stakeholders, effective strategies could be created, implemented, and maintained to improve sleep disruption in critically ill patients. 

Sleep Loss and Circadian Rhythm Disruption in the Intensive Care Unit.

Lung epithelial cell apoptosis is an important feature of hyperoxia-induced lung injury. Death receptor-associated extrinsic pathway and mitochondria-associated intrinsic pathway both mediate the development of lung epithelial cell apoptosis. Despite decades of research, molecular mechanisms of hyperoxia-induced epithelial cell apoptosis remain incompletely understood. Here we report a novel regulatory paradigm in response to hyperoxia-associated oxidative stress. Hyperoxia markedly up-regulated miR-15a/16 levels in lung epithelial cells, broncho-alveolar lavage fluid (BALF) and lung tissue. This effect was mediated by hyperoxia-induced reactive oxygen species (ROS). Functionally, miR-15a/16 inhibitors induced caspase 3-mediated lung epithelial cell apoptosis, in the presence of hyperoxia. MiR-15a/16 inhibitors robustly enhanced FADD level and down-regulated Bcl-2 expression. Consistently, cleaved caspase 8 and 9 were highly induced in the miR-15a/16 deficient cells, after hyperoxia. Using airway epithelial cell specific, miR-15a/16 mice, we found that Bcl-2 significantly reduced in lung epithelial cells  after hyperoxia. In contrast, caspase 3, 8 and Bcl-2 associated death promoter (BAD) were highly elevated in the miR-15a/16 epithelial cells . Interestingly, in lung epithelial malignant cells, rather than benign cells, deletion of miR-15a/16 prevented apoptosis. Furthermore, deletion of  in macrophages also prohibited apoptosis, opposite to what we have found in normal lung epithelial cells. Taken together, our data suggested that miR-15a/16 may exert differential roles in different cell types. MiR-15a/16 deficiency result in lung epithelial cell apoptosis in response to hyperoxia,  modulating both intrinsic and extrinsic apoptosis pathways.

MiR-15a/16 Regulates Apoptosis of Lung Epithelial Cells after Oxidative Stress.

The circadian clock orchestrates the timing of insulin and glucagon secretion, which is important for glycemic control.

Clocks stop sugar shock.

Patients with asthma experience circadian variations in their symptoms. However it remains unclear how specific aspects of this common airway disease relate to clock genes, which are critical to the generation of circadian rhythms in mammals. Here, we used a viral model of acute and chronic airway disease to examine how circadian clock disruption affects asthmatic lung phenotypes. Deletion of the core clock gene bmal1 or environmental disruption of circadian function by jet lag exacerbated acute viral bronchiolitis caused by Sendai virus (SeV) and influenza A virus in mice. Post-natal deletion of bmal1 was sufficient to trigger increased SeV susceptibility and correlated with impaired control of viral replication. Importantly, bmal1 mice developed much more extensive asthma-like airway changes post infection, including mucus production and increased airway resistance. In human airway samples from two asthma cohorts, we observed altered expression patterns of multiple clock genes. Our results suggest a role for bmal1 in the development of asthmatic airway disease via the regulation of lung antiviral responses to common viral triggers of asthma.

BMAL1 links the circadian clock to viral airway pathology and asthma phenotypes.

A new analytical method allows reconstruction of circadian gene expression in human biopsy samples.

An "eye" for rhythm.

The function of most human long noncoding RNAs (lncRNAs) remains unclear. Our studies identified a highly up-regulated mammalian lncRNA, , known as  in mice, in the setting of hyperoxia/reactive oxygen species (ROS)-induced lung injury. We found that ROS induced a robust expression of FOXD3 in mouse lung tissue. Functionally,  promoted oxidative stress-induced lung epithelial cell death. In human lung epithelial cells, the microRNA-150 (miR-150) was identified to interact with ; this finding was confirmed using the luciferase reporter assays. Consistently, mutation on the miR-150 pairing sequence in FOXD3-AS1 abolished the interactions between FOXD3-AS1 and miR-150. Additionally, miR-150 mimics suppressed the level of FOXD3-AS1. The antisense oligos of FOXD3-AS1 significantly augmented the intracellular level of miR-150, supporting the theory of sponging effects of FOXD3-AS1 on miR-150. We further investigated the cellular function of miR-150 in our lung injury models. MiR-150 conferred a cytoprotective role in lung epithelial cells after oxidative stress, whereas FOXD3-AS1 promoted cell death. Taken together, our studies indicated that FOXD3-AS1 serves as a sponge or as a competing endogenous noncoding RNA for miR-150, restricting its capability to promote cell growth and thereby exaggerating hyperoxia-induced lung epithelial cell death.-Zhang, D., Lee, H., Haspel, J. A., Jin, Y. Long noncoding RNA FOXD3-AS1 regulates oxidative stress-induced apoptosis  sponging microRNA-150.

Long noncoding RNA FOXD3-AS1 regulates oxidative stress-induced apoptosis  sponging microRNA-150.

A new type of biological clock regulates the unfolded protein response.

Surf's up for ER stress.

Motile cilia are characterized by dynein motor units, which preassemble in the cytoplasm before trafficking into the cilia. Proteins required for dynein preassembly were discovered by finding human mutations that result in absent ciliary motors, but little is known about their expression, function, or interactions. By monitoring ciliogenesis in primary airway epithelial cells and MCIDAS-regulated induced pluripotent stem cells, we uncovered two phases of expression of preassembly proteins. An early phase, composed of HEATR2, SPAG1, and DNAAF2, preceded other preassembly proteins and was independent of MCIDAS regulation. The early preassembly proteins colocalized within perinuclear foci that also contained dynein arm proteins. These proteins also interacted based on immunoprecipitation and Förster resonance energy transfer (FRET) studies. FRET analysis of HEAT domain deletions and human mutations showed that HEATR2 interacted with itself and SPAG1 at multiple HEAT domains, while DNAAF2 interacted with SPAG1. Human mutations in HEATR2 did not affect this interaction, but triggered the formation of p62/Sequestosome-1-positive aggregates containing the early preassembly proteins, suggesting that degradation of an early preassembly complex is responsible for disease and pointing to key regions required for HEATR2 scaffold stability. We speculate that HEATR2 is an early scaffold for the initiation of dynein complex assembly in motile cilia.

Establishment of the early cilia preassembly protein complex during motile ciliogenesis.

Circadian rhythms are a hallmark of physiology, but how such daily rhythms organize cellular catabolism is poorly understood. Here, we used proteomics to map daily oscillations in autophagic flux in mouse liver and related these rhythms to proteasome activity. We also explored how systemic inflammation affects the temporal structure of autophagy. Our data identified a globally harmonized rhythm for basal macroautophagy, chaperone-mediated autophagy, and proteasomal activity, which concentrates liver proteolysis during the daytime. Basal autophagy rhythms could be resolved into two antiphase clusters that were distinguished by the subcellular location of targeted proteins. Inflammation induced by lipopolysaccharide reprogrammed autophagic flux away from a temporal pattern that favors cytosolic targets and toward the turnover of mitochondrial targets. Our data detail how daily biological rhythms connect the temporal, spatial, and metabolic aspects of protein catabolism.

Diurnal Rhythms Spatially and Temporally Organize Autophagy.

Circadian rhythms help cells to organize complex processes, but how they shape the kinetics of protein catabolism is unclear. In a recent paper, we employed proteomics to map daily biological rhythms in autophagic flux in mouse liver, and correlated these rhythms with proteasome activity. We also explored the effect of inflammation caused by endotoxin on autophagy dynamics. Our data provide insight into how circadian rhythms serve as a framework for connecting the spatial, temporal, and metabolic aspects of autophagy at a system-wide level. Our observations also have implications for how to optimize autophagy-directed therapies in patients.

Article	Year
Sleep Loss and Circadian Rhythm Disruption in the Intensive Care Unit. Clinics in chest medicine\| PubMed ID: 26304279	2015
MiR-15a/16 Regulates Apoptosis of Lung Epithelial Cells after Oxidative Stress. Molecular medicine (Cambridge, Mass.)\| PubMed ID: 27257854	2016
Clocks stop sugar shock. Science translational medicine\| PubMed ID: 28381534	2017
BMAL1 links the circadian clock to viral airway pathology and asthma phenotypes. Mucosal immunology\| PubMed ID: 28401936	2018
An "eye" for rhythm. Science translational medicine\| PubMed ID: 28515338	2017
Long noncoding RNA FOXD3-AS1 regulates oxidative stress-induced apoptosis sponging microRNA-150. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 28655711	2017
Surf's up for ER stress. Science translational medicine\| PubMed ID: 28659440	2017
Establishment of the early cilia preassembly protein complex during motile ciliogenesis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29358401	2018
Diurnal Rhythms Spatially and Temporally Organize Autophagy. Cell reports\| PubMed ID: 30759397	2019
Adventures in spacetime: circadian rhythms and the dynamics of protein catabolism. Autophagy\| PubMed ID: 30894057	2019

Jeffrey A. Haspel

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