Deep RNA profiling identified CLOCK and molecular clock genes as pathophysiological signatures in collagen VI myopathy.

Collagen VI myopathies are genetic disorders caused by mutations in collagen 6 A1, A2 and A3 genes, ranging from the severe Ullrich congenital muscular dystrophy to the milder Bethlem myopathy, which is recapitulated by collagen-VI-null (Col6a1(-/-)) mice. Abnormalities in mitochondria and autophagic pathway have been proposed as pathogenic causes of collagen VI myopathies, but the link between collagen VI defects and these metabolic circuits remains unknown. To unravel the expression profiling perturbation in muscles with collagen VI myopathies, we performed a deep RNA profiling in both Col6a1(-/-)mice and patients with collagen VI pathology. The interactome map identified common pathways suggesting a previously undetected connection between circadian genes and collagen VI pathology. Intriguingly, Bmal1(-/-)(also known as Arntl) mice, a well-characterized model displaying arrhythmic circadian rhythms, showed profound deregulation of the collagen VI pathway and of autophagy-related genes. The involvement of circadian rhythms in collagen VI myopathies is new and links autophagy and mitochondrial abnormalities. It also opens new avenues for therapies of hereditary myopathies to modulate the molecular clock or potential gene-environment interactions that might modify muscle damage pathogenesis.

Drosophila Gut-A Nexus Between Dietary Restriction and Lifespan.

Aging is often defined as the accumulation of damage at the molecular and cellular levels which, over time, results in marked physiological impairments throughout the organism. Dietary restriction (DR) has been recognized as one of the strongest lifespan extending therapies observed in a wide array of organisms. Recent studies aimed at elucidating how DR promotes healthy aging have demonstrated a vital role of the digestive tract in mediating the beneficial effects of DR. Here, we review how dietary restriction influences gut metabolic homeostasis and immune function. Our discussion is focused on studies of the Drosophila digestive tract, where we describe in detail the potential mechanisms in which DR enhances maintenance of the intestinal epithelial barrier, up-regulates lipid metabolic processes, and improves the ability of the gut to deal with damage or stress. We also examine evidence of a tissue-tissue crosstalk between gut and neighboring organs including brain and fat body. Taken together, we argue that the Drosophila gut plays a critical role in DR-mediated lifespan extension.

Intrinsic muscle clock is necessary for musculoskeletal health.

KEY POINTS: The endogenous molecular clock in skeletal muscle is necessary for maintenance of phenotype and function. Loss of Bmal1 solely from adult skeletal muscle (iMSBmal1(-/-) ) results in reductions in specific tension, increased oxidative fibre type and increased muscle fibrosis with no change in feeding or activity. Disruption of the molecular clock in adult skeletal muscle is sufficient to induce changes in skeletal muscle similar to those seen in the Bmal1 knockout mouse (Bmal1(-/-) ), a model of advanced ageing. iMSBmal1(-/-) mice develop increased bone calcification and decreased joint collagen, which in combination with the functional changes in skeletal muscle results in altered gait. This study uncovers a fundamental role for the skeletal muscle clock in musculoskeletal homeostasis with potential implications for ageing. ABSTRACT: Disruption of circadian rhythms in humans and rodents has implicated a fundamental role for circadian rhythms in ageing and the development of many chronic diseases including diabetes, cardiovascular disease, depression and cancer. The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop with Bmal1 encoding a core molecular clock transcription factor. Germline Bmal1 knockout (Bmal1 KO) mice have a shortened lifespan, show features of advanced ageing and exhibit significant weakness with decreased maximum specific tension at the whole muscle and single fibre levels. We tested the role of the molecular clock in adult skeletal muscle by generating mice that allow for the inducible skeletal muscle-specific deletion of Bmal1 (iMSBmal1). Here we show that disruption of the molecular clock, specifically in adult skeletal muscle, is associated with a muscle phenotype including reductions in specific tension, increased oxidative fibre type, and increased muscle fibrosis similar to that seen in the Bmal1 KO mouse. Remarkably, the phenotype observed in the iMSBmal1(-/-) mice was not limited to changes in muscle. Similar to the germline Bmal1 KO mice, we observed significant bone and cartilage changes throughout the body suggesting a role for the skeletal muscle molecular clock in both the skeletal muscle niche and the systemic milieu. This emerging area of circadian rhythms and the molecular clock in skeletal muscle holds the potential to provide significant insight into intrinsic mechanisms of the maintenance of muscle quality and function as well as identifying a novel crosstalk between skeletal muscle, cartilage and bone.

OXR1 maintains the retromer to delay brain aging under dietary restriction.

Dietary restriction (DR) delays aging, but the mechanism remains unclear. We identified polymorphisms in mtd, the fly homolog of OXR1, which influenced lifespan and mtd expression in response to DR. Knockdown in adulthood inhibited DR-mediated lifespan extension in female flies. We found that mtd/OXR1 expression declines with age and it interacts with the retromer, which regulates trafficking of proteins and lipids. Loss of mtd/OXR1 destabilized the retromer, causing improper protein trafficking and endolysosomal defects. Overexpression of retromer genes or pharmacological restabilization with R55 rescued lifespan and neurodegeneration in mtd-deficient flies and endolysosomal defects in fibroblasts from patients with lethal loss-of-function of OXR1 variants. Multi-omic analyses in flies and humans showed that decreased Mtd/OXR1 is associated with aging and neurological diseases. mtd/OXR1 overexpression rescued age-related visual decline and tauopathy in a fly model. Hence, OXR1 plays a conserved role in preserving retromer function and is critical for neuronal health and longevity.

Dietary restriction and the transcription factor clock delay eye aging to extend lifespan in Drosophila Melanogaster.

Many vital processes in the eye are under circadian regulation, and circadian dysfunction has emerged as a potential driver of eye aging. Dietary restriction is one of the most robust lifespan-extending therapies and amplifies circadian rhythms with age. Herein, we demonstrate that dietary restriction extends lifespan in Drosophila melanogaster by promoting circadian homeostatic processes that protect the visual system from age- and light-associated damage. Altering the positive limb core molecular clock transcription factor, CLOCK, or CLOCK-output genes, accelerates visual senescence, induces a systemic immune response, and shortens lifespan. Flies subjected to dietary restriction are protected from the lifespan-shortening effects of photoreceptor activation. Inversely, photoreceptor inactivation, achieved via mutating rhodopsin or housing flies in constant darkness, primarily extends the lifespan of flies reared on a high-nutrient diet. Our findings establish the eye as a diet-sensitive modulator of lifespan and indicates that vision is an antagonistically pleiotropic process that contributes to organismal aging.

MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle.

In the present study we show that the master myogenic regulatory factor, MYOD1, is a positive modulator of molecular clock amplitude and functions with the core clock factors for expression of clock-controlled genes in skeletal muscle. We demonstrate that MYOD1 directly regulates the expression and circadian amplitude of the positive core clock factor Bmal1. We identify a non-canonical E-box element in Bmal1 and demonstrate that is required for full MYOD1-responsiveness. Bimolecular fluorescence complementation assays demonstrate that MYOD1 colocalizes with both BMAL1 and CLOCK throughout myonuclei. We demonstrate that MYOD1 and BMAL1:CLOCK work in a synergistic fashion through a tandem E-box to regulate the expression and amplitude of the muscle specific clock-controlled gene, Titin-cap (Tcap). In conclusion, these findings reveal mechanistic roles for the muscle specific transcription factor MYOD1 in the regulation of molecular clock amplitude as well as synergistic regulation of clock-controlled genes in skeletal muscle.

Muscle-specific loss of Bmal1 leads to disrupted tissue glucose metabolism and systemic glucose homeostasis.

BACKGROUND: Diabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence. This disease is characterized by hyperglycemia and glucose intolerance and symptoms caused by failure to produce and/or respond to insulin. The skeletal muscle is a key insulin-sensitive metabolic tissue, taking up ~80 % of postprandial glucose. To address the role of the skeletal muscle molecular clock to insulin sensitivity and glucose tolerance, we generated an inducible skeletal muscle-specific Bmal1 (-/-) mouse (iMSBmal1 (-/-)). RESULTS: Progressive changes in body composition (decreases in percent fat) were seen in the iMSBmal1 (-/-) mice from 3 to 12 weeks post-treatment as well as glucose intolerance and non-fasting hyperglycemia. Ex vivo analysis of glucose uptake revealed that the extensor digitorum longus (EDL) muscles did not respond to either insulin or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation. RT-PCR and Western blot analyses demonstrated a significant decrease in mRNA expression and protein content of the muscle glucose transporter (Glut4). We also found that both mRNA expression and activity of two key rate-limiting enzymes of glycolysis, hexokinase 2 (Hk2) and phosphofructokinase 1 (Pfk1), were significantly reduced in the iMSBmal1 (-/-) muscle. Lastly, results from metabolomics analyses provided evidence of decreased glycolytic flux and uncovered decreases in some tricarboxylic acid (TCA) intermediates with increases in amino acid levels in the iMSBmal1 (-/-) muscle. These findings suggest that the muscle is relying predominantly on fat as a fuel with increased protein breakdown to support the TCA cycle. CONCLUSIONS: These data support a fundamental role for Bmal1, the endogenous circadian clock, in glucose metabolism in the skeletal muscle. Our findings have implicated altered molecular clock dictating significant changes in altered substrate metabolism in the absence of feeding or activity changes. The changes in body composition in our model also highlight the important role that changes in skeletal muscle carbohydrate, and fat metabolism can play in systemic metabolism.

The endogenous molecular clock orchestrates the temporal separation of substrate metabolism in skeletal muscle.

BACKGROUND: Skeletal muscle is a major contributor to whole-body metabolism as it serves as a depot for both glucose and amino acids, and is a highly metabolically active tissue. Within skeletal muscle exists an intrinsic molecular clock mechanism that regulates the timing of physiological processes. A key function of the clock is to regulate the timing of metabolic processes to anticipate time of day changes in environmental conditions. The purpose of this study was to identify metabolic genes that are expressed in a circadian manner and determine if these genes are regulated downstream of the intrinsic molecular clock by assaying gene expression in an inducible skeletal muscle-specific Bmal1 knockout mouse model (iMS-Bmal1 (-/-) ). METHODS: We used circadian statistics to analyze a publicly available, high-resolution time-course skeletal muscle expression dataset. Gene ontology analysis was utilized to identify enriched biological processes in the skeletal muscle circadian transcriptome. We generated a tamoxifen-inducible skeletal muscle-specific Bmal1 knockout mouse model and performed a time-course microarray experiment to identify gene expression changes downstream of the molecular clock. Wheel activity monitoring was used to assess circadian behavioral rhythms in iMS-Bmal1 (-/-) and control iMS-Bmal1 (+/+) mice. RESULTS: The skeletal muscle circadian transcriptome was highly enriched for metabolic processes. Acrophase analysis of circadian metabolic genes revealed a temporal separation of genes involved in substrate utilization and storage over a 24-h period. A number of circadian metabolic genes were differentially expressed in the skeletal muscle of the iMS-Bmal1 (-/-) mice. The iMS-Bmal1 (-/-) mice displayed circadian behavioral rhythms indistinguishable from iMS-Bmal1 (+/+) mice. We also observed a gene signature indicative of a fast to slow fiber-type shift and a more oxidative skeletal muscle in the iMS-Bmal1 (-/-) model. CONCLUSIONS: These data provide evidence that the intrinsic molecular clock in skeletal muscle temporally regulates genes involved in the utilization and storage of substrates independent of circadian activity. Disruption of this mechanism caused by phase shifts (that is, social jetlag) or night eating may ultimately diminish skeletal muscle's ability to efficiently maintain metabolic homeostasis over a 24-h period.