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Phosphorylation of the N-terminal domain of the huntingtin (HTT) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that efficiently phosphorylates full-length and N-terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo. TBK1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

The nematode Caenorhabditis elegans is increasingly used as a model for human biology. However, in vivo culturing platforms for C. elegans allowing high-content phenotyping during their life cycle in an automated fashion are lacking so far. Here, a multiplexed microfluidic platform for the rapid high-content phenotyping of populations of C. elegans down to single animal resolution is presented. Nematodes are (i) reversibly and regularly confined during their life inside tapered channels for imaging fluorescence signal expression and to measure their growth parameters, and (ii) allowed to freely move in microfluidic chambers, during which the swimming behavior was video-recorded. The obtained data sets are analyzed in an automated way and 19 phenotypic parameters are extracted. Our platform is employed for studying the effect of bacteria dilution, a form of dietary restriction (DR) in nematodes, on a worm model of Huntington's disease and demonstrates the influence of DR on disease regression.

The nematode Caenorhabditis elegans is an important model organism for biomedical research and genetic studies relevant to human biology and disease. Such studies are often based on high-resolution imaging of dynamic biological processes in the worm body tissues, requiring well-immobilized and physiologically active animals in order to avoid movement-related artifacts and to obtain meaningful biological information. However, existing immobilization methods employ the application of either anesthetics or servere physical constraints, by using glue or specific microfluidic on-chip mechanical structures, which in some cases may strongly affect physiological processes of the animals. Here, we immobilize C. elegans nematodes by taking advantage of a biocompatible and temperature-responsive hydrogel-microbead matrix. Our gel-based immobilization technique does not require a specific chip design and enables fast and reversible immobilization, thereby allowing successive imaging of the same single worm or of small worm populations at all development stages for several days. We successfully demonstrated the applicability of this method in challenging worm imaging contexts, in particular by applying it for high-resolution confocal imaging of the mitochondrial morphology in worm body wall muscle cells and for the long-term quantification of number and size of specific protein aggregates in different C. elegans neurodegenerative disease models. Our approach was also suitable for immobilizing other small organisms, such as the larvae of the fruit fly Drosophila melanogaster and the unicellular parasite Trypanosoma brucei. We anticipate that this versatile technique will significantly simplify biological assay-based longitudinal studies and long-term observation of small model organisms.

Fast, label-free, high-resolution, three-dimensional imaging platforms are crucial for high-throughput in vivo time-lapse studies of the anatomy of Caenorhabditis elegans, one of the most commonly used model organisms in biomedical research. Despite the needs, methods combining all these characteristics have been lacking. Here, we present label-free imaging of live Caenorhabditis elegans with three-dimensional sub-micrometer resolution using visible optical coherence microscopy (visOCM). visOCM is a versatile optical imaging method which we introduced recently for tomography of cell cultures and tissue samples. Our method is based on Fourier domain optical coherence tomography, an interferometric technique that provides three-dimensional images with high sensitivity, high acquisition rate and micrometer-scale resolution. By operating in the visible wavelength range and using a high NA objective, visOCM attains lateral and axial resolutions below 1 μm. Additionally, we use a Bessel illumination offering an extended depth of field of approximately 40 μm. We demonstrate that visOCM's imaging properties allow rapid imaging of full sized living Caenorhabditis elegans down to the sub-cellular level. Our system opens the door to many applications such as the study of phenotypic changes related to developmental or ageing processes.

Mitochondrial dysfunction is at the core of many diseases ranging from inherited metabolic diseases to common conditions that are associated with aging. Although associations between aging and mitochondrial function have been identified using mammalian models, much of the mechanistic insight has emerged from Caenorhabditis elegans. Mitochondrial respiration is recognized as an indicator of mitochondrial health. The Seahorse XF96 respirometer represents the state-of-the-art platform for assessing respiration in cells, and we adapted the technique for applications involving C. elegans. Here we provide a detailed protocol to optimize and measure respiration in C. elegans with the XF96 respirometer, including the interpretation of parameters and results. The protocol takes ∼2 d to complete, excluding the time spent culturing C. elegans, and it includes (i) the preparation of C. elegans samples, (ii) selection and loading of compounds to be injected, (iii) preparation and execution of a run with the XF96 respirometer and (iv) postexperimental data analysis, including normalization. In addition, we compare our XF96 application with other existing techniques, including the eight-well Seahorse XFp. The main benefits of the XF96 include the limited number of worms required and the high throughput capacity due to the 96-well format., Competing Interests: The authors declare that they have no competing financial interests.

Across eukaryotic species, mild mitochondrial stress can have beneficial effects on the lifespan of organisms. Mitochondrial dysfunction activates an unfolded protein response (UPR(mt)), a stress signaling mechanism designed to ensure mitochondrial homeostasis. Perturbation of mitochondria during larval development in C. elegans not only delays aging but also maintains UPR(mt) signaling, suggesting an epigenetic mechanism that modulates both longevity and mitochondrial proteostasis throughout life. We identify the conserved histone lysine demethylases jmjd-1.2/PHF8 and jmjd-3.1/JMJD3 as positive regulators of lifespan in response to mitochondrial dysfunction across species. Reduction of function of the demethylases potently suppresses longevity and UPR(mt) induction, while gain of function is sufficient to extend lifespan in a UPR(mt)-dependent manner. A systems genetics approach in the BXD mouse reference population further indicates conserved roles of the mammalian orthologs in longevity and UPR(mt) signaling. These findings illustrate an evolutionary conserved epigenetic mechanism that determines the rate of aging downstream of mitochondrial perturbations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: While many biological studies can be performed on cell-based systems, the investigation of molecular pathways related to complex human dysfunctions - e.g. neurodegenerative diseases - often requires long-term studies in animal models. The nematode Caenorhabditis elegans represents one of the best model organisms for many of these tests and, therefore, versatile and automated systems for accurate time-resolved analyses on C. elegans are becoming highly desirable tools in the field., Results: We describe a new multi-functional platform for C. elegans analytical research, enabling automated worm isolation and culture, reversible worm immobilization and long-term high-resolution imaging, and this under active control of the main culture parameters, including temperature. We employ our platform for in vivo observation of biomolecules and automated analysis of protein aggregation in a C. elegans model for amyotrophic lateral sclerosis (ALS). Our device allows monitoring the growth rate and development of each worm, at single animal resolution, within a matrix of microfluidic chambers. We demonstrate the progression of individual protein aggregates, i.e. mutated human superoxide dismutase 1 - Yellow Fluorescent Protein (SOD1-YFP) fusion proteins in the body wall muscles, for each worm and over several days. Moreover, by combining reversible worm immobilization and on-chip high-resolution imaging, our method allows precisely localizing the expression of biomolecules within the worms' tissues, as well as monitoring the evolution of single aggregates over consecutive days at the sub-cellular level. We also show the suitability of our system for protein aggregation monitoring in a C. elegans Huntington disease (HD) model, and demonstrate the system's ability to study long-term doxycycline treatment-linked modification of protein aggregation profiles in the ALS model., Conclusion: Our microfluidic-based method allows analyzing in vivo the long-term dynamics of protein aggregation phenomena in C. elegans at unprecedented resolution. Pharmacological screenings on neurodegenerative disease C. elegans models may strongly benefit from this method in the near future, because of its full automation and high-throughput potential.

The most important physiological mechanism mediating enhanced exercise performance is increased sympathetic, beta adrenergic receptor (β-AR), and adenylyl cyclase (AC) activity. This is the first report of decreased AC activity mediating increased exercise performance. We demonstrated that AC5 disruption, that is, knock out (KO) mice, a longevity model, increases exercise performance. Importantly for its relation to longevity, exercise was also improved in old AC5 KO. The mechanism resided in skeletal muscle rather than in the heart, as confirmed by cardiac- and skeletal muscle-specific AC5 KO's, where exercise performance was no longer improved by the cardiac-specific AC5 KO, but was by the skeletal muscle-specific AC5 KO, and there was no difference in cardiac output during exercise in AC5 KO vs. WT. Mitochondrial biogenesis was a major mechanism mediating the enhanced exercise. SIRT1, FoxO3a, MEK, and the anti-oxidant, MnSOD were upregulated in AC5 KO mice. The improved exercise in the AC5 KO was blocked with either a SIRT1 inhibitor, MEK inhibitor, or by mating the AC5 KO with MnSOD hetero KO mice, confirming the role of SIRT1, MEK, and oxidative stress mechanisms. The Caenorhabditis elegans worm AC5 ortholog, acy-3 by RNAi, also improved fitness, mitochondrial function, antioxidant defense, and lifespan, attesting to the evolutionary conservation of this pathway. Thus, decreasing sympathetic signaling through loss of AC5 is not only a mechanism to improve exercise performance, but is also a mechanism to improve healthful aging, as exercise also protects against diabetes, obesity, and cardiovascular disease, which all limit healthful aging., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Studies of the real-time dynamics of embryonic development require a gentle embryo handling method, the possibility of long-term live imaging during the complete embryogenesis, as well as of parallelization providing a population's statistics, while keeping single embryo resolution. We describe an automated approach that fully accomplishes these requirements for embryos of Caenorhabditis elegans, one of the most employed model organisms in biomedical research. We developed a microfluidic platform which makes use of pure passive hydrodynamics to run on-chip worm cultures, from which we obtain synchronized embryo populations, and to immobilize these embryos in incubator microarrays for long-term high-resolution optical imaging. We successfully employ our platform to investigate morphogenesis and mitochondrial biogenesis during the full embryonic development and elucidate the role of the mitochondrial unfolded protein response (UPR(mt)) within C. elegans embryogenesis. Our method can be generally used for protein expression and developmental studies at the embryonic level, but can also provide clues to understand the aging process and age-related diseases in particular.

Mitochondria are semi-autonomous organelles regulated by a complex network of proteins that are vital for many cellular functions. Because mitochondrial modulators can impact many aspects of cellular homeostasis, their identification and validation has proven challenging. It requires the measurement of multiple parameters in parallel to understand the exact nature of the changes induced by such compounds. We developed a platform of assays scoring for mitochondrial function in two complementary models systems, mammalian cells and C. elegans. We first optimized cell culture conditions and established the mitochondrial signature of 1,200 FDA-approved drugs in liver cells. Using cell-based and C. elegans assays, we further defined the metabolic effects of two pharmacological classes that emerged from our hit list, i.e. imidazoles and statins. We found that these two drug classes affect respiration through different and cholesterol-independent mechanisms in both models. Our screening strategy enabled us to unequivocally identify compounds that have toxic or beneficial effects on mitochondrial activity. Furthermore, the cross-species approach provided novel mechanistic insight and allowed early validation of hits that act on mitochondrial function.

Dietary restriction (DR) is one of the most universal means of extending lifespan. Yet, whether and how DR specifically affects the metabolic changes associated with aging is essentially unknown. Here, we present a comprehensive and unbiased picture of the metabolic variations that take place with age at the whole organism level in Caenorhabditis elegans by using (1)H high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) analysis of intact worms. We investigate metabolic variations potentially important for lifespan regulation by comparing the metabolic fingerprint of two previously described genetic models of DR, the long-lived eat-2(ad465) and slcf-1(tm2258) worms, as single mutants or in combination with a genetic suppressor of their lifespan phenotype. Our analysis shows that significant changes in metabolite profiles precede the major physiological decline that accompanies aging and that DR protects from some of those metabolic changes. More specifically, low phosphocholine (PCho) correlates with high life expectancy. A mutation in the tumor suppressor gene PTEN/DAF-18, which suppresses the beneficial effects of DR in both C. elegans and mammals, increases both PCho level and choline kinase expression. Furthermore, we show that choline kinase function in the intestine can regulate lifespan. This study highlights the relevance of NMR metabolomic approaches for identifying potential biomarkers of aging.

The ability to respond to various intracellular and/or extracellular stresses allows the organism to adapt to changing environmental conditions and drives evolution. It is now well accepted that a progressive decline of the efficiency of stress response pathways occurs with aging. In this context, a correct proteostasis is essential for the functionality of the cell, and its dysfunction has been associated with protein aggregation and age-related degenerative diseases. Complex response mechanisms have evolved to deal with unfolded protein stress in different subcellular compartments and their moderate activation translates into positive effects on health. In this review, we focus on the mitochondrial unfolded protein response (UPR(mt)), a response to proteotoxic stress specifically in mitochondria, an organelle with a wide array of fundamental functions, most notably the harvesting of energy from food and the control of cell death. We compare UPR(mt) with the extensively characterized cytosolic heat shock response (HSR) and the unfolded protein response in endoplasmic reticulum (UPR(ER)), and discuss the current knowledge about UPR(mt) signaling pathways as well as their potential involvement in physiology.

Longevity is regulated by a network of closely linked metabolic systems. We used a combination of mouse population genetics and RNA interference in Caenorhabditis elegans to identify mitochondrial ribosomal protein S5 (Mrps5) and other mitochondrial ribosomal proteins as metabolic and longevity regulators. MRP knockdown triggers mitonuclear protein imbalance, reducing mitochondrial respiration and activating the mitochondrial unfolded protein response. Specific antibiotics targeting mitochondrial translation and ethidium bromide (which impairs mitochondrial DNA transcription) pharmacologically mimic mrp knockdown and extend worm lifespan by inducing mitonuclear protein imbalance, a stoichiometric imbalance between nuclear and mitochondrially encoded proteins. This mechanism was also conserved in mammalian cells. In addition, resveratrol and rapamycin, longevity compounds acting on different molecular targets, similarly induced mitonuclear protein imbalance, the mitochondrial unfolded protein response and lifespan extension in C. elegans. Collectively these data demonstrate that MRPs represent an evolutionarily conserved protein family that ties the mitochondrial ribosome and mitonuclear protein imbalance to the mitochondrial unfolded protein response, an overarching longevity pathway across many species.

Transcriptional coregulators control the activity of many transcription factors and are thought to have wide-ranging effects on gene expression patterns. We show here that muscle-specific loss of nuclear receptor corepressor 1 (NCoR1) in mice leads to enhanced exercise endurance due to an increase of both muscle mass and of mitochondrial number and activity. The activation of selected transcription factors that control muscle function, such as MEF2, PPARβ/δ, and ERRs, underpins these phenotypic alterations. NCoR1 levels are decreased in conditions that require fat oxidation, resetting transcriptional programs to boost oxidative metabolism. Knockdown of gei-8, the sole C. elegans NCoR homolog, also robustly increased muscle mitochondria and respiration, suggesting conservation of NCoR1 function. Collectively, our data suggest that NCoR1 plays an adaptive role in muscle physiology and that interference with NCoR1 action could be used to improve muscle function., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Dietary restriction (DR) is the most universal intervention known to extend animal lifespan. DR also prevents tumor development in mammals, and this effect requires the tumor suppressor PTEN. However, the metabolic and cellular processes that underly the beneficial effects of DR are poorly understood. We identified slcf-1 in an RNAi screen for genes that extend Caenorhabditis elegans lifespan in a PTEN/daf-18-dependent manner. We showed that slcf-1 mutation, which increases average lifespan by 40%, mimics DR in worms fed ad libitum. An NMR-based metabolomic characterization of slcf-1 mutants revealed lower lipid levels compared to wild-type animals, as expected for dietary-restricted animals, but also higher pyruvate content. Epistasis experiments and metabolic measurements support a model in which the long lifespan of slcf-1 mutants relies on increased mitochondrial pyruvate metabolism coupled to an adaptive response to oxidative stress. This response requires DAF-18/PTEN and the previously identified DR effectors PHA-4/FOXA, HSF-1/HSF1, SIR-2.1/SIRT-1, and AMPK/AAK-2. Overall, our data show that pyruvate homeostasis plays a central role in lifespan control in C. elegans and that the beneficial effects of DR results from a hormetic mechanism involving the mitochondria. Analysis of the SLCF-1 protein sequence predicts that slcf-1 encodes a plasma membrane transporter belonging to the conserved monocarboxylate transporter family. These findings suggest that inhibition of this transporter homolog in mammals might also promote a DR response.

The PTEN tumour suppressor encodes a phosphatase, and its daf-18 orthologue in Caenorhabditis elegans negatively regulates the insulin/IGF-1 DAF-2 receptor pathway that influences lifespan in worms and other species. In order to identify new DAF-18 regulated pathways involved in aging, we initiated a candidate RNAi feeding screen for clones that lengthen lifespan. Here, we report that smg-1 inactivation increases average lifespan in a daf-18 dependent manner. Genetic analysis is consistent with SMG-1 acting at least in part in parallel to the canonical DAF-2 receptor pathway, but converging on the transcription factor DAF-16/FOXO. SMG-1 is a serine-threonine kinase which plays a conserved role in nonsense-mediated mRNA decay (NMD) in worms and mammals. In addition, human SMG-1 has also been implicated in the p53-mediated response to genotoxic stress. The effect of smg-1 inactivation on lifespan appears to be unrelated to its NMD function, but requires the p53 tumour suppressor orthologue cep-1. Furthermore, smg-1 inactivation confers a resistance to oxidative stress in a daf-18-, daf-16- and cep-1-dependent manner. We propose that the role of SMG-1 in lifespan regulation is at least partly dependent on its function in oxidative stress resistance. Taken together, our results unveil a novel role for SMG-1 in lifespan regulation.

A major step for the validation of medical drugs is the screening on whole organisms, which gives the systemic information that is missing when using cellular models. Caenorhabditis elegans is a soil worm that catches the interest of researchers who study systemic physiopathology (e.g., metabolic and neurodegenerative diseases) because: (1) its large genetic homology with humans supports translational analysis; (2) worms are much easier to handle and grow in large amounts compared with rodents, for which (3) the costs and (4) the ethical concerns are substantial. Here, we demonstrate how multimodal optical imaging on such an organism can provide high-content information relevant to the drug development pipeline (e.g., mode-of-action identification, dose-response analysis), especially when combined with on-chip multiplexing capability. After designing a microfluidic array to select small separated populations of C. elegans, we combine fluorescence and brightfield imaging along with high-throughput feature recognition and signal detection to enable the identification of the mode-of-action of an antibiotic. For this purpose, we use a genetically encoded fluorescence reporter of mitochondrial stress, which we studied in living specimens during their entire development. Furthermore, we demonstrate real-time, very large field-of-view capability on multiplexed motility assays for the assessment of the dose-response relation of an anesthetic. [ABSTRACT FROM AUTHOR]

The nematode Caenorhabditis elegans is increasingly used as a model for human biology. However, in vivo culturing platforms for C. elegans allowing high-content phenotyping during their life cycle in an automated fashion are lacking so far. Here, a multiplexed microfluidic platform for the rapid high-content phenotyping of populations of C. elegans down to single animal resolution is presented. Nematodes are (i) reversibly and regularly confined during their life inside tapered channels for imaging fluorescence signal expression and to measure their growth parameters, and (ii) allowed to freely move in microfluidic chambers, during which the swimming behavior was video-recorded. The obtained data sets are analyzed in an automated way and 19 phenotypic parameters are extracted. Our platform is employed for studying the effect of bacteria dilution, a form of dietary restriction (DR) in nematodes, on a worm model of Huntington's disease and demonstrates the influence of DR on disease regression. [ABSTRACT FROM AUTHOR]