Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans

Background: Advances in automated image-based microscopy platforms coupled with high-throughput liquid workflows have facilitated the design of large-scale screens utilising multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high-throughput approaches, and a systematic way to analyse genetic interactions of essential genes in multicellular organisms has been lacking. Results: In C. elegans, non-conditional lethal mutations can be maintained in heterozygosity using chromosome balancers, commonly expressing green fluorescent protein (GFP) in the pharynx. However, gene expression or function is typically monitored by the use of fluorescent reporters marked with the same fluorophore, presenting a challenge to sort worm populations of interest, particularly at early larval stages. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we develop an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image analysis in a functional genomic RNA interference (RNAi) screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPR mt ). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both known and new PHB genetic interactors affecting the UPR mt and growth. Conclusions: The method presented here allows the study of balanced lethal mutations in a high-throughput manner. It can be easily adapted depending on the user's requirements and should serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.European Research Council ERC-2011-StG-281691Ministerio de Economía y Competitividad BFU2012–3550

Metformin promotes lifespan through mitohormesis via the peroxiredoxin PRDX-2

The antiglycemic drug metformin, widely prescribed as first-line treatment of type II diabetes mellitus, has lifespan-extending properties. Precisely how this is achieved remains unclear. Via a quantitative proteomics approach using the model organism Caenorhabditis elegans, we gained molecular understanding of the physiological changes elicited by metformin exposure, including changes in branched-chain amino acid catabolism and cuticle maintenance. We show that metformin extends lifespan through the process of mitohormesis and propose a signaling cascade in which metformin-induced production of reactive oxygen species increases overall life expectancy. We further address an important issue in aging research, wherein so far, the key molecular link that translates the reactive oxygen species signal into a prolongevity cue remained elusive. We show that this beneficial signal of the mitohormetic pathway is propagated by the peroxiredoxin PRDX-2. Because of its evolutionary conservation, peroxiredoxin signaling might underlie a general principle of prolongevity signaling

Deletion of heat shock protein 60 in adult mouse cardiomyocytes perturbs mitochondrial protein homeostasis and causes heart failure.

To maintain healthy mitochondrial enzyme content and function, mitochondria possess a complex protein quality control system, which is composed of different endogenous sets of chaperones and proteases. Heat shock protein 60 (HSP60) is one of these mitochondrial molecular chaperones and has been proposed to play a pivotal role in the regulation of protein folding and the prevention of protein aggregation. However, the physiological function of HSP60 in mammalian tissues is not fully understood. Here we generated an inducible cardiac-specific HSP60 knockout mouse model, and demonstrated that HSP60 deletion in adult mouse hearts altered mitochondrial complex activity, mitochondrial membrane potential, and ROS production, and eventually led to dilated cardiomyopathy, heart failure, and lethality. Proteomic analysis was performed in purified control and mutant mitochondria before mutant hearts developed obvious cardiac abnormalities, and revealed a list of mitochondrial-localized proteins that rely on HSP60 (HSP60-dependent) for correctly folding in mitochondria. We also utilized an in vitro system to assess the effects of HSP60 deletion on mitochondrial protein import and protein stability after import, and found that both HSP60-dependent and HSP60-independent mitochondrial proteins could be normally imported in mutant mitochondria. However, the former underwent degradation in mutant mitochondria after import, suggesting that the protein exhibited low stability in mutant mitochondria. Interestingly, the degradation could be almost fully rescued by a non-specific LONP1 and proteasome inhibitor, MG132, in mutant mitochondria. Therefore, our results demonstrated that HSP60 plays an essential role in maintaining normal cardiac morphology and function by regulating mitochondrial protein homeostasis and mitochondrial function

Mitochondrial Quality Control in Age-Related Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is age-related interstitial lung disease of unknown etiology. About 100,000 people in the U.S have IPF, with a 3-year median life expectancy post-diagnosis. The development of an effective treatment for pulmonary fibrosis will require an improved understanding of its molecular pathogenesis and the normal and pathological\u27 hallmarks of the aging lung. An important characteristic of the aging organism is its lowered capacity to adapt quickly to, and counteract, disturbances. While it is likely that DNA damage, chronic endoplasmic reticulum (ER) stress, and accumulation of heat shock proteins are capable of initiating tissue repair, recent studies point to a pathogenic role for mitochondrial dysfunction in the development of pulmonary fibrosis. These studies suggest that damage to the mitochondria induces fibrotic remodeling through a variety of mechanisms including the activation of apoptotic and inflammatory pathways. Mitochondrial quality control (MQC) has been demonstrated to play an important role in the maintenance of mitochondrial homeostasis. Different factors can induce MQC, including mitochondrial DNA damage, proteostasis dysfunction, and mitochondrial protein translational inhibition. MQC constitutes a complex signaling response that affects mitochondrial biogenesis, mitophagy, fusion/fission and the mitochondrial unfolded protein response (UPRmt) that, together, can produce new mitochondria, degrade the components of the oxidative complex or clearance the entire organelle. In pulmonary fibrosis, defects in mitophagy and mitochondrial biogenesis have been implicated in both cellular apoptosis and senescence during tissue repair. MQC has also been found to have a role in the regulation of other protein activity, inflammatory mediators, latent growth factors, and anti-fibrotic growth factors. In this review, we delineated the role of MQC in the pathogenesis of age-related pulmonary fibrosis

The transcription factor ATF5: role in cellular differentiation, stress responses, and cancer.

Activating transcription factor 5 (ATF5) is a cellular prosurvival transcription factor within the basic leucine zipper (bZip) family that is involved in cellular differentiation and promotes cellular adaptation to stress. Recent studies have characterized the oncogenic role of ATF5 in the development of several different types of cancer, notably glioblastoma. Preclinical assessment of a systemically deliverable dominant-negative ATF5 (dnATF5) biologic has found that targeting ATF5 results in tumor regression and tumor growth inhibition of glioblastoma xenografts in mouse models. In this review, we comprehensively and critically detail the current scientific literature on ATF5 in the context of cellular differentiation, survival, and response to stressors in normal tissues. Furthermore, we will discuss how the prosurvival role of ATF5 aides in cancer development, followed by current advances in targeting ATF5 using dominant-negative biologics, and perspectives on future research

Proteostasis and ageing: insights from long-lived mutant mice

The global increase in life expectancy is creating significant medical, social and economic challenges to current and future generations. Consequently, there is a need to identify the fundamental mechanisms underlying the ageing process. This knowledge should help develop realistic interventions capable of combatting age-related disease, and thus improving late-life health and vitality. While several mechanisms have been proposed as conserved lifespan determinants, the loss of proteostasis- where proteostasis is defined here as the maintenance of the proteome- appears highly relevant to both ageing and disease. Several studies have shown that multiple proteostatic mechanisms, including the endoplasmic reticulum (ER)-induced unfolded protein response (UPR), the ubiquitin-proteasome system (UPS) and autophagy, all appear indispensable for longevity in many long-lived invertebrate mutants. Similarly, interspecific comparisons suggest that proteostasis may be an important lifespan determinant in vertebrates. Over the last 20 years a number of long-lived mouse mutants have been described, many of which carry single-gene mutations within the growth-hormone, insulin/IGF-1 or mTOR signalling pathways. However, we still do not know how these mutations act mechanistically to increase lifespan and healthspan, and accordingly whether mechanistic commonality occurs between different mutants. Recent evidence supports the premise that the successful maintenance of the proteome during ageing may be linked to the increased lifespan and healthspan of long-lived mouse mutants

Depletion of mitochondrial protease OMA1 alters proliferative properties and promotes metastatic growth of breast cancer cells

Metastatic competence of cancer cells is influenced by many factors including metabolic alterations and changes in mitochondrial biogenesis and protein homeostasis. While it is generally accepted that mitochondria play important roles in tumorigenesis, the respective molecular events that regulate aberrant cancer cell proliferation remain to be clarified. Therefore, understanding the mechanisms underlying the role of mitochondria in cancer progression has potential implications in the development of new therapeutic strategies. We show that low expression of mitochondrial quality control protease OMA1 correlates with poor overall survival in breast cancer patients. Silencing OMA1 in vitro in patientderived metastatic breast cancer cells isolated from the metastatic pleural effusion and atypical ductal hyperplasia mammary tumor specimens (21MT-1 and 21PT) enhances the formation of filopodia, increases cell proliferation (Ki67 expression), and induces epithelial-mesenchymal transition (EMT). Mechanistically, loss of OMA1 results in alterations in the mitochondrial protein homeostasis, as reflected by enhanced expression of canonic mitochondrial unfolded protein response genes. These changes significantly increase migratory properties in metastatic breast cancer cells, indicating that OMA1 plays a critical role in suppressing metastatic competence of breast tumors. Interestingly, these results were not observed in OMA1-depleted non-tumorigenic MCF10A mammary epithelial cells. This newly identified reduced activity/levels of OMA1 provides insights into the mechanisms leading to breast cancer development, promoting malignant progression of cancer cells and unfavorable clinical outcomes, which may represent possible prognostic markers and therapeutic targets for breast cancer treatment

Life-span extension by axenic dietary restriction is independent of the mitochondrial unfolded protein response and mitohormesis in Caenorhabditis elegans

In Caenorhabditis elegans, a broad range of dietary restriction regimens extend life span to different degrees by separate or partially overlapping molecular pathways. One of these regimens, axenic dietary restriction, doubles the worm's life span but currently, almost nothing is known about the underlying molecular mechanism. Previous studies suggest that mitochondrial stress responses such as the mitochondrial unfolded protein response (UPRmt) or mitohormesis may play a vital role in axenic dietary restriction-induced longevity. Here, we provide solid evidence that axenic dietary restriction treatment specifically induces an UPRmt response in C elegans but this induction is not required for axenic dietary restriction-mediated longevity. We also show that reactive oxygen species-mediated mitohormesis is not involved in this phenotype. Hence, changes in mitochondrial physiology and induction of a mitochondrial stress response are not necessarily causal to large increases in life span