Mitochondrial p38 Mitogen-Activated Protein Kinase: Insights into Its Regulation of and Role in LONP1-Deficient Nematodes.

p38 Mitogen-Activated Protein Kinase (MAPK) cascades are central regulators of numerous physiological cellular processes, including stress response signaling. In C. elegans, mitochondrial dysfunction activates a PMK-3/p38 MAPK signaling pathway (MAPKmt), but its functional role still remains elusive. Here, we demonstrate the induction of MAPKmt in worms deficient in the lonp-1 gene, which encodes the worm ortholog of mammalian mitochondrial LonP1. This induction is subjected to negative regulation by the ATFS-1 transcription factor through the CREB-binding protein (CBP) ortholog CBP-3, indicating an interplay between both activated MAPKmt and mitochondrial Unfolded Protein Response (UPRmt) surveillance pathways. Our results also reveal a genetic interaction in lonp-1 mutants between PMK-3 kinase and the ZIP-2 transcription factor. ZIP-2 has an established role in innate immunity but can also modulate the lifespan by maintaining mitochondrial homeostasis during ageing. We show that in lonp-1 animals, ZIP-2 is activated in a PMK-3-dependent manner but does not confer increased survival to pathogenic bacteria. However, deletion of zip-2 or pmk-3 shortens the lifespan of lonp-1 mutants, suggesting a possible crosstalk under conditions of mitochondrial perturbation that influences the ageing process. Furthermore, loss of pmk-3 specifically diminished the extreme heat tolerance of lonp-1 worms, highlighting the crucial role of PMK-3 in the heat shock response upon mitochondrial LONP-1 inactivation.

Selective Destabilization of Transcripts by mRNA Decapping Regulates Oocyte Maturation and Innate Immunity Gene Expression during Ageing in C. elegans.

Removal of the 5' cap structure of RNAs (termed decapping) is a pivotal event in the life of cytoplasmic mRNAs mainly catalyzed by a conserved holoenzyme, composed of the catalytic subunit DCP2 and its essential cofactor DCP1. While decapping was initially considered merely a step in the general 5'-3' mRNA decay, recent data suggest a great degree of selectivity that plays an active role in the post-transcriptional control of gene expression, and regulates multiple biological functions. Studies in Caenorhabditis elegans have shown that old age is accompanied by the accumulation of decapping factors in cytoplasmic RNA granules, and loss of decapping activity shortens the lifespan. However, the link between decapping and ageing remains elusive. Here, we present a comparative microarray study that was aimed to uncover the differences in the transcriptome of mid-aged dcap-1/DCP1 mutant and wild-type nematodes. Our data indicate that DCAP-1 mediates the silencing of spermatogenic genes during late oogenesis, and suppresses the aberrant uprise of immunity gene expression during ageing. The latter is achieved by destabilizing the mRNA that encodes the transcription factor PQM-1 and impairing its nuclear translocation. Failure to exert decapping-mediated control on PQM-1 has a negative impact on the lifespan, but mitigates the toxic effects of polyglutamine expression that are involved in human disease.

The Thermal Stress Coping Network of the Nematode Caenorhabditis elegans.

Response to hyperthermia, highly conserved from bacteria to humans, involves transcriptional upregulation of genes involved in battling the cytotoxicity caused by misfolded and denatured proteins, with the aim of proteostasis restoration. C. elegans senses and responds to changes in growth temperature or noxious thermal stress by well-defined signaling pathways. Under adverse conditions, regulation of the heat shock response (HSR) in C. elegans is controlled by a single transcription factor, heat-shock factor 1 (HSF-1). HSR and HSF-1 in particular are proven to be central to survival under proteotoxic stress, with additional roles in normal physiological processes. For years, it was a common belief that upregulation of heat shock proteins (HSPs) by HSF-1 was the main and most important step toward thermotolerance. However, an ever-growing number of studies have shown that targets of HSF-1 involved in cytoskeletal and exoskeletal integrity preservation as well as other HSF-1 dependent and independent pathways are equally important. In this review, we follow the thermal stimulus from reception by the nematode nerve endings till the activation of cellular response programs. We analyze the different HSF-1 functions in HSR as well as all the recently discovered mechanisms that add to the knowledge of the heat stress coping network of C. elegans.

Organismal and Cellular Stress Responses upon Disruption of Mitochondrial Lonp1 Protease.

Cells engage complex surveillance mechanisms to maintain mitochondrial function and protein homeostasis. LonP1 protease is a key component of mitochondrial quality control and has been implicated in human malignancies and other pathological disorders. Here, we employed two experimental systems, the worm Caenorhabditis elegans and human cancer cells, to investigate and compare the effects of LONP-1/LonP1 deficiency at the molecular, cellular, and organismal levels. Deletion of the lonp-1 gene in worms disturbed mitochondrial function, provoked reactive oxygen species accumulation, and impaired normal processes, such as growth, behavior, and lifespan. The viability of lonp-1 mutants was dependent on the activity of the ATFS-1 transcription factor, and loss of LONP-1 evoked retrograde signaling that involved both the mitochondrial and cytoplasmic unfolded protein response (UPRmt and UPRcyt) pathways and ensuing diverse organismal stress responses. Exposure of worms to triterpenoid CDDO-Me, an inhibitor of human LonP1, stimulated only UPRcyt responses. In cancer cells, CDDO-Me induced key components of the integrated stress response (ISR), the UPRmt and UPRcyt pathways, and the redox machinery. However, genetic knockdown of LonP1 revealed a genotype-specific cellular response and induced apoptosis similar to CDDO-Me treatment. Overall, the mitochondrial dysfunction ensued by disruption of LonP1 elicits adaptive cytoprotective mechanisms that can inhibit cancer cell survival but diversely modulate organismal stress response and aging.

Biological implications of decapping: beyond bulk mRNA decay.

It is well established that mRNA steady-state levels do not directly correlate with transcription rate. This is attributed to the multiple post-transcriptional mechanisms, which control both mRNA turnover and translation within eukaryotic cells. One such mechanism is the removal of the 5' end cap structure of RNAs (decapping). This 5' cap plays a fundamental role in cellular functions related to mRNA processing, transport, translation, quality control, and decay, while its chemical modifications influence the fate of cytoplasmic mRNAs. Decapping is a highly controlled process, performed by multiple decapping enzymes, and regulated by complex cellular networks. In this review, we provide an updated synopsis of 5' end modifications and functions, and give an overview of mRNA decapping enzymes, presenting their enzymatic properties. Focusing on DCP2 decapping enzyme, a major component on the 5'-3' mRNA decay pathway, we describe cis-elements and trans-acting factors that affect its activity, substrate specificity, and cellular localization. Finally, we discuss current knowledge on the biological functions of mRNA decapping and decay factors, highlighting the major questions that remain to be addressed.

From Proteomic Mapping to Invasion-Metastasis-Cascade Systemic Biomarkering and Targeted Drugging of Mutant BRAF-Dependent Human Cutaneous Melanomagenesis.

Melanoma is classified among the most notoriously aggressive human cancers. Despite the recent progress, due to its propensity for metastasis and resistance to therapy, novel biomarkers and oncogenic molecular drivers need to be promptly identified for metastatic melanoma. Hence, by employing nano liquid chromatography-tandem mass spectrometry deep proteomics technology, advanced bioinformatics algorithms, immunofluorescence, western blotting, wound healing protocols, molecular modeling programs, and MTT assays, we comparatively examined the respective proteomic contents of WM115 primary (n = 3955 proteins) and WM266-4 metastatic (n = 6681 proteins) melanoma cells. It proved that WM115 and WM266-4 cells have engaged hybrid epithelial-to-mesenchymal transition/mesenchymal-to-epithelial transition states, with TGF-ß controlling their motility in vitro. They are characterized by different signatures of SOX-dependent neural crest-like stemness and distinct architectures of the cytoskeleton network. Multiple signaling pathways have already been activated from the primary melanoma stage, whereas HIF1α, the major hypoxia-inducible factor, can be exclusively observed in metastatic melanoma cells. Invasion-metastasis cascade-specific sub-routines of activated Caspase-3-triggered apoptosis and LC3B-II-dependent constitutive autophagy were also unveiled. Importantly, WM115 and WM266-4 cells exhibited diverse drug response profiles, with epirubicin holding considerable promise as a beneficial drug for metastatic melanoma clinical management. It is the proteome navigation that enables systemic biomarkering and targeted drugging to open new therapeutic windows for advanced disease.

mRNA decapping is an evolutionarily conserved modulator of neuroendocrine signaling that controls development and ageing.

Eukaryotic 5'-3' mRNA decay plays important roles during development and in response to stress, regulating gene expression post-transcriptionally. In Caenorhabditis elegans, deficiency of DCAP-1/DCP1, the essential co-factor of the major cytoplasmic mRNA decapping enzyme, impacts normal development, stress survival and ageing. Here, we show that overexpression of dcap-1 in neurons of worms is sufficient to increase lifespan through the function of the insulin/IGF-like signaling and its effector DAF-16/FOXO transcription factor. Neuronal DCAP-1 affects basal levels of INS-7, an ageing-related insulin-like peptide, which acts in the intestine to determine lifespan. Short-lived dcap-1 mutants exhibit a neurosecretion-dependent upregulation of intestinal ins-7 transcription, and diminished nuclear localization of DAF-16/FOXO. Moreover, neuronal overexpression of DCP1 in Drosophila melanogaster confers longevity in adults, while neuronal DCP1 deficiency shortens lifespan and affects wing morphogenesis, cell non-autonomously. Our genetic analysis in two model-organisms suggests a critical and conserved function of DCAP-1/DCP1 in developmental events and lifespan modulation.

Gene-Specific Intron Retention Serves as Molecular Signature that Distinguishes Melanoma from Non-Melanoma Cancer Cells in Greek Patients.

BACKGROUND: Skin cancer represents the most common human malignancy, and it includes BCC, SCC, and melanoma. Since melanoma is one of the most aggressive types of cancer, we have herein attempted to develop a gene-specific intron retention signature that can distinguish BCC and SCC from melanoma biopsy tumors. METHODS: Intron retention events were examined through RT-sqPCR protocols, using total RNA preparations derived from BCC, SCC, and melanoma Greek biopsy specimens. Intron-hosted miRNA species and their target transcripts were predicted via the miRbase and miRDB bioinformatics platforms, respectively. Ιntronic ORFs were recognized through the ORF Finder application. Generation and visualization of protein interactomes were achieved by the IntAct and Cytoscape softwares, while tertiary protein structures were produced by using the I-TASSER online server. RESULTS: c-MYC and Sestrin-1 genes proved to undergo intron retention specifically in melanoma. Interaction maps of proteins encoded by genes being potentially targeted by retained intron-accommodated miRNAs were generated and SRPX2 was additionally delivered to our melanoma-specific signature. Novel ORFs were identified in MCT4 and Sestrin-1 introns, with potentially critical roles in melanoma development. CONCLUSIONS: The property of c-MYC, Sestrin-1, and SRPX2 genes to retain specific introns could be clinically used to molecularly differentiate non-melanoma from melanoma tumors.

Neuronal function of the mRNA decapping complex determines survival of Caenorhabditis elegans at high temperature through temporal regulation of heterochronic gene expression.

In response to adverse environmental cues, Caenorhabditis elegans larvae can temporarily arrest development at the second moult and form dauers, a diapause stage that allows for long-term survival. This process is largely regulated by certain evolutionarily conserved signal transduction pathways, but it is also affected by miRNA-mediated post-transcriptional control of gene expression. The 5'-3' mRNA decay mechanism contributes to miRNA-mediated silencing of target mRNAs in many organisms but how it affects developmental decisions during normal or stress conditions is largely unknown. Here, we show that loss of the mRNA decapping complex activity acting in the 5'-3' mRNA decay pathway inhibits dauer formation at the stressful high temperature of 27.5°C, and instead promotes early developmental arrest. Our genetic data suggest that this arrest phenotype correlates with dysregulation of heterochronic gene expression and an aberrant stabilization of lin-14 mRNA at early larval stages. Restoration of neuronal dcap-1 activity was sufficient to rescue growth phenotypes of dcap-1 mutants at both high and normal temperatures, implying the involvement of common developmental timing mechanisms. Our work unveils the crucial role of 5'-3' mRNA degradation in proper regulation of heterochronic gene expression programmes, which proved to be essential for survival under stressful conditions.

Cytoplasmic mRNA turnover and ageing.

Messenger RNA (mRNA) turnover that determines the lifetime of cytoplasmic mRNAs is a means to control gene expression under both normal and stress conditions, whereas its impact on ageing and age-related disorders has just become evident. Gene expression control is achieved at the level of the mRNA clearance as well as mRNA stability and accessibility to other molecules. All these processes are regulated by cis-acting motifs and trans-acting factors that determine the rates of translation and degradation of transcripts. Specific messenger RNA granules that harbor the mRNA decay machinery or various factors, involved in translational repression and transient storage of mRNAs, are also part of the mRNA fate regulation. Their assembly and function can be modulated to promote stress resistance to adverse conditions and over time affect the ageing process and the lifespan of the organism. Here, we provide insights into the complex relationships of ageing modulators and mRNA turnover mechanisms.

Response of Caenorhabditis elegans to wireless devices radiation exposure.

PURPOSE: To examine the impact of electromagnetic radiation, produced by GSM (Global System for Mobile communications) mobile phones, Wi-Fi (Wireless-Fidelity) routers and wireless DECT (Digital Enhanced Cordless Telecommunications) phones, on the nematode Caenorhabditis elegans. MATERIALS AND METHODS: We exposed synchronized populations, of different developmental stages, to these wireless devices at E-field levels below ICNIRP's (International Commission on Non-Ionizing Radiation Protection) guidelines for various lengths of time. WT (wild-type) and aging- or stress-sensitive mutant worms were examined for changes in growth, fertility, lifespan, chemotaxis, short-term memory, increased ROS (Reactive Oxygen Species) production and apoptosis by using fluorescent marker genes or qRT-PCR (quantitative Reverse Transcription-Polymerase Chain Reaction). RESULTS: No statistically significant differences were found between the exposed and the sham/control animals in any of the experiments concerning lifespan, fertility, growth, memory, ROS, apoptosis or gene expression. CONCLUSIONS: The worm appears to be robust to this form of (pulsed) radiation, at least under the exposure conditions used.

Diverse functions of mRNA metabolism factors in stress defense and aging of Caenorhabditis elegans.

Processing bodies (PBs) and stress granules (SGs) are related, cytoplasmic RNA-protein complexes that contribute to post-transcriptional gene regulation in all eukaryotic cells. Both structures contain translationally repressed mRNAs and several proteins involved in silencing, stabilization or degradation of mRNAs, especially under environmental stress. Here, we monitored the dynamic formation of PBs and SGs, in somatic cells of adult worms, using fluorescently tagged protein markers of each complex. Both complexes were accumulated in response to various stress conditions, but distinct modes of SG formation were induced, depending on the insult. We also observed an age-dependent accumulation of PBs but not of SGs. We further showed that direct alterations in PB-related genes can influence aging and normal stress responses, beyond their developmental role. In addition, disruption of SG-related genes had diverse effects on development, fertility, lifespan and stress resistance of worms. Our work therefore underlines the important roles of mRNA metabolism factors in several vital cellular processes and provides insight into their diverse functions in a multicellular organism.

The general control nonderepressible-2 kinase mediates stress response and longevity induced by target of rapamycin inactivation in Caenorhabditis elegans.

The general control nonderepressible 2 (GCN2) kinase is a nutrient-sensing pathway that responds to amino acids deficiency and induces a genetic program to effectively maintain cellular homeostasis. Here we established the conserved role of Caenorhabditis elegans GCN-2 under amino acid limitation as a translation initiation factor 2 (eIF2) kinase. Using a combination of genetic and molecular approaches, we showed that GCN-2 kinase activity plays a central role in survival under nutrient stress and mediates lifespan extension conferred by dietary restriction (DR) or inhibition of the major nutrient-sensing pathway, the target of rapamycin (TOR). We also demonstrated that the GCN-2 and TOR signaling pathways converge on the PHA-4/FoxA transcription factor and its downstream target genes to ensure survival of the whole organism under a multitude of stress conditions, such as nutrient scarcity or environmental stresses. This is one step forward in the understanding of evolutionary conserved mechanisms that confer longevity and healthspan.

Effects of Lactobacillus salivarius, Lactobacillus reuteri, and Pediococcus acidilactici on the nematode Caenorhabditis elegans include possible antitumor activity.

This study examined the effects of three lactic acid bacteria (LAB) strains on the nematode Caenorhabditis elegans. Lactobacillus salivarius, Lactobacillus reuteri, and Pediococcus acidilactici were found to inhibit the development and growth of the worm. Compared to Escherichia coli used as the control, L. reuteri and P. acidilactici reduced the lifespan of wild-type and short-lived daf-16 worms. On the contrary, L. salivarius extended the lifespan of daf-16 worms when used live, but reduced it as UV-killed bacteria. The three LAB induced the expression of genes involved in pathogen response and inhibited the growth of tumor-like germ cells, without affecting DAF16 localization or increasing corpse cells. Our results suggest the possible use of C. elegans as a model for studying the antitumor attributes of LAB. The negative effects of these LAB strains on the nematode also indicate their potential use against parasitic nematodes.

Protein synthesis is a novel determinant of aging in Caenorhabditis elegans.

Protein synthesis is a tightly regulated cellular process that affects growth, reproduction, and survival in response to both intrinsic and extrinsic cues, such as nutrient availability and energy levels. A pronounced, age-related decline of the total protein synthesis rate has been observed in many organisms, including humans. The molecular mechanisms underlying this decline and their role in the aging process remain unclear. A series of recent studies in the nematode, Caenorhabditis elegans, have revealed a novel link between protein synthesis and aging. Remarkably, these research findings, in their totality, converge to indicate that reduction of mRNA translation prolongs life in worms. Signal transduction cascades implicated in aging, such as the insulin/insulin growth factor-1 pathway, interface with mechanisms regulating protein synthesis via a battery of key mRNA translation factors. Are the effects of these pathways on aging mediated, in part, by alterations in protein synthesis? This is an intriguing possibility in light of the latest discoveries. Whether attenuation of protein synthesis promotes longevity across different phyla is an additional important matter. Here, we survey work associating protein synthesis with aging and discuss the basis of life-span extension under conditions that attenuate protein synthesis.

eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans.

Regulation of protein synthesis is critical for cell growth and maintenance. Ageing in many organisms, including humans, is accompanied by marked alterations in both general and specific protein synthesis. Whether these alterations are simply a corollary of the ageing process or have a causative role in senescent decline remains unclear. An array of protein factors facilitates the tight control of messenger RNA translation initiation. The eukaryotic initiation factor 4E (eIF4E), which binds the 7-monomethyl guanosine cap at the 5' end of all nuclear mRNAs, is a principal regulator of protein synthesis. Here we show that loss of a specific eIF4E isoform (IFE-2) that functions in somatic tissues reduces global protein synthesis, protects from oxidative stress and extends lifespan in Caenorhabditis elegans. Lifespan extension is independent of the forkhead transcription factor DAF-16, which mediates the effects of the insulin-like signalling pathway on ageing. Furthermore, IFE-2 deficiency further extends the lifespan of long-lived age and daf nematode mutants. Similarly, lack of IFE-2 enhances the long-lived phenotype of clk and dietary-restricted eat mutant animals. Knockdown of target of rapamycin (TOR), a phosphatidylinositol kinase-related kinase that controls protein synthesis in response to nutrient cues, further increases the longevity of ife-2 mutants. Thus, signalling via eIF4E in the soma is a newly discovered pathway influencing ageing in C. elegans.

Signaling pathways regulating protein synthesis during ageing.

Ageing in many organisms, including humans, is accompanied by marked alterations in both general and specific protein synthesis. Protein synthesis is normally under tight control by a broad array of regulatory factors, which facilitate appropriate rates of mRNA translation. Are the wide changes in protein synthesis simply a corollary of the ageing process or do they have a causative role in senescent decline? The jury is still out on this important question. Nevertheless, recent studies reveal an intimate interface between mechanisms that govern the translation of mRNA and molecular pathways implicated in ageing. In our manuscript we consider these links, which potentially underlie age-associated changes in protein synthesis.

Lysosomal biogenesis and function is critical for necrotic cell death in Caenorhabditis elegans.

Necrotic cell death is defined by distinctive morphological characteristics that are displayed by dying cells (Walker, N.I., B.V. Harmon, G.C. Gobe, and J.F. Kerr. 1988. Methods Achiev. Exp. Pathol. 13:18-54). The cellular events that transpire during necrosis to generate these necrotic traits are poorly understood. Recent studies in the nematode Caenorhabditis elegans show that cytoplasmic acidification develops during necrosis and is required for cell death (Syntichaki, P., C. Samara, and N. Tavernarakis. 2005. Curr. Biol. 15:1249-1254). However, the origin of cytoplasmic acidification remains elusive. We show that the alkalization of endosomal and lysosomal compartments ameliorates necrotic cell death triggered by diverse stimuli. In addition, mutations in genes that result in altered lysosomal biogenesis and function markedly affect neuronal necrosis. We used a genetically encoded fluorescent marker to follow lysosome fate during neurodegeneration in vivo. Strikingly, we found that lysosomes fuse and localize exclusively around a swollen nucleus. In the advanced stages of cell death, the nucleus condenses and migrates toward the periphery of the cell, whereas green fluorescent protein-labeled lysosomal membranes fade, indicating lysosomal rupture. Our findings demonstrate a prominent role for lysosomes in cellular destruction during necrotic cell death, which is likely conserved in metazoans.

The vacuolar H+ -ATPase mediates intracellular acidification required for neurodegeneration in C. elegans.

Numerous studies implicate necrotic cell death in devastating human pathologies such as stroke and neurodegenerative diseases. Investigations in both nematodes and mammals converge to implicate specific calpain and aspartyl proteases in the execution of necrotic cell death. It is believed that these proteases become activated under conditions that inflict necrotic cell death. However, the factors that modulate necrosis and govern the erroneous activation of these otherwise benign enzymes are largely unknown. Here we show that the function of the vacuolar H(+)-ATPase, a pump that acidifies lysosomes and other intracellular organelles, is essential for necrotic cell death in C. elegans. Cytoplasmic pH drops in dying cells. Intracellular acidification requires the vacuolar H(+)-ATPase, whereas alkalization of endosomal and lysosomal compartments by weak bases protects against necrosis. In addition, we show that vacuolar H(+)-ATPase activity is required downstream of cytoplasmic calcium overload during necrosis. Thus, intracellular pH is an important modulator of necrosis in C. elegans. We propose that vacuolar H(+)-ATPase activity is required to establish necrosis-promoting, acidic intracellular conditions that augment the function of executioner aspartyl proteases in dying cells. Similar mechanisms may contribute to necrotic cell death that follows extreme acidosis-for example, during stroke-in humans.