Wild-type Worms Research Articles

Endosomal trafficking protein TBC-2 modulates stress resistance and lifespan through DAF-16-dependent and independent mechanisms.

The FOXO transcription factor, DAF-16, plays an integral role in insulin/IGF-1 signaling (IIS) and stress response. In conditions of stress or decreased IIS, DAF-16 moves to the nucleus where it activates genes that promote survival. To gain insight into the role of endosomal trafficking in resistance to stress, we disrupted tbc-2, which encodes a GTPase activating protein that inhibits RAB-5 and RAB-7. We found that tbc-2 mutants have decreased nuclear localization of DAF-16 in response to heat stress, anoxia, and bacterial pathogen stress, but increased nuclear localization of DAF-16 in response to chronic oxidative stress and osmotic stress. tbc-2 mutants also exhibit decreased upregulation of DAF-16 target genes in response to stress. To determine whether the rate of nuclear localization of DAF-16 affected stress resistance in these animals, we examined survival after exposure to multiple exogenous stressors. Disruption of tbc-2 decreased resistance to heat stress, anoxia, and bacterial pathogen stress in both wild-type worms and stress-resistant daf-2 insulin/IGF-1 receptor mutants. Similarly, deletion of tbc-2 decreases lifespan in both wild-type worms and daf-2 mutants. When DAF-16 is absent, the loss of tbc-2 is still able to decrease lifespan but has little or no impact on resistance to most stresses. Combined, this suggests that disruption of tbc-2 affects lifespan through both DAF-16-dependent and DAF-16-independent pathways, while the effect of tbc-2 deletion on resistance to stress is primarily DAF-16-dependent. Overall, this work demonstrates the importance of endosomal trafficking for the proper nuclear localization of DAF-16 during stress and that perturbation of normal endosomal trafficking is sufficient to decrease both stress resistance and lifespan.

Peroxiredoxin 2 is required for the redox mediated adaptation to exercise

Exercise generates a site-specific increase in Reactive Oxygen Species (ROS) within muscle that promotes changes in gene transcription and mitochondrial biogenesis, required for the beneficial adaptive response. We demonstrate that Peroxiredoxin 2 (Prdx2), an abundant cytoplasmic 2-Cys peroxiredoxin, is required for the adaptive hormesis response to physiological levels of H2O2 in myoblasts and following exercise in C. elegans. A short bolus addition of H2O2 increases mitochondrial capacity and improves myogenesis of cultured myoblasts, this beneficial adaptive response was suppressed in myoblasts with decreased expression of cytoplasmic Prdxs. Moreover, a swimming exercise protocol in C. elegans increased mitochondrial content, fitness, survival and longevity in wild type (N2) worms. In contrast, prdx-2 mutant worms had decreased fitness, disrupted mitochondria, reduced survival and lifespan following exercise. Global proteomics following exercise identified distinct changes in the proteome of N2 and prdx-2 mutants. Furthermore, a redox proteomic approach to quantify reversible oxidation of specific Cysteine residues revealed a more reduced redox state in the non-exercised prdx-2 mutant strain that become oxidized following exercise. In contrast, specific Cys residues from regulatory proteins become more reduced in the N2 strain following exercise, establishing the key regulatory role of PRDX-2 in a redox signalling cascade following endogenous ROS generation. Our results demonstrate that conserved cytoplasmic 2-Cys Peroxiredoxins are required for the beneficial adaptive response to a physiological redox stress.

The metabolite alpha-ketobutyrate extends lifespan by promoting peroxisomal function in C. elegans

Metabolism is intimately linked to aging. There is a growing number of studies showing that endogenous metabolites may delay aging and improve healthspan. Through the analysis of existing transcriptome data, we discover a link between activation of the transsulfuration pathway and a transcriptional program involved in peroxisome function and biogenesis in long-lived glp-1(e2141ts) mutant Caenorhabditis elegans worms. Subsequently, we show that supplementation with α-ketobutyrate, an intermediate of the transsulfuration pathway, extends lifespan in wild-type worms. Alpha-ketobutyrate augments the production of NAD+ via the lactate dehydrogenase LDH-1, leading to SIR-2.1/SIRT1-mediated enhanced peroxisome function and biogenesis, along with a concomitant increase in the expression of acox-1.2/ACOX1 in the peroxisomal fatty acid β-oxidation pathway. ACOX-1.2/ACOX1 promotes H2O2 formation, thereby resulting in activation of SKN-1/NRF2. This transcription factor in turn extends the lifespan of worms by driving expression of autophagic and lysosomal genes. Finally, we show that α-ketobutyrate also delays the cellular senescence in fibroblast cells through the SIRT1-ACOX1-H2O2-NRF2 pathway. This finding uncovers a previously unknown role for α-ketobutyrate in organismal lifespan and healthspan by coordinating the NAD+-SIRT1 signaling and peroxisomal function.

Comparison and agreement between two image analysis tools for quantifying GFP::SNB-1 puncta in fshr-1 mutants of C. elegans.

Quantitative imaging of synaptic vesicle localization and abundance using fluorescently labeled synaptic vesicle associated proteins like GFP::SNB-1 is a well-established method for measuring changes in synapse structure at neuromuscular junctions (NMJ) in C. elegans . To date, however, the ability to easily and reproducibly measure key parameters at the NMJ - maximum intensity, size of GFP::SNB-1 puncta, density of puncta - has relied on the use of expensive, customizable software that requires coding skills to modify, precluding widespread access and thus preventing standardization within the field. We carried out a comparative evaluation of a new, open-source Fiji puncta plugin versus traditional Igor-based analysis of GFP::SNB-1 imaging data taken of cholinergic motor neurons in the dorsal nerve cord of loss of function mutants in fshr-1 , which encodes a G protein-coupled receptor known to impact GFP::SNB-1 accumulation. We analyzed images taken on a widefield fluorescence microscope, as well as on a spinning disk confocal microscope. Our data demonstrate strong concordance between the differences in GFP::SNB-1 localization in fshr-1 mutants compared to wild type worms across both analysis platforms (Fiji and Igor), as well as across microscope types (widefield and confocal). These data also agree with previously published observations related to synapse number and GFP::SNB-1 intensity in fshr-1 and wild type worms. Based on these findings, we conclude that the Fiji platform is viable as a method for analyzing synaptic vesicle localization and abundance at cholinergic dorsal nerve cord motor NMJs and expect the Fiji puncta plugin to be of broad utility in imaging across a variety of imaging platforms and synaptic markers.

The impact of C. elegans ceramide glucosyltransferase enzymes on the beneficial effects of B. subtilis lifespan.

Ceramide glucosyltransferase (CGT) adds sugar moieties to ceramide, forming glucosylceramides that play roles in immune signaling, stress response, and host-bacterial interactions. Here, we examined whether mutations in cgt block the beneficial effects of Bacillus subtilis on C. elegans lifespan. We found that loss of cgt-1 or cgt-3 reduces lifespan compared to wildtype worms, but did not prevent the lifespan-extending phenotype of B. subtilis . However, cgt-1(ok1045) and cgt-3(tm504) did play a minor role in blocking stress resistance of 5-day old worms treated with B. subtilis . Further studying CGTs may elucidate potential roles of glucosylceramides in host-bacterial interaction.

Uncoupling Healthspan and Lifespan in Long-lived Daf-2, Pmk-3 and Sgk-1 Knockdown Caenorhabditis elegans

Modulation of genes expression in signaling pathways promotes longevity in Caenorhabditis elegans (C. elegans) but it is unclear whether this will improve healthspan. This study aimed to determine the healthspan of daf-2, pmk-3 and sgk-1 knocked-down worms. Health measures were determined at day 5, day 10 and day 15 of adult worms which were cultured in nematode growth media containing Escherichia coli. Heat resistance was determined by analysing the survival of worms at 37°C. Oxidative stress resistance was measured after exposure to 10mM hydrogen peroxide to analyse its survival capacity. Movement capacity was analysed by computing the distance travelled on solid media and the number of thrashing in liquid media. Results showed that the gene knockdown worms survived longer than wildtype worms. Daf-2 and pmk-3 knockdown increased movement capacity at day 5 and 10 in solid and liquid media but resulted in similar capacities to wildtype worms at day 15. Sgk-1 knockdown increased movement capacity only at day 5 in both media. The ability of gene knocked-down worms to withstand heat and oxidative stress significantly increased. In conclusion, knockdown of daf-2, pmk-3 and sgk-1 improved resistance to heat and oxidative stress but did not modulate movement incompetency at advanced ages C. elegans.

Bioassay-Guided Isolation of Anthelmintic Components from Semen pharbitidis, and the Mechanism of Action of Pharbitin.

Parasitic helminths continue to pose problems in human and veterinary medicine, as well as in agriculture. Semen pharbitidis, the seeds of Pharbitis nil (Linn.) Choisy (Convolvulaceae), is a well-known traditional Chinese medicinal botanical preparation widely used for treating intestinal parasites in China owing to its desirable efficacy. However, the anthelmintic compounds in Semen pharbitidis and their mechanism of action have not been investigated yet. This study aimed to identify the compounds active against helminths from Semen pharbitidis, and to establish the mechanism of action of these active compounds. Bioassay-guided fractionation was used to identify the anthelmintic compounds from Semen pharbitidis. The anthelmintic assay was performed by monitoring Caenorhabditis elegans (C. elegans) motility with a WMicrotracker instrument. Active compounds were identified by high-resolution mass spectrometry. Several (analogues of) fragments of the anthelmintic compounds were purchased and tested to explore the structure-activity relationship, and to find more potent compounds. A panel of C. elegans mutant strains resistant to major currently used anthelmintic drugs was used to explore the mechanism of action of the active compounds. The bioassay-guided isolation from an ethanol extract of Semen pharbitidis led to a group of glycosides, namely pharbitin (IC50: 41.0 ± 9.4 μg/mL). Hit expansion for pharbitin fragments yielded two potent analogues: 2-bromohexadecanoic acid (IC50: 1.6 ± 0.7 μM) and myristoleic acid (IC50: 35.2 ± 7.6 μM). One drug-resistant mutant ZZ37 unc-63 (x37) demonstrated a ~17-fold increased resistance to pharbitin compared with wild-type worms. Collectively, we provide further experimental scientific evidence to support the traditional use of Semen pharbitidis for the treatment of intestinal parasites. The anthelmintic activity of Semen pharbitidis is due to pharbitin, whose target could be UNC-63 in C. elegans.

Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents

BackgroundOne-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids.ResultsWe describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, β-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels.ConclusionsThis work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.

Oocyte mitophagy is critical for extended reproductive longevity.

Women's reproductive cessation is the earliest sign of human aging and is caused by decreasing oocyte quality. Similarly, C. elegans' reproduction declines in mid-adulthood and is caused by oocyte quality decline. Aberrant mitochondrial morphology is a hallmark of age-related dysfunction, but the role of mitochondrial morphology and dynamics in reproductive aging is unclear. We examined the requirements for mitochondrial fusion and fission in oocytes of both wild-type worms and the long-lived, long-reproducing insulin-like receptor mutant daf-2. We find that normal reproduction requires both fusion and fission, but that daf-2 mutants utilize a shift towards fission, but not fusion, to extend their reproductive span and oocyte health. daf-2 mutant oocytes' mitochondria are punctate (fissioned) and this morphology is primed for mitophagy, as loss of the mitophagy regulator PINK-1 shortens daf-2's reproductive span. daf-2 mutants maintain oocyte mitochondria quality with age at least in part through a shift toward punctate mitochondrial morphology and subsequent mitophagy. Supporting this model, Urolithin A, a metabolite that promotes mitophagy, extends reproductive span in wild-type mothers-even in mid-reproduction-by maintaining youthful oocytes with age. Our data suggest that promotion of mitophagy may be an effective strategy to maintain oocyte health with age.

The Human LRRK2 Modulates the Age-Dependent Effects of Developmental Methylmercury Exposure in Caenorhabditis elegans.

Methylmercury (MeHg) neurotoxicity exhibits age-dependent effects with a latent and/or persistent neurotoxic effect on aged animals. Individual susceptibility to MeHg neurotoxicity is governed by both exposure duration and genetic factors that can magnify or mitigate the pathologic processes associated with this exposure. We previously showed the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2) modulates the response of worms to high levels of MeHg, mitigating its effect on neuronal morphology in pre-vesicles in cephalic (CEP) dopaminergic neurons. Here we sought to better understand the long-term effects of MeHg exposure at low levels (100-fold lower than that in our previous report) and the modulatory role of the LRRK2 mutation. Worms exposed to MeHg (10 or 50nM) at the larval stage (L1 stage) were compared at adult stages (young age: day 1 adult; middle age: day 5 adult; old age: day 10 adult) for the swimming speeds in M9 buffer, moving speeds during locomotion on an OP50-seeded plate, and the numbers of CEP dopaminergic pre-vesicles, vesicular structures originating from the dendrites of CEP for exportation of cellular content. In addition, the expression levels of Caenorhabditis elegans homologs of dopamine transporter (dat-1) and tyrosine hydroxylase (cat-2) were also analyzed at these adult stages. Our data showed that swimming speeds were reduced in wild-type worms at the day 10 adult stage at 50nM MeHg level; yet, reduced swimming speeds were noted in the G2019S LRRK2 transgenic line upon MeHg exposures as low as 10nM. Compared to locomotor speeds, swimming speeds appear to be more sensitive to the behavioral effects of developmental MeHg exposures, as the locomotor speeds were largely intact and indistinguishable from controls following MeHg exposures. Furthermore, we showed an age-dependent modulation of dat-1 and cat-2 expressions, which could also be modified by the LRRK2 mutation. Although MeHg exposures did not change the number of pre-vesicles, the LRRK2 mutation was associated with increased numbers of pre-vesicles in aged worms. Our data suggest that the latent behavioral effects of MeHg are sensitized by the G2019S LRRK2 mutation, and the underlying mechanism likely involves age-dependent changes in dopaminergic signaling.

C. elegans ribosomal protein S3 protects against H2O2-induced DNA damage and suppresses spontaneous mutations in yeast

Carcinogenicity and cytotoxicity are severe consequences of DNA damage. Base Excision Repair (BER) is a conserved DNA repair pathway that replaces many damaged bases caused by oxidation. Aberrations in BER are associated with carcinogenesis, neurodegeneration, and aging. The nematode C. elegans is an attractive model system for studying BER. However, in this organism, the complete pathway is not fully delineated. To further explore the BER process in C. elegans, we used affinity tag chromatography and mass spectrometry to identify the interactome of uracil DNA glycosylase-1 (CeUNG-1), an enzyme that acts during the first step of the BER pathway. Our analysis identified that CeUNG-1 is associated with the 40 S ribosomal protein S3 (CeRPS-3), homologs of which have been shown to process 8-oxoguanine and abasic site lesions in other organisms. We report a strong in silico association between CeUNG-1 and CeRPS-3 and confirmed this interaction using the yeast two-hybrid system. Downregulation of the Cerps-3 gene reduced the viability of wild-type worms upon exposure to the chemical oxidant hydrogen peroxide. Further analysis shows that Cerps-3 knockdown significantly sensitized the AP endonuclease APN-1-deficient strain, apn-1, but to a lesser extent exo-3, to the lethal effects of hydrogen peroxide. A cross-species complementation experiment reveals that the expression of CeRPS-3 rescued the hydrogen peroxide sensitivity, and suppressed the high mutation frequency of the yeast AP endonuclease-deficient strain lacking Apn1 and Apn2. We propose that CeRPS-3 may function as an auxiliary DNA repair enzyme in C. elegans to process oxidative DNA lesions.

Mitochondrial thioredoxin system is required for enhanced stress resistance and extended longevity in long-lived mitochondrial mutants

Mild impairment of mitochondrial function has been shown to increase lifespan in genetic model organisms including worms, flies and mice. To better understand the mechanisms involved, we analyzed RNA sequencing data and found that genes involved in the mitochondrial thioredoxin system, trx-2 and trxr-2, are specifically upregulated in long-lived mitochondrial mutants but not other non-mitochondrial, long-lived mutants. Upregulation of trx-2 and trxr-2 is mediated by activation of the mitochondrial unfolded protein response (mitoUPR). While we decided to focus on the genes of the mitochondrial thioredoxin system for this paper, we identified multiple other antioxidant genes that are upregulated by the mitoUPR in the long-lived mitochondrial mutants including sod-3, prdx-3, gpx-6, gpx-7, gpx-8 and glrx-5. In exploring the role of the mitochondrial thioredoxin system in the long-lived mitochondrial mutants, nuo-6 and isp-1, we found that disruption of either trx-2 or trxr-2 significantly decreases their long lifespan, but has no effect on wild-type lifespan, indicating that the mitochondrial thioredoxin system is specifically required for their longevity. In contrast, disruption of the cytoplasmic thioredoxin gene trx-1 decreases lifespan in nuo-6, isp-1 and wild-type worms, indicating a non-specific detrimental effect on longevity. Disruption of trx-2 or trxr-2 also decreases the enhanced resistance to stress in nuo-6 and isp-1 worms, indicating a role for the mitochondrial thioredoxin system in protecting against exogenous stressors. Overall, this work demonstrates an important role for the mitochondrial thioredoxin system in both stress resistance and lifespan resulting from mild impairment of mitochondrial function.

Geranylgeranylacetone Ameliorates Beta-Amyloid Toxicity and Extends Lifespan via the Heat Shock Response in Caenorhabditis elegans.

Activation of a cytoprotective cellular pathway known as the heat shock response (HSR) is a promising strategy for the treatment of Alzheimer’s disease and other neurodegenerative diseases. Geranylgeranylacetone (GGA) is a commonly used anti-ulcer drug in Japan that has been shown to activate the HSR. Here, we establish C. elegans as a model system to investigate the effects of GGA. First, we show that GGA-mediated activation of the HSR is conserved in worms. Then, we show that GGA can ameliorate beta-amyloid toxicity in both muscle and neuronal worm Alzheimer’s disease models. Finally, we find that exposure to GGA is sufficient to extend the lifespan of wild-type worms. Significantly, the beneficial effects of GGA on both beta-amyloid toxicity and lifespan are dependent on HSR activation. Taken together, this research supports further development of GGA as a therapeutic for Alzheimer’s disease, provides evidence that HSR activation is a relevant therapeutic mechanism, and indicates that the beneficial effects of GGA are not limited to disease.

Phytochemical Composition and Anti-Aging Activity of Butanol Extract of Hedyotis diffusa in Caenorhabditis elegans.

Hedyotis diffusa Willd. (H. diffusa), a kind of traditional Chinese medicine, has been evaluated to potential display antioxidant and anti-aging effects in vitro experiments. In this work, we investigated the effects on lifespan and stress resistance of the butanol extract from H. diffusa (NHD) in vivo using a Caenorhabditis elegans (C. elegans) model. The phytochemicals of NHD were identified by UPLC-ESI-qTOF-MS/MS method. NHD-treated wild-type N2 worms showed an increase in survival time under both normal and stress conditions. Meanwhile, NHD promoted the healthspan of nematodes by stimulating growth and development, reducing the deposition of age pigment, increasing the activities of superoxide dismutase (SOD) and glutathione peroxidase dismutase (GSH-Px), and decreasing the level of ROS without impairing fertility. Moreover, the upregulating of the expression of daf-16, gst-4, sod-3, hsp12.6 genes and the downregulating of the expression of daf-2 were involved in the NHD-mediated lifespan extension. Finally, the increasing of the expression of GST-4::GFP in CL2166 transgenic nematodes and the life-span-extending activity of NHD was completely abolished in daf-2 and daf-16 mutants further revealed that the potential roles for these genes in NHD-induced longevity in C. elegans. Collectively, our findings suggest that NHD may have an active effect in healthy aging and age-related diseases.

Lycium barbarum Extracts Extend Lifespan and Alleviate Proteotoxicity in Caenorhabditis elegans.

Lycium barbarum berry (Ningxia Gouqi, Fructus lycii, goji berry, or wolfberry), as a traditional Chinese herb, was recorded beneficial for longevity in traditional Chinese medical scriptures and currently is a natural dietary supplement worldwide. However, under modern experimental conditions, the longevity effect of L. barbarum berry and the underlying mechanisms have been less studied. Here, we reported that total water extracts of L. barbarum berry (LBE), which contains 22% polysaccharides and other components, such as anthocyanins, extended the lifespan of Caenorhabditis elegans without side effects on worm fertility and pharyngeal pumping. Interestingly, we found that the lifespan extension effect was more prominent in worms with shorter mean lifespan as compared to those with longer mean lifespan. Furthermore, we showed that the lifespan extension effect of LBE depended on deacetylase sir-2.1. Remarkably, LBE rescued heat shock transcription factor-1 (hsf-1) deficiency in wild-type worms with different mean lifespans, and this effect also depended on sir-2.1. In addition, we found that LBE extended lifespan and alleviated toxic protein aggregation in neurodegenerative worms with hsf-1 deficiency. Our study suggested that LBE may be a potential antiaging natural dietary supplement especially to individuals with malnutrition or chronic diseases and a potential therapeutic agent for neurodegenerative diseases characterized by hsf-1 deficiency.

Linc-20 and linc-9 do not have compensatory fertility roles in C. elegans.

Long intergenic non-coding RNAs (lincRNAs) are transcripts longer than 200 nucleotides which are transcribed from regions that do not overlap with protein coding sequences. Reproductive organs express high levels of lincRNAs, yet removal of many lincRNA genes with high and dynamic germline expression did not lead to fertility defects. It was previously suggested this stems from redundant roles of different lincRNA genes. We previously reported engineering C. elegans strains in which we deleted lincRNA genes with high and dynamic expression in the gonad. The individual mutations did not lead to major effects on fertility. Two of those lincRNA genes, linc-9 and linc-20, are highly homologous, suggesting they could perform redundant roles. Here we report that in the double mutant linc-9; linc-20 the brood size and embryonic lethality do not significantly differ from wild-type worms. This could be explained by either lack of fertility roles, or redundancy with other lincRNA genes.

Effect of DMSO on lifespan and physiology in C. elegans : Implications for use of DMSO as a solvent for compound delivery.

Dimethyl sulfoxide (DMSO) is a solvent that has been used for basic and medical research based on its ability to dissolve both polar and non-polar compounds. In order to use DMSO to deliver compounds that may impact longevity or neurodegeneration, it is important to first determine the effects of DMSO on aging and physiology. We examined the effect of different concentrations of DMSO on lifespan and development time in C. elegans. We found that DMSO concentrations up to 2% DMSO did not affect longevity in wild-type worms, while concentrations of up to 0.5% DMSO were compatible with normal development times. 0.5% DMSO also had minimal effect on fertility and movement. In summary, our results show that concentrations of DMSO up to 0.5% can be safely used to deliver compounds to C. elegans with little or no modifying effects on lifespan or physiologic rates.

Multifaceted roles of mitochondrial stress responses under ETC dysfunction - repair, destruction and pathogenesis.

Electron transport chain (ETC) dysfunction is a common feature of mitochondrial diseases and induces severe cellular stresses, including mitochondrial membrane potential (Δψm ) reduction, mitochondrial matrix acidification, metabolic derangements and proteostatic stresses. Extensive studies of ETC dysfunction in yeast, Caenorhabditis elegans, cultured cells and mouse models have revealed multiple mitochondrial stress response pathways. Here, we summarise the current understanding of the triggers, sensors, signalling mechanisms and the functional outcomes of mitochondrial stress responses in different species. We highlight Δψm reduction as a major trigger of stress responses in different species, but the responses are species-specific and the outcomes are context-dependent. ETC dysfunction elicits a mitochondrial unfolded protein response (UPRmt ) to repair damaged mitochondria in C. elegans, and activates a global adaptive programme to maintain Δψm in yeast. Yeast and C. elegans responses are remarkably similar at the downstream responses, although they are activated by different signalling mechanisms. UPRmt generally protects ETC-defective worms, but its constitutive activation is toxic for wildtype worms and worms carrying mutant mtDNA. In contrast to lower organisms, ETC dysfunction in mammals mainly activates a mitochondrial integrated stress response (ISRmt ) to reprogramme metabolism and a PINK1-Parkin mitophagy pathway to degrade damaged mitochondria. Accumulating in vivo results suggest that the ATF4 branch of ISRmt exacerbates metabolic derangements to accelerate mitochondrial disease progression. The in vivo roles of mitophagy in mitochondrial diseases are also context-dependent. These results thus reveal the common and unique aspects of mitochondrial stress responses in different species and highlight their multifaceted roles in mitochondrial diseases.

MicroRNA Expression Influences Methylmercury-Induced Lipid Accumulation and Mitochondrial Toxicity in Caenorhabditis elegans.

Metabolic effects of methylmercury (MeHg) are gaining wider attention. We have previously shown that MeHg causes lipid dysregulation in Caenorhabditis elegans (C. elegans), leading to altered gene expression, increased triglyceride levels and lipid storage, and altered feeding behaviors. Transcriptional regulators, such as transcription factors and microRNAs (miRNAs), have been shown to regulate lipid storage, serum triglycerides, and adipogenic gene expression in human and rodent models of metabolic diseases. As we recently investigated adipogenic transcription factors induced by MeHg, we were, therefore, interested in whether MeHg may also regulate miRNA sequences to cause metabolic dysfunction. Lipid dysregulation, as measured by triglyceride levels, lipid storage sites, and feeding behaviors, was assessed in wild-type (N2) worms and in transgenic worms that either were sensitive to miRNA expression or were unable to process miRNAs. Worms that were sensitive to the miRNA expression were protected from MeHg-induced lipid dysregulation. In contrast, the mutant worms that were unable to process miRNAs had exacerbated MeHg-induced lipid dysregulation. Concurrent with differential lipid homeostasis, miRNA-expression mutants had altered MeHg-induced mitochondrial toxicity as compared to N2, with the miRNA-sensitive mutants showing mitochondrial protection and the miRNA-processing mutants showing increased mitotoxicity. Taken together, our data demonstrate that the expression of miRNAs is an important determinant in MeHg toxicity and MeHg-induced metabolic dysfunction in C. elegans.

Dityrosine Crosslinking of Collagen and Amyloid-β Peptides Is Formed by Vitamin B12 Deficiency-Generated Oxidative Stress in Caenorhabditis elegans.

(1) Background: Vitamin B12 deficiency in Caenorhabditis elegans results in severe oxidative stress and induces morphological abnormality in mutants due to disordered cuticle collagen biosynthesis. We clarified the underlying mechanism leading to such mutant worms due to vitamin B12 deficiency. (2) Results: The deficient worms exhibited decreased collagen levels of up to approximately 59% compared with the control. Although vitamin B12 deficiency did not affect the mRNA expression of prolyl 4-hydroxylase, which catalyzes the formation of 4-hydroxyproline involved in intercellular collagen biosynthesis, the level of ascorbic acid, a prolyl 4-hydroxylase coenzyme, was markedly decreased. Dityrosine crosslinking is involved in the extracellular maturation of worm collagen. The dityrosine level of collagen significantly increased in the deficient worms compared with the control. However, vitamin B12 deficiency hardly affected the mRNA expression levels of bli-3 and mlt-7, which are encoding crosslinking-related enzymes, suggesting that deficiency-induced oxidative stress leads to dityrosine crosslinking. Moreover, using GMC101 mutant worms that express the full-length human amyloid β, we found that vitamin B12 deficiency did not affect the gene and protein expressions of amyloid β but increased the formation of dityrosine crosslinking in the amyloid β protein. (3) Conclusions: Vitamin B12-deficient wild-type worms showed motility dysfunction due to decreased collagen levels and the formation of highly tyrosine-crosslinked collagen, potentially reducing their flexibility. In GMC101 mutant worms, vitamin B12 deficiency-induced oxidative stress triggers dityrosine-crosslinked amyloid β formation, which might promote its stabilization and toxic oligomerization.