Cisplatin-induced DNA crosslinks trigger neurotoxicity in C. elegans.

The anticancer drug cisplatin (CisPt) injures post-mitotic neuronal cells, leading to neuropathy. Furthermore, CisPt triggers cell death in replicating cells. Here, we aim to unravel the relevance of different types of CisPt-induced DNA lesions for evoking neurotoxicity. To this end, we comparatively analyzed wild-type and loss of function mutants of C. elegans lacking key players of specific DNA repair pathways. Deficiency in ercc-1, which is essential for nucleotide excision repair (NER) and interstrand crosslink (ICL) repair, revealed the most pronounced enhancement in CisPt-induced neurotoxicity with respect to the functionality of post-mitotic chemosensory AWA neurons, without inducing neuronal cell death. Potentiation of CisPt-triggered neurotoxicity in ercc-1 mutants was accompanied by complex alterations in both basal and CisPt-stimulated mRNA expression of genes involved in the regulation of neurotransmission, including cat-4, tph-1, mod-1, glr-1, unc-30 and eat-18. Moreover, xpf-1, csb-1, csb-1;xpc-1 and msh-6 mutants were significantly more sensitive to CisPt-induced neurotoxicity than the wild-type, whereas xpc-1, msh-2, brc-1 and dog-1 mutants did not distinguish from the wild-type. The majority of DNA repair mutants also revealed increased basal germline apoptosis, which was analyzed for control. Yet, only xpc-1, xpc-1;csb-1 and dog-1 mutants showed elevated apoptosis in the germline following CisPt treatment. To conclude, we provide evidence that neurotoxicity, including sensory neurotoxicity, is triggered by CisPt-induced DNA intra- and interstrand crosslinks that are subject of repair by NER and ICL repair. We hypothesize that especially ERCC1/XPF, CSB and MSH6-related DNA repair protects from chemotherapy-induced neuropathy in the context of CisPt-based anticancer therapy.

Mitochondria hormesis delays aging and associated diseases in Caenorhabditis elegans impacting on key ferroptosis players.

Excessive iron accumulation or deficiency leads to a variety of pathologies in humans and developmental arrest in the nematode Caenorhabditis elegans. Instead, sub-lethal iron depletion extends C. elegans lifespan. Hypoxia preconditioning protects against severe hypoxia-induced neuromuscular damage across species but it has low feasible application. In this study, we assessed the potential beneficial effects of genetic and chemical interventions acting via mild iron instead of oxygen depletion. We show that limiting iron availability in C. elegans through frataxin silencing or the iron chelator bipyridine, similar to hypoxia preconditioning, protects against hypoxia-, age-, and proteotoxicity-induced neuromuscular deficits. Mechanistically, our data suggest that the beneficial effects elicited by frataxin silencing are in part mediated by counteracting ferroptosis, a form of non-apoptotic cell death mediated by iron-induced lipid peroxidation. This is achieved by impacting on different key ferroptosis players and likely via gpx-independent redox systems. We thus point to ferroptosis inhibition as a novel potential strategy to promote healthy aging.

Neuroligin-mediated neurodevelopmental defects are induced by mitochondrial dysfunction and prevented by lutein in C. elegans.

Complex-I-deficiency represents the most frequent pathogenetic cause of human mitochondriopathies. Therapeutic options for these neurodevelopmental life-threating disorders do not exist, partly due to the scarcity of appropriate model systems to study them. Caenorhabditis elegans is a genetically tractable model organism widely used to investigate neuronal pathologies. Here, we generate C. elegans models for mitochondriopathies and show that depletion of complex I subunits recapitulates biochemical, cellular and neurodevelopmental aspects of the human diseases. We exploit two models, nuo-5/NDUFS1- and lpd-5/NDUFS4-depleted animals, for a suppressor screening that identifies lutein for its ability to rescue animals' neurodevelopmental deficits. We uncover overexpression of synaptic neuroligin as an evolutionarily conserved consequence of mitochondrial dysfunction, which we find to mediate an early cholinergic defect in C. elegans. We show lutein exerts its beneficial effects by restoring neuroligin expression independently from its antioxidant activity, thus pointing to a possible novel pathogenetic target for the human disease.

Identification of Modulators of the C. elegans Aryl Hydrocarbon Receptor and Characterization of Transcriptomic and Metabolic AhR-1 Profiles.

The Aryl hydrocarbon Receptor (AhR) is a xenobiotic sensor in vertebrates, regulating the metabolism of its own ligands. However, no ligand has been identified to date for any AhR in invertebrates. In C. elegans, the AhR ortholog, AHR-1, displays physiological functions. Therefore, we compared the transcriptomic and metabolic profiles of worms expressing AHR-1 or not and investigated the putative panel of chemical AHR-1 modulators. The metabolomic profiling indicated a role for AHR-1 in amino acids, carbohydrates, and fatty acids metabolism. The transcriptional profiling in neurons expressing AHR-1, identified 95 down-regulated genes and 76 up-regulated genes associated with neuronal and metabolic functions in the nervous system. A gene reporter system allowed us to identify several AHR-1 modulators including bacterial, dietary, or environmental compounds. These results shed new light on the biological functions of AHR-1 in C. elegans and perspectives on the evolution of the AhR functions across species.

Aryl Hydrocarbon Receptor-Dependent and -Independent Pathways Mediate Curcumin Anti-Aging Effects.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor whose activity can be modulated by polyphenols, such as curcumin. AhR and curcumin have evolutionarily conserved effects on aging. Here, we investigated whether and how the AhR mediates the anti-aging effects of curcumin across species. Using a combination of in vivo, in vitro, and in silico analyses, we demonstrated that curcumin has AhR-dependent or -independent effects in a context-specific manner. We found that in Caenorhabditis elegans, AhR mediates curcumin-induced lifespan extension, most likely through a ligand-independent inhibitory mechanism related to its antioxidant activity. Curcumin also showed AhR-independent anti-aging activities, such as protection against aggregation-prone proteins and oxidative stress in C. elegans and promotion of the migratory capacity of human primary endothelial cells. These AhR-independent effects are largely mediated by the Nrf2/SKN-1 pathway.

Prevention of anticancer therapy-induced neurotoxicity: Putting DNA damage in perspective.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe side effect of conventional anticancer therapeutics (cAT) that significantly impacts the quality of life of tumor patients. The molecular mechanisms of CIPN are incompletely understood and there are no effective preventive or therapeutic measures available to date. Here, we present a brief overview of the current knowledge about mechanisms underlying CIPN and discuss DNA damage-related stress responses as feasible targets for the prevention of CIPN. In addition, we discuss that the nematode Caenorhabditis elegans is a useful 3R-conform model organism to further elucidate molecular mechanisms of CIPN and to identify novel lead compounds protecting from cAT-triggered neuropathy.

Effects of Manganese on Genomic Integrity in the Multicellular Model Organism Caenorhabditis elegans.

Although manganese (Mn) is an essential trace element, overexposure is associated with Mn-induced toxicity and neurological dysfunction. Even though Mn-induced oxidative stress is discussed extensively, neither the underlying mechanisms of the potential consequences of Mn-induced oxidative stress on DNA damage and DNA repair, nor the possibly resulting toxicity are characterized yet. In this study, we use the model organism Caenorhabditis elegans to investigate the mode of action of Mn toxicity, focusing on genomic integrity by means of DNA damage and DNA damage response. Experiments were conducted to analyze Mn bioavailability, lethality, and induction of DNA damage. Different deletion mutant strains were then used to investigate the role of base excision repair (BER) and dePARylation (DNA damage response) proteins in Mn-induced toxicity. The results indicate a dose- and time-dependent uptake of Mn, resulting in increased lethality. Excessive exposure to Mn decreases genomic integrity and activates BER. Altogether, this study characterizes the consequences of Mn exposure on genomic integrity and therefore broadens the molecular understanding of pathways underlying Mn-induced toxicity. Additionally, studying the basal poly(ADP-ribosylation) (PARylation) of worms lacking poly(ADP-ribose) glycohydrolase (PARG) parg-1 or parg-2 (two orthologue of PARG), indicates that parg-1 accounts for most of the glycohydrolase activity in worms.

Cisplatin-induced neurotoxicity involves the disruption of serotonergic neurotransmission.

Neurotoxicity is a frequent side effect of cisplatin (CisPt)-based anticancer therapy whose pathophysiology is largely vague. Here, we exploited C. elegans as a 3R-compliant in vivo model to elucidate molecular mechanisms contributing to CisPt-induced neuronal dysfunction. To this end, we monitored the impact of CisPt on various sensory functions as well as pharyngeal neurotransmission by recording electropharyngeograms (EPGs). CisPt neither affected food and odor sensation nor mechano-sensation, which involve dopaminergic and glutaminergic neurotransmission. However, CisPt reduced serotonin-regulated pharyngeal pumping activity independent of changes in the morphology of related neurons. CisPt-mediated alterations in EPGs were fully rescued by addition of serotonin (5-HT) (≤ 2 mM). Moreover, the CisPt-induced pharyngeal injury was prevented by co-incubation with the clinically approved serotonin re-uptake inhibitory drug duloxetine. A protective effect of 5-HT was also observed with respect to CisPt-mediated impairment of another 5-HT-dependent process, the egg laying activity. Importantly, CisPt-induced apoptosis in the gonad and learning disability were not influenced by 5-HT. Using different C. elegans mutants we found that CisPt-mediated (neuro)toxicity is independent of serotonin biosynthesis and re-uptake and likely involves serotonin-receptor subtype 7 (SER-7)-related functions. In conclusion, by measuring EPGs as a surrogate parameter of neuronal dysfunction, we provide first evidence that CisPt-induced neurotoxicity in C. elegans involves 5-HT-dependent neurotransmission and SER-7-mediated signaling mechanisms and can be prevented by the clinically approved antidepressant duloxetine. The data highlight the particular suitability of C. elegans as a 3R-conform in vivo model in molecular (neuro)toxicology and, moreover, for the pre-clinical identification of neuroprotective candidate drugs.

Antioxidant and Anti-Inflammaging Ability of Prune (Prunus Spinosa L.) Extract Result in Improved Wound Healing Efficacy.

Prunus spinosa L. fruit (PSF) ethanol extract, showing a peculiar content of biologically active molecules (polyphenols), was investigated for its wound healing capacity, a typical feature that declines during aging and is negatively affected by the persistence of inflammation and oxidative stress. To this aim, first, PSF anti-inflammatory properties were tested on young and senescent LPS-treated human umbilical vein endothelial cells (HUVECs). As a result, PSF treatment increased miR-146a and decreased IRAK-1 and IL-6 expression levels. In addition, the PSF antioxidant effect was validated in vitro with DPPH assay and confirmed by in vivo treatments in C. elegans. Our findings showed beneficial effects on worms' lifespan and healthspan with positive outcomes on longevity markers (i.e., miR-124 upregulation and miR-39 downregulation) as well. The PSF effect on wound healing was tested using the same cells and experimental conditions employed to investigate PSF antioxidant and anti-inflammaging ability. PSF treatment resulted in a significant improvement of wound healing closure (ca. 70%), through cell migration, both in young and older cells, associated to a downregulation of inflammation markers. In conclusion, PSF extract antioxidant and anti-inflammaging abilities result in improved wound healing capacity, thus suggesting that PSF might be helpful to improve the quality of life for its beneficial health effects.

Use of C. elegans as a 3R-compliant in vivo model for the chemoprevention of cisplatin-induced neurotoxicity.

Anticancer therapeutics can provoke severe side effects that impair the patient's quality of life. A frequent dose-limiting side effect of platinum-based anticancer therapy is neurotoxicity. Its pathophysiology is poorly understood, and effective preventive or therapeutic measures are missing. Therefore, elucidation of the molecular mechanism of platinating drug-induced neurotoxicity and the development of preventive strategies is urgently needed. To this end, we aim to use C. elegans as a 3R-compliant in vivo model. The 3R principles were conceived for animal welfare in science concerning animal experiments, which should be replaced, reduced or refined. We can analytically demonstrate dose-dependent uptake of cisplatin (CisPt) in C. elegans, as well as genotoxic and cytotoxic effects based on DNA adduct formation (i.e., 1,2-GpG intrastrand crosslinks), induction of apoptosis, and developmental toxicity. Measuring the impairment of pharyngeal pumping as a marker of neurotoxicity, we found that especially CisPt reduces the pumping frequency at concentrations where basal and touch-provoked movement were not yet affected. CisPt causes glutathione (GSH) depletion and RNAi-mediated knockdown of the glutamate-cysteine ligase GCS-1 aggravates the CisPt-induced inhibition of pharyngeal pumping. Moreover, N-acetylcysteine (NAC) mitigated CisPt-triggered toxicity, indicating that GSH depletion contributes to the CisPt-induced pharyngeal damage. In addition to NAC, amifostine (WR1065) also protected the pharynx of C. elegans from the toxic effects of CisPt. Measuring pharyngeal activity by the electrophysiological recording of neurotransmission in the pharynx, we confirmed that CisPt is neurotoxic in C. elegans and that NAC is neuroprotective in the nematode. The data support the hypothesis that monitoring the pharyngeal activity of C. elegans is a useful surrogate marker of CisPt-induced neurotoxicity. In addition, a low GSH pool reduces the resistance of neurons to CisPt treatment, and both NAC and WR1065 are capable of attenuating platinum-induced neurotoxicity during post-incubation in C. elegans. Overall, we propose C. elegans as a 3R-compliant in vivo model to study the molecular mechanisms of platinum-induced neurotoxicity and to explore novel neuroprotective therapeutic strategies to alleviate respective side effects of platinum-based cancer therapy.

Dietary and environmental factors have opposite AhR-dependent effects on C. elegans healthspan.

Genetic, dietary, and environmental factors concurrently shape the aging process. The aryl hydrocarbon receptor (AhR) was discovered as a dioxin-binding transcription factor involved in the metabolism of different environmental toxicants in vertebrates. Since then, the variety of pathophysiological processes regulated by the AhR has grown, ranging from immune response, metabolic pathways, and aging. Many modulators of AhR activity may impact on aging and age-associated pathologies, but, whether their effects are AhR-dependent has never been explored. Here, using Caenorhabditis elegans, as an elective model organism for aging studies, we show for the first time that lack of CeAHR-1 can have opposite effects on health and lifespan in a context-dependent manner. Using known mammalian AhR modulators we found that, ahr-1 protects against environmental insults (benzo(a)pyrene and UVB light) and identified a new role for AhR-bacterial diet interaction in animal lifespan, stress resistance, and age-associated pathologies. We narrowed down the dietary factor to a bacterially extruded metabolite likely involved in tryptophan metabolism. This is the first study clearly establishing C. elegans as a good model organism to investigate evolutionarily conserved functions of AhR-modulators and -regulated processes, indicating it can be exploited to contribute to the discovery of novel information about AhR in mammals.

The Aryl Hydrocarbon Receptor (AhR) in the Aging Process: Another Puzzling Role for This Highly Conserved Transcription Factor.

Aging is the most important risk factor for the development of major life-threatening diseases such as cardiovascular disorders, cancer, and neurodegenerative disorders. The aging process is characterized by the accumulation of damage to intracellular macromolecules and it is concurrently shaped by genetic, environmental and nutritional factors. These factors influence the functionality of mitochondria, which play a central role in the aging process. Mitochondrial dysfunction is one of the hallmarks of aging and is associated with increased fluxes of ROS leading to damage of mitochondrial components, impaired metabolism of fatty acids, dysregulated glucose metabolism, and damage of adjacent organelles. Interestingly, many of the environmental (e.g., pollutants and other toxicants) and nutritional (e.g., flavonoids, carotenoids) factors influencing aging and mitochondrial function also directly or indirectly affect the activity of a highly conserved transcription factor, the Aryl hydrocarbon Receptor (AhR). Therefore, it is not surprising that many studies have already indicated a role of this versatile transcription factor in the aging process. We also recently found that the AhR promotes aging phenotypes across species. In this manuscript, we systematically review the existing literature on the contradictory studies indicating either pro- or anti-aging effects of the AhR and try to reconcile the seemingly conflicting data considering a possible dependency on the animal model, tissue, as well as level of AhR expression and activation. Moreover, given the crucial role of mitochondria in the aging process, we summarize the growing body of evidence pointing toward the influence of AhR on mitochondria, which can be of potential relevance for aging.

PMK-1 p38 MAPK promotes cadmium stress resistance, the expression of SKN-1/Nrf and DAF-16 target genes, and protein biosynthesis in Caenorhabditis elegans.

The mechanisms of cadmium (Cd) resistance are complex and not sufficiently understood. The present study, therefore, aimed at assessing the roles of important components of stress-signaling pathways and of ABC transporters under severe Cd stress in Caenorhabditis elegans. Survival assays on mutant and control animals revealed a significant promotion of Cd resistance by the PMK-1 p38 MAP kinase, the transcription factor DAF-16/FoxO, and the ABC transporter MRP-1. Transcriptome profiling by RNA-Seq on wild type and a pmk-1 mutant under control and Cd stress conditions revealed, inter alia, a PMK-1-dependent promotion of gene expression for the translational machinery. PMK-1 also promoted the expression of target genes of the transcription factors SKN-1/Nrf and DAF-16 in Cd-stressed animals, which included genes for molecular chaperones or immune proteins. Gene expression studies by qRT-PCR confirmed the positive effects of PMK-1 on DAF-16 activity under Cd stress and revealed negative effects of DAF-16 on the expression of genes for MRP-1 and DAF-15/raptor. Additional studies on pmk-1 RNAi-treated wild type and mutant strains provided further information on the effects of PMK-1 on SKN-1 and DAF-16, which resulted in a model of these relationships. The results of this study demonstrate a central role of PMK-1 for the processing of cellular responses to abiotic and biotic stressors, with the promoting effects of PMK-1 on Cd resistance mostly mediated by the transcription factors SKN-1 and DAF-16.

The aryl hydrocarbon receptor promotes aging phenotypes across species.

The ubiquitously expressed aryl hydrocarbon receptor (AhR) induces drug metabolizing enzymes as well as regulators of cell growth, differentiation and apoptosis. Certain AhR ligands promote atherosclerosis, an age-associated vascular disease. Therefore, we investigated the role of AhR in vascular functionality and aging. We report a lower pulse wave velocity in young and old AhR-deficient mice, indicative of enhanced vessel elasticity. Moreover, endothelial nitric oxide synthase (eNOS) showed increased activity in the aortas of these animals, which was reflected in increased NO production. Ex vivo, AhR activation reduced the migratory capacity of primary human endothelial cells. AhR overexpression as well as treatment with a receptor ligand, impaired eNOS activation and reduced S-NO content. All three are signs of endothelial dysfunction. Furthermore, AhR expression in blood cells of healthy human volunteers positively correlated with vessel stiffness. In the aging model Caenorhabditis elegans, AhR-deficiency resulted in increased mean life span, motility, pharynx pumping and heat shock resistance, suggesting healthier aging. Thus, AhR seems to have a negative impact on vascular and organismal aging. Finally, our data from human subjects suggest that AhR expression levels could serve as an additional, new predictor of vessel aging.

Iron-Starvation-Induced Mitophagy Mediates Lifespan Extension upon Mitochondrial Stress in C. elegans.

Frataxin is a nuclear-encoded mitochondrial protein involved in the biogenesis of Fe-S-cluster-containing proteins and consequently in the functionality of the mitochondrial respiratory chain. Similar to other proteins that regulate mitochondrial respiration, severe frataxin deficiency leads to pathology in humans--Friedreich's ataxia, a life-threatening neurodegenerative disorder--and to developmental arrest in the nematode C. elegans. Interestingly, partial frataxin depletion extends C. elegans lifespan, and a similar anti-aging effect is prompted by reduced expression of other mitochondrial regulatory proteins from yeast to mammals. The beneficial adaptive responses to mild mitochondrial stress are still largely unknown and, if characterized, may suggest novel potential targets for the treatment of human mitochondria-associated, age-related disorders. Here we identify mitochondrial autophagy as an evolutionarily conserved response to frataxin silencing, and show for the first time that, similar to mammals, mitophagy is activated in C. elegans in response to mitochondrial stress in a pdr-1/Parkin-, pink-1/Pink-, and dct-1/Bnip3-dependent manner. The induction of mitophagy is part of a hypoxia-like, iron starvation response triggered upon frataxin depletion and causally involved in animal lifespan extension. We also identify non-overlapping hif-1 upstream (HIF-1-prolyl-hydroxylase) and downstream (globins) regulatory genes mediating lifespan extension upon frataxin and iron depletion. Our findings indicate that mitophagy induction is part of an adaptive iron starvation response induced as a protective mechanism against mitochondrial stress, thus suggesting novel potential therapeutic strategies for the treatment of mitochondrial-associated, age-related disorders.