A Multisystemic Approach Revealed Aminated Polystyrene Nanoparticles-Induced Neurotoxicity.

Exposure to plastic nanoparticles has dramatically increased in the last 50 years, and there is evidence that plastic nanoparticles can be absorbed by organisms and cross the blood-brain-barrier (BBB). However, their toxic effects, especially on the nervous system, have not yet been extensively investigated, and most of the knowledge is based on studies using different conditions and systems, thus hard to compare. In this work, physicochemical properties of non-modified polystyrene (PS) and amine-functionalized PS (PS-NH2 ) nanoparticles are initially characterized. Advantage of a multisystemic approach is then taken to compare plastic nanoparticles effects in vitro, through cytotoxic readouts in mammalian cell culture, and in vivo, through behavioral readouts in the nematode Caenorhabditis elegans (C. elegans), a powerful 3R-complying model organism for toxicology studies. In vitro experiments in neuroblastoma cells indicate a specific cytotoxic effect of PS-NH2 particles, including a decreased neuronal differentiation and an increased Amyloid ß (Aß) secretion, a sensitive readout correlating with Alzheimer's disease pathology. In parallel, only in vivo treatments with PS-NH2 particles affect C. elegans development, decrease lifespan, and reveal higher sensitivity of animals expressing human Aß compared to wild-type animals. In summary, the multisystemic approach discloses a neurotoxic effect induced by aminated polystyrene nanoparticles.

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.

Insights into cisplatin-induced neurotoxicity and mitochondrial dysfunction in Caenorhabditis elegans.

Cisplatin is the most common drug in first-line chemotherapy against solid tumors. We and others have previously used the nematode Caenorhabditis elegans to identify genetic factors influencing the sensitivity and resistance to cisplatin. In this study, we used C. elegans to explore cisplatin effects on mitochondrial functions and investigate cisplatin-induced neurotoxicity through a high-resolution system for evaluating locomotion. First, we report that a high-glucose diet sensitizes C. elegans to cisplatin at the physiological level and that mitochondrial CED-13 protects the cell from cisplatin-induced oxidative stress. Additionally, by assessing mitochondrial function with a Seahorse XFe96 Analyzer, we observed a detrimental effect of cisplatin and glucose on mitochondrial respiration. Second, because catechol-O-methyltransferases (involved in dopamine degradation) are upregulated upon cisplatin exposure, we studied the protective role of dopamine against cisplatin-induced neurotoxicity. Using a Tierpsy Tracker system for measuring neurotoxicity, we showed that abnormal displacements and body postures in cat-2 mutants, which have dopamine synthesis disrupted, can be rescued by adding dopamine. Then, we demonstrated that dopamine treatment protects against the dose-dependent neurotoxicity caused by cisplatin.

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.

Mitophagy and iron: two actors sharing the stage in age-associated neuronal pathologies.

Aging is characterized by the deterioration of different cellular and organismal structures and functions. A typical hallmark of the aging process is the accumulation of dysfunctional mitochondria and excess iron, leading to a vicious cycle that promotes cell and tissue damage, which ultimately contribute to organismal aging. Accordingly, altered mitochondrial quality control pathways such as mitochondrial autophagy (mitophagy) as well as altered iron homeostasis, with consequent iron overload, can accelerate the aging process and the development and progression of different age-associated disorders. In this review we first briefly introduce the aging process and summarize molecular mechanisms regulating mitophagy and iron homeostasis. We then provide an overview on how dysfunction of these two processes impact on aging and age-associated neurodegenerative disorders with a focus on Alzheimer's disease, Parkinson's disease and Amyotrophic Lateral Sclerosis. Finally, we summarize some recent evidence showing mechanistic links between iron metabolism and mitophagy and speculate on how regulating the crosstalk between the two processes may provide protective effects against aging and age-associated neuronal pathologies.

The flavonoid 4,4'-dimethoxychalcone promotes autophagy-dependent longevity across species.

Ageing constitutes the most important risk factor for all major chronic ailments, including malignant, cardiovascular and neurodegenerative diseases. However, behavioural and pharmacological interventions with feasible potential to promote health upon ageing remain rare. Here we report the identification of the flavonoid 4,4'-dimethoxychalcone (DMC) as a natural compound with anti-ageing properties. External DMC administration extends the lifespan of yeast, worms and flies, decelerates senescence of human cell cultures, and protects mice from prolonged myocardial ischaemia. Concomitantly, DMC induces autophagy, which is essential for its cytoprotective effects from yeast to mice. This pro-autophagic response induces a conserved systemic change in metabolism, operates independently of TORC1 signalling and depends on specific GATA transcription factors. Notably, we identify DMC in the plant Angelica keiskei koidzumi, to which longevity- and health-promoting effects are ascribed in Asian traditional medicine. In summary, we have identified and mechanistically characterised the conserved longevity-promoting effects of a natural anti-ageing drug.

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.

BRCA1 and BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in Caenorhabditis elegans.

Interventions that promote healthy aging are typically associated with increased stress resistance. Paradoxically, reducing the activity of core biological processes such as mitochondrial or insulin metabolism promotes the expression of adaptive responses, which in turn increase animal longevity and resistance to stress. In this study, we investigated the relation between the extended Caenorhabditis elegans lifespan elicited by reduction in mitochondrial functionality and resistance to genotoxic stress. We find that reducing mitochondrial activity during development confers germline resistance to DNA damage-induced cell cycle arrest and apoptosis in a cell-non-autonomous manner. We identified the C. elegans homologs of the BRCA1/BARD1 tumor suppressor genes, brc-1/brd-1, as mediators of the anti-apoptotic effect but dispensable for lifespan extension upon mitochondrial stress. Unexpectedly, while reduced mitochondrial activity only in the soma was not sufficient to promote longevity, its reduction only in the germline or in germline-less strains still prolonged lifespan. Thus, in animals with partial reduction in mitochondrial functionality, the mechanisms activated during development to safeguard the germline against genotoxic stress are uncoupled from those required for somatic robustness and animal longevity.

Constitutive MAP-kinase activation suppresses germline apoptosis in NTH-1 DNA glycosylase deficient C. elegans.

Oxidation of DNA bases, an inevitable consequence of oxidative stress, requires the base excision repair (BER) pathway for repair. Caenorhabditis elegans is a well-established model to study phenotypic consequences and cellular responses to oxidative stress. To better understand how BER affects phenotypes associated with oxidative stress, we characterised the C. elegans nth-1 mutant, which lack the only DNA glycosylase dedicated to repair of oxidative DNA base damage, the NTH-1 DNA glycosylase. We show that nth-1 mutants have mitochondrial dysfunction characterised by lower mitochondrial DNA copy number, reduced mitochondrial membrane potential, and increased steady-state levels of reactive oxygen species. Consistently, nth-1 mutants express markers of chronic oxidative stress with high basal phosphorylation of MAP-kinases (MAPK) but further activation of MAPK in response to the superoxide generator paraquat is attenuated. Surprisingly, nth-1 mutants also failed to induce apoptosis in response to paraquat. The ability to induce apoptosis in response to paraquat was regained when basal MAPK activation was restored to wild type levels. In conclusion, the failure of nth-1 mutants to induce apoptosis in response to paraquat is not a direct effect of the DNA repair deficiency but an indirect consequence of the compensatory cellular stress response that includes MAPK activation.

C. elegans as a model organism for human mitochondrial associated disorders.

Mitochondria are small cytoplasmic organelles whose most important function is to provide the energy required by our cells and organism to live. To maintain an adequate mitochondrial homeostasis cells possess numerous mitochondrial quality controls and protective compensatory pathways, which can be activated to cope with a certain degree of mitochondrial dysfunction. However, when the mitochondrial damage is too severe and these defensive mechanisms are not anymore sufficient to deal with it, pathological signs arise. In the past few decades numerous genetic disorders ascribed to severe mitochondrial defects have been recognized with variable onset and symptomatology ranging from neuromuscular degeneration to cancer syndromes. Unfortunately, to date, only symptomatic and no curative therapies exist for most of these devastating, life-threatening disorders. Model organisms, and especially the nematode Caenorhabditis elegans, with its sequenced and highly conserved genome, and a simple but well-characterized nervous system, have enormously contributed in the past years to gain insight into the pathogenesis and treatment of different diseases. Here, we will summarize some of the advantages offered by the nematode system to model neurodegenerative diseases associated with mitochondrial electron transport chain defects and screen for therapeutic interventions.

Caenorhabditis elegans ATAD-3 modulates mitochondrial iron and heme homeostasis.

ATAD3 (ATPase family AAA domain-containing protein 3) is a mitochondrial protein, which is essential for cell viability and organismal development. ATAD3 has been implicated in several important cellular processes such as apoptosis regulation, respiratory chain function and steroid hormone biosynthesis. Moreover, altered expression of ATAD3 has been associated with several types of cancer. However, the exact mechanisms underlying ATAD3 effects on cellular metabolism remain largely unclear. Here, we demonstrate that Caenorhabditis elegans ATAD-3 is involved in mitochondrial iron and heme homeostasis. Knockdown of atad-3 caused mitochondrial iron- and heme accumulation. This was paralleled by changes in the expression levels of several iron- and heme-regulatory genes as well as an increased heme uptake. In conclusion, our data indicate a regulatory role of C. elegans ATAD-3 in mitochondrial iron and heme metabolism.

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.

Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.

Autophagy induction extends lifespan and reduces lipid content in response to frataxin silencing in C. elegans.

Severe mitochondria deficiency leads to a number of devastating degenerative disorders, yet, mild mitochondrial dysfunction in different species, including the nematode Caenorhabditis elegans, can have pro-longevity effects. This apparent paradox indicates that cellular adaptation to partial mitochondrial stress can induce beneficial responses, but how this is achieved is largely unknown. Complete absence of frataxin, the mitochondrial protein defective in patients with Friedreich's ataxia, is lethal in C. elegans, while its partial deficiency extends animal lifespan in a p53 dependent manner. In this paper we provide further insight into frataxin control of C. elegans longevity by showing that a substantial reduction of frataxin protein expression is required to extend lifespan, affect sensory neurons functionality, remodel lipid metabolism and trigger autophagy. We find that Beclin and p53 genes are required to induce autophagy and concurrently reduce lipid storages and extend animal lifespan in response to frataxin suppression. Reciprocally, frataxin expression modulates autophagy in the absence of p53. Human Friedreich ataxia-derived lymphoblasts also display increased autophagy, indicating an evolutionarily conserved response to reduced frataxin expression. In sum, we demonstrate a causal connection between induction of autophagy and lifespan extension following reduced frataxin expression, thus providing the rationale for investigating autophagy in the pathogenesis and treatment of Friedreich's ataxia and possibly other human mitochondria-associated disorders.

Preventing the ubiquitin-proteasome-dependent degradation of frataxin, the protein defective in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a devastating orphan disease, with no specific treatment. The disease is caused by reduced expression of the protein frataxin, which results in mitochondrial defects and oxidative damage. Levels of residual frataxin critically affect onset and progression of the disease. Understanding the molecular mechanisms that regulate frataxin stability and degradation may, therefore, be exploited for the design of effective therapeutics. Here we show that frataxin is degraded by the ubiquitin-proteasome system and that K(147) is the critical residue responsible for frataxin ubiquitination and degradation. Accordingly, a K(147)R substitution generates a more stable frataxin. We then disclose a set of lead compounds, computationally selected to target the molecular cleft harboring K(147), that can prevent frataxin ubiquitination and degradation, and increase frataxin levels in cells derived from FRDA patients. Moreover, treatment with these compounds induces substantial recovery of aconitase activity and adenosine-5'-triphosphate levels in FRDA cells. Thus, we provide evidence for the therapeutic potential of directly interfering with the frataxin degradation pathway.

A role for p53 in mitochondrial stress response control of longevity in C. elegans.

As in the case of aging, many degenerative disorders also result from progressive mitochondrial deterioration and cellular damage accumulation. Therefore, preventing damage accumulation may delay aging and help to prevent degenerative disorders, especially those associated with mitochondrial dysfunction. In the nematode Caenorhabditis elegans a mild mitochondrial dysfunction prolongs the lifespan. We previously proposed that, following a mild mitochondrial dysfunction, protective stress responses are activated in a hormetic-like fashion, and ultimately account for extended animal's lifespan. We recently showed that in C. elegans, lifespan extension induced by reduced expression of different mitochondrial proteins involved in electron transport chain functionality requires p53/cep-1. In this paper we find that reducing the expression of frataxin, the protein defective in patients with Friedreich's ataxia, triggers a complex stress response, and that the associated induction of the antioxidant glutathione-S-transferase is regulated by cep-1. Given the high percentage of homology between human and nematode genes and the conservation of fundamental intracellular pathways between the two species, identification of molecular mechanisms activated in response to frataxin suppression in C. elegans may suggest novel therapeutic approaches to prevent the accumulation of irreversible damage and the consequent appearance of symptoms in Friedreich's ataxia and possibly other human mitochondrial-associated diseases. The same pathways could be exploitable for delaying the aging process ascribed to mitochondrial degeneration.

p53/CEP-1 increases or decreases lifespan, depending on level of mitochondrial bioenergetic stress.

Mitochondrial pathologies underlie a number of life-shortening diseases in humans. In the nematode Caenorhabditis elegans, severely reduced expression of mitochondrial proteins involved in electron transport chain-mediated energy production also leads to pathological phenotypes, including arrested development and/or shorter life; in sharp contrast, mild suppression of these same proteins extends lifespan. In this study, we show that the C. elegans p53 ortholog cep-1 mediates these opposite effects. We found that cep-1 is required to extend longevity in response to mild suppression of several bioenergetically relevant mitochondrial proteins, including frataxin - the protein defective in patients with Friedreich's Ataxia. Importantly, we show that cep-1 also mediates both the developmental arrest and life shortening induced by severe mitochondrial stress. These findings support an evolutionarily conserved function for p53 in modulating organismal responses to mitochondrial dysfunction and suggest that metabolic checkpoint responses may play a role in longevity control and in human mitochondrial-associated diseases.

Long-lived C. elegans mitochondrial mutants as a model for human mitochondrial-associated diseases.

Mitochondria play a pivotal role in the life of cells, controlling diverse processes ranging from energy production to the regulation of cell death. In humans, numerous pathological conditions have been linked to mitochondrial dysfunction. Cancer, diabetes, obesity, neurodegeneration, cardiomyopathy and even aging are all associated with mitochondrial dysfunction. Over 400 mutations in mitochondrial DNA result directly in pathology and many more disorders associated with mitochondrial dysfunction arise from mutations in nuclear DNA. It is counter-intuitive then, that a class of mitochondrially defective mutants in the nematode Caenorhabditis elegans, the so called Mit (Mitochondrial) mutants, in fact live longer than wild-type animals. In this review, we will reconcile this paradox and provide support for the idea that the Mit mutants are in fact an excellent model for studying human mitochondrial associated diseases (HMADs). In the context of the 'Mitochondrial Threshold Effect Theory', we propose that the kinds of processes induced to counteract mitochondrial mutations in the Mit mutants (and which mediate their life extension), are very likely the same ones activated in many HMADs to delay disease appearance. The identification of such compensatory pathways opens a window of possibility for future preventative therapies for many HMADs. They may also provide a way of potentially extending human life span.