Benedetto, Alexandre et al. (2015) International Worm Meeting "Autophagy promotes heat resistance and intestinal biomass conversion in ageing hermaphrodites."

Benedetto, Alexandre, Gilliat, Ann, Cabreiro, Filipe, Au, Catherine, Tullet, Jennifer, Gems, David, Ezcurra, Marina

[International Worm Meeting, 2015]

In an attempt to understand causes of aging and organismal death C. elegans, we have established new tools for longitudinal studies in aging worm cohorts. We have characterized age-related pathologies and developed a high-throughput method for simultaneously performing survival assays on over a hundred age/genotype combinations. Applying these methodologies to the study of age-related changes in Age mutants, we have uncovered two new phenomena, both requiring autophagy. Using time-lapse recordings of blue death fluorescence (Coburn et al. PLoS Biology, 2013) to automate mortality scoring in worm populations, we performed semi-automated stress assays in multiwell plates, following daily changes in heat resistance in various Age mutants and conditions. Unexpectedly, we observed an age increase in resistance to acute heat (42degC) in N2 hermaphrodites, peaking between days 6 and 8 of adulthood, and decreasing thereafter. Mutation of daf-2 (insulin/IGF-1 receptor) markedly extended the period of maximum resistance, while mutation of daf-16 (FoxO transcription factor) shortened it. Overall, both amplitude and timing of the peak correlated positively with mutant longevity and this phenomenon required DAF-16, HSF-1 and an intact autophagy pathway. In a parallel line of investigation, we simultaneously assessed age-related pathologies in a range of Age mutants (see presentation by Ezcurra et al.). Two salient pathologies, extracellular yolk accumulation and intestinal atrophy, were correlated across genotypes and individuals, suggesting a causal link. In wild type N2, both phenomena occurred only in hermaphrodites, but they could be induced in males by feminization using mab-3 mutations. Autophagy mutants showed striking reductions in both extracellular yolk accumulation and intestinal atrophy. Accordingly, protein and lipid yolk components were also reduced. Intestinal-specific RNA interference of autophagy genes was sufficient to recapitulate these effects. Taken together, these results identify a likely mechanism of intestinal atrophy, a major pathology of aging in C. elegans: autophagy-dependent conversion of intestinal biomass into yolk. It implicates autophagy as a modulator of energy flux in C. elegans adulthood, generating energy and metabolites to satisfy the high energetic demand of yolk synthesis and afford optimal stress handling during the reproductive period. This work further suggests that early ageing in C. elegans is characterized by robust activity rather than metabolic decline.

Alexandre Benedetto et al. (2008) C.elegans Neuronal Development Meeting "Manganese and Dopamine: a perilous combination?"

Catherine Au, Alexandre Benedetto, Michael Aschner

[C.elegans Neuronal Development Meeting, 2008]

Manganese (Mn) is essential for normal human development and vital enzymatic activities. However, occupational exposure to high levels of Mn has been implicated in a Parkinsonian-like syndrome known as manganism, primarily affecting the dopaminergic (DAergic) system. We hypothesized that the commonalities in clinical and physiological symptoms exhibited in both Parkinson''s disease (PD) and manganism patients, at least partially, rely on common molecular pathways. In this study, we used C. elegans strains mutated for PD and DAergic circuitry-specific genes and evaluated their sensitivity to acute Mn exposure. We first show that acute Mn treatments specifically lead to DAergic neuron degeneration, while sparing other neurotransmitter systems, such as GABAergic or cholinergic neurons. We further establish that the dopamine (DA) transporter knock-out dat-1(ok157) and the DA receptor dop-2(vs105);dop-1(vs100);dop-3(vs106) triple mutant are hypersensitive to Mn exposure. Conversely, the vesicular monoamine transporter 2 cat-1(e1111) and the tyrosine hydroxylase cat-2(e1112) mutants are hyper-resistant. Combined with measurements of DA content in these strains, our results suggest that elevated DA levels sensitize the worm to Mn toxicity. Interestingly the serotonin transporter mutant mod-5(n3314) exhibited high DA levels comparable to dat-1(ok157), but was not hypersensitive to Mn. We propose that extracellular rather than intracellular dopamine is involved in Mn toxicity in C. elegans, which would explain the sensitivity of DAergic neurons to Mn exposure. Together, our data establish C. elegans as a suitable model for studies on genetic pathways involved in manganese toxicity and PD. This project is funded by R01 ES10563 to MA.

Alexandre Benedetto et al. (2008) European Worm Meeting "Investigation of Manganese Transport and Toxicity in C.elegans"

Michael Aschner, Alexandre Benedetto, Catherine Au

[European Worm Meeting, 2008]

Manganese (Mn) is an essential trace element for normal human development and vital enzymatic activities. Because of its reactivity, it is also widely used in industrial processes, such as welding. However, occupational exposure to high levels of Mn has been implicated in a Parkinsonian-like syndrome known as manganism, affecting the dopaminergic system. Since the initial discovery of Mn-induced pathogenesis, much effort has been put forth to study the etiology of manganism, yet the primary biochemical changes have to be elucidated. In the present study, we use C. elegans to understand mechanisms of Mn transport across biological membranes and its cytotoxic effects.. We first report on survival of wild-type C. elegans upon acute 30 min Mn treatment, which is characterized by an LD50~42 mM. As expected, lethality appeared to be due to major osmoregulation defects affecting most epidermal tissues including epidermis, intestine and the excretory cell. We cloned the three C. elegans divalent-metal transporters and found that smf-1 and smf-3 are involved in Mn toxicity. smf-1(eh5) and smf-3(ok1035) mutants are hyper-resistant to Mn exposure (LD50~83 mM and 122 mM, respectively, p<0.001)whereas smf-2(gk133) mutant is similar to control (LD50~41 mM). On the other hand, at sublethal doses of Mn (>0,5 mM), we found that C. elegans dopaminergic neurons are preferentially affected and in a dose-dependent manner. Furthermore, we demonstrate that dat-1(ok157) and dop-1(vs100);dop-2(vs105);dop-3(vs106) mutants which exhibit high dopamine levels are significantly more sensitive (LD50~9 mM and 27 mM, respectively, p<0.001), whereas the dopamine-depleted cat-1(e1111) and cat-2(e1112) mutants are more resistant to Mn toxicity (LD50~100 mM and 130 mM, respectively, p<0.001). Our results suggest that dopamine metabolism directly contributes to the toxicity mechanism, most likely by promoting reactive oxygen species production.. Taken together our data imply the existence of conserved mechanisms for Mn transport and toxicity from the nematode to humans, and show that C. elegans is a valuable model for dissecting out Mn toxicity mechanisms. This project is funded by R01 ES10563 to MA.

Alexandre Benedetto et al. (2005) International Worm Meeting "The v-ATPase is required for exocytosis at the apical membrane in epithelia, independently of its role as a proton pump"

Jean-Marie GARNIER, Alexandre Benedetto, Michel Labouesse, Samuel LIEGEOIS

[International Worm Meeting, 2005]

The vacuolar H+-ATPase is considered as the main proton pump responsible for osmoregulation in metazoan excretory systems, creating a H+ gradient used as a motor force for most solute exchanges. It is also crucial for pH regulation in all eukaryotic organelles. Mutations in its subunits lead to numerous diseases (for example a subunit disruption in osteoclasts is responsible for osteopetrosis) and prevent tumoral cells to further metastasize. In neurons the v-ATPase-generated H+ gradient is used to fill secretory vesicles with neurotransmitters, and is thus essential for their secretion. The v-ATPase comprises two sectors, V1 is cytoplasmic and responsible for ATP hydrolysis, V0 is transmembraneous and involved in H+ transport. Biochemical experiments and cell culture studies support the notion that V0 proteolipids mediate membrane fusion independently from V1. These observations show that the v-ATPase might play a dual role in secretion: either through its pumping activity, or via V0-facilitated membrane fusion. Our laboratory is analyzing secretion at the apical membrane in C. elegans epithelia. Compared to basolateral trafficking, apical secretion remains poorly understood, and our work provides new insights in this field. We previously showed that rdy-1 (a.k.a. vha-5) encodes the a subunit of the V0 sector that is expressed in hyp7, the excretory, rectal, vulval and support cells associated with sensory neurons. rdy-1 mutants display secretion and pH-regulation defects causing L2 lethality. To determine the role of the a subunit in apical secretion, we attempted to separate its two putative activities: proton pumping performed by the V1+V0 complex and membrane fusion due to V0 alone. We generated 66 site-directed mutations on a functional rdy-1 transgene and introduced mutated constructs in a rdy-1 null animal. We completed this approach by knocking-down V1 and V0 subunits by RNAi. We then assayed excretory canal formation (as a criterion for pumping), and alae formation as well as secretion of apically targeted proteins (as a criterion for apical exocytosis). We obtained mutations that specifically affect the excretory canal (W190A, R191A) and other that mostly affect secretion (L786S, E830Q, V844F). Besides, knocking-down V1 subunits by RNAi also leads to osmoregulation defects, without impairing secretion. Conversely, mutations such as L786S, E830Q and V844F have abnormal or absent alae and accumulate apical or secreted proteins in the hyp7 (RDY-1, CHE-14, WRT-2, WRT-8). We conclude that V1-associated pumping activity is not required for proper secretion to the apical membrane in epithelial cells, and propose that the V0 sector alone is a key player in apical secretion.

Alexandre Benedetto et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Manganese Uptake by C. elegans NRAMP2 Orthologues leads to Dopaminergic Neurodegeneration and Dopamine-dependant Toxicity"

Alexandre Benedetto, Catherine Au, Joel Anderson, Keith Erikson, Michael Aschner

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Manganese (Mn) is essential for development and vital enzymatic activities. However, occupational exposure to Mn has been implicated in a Parkinsonian-like syndrome called manganism, primarily affecting dopaminergic neurons. Here we demonstrate that the nematode C. elegans readily takes up Mn resulting in lethal osmoregulation defects at high concentrations. Among three divalent metal transporter 1 (DMT1, also called NRAMP2) orthologues, smf-1 and smf-3 mutants were found to take up less Mn than wildtype and showed hyper-resistance to the metal exposure, while smf-2 mutants were hypersensitive, exhibiting higher Mn content. Accordingly, expression pattern analysis revealed that smf-1 and smf-3 were strongly expressed in the intestine and localized at its apical membrane likely responsible for most Mn uptake, while smf-2 was found in epithelial pharyngeal cells and localized in intracellular compartments unlikely involved in Mn uptake. Concomitantly, we proved that sub-lethal doses of Mn lead to selective and dose-dependent degeneration of C. elegans dopaminergic neurons. Moreover, using mutants affecting dopamine metabolism or release, we established that high extracellular dopamine levels sensitize to manganese exposure, whereas low levels are protective, suggesting that dopamine directly contributes to manganese toxicity. Our work validates C. elegans as a suitable model for manganese toxicology studies. Moreover it unravels a potential toxic role for dopamine in dopaminergic system disorders such as manganism and Parkinson''s disease.

Zarate Potes, Alejandra et al. (2021) International Worm Meeting "Solving host-microbe interactions and gut dysbiosis"

Benedetto, Alexandre, Zarate Potes, Alejandra, Ali, Irtiqa, Martin, Jack

[International Worm Meeting, 2021]

Gut host-microbe interactions critically impact C. elegans health and ageing. Multiple pathways and interactions have already been characterised in probiotic and dysbiotic contexts. However, we are still to unravel the complex host-microbe core or pathogen-specific genetic networks that orchestrate these interactions. We recently observed that the kynurenine pathway, main catabolic route for tryptophan, is majorly involved in regulating host-microbe interactions in worms, while insulin/IGF1 signalling (IIS) is a major upstream determinant of C. elegans gut biology and reproduction, immunity and longevity trade-offs. To solve part of this picture, we are now performing microbial and host transcriptomics, proteomics, and Tryptophan metabolite measurements in daf-2(e1370) and wild-type worms exposed to E. faecalis or P. aeruginosa for up to 12h, combined with host genome-wide RNAi infection screens relying on Label-Free Survival Assays (Benedetto et al. Aging 2019). In this poster we present the result of our bioinformatic investigations, main experimental pipelines and project strategy.

Ali, Irtiqa et al. (2021) International Worm Meeting "Investigating the basis of severe stress resistance in ageing"

Ackerley, Jacob, Benedetto, Alexandre, Ali, Irtiqa, Zarate-Potes, Alejandra, Martin, Jack

[International Worm Meeting, 2021]

Key words Severe stress resistance, ageing, CeMbio, IIS, oxidative, heat. Abstract Stress resistance and longevity are robustly correlated across heat stress paradigms but not oxidative stress paradigms. In particular, severe stress resistance in ageing C. elegans markedly differs between exposure to oxidative and heat stress that kill wild type worms within a couple of hours (Benedetto et al. Aging Cell 2019). Severe stress resistance may engage multiple mechanisms such as basal stress handling vs adaptive stress response pathways, different stress transduction pathways, the ability to execute the organismal death program (Galimov et al. 2019), or protection/sensitisation conferred by the gut microbiota. To disentangle these and evaluate their relative roles in ageing worms, we are screening C. elegans mutant in combination with strains from CeMBio collection for resistance to 7% tert-butyl hydroperoxide (t-BHP) and 42oC heat-shock as severe oxidative stress and thermal stress paradigms, respectively, before contrasting these results with aged animals. We are performing these screens on young adults at first, before moving into aged worms. So far, we have found that the capacity to execute the organismal death pathway does not significantly impact severe stress resistance in young adults fed an OP50 diet. However, young adults fed on 48 different bacterial isolates displayed varying levels of severe heat and oxidative stress resistance, some of which were insulin/IGF1-signalling (IIS) pathway-dependent. We are currently investigating the basis of these differences. This poster will provide an overview of the methods used, current results and planned future work.

Ezcurra, Marina et al. (2015) International Worm Meeting "A massive increase in lipid and protein content in aging hermaphrodites."

Gems, David, Benedetto, Alexandre, Au, Catherine, Sornda, Thanet, Gilliat, Ann, Ezcurra, Marina

[International Worm Meeting, 2015]

One view of aging is as the set of endogenously-generated pathologies that increase in late life, some of which contribute to age-related mortality. This implies that to understand aging in C. elegans one needs to characterize age-related pathologies and their development, identify the processes that promote pathology development, and establish which pathologies cause death. To this end we have established methodologies for simultaneous assessment of a range of late-life pathologies of aging C. elegans. Using these we have identified a burst of rapid pathogenesis involving gross anatomical changes, which occurs mainly between day 4 and 10 of adulthood in N2 hermaphrodites. One such pathology results from the continuation of yolk production after reproduction has ceased, causing accumulation of yolk and in detrimental effects on the worm in later life. Yolk is a lipoprotein complex of yolk proteins (vitellogenins) and lipids, including phospholipids and triglycerides. Continued yolk synthesis leads to an eventual ~8-fold increase in the major yolk protein component YP170. Yolk proteins eventually constitute ~75% of total worm protein content, and there is an ~10-fold age increase in total protein content per worm. In old animals, up to 50% of the body cavity can be filled up with yolk pools, which stain with the lipophilic dye Bodipy, and there is a ~5-fold increase in overall triglyceride content. Thus, elderly hermaphrodites experience severe hyperproteinemia and steatosis. Knock down of vitellogenins using vit-5 + vit-6 RNAi suppressed yolk accumulation and the development of other age-related pathologies, and increased lifespan. Conversely, suppressing yolk uptake by oocytes by rme-2 RNAi increased yolk pool accumulation, altered other pathologies and decreased lifespan, suggesting that ectopic yolk deposition has toxic effects. In long-lived daf-2 insulin/IGF-1 receptor mutants this yolk hypertrophy is suppressed. Together, these data suggest that aging C. elegans hermaphrodites undergo an overgrowth phase during early adulthood, characterized by vigorous biological activity rather than loss of function. This overgrowth phase results in, among other things, excess biosynthesis and the accumulation of large pools containing vitellogenic proteins and lipids in the body cavity, which have toxic effects and result in the acceleration of age-related pathologies and shortening of lifespan.

Martin, Jack et al. (2021) International Worm Meeting "The kynurenine pathway is a major modulator of E. faecalis infection in C. elegans"

Whittingham-Dowd, Jayde, Cetnar, Kalina, Rezwana, Ruhi, Norvaisas, Povilas, Kosztelnik, Monika, Parry, Jackie, Au, Catherine, Martin, Jack, Cabreiro, Filipe, Zarate Potes, Alejandra, Urbaniak, Mick, Gems, David, Fathallah, Nadin, Hardgrave, Alex, Benedetto, Alexandre

[International Worm Meeting, 2021]

Key words Kynurenine pathway, E. faecalis, gut infection, microbiota, lysosome-related organelles autofluorescence. Abstract The kynurenine pathway (KP), main catabolic route for the essential amino-acid tryptophan, is well-known for its immunomodulatory role in mammals. While investigating death fluorescence in C. elegans, anthranilic acid (AA)-loaded lysosome-related organelles (LROs) were previously found responsible for the blue auto-fluorescence seen in the worm gut (Coburn et al. PLOS Biol. 2013). Given the bacteriostatic potential of AA and other kynurenine pathway compounds, we hypothesised that LROs and the KP play a key role in C. elegans gut microbial control. To test this idea, we exposed C. elegans to a worm-pathogenic strain of E. faecalis (OG1RF) and observed changes in gut morphology and autofluorescence dynamics upon infection. Transcriptomics and targeted metabolomics analyses further showed that KP activity is modulated upon E. faecalis infection. Using a combination of KP mutants from the Nollen lab (Van Der Goot et al. PNAS 2012), we observed that inhibition of various KP enzymes differentially affect C. elegans resistance to E. faecalis infection. E. faecalis growth on KP mutant worm extracts confirmed that resistant mutants produce bacteriostatic compounds, which we measured by HPLC. This was verified by the delayed or reduced gut colonisation of OG1RF-GFP (gifted by D. Garsin), and the ability for some mutants to thrive on OG1RF loans. We are currently investigating a broader role for the KP in C. elegans gut microbiota control, notably using newly generated CeMBio fluorescent strains.

Hsiung, Kuei Ching et al. (2021) International Worm Meeting "Are levels of autophagy increased or decreased in daf-2 insulin/IGF-1 mutants?"

Gems, David, Chapman, Hannah, Hsiung, Kuei Ching

[International Worm Meeting, 2021]

According to one theory, molecular damage is the principal cause of ageing, while somatic maintenance mechanisms act against damage accumulation and ageing. One such maintenance mechanism is autophagy, which can remove damaged cellular constituents. Autophagy is considered a likely mechanism protecting against ageing. Various findings support the view that autophagy levels are elevated in daf-2 insulin/IGF-1 receptor mutants in C. elegans, and that autophagy contributes to their extended lifespan. We have been investigating the causes of intestinal atrophy, a major senescent pathology in C. elegans. Our evidence supports the view that gut atrophy is the result of the intestine consuming its own biomass to support yolk synthesis in sperm-depleted hermaphrodites [1], an example of a programmatic mechanism of ageing [2]. This yolk is vented via the vulva after reproduction and can support larval growth [3] (see presentation by C.C. Kern). Such intestinal biomass repurposing is facilitated by autophagy, and greatly reduced in daf-2 mutants [1,4]. This suggests that autophagy is decreased in daf-2 mutants. Consistent with this, it has been reported that protein turnover is also reduced in daf-2 mutants. To try to resolve the conflicting conclusions about the relationship between IIS, autophagy and ageing in C. elegans we are re-examining effects of knockdown of genes encoding the machinery of autophagy on pathology and ageing, taking into consideration issues of timing and severity of knockdown, daf-2 allele class, temperature, bacterial status and other issues. Our interim data will be described. [1] M. Ezcurra, et al (2018) Curr. Biol. 28: 2544. [2] A.A Maklakov, T. Chapman (2019) Proc. Biol. Sci. 286: 20191604. [3] C.C. Kern et al (2020) BioRxiv doi.org/10.1101/ 2020.11.15.380253. [4] A. Benedetto, D. Gems (2019) Autophagy 15: 731.