Kyriakakis, Emmanouil et al. (2017) International Worm Meeting "A C. elegans model of Wolfram Syndrome type 2."

Tavernarakis, Nektarios, Ploumi, Christina, Kyriakakis, Emmanouil

[International Worm Meeting, 2017]

Wolfram syndrome (WS) is a rare autosomal neurodegenerative disorder, characterized by early juvenile diabetes mellitus, a gradual loss of vision due optic nerve atrophy, deafness and often behavioral and mental changes. The average life expectancy is 30 years due to systemic complications. Treatment is merely symptomatic and supportive. There are two genetics forms of WS; type 1 is caused by mutations in the WFS1 (wolframin) gene, whereas WS type 2 is caused by mutations in the CISD2 gene. The C. elegans cisd-1 gene encodes a homolog of the mammalian CISD2, based on sequence analysis and the presence of the conserved iron sulfur domain. Lesion of cisd-1 recapitulates key features of CISD2 loss associated with WS type 2, including lifespan shortening and neurodegeneration. Intriguingly, dietary restriction (DR) completely reverses the detrimental effects of CISD-1 deficiency on longevity. This effect is at least in part due to autophagy induction. In addition, cellular proteostasis mechanisms are deferentially affected by CISD-1. The mitochondrial unfolded protein response (UPRmito) is significantly suppressed, while the endoplasmic reticulum UPRER is upregulated during ER stress. The heat shock response remains unaffected. These findings suggest an involvement of CISD-1 in organelle-specific proteome homeostasis. The C. elegans WS model will facilitate elucidation of the molecular mechanisms underlying WS pathogenesis and provide a platform for drug discovery.

Ploumi, Christina et al. (2019) International Worm Meeting "A C. elegans model for Wolfram Syndrome type 2."

Kyriakakis, Emmanouil, Tavernarakis, Nektarios, Ploumi, Christina

[International Worm Meeting, 2019]

Wolfram Syndrome (WS) is a rare neurodegenerative disorder, the patients of which are very early diagnosed with diabetes, optic atrophy and deafness, while their life expectancy is around 30 years due to systemic complications. Two types of WS have been characterized; Type 1 is caused by mutations in the WFS1 gene encoding the ER-localized protein Wolframin, while Type 2 is caused by mutations in the Cisd2 gene. Cisd2 gene encodes a mitochondrial iron-sulfur cluster (ISC) binding protein which belongs to the NEET protein family. Importantly, it is not well understood how loss of the particular ISC binding protein leads to WS pathophysiology in humans. The C. elegans gene cisd-1 (W02B12.15) encodes a functional homolog of the mammalian NEET protein CISD2, based on sequence analysis and the presence of the conserved domain CDGSH for binding to ISCs. Downregulation of cisd-1 through RNAi recapitulates key features of CISD2 loss, associated with WS type 2, including short life expectancy and enhanced neurodegeneration. Based on our findings, CISD-1 exerts differential effects on longevity and healthspan, through its involvement in both the intrinsic apoptosis pathway and the autophagic process. The CISD-1- dependent effects on apoptosis and autophagy seem to be affected by intracellular iron levels, suggesting that CISD-1 deficiency may disturb intracellular iron homeostasis. Our work will facilitate the elucidation of the molecular mechanisms underlying WS pathogenesis and provide critical insights towards the development of effective therapeutic strategies.

Dallaire, A. et al. (2013) International Worm Meeting "Down regulation of miR-124 in both Werner syndrome DNA helicase mutant mice and mutant Caenorhabditis elegans wrn-1 reveals the importance of this microRNA in accelerated aging."

J. Mitchell, S., Garand, C., De Cabo, R., R. Paquet, E., Lebel, M., J. Simard, M., Dallaire, A.

[International Worm Meeting, 2013]

Small non-coding microRNAs are believed to be involved in the mechanism of aging but nothing is known on the impact of microRNAs in the progeroid disorder Werner syndrome (WS). WS is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN ortholog exhibit many phenotypic features of WS, including a pro-oxidant status and a shorter mean life span. Caenorhabditis elegans (C. elegans) with a nonfunctional wrn-1 DNA helicase also exhibit a shorter life span. Thus, both models are relevant to study the expression of microRNAs involved in WS. In this study, we show that miR-124 expression is lost in the liver of Wrn helicase mutant mice. Interestingly, the expression of this conserved miR-124 in whole wrn-1 mutant worms is also significantly reduced. The loss of mir-124 in C. elegans increases reactive oxygen species formation and accumulation of the aging marker lipofuscin, reduces whole body ATP levels and results in a reduction in life span. Finally, supplementation of vitamin C normalizes the median life span of wrn-1 and mir-124 mutant worms. These results suggest that biological pathways involving WRN and miR-124 are conserved in the aging process across different species.

Pham, B.T.T. et al. (2017) International Worm Meeting "The genetic interaction of wrn-1 with mre-11 or him-6 in embryonic development."

Ahn, Byungchan, Pham, B.T.T.

[International Worm Meeting, 2017]

Werner's syndrome (WS) is an autosomal recessive disorder in humans characterized by the premature development of age-associated pathologies. WRN, the gene defective in WS, plays a key role in protecting the genome. The cell derived from WS patients show genomic instabilities such as sister chromatid exchange and telomere shortening. Caenorhabditis elegans (C. elegans) with a WRN-1 ortholog also exhibits a shorter life span. Our results showed wrn-1 interacts with mre-11 or him-6 and affects during embryonic and larval development. When inhibiting the expression of mre-11 or him-6 in wrn-1 (gk99) by RNA interference (RNAi) resulted in a significant increase of embryonic lethality and the retarded larval development compared with control worms of N2 (RNAi) worms. In addition, wrn-1 (gk99):him-6 (RNAi) or wrn-1 (gk99):mre-11 (RNAi) worms displayed abnormal premeiotic nuclei and multiple chromosomal abnormalities in oocyte nuclei at the diakinesis stage. Furthermore, observation of embryo cell division showed the slower development. These results suggest that MRE-11 and HIM-6 may interact with WRN-1, which is involved in embryonic development.

Kyungjn Lee et al. (2007) International Worm Meeting "Caenorhabditis elegans WRN-1 protein is involved in response to DNA damaging agents:Paraquat and Camptothecin."

Hana Jung, Byungchan Ahn, Kyungjn Lee, Moonjung Hyun

[International Worm Meeting, 2007]

Human Werner syndrome (WS) is a rare autosomal recessive genetic disorder characterized by the premature aging in young adults. Cells from human WS patients show defects related to DNA metabolisms. In C. elegans, WRN homolog has been reported. In this study, the responses of the wrn-1 (RNAi) worms to DNA damaging agents were examined. When the RNAi worms were treated with Paraquat, inducing oxidative damage and Camptothecin (CPT), the division of embryonic cells was faster than that of untreated normal embryonic cells. However, the treated embryos were not able to grow to adult worms. The WRN-1 protein formed foci in response to the treatment of DNA damaging agents. In addition, the RNAi worms displayed small premeiotic nuclei in the gonad after CPT or Paraquat treatment. These results suggest that C. elegans WRN may play a role in base excision repair (BER) and DNA damage checkpoint. wrn-1.

Lees, Hayley et al. (2015) International Worm Meeting "Synthetic superviability: combining detrimental mutations can have unexpected lifespan enhancing consequences."

Lees, Hayley, Woollard, Alison, Cox, Lynne

[International Worm Meeting, 2015]

As geneticists, we take for granted that mutating a single gene can extend C. elegans lifespan way beyond that experienced by WT animals. Thus, the genetic approach to longevity research has revealed several important molecular pathways regulating lifespan, including insulin signalling and oxidative stress. This is very striking, given the inherent complexity of ageing phenotypes, which involve changes in many different biological and physiological processes. It seems likely that traditional genetic screens for lifespan mutants are at, or nearing, saturation, and yet our understanding of the biology of ageing is far from complete. Does this mean that longevity is controlled by just a few "master" regulators, or that complex genetic interactions are important in determining robust longevity outcomes, such that rather few single mutants display profoundly altered rates of ageing? Here, we have modeled the human monogenic premature ageing disorder Werner syndrome (WS) using mutation of wrn-1, homologous to the WS associated WRN gene. WS recapitulates many features of the normal ageing process, but these manifest at an early age. Furthermore, cells from WS patients are characterized by genomic instability. Although a growing body of research can explain how WRN maintains genome stability, it is not clear why functional WRN is so important for longevity, or whether the observed genomic instability in WS contributes to premature ageing. We have demonstrated the progeroid nature of C. elegans wrn-1 mutants, but, unexpectedly, we find that combining short-lived wrn-1 mutants with short lived p53/cep-1 mutants results in a very striking daf-16-dependent improvement in both lifespan and healthspan, greatly beyond that of WT worms - a phenomenon we call synthetic superviability (SSV). Furthermore, we show that genomic instability per se does not drive ageing, since these long-lived double mutants display high levels of genomic instability. Rather, it appears that the ability to sense and respond to stresses such as DNA damage (mediated by factors like p53) limits lifespan. Remove the stress responder (which normally perhaps "overreacts" to particular stresses, thus limiting lifespan), and the intrinsic stress pushes the physiological outcome into an enhanced longevity mode, akin to hormesis. Investigations are now underway to discover other genetic combinations which also induce SSV; the cep-1;wrn-1 double mutant may turn out to be the prototype of a new class of combinatorial mutant with improved longevity, despite the fact that each single mutant displays progeria. This completely novel type of genetic interaction may provide rich resources for a deeper understanding of the biology of ageing.

Se-Jin Lee et al. (2006) East Asia C. elegans Meeting "Werner syndrome protein homolog WRN-1, genetically interacts with a RNase D homolog in the DNA damage checkpoint pathway in Caenorhabditis elegans"

Se-Jin Lee, Hyeon-Sook Koo

[East Asia C. elegans Meeting, 2006]

Werner syndrome (WS) is a premature aging disorder characterized by genomic instability and increased cancer risk. The gene mutated in WS encodes a nuclear protein (WRN) which possesses exonuclease and helicase activities. The genomic instability associated with WS cells and the biochemical characteristics of WRN suggest that WRN plays a role in DNA metabolic pathways such as replication, recombination and repair. However, it is not well known whether both activities, helicase and exonuclease, are coordinated in DNA metabolic pathways. Previously, we suggested that C.elegans WRN-1 (Ce-WRN-1) acts as a checkpoint protein to DNA damage and replication blockage. Ce-WRN-1 contains helicase motifs but an exonuclease motif is absent. Instead, the ORF ZK1098.3 encoding a RNase D homolog is suggested to be the exonuclease partner of Ce-WRN-1, based on the following observations. After RNA interference of the C. elegans RNase D homolog (Ce-WRN-2), larval growth was accelerated irrespectively of γ-irradiation. In addition, similar reductions in the embryonic hatching rate due to DNA cross-linking were observed after either single RNAi of Ce-WRN-1 and Ce-WRN-2 or double RNAi. These results suggest that Ce-WRN-1 genetically interacts with Ce-WRN-2. For the further study, we will determine by co-immunoprecipitation whether Ce-WRN-1 physically interacts with Ce-WRN-2 to maintain genome stability. In addition, to determine in which step of DNA damage checkpoint pathway the WRN proteins work, we will investigate whether phosphorylation of checkpoint proteins including CHK-1 are affected by WRN proteins.

Edward Yeh et al. (2003) International Worm Meeting "A screen to identify factors involved in active zone formation."

Mei ZHEN, Edward YEH

[International Worm Meeting, 2003]

The active zone is a specialized presynaptic structure where vesicles containing neurotransmitters fuse with the plasma membrane, releasing the neurotransmitters to the post-synaptic membrane. At the EM level, electron dense active zones are surrounded by clusters of vesicles. A few components of the active zone have been identified such as UNC-10 (Koushika et al., 2001), UNC-13 (Richmond et al., 1999; Aravamudan et al., 1999) and SYD-2 (Zhen and Jin, 1999) but even fewer have been implicated in active zone establishment, differentiation or maintainance.syd-2 encodes a homolog of liprin- and has been shown to be a component of the active zone. Mutations in syd-2 give rise to larger active zones. At a previous meeting, we reported a SYD-2::GFP marker that could be used to visualize active zones in live animals. The SYD-2::GFP protein marker was placed under the promoter of unc-25 which expresses in GABAergic neurons. We have performed a SYD-2::GFP screen to identify a dditional factors involved in the formation of the active zone. An EMS mutagenesis screen was performed looking for mutations affecting the formation of the active zone. Approximately 2500 haploid genomes were screened and we are currently characterizing 5 isolated mutant alleles. hp77 and hp121 represent two loci on chromosome X, hp102 and hp198 represent two loci on chromosome IV and hp129 is on chromosome V. hp121 and hp102 have behavioural unc phenotypes, hp198 are slightly dumpy while hp77 and hp129 do not appear to have any behaviour or physical phenotypes. Initial characterization and phenotypic analysis will be presented on these mutants. It is our hope that the molecular characterization of these mutants will provide information on factors involved in the establishment, differentiation, and maintenance of active zones. Ultimately, this information will provide insight into the genetic pathways used to establish neural synapses in general.References: Aravamudan B, Fergestad T, Davis WS, Rodesch CK, and Broadie K. (1999) Nature Neuroscience. 2:965; Koushika SP, Richmond JE, Hadwiger G, Weimer RM, Jorgensen EM and Nonet M. (2001) Nature Neuroscience. 4:997-1005; Richmond JE, Davis WS and Jorgensen EM. (1999) Nature Neuroscience. 2:959-64; Zhen M and Jin Y. (1999) Nature. 401:371-5

Lees, Hayley et al. (2011) International Worm Meeting "Exploring the function of progeroid WRN gene homologues in C. elegans."

Cox, Lynne, Woollard, Alison, Lees, Hayley

[International Worm Meeting, 2011]

Understanding the molecular basis of ageing is a necessary precursor to developing therapies to counter deleterious aspects of human ageing including frailty and age-related diseases. The widely-studied premature ageing Werner syndrome (WS) phenocopies normal human ageing but is caused by mutation of a single gene, WRN, encoding both helicase and exonuclease activities. In order to dissect the distinct roles of WRN helicase and exonuclease throughout development and ageing, we have identified homologues of both the helicase and exonuclease activity of human WRN in C. elegans. Our comparative genomic analysis in C. elegans suggests that wrn-1 is the closest homologue of the human WRN helicase domain and mut-7 is the closest homologue of the human WRN exonuclease domain. Ageing phenotypes and shortened lifespan have been previously associated with reduction of function of wrn-1 by RNA interference [1], while mutation of mut-7 results in genetic instability by activating transposons in the germline [2], but there have been no reported lifespan studies. We suggest that together, wrn-1 and mut-7 constitute the same domain architecture as human WRN, as in other invertebrates (Drosophila) and plants (Arabidopsis). We have established out-crossed worm lines mutant for the WRN exonuclease, and for the WRN helicase (mut-7 and wrn-1, respectively), and have used these strains to generate wrn-1;mut-7 double mutants. Ageing phenotypes and lifespan of the mutant worms will be presented, as will the phenotype of our novel double mutants lacking both WRN helicase and exonuclease. In addition, further data highlighting how this worm model can be used to dissect at the molecular level the relative contributions of the WRN helicase and exonuclease to DNA damage responses and genome stability will also be given. References 1.Lee, SJ; Yook, JS; Han, SM; Koo, HS. A Werner syndrome protein homolog affects C. elegans development, growth rate, life span and sensitivity to DNA damage by acting at a DNA damage checkpoint. Development, 2004. 131(11): p. 2565-2575. 2.Ketting, RF; Haverkamp, TH; van Luenen, HG; Plasterk, RH. Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell, 1999. 99(2): p. 133-141.