The role of oxidative stress in C. elegans aging

The free radical theory of aging postulates that aging is caused by damage induced by oxidative stress. Such stress is present when the production of reactive oxygen species (ROS) exceeds the cellular antioxidant capacity. Hydrogen peroxide (H2O2) is one of the most abundant ROS. It is produced as a by-product by several enzymes and acts as second messenger controlling the activity of numerous cellular pathways. To maintain H2O2 levels that are sufficiently high to allow signaling to occur, but low enough to prevent damage of cellular macromolecules, the production and removal of H2O2 must be tightly regulated.rnWhen we investigated the effects of peroxide stress in the nematode C. elegans, we found that exogenous as well as endogenous peroxide stress causes age-related symptoms. We identified 40 target proteins of hydrogen peroxide that contain cysteines that get oxidized upon peroxide stress. Oxidation of redox-sensitive cysteines has been shown to regulate numerous cellular functions and likely contributes to the peroxide-mediated decrease in motility, fertility, growth rate and ATP levels. By monitoring the oxidation status of proteins over the lifespan of C. elegans, we discovered that many of the identified peroxide-sensitive proteins are heavily oxidized at distinct stages in life. As the free radical theory of aging predicts, we found oxidation to be significantly elevated in senescent worms. However, we were also able to identify numerous proteins that were significantly oxidized during the development of C. elegans. To investigate whether a correlation exists between developmental oxidative stress and lifespan, we monitored protein oxidation in long- and short-lived strains. We found that protein oxidation in short-lived C. elegans larvae was significantly increased. Additionally short-lived worms were incapable of recovering from the oxidative stress experienced during development which resulted in the inability to establish reducing conditions for the following reproductive phase. Long-lived C. elegans, on the other hand, did only experience a mild increase in protein oxidation in the developmental phase and were able to recover faster from oxidative stress than wild type worms. rnBecause many proteins that are sensitive to oxidation by H2O2 became oxidized in aging C. elegans, we monitored endogenous hydrogen peroxide concentrations over C. elegans lifespan and discovered that peroxide levels are significantly elevated in development. This suggests that the observed developmental protein oxidation is peroxide-mediated. The early onset of oxidative stress might be a result of increased metabolic activity in C. elegans development but could also represent the requirement of ROS dependent signaling events. Our results indicate that longevity is dependent on the worm’s ability to cope with this early boost of oxidants.rn

RAB3GAP1 und RAB3GAP2 (RAB3 GTPase-activating Protein 1/2) beeinflussen die Autophagie in C. elegans und humanen Zellen

Die Proteinhomöostase wird in der Zelle von drei Stoffwechselwegen reguliert: den molekularen Chaperonen, dem Ubiquitin-Proteasom-System und dem autophagosomalen Abbauweg. Die (Makro)Autophagie verpackt und transportiert zytosolische Komponenten in Autophagosomen zu den Lysosomen, wo sie abgebaut werden. Eine Störung dieses Abbauwegs wirkt auf die Proteostase.rnIn dieser Dissertation wurde C. elegans als Modellorganismus zur Erforschung von Proteinstabilität genutzt. In einer RNAi-vermittelten Proteostase-Analyse von Chromosom I und ausgewählter zusätzlicher Gene wurde ein Wurmstamm, der ein Luc::GFP-Konstrukt im Muskel exprimiert, genutzt. Dieses Reporterprotein aggregiert unter Hitzestressbedingungen und diese Aggregation kann durch Modulatoren der Proteostase beeinflusst werden. Dabei wurden mögliche neue Faktoren der Proteinhomöostase entdeckt. Durch weitere Experimente bei denen die Aggregation von PolyQ35::YFP im AM140-System, der Paralyse-Phänotyp und die Akkumulation Thioflavin S-gefärbter Aggregate von Aβ42 im CL2006-Wurmstamm und die Effekte auf die Autophagie mittels eines GFP::LGG1-Konstrukt analysiert wurden, konnten rbg-1 und rbg-2 als neue Modulatoren der Proteinhomöostase, insbesondere der Autophagie, identifiziert werden.rnIm Säuger bilden beide Orthologe dieser Gene, RAB3GAP1 und RAB3GAP2 den heterodimeren RAB3GAP-Komplex, der bisher nur bekannt war für die Stimulation der Umwandlung der GTP-gebundenen aktiven Form zur GDP-gebundenen inaktiven Form der RAB GTPase RAB3. In Immunoblot-Analysen und mikroskopischen Darstellungen im Säugersystem konnte gezeigt werden, dass die Effekte auf die Proteostase über den autophagosomalen Abbauweg wirken. RAB3GAP1/2 wirken als positive Stimulatoren, wenn die Lipidierung von LC3-I und der autophagische Flux von LC3-II und p62/SQSTM1 betrachtet werden. Diese Effekte werden aber nicht über die RAB GTPase RAB3 vermittelt. Die Proteine FEZ1 und FEZ2 haben einen antagonistischen Effekt auf die Autophagie und wenn alle vier Komponenten RAB3GAP1, RAB3GAP2, FEZ1 und FEZ2 zusammen herunter- oder hochreguliert werden, heben sich diese Effekte auf. In Co-Immunopräzipitationen und proteomischen Analysen konnte keine direkte Interaktion zwischen dem RAB3GAP-Komplex und FEZ1/2 oder zu anderen Autophagie-Genen nachgewiesen werden.rnHier konnte der RAB3GAP-Komplex funktionell mit Proteostase und Autophagie in C. elegans und Säugerzellen assoziiert werden. Dieser Komplex zeigt Einflüsse auf die autophagosomale Biogenese indem sie die Proteostase und die Bildung von (prä)autophagosomalen Strukturen in C. elegans und die Lipidierung von LC3 und damit den autophagischen Flux der Autophagiesubstrate LC3-II und p62/SQSTM1 in Säugerzellen beeinflusst. Darüber hinaus wirkt RAB3GAP der komplexen Autophagie-Unterdrückung durch FEZ1 und FEZ2 entgegen. Somit konnte gezeigt werden, dass RAB3GAP als neuartiger Faktor auf die autophagosomale Biogenese und somit auf die Proteostase wirkt.rn

Microscopic analysis of chromatin localization and dynamics in C. elegans

During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell analysis of gene positioning. Moreover, the dynamic behavior of chromatin has to be assessed on living samples. Combining fast stereotypic development with easy genetics and microscopy, the nematode C. elegans has become a model of choice in recent years to study changes in nuclear organization during cell fate acquisition. Here we present two complementary techniques to evaluate nuclear positioning of genes either by fluorescence in situ hybridization in fixed samples or in living worm embryos using the GFP-lacI/lacO chromatin-tagging system.

Nuclear organization in the nematode C. elegans.

With its invariant cell lineage, easy genetics and small genome, the nematode Caenorhabditis elegans has emerged as one of the prime models in developmental biology over the last 50 years. Surprisingly however, until a decade ago very little was known about nuclear organization in worms, even though it is an ideal model system to explore the link between nuclear organization and cell fate determination. Here, we review the latest findings that exploit the repertoire of genetic tools developed in worms, leading to the identification of important sequences and signals governing the changes in chromatin tridimensional architecture. We also highlight parallels and differences to other model systems.

Differential spatial and structural organization of the X chromosome underlies dosage compensation in C. elegans.

The adjustment of X-linked gene expression to the X chromosome copy number (dosage compensation [DC]) has been widely studied as a model of chromosome-wide gene regulation. In Caenorhabditis elegans, DC is achieved by twofold down-regulation of gene expression from both Xs in hermaphrodites. We show that in males, the single X chromosome interacts with nuclear pore proteins, while in hermaphrodites, the DC complex (DCC) impairs this interaction and alters X localization. Our results put forward a structural model of DC in which X-specific sequences locate the X chromosome in transcriptionally active domains in males, while the DCC prevents this in hermaphrodites.

Modern techniques for the analysis of chromatin and nuclear organization in C. elegans.

In recent years, Caenorhabditis elegans has emerged as a new model to investigate the relationships between nuclear architecture, cellular differentiation, and organismal development. On one hand, C. elegans with its fixed lineage and transparent body is a great model organism to observe gene functions in vivo in specific cell types using microscopy. On the other hand, two different techniques have been applied in nematodes to identify binding sites for chromatin-associated proteins genome-wide: chromatin immunoprecipitation (ChIP), and Dam-mediated identification (DamID). We summarize here all three techniques together as they are complementary. We also highlight strengths and differences of the individual approaches.

A dual oxidase generates a protective oxidative burst during infection in C. elegans

Caenorhabditis elegans has recently been developed as a model system to study both pathogen virulence mechanisms and host defense responses. We have shown that C. elegans produces reactive oxygen species (ROS) in response to exposure to the important Gram-positive, noscomial pathogen, Enterococcus faecalis. We have also shown evidence of oxidative stress and upregulation of stress response after exposure to the pathogen. As in mammalian systems, this work shows that production of ROS for innate immune functions occurs via an NADPH oxidase. Specifically, reducing expression of a dual oxidase, Ce-duox1/BLI-3 causes a decrease in ROS production in response to E. faecalis. We also present evidence that reduction of expression of Ce-duox1/BLI-3 increases susceptibility to this pathogen, specifically when expression is reduced in the intestine and the hypodermis. This dual oxidase has previously been localized to the hypodermis, but we show that it is additionally localized to the intestine of C. elegans. To further demonstrate the protective effects of the pathogen-induced ROS production, we demonstrate that antioxidants that scavenge ROS, increase the sensitivity of the nematode to the infection, in stark contrast to their longevity-promoting effects under non-pathogenic conditions. In conclusion, we postulate that the generation of ROS by NADPH oxidases in the barrier epithelium is an ancient, highly conserved innate immune defense mechanism.^

A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans

Drosophila shibire and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a Caenorhabditis elegans dynamin gene, dyn-1, based upon hybridization to the Drosophila gene. The dyn-1 RNA transcripts are trans-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830- or 838-amino acid protein. These dyn-1 proteins are highly similar in amino acid sequence, structure, and size to the Drosophila and mammalian dynamins: they contain an N-terminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive dyn-1 mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to D. shibire mutants. When maintained at higher temperatures, dyn-1 mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a dyn-1::lacZ gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that dyn-1 function is required during development and for normal locomotion.

Functionality, Robustness and Control of Nonlinear Network Dynamics: Modeling and Understanding the C. elegans Connectome

Thesis (Ph.D.)--University of Washington, 2016-06

A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport

BACKGROUND: Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics. RESULTS: Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes. CONCLUSIONS: This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.