Gregg Czerwieniec

Buck Institute For Research on Aging; Novato CA, United States of America

PS1259

Caenorhabditis elegans

Rodrigues, Pedro Reis et al. (2009) International Worm Meeting "A variety of age-dependant aggregating proteins determine lifespan."

Peters, Theodore, Gibson, Brad, Lithgow, Gordon, Hughes, Robert, Alavez, Silvestre, Rodrigues, Pedro Reis, Czerwieniec, Gregg

[International Worm Meeting, 2009]

Protein aggregation has for long been hypothesised as a determinant of lifespan. Briefly, normal cellular activity may give rise to damaged proteins causing them to become insoluble, missfold and aggregate. To test this hypothesis we adapted a protocol in order to extract insoluble proteins from synchronously aging populations of C. elegans. Proteins were separated based on their aqueous and detergent solubility and the insoluble fraction was resolubilized in 70%; formic acid. Insoluble proteins were chemically labelled, identified and quantified by liquid chromatography coupled with mass spectrometry (LC- ESI-MS/MS). We identified a range of proteins with roles in various cellular processes and possibly from a range of cellular compartments. 27%; of the proteins identified as forming aggregates have previously been shown to be important in keeping low levels of polyglutamine aggregation1. This suggests that reduced soluble levels of these proteins caused by age-related aggregation may cause increased risk of polyglutamine aggregation. We then considered whether proteins that appear to form aggregates during normal aging influenced lifespan. To test this notion we, reduced their expression in adult animals (from 4 days old) by RNA interference (RNAi). 34%; of the RNAi treatments were found to significantly extend mean lifespan in C. elegans suggesting that a variety of age-dependant aggregating proteins determine lifespan. Among the insoluble proteins, DAF-21, an ortholog of the mammalian HSP-90, showed age-dependant aggregation and is being used as a marker to study the role of several molecular pathways in protein aggregation. Taken together our results suggest that protein aggregation may play a common and key role in aging and age-related disease. 1 - Nollen, E., Garcia, S., Haaften, G., Kim, S., Chavez, A., Morimoto, R., Plasterk, R., Genome-wide RNA interference screen identified previously undescribed regulators of polyglutamine aggregation. Proc Natl. Acad. Sci., 101, 6403-6408, 2004.

Rogers, Aric N. et al. (2009) International Worm Meeting "eIF-4G post-transcriptionally remodels stress response and cellular homeostasis gene expression in C. elegans."

Chen, Di, Hubbard, Alan, McColl, Gawain, Kapahi, Pankaj, Lithgow, Gordon, Rogers, Aric N., Melov, Simon, Gibson, Brad, Czerwieniec, Gregg

[International Worm Meeting, 2009]

Attenuation of mRNA translation extends lifespan in metazoans by as yet unknown mechanisms. Eukaryotic translation initiation factor (eIF)-4G is an mRNA cap-binding complex member that positively regulates translation by acting as a scaffold between transcript 5'' and 3'' ends via eIF-4E and poly A binding protein, respectively. Here we show that reducing translation via suppression of eIF-4G (ifg-1 in C. elegans) also differentially regulates expression of genes involved in responding to stress. Furthermore, we observe a positive correlation between mRNA size and translation as measured by ribosome loading when ifg-1 is inhibited. This correlation extends to overall protein levels assayed using quantitative mass spectrometry. Genome-wide analysis shows that longer mRNAs are enriched with genes important for stress responses and that positively regulate lifespan and cellular homeostasis. Inhibition of some of these stress response genes substantially diminishes the increased lifespan under eIF-4G inhibition and include the FOXO transcription factor daf-16 and the energy sensor AMP kinase catalytic subunit aak-2. Expression of SCA-1, a Calcium ATPase involved in ER homeostasis, is essential for lifespan extension in an ifg-1 mutant. Our results indicate that ifg-1 controls a switch in mRNA translation between growth and development and somatic maintenance, which provides a novel mechanism for the tradeoffs between growth and longevity.

Rogers, Aric N. et al. (2011) International Worm Meeting "Lifespan extension via eIF4G inhibition is mediated by post-transcriptional remodeling of stress response gene expression in C. elegans."

Kapahi, Pankaj, Hubbard, Alan, Melov, Simon, Chen, Di, Gibson, Bradford W., Rogers, Aric N., Felkey, Krysta, Lithgow, Gordon, McColl, Gawain, Czerwieniec, Gregg

[International Worm Meeting, 2011]

Reducing protein synthesis slows growth and development but can increase adult lifespan. We demonstrate that knock-down of eukaryotic translation initiation factor 4G (eIF4G), which is down-regulated during starvation, results in differential translation of genes important for growth and longevity in C. elegans. Genome-wide mRNA translation state analysis showed that inhibition of IFG-1, the C. elegans ortholog of eIF4G, results in a relative increase in ribosomal loading and translation of stress response genes. Some of these genes are required for lifespan extension when IFG-1 is inhibited and are novel determinants of longevity. Furthermore, enhanced ribosomal loading of certain mRNAs upon IFG-1 inhibition was correlated with increased mRNA length. This association was supported by changes in the proteome assayed via quantitative mass spectrometry. Our results support a role for IFG-1 in mediating the antagonistic effects on growth and somatic maintenance by modulating translation of a specific class of mRNA based on transcript length.

Rodrigues, Pedro R. et al. (2011) International Worm Meeting "Proteomic Analysis of Age-dependent Changes in Protein Solubility Identifies Genes that Modulate Lifespan."

Czerwieniec, Gregg, Gaman, Emily A., Lithgow, Gordon J., Hughes, Robert E., Rodrigues, Pedro R., Evani, Uday S., Peters, Theodore, Lucanic, Mark, Alavez, Silvestre, Mooney, Sean D., Gibson, Bradford W.

[International Worm Meeting, 2011]

While it is generally recognized that misfolding of specific proteins can cause late-onset disease states, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging. SDS Insoluble proteins purified from young and aged C. elegans were chemically labelled by isobaric tagging for relative and absolute quantitation (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Transgenic nematodes expressing three proteins identified in the insoluble fraction, DAF-21, RPS-0 and EFT-3 fused to Green Fluorescent Protein (GFP) showed the formation of visible aggregates by fluorescence microscopy. In the case of RPS-0 and EFT-3, these aggregates appeared immobile as measured by Fluorescence Recovery after Photobleaching (FRAP). Expression of genes encoding insoluble proteins observed in aged nematodes was knocked-down in using RNAi and effects on lifespan were measured. Forty of 100 genes tested were shown to extend lifespan after RNAi. These data indicate that genes encoding proteins that become insoluble with age are modifiers of lifespan. These data also demonstrate that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that aggregation of a diverse range of proteins may be a general feature of aging.

Shivakumar S et al. (1994) Worm Breeder's Gazette "wnt Homologs in Caenorhabditis elegans"

Kenyon CJ, Shivakumar S, Varmus HE, Jongeward GD

[Worm Breeder's Gazette, 1994]

wnt homologs in Caenorhabditis elegans Supriya Shivakumar, Gregg Jongeward, Cynthia Kenyon and Harold Varmus. University of California, San Francisco, CA 94143 The wnt gene family encodes cysteine-rich secreted proteins which have been shown to act as important regulators of early development in the frog, fly and mouse. wnt genes have a crucial role in intercellular signaling in the mouse CNS development and in pattern formation in the fly. We chose to study the normal role of Ce-wnt-Z in signaling in C. elegans development. Two members of the wnt gene family have been identified in C elegans, Cc-wnt 1 and Ce-wnt-22. The Ce-wnt-1 gene encodes a 372 amino acid protein and shares 22 of the 24 cysteines found among other members of the wnt family. A 1.4 kb transcript (including an SL1 transpliced leader sequence) is detected at high levels in embryos and at lower levels at all other stages. The 1.4 kb Ce-wnt-2 cDNA is also expressed in a similar temporal pattern to Ce-wnt-1. The Cc-wnt-2 cDNA encodes a protein of 362 amino acids with 22 of the cysteines shared among other Wnt proteins. Neither Ce-wnt 1 and Ce-wnt-2 are direct homologs of any specific wnt genes in other organisms. To elucidate the functions of the Ce-wnt-Z gene product, we have isolated mutations in the gene. The Ce- wnt-l locus maps to the left arm of chromosome II, but does not appear to correspond to any existing mutations. We made the assumption that the Ce-wnt-1 gene is required for viability based on wnt mutant phenotypes in other species. We screened for psoralen induced lethal mutations balanced by an extrachromosomal array comprising a 15 kb lambda clone containing the Ce-wnt-1 gene and a marker (rol-6D) in a F2 clonal screen (see diagram below). 1500 F2 animals were screened and 2 embryonic lethal alleles were isolated. Both contain deletions in the Ce wnt-1 coding region. These mutations are rescued by the lambda done, but not by rol-6. A 53 kb subdone also rescues the lethality. We are now trying to demonstrate conclusively that this early embryonic lethality is due to the disruption of the Ce-wnt-1 gene. We have also screened for psoralen induced mutations (in an identical screen) that require a cosmid clone containing Ce- wnt-2. We have identified a number of mutations in the screen which we are nDw analyzing in more detail. 1Shackleford et al., Oncogene 8:1857 (1993) 2Waterson et al., Nature genetics 1:114 (1992)

Liu KK et al. (1990) Worm Breeder's Gazette "In Search of Celibate Old Dullards"

Chamberlin HM, Sternberg PW, Liu KK, Hajdu-Cronin YM

[Worm Breeder's Gazette, 1990]

Behavioral mutants have been useful in studying the function and development of the C. elegans nervous system. We're using this behavioral genetic approach to study the 79 neurons specific to the male. On the assumption that these additional neurons mediate mating behavior, which is seen in males but not hermaphrodites, we have been screening for mutant strains defective in this behavior using the screen first described by Hodgkin (Gen. 103:43-64, 1983). him-5( e1490) worms are mutagenized with EMS; 20 P0 L4 hermaphrodites are cloned; 10 F1 worms are picked per mutagenized P0; and 10 F2 L4 hermaphrodites are cloned per P0 and their male progeny tested for mating efficiency via a qualitative mating test (6 males x 6 unc-52 hermaphrodites). Mutations that would reduce mating efficiency in a non-specific manner (such as Uncs, Dpys, Lons) are discarded. Those strains that appear morphologically normal under the dissecting scope yet fail to mate or mate at a very low efficiency (1-5% cross progeny compared to wild type) are screened under Nomarski optics for defects in male reproductive structures. Thus far,we have screened 2,123 haploid genomes. Although the clonal nature of this screen is designed to pick up behavioral (i.e. neural) defects, screening by mating efficiency will of course also pick up mutants defective in male copulatory structures. This is a welcome side effect as we are also interested in genes that affect the development of the copulatory structures - particularly the spicules (see below; Chamberlin & Sternberg, WBG 11(2) :103). Wild type males normally respond to a hermaphrodite by backing along the length of the hermaphrodite with its tail in contact (avoiding the head and tail of the hermaphrodite by turning either under or over the hermaphrodite body near but before it reaches the ends) until it locates the vulva. At this point the male stops, slides back and forth until it inserts its spicules into the vulva and finally injects sperm. To date, we have backcrossed nineteen strains (about a 25% success rate) which appear to be wild type in morphology yet are unable to perform the normal mating behavior. We call these strains Cod for copulation defective (or Celibate old dullards). sy155, sy166, sy172, and sy178 males respond only weakly to a hermaphrodite if at all. Three mutants, sy35, sy174, and sy181, are unable to turn normally around the head and tail of the hermaphrodite (backing all the way to the tip and stopping or turning unsuccessfully). Two of the turning defective mutants, sy174 and sy181, plus sy157, are unable to locate the vulva. Eight mutants, sy38, sy43, sy153, sy156, sy158, sy165, sy176, and sy177, are unable to insert their spicules. And lastly, three mutants, sy36, sy179, and sy180, appear to mate normally but sire very few or no progeny. Since the hermaphrodite brood sizes are normal in these strains, they appear to be defective in sperm transfer. So far, we have characterized twelve mating defective mutants with morphological defects. Mutations that disrupt gonad, ray, and/or spicule development have been isolated due to the reduced male mating efficiency phenotype they confer. The gonads of sy189 males exhibit abnormal linker cell migration and failure to attach to the cloaca. Four mutations, sy31, sy68, sy173, and sy183, affect ray formation. sy183 causes variably swollen rays and tail structures. sy31 causes a swollen ray, Ram phenotype (ray morphology, Scott Baird, personal communication), but does not affect the spicules. sy68 males have crumpled spicules and also retain the larval tail spike, usually at the expense of rays 7-9. sy173 males lack rays 1-6, have posterior alae, and wrinkled spicules. This mutation maps to LGIII and fails to complement mab-5(e1239) for the ray and spicule abnormality. Four other mutations also cause crumpled or wrinkled spicules: bx31, sy29, sy32, and sy66. Most of these are pleiotropic in their phenotype. bx31 hermaphrodites are variably Muv. bx31 maps to the left arm of LGII and fails to complement lin-31(n301) for both the vulval and the spicule phenotype. sy29 hermaphrodites are Vul, and sy66 worms, both males and hermaphrodites, are notch-head Vab. Only sy32 exhibits no pleiotropies at the plate level. bx31, sy29, sy32, and sy66 all exhibit defects in the B cell (spicule) lineage (work in progress). Three mutations, sy170, sy187, and sy188 result in spicules that appear to be normal except that they lack the autofluorescent properties of wild-type spicules, and they are extremely flexible. If a male with one of these mutations extrudes his spicules, they flatten out and 'flap in the breeze'. Finally, one mutation, sy182, results in the absence of male progeny despite the him-5(e1490) background of the parental strain. We are currently characterizing sy182 in order to determine the nature of this phenotype. Special thanks to Scott Baird and Scott Emmons for bx31 and to Gregg Jongeward for the title.