Davis RE et al. (1991) International C. elegans Meeting "EXTRACELLULAR RECORDINGS AND EXCITATORY AMINO ACID STUDIES IN THE MOTORNERVOUS SYSTEM OF ASCARIS."

Stretton AOW, Davis RE

[International C. elegans Meeting, 1991]

Previous intracellular recordings from Ascaris motorneurons and some interneurons revealed that these cells do not show the classical voltagesensitive action potentials characteristic of other nervous systems (Davis, R.E. and A.O.W. Stretton, J. Neurosci., 9:415, 1989; Angstadt, J.D. et al., J. Comp. Neurol., 284:374, 1989). The existence of discrete EPSPs and IPSPs in some classes of motorneurons, however, suggested that spikes are being generated in some presynaptic interneurons. Extracellular suction electrode recordings made over the nerve cords and over muscle cells have revealed fast spike activity of two general categories: (l) small amplitude, short duration, relatively simple biphasic spikes exclusively associated with the ventral nerve cord and (2) larger amplitude, longer duration spikes which are frequently multiphasic. The small spike population can show a variety of patterns of firing activity, has been correlated with motorneuron PSPs, and is not correlated with intracellularly recorded ventral muscle events. This population of signals probably originates in interneurons responsible for the motorneuron PSPs and, by means of those PSPs, may play a role in the generation of patterned motorneuronal, and ultimately behavioral, activity. The large spike population is relatively infrequent in occurrence, is correlated with intracellularly recorded muscle events and can be manipulated by polarizations of presynaptic motorneurons as would be expected if this population originates in muscle. Pharmacological experiments involving excitatory amino acids (EAA) and their analogs reveal significant depolarizing responses in DE2 (DB) motorneurons to application of glutamate and other EAA analogs. At best, only very weak responses are seen in DEl (AS) or DI (DD) to such applications. We are further characterizing these various spike events and the EAA pharmacology to determine their role in locomotory behavior. (USPHS Grant 15429)

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.

Susana M Garcia et al. (2005) International Worm Meeting "Novel Molecular Modulators of Protein Homeostasis in C. Elegans"

Susana M Garcia, Margarida Amaral, Richard Morimoto

[International Worm Meeting, 2005]

Protein folding in the cells highly crowded macromolecular environment is a complex process. Many factors can affect the folded state of proteins, these include alterations of the cellular environment and the protein folding machinery. Disruptions in the folding process result in accumulation of misfolded proteins and the formation of aggregates, which have been linked to a number of human diseases such as polyglutamine (polyQ) expansion disorders. Our lab has previously expressed polyQ repeats as YFP (yellow fluorescent protein) fusion proteins in body wall muscle cells in C. elegans (Satyal et al. 2000; Morley et al 2002). These animals showed a polyQ length dependent aggregation phenotype. At intermediate polyQ length, animals show age-dependent transition from soluble to aggregated polyQ, optimal for screening. Previously, our C. elegans strains expressing different polyQ lengths were used in a genome-wide RNAi screen and 186 modulators of polyQ aggregation were identified (Nollen et al 2004). Recently, we performed a forward genetic screen, using EMS mutagenesis, to identify additional modifiers of protein homeostasis, including weaker alleles. Two recessive mutations found in our screen, rm7 and rm8, cause an enhancement of the aggregation phenotype in our polyQ C. elegans strains. These were mapped to chromosomes IV and X respectively. rm7 is a deletion in the C. elegans gene unc-30. UNC-30 is a neuronal transcription factor that regulates GABA expression in a subset of motor neurons. The effect of rm7 and other mutants of this pathway in modulating polyQ aggregation establishes a link between neuronal signaling and protein homeostasis in muscle cell.

Ellen Nollen et al. (2006) European Worm Meeting "C. elegans Models of Protein Misfolding Diseases"

Ellen Nollen, Tjakko van Ham, Ronald H. A. Plasterk.

[European Worm Meeting, 2006]

Ellen Nollen, Tjakko van Ham & Ronald H. A. Plasterk. Aggregation of misfolded proteins occurs in various age-related neurodegenerative disorders, including Parkinsons, Alzheimers, and Huntingtons disease. To understand how cells protect themselves against misfolded proteins, we search for genes that enhance or prevent protein aggregation. C. elegans strains expressing polyglutamine stretches fused to YFP with visible, age-dependend protein aggregation are used as a genetic model. Using a genome-wide RNAi screen, we have previously identified 186 genes that, when knocked down, cause premature protein aggregation. These genes include genes involved in protein synthesis, folding, degradation and RNA synthesis and processing. 1. Conversely, we performed a forward mutagenesis screen to identify genes that, when mutated, suppress age-dependent polyglutamine aggregation. For one suppressor mutant, in which aggregation is suppressed by more than 75%, we have now identified the responsible mutation. This mutation is a missense mutation in a gene encoding a protein of unknown function that is highly conserved between C. elegans and humans. Knock-down by RNAi of the same gene in wild-type worms yielded a similar reduction in aggregation, suggesting a loss-of-function mutation. We are currently further characterizing this mutant and the remaining suppressor mutants. In addition, to establish whether the genes we have identified are specific for polyglutamine aggregation or whether they comprise of a general protein homeostatic buffer, we have developed a worm model for aggregation of alpha synuclein, which occurs in Parkinson''s disease. Altogether our results will provide insight into cellular protection against misfolded proteins and yield targets for therapy against protein misfolding diseases.. 1Nollen E.A.A., Garcia S.M., van Haaften G., Kim S., Chavez A., Morimoto R.I., Plasterk R.H. (2004) Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. Proc. Natl. Acad. Sci. U.S.A. 101(17):6403-8.

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.

Mats Holmberg et al. (2008) European Worm Meeting "Modifiers of aggregation in neurodegenerative diseases"

Tjakko van Ham T, Ellen Nollen, Jeroen van Rheenen, Mats Holmberg

[European Worm Meeting, 2008]

Inheritable expansions of polyglutamine tracts lead to misfolding of the. affected protein in at least nine neurodegenerative diseases. Although all. are characterized by the accumulation of inclusions, increasing evidence. point to smaller, monomeric or oligomeric precursors as the toxic species. and suggest an active, protective sequestration of misfolded protein. It. has previously been established that genetic factors, other then the. unstable extended CAG repeat, affect the age of onset as well as disease. progression1. Screens for modifiers of polyglutamine, by for example RNAi,. makes it increasingly clear that disease related misfolding is influenced. by a large network of genes involved in different cellular processes2. We. aim to identify and characterise modifiers of polyglutamine aggregation and. toxicity in C. elegans expressing YFP-tagged polyglutamine. Aggregation can. be followed in time and screened for by observing fluorescent foci in. polyglutamine strains. Cytotoxicity has been shown in these strains to. correlate with longer glutamine tracts. In a forward mutagenesis screen in. a C. elegans model of polyglutamine disorders, we identified a missense. mutation in Moag-4 (modifier of aggregation), causing a reduction of. aggregation and a polyglutamine threshold specific increase in toxicity.. The influence on inclusion formation is confirmed by gene knockout and RNAi. knockdown. Our results implicate moag-4 in the active sequestration of. misfolded protein under cellular stress and therefore in a novel category. of stress response. 1 Rosenblatt et al. Am J Med Genet. 2001 Jul 8;105(5):399-403. Familial. influence on age of onset among siblings with Huntington disease.. 2 Nollen et. al. Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6403-8.. Genome-wide RNA interference screen identifies previously undescribed. regulators of polyglutamine aggregation.

T.J. van Ham et al. (2007) International Worm Meeting "A C. elegans model links novel modifiers of a-synuclein inclusion formation to aging."

K.L. Thijssen, E.A.A. Nollen, T.J. van Ham, R.H.A. Plasterk

[International Worm Meeting, 2007]

Accumulation and aggregation of <font face=symbol>a</font>-synuclein in the brain is characteristic of, and considered to be causatively related to, several age-related neurodegenerative diseases such as Parkinsons disease (PD). To study early stages of <font face=symbol>a</font>S accumulation and to identify modifier genes, we created transgenic C. elegans expressing the human <font face=symbol>a</font>S gene fused to YFP in the body wall muscle. These transgenic animals show localization of <font face=symbol>a</font>S-YFP to discrete foci during aging, which is not seen for YFP alone, indicating specificity to <font face=symbol>a</font>S. These foci are present as early as day two after hatching, and become more abundant during aging. By fluorescence recovery after photobleaching (FRAP) we determined that the foci contain mobile material until day 11. However, at old age also immobilized aggregated protein is present. The amount of <font face=symbol>a</font>S-foci increases to day 11 and decreases thereafter. Interestingly, this decrease in foci formation is associated with a relative increase in the amount of immobilized aggregates. We performed a genome-wide RNAi screen for modifiers of foci formation, and found 108 genes that repeatedly showed increased foci formation when silenced. Remarkably, none of these genes overlaps with genes found in a screen for polyglutamine aggregation we performed in C. elegans1. This might indicate that different mechanisms are involved in these diseases. To further characterize genes found in the screen, deletion strains of a selection of the genes representing different functional classes were crossed into the <font face=symbol>a</font>S-YFP strain. Deletion of a gene - involved in aging and encoding one of the most potent suppressors of foci-formation - resulted in a twofold increase in the amount of foci on day thirteen. We are currently further characterizing the deletion strains. In conclusion, we developed a new animal model that captures features of age-related PD. We used the model to dentify novel modifiers of <font face=symbol>a</font>S-YFP foci formation, of which we are further characterizing the effect of deletion of these genes on <font face=symbol>a</font>S-YFP foci formation. These results can provide insight into the early cellular mechanisms involved in <font face=symbol>a</font>S accumulation, and could thereby yield molecular targets for therapy. 1 Nollen E.A.A., Garcia S.M., van Haaften G., Kim S., Chavez A., Morimoto R.I., Plasterk R.H.A. (2004) Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. Proc. Natl. Acad. Sci. U.S.A. 101(17):6403-8.