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.

Ellen Nollen et al. (2003) International Worm Meeting "Genome-wide RNAi screen for genes involved in polyglutamine aggregate formation"

Ronald H Plasterk, Richard I Morimoto, Gijs van Haaften, Susana Garcia, Ellen Nollen

[International Worm Meeting, 2003]

Accumulation of polyglutamine containing proteins into intracellular aggregates is associated with various CAG trinucleotide expansion disorders, including neurodegenerative diseases such as Huntingtons disease and spinocerebellar ataxias. The aggregation properties of polyglutamine proteins are directly related to the length of the polyglutamine stretch. With polyglutamine stretches above a threshold of approximately 30 glutamine residues, the aggregation rate increases with increasing numbers of glutamine residues. The length-dependent kinetics of aggregation recapitulates the length-dependent increase in cellular toxicity and age of onset of disease. Expression of polyglutamine stretches of 0, 24, 33, 35, 40, 44, and 82 glutamine residues as YFP-fusion proteins under control of the muscle specific unc-54 promoter in C. elegans reconstitutes the length and age dependence of aggregation. 1 Whereas worms expressing YFP-fusions with polyglutamine stretches up to 24 (Q24) show a diffuse YFP staining in all muscle cells, Q82 animals show a punctate staining in most of the cells. Interestingly, all Q lengths show variability in aggregation within individual animals, depending on the cell and the age of the worm, which is influenced by the genetic background of the worms. For example, aggregation of Q82-YFP is greatly delayed in the aging mutant age-11. This heterogeneity of aggregation suggests that genes exist that influence the formation of polyglutamine aggregates. We are using a genome-wide RNAi screen to identify genes involved in polyglutamine aggregation. In a candidate gene approach with RNAi against genes encoding molecular chaperones and molecules involved in proteins degradation, we have already identified genes that may play a role in aggregate formation. 1Morley JF, Brignull HR, Weyers JJ, Morimoto RI. Proc Natl Acad Sci U S A 2002 99(16):10417-22

Ellen Nollen et al. (2001) International C. elegans Meeting "BAG1, AN HSP70 CO-CHAPERONE INVOLVED IN CELL GROWTH AND CELL STRESS"

Ellen Nollen, Richard I Morimoto, Frederic Gabriel, Kyle Duke, Jim Morley, Stuart Kim, Sanjeev Satyal

[International C. elegans Meeting, 2001]

Bag1 is a multifunctional protein that interacts with a variety of partner proteins. In mammalian cells, overexpression of Bag1 influences different signal transduction pathways, which in general leads to prevention of apoptosis or inhibition of cell cycle arrest. Interaction partners of Bag1 include Bcl-2, Raf-1 kinase, hormone receptors, and Hsp70. Bag1 enhances the anti-apoptotic function of Bcl-2, stimulates of the Raf-1 kinase activity, and inhibits Hsp70 chaperone activities. We are using C. elegans as a model to investigate the function of Bag1 during development and survival of a complex multicellular organism. We have created and analyzed a deletion mutant of Bag1 (F57B10.11). Null mutants appear similar to the wild type N2 strain with respect to viability, anatomy, timing of development, and life span. This indicates that Bag1 is not an essential gene and, moreover that Bag1 is not essential for Hsp70 activity under physiological conditions. Interestingly, the Bag1 null hermaphrodites show a 20% increase in the number of progeny compared to wild type N2 worms. Given the effect of overexpression of Bag1 on signal transduction pathways in mammalian cells, often reflected in changes in transcriptional activities, we asked whether phenotypic changes in C.elegans caused by deletion of Bag1 could be explained by changes in the mRNA expression profile. We are currently analyzing the mRNA expression profile of adult Bag1 null worms, compared to adult wild type N2 worms, using full genome microarrays. We will report on our progress on the microarray analysis and on the characterization of the phenotype of the Bag1 deletion strain.

Koopman, Mandy et al. (2021) International Worm Meeting "Optogenetic manipulation of individual or whole population Caenorhabditis elegans worms with an under hundred-dollar tool: the OptoArm"

Koopman, Mandy, Janssen, Leen, Nollen, Ellen

[International Worm Meeting, 2021]

Optogenetic tools have revolutionized the study of neuronal circuits in Caenorhabditis elegans. The expression of light-sensitive ion channels or pumps under specific promotors allows researchers to modify the behavior of excitable cells. Several optogenetic systems have been developed to spatially and temporally photoactivate light-sensitive actuators in C. elegans. Nevertheless, their high costs and low flexibility have limited access to optogenetics for a broad public. Here, we developed an inexpensive, easy-to-build and highly adjustable optogenetics device for use with different microscopes and wormtrackers, called the OptoArm. The OptoArm allows for single-and multiple-worm illumination during imaging and is adaptable in terms of light intensity, lighting profiles and light-color. We demonstrate the system's versatility by performing multi-parameter behavioral analysis upon cholinergic and GABAergic stimulation. Furthermore, we leveraged the OptoArm's power in a population-based study to dissect the contributions of motor circuit cells to age-related motility decline. We discovered that the functional decline of cholinergic neurons corresponds with motor decline, while GABAergic neurons and muscle cells appear relatively age-resilient. This would suggest a rate-limiting, cell type-specific vulnerability to ageing, which may underlie neuronal circuit aging.

Gungordu, Lale et al. (2021) International Worm Meeting "Modifiers of TDP-43 Toxicity"

Nollen, Ellen, Gungordu, Lale, Koopman, Mandy, Seinstra, Renee

[International Worm Meeting, 2021]

Protein toxicity is thought to underlie several, yet incurable, age-related neurodegenerative diseases, including Parkinson's disease and Amyotrophic Lateral Sclerosis (ALS). TDP-43 aggregation is the major pathological hallmark of ALS and present in 97% of all cases, suggesting that TDP-43 contributes to in the disease mechanism. How protein toxicity triggers cell-and physical dysfunction and leads to degeneration is still not understood. This project aims to find disease mechanisms and uncover targets to suppress ALS-related TDP-43 toxicity. For this aim, a combination of genetic- and phenotypic screens in a Caenorhabditis elegans model for disease are being used. We make use of a C.elegans strain with overexpressed human TDP-43, which shows several cellular- and behavioral ALS disease phenotypes, including age-related motor impairment is used as a model. We performed a genetic screen, which identified 22 mutant animals that show a suppression of this impairment. The strongest suppressor mutant, called MOTT-22 (Modifier of TDP-43 Toxicity 22), was selected for further experiments. We are currently verifying and characterizing a candidate gene that may be responsible for the suppression of motor impairment in MOTT-22. After finding a candidate gene for MOTT-22, gene functions in the cell will be studied to find new mechanisms involved in protein toxicity.

van der Goot, Annemieke T et al. (2009) International Worm Meeting "Elucidating aggregation pathways in a C. elegans model for a-synuclein inclusion formation."

Dobson, Christopher M, van der Goot, Annemieke T, Kumita, Janet, Nollen, Ellen A A, Bertoncini, Carlos W, van Ham, Tjakko J

[International Worm Meeting, 2009]

Fibrillar protein aggregates of misfolded proteins are the pathological hallmark of many ageing-associated neurodegenerative diseases, including Parkinson''s disease (PD). The main component of these inclusions is fibrillar alpha-synuclein (AS). How these inclusions are formed and how this links to disease is poorly understood. Although these inclusions were initially thought to be pathogenic, more recent evidence suggests that they have a protective role and that it is the microscopically invisible precursors to these aggregates that are toxic to cells. We have previously established a C. elegans model expressing an AS-YFP fusion protein in body wall muscle cells, which allows us to monitor AS accumulation in living and ageing animals. By fluorescence recovery after photobleaching (FRAP) we have shown that, at old age, these inclusions contain immobile AS, resembling a key pathological feature in PD patients. By performing a genome-wide RNAi screen, we identified 80 genetic modifiers whose knockdown resulted in a premature increase in the number of microscopically visible inclusions (van Ham et al. 2008). We currently attempt to elucidate the effects that these genetic modifiers have on steps preceding inclusion formation. Studying the intermediates of aggregation in vivo has proven difficult due to the lack of methods to detect their presence and due to their transient and heterogeneous nature. Here, we show by several biophysical methods, including standard aggregation assays and transmission electron microscopy, that the AS-YFP fusion protein, in vitro, has similar amyloid forming properties as non-fused wild-type AS. We will report on our progress in elucidating AS aggregation pathways.

Sin, Olga et al. (2015) International Worm Meeting "Identification of MOAG-2/LIR-3 as a regulator of protein aggregation."

Guryev, Victor, Reinke, Valerie, Sin, Olga, Willinge Prins, Romeo, Wang, Hai Hui, Martineau, Celine, Mata Cabana, Alejandro, Seinstra, Renee, Nollen, Ellen, Kudron, Michelle, de Jong, Tristan

[International Worm Meeting, 2015]

Aging-related protein aggregation is one of the hallmarks of neurodegenerative disorders such as Alzheimer, Parkinson and polyglutamine diseases. The cellular processes that drive protein aggregation in these diseases have remained largely unknown. Using a genetic screen in a C. elegans model for polyglutamine aggregation, we here identified modifier of aggregation 2 (moag-2). Mutation or partial deletion in this gene decreased the number of aggregates in our model. Additionally, moag-2/lir-3 mutants reduce the amount of SDS-resistant aggregates without changing total polyglutamine expression levels. We discovered that the causative gene of moag-2 is lir-3. moag-2/lir-3 encodes a protein with a predicted nuclear localization signal and two non-canonical C2H2 domains, which are homologous to the transcription factor for RNA polymerase III. The molecular function of MOAG-2/LIR-3 is unknown. Chromatin immunoprecipitation followed by sequencing data revealed that MOAG-2/LIR-3 is preferentially bound to promoter regions of non-coding RNA, namely tRNAs and snoRNAs. The consensus DNA sequence to which MOAG-2/LIR-3 is bound to corresponds to Box A and Box B motifs, which are recognized by the RNA Polymerase III machinery to initiate tRNA and snoRNA transcription. In fact, MOAG-2/LIR-3 is positioned in the same binding positions as the RNA Pol III complex, further suggesting a role for LIR-3 in non-coding RNA transcription. We are currently performing total RNA sequencing to reveal whether the aggregation phenotype displayed by the MOAG-2/LIR-3 mutants can be due to imbalanced RNA metabolism. By unraveling the role of moag-2/lir-3 we hope to get insight into how cells cope with aggregation-prone proteins.

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.

Perni, Michele et al. (2017) International Worm Meeting "A natural product inhibits the initiation of alpha-synuclein aggregation by displacing it from lipid membranes and suppresses its toxicity in cells and in C. elegans."

Nollen, Ellen AA, Cascella, Roberta, Bax, Ad, Cremades, Nunilo, Challa, Pavan K, Zasloff, Michael, Sormanni, Pietro, Perni, Michele, Dobson, Christopher, Knowles, Tuomas PJ, Kirkegaard, Julius B, Muller, Martin BD, Chiti, Fabrizio, Galvagnion, Celine, Vendruscolo, Michele, Limbocker's, Ryan, Maltsev, Alexander S

[International Worm Meeting, 2017]

The self-assembly of a-synuclein is closely associated with Parkinson's disease (PD) and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, isolated originally from the liver of the dogfish shark by Dr Michael Zasloff in 1993, dramatically affects a-synuclein aggregation in vitro and in vivo, both in neuronal cells and in worms (Perni et al PNAS, 2017). We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation and find that this compound displaces a-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of a-synuclein oligomers in human neuroblastoma cells by preventing their interactions with lipid membranes. We further examine the effects of squalamine in a C. elegans strain overexpressing a-synuclein, observing a dramatic reduction of a-synuclein aggregation and an almost complete elimination of muscle paralysis. In order to reduce the human bias of those experiments we also developed novel an automated high-throughput worm-screening procedure, which improved notably the throughput and sensitivity of currently avaialble methods for C. elegans drug screenings (Perni, Challa, Kirkegaard et al, Nat Comm, Under review). These findings suggest that squalamine and related compounds could be a means of therapeutic intervention in PD and other a-synucleionpathies, and indeed this possiblity is being explored by a recently-founded company.