Arnold JM et al. (1995) International C. elegans Meeting "PES-2: A GENE EXPRESSED IN C.ELEGANS EMBRYOS."

Arnold JM, Hope IA

[International C. elegans Meeting, 1995]

Pes-2 (pattern expression site) was isolated during a promoter trap screen of gene expression patterns in C.elegans. Preliminary characterisation of the pes-2/lacZ pattern using immunofluorescence microscopy has revealed that expression is first detected at the 14 cell stage. From the 14 to the 28 cell stage, all cells except those of the germline and D and C cell lineages appear to show staining. The pattern becomes more restricted as development continues and by 260 minutes staining may be limited to hypodermal cells. Staining has not been observed in larvae or adults.

Arnold ML et al. (2018) BMC Biol "Q&A: Trash talk: disposal and remote degradation of neuronal garbage."

Arnold ML, Driscoll M, Smart AJ, Melentijevic I

[BMC Biol, 2018]

Caenorhabditis elegans neurons have recently been found to throw out cellular debris for remote degradation and/or storage, adding an "extracellular garbage elimination" option to known intracellular protein and organelle degradation pathways. This Q&A describes initial insights into the biology of seemingly selective protein and organelle elimination by challenged neurons, highlighting mysteries of how garbage is distinguished and sorted in the sending neuron, how the garbage-filled "exophers" appear to elicit degradative responses as they transit neighboring tissue, and how non-digestible materials get thrown out of cells again via processes that may be highly relevant to human neurodegenerative disease mechanisms.

Arnold, Meghan Lee et al. (2021) International Worm Meeting "An aggresome-like collection mechanism functions in neuronal expulsion of disease-aggregates"

Guasp, Ryan, Smart, Joelle, Grant, Barth, Melentijevic, Ilija, Hall, Dave, Cooper, Jason, Nyguen, Ken, Arnold, Meghan Lee, Driscoll, Monica, Ardeshna, Sohil

[International Worm Meeting, 2021]

The accumulation of aggregated proteins is associated with aging and neurodegeneration. Accumulation and subsequent spread of misfolded proteins causes toxicity that can induce loss of neurological function. Given that aggregate accumulation and spread is a prominent feature in neurodegenerative disease pathology and functional decline, a major goal in aging biology is thus to understand the mechanisms of how cells deal with protein aggregation, accumulation, and spread. In mammalian biology, cells can handle aggregated proteins to maintain proteostasis via numerous pathways -- central aggregate collection, degradation via the ubiquitin-proteasome system or autophagy-lysosome pathway. In addition, neurons can identify, collect, and eject aggregates in large membrane-bound packages, "exophers". Mammalian and fly neurons also throw out aggregated-trash, which contributes to aggregate spreading via an unknown mechanism and is thought to promote pathology in human neurodegenerative disease. While we have documented the dynamic aggregate movement from the soma into the exopher domain, followed by a dramatic budding of neuronal contents into the exopher as key hallmarks of exopher formation, we know little about the molecular requirements for these complex tasks. I will describe the novel aggresome-like organelle in C. elegans neurons, an organelle that hosts disease aggregates. We discovered that many proteins important for aggresome-formation are also important for trash-expulsion via the exopher mechanism. Together, both aggresome-formation and exophergenesis-mechanisms represent intriguing molecular targets for improper aggregate handling and aggregate spread as seen in neurodegenerative disease pathology.

Arnold, Meghan et al. (2019) International Worm Meeting "How do cells throw out the trash? Classical and unexpected cytoskeletal contributions to neuronal garbage ejection."

Nguyen, K., Ardeshna , S., Melentijevic, I., Grant , B., Driscoll , M., Cooper , J., Guasp , R., Hall , D., Arnold, Meghan

[International Worm Meeting, 2019]

Cellular increase of aggregated proteins is a hallmark feature of aging and neurodegeneration. Accumulation and spread of misfolded proteins causes toxicity that can induce loss of neurological function. Given that aggregate accumulation and spread is a prominent feature in human neurodegenerative disease pathology and functional decline, a major goal in aging biology is to understand the mechanisms of how cells deal with protein aggregation and accumulation, both as individual neurons and as communities of cells. In mammalian biology, cells attempt to handle aggregated proteins to maintain proteostasis via central aggregate collection, degradation via the ubiquitin-proteasome system, or autophagy-lysosome pathway. We discovered another mechanism of neurotoxic-trash handling- C. elegans adult neurons can identify, collect, and selectively eject toxic aggregates in large membrane-bound packages called "exophers" (Nature 2017). Extruded materials can be delivered directly to other tissues to handle (hypodermis in the case of touch-neuron-derived exophers). Mammalian cells and fly neurons also appear to throw out aggregate-trash for their neighbors to deal with. We speculate that the C. elegans exopher mechanism is analogous to the mysterious process by which neurodegenerative disease-associated aggregates spread to promote pathology in human neurodegenerative disease. If so, the mechanistic dissection of this process using powerful C. elegans genetics approaches can be of great value. We have documented the dynamic aggregate movement from the soma into the pre-exopher domain, followed by a dramatic budding out of large membrane-bound exophers which selectively include damaged organelles and toxic proteins, yet we know little about the molecular requirements or molecular machinery that execute these complex tasks - trash selection, collection, and physical ejection. We have used a combination of genetics, fluorescence microscopy, and electron microscopy to begin deciphering how exophers are made. I will present our current understanding of the cell biology of neuronal trash handling and removal via exopher ejection, focusing on the cell biology of cytoskeletal components such as actin, microtubules, and intermediate filaments. Particularly of interest are the roles of intermediate filament proteins as they 1) are generally implicated in neuronal health and disease and 2) have specific roles in mammalian aggresome/aggregate management. I show that IFDs have unique and distinct localization in the neuron and that intermediate filament proteins IFD-1 and IFD-2 are important for exopher-genesis in a cell autonomous manner. Work presented will include some of the first known steps of exopher-related cell biology mechanisms, informing on a newly discovered branch of proteostasis that works to preserve neuronal functionality into late age.

Arnold A et al. (2014) Nucleic Acids Res "Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes."

Boag PR, Lee MC, Gaidatzis D, Rahman MM, Katic I, Stetak A, Hess D, Muehlhaeusser S, Ciosk R, Arnold A, Wright JE, Scheckel C

[Nucleic Acids Res, 2014]

The cold shock domain is one of the most highly conserved motifs between bacteria and higher eukaryotes. Y-box-binding proteins represent a subfamily of cold shock domain proteins with pleiotropic functions, ranging from transcription in the nucleus to translation in the cytoplasm. These proteins have been investigated in all major model organisms except Caenorhabditis elegans. In this study, we set out to fill this gap and present a functional characterization of CEYs, the C. elegans Y-box-binding proteins. We find that, similar to other organisms, CEYs are essential for proper gametogenesis. However, we also report a novel function of these proteins in the formation of large polysomes in the soma. In the absence of the somatic CEYs, polysomes are dramatically reduced with a simultaneous increase in monosomes and disomes, which, unexpectedly, has no obvious impact on animal biology. Because transcripts that are enriched in polysomes in wild-type animals tend to be less abundant in the absence of CEYs, our findings suggest that large polysomes might depend on transcript stabilization mediated by CEY proteins.

Arnold ML et al. (2023) Nat Commun "Intermediate filaments associate with aggresome-like structures in proteostressed C. elegans neurons and influence large vesicle extrusions as exophers."

Nguyen KCQ, Androwski R, Grant BD, Melentijevic I, Bai G, Arnold ML, Ardeshna S, Driscoll M, Cooper J, Guasp RJ, Smart J, Hall DH

[Nat Commun, 2023]

Toxic protein aggregates can spread among neurons to promote human neurodegenerative disease pathology. We found that in C. elegans touch neurons intermediate filament proteins IFD-1 and IFD-2 associate with aggresome-like organelles and are required cell-autonomously for efficient production of neuronal exophers, giant vesicles that can carry aggregates away from the neuron of origin. The C. elegans aggresome-like organelles we identified are juxtanuclear, HttPolyQ aggregate-enriched, and dependent upon orthologs of mammalian aggresome adaptor proteins, dynein motors, and microtubule integrity for localized aggregate collection. These key hallmarks indicate that conserved mechanisms drive aggresome formation. Furthermore, we found that human neurofilament light chain (NFL) can substitute for C. elegans IFD-2 in promoting exopher extrusion. Taken together, our results suggest a conserved influence of intermediate filament association with aggresomes and neuronal extrusions that eject potentially toxic material. Our findings expand understanding of neuronal proteostasis and suggest implications for neurodegenerative disease progression.

Arnold MC et al. (2013) Arch Environ Contam Toxicol "Cerium oxide nanoparticles are more toxic than equimolar bulk cerium oxide in Caenorhabditis elegans."

Arnold MC, Di Giulio RT, Badireddy AR, Meyer JN, Wiesner MR

[Arch Environ Contam Toxicol, 2013]

Engineered cerium oxide nanoparticles (CeO2 NPs) are widely used in biomedical and engineering manufacturing industries. Previous research has shown the ability of CeO2 NPs to act as a redox catalyst, suggesting potential to both induce and alleviate oxidative stress in organisms. In this study, Caenorhabditis elegans and zebrafish (Danio rerio) were dosed with commercially available CeO2 NPs. Non-nano cerium oxide powder (CeO2) was used as a positive control for cerium toxicity. CeO2 NPs suspended in standard United States Environmental Protection Agency reconstituted moderately hard water, used to culture the C. elegans, quickly formed large polydisperse aggregates. Dosing solutions were renewed daily for 3 days. Exposure of wild-type nematodes resulted in dose-dependent growth inhibition detected for all 3 days (p < 0.0001). Non-nano CeO2 also caused significant growth inhibition (p < 0.0001), but the scale of inhibition was less at equivalent mass exposures compared with CeO2 NP exposure. Some metal and oxidative stress-sensitive mutant nematode strains showed mildly altered growth relative to the wild-type when dosed with 5 mg/L CeO2 NPs on days 2 and 3, thus providing weak evidence for a role for oxidative stress or metal sensitivity in CeO2 NP toxicity. Zebrafish microinjected with CeO2 NPs or CeO2 did not exhibit increased gross developmental defects compared with controls. Hyperspectral imaging showed that CeO2 NPs were ingested but not detectable inside the cells of C. elegans. Growth inhibition observed in C. elegans may be explained at least in part by a non-specific inhibition of feeding caused by CeO2 NPs aggregating around bacterial food and/or inside the gut tract.

Arnold ML et al. (2020) J Vis Exp "Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans."

Arnold ML, Driscoll M, Grant BD, Cooper J

[J Vis Exp, 2020]

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease. C. elegans adult neurons that express aggregating proteins can extrude large (~4 m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called "exophers" and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases. While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons. Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Aeschimann F et al. (2015) Methods "Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling."

Arnold A, Grosshans H, Aeschimann F, Xiong J, Dieterich C

[Methods, 2015]

Gene expression profiling provides a tool to analyze the internal states of cells or organisms, and their responses to perturbations. While global measurements of mRNA levels have thus been widely used for many years, it is only through the recent development of the ribosome profiling technique that an analogous examination of global mRNA translation programs has become possible. Ribosome profiling reveals which RNAs are being translated to what extent and where the translated open reading frames are located. In addition, different modes of translation regulation can be distinguished and characterized. Here, we present an optimized, step-by-step protocol for ribosome profiling. Although established in Caenorhabditis elegans, our protocol and optimization approaches should be equally usable for other model organisms or cell culture with little adaptation. Next to providing a protocol, we compare two different methods for isolation of single ribosomes and two different library preparations, and describe strategies to optimize the RNase digest and to reduce ribosomal RNA contamination in the libraries. Moreover, we discuss bioinformatic strategies to evaluate the quality of the data and explain how the data can be analyzed for different applications. In sum, this article seeks to facilitate the understanding, execution, and optimization of ribosome profiling experiments.

Hoareau M et al. (2023) Bioessays "DREAM a little dREAM of DRM: Model organisms and conservation of DREAM-like complexes: Model organisms uncover the mechanisms of DREAM-mediated transcription regulation."

Gaumer S, Hoareau M, Gué\;nal I, Rincheval-Arnold A

[Bioessays, 2023]

DREAM complexes are transcriptional regulators that control the expression of hundreds to thousands of target genes involved in the cell cycle, quiescence, differentiation, and apoptosis. These complexes contain many subunits that can vary according to the considered target genes. Depending on their composition and the nature of the partners they recruit, DREAM complexes control gene expression through diverse mechanisms, including chromatin remodeling, transcription cofactor and factor recruitment at various genomic binding sites. This complexity is particularly high in mammals. Since the discovery of the first dREAM complex (drosophila Rb, E2F, and Myb) in Drosophila melanogaster, model organisms such as Caenorhabditis elegans, and plants allowed a deeper understanding of the processes regulated by DREAM-like complexes. Here, we review the conservation of these complexes. We discuss the contribution of model organisms to the study of DREAM-mediated transcriptional regulatory mechanisms and their relevance in characterizing novel activities of DREAM complexes.