Sloan, Dillon E. et al. (2020) MicroPubl Biol

Bembenek, Joshua N., Sloan, Dillon E.

[MicroPubl Biol, 2020]

Caveolins are integral membrane proteins responsible for the formation of caveolae, invaginations of the plasma membrane linked to various disease states (Parton et al. 2020). In C. elegans, there are two caveolin proteins, CAV-1 and CAV-2. The cav-1 gene shares homology with all three mammalian caveolin genes (Tang et al. 1997). C. elegans CAV-1 protein does not appear to form caveolae, but a double-knockout mutant of cav-1 and cav-2 affects egg laying, and knockdown of cav-1 affects locomotion in a dynamin mutant background (Parker et al. 2007, Kirkham et al. 2008, Sato et al. 2008). Based on exogenous expression, CAV-1::GFP is known to localize to cortical granules and the plasma membrane in oocytes and the early embryo, to the plasma membrane in later embryos, as well as to the neuromuscular system in larvae and adult worms (Sato et al. 2006, Bembenek et al. 2007, Parker et al. 2007).

Cendrine Tourette et al. (2007) International Worm Meeting "Classification of oligonucleotide microarray data for mutant polyglutamine toxicity in cell-sorted neurons using gene networks C.elegans."

Cendrine Tourette, Christian Neri, Alex, J Parker, Anne Louise

[International Worm Meeting, 2007]

The limitations of cellular and rodent models preclude the detailed analysis of molecular and genetic factors involved in polyglutamine neuronal toxicity in vivo. Invertebrates provide convenient and robust experimental systems to study the regulators of polyglutamine neuronal toxicity in whole organisms and at the genomic level. We have previously described C.elegans models of the early phases of mutant polyglutamine (polyQ) cytotoxicity in neurons, namely the dysfunction of touch receptor neurons before cell death produced by the expression of N-ter huntingtin fused to a fluorescent protein under the control of the mec-3 promoter (Parker et al., PNAS 2001). We have illustrated the value of these animals to defining disease targets and active compounds for neuroprotection in Huntingtons disease (Parker et al., Nature Genet 2005). We analyzed the effects of mutant polyQ pathogenicity using FACS-purified neurons and oligonucleotide microarrays from Agilent. Data classification using C. elegans gene networks and the comparison of our data to microarray data from other models of mutant huntingtin neurotoxicity will be presented.

Rieckher M et al. (2017) Bio Protoc "P-body and Stress Granule Quantification in Caenorhabditis elegans."

Rieckher M, Tavernarakis N

[Bio Protoc, 2017]

Eukaryotic cells contain various types of cytoplasmic, non-membrane bound ribonucleoprotein (RNP) granules that consist of non-translating mRNAs and a versatile set of associated proteins. One prominent type of RNP granules are Processing bodies (P bodies), which majorly harbors translationally inactive mRNAs and an array of proteins mediating mRNA degradation, translational repression and cellular mRNA transport (Sheth and Parker, 2003). Another type of RNP granules, the stress granules (SGs), majorly contain mRNAs associated with translation initiation factors and are formed upon stress-induced translational stalling (Kedersha et al., 2000 and 1999). Multiple evidence obtained from studies in unicellular organisms supports a model in which P bodies and SGs physically interact during cellular stress to direct mRNAs for transport, decay, temporal storage or reentry into translation (Anderson and Kedersha, 2008; Decker and Parker, 2012). The quantification, distribution and colocalization of P bodies and/or SGs are essential tools to study the composition of RNP granules and their contribution to fundamental cellular processes, such as stress response and translational regulation. In this protocol we describe a method to quantify P bodies and SGs in somatic tissues of the nematode Caenorhabditis elegans.

Alex Parker et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Studying the modifier genes of neuronal cell response to mutant huntingtin expression in C. elegans"

Christian Neri, Alex Parker, Maryline Mage, Emmanuel Lambert, Sabine Cloax, Cendrine Tourette, Salima Abderrahmane, Beate Gertsbrein

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Huntington"s disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in huntingtin. Although HD is inherited, its age of onset shows great variability, suggesting neuronal dysfunction and death in HD is influenced by modifier genes. To study how the neuronal cell may respond to mutant htt, we have developed C. elegans transgenics that overexpress mutant N-terminal htt fused to fluorescent proteins in touch receptor neurons. This model shows neuronal dysfunction without cell death, an effect accompanied by aggregate formation and dystrophy of neuronal processes (Parker et al., PNAS 2001). To study the pathways underlying these transgenic phenotypes, we use a combination of C. elegans mutants, RNAi screens, microarray analysis, and physiological analysis. This effort has led us to identify sirtuin activation as a neuroprotective mechanism against the effects of mutant htt expression, an effect mediated by daf-16/FOXO, (Parker et al., Nature Genetics 2005). Our findings indicated that modulators of organismal longevity like sir2 and FOXO may modify early-stage pathogenesis of neurodegenerative diseases, defining a new strategy with therapeutic potential. We will present an update on the progress made with our models in better understanding how mutant htt may be toxic to the neuronal cell and how neuroprotection may be achieved.

Vazquez-manrique, Rafael et al. (2011) International Worm Meeting "AMPK activation is protective in models of neuron dysfunction and death in Huntington's disease."

Deglon, Nicole, Neri, Christian, Offner, Nicolas, Vazquez-manrique, Rafael, Cambon, Karine, Orfila, Anne-marie, Darbois, Aurelie

[International Worm Meeting, 2011]

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the huntingtin protein (Htt). Expression of the first exon of Htt containing an expanded polyQ produces neuronal dysfunction and axonal dystrophy in mechanosensory neurons in C. elegans (Parker et al., 2001). Genetic pathway analysis using this nematode model has emphasized a role for the longevity-promoting factor daf-16/FoxO in the protection of neurons from expanded polyQ toxicity (Parker et al., 2005). Here, we investigated the role of AMP-activated protein kinase (AMPK), a well-known energy sensor involved in lifespan and health span extension, on neuronal dysfunction in expanded-polyQ nematodes and vulnerability to cell death in mutant Htt striatal cells (HdhQ111 knock-in mice). In C. elegans, activating this enzyme (aak-2/AMPK) with metformin reduces the neuronal dysfunction caused by expanded polyQ expression. In contrast, aak-2 mutants show enhanced neuronal impairment. In striatal cells from HdhQ111 knock-in mice, reducing AMPK levels by siRNA treatment enhances the susceptibility to cell death of mutant htt cells, and metformin treatment has the opposite effect. Additionally, overexpressing AMPK reduced striatal neurodegeneration in a rat lentiviral-based fragment model of HD pathogenesis. Collectively, our results suggest that AMPK activation has therapeutic potential to protect from neuron dysfunction and degeneration in HD.

Nichols, Courtney Rose et al. (2013) International Worm Meeting "A role for the insulin signaling pathway in development of neuronal aging markers in a polyglutamine protein aggregation C. elegans model."

Parker, J. Alex, Nichols, Courtney Rose, Driscoll, Monica, Neri, Christian, Vayndorf, Elena, Taylor, Barbara

[International Worm Meeting, 2013]

Neurodegenerative diseases, such as Huntington's, Alzheimer's, and Parkinson's disease, result in the progressive loss of neuronal structure and function with age. Many neurodegenerative disorders are caused by genetic mutations and characterized by toxic aggregation of proteins within neurons. We explored the mechanism of accelerated neuronal aging in a polyglutamine (polyQ) protein aggregation model through genetic manipulation. Caenorhabditis elegans expressing the first 57 amino acids of human huntingtin protein with a toxic polyglutamine chain (polyQ128) fluorescently labeled with CFP in YFP-labeled mechanosensory neurons (Parker et al, 2001) were used to monitor neuronal aging phenotypes. We found an age-associated increase in aberrant neuronal morphology, protein aggregation, and functional impairment of the mechanosensory neurons expressing toxic 128 polyQ. We also tested the hypothesis that the insulin signaling pathway is involved in morphological and functional health of aging mechanosensory neurons using neuron-specific RNA interference (RNAi). Furthermore, we measured levels of endogenous oxidative stress and lifespan of aging animals that contain toxic polyQ128 repeats following RNAi manipulation of the insulin signaling pathway. Overall, we found that DAF-16/FOXO is neuroprotective in this accelerated aging model, which corroborates previous findings on the role of DAF-16/FOXO in polyQ128 young adults (Parker et al. 2005). To further characterize overall muscle and neuronal health, we measured action potentials of pharyngeal muscle contractions and neurons that innervate the pharynx using microfluidic electropharyngeography (EPG). In total, our observations support that insulin signaling via DAF-16/FOXO is a mechanism through which accelerated aging and neurodegeneration occurs in this model.

Mills, Joslyn et al. (2021) International Worm Meeting "Lipid droplets modulate lifespan and selective autophagy receptor p62/SQST-1 dynamics"

Kumar, Anita, Leitao, Joshua, Wong, Shi Quan, Ng, Celeste, Parker, Wesley, Lapierre, Louis, Mills, Joslyn

[International Worm Meeting, 2021]

Organismal health and longevity depend largely on the maintenance of proteome stability, and age-associated proteostatic decline contributes to protein aggregation-induced pathologies found in many neurodegenerative diseases. Proteostasis is maintained via cellular mechanisms including the autophagy and the proteosome degradation machineries. A key regulator of the autophagic degradation of specific organelle and aggregating proteins that are prone to damage and ubiquitination is the selective autophagy receptor p62/SQST-1/SQSTM1. We tested whether increasing p62 levels (p62 OE) would improve proteostasis and consequently increase the lifespan of C. elegans. Unexpectedly, we found that simply overexpressing p62 is not sufficient to extend lifespan in C. elegans at the standard growth temperature of 20C. Instead, we found that p62 OE is detrimental to the lifespan of wild-type (WT) animals under modest heat stress at 25C, which, similar to aging, caused abnormal accumulation of p62 aggregates. To systematically identify modulators of p62 levels under these conditions, we employed p62 OE strains with fluorescent reporters in an unbiased genome-wide RNAi screen and identified several p62 modulators that coded for proteins associated with lipid droplets and those prone to aggregate with age. A role for lipid droplets (LDs) in lifespan extension has emerged, as several long-lived C. elegans strains, including Insulin/IGF-1 receptor daf-2 mutants, maintain elevated intestinal lipid stores throughout life, but a mechanistic understanding of the LD role in longevity is still lacking. We demonstrate that the detrimental effects of unprocessed p62 is rescued in daf-2 mutants, underscoring the need for elevated lipid storage for proper p62 dynamics. To further support this point, we show that expansion of the intestinal LDs by silencing the cytosolic triacylglycerol lipase gene atgl-1/ATGL was sufficient to extend lifespan in WT animals and in comparatively short-lived animals exhibiting impaired proteostasis or p62 accumulation, as well as mitigate the age-related p62 accumulation and reduced overall ubiquitination of proteins. Conversely, depleting LDs accelerated the age-dependent accumulation of p62 and decreased lifespan at 25C. Altogether, our study supports the notion that LDs serve as a buffer for proteostasis under proteostatic strain, which reduces protein ubiquitination and ultimately unburdens p62-selective autophagy and promotes longevity in C. elegans.

Tauffenberger A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Glucose rescues age-dependent proteotoxicity in C.elegans"

Vaccaro A, Parker A, Tauffenberger A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

As the population shifts to an older demographic, it has become important to find new therapeutic ways to combat neurological disease. To model neurodegeneration we use a C. elegans model for Huntington's Disease (HD). HD is a late-onset fatal neurodegenerative disease caused by an expanded CAG trinucleotide repeat that codes for glutamine (Q), and is characterized by the intracellular accumulation of mutant proteins. Our transgenic C. elegans strains expressing normal (19Q) and expanded mutant (128Q) polyQ in mechanosensory neurons show a progressive and polyQ size dependent loss-of-mechanosensation and increased-aggregation phenotypes in vivo1. The difference between mutant (128Q) and controls (19Q) is easily distinguished and the effects of genetic and pharmacological modifiers can be rapidly derived from small populations of animals2. Disrupted metabolism is one of a number of mechanisms that may contribute to neurodegeneration. Indeed, conditions that reprogram metabolism like dietary restriction are active areas of investigation in efforts to identify new therapeutic targets. We have observed that opposite to the effects on organismal lifespan, excess glucose suppresses toxicity in worm models of proteotoxicity. Furthermore, many of the genes essential for glucose-mediated suppression of proteotoxicity are in common with genes required for dietary restriction. These data demonstrate that glucose enrichment and dietary restriction utilize many of the same genetic pathways with opposite metabolic endpoints both of which are beneficial in models of neurodegeneration. A summary of our recent data will be presented. 1.Parker, J.A. et al. Expanded polyglutamines in Caenorhabditis elegans cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death. Proc Natl Acad Sci U S A 98, 13318-23 (2001). 2.Parker, J.A. et al. Resveratrol rescues mutant polyglutamine cytotoxicity in nematode and mammalian neurons. Nat Genet 37, 349-50 (2005).

Nicholas M Johnson et al. (2003) International Worm Meeting "Wormgate: a high-throughput cloning system for heritable and inducible RNA interference in C. elegans"

Stephen C Trowell, Nicholas M Johnson, Carolyn A Behm

[International Worm Meeting, 2003]

RNA interference is being used to investigate gene function particularly in those animals with sequenced genomes such as C. elegans. RNAi is performed widely in C. elegans by administering exogenous dsRNA via microinjection, feeding or soaking. It has however been shown that neurally expressed genes are somewhat resistant to the effects of this exogenous dsRNA (Kamath et al 2003, Nature 421: 231-237). One approach for overcoming this problem is to transform nematodes with a transgene expressing dsRNA as a hairpin (hpRNAi) construct. While this technique has advantages over dsRNA soaking and feeding, it is far more labour intensive. We have therefore modified the high throughput hpRNAi cloning system generated for plants (Wesley et al 2001, The Plant Journal 27: 581-590), for use in C. elegans. This system, called Wormgate, produces hpRNAi constructs via Gateway (Invitrogen) recombination reactions. Wormgate allows efficient single-step production of hpRNAi constructs for any C. elegans gene especially used in conjunction with the C. elegans ORFeome project. Transformation of nematodes with these hpRNAi transgenes has been facilitated with a recently described particle bombardment system (Praitis et al 2001, Genetics 157: 1217-1226). The synergistic use of these three recently developed technologies (Wormgate, ORFeome and Biolistics) has provided a powerful platform for investigating C. elegans gene function through inducible and heritable hpRNAi, particularly for neurally expressed genes. Silencing of genes of various functions including unc-22, pos-1, deg-3 and gfp will be reported.

Arzu Atalay et al. (2005) International Worm Meeting "Functional analysis of CGH-1-associated proteins in C.elegans"

T Keith Blackwell, Arzu Atalay, Peter Boag, Rosa E Navarro, Jiayun Lu

[International Worm Meeting, 2005]

Two conserved aspects of oogenesis are storage of translationally repressed mRNA and a high frequency of apoptosis. In C.elegans approximately half of the developing oocytes die through a germline-specific physiological apoptosis pathway (1). Previous studies in our laboratory reported that cgh-1 (Conserved Germline Helicase-1) is required for gametogenesis and protection from physiological germline apoptosis in C.elegans (2). cgh-1 RNAi leads to markedly increased physiological germline cell death and sterility. CGH-1 is specifically expressed in the germline and early embryo. It associates with P granules, and is present in additional predicted RNA storage particles. In Drosophila and Xenopus CGH-1 orthologs are involved in translational control of oocyte mRNA. In yeast, the CGH-1 ortholog Dhh1p is required for decapping- and deadenylation-mediated RNA degradation, which occurs in discrete cytoplasmic foci called P (processing) bodies. Yeast Dhh1p is concentrated in P bodies and is critical for their decapping and subsequent degradation functions (3). Biochemical studies in yeast and two-hybrid screens in yeast and C.elegans have identified numerous other proteins including P-body associated proteins- that interact with CGH-1 either directly or indirectly. We are investigating whether RNAi knockdown of these genes influences the frequency of physiological apoptosis, or affects other phenotypes associated with CGH-1. The vast majority of these genes do not affect the frequency of germ cell death, supporting the idea that this process responds to specific cues during oogenesis. We are particularly interested in studying how CGH-1 interacts functionally with C.elegans counterparts of other proteins that are present in P bodies in yeast or other organisms. For example, we are investigating how the decapping/degradation machinery influences the localization and levels of CGH-1 RNA-protein particles in the embryo. Since there are several parallels between deadenylation-dependent mRNA degradation pathways and regulation of maternal RNA storage events, we are also investigating whether CGH-1 functions in the regulation of specific maternal mRNAs. (1)Gumienny et al. (1999) Development 126: 1011-22 (2)Navarro et al. (2001) Development 128: 3221-32 (3)Sheth and Parker (2003) Science 300: 805-8