Caloric Restriction and Autophagy in Caenorhabditis elegans

Autophagy is a catabolic process in which long-lived proteins and organelles are degraded for recycling in the cytoplasm. In the nematode Caenorhabditis elegans autophagy is associated with formation of the dauer larva, an alternative developmental stage that worms can enter under poor growth conditions. We have shown that C. elegans mutants that experience caloric restriction because they are feeding-defective also exhibit elevated autophagy and decreased levels of fat deposits, as well as smaller cells and, consequently, a smaller body size. Our results suggest novel relationships between caloric restriction, longevity, body size and autophagy.

Addendum to:

C. elegans Feeding Defective Mutants Have Shorter Body Lengths and Increased Autophagy

C. Mörck and M. Pilon

BMC Dev Biol 2006; 6:39     

Proteins interacting with Caenorhabditis elegans Galpha subunits

To identify novel components in heterotrimeric G-protein signalling, we performed an extensive screen for proteins interacting with Caenorhabditis elegans Galpha subunits. The genome of C. elegans contains homologues of each of the four mammalian classes of Galpha subunits (Gs, Gi/o, Gq and G12), and 17 other Galpha subunits. We tested 19 of the GGalpha subunits and four constitutively activated Galpha subunits in a largescale yeast two-hybrid experiment. This resulted in the identification of 24 clones, representing 11 different proteins that interact with four different Galpha subunits. This set includes C. elegans orthologues of known interactors of Galpha subunits, such as AGS3 (LGN/PINS), CalNuc and Rap1Gap, but also novel proteins, including two members of the nuclear receptor super family and a homologue of human haspin (germ cell-specific kinase). All interactions were found to be unique for a specific Galpha subunit but variable for the activation status of the Galpha subunit. We used expression pattern and RNA interference analysis of the G-protein interactors in an attempt to substantiate the biological relevance of the observed interactions. Furthermore, by means of a membrane recruitment assay, we found evidence that GPA-7 and the nuclear receptor NHR-22 can interact in the animal.     

Genes regulating touch cell development in Caenorhabditis elegans

To identify genes regulating the development of the six touch receptor neurons, we screened the F(2) progeny of mutated animals expressing an integrated mec-2::gfp transgene that is expressed mainly in these touch cells. From 2638 mutated haploid genomes, we obtained 11 mutations representing 11 genes that affected the production, migration, or outgrowth of the touch cells. Eight of these mutations were in known genes, and 2 defined new genes (mig-21 and vab-15). The mig-21 mutation is the first known to affect the asymmetry of the migrations of Q neuroblasts, the cells that give rise to two of the six touch cells. vab-15 is a msh-like homeobox gene that appears to be needed for the proper production of touch cell precursors, since vab-15 animals lacked the four more posterior touch cells. The remaining touch cells (the ALM cells) were present but mispositioned. A similar touch cell phenotype is produced by mutations in lin-32. A more severe phenotype; i.e., animals often lacked ALM cells, was seen in lin-32 vab-15 double mutants, suggesting that these genes acted redundantly in ALM differentiation. In addition to the touch cell abnormalities, vab-15 animals variably exhibit embryonic or larval lethality, cell degenerations, malformation of the posterior body, uncoordinated movement, and defective egg laying.     

Autophagy activity contributes to programmed cell death in Caenorhabditis elegans

The physiological relationship between autophagy and programmed cell death during C. elegans development is poorly understood. In C. elegans, 131 somatic cells and a large number of germline cells undergo programmed cell death. Autophagy genes function in the removal of somatic cell corpses during embryogenesis. Here we demonstrated that autophagy activity participates in germ-cell death induced by genotoxic stress. Upon γ ray treatment, fewer germline cells execute the death program in autophagy mutants. Autophagy also contributes to physiological germ-cell death and post-embryonic cell death in ventral cord neurons when ced-3 caspase activity is partially compromised. Our study reveals that autophagy activity contributes to programmed cell death during C. elegans development.     

Autophagy is required for necrotic cell death in Caenorhabditis elegans

Autophagy is the main process for bulk protein and organelle recycling in cells under extracellular or intracellular stress. Deregulation of autophagy has been associated with pathological conditions such as cancer, muscular disorders and neurodegeneration. Necrotic cell death underlies extensive neuronal loss in acute neurodegenerative episodes such as ischemic stroke. We find that excessive autophagosome formation is induced early during necrotic cell death in C. elegans. In addition, autophagy is required for necrotic cell death. Impairment of autophagy by genetic inactivation of autophagy genes or by pharmacological treatment suppresses necrosis. Autophagy synergizes with lysosomal catabolic mechanisms to facilitate cell death. Our findings demonstrate that autophagy contributes to cellular destruction during necrosis. Thus, interfering with the autophagic process may protect neurons against necrotic damage in humans.     

Autophagy genes protect against disease caused by polyglutamine expansion proteins in Caenorhabditis elegans

Expanded polyglutamine (polyQ) proteins aggregate intracellularly in Huntington's disease and other neurodegenerative disorders. The lysosomal degradation pathway, autophagy, is known to promote clearance of polyQ protein aggregates in cultured cells. Moreover, basal autophagy in neuronal cells in mice prevents neurodegeneration by suppressing the accumulation of abnormal intracellular proteins. However, it is not yet known whether autophagy genes play a role in vivo in protecting against disease caused by mutant aggregate-prone, expanded polyQ proteins. To examine this question, we used two models of polyQ-induced toxicity in C. elegans, including the expression of polyQ40 aggregates in muscle and the expression of a human huntingtin disease fragment containing a polyQ tract of 150 residues (Htn-Q150) in ASH sensory neurons. Here, we show that genetic inactivation of autophagy genes accelerates the accumulation of polyQ40 aggregates in C. elegans muscle cells and exacerbates polyQ40-induced muscle dysfunction. Autophagy gene inactivation also increases the accumulation of Htn-Q150 aggregates in C. elegans ASH sensory neurons and results in enhanced neurodegeneration. These data provide in vivo genetic evidence that autophagy genes suppress the accumulation of polyQ aggregates and protect cells from disease caused by polyQ toxicity.     

Genes required for systemic RNA interference in Caenorhabditis elegans

RNA interference (RNAi) in the nematode worm, Caenorhabditis elegans, occurs systemically. Double-stranded RNA (dsRNA) provided in the diet can be absorbed from the gut lumen and distributed throughout the body, triggering RNAi in tissues that are not exposed to the initial dsRNA trigger. This is in marked contrast to other animals, in which RNAi does not spread from targeted tissues to neighboring cells. Here, we report the characterization of mutants defective in the systemic aspect of RNAi, but not in the core RNAi process itself. Analysis of these mutants suggests that dsRNA uptake is a specific process involving several unique proteins.     

Genes implicated in Caenorhabditis elegans and human health regulate stress resistance and physical abilities in aged Caenorhabditis elegans

Recently, nine Caenorhabditis elegans genes, grouped into two pathways/clusters, were found to be implicated in healthspan in C. elegans and their homologues in humans, based on literature curation, WormBase data mining and bioinformatics analyses. Here, we further validated these genes experimentally in C. elegans. We downregulated the nine genes via RNA interference (RNAi), and their effects on physical function (locomotion in a swim assay) and on physiological function (survival after heat stress) were analysed in aged nematodes. Swim performance was negatively affected by the downregulation of acox-1.1, pept-1, pak-2, gsk-3 and C25G6.3 in worms with advanced age (twelfth day of adulthood) and heat stress resistance was decreased by RNAi targeting of acox-1.1, daf-22, cat-4, pig-1, pak-2, gsk-3 and C25G6.3 in moderately (seventh day of adulthood) or advanced aged nematodes. Only one gene, sad-1, could not be linked to a health-related function in C. elegans with the bioassays we selected. Thus, most of the healthspan genes could be re-confirmed by health measurements in old worms.     

Small Collagenous Proteins Present during the Molt in Caenorhabditis elegans

Immunoblotting experiments using antibodies directed against the large collagenous cuticle proteins of Caenorhabditis elegans revealed a class of small collagenous proteins (CP) of apparent molecular weight 38,000-52,000 present during the L4 to adult molt. These CP are smaller than most vertebrate collagens characterized to date and share many characteristics with the small collagenous products translated in vitro from RNA isolated at this molt. C. elegans collagen genes, collagen-coding mRNA, and collagenous in vitro products that have been characterized are also small. Detection of small CP in vivo in C. elegans thus lends further support to the hypothesis that such small collagenous proteins are the primary gene product precursors to the larger collagenous proteins isolated from the C. elegans cuticle.     

On the Paucity of Duplicated Genes in Caenorhabditis elegans Operons

Spliced leader trans-splicing is an mRNA maturation process used by a small set of eukaryotes, including the nematode C. elegans, to cap the downstream genes of operons. We analyzed the frequency of duplication of operonic genes in C. elegans and confirmed that they are duplicated less often in the genome than monocistronic genes. Because operons account for about 15% of the genes in C. elegans, this lower duplication frequency might place a large constraint on the plasticity of the genome. Further analyses suggest that this paucity of duplicated genes results from operon organization hindering specific types of gene duplication. [Reviewing Editor: Dr. Yves van de Peer]     

Genes required for osmoregulation and apical secretion in Caenorhabditis elegans

Few studies have investigated whether or not there is an interdependence between osmoregulation and vesicular trafficking. We previously showed that in Caenorhabditis elegans che-14 mutations affect osmoregulation, cuticle secretion, and sensory organ development. We report the identification of seven lethal mutations displaying che-14-like phenotypes, which define four new genes, rdy-1-rdy-4 (rod-like larval lethality and dye-filling defective). rdy-1, rdy-2, and rdy-4 mutations affect excretory canal function and cuticle formation. Moreover, rdy-1 and rdy-2 mutations reduce the amount of matrix material normally secreted by sheath cells in the amphid channel. In contrast, rdy-3 mutants have short cystic excretory canals, suggesting that it acts in a different process. rdy-1 encodes the vacuolar H+-ATPase a-subunit VHA-5, whereas rdy-2 encodes a new tetraspan protein. We suggest that RDY-1/VHA-5 acts upstream of RDY-2 and CHE-14 in some tissues, since it is required for their delivery to the epidermal, but not the amphid sheath, apical plasma membrane. Hence, the RDY-1/VHA-5 trafficking function appears essential in some cells and its proton pump function essential in others. Finally, we show that RDY-1/VHA-5 distribution changes prior to molting in parallel with that of actin microfilaments and propose a model for molting whereby actin provides a spatial cue for secretion.     

Targeted Mutagenesis of Duplicated Genes in Caenorhabditis elegans Using CRISPR-Cas9

正CRISPR(clustered regularly interspaced short palindromic repeats)-Cas9-based genome editing has revolutionized functional genomics in many biological research fields.The specificity and potency of CRISPR-Cas9 genome editing make it ideal for investigating the function of genes in vivo(Hsu     

Genes required for the common miracle of fertilization in Caenorhabditis elegans

Fertilization involves multiple layers of sperm-egg interactions that lead to gamete fusion and egg activation. There must be specific molecules required for these interactions. The challenge is to determine the identity of the genes encoding these molecules and how their protein products function. The nematode worm Caenorhabditis elegans has emerged as an efficient model system for gene discovery and understanding the molecular mechanisms of fertilization. The primary advantage of the C. elegans system is the ability to isolate and maintain mutants that affect sperm or eggs and no other cells. In this review we describe progress and challenges in the analysis of genes required for gamete interactions and egg activation in the worm.     

Caenorhabditis elegans PAQR-2 and IGLR-2 Protect against Glucose Toxicity by Modulating Membrane Lipid Composition

In spite of the worldwide impact of diabetes on human health, the mechanisms behind glucose toxicity remain elusive. Here we show that C. elegans mutants lacking paqr-2, the worm homolog of the adiponectin receptors AdipoR1/2, or its newly identified functional partner iglr-2, are glucose intolerant and die in the presence of as little as 20 mM glucose. Using FRAP (Fluorescence Recovery After Photobleaching) on living worms, we found that cultivation in the presence of glucose causes a decrease in membrane fluidity in paqr-2 and iglr-2 mutants and that genetic suppressors of this sensitivity act to restore membrane fluidity by promoting fatty acid desaturation. The essential roles of paqr-2 and iglr-2 in the presence of glucose are completely independent from daf-2 and daf-16, the C. elegans homologs of the insulin receptor and its downstream target FoxO, respectively. Using bimolecular fluorescence complementation, we also show that PAQR-2 and IGLR-2 interact on plasma membranes and thus may act together as a fluidity sensor that controls membrane lipid composition.     

Autophagy and Insulin/TOR Signaling in Caenorhabditis elegans pcm-1 Protein Repair Mutants

Biological responses due to nutrient deprivation in the nematode Caenorhabditis elegans, including L1 diapause and autophagy during dauer formation, can be mediated through the linked DAF-2/insulin/IGF receptor and target-of-rapamycin (TOR) kinase pathways. Here we discuss how altered insulin/TOR signaling may underlie the previously reported phenotypes of worms with a null mutation in the pcm-1 gene that results in reduced autophagy during dauer formation and decreased L1 arrest survival. PCM-1 encodes a protein repair methyltransferase and mutants of the encoding pcm-1 gene are incapable of converting spontaneously damaged l-isoaspartyl residues in cellular proteins to normal forms by this pathway. We speculate that PCM-1 may function either directly or indirectly as an inhibitor of insulin/TOR signaling, perhaps in a role to balance autophagy with alternative protein degradation pathways that may be more specific for recognizing age-damaged proteins.

Addendum to:

The L-Isoaspartyl-O-Methyltransferase in Caenorhabditis elegans Larval Longevity and Autophagy

T.A. Gomez, K.L. Banfield, D.M. Trogler and S.G. Clarke

Developmental Biol 2007; 303:493-500