N Tavernarakis et al. (1999) International C. elegans Meeting "Knockout of Elongation Factor-2 Kinase Extends Lifespan in C. elegans"

AG Ryazanov, L Zhang, CE Mendola, M Driscoll, KS Pavur, Z Altun-Gultekin, W Wadsworth, N Tavernarakis

[International C. elegans Meeting, 1999]

Elongation factor-2 kinase (EF-2 kinase) is a ubiquitous protein kinase that regulates protein synthesis by phosphorylating and inactivating elongation factor-2 (EF-2), which catalyzes movement of the ribosome along mRNA during translation (Nature 334:170-173). EF-2 kinase belongs to an emerging new class of protein kinases called alpha-kinases that are structurally and evolutionarily unrelated to conventional eukaryotic protein kinases (Proc. Natl. Acad. Sci. 94: 4884-4889; Curr. Biol. 9: R43-R45). The physiological role of EF-2 phosphorylation remains a mystery. To investigate the role of EF-2 kinase, we isolated a TC1 insertion mutant for C. elegans EF-2 kinase. The TC1 insertion is in the middle of exon 2 which contains the EF-2 kinase catalytic domain. The TC1 insertion completely abolishes EF-2 kinase activity. Mutant worms lacking functional EF-2 kinase showed slightly decreased locomotion, pharyngeal pumping, defecation, and developmental rates. The mutant phenotypes were rescued by injection of the wild-type EF-2 kinase gene. Intriguingly, C. elegans eEF-2 kinase loss of function mutants live 25% longer than do wild type animals. These results provide the first direct evidence that regulation of protein synthesis and longevity may be intimately related.

Charmpilas, N. et al. (2019) International Worm Meeting "Mitochondrial maturation drives germline stem cell differentiation in C. elegans."

Tavernarakis, N., Charmpilas, N.

[International Worm Meeting, 2019]

The C. elegans germline recapitulates mammalian stem cell niches, and provides an effective platform for investigating key aspects of stem cell biology. However, the molecular and physiological requirements for germline stem cell homeostasis remain largely elusive. Here, we report that mitochondrial biogenesis and function are crucial for germline stem cell identity. We show that general transcription activity in germline mitochondria is highly compartmentalized, and determines mitochondrial maturation. RPOM-1, the mitochondrial RNA polymerase, is differentially expressed as germ nuclei progress from the distal to the proximal gonadal arm to form oocytes. Mitochondria undergo changes from globular to tubular morphology and become polarized, as they approach the proximal gonad arm. Concomitantly, ATP and ROS production increases sharply during maturation. Impaired mitochondrial bioenergetics causes gonad syncytium tumour formation by disrupting the balance between mitosis and differentiation to oocytes, which results in a marked reduction of fecundity. Consequently, compensatory apoptosis is induced in the germline. Sperm-derived signals promote mitochondrial maturation and proper germ cell differentiation via the MEK/ERK kinase pathway. Germ cell fate decisions are determined by a crosstalk between Insulin/IGF-1 and TGF-? signaling, mitochondria and protein synthesis. Our findings demonstrate that mitochondrial transcription activity determines a shift in mitochondrial bioenergetics, which in turn regulates germline stem cell survival and differentiation. Perturbation of mitochondrial transcription hinders germ cell differentiation and causes germline tumour development.

Petratou, D. et al. (2019) International Worm Meeting "The role of neuronal DEG/ΕNaC ion channel family members in organismal stress responses."

Petratou, D., Tavernarakis, N.

[International Worm Meeting, 2019]

Organisms receive and process external information to adapt their behavior to an ever-changing environment. The molecular mechanisms that underlie neuronal integration of sensory information towards motor adaptation are not fully understood. Dopamine signaling is involved in several forms of behavioral plasticity, in reward processing and in the control of motor output. In C. elegans, the functionality of dopamine pathway can be easily assessed by monitoring a specific locomotory response to environmental food availability cues, termed basal slowing. By implementing molecular genetic manipulation technics and behavioral assays we identified three degenerin ion channel proteins to participate in sensory integration through modulation of the dopaminergic pathway. Utilizing advanced imaging technics, we found that degenerins DEL-2, DEL-3 and DEL-4 are expressed in mechanosensory, chemosensory and motor neurons. These ion channel proteins modulate basal slowing response and respond to gustatory stimuli. They affect neurotransmission efficiency of dopaminergic and motor neurons, even though they do not adopt a synaptic localization pattern. Moreover, they act through the dopamine receptors, DOP-1, DOP-2 and DOP-3, mainly DOP-2. We also demonstrated that PKC-1, but not PKC-2, plays a crucial role downstream of these receptors. Furthermore, degenerin effects are largely influenced by stress conditions, such as heat and starvation. Notably, the stress response transcription factors DAF-16/FOXO and SKN-1/Nrf couple degenerin ion channel function to environmental conditions and behavioral output. Our results provide new insights into how sodium channels participate in the mechanism of neuronal integration.

Papandreou, M.E. et al. (2019) International Worm Meeting "The role of nuclear envelope components in the regulation of autophagy."

Tavernarakis, N., Papandreou, M.E.

[International Worm Meeting, 2019]

The crosstalk between autophagy and the nucleus in the context of physiology and pathology remains largely elusive. We investigated this potential association by dissecting the involvement of nuclear membrane components in the autophagic process. To this end, we examined the role of the highly conserved, C. elegans nuclear envelope anchorage protein 1 (ANC-1) and its mammalian orthologues Nesprin 1 and 2 in autophagy. These are large, multi-domain, outer nuclear membrane proteins that maintain nuclear integrity. Nesprin 1 and 2 isoforms are highly abundant in multiple tissues, including the brain, heart, liver and kidney. In humans, polymorphisms in Nesprin 1 and 2 have been implicated in neurodevelopmental disorders, such as learning disability, autism and ataxia, as well as, in muscular dystrophies, in cancer and ageing. Using both C. elegans and mouse models we uncovered a novel link between these nuclear envelope components and autophagic processes. We find that autophagy regulates Nesprin 1 and 2 protein levels under both basal conditions and nutrient stress. Conversely, both C. elegans ANC-1 and mouse Nesprins interact with, and regulate the abundance and localization of general and selective autophagic machinery components such as LGG-1/LC3B and SQST-1/p62. Notably, these nuclear envelope components act downstream of autophagy under nutrient stress to maintain nucleolar shape and integrity. Thus, cross-regulation between autophagy and ANC-1/Nesprin proteins promotes nuclear homeostasis and safeguards the nucleolus and nuclear proteins under stress and during ageing.

Palikaras, K. et al. (2017) International Worm Meeting "Mitophagy and neuronal homeostasis in C. elegans."

Tavernarakis, N., Palikaras, K.

[International Worm Meeting, 2017]

Mitochondria are essential for energy production and have vital roles in calcium signalling and storage, metabolite synthesis and apoptosis in eukaryotic cells. Neuronal cells depend, perhaps more than any other cell type, on proper mitochondrial function. Thus, maintenance of neuronal homeostasis necessitates a tight regulation of mitochondrial biogenesis, as well as, the elimination of damaged or superfluous mitochondria. Mitochondrial impairment has been implicated in several age-related neurodegenerative diseases. Mitophagy is a selective type of autophagy mediating elimination of damaged mitochondria, and the major degradation pathway, by which cells regulate mitochondrial number in response to metabolic state. However, little is known about the effects of mitophagy deficiency on neuronal physiology. To address this question, we developed two composite, in vivo imaging systems to monitor mitophagy in neurons. We find that neuronal mitophagy is induced in response to oxidative stress. Mitochondrial dysfunction leads to transport of axonal mitochondria towards the neuronal cell body, in a calcium- and an AMPK/AAK-2-dependent manner. Impairment of autophagy increases the number of mitochondria in neurons of age-matched nematodes, and abolishes mitochondrial axonal transport upon stress. Additionally, mitophagy deficiency results in enhanced cell death in C. elegans models of neurodegeneration. Our findings indicate that mitophagy contributes critically to the preservation of mitochondrial homeostasis and neuronal health.

Papandreou, M.E. et al. (2017) International Worm Meeting "DNA damage-induced autophagy and necrotic neurodegeneration during ageing in C. elegans."

Tavernarakis, N., Papandreou, M.E.

[International Worm Meeting, 2017]

Accumulation of DNA damage is a key determinant of ageing and has been implicated in neurodegeneration. Although it is well known that ultraviolet (UV) radiation induces apoptosis, the contribution of necrotic cell death to DNA damage-related pathology remains elusive. To address this question, we developed a nematode model for DNA damage-induced neurodegeneration by using UV-C irradiation to trigger DNA damage in C. elegans neurons. Initial observations using this model show a marked increase of cytoplasmic calcium concentration upon UV irradiation. To examine whether this acute cytoplasmic calcium elevation triggers necrosis in neurons, we exposed DNA repair-defective mutants to UV light. These mutant animals are hypersensitive to UV irradiation and exhibit widespread necrotic cell death in somatic tissues upon exposure, while neurons are particularly affected. Runaway autophagy has previously been implicated in necrotic neurodegeneration. In this context, we investigated the contribution of autophagy in DNA damage-induced cellular pathology and nuclear dynamics. Notably, we found that DNA damage induces autophagic flux and alters nuclear dynamics both in nematodes and mouse cells. We are currently dissecting the interplay between DNA damage-induced autophagy, nuclear membrane alterations and necrotic cell death, aiming to identify evolutionarily conserved molecular mechanisms interfacing these processes.

Charmpilas, N. et al. (2017) International Worm Meeting "The adiponectin receptor PAQR-1 couples ER stress resistance with lipid homeostasis during ageing in C. elegans."

Tavernarakis, N., Kyriakakis, E., Charmpilas, N.

[International Worm Meeting, 2017]

In addition to serving fat and energy storage functions, the adipose tissue is a highly active metabolic and endocrine organ which exerts its systemic effects through secretion of adipose derived hormones (adipokines). The adipokine adiponectin affects organismal homeostasis through binding to its cognate receptors. Impairment of this signaling axis has been associated with human disease, particularly with type II diabetes and obesity. Manipulation of hormonal signaling pathways may influence lifespan by regulating survival under stress conditions. We examined the involvement of adiponectin signaling in the ER unfolded protein response (UPRER) and the regulation of lifespan in C. elegans. We find that animals with lesions in PAQR-1, one of the three adiponectin receptor homologues in C. elegans, are resistant to ER stressors. Resistance depends on the XBP-1 and PERK UPRER branches. paqr-1(tm3262) mutant animals exhibit elevated HSP-4/BiP baseline levels, and robustly induce chaperone expression and canonical UPRER upon ER stress, in a manner reminiscent of hormesis. PAQR-1 deficiency differentially affects lipid content in ER-stressed animals in an age-dependent manner. Our findings indicate that ER stress triggers lipid macroautophagy (i.e lipophagy) and that PAQR-1-mediated signaling negatively regulates the expression of the ATGL-1 lipase. The selective ability of paqr-1 mutant animals to degrade lipids through lipophagy and ATGL-1-mediated lipolysis may underlie their enhanced survival when challenged with ER stressors. Elucidation of the molecular circuitry that links adiponectin signaling with lipid turnover may contribute to the development of effective therapeutic strategies for the treatment of obesity.

Katidou, M. et al. (2009) International Worm Meeting "The contactin homolog rig-6 is involved in axon guidance and branching in C. elegans."

Katidou, M., Tavernarakis, N., Karagogeos, D.

[International Worm Meeting, 2009]

The Immunoglobulin Superfamily (IgSF) is a conserved family of proteins, playing a leading role in many developmental processes, including nervous system patterning. The contactin subgroup of the IgSF consists of glycosylphosphatidylinositol (GPI) anchored glycoproteins, known to be essential for axon growth, guidance and fasciculation, neuronal migration and myelination in vertebrates. Aiming to further understand the role of contactins, we are characterizing RIG-6 (C33F10.5), the only member of the contactin subfamily in C. elegans. We have determined rig-6 spatiotemporal expression pattern; it is expressed in head neurons, ventral cord motorneurons and commissures, HSN and CAN neurons, in muscle cells, spermatheca and hypodermis. rig-6 expression begins in embryonic stages, and it is maintained throughout adulthood. To further investigate the role of RIG-6, we have used rig-6 RNAi knockdown to study effects on neuronal development and axonal migration. Our data show that downregulation of rig-6 expression leads to the formation of ectopic branches in ALM axons. Moreover, we have observed abnormal crossing of axons in the ventral nerve cord (VNC) To over-express rig-6, we have generated a plasmid that encompasses the promoter, the complete coding sequence and the UTRs of the gene. Transgenic animals show cross defects in the VNC, revealing that the level of expression of rig-6 is critical for normal axon guidance in the VNC. In addition, commissures are misguided and tend to form branches. Several behavioral abnormalities have been observed in the gain of function mutants of rig-6, namely in locomotion, defecation and fertility. Mutations in UNC-53, a cytoskeleton binding protein involved in anteroposterior cell migration and axon guidance, cause the formation of ectopic branches in ALM axons, abnormal commissure branches as well as cross defects in the VNC. To test whether unc-53 and rig-6 function in the same pathway, we have used rig-6 RNAi in unc-53(n152) mutant animals. rig-6 downregulation enhances the formation of commissure branches in unc-53(n152) mutants. Moreover, rig-6 overexpression in unc-53(n152) animals leads to increased frequency of commissure branches and cross defects compared to unc-53(n152) or rig-6 gain of function mutants alone. Our data suggest that rig-6 affects axon guidance and branching in C. elegans. UNC-53 could act downstream of RIG-6, converting an extra-cellular signal to an intracellular response.

Rieckher, M. et al. (2013) International Worm Meeting "Regulation of eIF4E compartmentalization by the heat shock response pathway during ageing in C. elegans."

Rieckher, M., Princz, A., Tavernarakis, N.

[International Worm Meeting, 2013]

Increased lifespan is often correlated with enhanced resistance against intrinsic and extrinsic stress. The eukaryotic initiation factor 4E (eIF4E) is known to regulate ageing and cellular stress resistance through the control of global protein synthesis. Recent studies reveal that eIF4E performs additional regulatory functions by shuttling specific mRNAs from the nucleus to the cytoplasm, and sequesters mRNAs in cytoplasmic aggregates, predominantly during stress. Genetic studies show that the heat shock transcription factor HSF-1 regulates stress responses and modulates lifespan in C. elegans. Here, we present evidence that HSF-1 regulates multiple cellular functions of IFE-2, an isoform of eIF4E expressed in the soma. We find that IFE-2 protein levels decrease during ageing. Loss of HSF-1, but not SKN-1 or DAF-16, results in substantially increased ife-2 mRNA and protein levels in aged animals. Notably, IFE-2 localizes primarily to the nuclei of most somatic cells in animals lacking HSF-1. Moreover, a significantly higher percentage of IFE-2 was found to reside in the nuclei of young animals, compared to aged nematodes. In these animals, IFE-2 mainly granulizes in the perinuclear space and within the nucleus. Granular formation is also observed throughout the cytoplasm. These granules co-localize with well-characterized sites of mRNA degradation, which have been shown to be involved in transcription regulation in the nucleus. Consistently, we observe decreased localization of IFE-2 in the nucleus, upon depletion of specific mRNA degradation factors. Our findings suggest that HSF-1 modulates IFE-2 function and localization during ageing, and that IFE-2 also serves as mRNA transport protein in C. elegans. Thus, IFE-2 likely mediates the effects of the heat stress response on both mRNA translation and degradation to influence ageing.

Ploumi, C. et al. (2017) International Worm Meeting "The NEET homolog CISD-1 modulates ageing through mechanisms involving autophagy and components of the intrinsic apoptosis pathway."

Ploumi, C., Tavernarakis, N., Kyriakakis, E.

[International Worm Meeting, 2017]

The NEET family of proteins comprises a special type of Iron Sulfur Cluster (ISC) binding proteins implicated in various human pathologies ranging from neurodegeneration to cancer and age-related diseases. Despite the well known structural properties of the mammalian NEETs, the mechanisms by which they influence longevity remain largely enigmatic. The C.elegans gene cisd-1 (W02B12.15) encodes a functional homolog of the mammalian CISD1 (CDGSH Iron Sulfur Domain 1) and CISD2 (CDGSH Iron Sulfur Domain 2) proteins, based on sequence analysis and the presence of the conserved domain CDGSH for binding to ISCs. Similar to its mammalian homologs, CISD-1 is an outer mitochondrial membrane protein, ubiquitously expressed in neuronal, intestinal and muscle cells. Downregulation of cisd-1 shortens animal lifespan, boosts mitochondrial activity and induces germline apoptosis. Our findings indicate that CISD-1 exerts differential effects on ageing through its involvement in both the autophagic degradation process and the intrinsic apoptosis pathway. We aim to further elucidate the mechanisms through which CISD-1 function in mitochondria modulates ageing, and gain insight into the functional interplay between autophagy and apoptosis that influences longevity.