Monitoring Autophagic Responses in Caenorhabditis elegans.

Autophagy, from the Greek auto (self) and phagy (eating), is a self-degradative process critical for eukaryotic cell homeostasis. Its rapidly responsive, highly dynamic nature renders this process essential for adapting to and offsetting acute/harsh conditions such as starvation, organelle dysfunction, and deoxyribonucleic acid (DNA) damage. Autophagy involves an intricate network of interacting factors with multiple levels of control. Importantly, dysregulation of autophagy has been linked to numerous debilitating pathologies, including cancer and neurodegenerative conditions in humans. Methods to monitor and quantify autophagic activity reliably are essential both for studying the basic mechanisms of autophagy and for dissecting its involvement in disease. The nematode Caenorhabditis elegans is a particularly suitable model organism to effectively visualize and study autophagy, in vivo, in a physiological and pathological context due to its optical transparency, experimental malleability, and precise developmental and anatomical characterization. Here, we outline the main tools and approaches to monitor and measure autophagic responses in C. elegans.

Autophagy in the physiology and pathology of the central nervous system.

Neurons are highly specialized postmitotic cells that depend on dynamic cellular processes for their proper function.These include among others, neuronal growth and maturation, axonal migration, synapse formation and elimination, all requiring continuous protein synthesis and degradation. Therefore quality-control processes in neurons are directly linked to their physiology. Autophagy is a tightly regulated cellular degradation pathway by which defective or superfluouscytosolic proteins, organelles and other cellular constituents are sequestered in autophagosomes and delivered to lysosomes for degradation. Here we present emerging evidence indicating that constitutive autophagic fluxin neurons has essential roles in key neuronal processes under physiological conditions.Moreover, we discuss how perturbations of the autophagic pathway may underlie diverse pathological phenotypes in neurons associated with neurodevelopmental and neurodegenerative diseases.

The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca(2+) levels to α-synuclein toxicity in Parkinson's disease models.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons, which arises from a yet elusive concurrence between genetic and environmental factors. The protein α-synuclein (αSyn), the principle toxic effector in PD, has been shown to interfere with neuronal Ca(2+) fluxes, arguing for an involvement of deregulated Ca(2+) homeostasis in this neuronal demise. Here, we identify the Golgi-resident Ca(2+)/Mn(2+) ATPase PMR1 (plasma membrane-related Ca(2+)-ATPase 1) as a phylogenetically conserved mediator of αSyn-driven changes in Ca(2+) homeostasis and cytotoxicity. Expression of αSyn in yeast resulted in elevated cytosolic Ca(2+) levels and increased cell death, both of which could be inhibited by deletion of PMR1. Accordingly, absence of PMR1 prevented αSyn-induced loss of dopaminergic neurons in nematodes and flies. In addition, αSyn failed to compromise locomotion and survival of flies when PMR1 was absent. In conclusion, the αSyn-driven rise of cytosolic Ca(2+) levels is pivotal for its cytotoxicity and requires PMR1.

Imaging Caenorhabditis elegans embryogenesis by third-harmonic generation microscopy.

In this study, third-harmonic generation (THG) imaging measurements were performed to characterize different developmental stages of the nematode Caenorhabditis elegans (C. elegans) embryos. Femtosecond laser pulses (1028 nm) were utilized for excitation. THG image contrast modality proved as a powerful diagnostic tool, providing valuable information and offering new insights into the complex developmental process of C. elegans embryogenesis.

Caloric restriction and resveratrol promote longevity through the Sirtuin-1-dependent induction of autophagy.

Caloric restriction and autophagy-inducing pharmacological agents can prolong lifespan in model organisms including mice, flies, and nematodes. In this study, we show that transgenic expression of Sirtuin-1 induces autophagy in human cells in vitro and in Caenorhabditis elegans in vivo. The knockdown or knockout of Sirtuin-1 prevented the induction of autophagy by resveratrol and by nutrient deprivation in human cells as well as by dietary restriction in C. elegans. Conversely, Sirtuin-1 was not required for the induction of autophagy by rapamycin or p53 inhibition, neither in human cells nor in C. elegans. The knockdown or pharmacological inhibition of Sirtuin-1 enhanced the vulnerability of human cells to metabolic stress, unless they were stimulated to undergo autophagy by treatment with rapamycin or p53 inhibition. Along similar lines, resveratrol and dietary restriction only prolonged the lifespan of autophagy-proficient nematodes, whereas these beneficial effects on longevity were abolished by the knockdown of the essential autophagic modulator Beclin-1. We conclude that autophagy is universally required for the lifespan-prolonging effects of caloric restriction and pharmacological Sirtuin-1 activators.

In vivo imaging of cell morphology and cellular processes in Caenorhabditis elegans, using non-linear phenomena.

We present the detailed imaging of structures and processes of the nematode Caenorhabditis elegans (C. elegans) using non-linear microscopy. Complementary information about the anatomy of the nematode was collected by implementing a combination of two photon excitation fluorescence (TPEF), second and third harmonic generation (SHG and THG) image contrast modes on the same microscope. Three-dimensional (3D) reconstructions of TPEF, SHG and THG images were also performed. Moreover, THG imaging technique has been tested as a potential, novel, non-destructive diagnostic tool for monitoring cellular processes in vivo, such as neuronal degeneration.

Autophagy and cell death in model organisms.

Autophagy evolved in unicellular eukaryotes as a means for surviving nutrient stress. During the course of evolution, as multicellular organisms developed specialized cell types and complex intracellular signalling networks, autophagy has been summoned to serve additional cellular functions. Numerous recent studies indicate that apart from its pro-survival role under nutrient limitation, autophagy also participates in cell death. However, the precise role of this catabolic process in dying cells is not fully understood. Although in certain situations autophagy has a protective function, in other types of cell death it actually contributes to cellular destruction. Simple model organisms ranging from the unicellular Saccharomyces cerevisiae to the soil amoeba Dictyostelium discoideum and the metazoans Caenorhabditis elegans and Drosophila melanogaster provide clearly defined cell death paradigms that can be used to dissect the involvement of autophagy in cell death, at the molecular level. In this review, we survey current research in simple organisms, linking autophagy to cell death and discuss the complex interplay between autophagy, cell survival and cell death.

In vivo imaging of neurodegeneration in Caenorhabditis elegans by third harmonic generation microscopy.

In this study, neurodegeneration phenomena were investigated, by performing third harmonic generation imaging measurements on the nematode Caenorhabditis elegans, in vivo. The in vivo, precise identification of the contour of the degenerating neurons in the posterior part of the nematode and the monitoring, in real time, of the progression of degeneration in the worm, through third harmonic generation imaging measurements, were achieved. Femtosecond laser pulses (1028 nm) were utilized for excitation. Thus, the THG image contrast modality comprises a powerful diagnostic tool, providing valuable information and offering new insights into morphological changes and complex developmental processes in live biological specimens.

No death without life: vital functions of apoptotic effectors.

As a result of the genetic experiments performed in Caenorhabditis elegans, it has been tacitly assumed that the core proteins of the 'apoptotic machinery' (CED-3, -4, -9 and EGL-1) would be solely involved in cell death regulation/execution and would not exert any functions outside of the cell death realm. However, multiple studies indicate that the mammalian orthologs of these C. elegans proteins (i.e. caspases, Apaf-1 and multidomain proteins of the Bcl-2 family) participate in cell death-unrelated processes. Similarly, loss-of-function mutations of ced-4 compromise the mitotic arrest of DNA-damaged germline cells from adult nematodes, even in a context in which the apoptotic machinery is inoperative (for instance due to mutations of egl-1 or ced-3). Moreover, EGL-1 is required for the activation of autophagy in starved nematodes. Finally, the depletion of caspase-independent death effectors, such as apoptosis-inducing factor (AIF) and endonuclease G, provokes cell death-independent consequences, both in mammals and in yeast (Saccharomyces cerevisiae). These results corroborate the conjecture that any kind of protein that has previously been specifically implicated in apoptosis might have a phylogenetically conserved apoptosis-unrelated function, most likely as part of an adaptive response to cellular stress.

In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy.

In this study, we use combined two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG) and third-harmonic generation (THG) measurements to image cellular structures of the nematode Caenorhabditis elegans, in vivo. To our knowledge, this is the first time that a THG modality is employed to image live C. elegans specimens. Femtosecond laser pulses (1028 nm) were utilized for excitation. Detailed and specific structural and anatomical features can be visualized, by recording THG signals. Thus, the combination of three image-contrast modes (TPEF-SHG-THG) in a single instrument has the potential to provide unique and complementary information about the structure and function of tissues and individual cells of live biological specimens.

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.

Germ line transformation of the olive fly Bactrocera oleae using a versatile transgenesis marker.

The olive fruit fly (olive fly) Bactrocera oleae (Dacus), recently introduced in North America, is the most destructive pest of olives worldwide. The lack of an efficient gene transfer technology for olive fly has hampered molecular analysis, as well as development of genetic techniques for its control. We have developed a Minos-based transposon vector carrying a self-activating cassette which overexpresses the enhanced green fluorescent protein (EGFP). Efficient transposase-mediated integration of one to multiple copies of this vector was achieved in the germ line of B. oleae by coinjecting the vector along with in vitro synthesized Minos transposase mRNA into preblastoderm embryos. The self-activating gene construct combined with transposase mRNA present a system with potential for transgenesis of very diverse species.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca(2+) release from the endoplasmic reticulum.

In C. elegans, a hyperactivated MEC-4(d) ion channel induces necrotic-like neuronal death that is distinct from apoptosis. We report that null mutations in calreticulin suppress both mec-4(d)-induced cell death and the necrotic cell death induced by expression of a constitutively activated Galpha(S) subunit. RNAi-mediated knockdown of calnexin, mutations in the ER Ca(2+) release channels unc-68 (ryanodine receptor) or itr-1 (inositol 1,4,5 triphosphate receptor), and pharmacological manipulations that block ER Ca(2+) release also suppress death. Conversely, thapsigargin-induced ER Ca(2+) release can restore mec-4(d)-induced cell death when calreticulin is absent. We conclude that high [Ca(2+)](i) is a requirement for necrosis in C. elegans and suggest that an essential step in the death mechanism is release of ER-based Ca(2+) stores. ER-driven Ca(2+) release has previously been implicated in mammalian necrosis, suggesting necrotic death mechanisms may be conserved.

Degenerins. At the core of the metazoan mechanotransducer?

Mechanosensory signaling, believed to be mediated by mechanically gated ion channels, constitutes the basis for the senses of touch and hearing, and contributes fundamentally to the development and homeostasis of all organisms. Despite this profound importance in biology, little is known of the molecular identities or functional requirements of mechanically gated ion channels. Genetic analyses of touch sensation and locomotion in Caenorhabditis elegans have implicated a new class of ion channels, the degenerins (DEG) in nematode mechanotransduction. Related fly and vertebrate proteins, the epithelial sodium channel (ENaC) family, have been implicated in several important processes, including transduction of mechanical stimuli, pain sensation, gametogenesis, sodium reabsorption, and blood pressure regulation. Still-to-be-discovered DEG/ENaC proteins may compose the core of the elusive human mechanotransducer.

Mechanotransduction in Caenorhabditis elegans: the role of DEG/ENaC ion channels.

One of the looming mysteries in signal transduction today is the question of how mechanical signals, such as pressure or mechanical force delivered to a cell, are interpreted to direct biological responses. All living organisms, and probably all cells, have the ability to sense and respond to mechanical stimuli. At the single-cell level, mechanical signaling underlies cell-volume control and specialized responses such as the prevention of poly-spermy in fertilization. At the level of the whole organism, mechanotransduction underlies processes as diverse as stretch-activated reflexes in vascular epithelium and smooth muscle; gravitaxis and turgor control in plants; tissue development and morphogenesis; and the senses of touch, hearing, and balance. Intense genetic, molecular, and elecrophysiological studies in organisms ranging from nematodes to mammals have highlighted members of the recently discovered DEG/ENaC family of ion channels as strong candidates for the elusive metazoan mechanotransducer. Here, we discuss the evidence that links DEG/ENaC ion channels to mechanotransduction and review the function of Caenorhabditis elegans members of this family called degenerins and their role in mediating mechanosensitive behaviors in the worm.

Caenorhabditis elegans degenerins and vertebrate ENaC ion channels contain an extracellular domain related to venom neurotoxins.

The DEG/ENaC (DEGenerin/Epithelial Na+ Channel) superfamily includes closely related ion channel subunits from divergent species ranging from the simple nematode Caenorhabditis elegans to humans. Members of this protein group play roles in several important processes including transduction of mechanical stimuli, sodium re-absorption and blood pressure regulation. Structure/function relationships in members of this superfamily are just beginning to be elaborated. Using a bio-informatics approach, we identified a novel structural element in the extracellular region of DEG/ENaC proteins that exhibits significant similarity to venom neurotoxins. Since venom neurotoxins bind to sodium channels at high affinity, we suggest that the related domain embedded in DEG/ENaC channels may interact with other regions of the channel or channel complex to modulate channel function.