Regulation of Caenorhabditis elegans p53/CEP-1-dependent germ cell apoptosis by Ras/MAPK signaling

Maintaining genome stability in the germline is thought to be an evolutionarily ancient role of the p53 family. The sole Caenorhabditis elegans p53 family member CEP-1 is required for apoptosis induction in meiotic, late-stage pachytene germ cells in response to DNA damage and meiotic recombination failure. In an unbiased genetic screen for negative regulators of CEP-1, we found that increased activation of the C. elegans ERK orthologue MPK-1, resulting from either loss of the lip-1 phosphatase or activation of let-60 Ras, results in enhanced cep-1-dependent DNA damage induced apoptosis. We further show that MPK-1 is required for DNA damage-induced germ cell apoptosis. We provide evidence that MPK-1 signaling regulates the apoptotic competency of germ cells by restricting CEP-1 protein expression to cells in late pachytene. Restricting CEP-1 expression to cells in late pachytene is thought to ensure that apoptosis doesn't occur in earlier-stage cells where meiotic recombination occurs. MPK-1 signaling regulates CEP-1 expression in part by regulating the levels of GLD-1, a translational repressor of CEP-1, but also via a GLD-1-independent mechanism. In addition, we show that MPK-1 is phosphorylated and activated upon ionising radiation (IR) in late pachytene germ cells and that MPK-1-dependent CEP-1 activation may be in part direct, as these two proteins interact in a yeast two-hybrid assay. In summary, we report our novel finding that MAP kinase signaling controls CEP-1-dependent apoptosis by several different pathways that converge on CEP-1. Since apoptosis is also restricted to pachytene stage cells in mammalian germlines, analogous mechanisms regulating p53 family members are likely to be conserved throughout evolution.     

Impact of Cigarette Smoke Exposure on Innate Immunity: A Caenorhabditis elegans Model

Background

Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and lung cancer. Respiratory bacterial infections have been shown to be involved in the development of COPD along with impaired airway innate immunity.

Methodology/Principal Findings

To address the in vivo impact of cigarette smoke (CS) exclusively on host innate defense mechanisms, we took advantage of Caenorhabditis elegans (C. elegans), which has an innate immune system but lacks adaptive immune function. Pseudomonas aeruginosa (PA) clearance from intestines of C. elegans was dampened by CS. Microarray analysis identified 6 candidate genes with a 2-fold or greater reduction after CS exposure, that have a human orthologue, and that may participate in innate immunity. To confirm a role of CS-down-regulated genes in the innate immune response to PA, RNA interference (RNAi) by feeding was carried out in C. elegans to inhibit the gene of interest, followed by PA infection to determine if the gene affected innate immunity. Inhibition of lbp-7, which encodes a lipid binding protein, resulted in increased levels of intestinal PA. Primary human bronchial epithelial cells were shown to express mRNA of human Fatty Acid Binding Protein 5 (FABP-5), the human orthologue of lpb-7. Interestingly, FABP-5 mRNA levels from human smokers with COPD were significantly lower (p = 0.036) than those from smokers without COPD. Furthermore, FABP-5 mRNA levels were up-regulated (7-fold) after bacterial (i.e., Mycoplasma pneumoniae) infection in primary human bronchial epithelial cell culture (air-liquid interface culture).

Conclusions

Our results suggest that the C. elegans model offers a novel in vivo approach to specifically study innate immune deficiencies resulting from exposure to cigarette smoke, and that results from the nematode may provide insight into human airway epithelial cell biology and cigarette smoke exposure.     

Stress-induced germ cell apoptosis by a p53 independent pathway in Caenorhabditis elegans

In Caenorhabditis elegans, several distinct apoptosis pathways have been characterized in the germline. The physiological pathway is though to eliminate excess germ cells during oogenesis to maintain gonad homeostasis and it is activated by unknown mechanisms. The DNA damage-induced germ cell apoptosis occurs in response to genotoxic agents and involves the proteins EGL-1 and CED-13, and the DNA damage response protein p53. Germ cell apoptosis can also be induced in response to pathogen infection through an EGL-1 dependent pathway. To gain insight into the mechanism and functions of germ cell apoptosis, we investigated whether and how other forms of stress induce this cell death. We found that oxidative, osmotic, heat shock and starvation stresses induce germ cell apoptosis through a p53 and EGL-1 independent pathway. We also learned that the MAPK kinases MEK-1 and SEK-1, and the p53 antagonist protein ABL-1, are essential for stress-induced germ cell apoptosis. We conclude that in C. elegans responses to various stresses that do not involve genotoxicity include an increase in germ cell apoptosis through the physiological pathway.     

p53/CEP-1 increases or decreases lifespan, depending on level of mitochondrial bioenergetic stress

Mitochondrial pathologies underlie a number of life-shortening diseases in humans. In the nematode Caenorhabditis elegans, severely reduced expression of mitochondrial proteins involved in electron transport chain-mediated energy production also leads to pathological phenotypes, including arrested development and/or shorter life; in sharp contrast, mild suppression of these same proteins extends lifespan. In this study, we show that the C. elegans p53 ortholog cep-1 mediates these opposite effects. We found that cep-1 is required to extend longevity in response to mild suppression of several bioenergetically relevant mitochondrial proteins, including frataxin – the protein defective in patients with Friedreich's Ataxia. Importantly, we show that cep-1 also mediates both the developmental arrest and life shortening induced by severe mitochondrial stress. These findings support an evolutionarily conserved function for p53 in modulating organismal responses to mitochondrial dysfunction and suggest that metabolic checkpoint responses may play a role in longevity control and in human mitochondrial-associated diseases.     

The phosphoinositide kinase PIKfyve/Fab1p regulates terminal lysosome maturation in Caenorhabditis elegans

Membrane dynamics is necessary for cell homeostasis and signal transduction and is in part regulated by phosphoinositides. Pikfyve/Fab1p is a phosphoinositide kinase that phosphorylates phosphatidylinositol 3-monophosphate into phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and is implicated in membrane homeostasis in yeast and in mammalian cells. These two phosphoinositides are substrates of myotubularin phosphatases found mutated in neuromuscular diseases. We studied the roles of phosphatidylinositol phosphate kinase 3 (PPK-3), the orthologue of PIKfyve/Fab1p, in a multicellular organism, Caenorhabditis elegans. Complete loss of ppk-3 function induces developmental defects characterized by embryonic lethality, whereas partial loss of function leads to growth retardation. At the cellular level, ppk-3 mutants display a striking enlargement of vacuoles positive for lysosome-associated membrane protein 1 in different tissues. In the intestine, RAB-7-positive late endosomes are also enlarged. Membranes of the enlarged lysosomes originate at least in part from smaller lysosomes, and functional and genetic analyses show that the terminal maturation of lysosomes is defective. Protein degradation is not affected in the hypomorphic ppk-3 mutant and is thus uncoupled from membrane retrieval. We measured the level of PtdIns(3,5)P2 and showed that its production is impaired in this mutant. This work strongly suggests that the main function of PPK-3 is to mediate membrane retrieval from matured lysosomes through regulation of PtdIns(3,5)P2.     

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.     

Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity

We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity.     

The Caenorhabditis elegans ing-3 Gene Regulates Ionizing Radiation-Induced Germ-Cell Apoptosis in a p53-Associated Pathway

The inhibitor of growth (ING) family of type II tumor suppressors are encoded by five genes in mammals and by three genes in Caenorhabditis elegans. All ING proteins contain a highly conserved plant homeodomain (PHD) zinc finger. ING proteins are activated by stresses, including ionizing radiation, leading to the activation of p53. ING proteins in mammals and yeast have recently been shown to read the histone code in a methylation-sensitive manner to regulate gene expression. Here we identify and characterize ing-3, the C. elegans gene with the highest sequence identity to the human ING3 gene. ING-3 colocalizes with chromatin in embryos, the germline, and somatic cells. The ing-3 gene is part of an operon but is also transcribed from its own promoter. Both ing-3(RNAi) and ing-3 mutant strains demonstrate that the gene likely functions in concert with the C. elegans p53 homolog, cep-1, to induce germ-cell apoptosis in response to ionizing radiation. Somatically, the ing-3 mutant has a weak kinker uncoordinated (kinker Unc) phenotype, indicating a possible neuronal function.     

Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning

In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (egl-9) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.