Autophagy of germ-granule components, PGL-1 and PGL-3, contributes to DNA damage-induced germ cell apoptosis in C. elegans.

Germ granules, termed P granules in nematode C. elegans, are the germline-specific cytoplasmic structures widely observed from worms to humans. P granules are known to have critical functions for postembryonic germline development likely through regulating RNA metabolism. They are localized at the perinuclear region of germ cells during most of the developmental stages. However, the biological significance of this specific localization remains elusive. PGL-1 and PGL-3, the defining components of P granules, were shown to be lost from the perinuclear region prior to germ cell apoptosis. Furthermore, this loss was shown to be significantly enhanced upon DNA damage. Here, we show that the removal of PGL-1 and PGL-3 from the perinuclear region following UV-induced DNA damage is significantly reduced in autophagy mutants. Autophagy was previously shown to be required for DNA damage-induced germ cell apoptosis. We show that the apoptosis defect of autophagy mutants is bypassed by depletion of pgl-1 or pgl-3. These findings are consistent with time-lapse observations of LGG-1 foci formation, showing that autophagy is activated following UV irradiation and that maximal accumulation of LGG-1 foci occurs before PGL-1 removal. We also show that some of the autophagy genes are transcriptionally activated following UV irradiation by CEP-1, the worm p53-like protein. Taken together, our results indicate that autophagy is required to remove the major P granule components, PGL-1 and PGL-3, and that their removal is required for the full induction of DNA damage-induced germ cell apoptosis. Our study contributes to a better understanding of germ cell apoptosis, a process that leads to the elimination of the vast majority of germ cells in various animals from worms to mammals.

The B-type cyclin CYB-1 maintains the proper position and number of centrosomes during spermatogenesis in Caenorhabditis elegans.

Depletion of cyb-1, a major B-type cyclin expressed during Caenorhabditis elegans spermatogenesis, causes a meiotic division arrest in diakinesis-stage spermatocytes with multiple and mispositioned centrosomes. Association of the two nuclear membrane proteins SUN-1 and ZYG-12 is essential for centrosome-nuclear envelope attachment. We found that depletion of sun-1 causes centrosome defects similar to those caused by cyb-1 depletion in diakinesis-stage spermatocytes. In addition, Ser8 and Ser43 residues in SUN-1 are dephosphorylated in cyb-1-depleted diakinesis-stage spermatocytes. Nevertheless, dephosphorylation of these residues was not sufficient to reproduce the cyb-1-related centrosome defects. We then found that the ZYG-12::GFP signal in the nuclear envelope was significantly reduced in the cyb-1-depleted diakinesis-stage spermatocytes. However, only mispositioned but not multiplied centrosomes were observed in zyg-12 mutant diakinesis-stage spermatocytes, suggesting that zyg-12 is not involved in the centrosome duplication at this stage. Our results suggest that CYB-1 functions to maintain proper positioning of centrosomes during spermatogenesis by regulating phosphorylation of SUN-1, which is possibly crucial for the association between SUN-1 and ZYG-12. This phosphorylation of SUN-1 may also regulate centrosome duplication independently of ZYG-12.

Loss of PGL-1 and PGL-3, members of a family of constitutive germ-granule components, promotes germline apoptosis in C. elegans.

In Caenorhabditis elegans, the mechanisms regulating germline apoptosis remain largely unknown, except for the core machinery. Here, we found that mutants of pgl-1 and pgl-3, encoding members of a family of constitutive protein components of germline-specific P granules, showed increased germline apoptosis under both physiological and DNA-damaged conditions. We also found that the number of germ cells that lost PGL proteins increased significantly following UV irradiation, and that only those PGL-absent germ cells were selectively engulfed by gonadal sheath cells in adult hermaphrodite gonads. We further revealed that CEP-1, the p53 homolog, and the caspase CED-3 promoted elimination of PGL-1 from germ cells following UV irradiation. Furthermore, protein levels of CED-4, the Apaf-1 homolog, and cytoplasmic translocation of SIR-2.1, a Sirtuin homolog, significantly increased in pgl mutants and increased even more following UV irradiation. CED-4 and SIR-2.1 were essential for high levels of germline apoptosis in pgl mutants. We conclude that PGL proteins suppress excessive germline apoptosis by repressing both the protein levels of CED-4 and the cytoplasmic translocation of SIR-2.1. Our study has revealed new roles for PGL-1 and PGL-3 in the control of germline apoptosis.

Gliadin intake induces oxidative-stress responses in Caenorhabditis elegans.

Clinical attention to gluten-related disorders, such as celiac disease and nonceliac gluten sensitivity, is on the rise. However, identifying the pathophysiological mechanisms of gluten-related disorders remains elusive. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. Caenorhabditis elegans has been widely used as the predominant experimental animal model to study toxicity and stress response in biomedical research. We investigated the stress response induced by gliadin intake in C. elegans to evaluate its toxicity and found brood size, body bending, and pumping rates to be significantly altered in response to gliadin. Notably, reactive oxygen species (ROS) production and Pgst-4::GFP transgene expression, an indicator of the oxidative-stress response, were significantly increased after gliadin intake. Reduced pumping rates were most likely caused by gliadin-induced oxidative stress, since pumping rates in oxidative stress-sensitive mev-1 mutants were more severely reduced than in oxidative stress-resistant daf-2 mutants following gliadin intake. Our results indicated that gluten/gliadin intake in C. elegans triggered ROS production and induced an oxidative stress response that reduced pumping rates and decreased brood size. We suggest C. elegans to be a useful model system for studying gluten/gliadin toxicity.

Transgenerational effects of proton beam irradiation on Caenorhabditis elegans germline apoptosis.

When treating cancer using radiation therapy, it is critical to increase patient survival rates and to reduce side effects. In this respect, proton beam radiation treatment performs better than other radiation treatments because of its high target specificity. However, complications still remain after proton beam radiation treatment. Among them, the risk to progeny after irradiation of their parents is a major concern. In this study, we analyzed the transgenerational effects of proton beam irradiation using the model organism Caenorhabditis. elegans. We found that germline apoptosis increased after proton beam irradiation and its effects were sustained transgenerationally. Moreover, we identified that a germline-specific histone methyltransferase component, SET-2, has a critical role in transmitting the transgenerational effect on germline apoptosis to the next generation after proton beam irradiation.

cdc-25.2, a Caenorhabditis elegans ortholog of cdc25, is required for male tail morphogenesis.

Cell division cycle 25 (Cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression by activating cyclin-dependent kinases (Cdks) which are inactivated by Wee1/Myt1 kinases. It was previously reported that cdc-25.2 promotes oocyte maturation and intestinal cell divisions in Caenorhabditis elegans hermaphrodites. Here, we report a novel function of cdc-25.2 in male tail development which was significantly deformed by cdc-25.2 RNAi depletion and in cdc-25.2 mutant males. The deformation was also observed after RNAi depletion of other cell cycle regulators, cdk-1, cyb-3, cyd-1, and cyl-1. Furthermore, wee-1.3 counteracted cdc-25.2 in male tail development as observed in oocyte maturation and intestine development. The number of cells in ray precursor cell lineages was significantly reduced in cdc-25.2 depleted males. These results indicate that CDC-25.2 is essential for cell divisions in ray precursor cell lineages for proper male tail development.

MARC-3, a membrane-associated ubiquitin ligase, is required for fast polyspermy block in Caenorhabditis elegans.

In many sexually reproducing organisms, oocytes are fundamentally fertilized with one sperm. In Caenorhabditis elegans, chitin layer formation after fertilization by the EGG complex is one of the mechanisms of polyspermy block, but other mechanisms remain unknown. Here, we demonstrate that MARC-3, a membrane-associated RING-CH-type ubiquitin ligase that localizes to the plasma membrane and cortical puncta in oocytes, is involved in fast polyspermy block. During polyspermy, the second sperm entry occurs within approximately 10 s after fertilization in MARC-3-deficient zygotes, whereas it occurs approximately 200 s after fertilization in egg-3 mutant zygotes defective in the chitin layer formation. MARC-3 also functions in the selective degradation of maternal plasma membrane proteins and the transient accumulation of endosomal lysine 63-linked polyubiquitin after fertilization. The RING-finger domain of MARC-3 is required for its in vitro ubiquitination activity and polyspermy block, suggesting that a ubiquitination-mediated mechanism sequentially regulates fast polyspermy block and maternal membrane protein degradation during the oocyte-to-embryo transition.

cdc-25.2, a C. elegans ortholog of cdc25, is required to promote oocyte maturation.

Cdc25 is an evolutionarily conserved protein phosphatase that promotes progression through the cell cycle. Some metazoans have multiple isoforms of Cdc25, which have distinct functions and different expression patterns during development. C. elegans has four cdc-25 genes. cdc-25.1 is required for germline mitotic proliferation. To determine if the other members of the cdc-25 family also contribute to regulation of cell division in the germ line, we examined phenotypes of loss-of-function mutants of the other cdc-25 family genes. We found that cdc-25.2 is also essential for germline development. cdc-25.2 homozygous mutant hermaphrodites exhibited sterility as a result of defects in oogenesis: mutant oocytes were arrested as endomitotic oocytes that were not fertilized successfully. Spermatogenesis and male germline development were not affected. Through genetic interaction studies, we found that CDC-25.2 functions upstream of maturation-promoting factor containing CDK-1 and CYB-3 to promote oocyte maturation by counteracting function of WEE-1.3. We propose that cdc-25 family members function as distinct but related cell cycle regulators to control diverse cell cycles in C. elegans germline development.

A circulatory transcriptional regulation among daf-9, daf-12, and daf-16 mediates larval development upon cholesterol starvation in Caenorhabditis elegans.

C. elegans shows dauer-like larvae formation upon cholesterol starvation (CS), but the genetic epistasis among abnormal dauer formation (daf) genes during the process remains unclear. To clarify the genetic interactions among daf-9, daf-12, and daf-16 in this process, mRNA levels of these genes upon CS were measured. CS increased the mRNA levels of daf-9, daf-12, and daf-16. CS also induced DAF-16 nuclear localization, which was positively and negatively regulated by DAF-12 and DAF-9 activities, respectively. Activated DAF-16, a FOXO transcription factor, enhanced daf-12 but suppressed daf-9 expression, whereas DAF-9 inhibited daf-12 expression. Concomitantly, CS-induced larval arrest was regulated positively by DAF-12 and DAF-16, but negatively by DAF-9. The larval arrest in daf-9 mutant was suppressed by daf-12 RNAi, placing DAF-12 downstream of DAF-9. These results altogether suggest that circulatory mutual regulation among daf-9, daf-12, and daf-16 at the expression level mediates cholesterol signal to control larval development upon CS.

ASB-1, a germline-specific isoform of mitochondrial ATP synthase b subunit, is required to maintain the rate of germline development in Caenorhabditis elegans.

The developmental timing of all types of cells must be synchronized and spatially coordinated to achieve the organized development of a multicellular organism. Previously, we found RNAi of asb-1, encoding a germline-specific isoform of mitochondrial ATP synthase b subunit, caused 100% penetrant sterility in Caenorhabditis elegans. ATP synthase is one of the five complexes of the mitochondrial respiratory chain, and defects in some of the components of the chain are known to slow the growth and extend the lifespan of worms. We found that development of asb-1 mutant germ line was not arrested at any stage, but did slow to half the rate of wild type, whereas the rate of somatic development was the same in asb-1 mutants as that of wild type, indicating that asb-1 is required to maintain the rate of germline development but has no effect on somatic development. Among ATP synthase subunit genes, RNAi of asg-1, encoding a germline-specific isoform of the g subunit, also caused asb-1-like sterility, indicating that some other germline-specific components are also required to maintain the rate of germline development. Both asb-1 and asg-1 are located on autosomes while they possess counterparts, asb-2 and asg-2, respectively, on X chromosome, which are both required for somatic development. Chromosomal locations of the genes may be the basis of the segregation of germline/somatic functions of each gene, as were demonstrated for other autosomal/X-linked duplicated gene pairs.

Inhibition of developmental processes by flavone in Caenorhabditis elegans and its application to the pinewood nematode, Bursaphelenchus xylophilus.

Flavone (2-phenyl chromone) is a well-known plant flavonoid, but its bioactivity has been little explored. Treatment of Caenorhabditis elegans or C. brissage with flavones induced embryonic and larval lethality that was pronounced in early larval stages. This anti-nematodal effect was also observed in the pinewood nematode, B. xylophilus. LD(50) values were approximately 100 muM for both B. xylophilus and C. elegans. Our results indicate that flavone is an active nematicidal compound that should be further investigated with the aim of developing a potent drug against B. xylophilus.

The micronucleus test and erythropoiesis: effects of cyclic adenosine monophosphate (cAMP) on micronucleus formation.

The aim of this study was to determine the mechanism of the rodent bone marrow micronucleus test in relation to erythropoiesis. We have previously reported that an acceleration of erythropoiesis increases the frequency of micronucleated polychromatic erythrocytes (MPCE) induced by mutagens. The blood plasma erythropoietin level increased after the injection of N6-2-O-dibutyladenosine-3',5'-cyclic monophosphate into adenosine 3',5'-cyclic monophosphate (cAMP) at a dose of 500 mg/kg. A peak of erythropoietin induction was observed 3 h after the injection of cAMP. cAMP itself did not induce any micronuclei in erythroblasts of BALB/c mice. So, the frequency of MPCE did not increase after injection of cAMP. The highest frequency of MPCE and the dose-response relationship between the cAMP doses and micronucleus frequency were observed 30 h after injection of mitomycin C (MMC) in mice which had been administered cAMP 24 h previously. The highest effect of cAMP on the increase of MPCE was observed when cAMP was given 24 h before MMC injection, thus indicating that accelerating the multiplication of erythroblasts increases the frequency of MPCE induced by mutagens. The induction of MPCE in the bone marrow by three other chemicals (carboquone, 5-fluorouracil, and vincristine) also increased after pretreatment with cAMP. Our results suggest that the increase of MPCE induced by mutagens can be amplified following the acceleration of erythropoiesis by pretreatment with cAMP.

Depletion of cdc-25.3, a Caenorhabditis elegans orthologue of cdc25, increases physiological germline apoptosis.

In Caenorhabditis elegans hermaphrodites, physiological germline apoptosis is higher in cdc-25.3 mutants than in wild-type. The elevated germline apoptosis in cdc-25.3 mutants seems to be induced by accumulation of double-stranded DNA breaks (DSBs). Both DNA damage and synapsis checkpoint genes are required to increase the germline apoptosis. Notably, the number of germ cells that lose P-granule components, PGL-1 and PGL-3, increase in cdc-25.3 mutants, and the increase in germline apoptosis requires the activity of SIR-2.1, a Sirtuin orthologue. These results suggest that elevation of germline apoptosis in cdc-25.3 mutants is induced by accumulation of DSBs, leading to a loss of PGL-1 and PGL-3 in germ cells, which promotes cytoplasmic translocation of SIR-2.1, and finally activates the core apoptotic machinery.

Caffeine-induced food-avoidance behavior is mediated by neuroendocrine signals in Caenorhabditis elegans.

High-dose caffeine uptake is a developmental stressor and causes food-avoidance behavior (aversion phenotype) in C. elegans, but its mode of action is largely unknown. In this study, we investigated the molecular basis of the caffeineinduced aversion behavior in C. elegans. We found that aversion phenotype induced by 30 mM caffeine was mediated by JNK/MAPK pathway, serotonergic and dopaminergic neuroendocrine signals. In this process, the dopaminergic signaling appears to be the major pathway because the reduced aversion behavior in cat-2 mutants and mutants of JNK/MAPK pathway genes was significantly recovered by pretreatment with dopamine. RNAi depletion of hsp-16.2, a cytosolic chaperone, and cyp-35A family reduced the aversion phenotype, which was further reduced in cat-2 mutants, suggesting that dopaminergic signal is indeed dominantly required for the caffeine-induced food aversion. Our findings suggest that aversion behavior is a defense mechanism for worms to survive under the high-dose caffeine conditions. [BMB Reports 2017; 50(1): 31-36].

LIN-23, an E3 Ubiquitin Ligase Component, Is Required for the Repression of CDC-25.2 Activity during Intestinal Development in Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans) utilizes two different cell-cycle modes, binucleations during the L1 larval stage and endoreduplications at four larval moltings, for its postembryonic intestinal development. Previous genetic studies indicated that CDC-25.2 is specifically required for binucleations at the L1 larval stage and is repressed before endoreduplications. Furthermore, LIN-23, the C. elegans ß-TrCP ortholog, appears to function as a repressor of CDC-25.2 to prevent excess intestinal divisions. We previously reported that intestinal hyperplasia in lin-23(e1883) mutants was effectively suppressed by the RNAi depletion of cdc-25.2. Nevertheless, LIN-23 targeting CDC-25.2 for ubiquitination as a component of E3 ubiquitin ligase has not yet been tested. In this study, LIN-23 is shown to be the major E3 ubiquitin ligase component, recognizing CDC-25.2 to repress their activities for proper transition of cell-cycle modes during the C. elegans postembryonic intestinal development. In addition, for the first time that LIN-23 physically interacts with both CDC-25.1 and CDC-25.2 and facilitates ubiquitination for timely regulation of their activities during the intestinal development.

CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions.

Intestinal divisions in Caenorhabditis elegans take place in 3 stages: (1) cell divisions during embryogenesis, (2) binucleations at the L1 stage, and (3) endoreduplications at the end of each larval stage. Here, we report that CDC-25.2, a C. elegans ortholog of Cdc25, is required for these specialized division cycles between the 16E cell stage and the onset of endoreduplication. Results of our genetic analyses suggest that CDC-25.2 regulates intestinal cell divisions and binucleations by counteracting WEE-1.3 and by activating the CDK-1/CYB-1 complex. CDC-25.2 activity is then repressed by LIN-23 E3 ubiquitin ligase before the onset of intestinal endoreduplication, and this repression is maintained by LIN-35, the C. elegans ortholog of Retinoblastoma (Rb). These findings indicate that timely regulation of CDC-25.2 activity is essential for the progression of specialized division cycles and development of the C. elegans intestine.

Caffeine Induces the Stress Response and Up-Regulates Heat Shock Proteins in Caenorhabditis elegans.

Caffeine has both positive and negative effects on physiological functions in a dose-dependent manner. C. elegans has been used as an animal model to investigate the effects of caffeine on development. Caffeine treatment at a high dose (30 mM) showed detrimental effects and caused early larval arrest. We performed a comparative proteomic analysis to investigate the mode of action of high-dose caffeine treatment in C. elegans and found that the stress response proteins, heat shock protein (HSP)-4 (endoplasmic reticulum [ER] chaperone), HSP-6 (mitochondrial chaperone), and HSP-16 (cytosolic chaperone), were induced and their expression was regulated at the transcriptional level. These findings suggest that high-dose caffeine intake causes a strong stress response and activates all three stress-response pathways in the worms, including the ER-, mitochondrial-, and cytosolic pathways. RNA interference of each hsp gene or in triple combination retarded growth. In addition, caffeine treatment stimulated a food-avoidance behavior (aversion phenotype), which was enhanced by RNAi depletion of the hsp-4 gene. Therefore, up-regulation of hsp genes after caffeine treatment appeared to be the major responses to alleviate stress and protect against developmental arrest.

Somatically expressed germ-granule components, PGL-1 and PGL-3, repress programmed cell death in C. elegans.

We previously reported that germline apoptosis in C. elegans increased by loss of PGL-1 and PGL-3, members of a family of constitutive germ-granule components, from germ cells in adult hermaphrodite gonads. In this study, we found that somatic apoptosis was reduced in synthetic multivulva class B (synMuv B) mutants due to ectopic expression of PGL-1 and PGL-3 in the soma. In synMuv B-mutant somatic cells, CED-4 expression level was reduced due to ectopic expression of PGL-1. Furthermore, in contrast to wild type, somatic apoptosis in synMuv B mutants increased following DNA damage in a SIR-2.1-dependent manner. Intriguingly, somatic apoptosis was repressed not only in synMuv B mutants but also by ectopically expressing pgl-1 and/or pgl-3 transgenes in wild-type somatic cells. Our study demonstrates that germ-granule components, PGL-1 and PGL-3, can serve as negative regulators of apoptosis not only in the germline but also in the soma in C. elegans.

cdc-25.4, a Caenorhabditis elegans Ortholog of cdc25, Is Required for Male Mating Behavior.

Cell division cycle 25 (cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression. Among the four cdc25 orthologs in Caenorhabditis elegans, we found that cdc-25.4 mutant males failed to produce outcrossed progeny. This was not caused by defects in sperm development, but by defects in male mating behavior. The cdc-25.4 mutant males showed various defects during male mating, including contact response, backing, turning, and vulva location. Aberrant turning behavior was the most prominent defect in the cdc-25.4 mutant males. We also found that cdc-25.4 is expressed in many neuronal cells throughout development. The turning defect in cdc-25.4 mutant males was recovered by cdc-25.4 transgenic expression in neuronal cells, suggesting that cdc-25.4 functions in neurons for male mating. However, the neuronal morphology of cdc-25.4 mutant males appeared to be normal, as examined with several neuronal markers. Also, RNAi depletion of wee-1.3, a C. elegans ortholog of Wee1/Myt1 kinase, failed to suppress the mating defects of cdc-25.4 mutant males. These findings suggest that, for successful male mating, cdc-25.4 does not target cell cycles that are required for neuronal differentiation and development. Rather, cdc-25.4 likely regulates noncanonical substrates in neuronal cells.

Anti-aging treatments slow propagation of synucleinopathy by restoring lysosomal function.

Aging is the major risk factor for neurodegenerative diseases that are also associated with impaired proteostasis, resulting in abnormal accumulation of protein aggregates. However, the role of aging in development and progression of disease remains elusive. Here, we used Caenorhabditis elegans models to show that aging-promoting genetic variations accelerated the rate of cell-to-cell transmission of SNCA/α-synuclein aggregates, hallmarks of Parkinson disease, and the progression of disease phenotypes, such as nerve degeneration, behavioral deficits, and reduced life span. Genetic and pharmacological anti-aging manipulations slowed the spread of aggregates and the associated phenotypes. Lysosomal degradation was significantly impaired in aging models, while anti-aging treatments reduced the impairment. Transgenic expression of hlh-30p::hlh-30, the master controller of lysosomal biogenesis, alleviated intercellular transmission of aggregates in the aging model. Our results demonstrate that the rate of aging closely correlates with the rate of aggregate propagation and that general anti-aging treatments can slow aggregate propagation and associated disease progression by restoring lysosomal function.