Identification of the AFD neuron as the site of action of the CREB protein in Caenorhabditis elegans thermotaxis

Behaviour is a consequence of computation in neural circuits composed of massive synaptic connections among sensory neurons and interneurons. The cyclic AMP response element-binding protein (CREB) responsible for learning and memory is expressed in almost all neurons. Nevertheless, we find that the Caenorhabditis elegans CREB orthologue, CRH-1, is only required in the single bilateral thermosensory neuron AFD, for a memory-related behaviour. Restoration of CRH-1 in AFD of CREB-depleted crh-1 mutants rescues its thermotactic defect, whereas restorations in other neurons do not. In calcium-imaging analyses, the AFD neurons of CREB-depleted crh-1 mutants exhibit an abnormal response to temperature increase. We present a new platform for analysing the mechanism of behavioural memory at single-cellular resolution within the neural circuit.     

秀丽隐杆线虫（Caenorhabditis elegans）在药物筛选中的应用

基于靶点的体外药物筛选操作相对简单,成本较低,但是由于药物在体内的作用并不仅仅取决于其与靶点的作用程度,吸收、分布、代谢、排泄特征和毒性均会对早期先导物能否进入临床使用产生极大的影响,因此,药物的体内筛选受到重视。本文重点综述了秀丽隐杆线虫（C.elegans）在抗衰老、抗感染药物筛选中的应用情况。秀丽隐杆线虫结构简单、易于培养和可实现高通量筛选,在未来的药物筛选中必将发挥更重要的作用。     

Distinct thermal migration behaviors in response to different thermal gradients in Caenorhabditis elegans

The nematode Caenorhabditis elegans exhibits a complex behavior called thermotaxis in response to temperature. This behavior is defined as a form of associative learning, in which temperature pairs with the presence or absence of food. Different interpretations have been drawn from the diverse results obtained by several groups, mainly because of the application of different methodologies for the analysis of thermotaxis. To clarify the discrepancies in behavioral observations and subsequent interpretations by different laboratories, we attempted to systematize several parameters to observe thermotaxis behavior as originally defined by Hedgecock and Russell in 1975. In this study, we show clearly how C. elegans can show a conditioned migration toward colder or warmer areas on a thermal gradient, given certain criteria necessary for the observation of thermotaxis. We thus propose to distinguish thermotaxis from other temperature-related behaviors, such as the warm avoidance response displayed at temperature gradients of 1°C/cm and steeper.     

Studies of synaptic vesicle endocytosis in the nematode C. elegans

After synaptic vesicle exocytosis, synaptic vesicle proteins must be retrieved from the plasma membrane, sorted away from other membrane proteins, and reconstituted into a functional synaptic vesicle. The nematode Caenorhabditis elegans is an organism well suited for a genetic analysis of this process. In particular, three types of genetic studies have contributed to our understanding of synaptic vesicle endocytosis. First, screens for mutants defective in synaptic vesicle recycling have identified new proteins that function specifically in neurons. Second, RNA interference has been used to quickly confirm the roles of known proteins in endocytosis. Third, gene targeting techniques have elucidated the roles of genes thought to play modulatory or subtle roles in synaptic vesicle recycling. We describe a molecular model for synaptic vesicle recycling and discuss how protein disruption experiments in C. elegans have contributed to this model.     

Caenorhabditis elegans integrates food and reproductive signals in lifespan determination

Dietary restriction extends lifespan and inhibits reproduction in many species. In Caenorhabditis elegans, inhibiting reproduction by germline removal extends lifespan. Therefore, we asked whether the effect of dietary restriction on lifespan might proceed via changes in the activity of the germline. We found that dietary restriction could increase the lifespan of animals lacking the entire reproductive system. Thus, dietary restriction can extend lifespan independently of any reproductive input. However, dietary restriction produced little or no increase in the long lifespan of animals that lack germ cells. Thus, germline removal and dietary restriction may potentially activate lifespan-extending pathways that ultimately converge on the same downstream longevity mechanisms. In well-fed animals, the somatic reproductive tissues are generally completely required for germline removal to extend lifespan. We found that this was not the case in animals subjected to dietary restriction. In addition, in these animals, loss of the germline could either further lengthen lifespan or shorten lifespan, depending on the genetic background. Thus, nutrient levels play an important role in determining how the reproductive system influences longevity.     

Endocrine targets for pharmacological intervention in aging in Caenorhabditis elegans

Studies in the nematode Caenorhabditis elegans have been instrumental in defining genetic pathways that are involved in modulating lifespan. Multiple processes such as endocrine signaling, nutritional sensing and mitochondrial function play a role in determining lifespan in the worm and these mechanisms appear to be conserved across species. These discoveries have identified a range of novel targets for pharmacological manipulation of lifespan and it is likely that the nematode model will now prove useful in the discovery of compounds that slow aging. This review will focus on the endocrine targets for intervention in aging and the use of C. elegans as a system for high throughput screens of compounds for their effects on aging.     

C.elegans中dyn-1基因对寿命和发育的影响

细胞极性对于细胞的多样性起着很重要的作用。发动蛋白是一个大的GTP酶,作用于胞吞作用和肌动蛋白的动力学过程。C.elegans中发动蛋白的同源基因dyn-1起着维持早期细胞极性的功能。我们对C.elegans中dyn-1基因进行了克隆,并构建到表达载体和RNAi载体中。经IPTG诱导表达得到了约90 kDa的DYN-1融合蛋白。同时,利用RNAi方法研究了dyn-1基因沉默后对三种线虫虫株N2、daf-2（e1370）和daf-16（e1038）寿命的影响。C.elegans在喂食dyn-1 RNAi食物后寿命明显缩短,也会导致严重的不育和胚胎致死。     

High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics

The phenomenon of RNA-mediated interference (RNAi) was first discovered in the nematode Caenorhabditis elegans, in which introduction of double-stranded RNA causes specific inactivation of genes with corresponding sequences. Technical advances in RNAi methodology and the availability of the complete genome sequence have enabled the high-throughput, genome-wide RNAi analysis of this organism. Several groups have used large-scale RNAi to systematically examine every C. elegans gene for knock-down phenotypes, providing basal information to be mined in more detailed studies. Now, in addition to functional genomic RNAi analyses, high-throughput RNAi is also routinely used for rapid, genome-wide screens for genes involved in specific biological processes. The integration of high-throughput RNAi experiments with other large-scale data, such as DNA microarrays and protein-protein interaction maps, enhances the speed and reliability of such screens. The accumulation of RNAi phenotype data dramatically accelerates our understanding of this organism at the genetic level.     

Glucose-induced abnormal egg-laying rate in Caenorhabditis elegans

High glucose reduced the egg-laying rate of the nematode Caenorhabditis elegans and was dependent on serotonergic signaling. Antidiabetic drugs of the biguanide and thiazolidine classes ameliorated the detrimental effect of glucose on egg-laying rate, suggesting the possibility that this quick and easy assay system may be applicable to whole-animal screening for novel antidiabetic drugs, at least, of these classes.     

CDC-25.1 regulates germline proliferation in Caenorhabditis elegans

The cell cycles in C. elegans are tightly controlled but appear to use the same regulators found in other organisms. Four homologues of the dual-specificity phosphatase Cdc25 are present in the C. elegans genome. In our study, we have characterized a deletion mutant for one of these orthologues. We show that embryonic defects are absent in cdc-25.1 homozygous mutants, presumably because of maternally contributed CDC-25.1 product. These embryos hatch and develop into sterile adults. The adults do not appear to have any somatic defects. The sterility results from inadequate germline proliferation. Germline precursors divide slowly and produce abnormally sized daughter cells. Only three to four rounds of germ-cell division occur before they die during the L3 and L4 larval stages.     

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.     

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.     

Longevity determined by developmental arrest genes in Caenorhabditis elegans

The antagonistic pleiotropy theory of aging proposes that aging takes place because natural selection favors genes that confer benefit early on life at the cost of deterioration later in life. This theory predicts that genes that impact development would play a key role in shaping adult lifespan. To better understand the link between development and adult lifespan, we examined the genes previously known to be essential for development. From a pool of 57 genes that cause developmental arrest after inhibition using RNA interference, we have identified 24 genes that extend lifespan in Caenorhabditis elegans when inactivated during adulthood. Many of these genes are involved in regulation of mRNA translation and mitochondrial functions. Genetic epistasis experiments indicate that the mechanisms of lifespan extension by inactivating the identified genes may be different from those of the insulin/insulin-like growth factor 1 (IGF-1) and dietary restriction pathways. Inhibition of many of these genes also results in increased stress resistance and decreased fecundity, suggesting that they may mediate the trade-offs between somatic maintenance and reproduction. We have isolated novel lifespan-extension genes, which may help understand the intrinsic link between organism development and adult lifespan.     

rBTI及其突变体对C. elegans寿命的影响

重组荞麦胰蛋白酶抑制剂（recombinant buckwheat trypsin inhibitor,rBTI）是一种来源于荞麦Potato Ⅰ抑制剂家族的丝氨酸蛋白酶抑制剂,具有很好的生物活性及功能。先前的研究表明,rBTI在秀丽隐杆线虫（Caenorhabditis elegans）中具有很好的延长寿命的性质,但其具体的作用机制还不太清楚。本文的研究证明,rBTI能够调节转录因子DAF-16的转录活性,进而影响线虫的寿命,且该性质与其胰蛋白酶抑制活性密切相关。通过定点突变技术,分别对rBTI的45位、53位和44位氨基酸活性位点进行突变,获得了4种不同胰蛋白酶抑制活性的rBTI突变体,分别命名为rBTI-R45A,rBTI-R45F,rBTI-W53R和rBTI-P44T。经典模式生物秀丽隐杆线虫寿命检测实验显示,野生型rBTI可以明显延长C.elegans的寿命,且在0～10 μmol/L 范围内具有浓度依赖性。和未处理对照组相比,10 μmol/L 野生型rBTI延长寿命幅度可达到14.5%,但是突变体rBTI-R45A,rBTI-R45F和rBTI W53R均不同程度失去了延长寿命的功能。利用荧光显微观察及qRT-PCR等方法进一步研究发现,野生型rBTI 可增强寿命调控转录因子DAF-16的转录活性。与寿命检测实验结果一致,4种 rBTI突变体均不能使DAF-16转录活性增强。上述结果表明,在C.elegans中,rBTI可增强长寿因子DAF 16的转录活性,进而延长虫体寿命,且该功能的发挥依赖于其适当的胰蛋白酶抑制活性。本文的结果为进一步研究开发rBTI的功能提供了实验支持和理论基础。     

Chemosensory behavior of semi‐restrained Caenorhabditis elegans

A new behavioral assay is described for studying chemosensation in the nematode Caenorhabditis elegans. This assay presents three main characteristics: (1) the worm is restrained by gluing, preserving correlates of identifiable behaviors; (2) the amplitude and time course of the stimulus are controlled by the experimenter; and (3) the behavior is recorded quantitatively. We show that restrained C. elegans display behaviors comparable to those of freely moving worms. Moreover, the chemosensory response of wild‐type glued animals to changes in salt concentration is similar to that of freely moving animals. This glued‐worm assay was used to reveal new chemosensory deficits of the potassium channel mutant egl‐2. We conclude that the glued worm assay can be used to study the chemosensory regulation of C. elegans behavior and how it is affected by neuronal or genetic manipulations. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Inhibitory effects of caffeine on gustatory plasticity in the nematode Caenorhabditis elegans

The effects of caffeine on salt chemotaxis learning were investigated using the nematode Caenorhabditis elegans. To estimate the degree of salt chemotaxis learning, nematodes were placed in a mixed solution of NaCl and caffeine, and then the chemotaxis index of NaCl was obtained from the nematodes placed on agar medium after pre-exposure to caffeine concentrations of 0.01, 0.1, 0.3, and 1.0%. Locomotor activity and preference behavior for caffeine were also estimated under these caffeine conditions. Nematodes pre-exposed to 0.3% caffeine showed inhibition of salt chemotaxis learning. Additional experiments indicated that nematodes showed a preference response to the middle concentration of caffeine (0.1%), with preference behavior declining in the 0.3% caffeine condition. Stable locomotor activity was observed under 0.01–0.3% caffeine conditions. These results suggest that salt chemotaxis learning with 0.3% caffeine is useful for investigating the effects of caffeine on learning in nematodes.     

A glial K+/Cl− cotransporter modifies temperature‐evoked dynamics in Caenorhabditis elegans sensory neurons

K⁺/Cl^? cotransporters (KCCs) are known to be crucial in the control of neuronal electrochemical Cl^? gradient. However, the role of these proteins in glial cells remains largely unexplored despite a number of studies showing expression of KCC proteins in glial cells of many species. Here, we show that the Caenorhabditis elegans K⁺/Cl^? cotransporter KCC‐3 is expressed in glial‐like cells and regulates the thermosensory behavior through modifying temperature‐evoked activity of a thermosensory neuron. Mutations in the kcc‐3 gene were isolated from a genetic screen for mutants defective in thermotaxis. KCC‐3 is expressed and functions in the amphid sheath glia that ensheathes the AFD neuron, a major thermosensory neuron known to be required for thermotaxis. A genetic analysis indicated that the regulation of the thermosensory behavior by KCC‐3 is mediated through AFD, and we further show that KCC‐3 in the amphid sheath glia regulates the dynamics of the AFD activity. Our results show a novel mechanism by which the glial KCC‐3 protein non‐cell autonomously modifies the stimulus‐evoked activity of a sensory neuron and highlights the functional importance of glial KCC proteins in modulating the dynamics of a neural circuitry to control an animal behavior.     

Regulation of Dauer Formation by O-GlcNAcylation in Caenorhabditis elegans

Modification of proteins at serine or threonine residues with N-acetylglucosamine, termed O-GlcNAcylation, plays an important role in most eukaryotic cells. To understand the molecular mechanism by which O-GlcNAcylation regulates the entry of Caenorhabditis elegans into the non-aging dauer state, we performed proteomic studies using two mutant strains: the O-GlcNAc transferase-deficient ogt-1(ok430) strain and the O-GlcNAcase-defective oga-1(ok1207) strain. In the presence of the dauer pheromone daumone, ogt-1 showed suppression of dauer formation, whereas oga-1 exhibited enhancement of dauer formation. Consistent with these findings, treatment of wild-type N2 worms with low concentrations of daumone and the O-GlcNAcase inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) enhanced dauer formation, which was dependent on intact O-GlcNAcylation metabolism. We also found that the treatment of daumone enhanced O-GlcNAcylation in vivo. Seven proteins, identified by coupled two-dimensional electrophoresis/liquid chromatography-mass spectroscopy (LC-MS) analysis, were differentially expressed in oga-1(ok1207) worms compared with wild-type N2 worms. The identities of these proteins suggest that O- GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. Using O-GlcNAc labeling with fluorescent dye combined with two-dimensional electrophoresis/LC-MS analysis, we also identified five proteins that were differentially O-GlcNAcylated during dauer formation. Analysis of these candidate O-GlcNAcylated proteins suggests that O-GlcNAcylation may regulate cytoskeleton modifications and protein turnover during dauer formation.