| Background:Caenorhabditis elegans is a classic model organism which has been widely used in life science since the last century.It has many advantages that other model animals do not have,such as convenient culture conditions,short life cycle,transparent body,constant number of cells and even every cell can trace origin source of its occurrence.The study of the neuroligin gene(NLG-1)can help us have some insight into Neuroligins famliy and their biological functions.As an important component of the synapses,it is associated with various neuropsychiatric disorders.Graphene oxide(GO)is a derivative of graphene,and has some special and excellent physical and chemical properties.It has been widely used in many fields,such as drug carriers,biosensors,and biological imaging,and so on.There are some in vitro and in vivo evidences that GO is potentially toxic to organisms.However,the underlying mechanism for the neuronal regulation of GO toxicity is still not clear.The aim of this study is to investigate the function of NLG-1 in the regulation of GO toxicity and the underlying mechanisms,which will help us understand the mechanism of neural regulation of GO toxicity.At the same time,this study will provide some suggestions for the biomedical use of nanomaterials in special populations.Methods:The loss-of-fucntion mutants or RNAi nematodes was exposed to GO.Intestinal ROS productions and locomotion behaviors was used as endpoints to investigate the function of specific genes in the regulation of GO toxicity.Genetic methods are used to overexpress,knockdown or rescue genes in widetype or mutant nematodes.Genetically modified nematodes was exposed to GO and its toxic effect was analzyed to investigate the functional neurons or determine the upstream and downstream targeting relationship of genes in the regulation of GO toxicity.qRT-PCR,GFP fluorescence intensity analysis,bioinformatics analysis,COIP,and Western Blot were used to determine the molecular mechanism.RhoB,a kind of molecular probe,was used to examine the translocation and distribution of GO in nematodes.We also established a behavioral model to explore the perceptive and avoidant behavior of Caenorhabditis elegans to GO.Results:1.Graphene oxide dysregulates neuroligin/NLG-1-mediated molecular signaling in interneurons in Caenorhabditis elegansUsing assay system of Caenorhabditis elegans,we identifed the NLG-1/Neuroligin-mediated neuronal signaling dysregulated by GO exposure.In nematodes,GO exposure signifcantly decreased the expression of NLG-1,a postsynaptic cell adhesion protein.Loss-of-function mutation of nlg-1 gene resulted in a susceptible property of nematodes to GO toxicity.Rescue experiments suggested that NLG-1 could act in AIY interneurons to regulate the response to GO exposure.In the AIY interneurons,PKC-1,a serine/threonine protein kinase C(PKC)protein,was identifed as the downstream target for NLG-1 in the regulation of response to GO exposure.LIN-45,a Raf protein in ERK signaling pathway,was further identifed as the downstream target for PKC-1 in the regulation of response to GO exposure.Therefore,GO may dysregulate NLG-1-mediated molecular signaling in the interneurons,and a neuronal signaling cascade of NLG-1-PKC-1-LIN-45 was raised to be required for the control of response to GO exposure.More importantly,intestinal RNAi knockdown of daf-16 gene encoding a FOXO transcriptional factor in insulin signaling pathway suppressed the resistant property of nematodes overexpressing NLG-1 to GO toxicity,suggesting the possible link between neuronal NLG-1 signaling and intestinal insulin signaling in the regulation of response to GO exposure.2.Postsynaptic complex molecules DLG-1 and MAGI-1 bind to NLG-1 and act as downstream targets of NLG-1 to regulate the response to GO exposure.We rescued the expression of NLG-1 in the neurons or muscles of nlg-1(ok259)by using tissues-specific promoter Punc-14 or Pmyo-3,and analyzed the GO toxicity.It implied that NLG-1 regulates GO toxicity only in neurons of nematodes.Using bioinformatics analysis,dlg-1,lin-2,magi-1,nmr-1,nmr-2 were identified as homologous of postsynaptic complex molecules in mammals.These mutants or RNAi nematodes were exposed to GO,and the toxic effects were analzyed.Among these,only dlg-1 RNAi or mutation of magi-1 gene resulted in a susceptibility of nematodes to GO toxicity.Moreover,dlg-1 RNAi or mutation of magi-1 gene obviously suppressed the resistance of transgenic nematodes overexpressing NLG-1 in neurons to GO toxicity.The potential binding domains of DLG-1 or MAGI-1 were analyzed,and we expressed these domains in prokaryotic expression system.By coexpressing NLG-1-C terminal with GST-tag,and coexpressing candidate binding domains with 6×His-tag,COIP was performed.We identified DLG-1-PDZ-3,MAGI-1-WW(1-2)and MAGI-1-PDZ-3 could bind to NLG-1-C terminal.Moreover,by adding GO to COIP system,we found that GO could suppress the binding ratio of DLG-1-PDZ-3,MAGI-1-WW(1-2),MAGI-1-PDZ-3 to NLG-1-C terminal domain.3.Identification of neurons regulating the avoidance behavior of nematodes to GOBased on the established behavioral model,it was found that wild-type nematodes exhibited behavioral avoidance to GO.Mutations of nlg-1 gene(nlg-1(ok259)or nlg-1(tm474))exhibited defective in the avoidance to GO.Neuron-specific expression of NLG-1 in AIB or AIY interneurons in nlg-1(ok259)could rescue the avoidance defect,implying that AIB or AIY interneurons could regulate the behavioral avoidance to GO in Caenorhabditis elegans.ConclusionNeuronal adhesion molecule NLG-1 plays an important role in the regulation of GO toxicity in C.elegans.The NLG-1-PKC-1-LIN-45 signaling cascade is raised in the AIY interneurons to be required for induction of GO toxicity.Direct binding between NLG-1 and DLG-1 or between NLG-1 and MAGI-1 is required for regulation of GO toxicity.Moreover,AIB or AIY interneurons could regulate the avoidance to GO.Our results will provide an important neuronal basis for the induction of GO toxicity in organisms. |
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