Dysregulation of Neuronal Gαo Signaling by Graphene Oxide in Nematode Caenorhabditis elegans

Exposure to graphene oxide (GO) induced some dysregulated microRNAs (miRNAs), such as the increase in mir-247, in nematode Caenorhabditis elegans. We here further identified goa-1 encoding a Gαo and pkc-1 encoding a serine/threonine protein kinase as the targets of neuronal mir-247 in the regulation of GO toxicity. GO exposure increased the expressions of both GOA-1 and PKC-1. Mutation of goa-1 or pkc-1 induced a susceptibility to GO toxicity, and suppressed the resistance of mir-247 mutant to GO toxicity. GOA-1 and PKC-1 could also act in the neurons to regulate the GO toxicity, and neuronal overexpression of mir-247 could not affect the resistance of nematodes overexpressing neuronal goa-1 or pkc-1 lacking 3′-UTR to GO toxicity. In the neurons, GOA-1 acted upstream of diacylglycerol kinase/DGK-1 and PKC-1 to regulate the GO toxicity. Moreover, DGK-1 and GOA-1 functioned synergistically in the regulation of GO toxicity. Our results highlight the crucial role of neuronal Gαo signaling in response to GO in nematodes.


Figure 1.
Genetic interaction between mir-247 and goa-1 or pkc-1 in the regulation of GO toxicity. (a) Genetic interaction between mir-247 and goa-1 or pkc-1 in the regulation of GO toxicity in inducing intestinal ROS production. (b) Genetic interaction between mir-247 and goa-1 or pkc-1 in the regulation of GO toxicity in decreasing locomotion behavior. GO exposure concentration was 10 mg/L. Prolonged exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. ** P < 0.01 vs wild-type (if not specially indicated).
Neuronal overexpression of mir-247 could not affect the resistance of nematodes overexpressing neuronal goa-1 or pkc-1 lacking 3′-UtR to Go toxicity. To further confirm the roles of GOA-1 and PKC-1 as the target of neuronal mir-247, we next investigated the genetic interaction between mir-247 and goa-1 or pkc-1 in the neurons to regulate the GO toxicity. We introduced the goa-1 or pkc-1 lacking 3′-UTR driven by unc-14 promoter into the nematodes overexpressing neuronal mir-247. After GO exposure, the transgenic strain Is(Punc-14-goa-1-3′-UTR);Ex(Punc-14-mir-247) exhibited the similar resistance to GO toxicity to that in the transgenic strain Is(Punc-14-goa-1-3′-UTR) (Fig. 2). Additionally, the transgenic strain Is(Punc-14-pkc-1-3′-UTR);Ex(Punc-14-mir-247) showed the similar resistance to GO toxicity to that in the transgenic strain Is(Punc-14-pkc-1-3′-UTR) (Fig. 2). www.nature.com/scientificreports www.nature.com/scientificreports/ Tissue-specific activity of goa-1 in the regulation of Go toxicity. goa-1 gene is expressed in the pharynx, the neurons, and the muscle 42,43 . pkc-1 is only expressed in the neurons 44 . Using tissue-specific promoters, we investigated the tissue-specific activity of goa-1 in the regulation of GO toxicity. Rescue assay by expression of goa-1 in the pharynx or the muscle did not significantly influence the susceptibility of goa-1(sa734) mutant to GO toxicity (Fig. 3). Different from these, expression of goa-1 in the neurons could significantly suppress the susceptibility of goa-1(sa734) mutant to GO toxicity (Fig. 3). These results suggest that both GOA-1 and PKC-1 may act in the neurons to regulate the GO toxicity.

Identification of downstream targets for GOA-1 in the Gαo signaling pathway in the regulation of Go toxicity.
In the Gαo signaling pathway, DGK-1 is a downstream target for GOA-1, and dgk-1 encodes an ortholog of mammalian diacylglycerol kinase theta (DGKQ) 45 . In GO (10 mg/L) exposed goa-1 mutant, we detected the significant decrease in expressions of both pkc-1 and dgk-1 compared with those in GO (10 mg/L) exposed wild-type nematodes (Fig. 4a), which implies that both PKC-1 and DGK-1 may act as important downstream targets for GOA-1 during the control of GO toxicity.
Using induction of intestinal ROS production and locomotion behavior as the toxicity assessment endpoints, we found that the dgk-1(sy428) mutant was susceptible to GO toxicity (Fig. 4b,c), suggesting that GOA-1 may positively regulate GO toxicity by affecting functions of PKC-1 and DGK-1.

Genetic interaction between GOA-1 and PKC-1 or DGK-1 in the regulation of GO toxicity.
To determine the genetic interaction between goa-1 and dgk-1 or pkc-1 in the regulation of GO toxicity, we examined the effects of mutation of dgk-1 or pkc-1 on GO toxicity in transgenic strain overexpressing the neuronal goa-1. The nematodes overexpressing neuronal goa-1 exhibited the resistance to GO toxicity (Fig. 5). In contrast, after the GO exposure, dgk-1 or pkc-1 mutation suppressed the resistance of nematodes overexpressing neuronal goa-1 to GO toxicity (Fig. 5). Therefore, neuronal GOA-1 may act upstream of both DGK-1 and PKC-1 to regulate the GO toxicity. www.nature.com/scientificreports www.nature.com/scientificreports/ Genetic interaction between PKC-1 and DGK-1 in the regulation of GO toxicity. We further investigated the genetic interaction between the PKC-1 and DGK-1. After GO exposure, we observed the more severe GO toxicity in double mutant of pkc-1(ok563);dgk-1(sy428) compared with that in single mutant of pkc-1(ok563) or dgk-1(sy428) (Fig. 6a,b).

Discussion
In this study, we first provide several lines of evidence to indicate the potential role of GOA-1 and PKC-1 as the targets for neuronal mir-247 in the regulation of GO toxicity. First of all, expressions of both GOA-1 and PKC-1 could be suppressed by GO exposure, and their expressions could be further decreased by overexpression of neuronal mir-247 in GO exposed nematodes (Fig. S1). Secondly, in nematodes, the phenotypes in GO exposed goa-1(sa734) or pkc-1(ok563) mutant were opposite to those in GO exposed mir-247/797(n4505) mutant (Fig. S2). Thirdly, we found that mutation of goa-1 or pkc-1 could effectively inhibit the resistance of mir-247/797(n4505) mutant to GO toxicity (Fig. 1). Moreover importantly, we observed that neuronal overexpression of mir-247 did not influence the resistance of transgenic strain overexpressing neuronal goa-1 lacking 3′-UTR or pkc-1 lacking 3′-UTR to GO toxicity (Fig. 2), implying the binding of mir-247 to the 3-UTR of goa-1 or pkc-1. Previous study has identified the EGL-5 as the target for mir-247 in the control of male tail development 46 . In this study, we identified the GOA-1 and the PKC-1 as the potential targets for mir-247 during the control of GO toxicity formation in hermaphrodite nematodes.
GOA-1 activity is required for the regulation of asymmetric cell division in the early embryo, innate immunity, olfactory-mediated behaviors, and decision-making 42,43,47,48 . In this study, we further found the novel function of Gαo signaling in the control of nanotoxicity. In C. elegans, goa-1 mutation induced a susceptibility of nematodes to GO toxicity (Fig. S2), implying that goa-1-encoded Gαo signaling negatively regulates GO toxicity.
The tissue-specific activity assays indicated that the neuronal GOA-1 regulates the GO toxicity (Fig. 3). In organisms, G protein coupled receptors (GPCRs), seven-transmembrane receptors, can sense the environmental signals or molecules outside the cell and activate the inside signal transduction pathways and, ultimately, the cellular responses by coupling with the G proteins 49 . The function of goa-1-encoded Gαo signaling in the neurons www.nature.com/scientificreports www.nature.com/scientificreports/ implies that certain GPCRs in the neurons may be activated or suppressed by GO exposure, and the affected neuronal GPCRs may further function through the goa-1/Gαo-mediated signaling cascade to regulate the GO toxicity.
In this study, GOA-1 could further act upstream of diacylglycerol kinase/DGK-1 and PKC-1 to regulate the GO toxicity. Under the condition of GO exposure, goa-1 mutation decreased dgk-1 and pkc-1 expressions (Fig. 4a). Additionally, dgk-1 or pkc-1 mutation inhibited the resistance of transgenic strain overexpressing neuronal goa-1 to GO toxicity (Fig. 5). dgk-1 gene is expressed in most of the neurons. Therefore, a corresponding signaling cascade of GOA-1-DGK-1/PKC-1 can be raised to explain the molecular basis for neuronal mir-247 in response to GO exposure (Fig. 6c).
Prolonged exposure to GO (≥10 μg/L) increased the mir-247 expression 18 . Meanwhile, neuronal overexpression of mir-247 induced a susceptibility to GO toxicity 18 . Therefore, the raised neuronal signaling cascade of mir-247-GOA-1-DGK-1/PKC-1 provides an important molecular mechanism for the potential GO toxicity induction in nematodes.
In this study, we further found that DGK-1 and PKC-1 functioned synergistically to regulate GO toxicity (Fig. 6a,b). PKC-1 plays a role in regulating function of nervous system, such as the neurotransmission 50 . This observation implies the possibility that, besides the normally considered downstream diacylglycerol kinase/ DGK-1 signaling, the neuronal GOA-1/Gαo signaling may also regulate the GO toxicity by influencing the neurotransmission process. Our previous study has identified the NLG-1-PKC-1 signaling cascade in the regulation of GO toxicity 39 . Our results suggest that PKC-1 may act as an important link between the Gαo/GOA-1 signaling and the NLG-1 signaling in the regulation of GO toxicity. Additionally, PKC-1 may further act as the direct target for mir-247 in the regulation of GO toxicity (Fig. 2). These results imply the potential crucial role of neurotransmission process in the toxicity induction in GO exposed nematodes.
Head thrash and body bend were used to assess the locomotion behavior. The method was performed under the dissecting microscope by eyes as described previously 6,53 . Fifty nematodes were examined per treatment.
Reverse-transcription and quantitative real-time polymerase chain reaction (PCR). Total RNA was isolated from the nematodes using Trizol reagent (Invitrogen, UK) according manufacturer's protocol. Purity and concentration of RNA were evaluated by a ratio of OD260/280 using a spectrophotometer. The extracted RNA was used for the cDNA synthesis. After the cDNA synthesis, the relative expression levels of targeted genes were determined by real-time PCR in an ABI 7500 real-time PCR system with Evagreen (Biotium, USA). All reactions were performed in triplicate. Relative quantification of targeted gene was expressed as the ratio (targeted gene/reference gene tba-1 encoding a tubulin). The related primer in formation is shown in Table S1.

DNA constructs and germline transformation.
To generate entry vector carrying promoter sequence, promoter region for myo-2 gene specially expressed in pharynx, promoter region for myo-3 gene specially expressed in muscle, or promoter region for unc-14 gene specially expressed in neurons was amplified by PCR from wild-type C. elegans genomic DNA. The promoter fragment was inserted into pPD95_77 vector in the sense orientation. goa-1/C26C6.2.1 cDNA containing or lacking 3′-UTR was amplified by PCR, and inserted into corresponding entry vector carrying the myo-2, myo-3, or unc-14 promoter sequence. Transformation was performed by coinjecting testing DNA (10-40 μg/mL) and marker DNA of Pdop-1::rfp (60 μg/mL) into the gonad of nematodes as described 55 . The related primer information for DNA constructs was shown in Table S2.

RNA interference (RNAi).
RNAi assay was performed basically as described 54 . The nematodes were fed with E. coli strain HT115 (DE3) expressing double-stranded RNA for the examined gene. After grown in LB broth containing ampicillin (100 μg/mL), E. coli HT115 (DE3) expressing double-stranded RNA for the examined gene was plated onto NGM containing ampicillin (100 μg/mL) and isopropyl 1-thio-β-D-galactopyranoside (IPTG, 5 mM). L1 larvae were transferred onto certain RNAi plates until the nematodes became the gravid. The gravid adults were transferred to fresh RNAi-expressing bacterial lawns to let them lay eggs to obtain the second generation of RNAi population. The eggs were allowed to develop into L1-larvae for the toxicity assessment. statistical analysis. Data in this article were expressed as means ± standard deviation (SD). Statistical analysis was performed using SPSS 12.0 software (SPSS Inc., Chicago, USA). Differences between groups were determined using analysis of variance (ANOVA), and probability levels of 0.05 and 0.01 were considered statistically significant.