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

Abstract

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).

Figure 2

Effects of neuronal overexpression of mir-247 on GO toxicity in nematodes overexpressing neuronal goa-1 or pkc-1 lacking 3′-UTR. (a) Effects of neuronal overexpression of mir-247 on GO toxicity in inducing intestinal ROS production in nematodes overexpressing neuronal goa-1 or pkc-1 lacking 3′-UTR. (b) Effects of neuronal overexpression of mir-247 on GO toxicity in decreasing locomotion behavior in nematodes overexpressing neuronal goa-1 or pkc-1 lacking 3′-UTR. 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.

Figure 3

Tissue-specific activity of goa-1 in the regulation of GO toxicity in nematodes. (a) Tissue-specific activity of goa-1 in the regulation of GO toxicity in inducing intestinal ROS production. (b) Tissue-specific activity of goa-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 goa-1.

Figure 4

Effects of dgk-1 mutation on GO toxicity in nematodes. (a) Effect of goa-1 mutation on expressions of pkc-1 and dgk-1 in GO exposed nematodes. Bars represent means ± SD. ^**P < 0.01 vs wild-type (GO). (b) Effect of dgk-1 mutation on GO toxicity in inducing intestinal ROS production. Bars represent means ± SD. ^**P < 0.01 vs wild-type. (c) Effect of dgk-1 mutation on 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.

Figure 5

Genetic interaction between GOA-1 and PKC-1 or DGK-1 in the regulation of GO toxicity. (a) Genetic interaction between GOA-1 and PKC-1 or DGK-1 in the regulation of GO toxicity in inducing intestinal ROS production. (b) Genetic interaction of GOA-1 and PKC-1 or DGK-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 Is (Punc-14-goa-1).

Figure 6

Genetic interaction of PKC-1 and DGK-1 in the regulation of GO toxicity. (a) Genetic interaction of PKC-1 and DGK-1 in the regulation of GO toxicity in inducing ROS production. 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. (b) Genetic interaction of PKC-1 and DGK-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. (c) A diagram showing the molecular basis for neuronal Gαo signaling in the regulation of GO toxicity in nematodes. A neuronal signaling cascade of mir-247-GOA-1-DGK-1/PKC-1 was raised to explain the molecular mechanism for GO toxicity induction in nematodes.