Drp-1-Dependent Mitochondrial Fragmentation Contributes to Cobalt Chloride-Induced Toxicity in Caenorhabditis elegans

Abstract

Excess cobalt may lead to metallosis, characterized by sensorineural hearing loss, visual, and cognitive impairment, and peripheral neuropathy. In the present study, we sought to address the molecular mechanisms of cobalt-induced neurotoxicity, using Caenorhabditis elegans as an experimental model. Exposure to cobalt chloride for 2 h significantly decreased the survival rate and lifespan in nematodes. Cobalt chloride exposure led to increased oxidative stress and upregulation of glutathione S-transferase 4. Consistently, its upstream regulator skn-1, a mammalian homolog of the nuclear factor erythroid 2-related factor 2, was activated. Among the mRNAs examined by quantitative real-time polymerase chain reactions, apoptotic activator egl-1, proapoptotic gene ced-9, autophagic (bec-1 and lgg-1), and mitochondrial fission regulator drp-1 were significantly upregulated upon cobalt exposure, concomitant with mitochondrial fragmentation, as determined by confocal microscopy. Moreover, drp-1 inhibition suppressed the cobalt chloride-induced reactive oxygen species generation, growth defects, and reduced mitochondrial fragmentation. Our novel findings suggest that the acute toxicity of cobalt is mediated by mitochondrial fragmentation and drp-1 upregulation.

Keywords: cobalt; drp-1; mitochondria fragmentation; oxidative stress; toxicity.

Figure 1.

CoCl₂ exposure induces lethality and oxidative stress in Caenorhabditis elegans. A, L1 nematodes were exposed to CoCl₂ at various doses for 2 h. N = 3 with more than 30 animals per plate. Individual experiment was repeated for 5 times. B, L1 animals exposed to CoCl₂ (0, 2.5, 5, and 10 mM), were detected by 2′,7′-dichlorodihydrofluorescein diacetate/2′,7′-dichlorofluorescein assay. Fluorescence was followed every 0.5 h for 6 h, with representative 2 and 4 h shown here (N = 3). Individual experiments were independently repeated for 3 times. *p < .05 compared with 0 mM CoCl₂. ^#p < .05 compared with 5 mM Co group.

Figure 2.

Oxidative stress defense system is turned on by CoCl₂. A, Worms with oxidative stress inducible gst-4p::GFP were exposed to CoCl₂ for 2 h and detected by confocal microscopy. Bar indicates 50 µm. B, Fluorescent intensities of at least 30 animals were quantified by Fiji. C, Skn-1 mRNA, the activator of gst-4, is increased (N = 3 with 10 000 animals per sample). Individual experiments were independently repeated for 4 times. *p < .05 compared with 0 mM CoCl₂.

Figure 3.

Apoptotic, autophagic, and mitochondrial morphologic regulators are activated upon CoCl₂ exposure. mRNAs were isolated from L1 animals exposed to CoCl₂ (0, 2.5, 5, and 10 mM) for 2 h. Apoptosis, autophagy, and mitochondrial morphology-associated genes were examined by quantitative real-time polymerase chain reaction. mRNA levels were normalized to tba-1 (N = 3, 10 000 animals per sample). Individual experiments were independently repeated for 4 times. *p < .05 compared with 0 mM CoCl₂. Experiments were performed in L4 animals with analogous results.

Figure 4.

CoCl₂ induces mitochondrial fission. L4 nematodes were exposed to CoCl₂ at various doses for 2 h, then examined by fluorescent microscopy (N > 30). Mitochondrial morphology was classified into 3 categories: tubular, intermediate, and fragmented. All exposure groups are significantly different to control group (p < .01), examined by Chi-square test. Individual experiments were independently repeated for 3 times.

Figure 5.

Inhibition of mitochondrial fission regulator DRP1 rescues cell growth and extends life span in nematodes upon CoCl₂ exposure. L4 drp-1 (tm1108) null mutant strain was exposed to CoCl₂ of various doses for 2 h, in line with WT strain. A, Drp-1 inhibition rescued lethality in 50 mM CoCl₂ exposure group (N = 3, at least 30 animals per replicate). *p < .05 compared with unexposed nematodes. ^#p < .05 compared WT-CoCl₂ with drp-1-CoCl₂. B, Drp-1 suppression extended lifespan in response to 10 mM CoCl₂ exposure (N = 106, 105, 108, and 107 for WT-0, WT-10, drp-1-0, and drp-1-10, respectively). *p < .0001 compared with unexposed nematodes. ^#p = .0042 compared WT-CoCl₂ with drp-1-CoCl₂, examined by log-rank method (Mantel-Cox method). Individual experiments were independently repeated for 3 times for survival and 4 times for lifespan.

Figure 6.

Reduced mitochondrial fission and elevated reactive oxygen species production in drp-1 (tm1108) mutant upon CoCl₂ exposure. A, WT and drp-1 mutant strains were stained with MitoTracker red CMXRos overnight, followed by CoCl₂ exposure of 0, 10, and 25 mM. Mitochondrial morphology was examined using fluorescent microscopy. Bar indicates 5 µm. The lower panel shows the enlarged areas as boxed in the upper panel within each strain (N > 30). B, Quantification of mitochondrial morphology to tubular, intermediate, and fragmented. WT-10 and WT-25 are significantly different to WT-0, whereas drp-1-25 is significantly different to WT-0 and drp-1-0 (p < .01), examined by Chi-square test. Individual experiments were independently repeated for 3 times. C, L4 animals exposed to CoCl₂ (0, 10, and 25 mM), were detected by 2′,7′-dichlorodihydrofluorescein diacetate/2′,7′-dichlorofluorescein assay. Fluorescence was followed with 2 h RLU data shown here (cobalt-untreated group was set to 1) (N = 3). Individual experiments were independently repeated in triplicates. *p < .05 compared with 0 mM CoCl₂.

Figure 7.

A model for CoCl₂-induced toxicity in C. elegans. CoCl₂ induces oxidative stress, followed by altered apoptosis, increased lethality, and reduced lifespan. Increased reactive oxygen species (ROS) generation leads to skn-1 (Nrf2 homolog) activation and its downstream target gst-4, corroborating the involvement of oxidative stress defense systems (in green). CoCl₂ upregulates drp-1 and induces mitochondrial fragmentation. Mitochondrial impairment is likely caused by oxidative stress from ROS generation. When drp-1 is inhibited, mitochondrial fragmentation and ROS generation are suppressed, rescuing the CoCl₂-induced growth defects.