Exposure to mitochondrial genotoxins and dopaminergic neurodegeneration in Caenorhabditis elegans

Abstract

Neurodegeneration has been correlated with mitochondrial DNA (mtDNA) damage and exposure to environmental toxins, but causation is unclear. We investigated the ability of several known environmental genotoxins and neurotoxins to cause mtDNA damage, mtDNA depletion, and neurodegeneration in Caenorhabditis elegans. We found that paraquat, cadmium chloride and aflatoxin B1 caused more mitochondrial than nuclear DNA damage, and paraquat and aflatoxin B1 also caused dopaminergic neurodegeneration. 6-hydroxydopamine (6-OHDA) caused similar levels of mitochondrial and nuclear DNA damage. To further test whether the neurodegeneration could be attributed to the observed mtDNA damage, C. elegans were exposed to repeated low-dose ultraviolet C radiation (UVC) that resulted in persistent mtDNA damage; this exposure also resulted in dopaminergic neurodegeneration. Damage to GABAergic neurons and pharyngeal muscle cells was not detected. We also found that fasting at the first larval stage was protective in dopaminergic neurons against 6-OHDA-induced neurodegeneration. Finally, we found that dopaminergic neurons in C. elegans are capable of regeneration after laser surgery. Our findings are consistent with a causal role for mitochondrial DNA damage in neurodegeneration, but also support non mtDNA-mediated mechanisms.

Figure 1. Exposures to aflatoxin B₁, paraquat, cadmium chloride and 6-OHDA caused detectable DNA damage.

p<.0001, p<.0001, p = 0.028, p<.0001 respectively for main effects of dose. Aflatoxin B₁, paraquat and cadmium chloride caused more damage to the mitochondrial than nuclear genomes (p<.0001, p<.0001, and p = 0.0125 respectively for main effect of genome). Data analyzed by Two-way ANOVA. Bars ± SEM.

Figure 2. Manganese chloride and cadmium chloride resulted in a decrease in the relative mtDNA:nDNA ratio.

Ratio defined as 100% in controls. For MnCl₂, one-way ANOVA, effect of concentration p = 0.03; Fisher's PLSD, p = 0.0231 for 7.5 mM and p = 0.0015 for 25 mM. For CdCl₂, one-way ANOVA, effect of concentration p = 0.0091; Fisher's PLSD, p = 0.0032 for 1 mM. Bars ± SEM.

Figure 3. Representation of progressive damage in dopaminergic neurons after 6-OHDA exposure.

The circled areas include typically observed abnormalities, referred to as blebs and breaks in this publication.

Figure 4. Fasting protects against 6-OHDA–induced dopaminergic neurodegeneration.

Neuronal damage was scored from 0 (lowest) to 2.5 (highest) and assessed statistically using the Fisher's Exact Test.

Figure 5. Damage to dopaminergic neurons caused by laser ablation is repaired.

A, After laser ablation, 81% of vtIs7[Pdat-1::GFP] worms showed neuronal anterior growth, compared to 17% of the vtIs7[Pdat-1::GFP];mkk-4(ju91) worms tested (p = 0.0016, Fisher's Exact Test, bars represent 95% CI). B, From left to right, images are: uncut (with dashed line representing cut site), no response, and response. Scale bars are 10 µm.