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. 2010 Mar;10(2):125-36.
doi: 10.1016/j.mito.2009.11.003. Epub 2009 Nov 10.

Mitochondrial respiratory chain dysfunction variably increases oxidant stress in Caenorhabditis elegans

Stephen Dingley  1 , Erzsebet Polyak, Richard Lightfoot, Julian Ostrovsky, Meera Rao, Todd Greco, Harry Ischiropoulos, Marni J Falk
Affiliations

Mitochondrial respiratory chain dysfunction variably increases oxidant stress in Caenorhabditis elegans

Stephen Dingley et al. Mitochondrion. 2010 Mar.

Abstract

Mitochondrial dysfunction and associated oxidant stress have been linked with numerous complex diseases and aging largely by in vitro determination of mitochondria oxidant production and scavenging. We applied targeted in vivo fluorescence analyses of mitochondria-dense pharyngeal tissue in Caenorhabditis elegans to better understand relative mitochondrial effects, particularly on matrix oxidant burden, of respiratory chain complex, MnSOD, and insulin receptor mutants displaying variable longevity. The data demonstrate significantly elevated in vivo matrix oxidant burden in the short-lived complex I mutant, gas-1(fc21), which was associated with limited superoxide scavenging capacity despite robust MnSOD induction, as well as decreased mitochondria content and membrane potential. Significantly increased MnSOD activity was associated with in vivo matrix oxidant levels similar to wild-type in the long-lived respiratory chain complex III mutant, isp-1(qm150). Yet, despite greater superoxide scavenging capacity in the complex III mutant than in the significantly longer-lived insulin receptor mutant, daf-2(e1368), only the former showed modest oxidative stress sensitivity. Furthermore, increased longevity was seen in MnSOD knockout mutants (sod-2(ok1030) and sod-2(gk257)) that had decreased MnSOD scavenging capacity and increased in vivo matrix oxidant burden. Thus, factors beside oxidant stress must underlie RC mutant longevity in C. elegans. This work highlights the utility of the C. elegans model as a tractable means to non-invasively monitor multi-dimensional in vivo consequences of primary mitochondrial dysfunction.

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Figures

Figure 1
Figure 1. Nematode terminal pharyngeal bulb labeling with mitochondria-targeted dyes
a) Light micrograph of young adult wild-type (N2) nematode, where cephalad portion (red circle) highlights the two pharyngeal bulbs (m, metacorpus and tb, terminal bulb) and initial portion of the gastrointestinal tract (g); b) MitoSOX fluorescence in N2 demonstrates consistent labeling within the C. elegans pharynx, as well as in lipid-rich gut granules that non-specifically bind lipophilic dyes. The top worm shows nuclear sparing of the mitochondria-localized dye both in the terminal pharyngeal bulb (tb) and in cells of the gastrointestinal tract (g); c) MitoSOX fluorescence of N2 pharynx overlay with DIC image demonstrates preferential labeling in terminal pharyngeal bulb; d) Threshholding permits focused analysis of terminal pharyngeal bulb (tb) fluorescence. Similar fluorescent patterns were observed by confocal microscopy maximal projection images whether animals are fed: e) MitoTracker Green in N2, f) MitoTracker Red in N2, g) Overlay of e and f, h) Typical MitoSOX terminal pharyngeal bulb fluorescent pattern in sod-3 knockout mutant, i) 1000x magnification image taken with CY3 filter of N2 fed MitoTracker Red demonstrated individual cytoplasmic vesicle labeling on the order of ~800 nm diameter fluorescent foci.
Figure 2
Figure 2. In vivo relative oxidant quantitation in C. elegans terminal pharyngeal bulbs
Steady-state oxidant levels in young adult nematodes at baseline as determined by mean terminal pharyngeal bulb (PB) fluorescence at 160X magnification at baseline and following 24 hour exposure to 10 uM MitoSOX Red (“S” = baseline, blue bars) ± 1 mM paraquat (“S+P” = oxidant stress, red bars) at 20°C. Steady-state oxidant levels were significantly increased only in the complex I and both MnSOD paralog knockout mutants. With paraquat exposure, in vivo relative oxidant levels rose significantly in all three RC mutants (gas-1, mev-1, isp-1). Statistical analyses were performed by ANOVA, where ** p<0.01, *** p<0.001). Box length represents 25th to 75th percentile inter-quartile range, interior horizontal line represents median, vertical lines issuing from the box extend to minimum and maximum values of the adjacent analysis variable [calculated as third quartile ± (1.5*interquartile range)], open red circles indicate outliers defined as values exceeding the upper adjacent level, and asterisks represent severe outliers (exceed 3 box lengths above the upper adjacent level). N, total animal number studied over three replicate experiments per strain, as indicated within boxes. sod-2 strain presented is sod-2(gk235).
Figure 3
Figure 3. In vivo quantitation of relative membrane potential and mitochondria density in C. elegans terminal pharyngeal bulbs
A) Mitochondria membrane potential (ΔΨm) in living nematodes at baseline as determined by mean fluorescence at 160X magnification following 24 hour exposure to 1 uM TMRE at 20°C. ΔΨm was most dramatically reduced relative to wild-type (N2) in the complex I mutant (gas-1, mean PB fluorescence decreased by 63%). Mean TMRE PB fluorescence was also substantially reduced in the complex III mutant (isp-1) and both sod-2 mutants, but was only decreased by 12% in the complex II mutant (mev-1) and by 9% in the insulin receptor mutant (daf-2). Statistical analyses were performed by ANOVA, where *** p<0.001). Box plot components indicate values as detailed in Figure 2 legend. N, total animal numbers studied across three replicate experiments per strain, as indicated within boxes. B) Decreased mitochondria density in complex I mutant worms is suggested by a 48% decrease in mean PB fluorescence intensity in gas-1 relative to N2 following 24 hour exposure of synchronous young adult worms to 10 uM MitoTracker Green FM dye, whose mitochondria localization is largely independent of membrane potential. Statistical analysis by student’s t-test, where *** p<0.001. N, animal number studied per strain. PB, pharyngeal bulb.
Figure 4
Figure 4. Whole worm MnSOD expression and protein levels in mitochondria mutants
A) Whole worm MnSOD total RNA levels were measured by qPCR. sod-3 upregulation dramatically exceeded sod-2 upregulation in primary RC dysfunction, both at baseline and following oxidant stress. B) MnSOD western blot analysis in isolated mitochondria from RC mutant strains confirmed that total MnSOD protein is upregulated (22kDa band) in RC mutants (complex I, gas-1; complex II, mev-1, and complex III, isp-1). By contrast, MnSOD protein was not increased in the insulin receptor mutant (daf-2), despite its 4-fold increased sod-2 transcript level (Figure 4A, above). MnSOD protein levels were diminished relative to N2 in the sod-3 MnSOD knockout mutant, suggesting both sod-2 and sod-3 contribute to total MnSOD protein levels. C) MnSOD western blot analysis in isolated mitochondria from three distinct knockout alleles of two sod-2 paralogs again demonstrated decreased MnSOD in an independent mitochondria preparation of sod-3(gk235), mutant as well as decreased MnSOD in the sod-2(gk257) compound heterozygous deletion/insertion allele. However, MnSOD protein levels were increased relative to wild-type in the long-lived sod-2(ok1030) homozygous deletion allele. Relative changes are reported in Table 2A.
Figure 5
Figure 5. MnSOD enzyme activity assessment in isolated mitochondria of RC mutants
Only the complex III mutant (isp-1) had significantly elevated MnSOD enzyme activity in isolated mitochondria. Trends toward upregulation observed in gas-1, daf-2, and sod-3 did not reach statistical significance, perhaps because of small sample size. No discrimination was made by this assay between protein encoded by sod-2 or sod-3. Bars represent mean ± standard deviation of two to five biological replicates per strain. Single mitochondria preparations were assessed for each of the sod-2 knockout alleles, which demonstrated similar reductions in MnSOD activity relative to N2. *, p < 0.05. Percent change in each mutant strain relative to N2 is reported in Table 2A.
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