Examination of the requirement for ucp-4, a putative homolog of mammalian uncoupling proteins, for stress tolerance and longevity in C. elegans

Abstract

Reactive oxygen species (ROS) are generated by mitochondrial respiration and can react with and damage cellular components. According to the free radical theory of aging, oxidative damage from mitochondrial ROS is a major cause of cellular decline during aging. Mitochondrial uncoupling proteins (UCPs) uncouple ATP production from electron transport and can be stimulated by free radicals, suggesting UCPs may perform a cytoprotective function. The nematode, Caenorhabditis elegans, contains one UCP-like protein, encoded by the ucp-4 gene. We have investigated the genetic requirement for ucp-4 in normal aging and stress resistance. Consistent with the hypothesis that ucp-4 encodes a putative uncoupling protein, animals lacking ucp-4 function contained elevated ATP levels. However, the absence of ucp-4 function did not affect adult lifespan or survival in the presence of thermal or oxidative stress. Together, these results demonstrate that ucp-4 is a negative regulator of ATP production in C. elegans, but is not required for normal lifespan.

Fig. 1

(a) Genomic organization of the ucp-4 locus, within a 5-kb fragment of chromosome V. The position of the 1.5-kb deleted region in ok195 is shown as a gray bar. The 1.8-kb promoter fragment used for the transcriptional fusion to GFP is shown below, (b) Genotypic analysis by PCR using primers flanking the ok195 deletion produced a 1.5 kb smaller product from the ok195 deletion allele than from wildtype genomic DNA.

Fig. 2

Expression analysis of ucp-4. (a) Nomarski image of three larvae at approximately the third larval stage, (b) Same animals as (a) showing fluorescence from ucp-4p::GFP expression in one transgenic animal (center), but not in two non-transgenic control animals (left and right), ucp-4p:GFP fluorescence was detected in the pharynx (large arrow) and head muscles in transgenic animals carrying the ucp-4p:GFP construct, but not in non-transgenic controls (large white arrowhead), (c) Higher magnification view of ucp-4p::GFP expression in head showing fluorescence in the pharyngeal corpus (C), isthmus (I) and terminal bulb (TB). Arrows point to UCP-4p:GFP expression in head muscles, (d) UCP-4p:GFP fluorescence in head muscles. All scale bars represent 20 μm.

Fig. 3

Elevated ATP levels in ucp-4(ok195) animals. ATP levels in wildtype (filled) and ucp-4(ok195) (open) eggs (a) and L4 larvae (b). The error bars represent S.E.M. from two experiments, and four replicates of each genotype were performed, (c) JC-1 staining was performed to compare mitochondrial membrane potential of wildtype and ucp-4(ok195) animals. JC-1 mitochondrial accumulation is mitochondrial membrane potential dependent, and greater JC-1 concentrations promote dye aggregation, shifting fluorescence from green to red. The red:green ratio of mitochondrial JC-1 fluorescence reflects mitochondrial membrane potential and tended to be slightly greater in ucp-4(ok195) animals than in wildtype animals, although this difference was only statistically significant at 0.66 μg/mL (p = 0.01, t-test).

Fig. 4

Lifespan and development in ucp-4(ok195) animals, (a) Adult lifespan of wildtype (filled symbols, n = 167) and ucp-4(ok195) (open symbols, n = 116) animals at 20 °C. Curve shows the average of two independent trials; consistent results were obtained in a third trial, (b) Survival at high temperature (35 °C) of wildtype (filled symbols) and ucp-4(ok195) (open symbols). Shown is average of two trials with 51–56 animals/trial, (c) Survival of wildtype and ucp-4(ok195) young adults after 24 h incubation at 2.5 °C; average survival for six trials ± S.E.M., n = 40–100 animals/trial; p = 0.046, t-test.

Fig. 5

Oxidative stress tolerance of ucp-4(ok195) animals, (a) Survival of adult wildtype (filled) or ucp-4(ok195) (open) animals in the presence of the free-radical generating compound, paraquat (10 mM) at 25 °C. Results are average of four trials; (n) wildtype, 73 animals total in all trials; ok195, 72 animals in all trials, (b) Larval development in the presence of paraquat. Eggs were laid onto NGM agar medium containing 0, 0.2 or 0.8mM paraquat and allowed to develop for 48 h at 25 °C. Development of wildtype (left, average n = 140 ± 61 animals/dose/trial), ucp-4(ok195) (center, average n = 102 ± 45 animals/dose/trial), and mev-1(kn1) (right, average n = 46 ± 24 animals/dose/trial) animals is shown. Graphs show the means and standard deviations of results from four trials conducted on two different days. Paraquat concentrations greater than 0.8 mM severely impaired larval development in all strains.