Sci. Aging Knowl. Environ., 27 November 2002
Vol. 2002, Issue 47, p. pe18
[DOI: 10.1126/sageke.2002.47.pe18]

PERSPECTIVES

Spotlight on Nematode Mitochondria in RNAi Mega-Screen

Simon Melov

The author is at the Buck Institute for Age Research, Novato, CA 94945, USA. E-mail: smelov{at}buckinstitute.org

http://sageke.sciencemag.org/cgi/content/full/sageke;2002/47/pe18

Key Words: C. elegans • RNAi • mitochondria • life-span

In the current Advance Online Publication section of Nature Genetics (1), Lee and colleagues report the results of an RNA interference (RNAi) screen for genes that, when down-regulated, produce enhanced longevity in the nematode Caenorhabditis elegans (Fig. 1). This report is interesting from a number of perspectives. The approach is unique in that it uses a specialized genetic screen on a massive scale to identify genes that affect aging in C. elegans (I use "affect" because I bristle at the term "regulate," as this implies selection for so-called "aging genes," which runs counter to established theory). In order to identify potential gerontogenes (see Johnson Review for a derivation of this term), these investigators used the RNAi technique--a method currently being refined and developed for practical use in other organisms--to systematically reduce the endogenous levels of 5690 individual gene transcripts, representing approximately 25 to 30% of all the genes in the nematode. This report concentrates on the group's results with chromosome I; presumably the results for chromosome II will be reported elsewhere. The study is somewhat analogous to making about 5500 lines of knockout mice and looking for effects on life-span--not a project many mammalian biologists would be willing to undertake. It should be noted that the RNAi technique is more properly described as causing a "knock down" of gene function, rather than a total absence of the gene product in question, as discussed in the article by Lee et al. Lee and colleagues clearly use the "awesome power of the worm" to its best effect in the rapidly growing field of "nematode gerontology."



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Fig. 1. The RNAi technique. Each well contains a colony of E. coli that expresses double-stranded RNA representing the worm gene that is to be down-regulated. Worms at the L1 larval stage were placed in each well and allowed to feed on bacteria expressing the gene of interest, which results in lowered expression of that gene.

 
The authors found that of the 2663 genes on chromosome I that were studied, down-regulation via RNAi of 52 (1.8%) genes resulted in worms that live longer than the wild type, the so-called "AGE" phenotype. Of these, the single largest functional class was found to be involved in mitochondrial function (see the Nicholls Perspective and "The Two Faces of Oxygen"). Simply put, Lee et al. discovered that down-regulating mitochondrial function via a number of different pathways can result in longer-lived worms. This result is surprising, because the impairment of mitochondrial function in mammals is associated with the development of certain diseases, such as Parkinson's disease (see Andersen Review) and a variety of mitochondrial disorders (for example, progressive external opthalmoplegia; see Hoopes Viewpoint).

RNAi-mediated repression of genes related to mitochondrial metabolism did not always result in an AGE phenotype. The adenine nucleotide translocator (ANT) is critical for the import of adenosine diphosphate (ADP) into the mitochondrion and the export of adenosine triphosphate (ATP) to the cytoplasm. Surprisingly, when RNAi repressed ANT gene expression, the worms exhibited a normal life-span. The same result was obtained for inhibition of an ATP synthase gene, which is required for ATP synthesis. Taken together, these results imply either that complementary mechanisms exist for the import/export of ADP/ATP or that ATP levels exert a minimal influence on longevity in the nematode.

Which brings us to an intriguing area of study uncovered by the Lee et al. screen: the counterintuitive notion that the suppression of certain genes required for critical aspects of energy metabolism (at least in mammals) results in increased life-span in the worm. One example of this is the phosphoglycerate mutase gene, which encodes a protein required for glycolysis. Worms with an RNAi down-regulated phosphoglycerate mutase gene show enhanced life-span, with no developmental or mitochondrial morphological defects. It is intriguing that this increased life-span phenotype is suppressed by daf-16, which encodes a transcription factor that is modulated by the longevity-related insulin/IGF-1 signaling pathway (see Johnson Review and Sonntag Perspective). These mutations may result in an alteration in glycolysis and a synergistic interaction with insulin signaling, which perhaps results in a longer life-span. This finding is surprising, because it is well known that in humans, mutations in the glycolytic pathway result in disease (2). Similarly, mutations that affect the respiratory chain of humans also result in shortened life-span and severe pathology (3).

There are some other surprises in this substantial analysis for those who study mitochondrial bioenergetics, physiology, and aging. First, the observed extension of life-span caused by RNAi down-regulation of a number of genes was coupled with resistance to some forms of stress and sensitivity to others. This is illustrated by the fact that a majority of the animals with RNAi down-regulated mitochondrial genes (nuclear-encoded) were resistant to the reactive oxygen species H2O2 (which technically is not a free radical), but were sensitive to the redox cycler paraquat. This result poses a number of conundrums, not the least being which pathways and/or proteins are differentially labile to these different stressors, and how does that lability relate to aging in a functional sense? Although the authors have used a number of elegant high-throughput approaches to screen for new genes that modulate aging in C. elegans, they have also used some techniques that are, at best, controversial. Perhaps the most obvious of these are the methods used to measure ATP concentrations and oxygen consumption. A detailed discussion of these issues is beyond the scope of this Perspective; however, I refer those interested to issue 2 of the new journal Aging Cell, which contains an excellent series of articles that address these issues in detail.

The general finding of Lee et al., that a number of the long-lived animals with RNAi down-regulated mitochondrial genes show decreased oxygen consumption, compounded with likely impaired mitochondrial function, begs the question of whether this class of longevity mutants is living longer simply because they are "living slower." A perhaps trivial analogy would be to make worms live longer by rearing them at a lower temperature. Certainly, general metabolic function is lowered in worms that are grown at 15°C, and such worms can live substantially longer than worms grown at 20°. Is this an inherently interesting process that, if understood in molecular detail, would further our knowledge about the molecular mechanisms that drive aging? (I think not!) It seems clear, a priori, that there will be mutations in genes that interfere with metabolism (by any one of a number of different mechanisms), decrease metabolic rate, and prolong life-span. I would argue that such genes are inherently uninteresting from the perspective of furthering our understanding of the molecular etiology of aging. Clearly this is debatable, but it is a perspective that we should all consider, given the common theme of energy metabolism that is appearing with increasing frequency in studies on the growing numbers of gerontogenes.

As one of the premier models for the study of aging, C. elegans has tremendous strengths and advantages (well enumerated in the Review by Johnson). However, the worm also has some unique aspects to its biology, particularly with regard to mitochondrial function and respiration, that must be better understood before knowledge gleaned from powerful mutant screens such as the one used by Lee et al. can be understood in the context of mammalian aging. Unlike ourselves, C. elegans does not rely so heavily on Oxygen. This is exemplified by the observation that 50% of wild-type C. elegans can survive for nearly 100 hours under 0% Oxygen (4). This is extremely important to remember when we are tempted to generalize results from nematodes to mammals. Studies such as the one discussed here, which show that mutations that clearly impair mitochondrial function also increase life-span in the nematode, should resolve why orthologous mutations in the mammal drastically shorten life-span and/or cause severe disease. When genomic techniques, used in such a powerful way by Lee et al., are coupled with a sophisticated bioenergetic approach (see, for example, the Nicholls Perspective) to the study of aging in invertebrate models par excellence, the answers to such questions will begin to unfold. The next few years should be very exciting indeed.


November 27, 2002
  1. S. S. Lee, R. Y. N. Lee, A. G. Fraser, R. S. Kamath, J. Ahringer, Gary Ruvkun, A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat. Genet. 25 November 2002 (10.1038/ng1056).
  2. R. C. Nichols, O. Rudolphi, B. Ek, R. Exelbert, P. H. Plotz, N. Raben, Glycogenosis type VII (Tarui disease) in a Swedish family: two novel mutations in muscle phosphofructokinase gene (PFK-M) resulting in intron retentions. Am. J. Hum. Genet. 59, 59-65 (1996).[Medline]
  3. E. A. Shoubridge, Nuclear genetic defects of oxidative phosphorylation. Hum. Mol. Genet. 10, 2277-2284 (2001).[Abstract/Free Full Text]
  4. W. A. Van Voorhies, S. Ward, Broad oxygen tolerance in the nematode Caenorhabditis elegans. J. Exp. Biol. 203, 2467-2478 (2000).[Abstract]
Citation: S. Melov, Spotlight on Nematode Mitochondria in RNAi Mega-Screen. Science's SAGE KE (27 November 2002), http://sageke.sciencemag.org/cgi/content/full/sageke;2002/47/pe18








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