Hormesis A New Hope for Aging Studies or a Poor Second to Genetics?
Gordon J. Lithgow Ph.D.
The School of Biological Sciences
3.239 Stopford Building, University of Manchester, Oxford Road
Manchester M13 9PT
and the Buck Institute, 8001 Redwood Blvd
Novato, CA 94945
Tel: 0161 275 5215
Fax: 0161 275 5654
Hardly a week goes by without the publication of a new genetic variant with greatly extended lifespan. The genetics of longevity is a flourishing science that is leading to the identification of signalling pathways controlling the rate of ageing and prompting some of the first rational attempts to describe the physiological and molecular processes determining lifespan (1). Whilst much of this research has been undertaken on invertebrates, there are hints that mammalian ageing can be studied using similar genetic approaches (2).
There is also a growing list of environmental interventions that prolong organismal lifespan (Rattan, this volume). Because such treatments have wide ranging effects, they do little to help define specific ageing mechanisms. But when combined with genetic analysis and microarray technologies they may provide pointers for experiments that address causality more directly.
Aging and Hormesis
Ageing is clearly a complex biological phenomenon that is greatly influenced by both genes and environmental conditions. A number of environmental manipulations have quite startling effects on lifespan. The most dramatic and robust being caloric restriction (CR) of laboratory rodents where reducing caloric intake by 40% leads to a similar increase in mean lifespan. CR has been studied for 40 years without any resolution of the mechanism acting to prolong life. Indeed, CR may be a hormetic effect (3). Hormesis is where a sub-lethal stressful event can lead to improved survival when the organism is challenged by a lethal stress. Of particular interest to biogerontologist are those mild stresses that lead to an increase in mean and maximum lifespan (4). Rattan has pointed to the potential for hormesis for the study of ageing and as a possible intervention in age-related disease. Let us examine the example of heat shock induced longevity extension and the opportunities and limitations of this approach.
Thermotolerance and Longevity
Animals inhabit a temperature "tolerance zone" where development and reproduction is sustainable. If the organism is pushed to the limits of the tolerance zone, reproduction will decline and death may result. In natural conditions temperature can fluctuate considerably and hence organisms have a range of behavioural and physiological responses to buffer the resulting trauma. Following acute temperature elevation all prokaryotes and eukaryotes repress normal protein synthesis and instead accumulate a set of specialised heat shock proteins (5). This is the classical heat shock response that protects cells from subsequent damage.
Long before the discovery of the molecular details of the heat shock response, hormetic effects of heat shock on lifespan had been observed. The evolutionary biologist Maynard Smith published a seminal paper in which he demonstrated a large increase in lifespan of female Drosophila fruit flies subjected to transient heat shock at 30.50C (6;7). Maynard Smith proposed that the heat-induced longevity was due to a reduction in reproduction, as heat treated female flies also exhibited a permanent reduction in egg laying. Thus the "hormetic" treatment may have increased lifespan but it was also clearly detrimental for overall fitness.
Many years later, Curtsinger and co-workers demonstrated a reduction of age-specific mortality following transient elevated temperature by exposure of fruit fly cohorts to heat shock (8). The resulting flies were highly resistant to lethal heat shock and, just as with Maynard Smith's flies, their survival was increased. However, no inverse correlation between lifespan and the number of eggs laid was apparent. This was at odds with Maynard Smith's interpretation that lifespan was a trade-off with reproductive output and suggested something else was going on.
A similar picture emerged from studies on the nematode Caenorhabditis elegans where exposure to elevated temperatures resulted in significant increases in thermotolerance (so called "acquired thermtolerance") and also in lifespan under non-stress conditions (9). The C. elegans studies went one step further because of the availability of single gene mutations that extend C. elegans lifespan (Age mutations). Mutations in genes, such as that encoding the insulin receptor-like protein DAF-2, double mean and maximum lifespan in this organism. When these mutant worms were examined for their resistance to heat, they were found to be highly thermotolerent (9;10). Here we have a strong correlation between thermotolerance and longevity for both environmental and genetic manipulations. But what is the mechanism linking these characters?
A great deal is known about the molecular mechanisms leading to acquired thermotolerance through the extensive studies of heat shock proteins (11). Previous studies have demonstrated that certain heat shock proteins (hsps) are critical for acquired thermotolerance. For example, Drosophila lines engineered to maintain extra copies of the gene encoding HSP-70, exhibit enhanced acquired thermotolerance (12). Thus there was clearly an opportunity to move away from non-specific hormetic treatments toward the direct testing of the roles of specific stress factors.
Following thermotolerance/longevity observations made in fruit flies and nematode worms, the role of specific molecular chaperones was assessed. The first and most extensive study was undertaken by Tatar and co-workers who showed that strains of Drosophila maintaining extra copies of the inducible hsp-70 gene was sufficient to increase survival following a mild heat treatment (13). In other words, a mild heat shock early in life is beneficial for longevity but additional hsp-70 increases that effect. More recently in C. elegans, a small hsp known to be upregulated in long-lived, thermotolerant Age mutants has also been shown to effect on normal ageing. Strains of worms engineered to maintain extra copies of the hsp-16 gene also exhibit increased acquired longevity and heat-induced longevity (Walker and Lithgow, unpublished data). Some Age mutations enhance this effect.
Explaining Hormesis (and Aging?)
The demonstration that over-expression of a molecular chaperone is sufficient to confer hormetic-like effects suggests molecular-level explanations are possible by undertaking direct genetic manipulations. However, these studies have only been made possible by the considerable body of knowledge of the expression and action of molecular chaperones. Hormesis treatments, such as mild heat shock, alter the expression of hundreds if not thousands of genes. It is difficult to imagine a physiological process that is not altered by even mild treatments. With regard to the effects on ageing, there are two scenarios; the first is that there are some key changes that bring about slowed ageing. In this case it may be difficult, by taking a candidate gene approach, to determine which of these hundreds of changes are responsible. The second scenario is that slowed ageing is the consequence of most or all of the hormetic-induced changes together. This may be of limited value as causality cannot be addressed by genetic means. In other words, no matter how hormesis works, in comparison to more direct genetic based interventions, studies utilising hormesis are somewhat unfocused.
All is not lost and there is indeed a way forward. The simultaneous measure of the levels of all proteins and mRNAs through microarray and proteomic technologies should eventually provide a "fingerprint" of hormesis. It may be possible to determine the co-ordinate regulators of stress response through such studies. It is very likely that genetic approaches have already identified such regulating genes. Long-lived fruit fly and nematode mutants are resistant not just to heat, but to a whole range of environmental stresses (14) and over-express a range of stress response genes not just those encoding hsps (15). The Age mutations themselves tend to be in genes encoding components of signalling pathways.
Only by combining the hormesis concept with reductionist genetic interventions in lifespan, can progress be made. However, as the massively parallel methods of analysis of gene expression become routine, many of the aims set out by Rattan will be within reach. Until then, establishing the exact mechanisms at work will probably remain the subject of educated guesswork.
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