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A 10-wk-old infant girl with severe hypertrophy of the septal and atrial walls by cardiac ultrasound, developed progressive ventricular wall thickening and died of aspiration pneumonia at 5 mo of age. Postmortem examination revealed ventricular hypertrophy and massive atrial wall thickening due to glycogen accumulation. A skeletal muscle biopsy showed increased free glycogen and decreased activity of phosphorylase b kinase (PHK). The report of a pathogenic mutation (R531Q) in the gene (PRKAG2) encoding the gamma2 subunit of AMP-activated protein kinase (AMPK) in three infants with congenital hypertrophic cardiomyopathy, glycogen storage, and "pseudo PHK deficiency" prompted us to screen this gene in our patient. We found a novel (R384T) heterozygous mutation in PRKAG2, affecting an arginine residue in the N-terminal AMP-binding domain. Like R531Q, this mutation reduces the binding of AMP and ATP to the isolated nucleotide-binding domains, and prevents activation of the heterotrimer by metabolic stress in intact cells. The mutation was not found in DNA from the patient's father, the only available parent, and is likely to have arisen de novo. Our studies confirm that mutations in PRKAG2 can cause fatal infantile cardiomyopathy, often associated with apparent PHK deficiency.

Summary The lifespan of Caenorhabditis elegans can be extendedby the administration of synthetic superoxide dismutase/catalase mimetics (SCMs) without any effects on develop-ment or fertility. Here we demonstrate that the mimetics,Euk-134 and Euk-8, confer resistance to the oxidativestress-inducing agent, paraquat and to thermal stress. Theprotective effects of the compounds are apparent withtreatments either during development or during adult-hood and are independent of an insulin/IGF-I-like signal-ling pathway also known to affect thermal and oxidativestress resistance. Worms exposed to the compounds donot induce a cellular stress response and no detrimentaleffects are observed.Key words: antioxidants; Caenorhabditis elegans ; oxidativestress; SOD/catalase mimetics; stress resistance. Introduction Accumulation of macromolecular damage caused by reactiveoxygen species (ROS) is believed to be one of the main under-lying causes of aging and is implicated in a number of diseasestates (Harman, 1956, 1992; Martin

To date, more than 40 genes have been identified in the nematode Caenorhabditis elegans, which, when mutated, lead to an increase in lifespan. Of those tested, all confer an increased resistance to oxidative stress. In addition, the lifespan of C. elegans can also be extended by the administration of synthetic superoxide dismutase/catalase mimetics. These compounds also appear to confer resistance to oxidative damage, since they protect against paraquat treatment. The protective effects of these compounds are apparent with treatment during either development or adulthood. These findings have demonstrated that pharmacological intervention in the aging process is possible and that these compounds can provide important information about the underlying mechanisms. To date, such interventions have targeted known processes rather than screening compound libraries because of the limitations of assessing lifespan in nematodes. However, we have recently developed a microplate-based assay that allows for a rapid and objective score of nematode survival at rates many times higher than previously possible. This system now provides the opportunity to perform high-throughput screens for compounds that affect nematode survival in the face of acute oxidative stress and will facilitate the identification of novel drugs that extend nematode lifespan.

The observation that long-lived and relatively healthy animals can be obtained by simple genetic manipulation prompts the search for chemical compounds that have similar effects. Since aging is the most important risk factor for many socially and economically important diseases, the discovery of a wide range of chemical modulators of aging in model organisms could prompt new strategies for attacking age-related disease such as diabetes, cancer and neurodegenerative disorders [Collins, J.J., Evason, K., Kornfeld, K., 2006. Pharmacology of delayed aging and extended lifespan of Caenorhabditis elegans. Exp. Gerontol.; Floyd, R.A., 2006. Nitrones as therapeutics in age-related diseases. Aging Cell 5, 51-57; Gill, M.S., 2006. Endocrine targets for pharmacological intervention in aging in Caenorhabditis elegans. Aging Cell 5, 23-30; Hefti, F.F., Bales, R., 2006. Regulatory issues in aging pharmacology. Aging Cell 5, 3-8]. Resistance to multiple types of stress is a common trait in long-lived genetic variants of a number of species; therefore, we have tested compounds that act as stress response mimetics. We have focused on compounds with antioxidant properties and identified those that confer thermal stress resistance in the nematode Caenorhabditis elegans. Some of these compounds (lipoic acid, propyl gallate, trolox and taxifolin) also extend the normal lifespan of this simple invertebrate, consistent with the general model that enhanced stress resistance slows aging.

Prompted by successful genetic interventions in aging we now consider the utility of pharmacological interventions. A few clear cases of lifespan extension resulting from exposure to compounds suggest that aging can be slowed and that the pharmacology of lifespan extension is a feasible area for research. Compounds that slow aging may prompt the rational design of therapeutics against age-related diseases. Here we evaluate invertebrate models for drug discovery in aging and outline how the field may develop from these simple first steps.

Relative to iron and copper we know very little about the cellular roles of manganese. Some studies claim that manganese acts as a radical scavenger in unicellular organisms, while there have been other reports that manganese causes Parkinson's disease-like syndrome, DNA fragmentation, and interferes with cellular energy production. The goal of this study was to uncover if manganese has any free radical scavenging properties in the complex multicellular organism, Caenorhabditis elegans. We measured internal manganese in supplemented worms using inductively coupled plasma mass spectrometry (ICP-MS) and the data obtained suggest that manganese supplemented to the growth medium is taken up by the worms. We found that manganese did not appear to be toxic as supplementation did not negatively effect development or fertility. In fact, supplementation at higher levels accelerated development and increased total fertility of wild type worms by 16%. Manganese-supplemented wild type worms were found to be thermotolerant and, under certain conditions, long-lived. In addition, the oxidatively challenged C. elegans strain mev-1's short life span was significantly increased after manganese supplementation. Although manganese appears to be beneficial to C. elegans, the mode of action remains unclear. Manganese may work directly as a free radical scavenger, as it has been postulated to do so in unicellular organisms, or may work indirectly by up regulating several protective factors.

The accumulation of damage caused by reactive oxygen species (ROS) is held to be one of the underlying causes of age-related decline and has been shown to be involved in a number of pathological states. Inherent defense mechanisms have evolved to limit this damage by reducing the levels of ROS, which are produced mainly by the mitochondria in aerobic organisms. One such defense is superoxide dismutase 1 (SOD1). It is well established that oxidative stress results in increased transcription and translation of the SOD1 gene, but it is now known that an additional level of posttranslational control exists. A recent paper describes the presence of an inactive pool of SOD1 whose activation is wholly reliant on the presence of superoxide or oxygen and a specific copper-containing chaperone. This mechanism highlights the importance of rapid responses in the fight against oxidative stress.

Many genetic or environmental manipulations that extend life span in the nematode Caenorhabditis elegans (C. elegans) also enhance survival following acute stresses such as oxidative damage and thermal stress. This coupling of stress response and aging mechanisms has proved a useful tool in identifying new genes that affect the aging process without the need for performing lengthy life span analyses. Therefore, it is likely that this approach may also be applied to the identification of pharmacological agents that extend life span through enhanced resistance to oxygen radicals or other stressors. To facilitate high-throughput drug screens in the nematode, we have developed a microtitre plate survival assay that uses uptake of the fluorescent dye SYTOX green as a marker of nematode death. An increase in throughput compared with the conventional survival assay was achieved by combining automated worm-handling technology with automated real-time fluorescence detection. We have validated this assay by examining survival during acute heat stress and protection against oxidative stress with the superoxide dismutase/catalase mimetic Euk-134. We propose that this novel method of survival analysis will accelerate the discovery of new pharmacological interventions in aging and oxidative stress.

In the last decade of the 20th century one model organism has received more attention from gerontologists than any other, namely the free-living soil nematode Caenorhabditis elegans (C. elegans). We owe much of our general view of the genetic determination of lifespan to this organism. C. elegans became a popular organism for the analysis of complex biological problems following the pioneering work of Sidney Brenner and Sir John Sulston who determined the developmental cellular fate map [1]. It is also noted for the early work of Robert Horvitz and colleagues who used the cell lineage map to begin a genetic dissection of programmed cell death [2].