Stephen L. Helfand

Sirtuin activators mimic caloric restriction and delay ageing in metazoans.

Caloric restriction extends lifespan in numerous species. In the budding yeast Saccharomyces cerevisiae this effect requires Sir2 (ref. 1), a member of the sirtuin family of NAD+-dependent deacetylases. Sirtuin activating compounds (STACs) can promote the survival of human cells and extend the replicative lifespan of yeast. Here we show that resveratrol and other STACs activate sirtuins from Caenorhabditis elegans and Drosophila melanogaster, and extend the lifespan of these animals without reducing fecundity. Lifespan extension is dependent on functional Sir2, and is not observed when nutrients are restricted. Together these data indicate that STACs slow metazoan ageing by mechanisms that may be related to caloric restriction.

Survival and development in culture of dissociated parasympathetic neurons from ciliary ganglia

Abstract Parasympathetic neurons from avian embryonic ciliary ganglia survive in low density culture when neurons are free from contact with other cells. A charged substratum, polyornithine, and a conditioned medium permit cell survival and vigorous neurite formation. The heart-conditioned medium must be present continuously and is active after dialysis. Neurites elongate rapidly, branch extensively, and follow patterns of charged substratum provided in the culture dish.

Conditional tradeoffs between aging and organismal performance of Indy long-lived mutant flies

Alterations that extend the life span of animals and yeast typically involve decreases in metabolic rate, growth, physical activity, and/or early-life fecundity. This negative correlation between life span and the ability to assimilate and process energy, to move, grow, and reproduce, raises questions about the potential utility of life span extension. Tradeoffs between early-life fitness and longevity are central to theories of the evolution of aging, which suggests there is necessarily a price to be paid for reducing the rate of aging. It is not yet clear whether life span can be extended without undesirable effects on metabolism and fecundity. Here, we report that the long-lived Indy mutation in Drosophila causes a decrease in the slope of the mortality curve consistent with a slowing in the rate of aging without a concomitant reduction in resting metabolic rate, flight velocity, or age-specific fecundity under normal rearing conditions. However, Indy mutants on a decreased-calorie diet have reduced fecundity, suggesting that a tradeoff between longevity and this aspect of performance is conditional, i.e., the tradeoff can occur in a stressful environment while being absent in a more favorable environment. These results provide evidence that there do exist mechanisms, albeit conditional, that can extend life span without significant reduction in fecundity, metabolic rate, or locomotion.

Non-equivalence of conditioned medium and nerve growth factor for sympathetic, parasympathetic, and sensory neurons.

Abstract Conditioned medium derived from embryonic chick heart cell cultures (heart conditioned medium; HCM) supports vigorous neurite outgrowth from embryonic sympathetic, sensory, and ciliary neurons. Antiserum to nerve growth factor (NGF) does not reduce neurite outgrowth in response to HCM, and a displacement assay fails to detect binding by components of HCM to the NGF receptor. The HCM activity is resistant to acid treatment but is destroyed by alkali. In both these respects the HCM activity differs from NGF. The activity can be inactivated by heating to 90 °C, and is retained by Amicon PM-50 filters. It is concluded that HCM stimulation of peripheral nervous system cells may not operate via NGF.

Neuronal Expression of p53 Dominant-Negative Proteins in Adult Drosophila melanogaster Extends Life Span

Hyperactivation of p53 leads to a reduction in tumor formation and an unexpected shortening of life span in two different model systems . The decreased life span occurs with signs of accelerated aging, such as osteoporosis, reduction in body weight, atrophy of organs, decreased stress resistance, and depletion of hematopoietic stem cells. These observations suggest a role for p53 in the determination of life span and the speculation that decreasing p53 activity may result in positive effects on some aging phenotypes . In this report, we show that expression of dominant-negative versions of Drosophila melanogaster p53 in adult neurons extends life span and increases genotoxic stress resistance in the fly. Consistent with this, a naturally occurring allele with decreased p53 activity has been associated with extended survival in humans . Expression of the dominant-negative Drosophila melanogaster p53 constructs does not further increase the extended life span of flies that are calorie restricted, suggesting that a decrease in p53 activity may mediate a component of the calorie-restriction life span-extending pathway in flies.

Neuronal specificity and growth cone guidance in grasshopper and Drosophila embryos

Abstract Studies on a variety of vertebrate and invertebrate nervous systems point to the existence of specific chemical cues for both growth cone guidance and target recognition. Open questions, however, include how many recognition labels and how specific is their expression? Here we review our recent studies on insect embryos aimed at trying to answer these questions, focusing in particular on the specificity of two identified growth cones (aCC and pCC) in the CNS of the grasshopper and Drosophila embryos. Ablation experiments demonstrate the selective affinities of the aCC and pCC growth cones for particular axonal surfaces; in some cases such ablations result in temporal and spatial transplants which further demonstrate this exquisite specificity. Our results suggest the differential expression of many different surface recognition molecules in the developing CNS (labelled pathways hypothesis), a notion reminiscent of Sperrys chemoaffinity hypothesis. The SOX2 monoclonal antibody reveals an antigen in the Drosophila embryo that correlates with this model: SOX2 is expressed by the aCC and the small subset of neurons whose axons fasciculate with it.

Behavioral, physical, and demographic changes in Drosophila populations through dietary restriction

Dietary restriction (DR) is a valuable experimental tool for studying the aging process. Primary advancement of research in this area has relied on rodent models, but attention has recently turned toward Drosophila melanogaster. However, little is known about the baseline effects of DR on wild‐type Drosophila and continued experimentation requires such information. The findings described here survey the effects of DR on inbred, wild‐type populations of Canton‐S fruit flies and demonstrate a robust effect of diet on longevity. Over a circumscribed range of dietary conditions, healthy lifespan varies by as much as 121% for wild‐type Drosophila females. Significant differences are also observed for male flies, but the magnitude of the DR effect is less robust. Mortality analyses of the survivorship data reveal that this variation in lifespan can be attributed to a modulation of the rate parameter for the mortality function – a change in the demographic rate of aging. Since the feeding of fruit flies is less easily controlled than that of rodents, this research also addresses the validity of applying a DR model to Drosophila populations. Feeding and body weight data for flies given the various dietary conditions surveyed indicate that Drosophila on higher‐calorie diets consume a similar volume of food to those on a low‐calorie diet, resulting in different levels of calorie intake. Fertility and activity levels demonstrate that the diets surveyed are comparable, and that increasing the calorie content of laboratory food up to twice the normal concentration is not pathologic for experimental fly populations.

Chromatin remodeling in the aging genome of Drosophila

Chromatin structure affects the accessibility of DNA to transcription, repair, and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. We examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole‐genome tiling microarrays of activation and repressive chromatin marks, whole‐genome transcriptional microarrays and single‐cell immunohistochemistry. We found dramatic reorganization of chromosomal regions with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome, and islands of facultative heterochromatin dispersed throughout the genome. With age, there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin, and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age‐related gene expression. Large‐scale changes in repressive marks with age are further substantiated by single‐cell immunohistochemistry that shows changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair, and DNA replication. The combination of genome‐wide approaches such as whole‐genome chromatin immunoprecipitation and transcriptional studies in conjunction with single‐cell immunohistochemistry as shown here provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans.

Calorie restriction delays lipid oxidative damage in Drosophila melanogaster

The oxidative stress hypothesis predicts that the accumulation of oxidative damage to a variety of macromolecules is the molecular trigger driving the process of aging. Although an inverse relationship between oxidative damage and lifespan has been established in several different species, the precise relationship between oxidative damage and aging is not fully understood. Drosophila melanogaster is a favored model organism for aging research. Environmental interventions such as ambient temperature and calorie restriction can alter adult lifespan to provide an excellent system to examine the relationship between oxidative damage, aging and lifespan. We have developed an enzyme‐linked immunosorbent assay (ELISA) using commercially available reagents for measuring 4‐hydroxy‐2‐nonenal (HNE) in proteins, a marker for oxidative damage to lipids, and present data in flies to show that HNE adducts accumulate in an age‐dependent manner. With immunohistology, we also find the primary site of HNE accumulation is the pericerebral fat body, where induction of dFOXO was recently shown to retard aging. When subjected to environmental interventions that shorten lifespan, such as elevated ambient temperature, the chronological accumulation of HNE adduct is accelerated. Conversely, interventions that extend lifespan, such as lower ambient temperature or low calorie diets, slow the accumulation of HNE adduct. These studies associate damage from lipid peroxidation with aging and lifespan in Drosophila and show that calorie restriction in flies, as in mammals, slows the accumulation of lipid related oxidative damage.

Expression of dominant-negative Dmp53 in the adult fly brain inhibits insulin signaling

In Drosophila melanogaster, p53 (Dmp53) is an important mediator of longevity. Expression of dominant-negative (DN) forms of Dmp53 in adult neurons, but not in muscle or fat body cells, extends lifespan. The lifespan of calorie-restricted flies is not further extended by simultaneously expressing DN-Dmp53 in the nervous system, indicating that a decrease in Dmp53 activity may be a part of the CR lifespan-extending pathway in flies. In this report, we show that selective expression of DN-Dmp53 in only the 14 insulin-producing cells (IPCs) in the brain extends lifespan to the same extent as expression in all neurons and this lifespan extension is not additive with CR. DN-Dmp53-dependent lifespan extension is accompanied by reduction of Drosophila insulin-like peptide 2 (dILP2) mRNA levels and reduced insulin signaling (IIS) in the fat body, which suggests that Dmp53 may affect lifespan by modulating insulin signaling in the fly.