Functional Genomic Study of Aging and Aging Intervention

本项目旨在从分子和组织水平鉴定衰老调控基因，探讨长寿基因延长寿命的分子机制，寻找有效的延长寿命干预措施。为了实现我们的目标，我们正在利用三种无脊椎动物系统，墨西哥果蝇（A. ludens）和果蝇（D. melanogaster）以及线虫（C. elegans）。寿命受许多遗传和环境因素的影响。饮食限制（DR）是影响寿命的最有力的环境因素之一。从无脊椎动物到哺乳动物，DR已被证明可以延长许多物种的寿命，这表明如果将DR应用于人类，可能会延缓衰老。然而，对人类实施长期的DR将是一项挑战。另一种策略是使用药物或营养品化合物来诱导类似dr的反应，一些化合物已被证明在模式生物中具有这种效果。然而，这一数字仍然很小，人们对这些化合物延长寿命的机制知之甚少。模式生物的遗传分析揭示了一些影响寿命的基因突变。在许多生物体中，包括蠕虫、苍蝇、啮齿动物、灵长类动物和人类，都观察到了衰老过程中基因表达的变化。然而，对于不同的组织如何衰老，以及长寿基因和延长寿命的干预措施如何影响衰老，人们知之甚少。为了解决组织特异性衰老问题，我们系统地研究了影响寿命和衰老过程的组织特异性因素。我们测量了果蝇大脑、肌肉、消化系统和生殖系统等7个组织的衰老表达谱，这些组织代表着不同的生理功能。数百个基因已被确定，在每个组织的衰老过程中，在转录水平上显示出显著的变化。这些组织特异性的变化很少在所有组织中共享，这表明不同的组织以独特的方式衰老。然而，一些与衰老相关的变化在两个或多个组织中是共同的，这表明不同组织在衰老过程中确实存在共同的分子特征。例如，我们发现涉及主要代谢途径TCA循环的基因在大脑、肠道、肌肉和睾丸中下调，但在脂肪组织、副腺和马氏小管中不下调。为了在组织和分子水平上研究长寿基因延长寿命的机制，我们选择了长寿基因突变体玛土撒拉果蝇作为研究对象。我们已经测量了上述七种组织中这种突变体在不同年龄的分子变化。我们在分子和组织水平上将这些变化与野生型蝇株进行了比较。数百个基因已经被确定在野生型和玛士撒拉蝇之间有组织特异性的变化。这一评估将阐明甲基撒拉基因如何在组织水平上调控寿命的分子和细胞机制。类似的方法将应用于研究机制，通过延长寿命的干预措施延长寿命在组织水平在未来。膳食补充剂被广泛使用，人们相信它们可以预防疾病，延长寿命。很少有系统的尝试来证实延长寿命的说法或调查潜在有效的干预措施。我们目前正在使用mexfly开发一个高通量系统。使用mexflies的主要原因是，我们可以很容易地在墨西哥恰帕斯州塔帕丘拉的Moscafrut大规模饲养设施中每天获得数百万只mexfly。这最大限度地减少了我们为高通量寿命筛选所需的大量动物死亡率分析而付出的努力。还有其他一些优点。蝇类的体型相对于黑腹巨蝇要大一些，因此很容易按性别分类和测量食物摄入量，后者对于评估化合物的剂量效应至关重要。此外，与黑腹蝇不同的是，麻蝇和麻蝇可以以干燥的食物来源为食，但在不同的媒介（如有机网）上产卵，这有助于研究化合物对生殖的影响，而不依赖于饮食。最后，正常饮食的苍蝇的预期寿命约为2个月，这对于进行寿命筛选来说足够短，但对于老龄化的人口统计学研究来说足够长。作为一个例子，我们已经评估了补充两种相关抗氧化剂，α -生育酚和γ -生育酚对果蝇寿命的影响。我们发现这两种抗氧化剂对延长寿命有边际作用，这与我们在D. melanogaster中观察到的一致。利用高通量系统将为老龄化干预的效果提供可靠和统计上令人信服的结果。对延长寿命的干预措施进行系统评估，不仅可以识别有效的抗衰老化合物，还可以揭示通过膳食补充剂延长寿命的机制。这种方法对于推进实验老年学研究和开发哺乳动物衰老干预措施的目标是有价值的。我们对DR的分子机制的理解主要来自对遗传可适应系统的研究，包括酵母、蠕虫和苍蝇，其中DR是通过稀释食物来源或使用降低摄食效率的基因突变来施加的。然而，这些方法的一个主要缺点是，在确定这些DR范式下个体的确切热量摄入方面仍然存在很大的不确定性，这与研究高等生物的能力不同。这使我们发现并开发了一种可以延长秀丽隐杆线虫寿命的替代饮食模式。我们发现，饮食剥夺（DD）方案，即完全从成年人的食物来源中移除，可以使成年人的寿命延长45%。由于这一方案涉及到完全去除食物来源，控制食物摄入的问题得到了缓解，这一问题阻碍了对过去研究的解释。使用这种明确的方法，我们已经开始研究通过饮食延长寿命所必需的遗传途径。测量DD反应的全基因组转录谱，并将其与DR进行比较，以揭示DD和DR范式之间的相似性。为了确定DD反应所需的遗传途径，正在对从基因组研究中发现的DD相关基因以及已知延长寿命的基因进行遗传筛选。这一分析将揭示不同环境特别是饮食条件下的长寿机制。考虑到DD和DR之间的相似性，一些DD机制应该是进化保守的，这将推进对饮食对哺乳动物衰老和长寿影响的认识。总之，我们应用了三种不同的无脊椎动物物种，通过利用每个系统的独特特征来解决与饮食调节寿命相关的问题。我们正在研究延长寿命干预和长寿基因在分子和组织水平上延长寿命的机制。我们正在使用mexflies来确定有效的延长寿命干预措施，这将为进一步研究哺乳动物的衰老干预措施提供指导。通过利用秀丽隐杆线虫独特而稳健的饮食方案，我们正在剖析饮食调节寿命的分子机制。确定衰老的保守特征和有效的延长寿命干预措施显然对我们至关重要

The aim of this project is to identify aging-regulated genes at the molecular and tissue levels, to investigate molecular mechanisms of lifespan extension by longevity genes, and to identify efficient prolongevity interventions. To achieve our goals, we are utilizing three invertebrate systems, the Mexican fruit fly (mexfly), A. ludens, and the fly, D. melanogaster, and the nematode, C. elegans. Lifespan is influenced by a number of genetic and environmental factors. One of the most robust environmental manipulations of lifespan is dietary restriction (DR). DR has been shown to extend lifespan in many species, ranging from invertebrates to mammals, indicating that DR might retard aging if applied in humans. However, it would be challenging to impose long-term DR in humans. An alternative strategy would be to apply pharmaceutical or nutraceutical compounds to induce responses that would mimic DR. A few compounds have been shown to have this effect in model organisms. However, the number is still small and little is known about mechanisms by which these compounds extend lifespan. Genetic analyses of model organisms have uncovered mutations in a number of genes that can affect lifespan. Changes in gene expression in aging have been observed in a number of organisms, including worms, flies, rodents, primates and human beings. However, little is known about how different tissues age, and how longevity genes and prolongevity interventions influence aging. To address tissue-specific aging, we have systematically investigated tissue-specific factors that affect lifespan and aging processes. We have measured the expression profile of aging for seven tissues from fly, including brain, muscle and tissues in the digestive and reproductive systems, which represent different physiological functions. Hundreds of genes have been identified to show significant changes at the transcript level in aging in each tissue. Very few of these tissue-specific changes were shared among all the tissues, suggesting that different tissues age in unique ways. However, some of the aging-related changes are shared among two or more tissues, suggesting that common molecular features do exist among different tissues in aging. As an example, we have found genes involved a major metabolic pathway, TCA cycle, are down-regulated in brain, gut, muscle and testis but not fat tissue, accessory gland and malpighian tubule in aging. To study the mechanisms of lifespan extension by the longevity genes at the tissue and molecular levels, we have chosen to study the methuselah mutant fly, which has an extended lifespan. We have measured molecular changes of this mutant across age for the seven tissues described above. We have compared these changes to those in the wild type fly strain at the molecular and tissue levels. Several hundreds of genes have been identified to have tissue-specific changes between wild type and methuselah flies. This assessment will elucidate molecular and cellular mechanisms on how the methuselah gene regulates lifespan at the tissue level. Similar approaches will be applied to study mechanisms by which prolongevity interventions extend lifespan at the tissue levels in the future. Dietary supplements are widely used with the belief that they can forestall disease and increase longevity. Few systematic attempts have been made to confirm prolongevity claims made or to investigate potentially effective interventions. We are currently developing a high-throughput system by using mexfly. The main reason to use mexflies is that we have easy access to millions of mexflies available daily in the Moscafrut mass-rearing facility at Tapachula, Chiapas, Mexico. This minimizes our efforts to obtain a large number of animals for mortality analysis required for the high-throughput lifespan screen. There are several other advantages as well. The size of mexflies is relatively large relative to D. melanogaster so that it is easy to sort out by sex and to measure food intake, the latter of which is critical for assessing dosage effects of compounds. Moreover, unlike D. melanogaster, mexflies and medflies can feed on a dry food source but lay their eggs on a different medium such as organdy mesh, which facilitates investigation of effects of compounds on reproduction independent of diet. Finally, the expected lifespan of mexflies on a regular diet is approximately 2 months, which is short enough for conducting a lifespan screen and but long enough for demographic studies of aging. As an example, we have assessed the effects of supplementation of two related antioxidants, alpha-tocopherol and gamma-tocopherol, on lifespan of mexflies. We have found that these two antioxidants have marginal effects on lifespan extension, which is consistent with what we have observed in D. melanogaster. Utilization of the high-throughput system will provide reliable and statistically convincing results on the effects of aging interventions. Systematic evaluation of prolongevity interventions will not only allow identification of effective anti-aging compounds but also uncover mechanisms of lifespan extension by dietary supplementation. This approach should prove valuable to advance the objective of experimental gerontology to investigate and develop aging interventions in mammals. Our understanding of molecular mechanisms of DR comes primarily from studies of genetically amenable systems including yeast, worms, and flies, where DR has been imposed by either diluting the food source or by using genetic mutations that reduce feeding efficiency. However, a major drawback of these approaches is that there remains substantial uncertainty in determining the exact caloric intake of individuals under these DR paradigms, unlike this ability in studies of higher organisms. This led us to discover and develop an alternative dietary paradigm that can extend lifespan in C. elegans. We have found that a dietary deprivation (DD) regimen, in which the food source is completely removed from adults, can prolong adult lifespan by 45%. Since this regimen involves complete removal of the food source, the problem of controlling food intake, which has hampered interpretation of past studies, is alleviated. Using this unambiguous method, we have started investigating the genetic pathways necessary for lifespan extension by diet. Genome-wide transcript profiles of DD response are measured and compared to that of DR to reveal similarities between DD and DR paradigm. To identify which genetic pathways are required for the DD response, a genetic screen is being conducted for DD-associated genes identified from genomic studies as well as for genes known to extend lifespan. This analysis should reveal mechanisms governing longevity under different environmental especially dietary conditions. Considering the similarities between DD and DR, some of the DD mechanisms should be evolutionarily conserved, which will advance knowledge about effects of diet on aging and longevity in mammals. In summary, we have applied three different invertebrate species to address issues related to dietary regulation of lifespan by taking advantage of unique features of each system. With D. melanogaster, we are studying mechanisms by which prolongevity interventions and longevity genes extend lifespan at molecular and tissue levels. We are using mexflies to identify effective prolongevity interventions, which should provide guidance for further investigation of aging interventions in mammals. By utilizing a unique and robust dietary regimen in C. elegans, we are dissecting molecular mechanisms of dietary regulation of lifespan. Identification of the conserved features in aging and efficient prolongevity interventions are clearly critical for us