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T
hanks to medical and environmental advances over the last quarter century, the 65-year-and-older crowd is the most rapidly growing segment of the U.S. population. Regrettably, however, this group confronts physical decline and increased risk for debilitating diseases, which has a profound impact on individuals and on the medical economy. Thus, a critical research priority is to understand the basic biology of the aging process and to define plausible strategies for the delay (or better yet, the prevention) of components of age-related decline.
Obviously, this is a problem in human biology—but deciphering clear answers against the background of diverse genetics, widely variable environments and behavioral differences among people is challenging, if not impossible. Quite remarkably, research to date indicates that although aging is a complex process, many components of age-related decline are shared—by organisms as small as single-celled yeast to humans. For this reason, research on simple animal models can provide fundamental insights into the biology of aging, and findings can be exploited to reveal molecular and pharmacological strategies by which humans might age well. Once specific molecular strategies are revealed in simple models, it is straightforward to formulate and test hypotheses regarding their analogous function in higher organisms.
In the Driscoll laboratory we use the simple roundworm C. elegans to determine molecular strategies that extend lifespan and healthspan — the period of youthful vigor that precedes age-related decline. C. elegans is a free-living invertebrate soil nematode widely used to decipher basic mechanisms in biology. The adult C. elegans is only 1mm in size—about the size of a fleck of dust. The simple body, which includes neurons, muscle, kidney, polarized intestinal epithelia, and hypodermal (skin) cells, is made up of only 959 somatic cells. The skin is transparent, so we can directly examine cells within the living animal. The animal reproduces by self-fertilization, making sperm early in development and eggs later as a young adult. The hermaphroditic reproductive strategy means we can rear large numbers of animals without labor-intensive mating. C. elegans develops from fertilized egg to adult in only 2.5 days! It then produces offspring in 4.5 days (each parent produces about 300 progeny, all of which are genetically identical to the parent). The animal lives for about 3 weeks.
C. elegans is highly amenable to experimental manipulation. Its genome encodes about 20,000 genes (perhaps 1/3 as complex as humans) and a high percentage of these have homologous (related) genes encoded in the human genome. Mutations affecting many of these genes are known, and an effort toward knocking out each gene of the organism is underway. In the absence of a true genetic deletion, we can accomplish individual gene knockdown by double-stranded RNA-mediated interference (dsRNAi). More specifically, if C. elegans ingests a bacterial meal in which the bacteria synthesize double-stranded RNA corresponding to a specific nematode gene, that dsRNA will travel throughout the animal, pair with its homologous RNA transcript and initiate the degradation of that specific transcript—producing a phenocopy of a genetic deficiency. Libraries of bacteria that house clones of each of the 20,000 genes are available — so simply by feeding the animal its bacterial diet we can systematically evaluate the consequences of gene-by-gene elimination. Conversely, it is easy to over-express C. elegans genes by reintroducing DNA back into the germline. Thousands of transgenic animals can be made within a few days’ time for relatively little cost. The bottom line is that genes can be introduced and eliminated with ease—allowing extensive evaluation of the importance of a given gene on a specific process. The transparent body permits direct observations of the consequences of such genetic manipulations in vivo—in the fully physiological context of its native environment.
The three-week lifespan of C. elegans is a great advantage for the practical study of lifespan. More than 200 mutations or RNAi manipulations are known to extend lifespan. Certain mutations can double the lifespan; and combinations of lifespan-extending manipulations have been found that increase it to more than 5 times its maximum. Lifespan genes affect a range of processes including metabolism, repair and maintenance, mitochondrial function and insulin signaling.
Although the longevity phenotype is a focal point for much of the work in the field, much less is understood about whether these genes actually act to extend healthspan (the period of healthy mid-life that precedes significant senescent decline), enabling the animal to live a high quality life for longer. Our basic goal is to exploit biomarkers of aging we have characterized to identify genes that, when disrupted, can extend healthspan. Manipulation of the homologs of these genes in humans would be predicted to exert effects similar to those in nematodes, and thus the identification of such genes is of considerable therapeutic interest. We focus on two phenotypes that change with time — muscle integrity and cellular accumulation of autofluorescent material.
Sarcopenia biology: defining the molecular bases of age-related muscle decline
Age-related muscle decline, a condition referred to as sarcopenia and defined as loss in muscle mass and muscle strength over time, is one of the most pervasive problems of the elderly, such that significant declines in strength and mobility affect essentially every old person. Although the rate of decline is relatively slow (estimated to be only 1% loss annually), ultimate losses are substantial, such that nearly a 50% loss of muscle mass can occur by age 90. Decreased physical strength is a central contributor to loss of
independence. As individuals are not sufficiently mobile or adequately strong to conduct routine daily tasks, extensive assisted care must be arranged. There are no existing therapies that specifically address this problem.
We have found that aging C. elegans bodywall muscle undergoes a process remarkably reminiscent of human sarcopenia. In both organisms,
sarcopenia has a mid-life onset and is characterized by progressive loss of
sarcomeres and cytoplasmic volume within muscle cells; both are associated
with locomotory decline. Our observations present the exciting (but prev-
iously unappreciated) opportunity to exploit the powerful genetics of
C. elegans to identify and characterize physiological influences on sarcopenia.
We have found that genetically lowering the strength of insulin signaling, a condition known to prolong life, can also significantly delay age-related muscle decline. We identified several biomarkers that can be used to evaluate aging and found that mutations that down-regulate insulin receptor daf-2 and signal transducer age-1 PI3kinase delay age-associated muscle decline, although they do not delay the onset of some other aging biomarkers. This work has two important implications. First, it directly suggests that modulating insulin signaling might defer muscle aging in higher organisms. Second, showing that we can alter just one molecule and elicit a significant “youthenizing” boost to muscle integrity and function, despite the fact that deterioration in other tissues still occurs, tells us we can exert tissue-specific healthspan effects without the technical challenge of remodeling the aging of the entire organism.
Figure 1. Sarcopenia, age-related muscle decline, in C. elegans. Top: Sarcomers,
structural elements of muscle, become frayed and disorganized as animals age as
visualized by a fluorescent sarcomere tag. Bottom row: Nuclear structure also
deteriorates over time.
Our initial work in this area leaves us well positioned to screen the entire genome to identify all the genes that contribute to age-related muscle decline. We are pursuing this goal by systematically knocking down each gene activity using RNAi in collaboration with Dimitris Metaxas, PhD, Rutgers professor of computer science, who is developing bioimaging analysis to quantify locomotion features of aging animals.
Age pigment levels reflect the quality of aging and can be used to
identify genes that promote healthy aging.
Naturally autofluorescing components include advanced glycation end-products and lipofuscin, which accumulate with age in humans and many other species. We used in vivo autofluorescence spectroscopy to measure age pigment accumulation in live, senescing nematodes. We found that age-related fluorescence clearly increases over the lifespan of C. elegans and that long-lived mutants of the insulin-like signaling pathway in worms (daf-2/insulin-like receptor and age-1/PI3 kinase) accumulate age pigments at a much lower rate than wild type, consistent with their healthspan effects. The most exciting work on these age pigments, however, is based on an interesting observation we have previously made about the biology of aging.
C. elegans siblings that have identical genes and are reared in the same environment surprisingly exhibit an amazing variation in how “well” individuals age. Within a population of same-age mid-life animals, we could find animals that are youthful by locomotory criteria (A class), animals that are impaired (B class), animals that are paralyzed (C class), and animals that are dead. This data indicates that factors other than genes and environment must hold a significant influence on aging. These factors are likely to have random chance occurrence and may include mitochondrial dysfunction and disrupted protein folding. However, the key point here is that we can distinguish animals that have aged well and those that have aged poorly—what are the differences between these animals that have precisely the same genes and environments? We have found that decrepit C Class animals have markedly elevated levels of age pigments as compared to their same-age, same-environment siblings that have aged well (A class). This suggests that age pigment level actually reflects how well or how poorly an animal has aged. We are currently taking advantage of genetic approaches applicable in C. elegans to identify both genetic and pharmacological factors that shift the population toward a more youthful state. Our ultimate goal is to describe concrete genetic or drug therapies that will enable nematodes and humans to age gracefully.
Figure 2. Example of muscle healthspan extension.
Muscle nuclei from an age-1 mutant appear much more youthful than same-aged wild type
animals and age-1 mutants swim much more vigorously than their same age counterparts.
The age-1 mutation affects a P13 kinase and lowers overall insulin signaling.
Monica Driscoll earned a PhD from Harvard University in 1985 for her molecular genetic studies of gene regulation in yeast and did postdoctoral work at Columbia University, investigating mutant genes that induce inappropriate neuronal degeneration in the nematode Caenorhabditis elegans. She has continued to research the molecular genetics of necrotic neurodegeneration in C. elegans and has moved into the field of aging. Dr. Driscoll is a professor of molecular biology and biochemistry at Rutgers University and a member of The Cancer Institute of New Jersey (CINJ). She teaches in several joint graduate programs of UMDNJ and Rutgers. She has served as a fellow of the Alfred P Sloan Foundation and is currently a Senior Scientist of the Ellison Medical Foundation.
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