Gerontology is a branch of the biological sciences devoted to the study of the aging process and its effects on cells and organisms. Philosophers and scientists have been interested in this subject for thousands of years, but this history will be confined to the modern era, extending back no further than the late 1800s. The history of gerontology, like many other branches of biological research, may be divided into four epochs. The first, covering the early years, began around 1870, with the invention of the compound microscope and ended in the 1950s. The second epoch began with the discovery of the DNA double helix in 1952 and extended to the early 1970s. The third epoch began with the introduction of recombinant DNA technology in 1973, ending in the early 1990s. The current epoch, known as the post-genomic era, began with the formation of a genome-sequencing consortium in 1990 and continues to the present day.
Gerontological research has always been driven by the same questions: Why do people grow old? Why do they change with time? Can the effects of age be reversed? Gerontologists have tried to answer these questions using a variety of techniques, but with the approach of the third and fourth epochs, the questions became more numerous, more specific, and much more complex.
The early years
In 1868 the German physicist Ernst Abbe perfected the design of the compound microscope and in so doing made it possible for scientists to study the structure and function of individual cells in a way that was never before possible. While many microbiologists of the time concentrated on studying the link between disease and microbes, many others began studying the life cycle of bacteria and protozoa in the hope that it would shed some light on the aging process. These studies were descriptive in nature; that is, the researcher observed the behavior of the cells and recorded it without subjecting the system to experimental procedures that would modulate the rate of the aging process.
During this period scientists realized that senescence is not universal; it occurs in multicellular creatures only. Bacteria and protozoans do not grow old and die, but rejuvenate themselves every hour or so by dividing into two new cells. A lifestyle such as this can hardly serve as a model system for gerontological research. Consequently, scientists all but abandoned the use of these cells to gain insights into the cellular mechanisms of the aging process (see the error catastrophe theory below for two exceptions).
In 1882 August Weismann, a German embryologist, proposed the first theory of senescence that tried to link life span to natural selection. Weismann argued that the termination of life may have a selective advantage, and that there is a connection between a species’ life span and its ecological niche, body size, and intelligence. During this same period German chemists were developing the first biochemical techniques that allowed Hans Krebs to work out the cyclic details of energy metabolism that now bear his name (Krebs cycle, also known as the citric acid cycle). The new biochemical techniques were used by chemists to begin cataloging the many molecules of the cell, and by the time the citric acid cycle had been worked out in 1937, DNA had been identified and localized to the cell nucleus. During the last three decades of the 1800s, European scientists, most notably Anton Schneider, Paul Ehrlich, Santiago Ramon y Cajal, and Camillo Golgi, were developing special dyes and procedures that could be used to stain cells in order to better study the nucleus, cell division, and cytoplasmic organelles, giving birth to histochemistry and histology.
Thus it was that light microscopy, biochemistry, histochemistry, and histology became the basic tool kit for gerontologists during the early years of scientific research in this field. Scientists at that time believed they had all the techniques that were needed to fully understand the structure and the function of cells and animals. They were only partly right. The techniques of that day made it possible for scientists to gain a basic understanding of cell structure and, to some extent, how that structure changes with time, but they learned very little about the functional significance of those changes or how their knowledge could be used to form a physiological theory of the aging process. Much of this was due to the limited resolution of the techniques available at the time. Camillo Golgi, the Italian microbi-ologist, had discovered an unusual cellular structure that now bears his name (the Golgi apparatus), but no one had a clue as to the functional significance of this organelle nor were they able to explore the question with the methods at hand. Elie Metchnikoff, winner of the 1908 Nobel Prize for physiology or medicine for his work on the human immune system, attempted to form a physiological theory of the aging process by suggesting that lactic-acid bacteria (such as Bacillus acidophilus) in the digestive tract could prolong life by preventing putrefaction (decay). He noted that Bulgarian villagers, who eat large quantities of curded milk and yogurt, were known for their longevity. Other scientists of the time believed the secret of long life depended on hormones and, in particular, claimed that an extract of dog endocrine glands could reverse the signs of age. Studies such as these make it clear that the early gerontologists had only vague notions about the mechanisms of cellular senescence.
Metchnikoff’s theory and the interest in hormone extracts was part of a tendency among scientists of the era to believe in magic potions that could cure many maladies at once or even reverse all signs of the aging process. This is an ancient idea that can be found in the medical practices of Egyptian physicians 4,000 years ago, and the witchcraft of the Middle Ages. The ancient Egyptians had magical spells and potions that were reputed to be powerful rejuvena-tors. Ancient Chinese physicians had a similar potion in the form of a broth produced from the ginseng root.The "stone" was a mineral, or mineral concoction, of mythical powers that was discovered by Nicholas Flamel, a French alchemist who lived in the 14th century. Flamel and his followers claimed that in addition to transmuting mercury and silver to gold, the philosopher’s stone could also reverse the aging process. Potions and elixirs of this kind have never been authenticated, but the allure of a quick fix is always tempting, even to scientists.
DNA structure inspires new theories
On April 25, 1953, James Watson and Francis Crick published a classic paper on DNA in the journal Nature: "A Structure for De-oxyribose Nuclei Acid" not only proposed a structural model for the DNA molecule but also showed how DNA could store a genetic code, specifying a unique protein, and how that code could be duplicated, in a process now known as DNA replication. Watson and Crick were also the first to propose the existence of a molecular intermediary (messenger RNA) between DNA and protein synthesis, and special adaptor molecules (transfer RNA) that were part of the protein synthesis machinery. By 1966, using synthetic messenger RNAs, other scientists had worked out the complete genetic code thereby establishing the one-gene-one-protein hypothesis and describing the functional relationships between replication, transcription, and translation.
The discoverers of the structure of DNA. James Watson (b. 1928) at left and Francis Crick (1916-2004), seen with their model of part of a DNA molecule in 1953. Crick and Watson met at the Cavendish Laboratory, Cambridge, in 1951. Their work on the structure of DNA was performed with a knowledge of Chargaff’s ratios of the bases in DNA and some access to the X-ray crystallography of Maurice Wilkins and Rosalind Franklin at King’s College London. Combining all of this work led to the deduction that DNA exists as a double helix, thus to its structure. Crick, Watson, and Wilkins shared the 1962 Nobel Prize in physiology or medicine, Franklin having died of cancer in 1958.
Gerontologists of the second epoch quickly realized that the genetic code and the events of protein synthesis gave them, for the first time, testable theories of the aging process. The first, proposed by Denham Harman in 1956, was the free radical theory, and the second, proposed by Leslie Orgel in 1963, was the error catastrophe theory. Both of these theories suggest that aging is due to errors in biosynthesis, due either to free radicals or to inherent error frequencies associated with transcription and translation. In either case, according to the theories, the result is a buildup of dysfunctional proteins that damage normal cellular functions, thus reducing cell viability with time. The error catastrophe theory was first tested on bacteria, experimental organisms introduced to gerontology during the early years. To further test this theory and the free radical theory, gerontologists of the second epoch began using baker’s yeast (Saccharomyces cerevisiae), the housefly (Musca domestical the fruit fly (Drosophila melanogaster), the rat, and the mouse (mus musculus). Experiments on all of these organisms, though offering some support for the free radical theory, failed to substantiate the original formulation of the error catastrophe theory.
Many investigators, however, realized that even though induced errors in protein synthesis had no effect on the rate of aging, other errors, involving replication or the repair of the DNA molecule, could still be an important, if not primary, cause of the aging process. Testing the revised catastrophe theory required detailed information about the gene, but at the time there was no way to sequence DNA or to infer the sequence of messenger RNA. Throughout the 1960s physicists were busy perfecting the electron microscope, which offered unparalleled resolution of cellular organ-elles and tissue ultrastructure. Consequently, many gerontologists turned their attention to refining the structural and biochemical analysis of age-related changes that was begun by scientists of the first epoch. These studies, carried out on the housefly, Drosophila, and mouse, introduced methods for modulating the life span of the organism. The life span of houseflies, for example, was tripled when they were reared in tiny cages that minimized flight activity. Caloric restriction was also introduced, which could extend the life span of a mouse by 30 to 40 percent. Finally, with extensive genetic data available for Drosophila, many researchers conducted studies on long-lived or short-lived mutants in an attempt to correlate their life span with changes at the cellular or biochemical level. Although the research in the second epoch used more powerful techniques than were available during the first epoch, the results were still largely descriptive in nature and generally fell far short of achieving a deeper understanding of the aging process.
Biotechnology revolutionizes the field
In 1973 Paul Berg, a professor of biochemistry at Stanford University, produced the first recombinant DNA molecule, consisting of a piece of mammalian DNA joined to a bacterial plasmid (a bacterial mini-chromosome). Bacteria have a natural tendency to take up plasmids from the medium they are growing in; once they do, the plasmid DNA, with any insert it may contain, is replicated along with the bacterial chromosome each time the cell divides. This proliferation of a segment of DNA is called amplification.
To amplify a mammalian gene, bacteria are coaxed to take up a recombinant plasmid in a small test tube containing a special medium, after which they are transferred to a large flask containing nutrient broth and allowed to grow for 24 hours. By the end of the culturing period, the amount of cloned insert has increased more than a million fold. In 1977 Fred Sanger, a professor at Cambridge University, and Walter Gilbert, a professor at Harvard, developed methods for sequencing DNA. The production of recombinant clones, combined with the new sequencing technology, made it possible to isolate any gene and to produce enough of it for sequencing and expression studies.
Expression studies observe the transcription of a gene to produce messenger RNA (mRNA), and the resulting translation of mRNA into protein. Because most mRNA is automatically translated into protein, conducting an expression study involves determining the amount of mRNA being produced by a specific gene. The information gained by doing so is extremely important because all cellular processes are ultimately controlled by the differential expression of various genes. Some genes in some cells always stay off, whereas some are always on (constitutive expression), and some turn on or off, as conditions demand (regulative expression). One theory of aging suggests that the aging process is caused by subtle disruptions in the normal control of gene expression. At first gerontologists tried to test this assumption by examining the protein products of translation with protein electrophoresis, a technology introduced in the 1960s and refined in 1977. In this procedure proteins are isolated from the tissue of interest and then separated on a small gel slab subjected to an electric field.After separation the gel is stained, dried, and photographed. Proteins of different sizes appear as blue bands in the photograph.
But protein electrophoresis can detect only a few hundred proteins; a typical cell is capable of producing thousands of different proteins. Despite its limitations, many studies were conducted with this procedure throughout the 1980s on wild-type (normal) or mutant Drosophila. The hope was that electrophoresis would show that old animals were completely missing a protein present in young animals or that a new protein would appear in old animals that might be responsible for the age-related changes. But no such results were ever obtained, at least not on a consistent basis. The studies failed to show a consistent change in any of the proteins that could be visualized with this technique. The animals were clearly aging, but they seemed to be making the same proteins when they were old as when they were young.
Protein electrophoresis. In this procedure, proteins are extracted from cells of interest and then fractionated by electrophoresis on a polyacrylamide gel. After the gel is stained, or exposed to X-ray film, the proteins appear as bands. In the example shown, approximately 30 different proteins (bands) have been identified. Lanes 1 to 3 are proteins extracted from housefly flight muscle at one, four, and eight days of age. Lanes 4 and 5 are size markers, which decrease in size from top to bottom. In a different form of this procedure, called two-dimensional protein electrophoresis, the proteins appear as spots over the face of the gel. Two-dimensional protein gels have a higher resolution and can detect about 1,000 different proteins, but this is still much less than the more than 20,000 proteins a typical animal cell can produce.
To address the question of whether the absence or presence of a given protein influenced aging, scientists abandoned protein electrophoresis in favor of recombinant technology. With this technology it is possible to study the mRNA expression of every gene in the cell. Consequently, gerontologists of the third epoch conducted a large number of expression studies involving genes coding for glo-bin, actin, liver enzymes, microtubules, apolipoprotein (a protein that carries lipids in the blood), brain- and kidney-specific proteins, and several oncogenes. In most cases, the choice of which gene to study was an equal mix of educated guess and common practicality. If an investigator had a hunch that a particular liver enzyme was responsible for some aspect of cellular aging, the expression of the gene could be studied, but only if it had already been cloned.Since no one at the time had a clear idea of which genes were responsible for the aging process, virtually any gene for which a probe was available made a good candidate for an expression study.
It was during this epoch that Daniel Rudman and his colleagues at Emory University Hospital in Atlanta, Georgia, demonstrated the striking age-related decline in the expression of growth hormone (GH) in humans. Soon after, many other investigators demonstrated an age-related decline in a number of other hormones, such as thyroid hormone, dehyroepiandrosterone (DHEA), estrogen, and insulin-like growth factor (IGF). These studies, conducted on humans, rats, and mice, all showed a similar trend. Other expression studies, however, carried out on rat, Drosophila, and housefly tissues did not produce the striking results that most scientists were expecting. The expression of some genes was shown to increase with age while others decreased, but there was no obvious connection to cellular senescence. Even worse, the expression of some genes was shown to decrease with age in the rat, but not in Drosophila or the mouse. Since the aging process should be similar for all animals, those genes could not be the cause of a universal aging mechanism. When all expression studies were taken together, there appeared to be a general decline in the rate of gene expression with age, with the hormones mentioned above (GH, thyroid hormone, DHEA, estrogen, and IGF) showing the most consistent trend.
Scientists interested in chromatin structure and the role it plays in regulating gene expression adopted a different approach to the study of the aging process. Eukaryote chromosomes are a complex of DNA and proteins, called histones, that are arranged on the DNA like beads on a string. Each bead, consisting of several different kinds of histone, is called a nucleosome. This complex of DNA and histones is known as chromatin. The histones are essential for packing up the chromosomes in preparation for cell division. Phosphorylating the nucleosomes (adding phosphate groups to the proteins) is like releasing a stretched rubber band: The chromosome contracts to form a compact structure that is 10,000 times shorter than the bare piece of DNA. Just as a suitcase makes it possible for us to take our clothes on a trip, histones and the chromatin structure they produce make it possible for the cell to package its genes in preparation for cell division.
Chromatin compaction, or condensation, is also used during in-terphase (the period between cell divisions) to help manage the chromosomes. It is also one mechanism for controlling gene expression. The packing ratio of interphase chromatin (condensed length divided by relaxed length) is about 1:1,000 overall, but there are highly condensed regions where it can be as low as 1:10,000. This variation in the density of the chromatin accounts for the blotchy appearance that most interphase nuclei have. Areas of the nucleus that are very dark represent highly compacted chromatin, whereas the lighter regions contain chromatin in a more relaxed state. At the molecular level, chromatin condensation is an extremely dynamic process that is used to close down single genes or whole neighborhoods consisting of hundreds of genes. The mechanism by which this occurs is fairly straightforward: Highly condensed chromatin blocks the transcription machinery so it cannot get access to the gene.
Pattern analysis of cell nuclei. A cell nucleus (A) is processed by a computer to show low- (LDC), medium- (MDC), and high- (HDC) density chromatin components. (B) Each component is analyzed for quantity and spatial distribution. This type of analysis was used to characterize nuclei from young and old houseflies. The computer then selected images from the data files that best represented young (C) and old (D) flies. These images show a dramatic decrease in total nuclear area, an increase in the amount of HDC, a change in the HDC spatial distribution, and a decrease in the number of MDC clusters. LDC (pale blue), MDC (blue), HDC (black).
Many gerontologists of the third epoch studied chromatin condensation as a function of age. These studies were either biochemical or they relied on computerized histochemistry. The biochemical analysis depended on the fact that uncondensed chromatin is easy to dissociate (i.e., it is easy to separate the histones from the DNA) in certain buffers, whereas highly condensed chromatin is either very difficult to dissociate or does not dissociate at all. Studies such as these invariably showed that chromatin became more condensed with age. Consequently, condensed chromatin was believed to be responsible for the age-related reduction in transcriptional activity. Computerized histochemical analysis of intact nuclei supported the biochemical results and in addition, provided a way to visualize the progressive condensation of cell nuclei. Scientists produced a model of this event by analyzing the condensation pattern over the surface of the nucleus, and then, with the aid of computer algorithms, selecting nuclei that best represent the young and old groups.
The third epoch was a productive period for gerontological research that provided many insights into the mechanisms controlling the aging process. But many scientists came to realize that the available DNA sequence data was inadequate. They needed more in order to expand the expression profiles for the organisms being studied. Indeed, they needed the complete genomic sequence for humans and for all organisms for which age-related studies were under way.
The post-genomic era
An international genome-sequencing consortium was formed in 1990 to sequence the human, bacteria, yeast, nematode (Cae-norhabditis elegans, or simply C. elegans), Drosophila, and mouse genomes. This project was initiated by the U.S. Department of Energy and the National Research Council and is coordinated by the Human Genome Organization (HUGO). The principal consortium members include the United States, United Kingdom, France, Germany, Japan, and China. Sequencing of the human genome was completed in early 2003, and work on the other organisms was completed in 2008.
In 1993 the American National Institute of Aging (NIA) started a program to identify longevity genes in yeast, nematode, Drosophi-la, and mice. This program provided research funding for scientists at NIA, as well as other scientists working in university laboratories around the country. The main interest of this program is single-gene mutants that may be used to identify genes and physiological factors that favor longevity in all animal species. These include the insulinlike signaling pathway, stress resistance, and most recently, chromosome and nuclear architecture. The ultimate goal is to use information gathered from lower animals (i.e., invertebrates and insects) to identify longevity genes in humans.
In addition to financial support, the NIA program and the genome-sequencing consortium provided encouragement and focus to the gerontological community. Research focus came in two forms. First, by settling on just four research organisms, different research groups could easily compare results. Gerontology of previous epochs was carried out to a great extent on houseflies and rats, neither of which are genetically defined (i.e., mutants have not been identified or characterized). The four organisms chosen by NIA are well characterized genetically, and there are many long- and short-lived mutants available that greatly expedite aging research. Second, aging research shifted from projects aimed at testing one of the many theories of the aging process to a narrower, thus more practical approach involving the search for longevity genes. This was done by selecting for long-lived individuals or by searching for naturally occurring short-lived mutants. In some cases exposing the animals to chemical mutagens generated short-lived mutants.
There is also a great deal of interest in Werner’s syndrome, a human disease that is characterized by a greatly accelerated rate of aging. Individuals suffering from this disease age so rapidly that they appear to be in their 70s or 80s by the time they are 10 years old.
The great value of the sequencing consortium in the effort to identify aging genes lies in the fact that all of the organisms under study, including humans, share a common cellular and genetic heritage. Thus, if a longevity gene is discovered in Drosophila, its homo-log (a gene having a similar or identical sequence) can be identified in humans simply by searching the human database for a gene that matches the Drosophila sequence. Research of this kind is bringing us closer to identifying the physiological processes and molecular mechanisms that are important for longevity. Reversal of the aging process and treatment of its clinical symptoms will become a practical reality after all of the genes controlling these processes have been identified and their functions clearly defined.
