Download Using Animal Models to Understand Aging

LENScience Senior Biology Seminar Series Eat Yourself Old: Using Animal Models to Understand Aging Michal Denny, Sarah Morgan and Peter Dearden
Aging Hundreds of thousands of dollars are spent every year in the fight against aging. In humans aging is a complex but natural process that affects every cell and organ in our bodies. Aging is not one process but many, including the decline in organ function and body systems during adulthood, the decline in ability to successfully reproduce and the increased likelihood of death due to systems failure. With modern medicine and improvements in our living environment humans are living longer than ever. This means that more people are reaching old age and therefore suffering from age‐related diseases such as cardiovascular disease, cancer and dementia. But aging is not inevitable for all living things and there are organisms that age very slowly or not at all, for example rock fish and turtles. Scientists have long been interested in aging and longevity, particularly in what can be done to lengthen life span and to reduce the impact of age‐related diseases. One area of research relates to nutrition. Did you know that in many animals by limiting food you can extend lifespan? – for example, skinny rats live longer than fat rats. This is perhaps opposite to what you’d normally expect, that a rat with as much of everything as it could want would be better off. University of Otago PhD student Sarah Morgan has spent the last 2 ½ years looking at the impact of diet on lifespan; in particular the links between aging and the environmental effects of diet on the genome. Fig 1. Sarah with her colonies of fruit flies. 1 To find out more Sarah is using fruit flies (Drosophila melanogaster). Her fruit flies have an extended lifespan on a diet high in carbohydrate, and a restricted one on a diet with high levels of protein. Sarah is also looking for a genetic link between this differing intake of diet nutrients and the extended lifespan phenotype Sarah uses fruit flies as a model to understand what might be happening inside the human body as we age. Most of the research into aging has been carried out in animals such as rats, fruit flies and nematodes. Scientific models, including animal models are an important part of the scientific process. What are Scientific Models? You have come across many different scientific models in your study of science and may not have realised that they are models. A scientific model is a representation of an object or system, which is often a simplified view of something that is quite complex in reality. Models are used because they are convenient substitutes for the real thing. Two common examples where we use models are the atom and DNA. We can’t physically see what is inside an atom or exactly what DNA looks like, but the models we use are based on scientific evidence about their properties. Diagrams of DNA and the atom that you’ve seen in textbooks summarise this evidence in a way that best represents our current scientific understanding. Models also change over time as our knowledge of the system or object they represent Fig 2. Two models of the changes. The two pictures in Fig. 2 show two models of the atom. Do atom you know which is the most up‐to‐date version? The introduction to this paper described another example of a scientific model — the use of rats to determine whether diet affects lifespan; here the rats are a used as model of what goes on inside the human body. Our current understanding of how the human body works owes a lot to the use of animal models. How processes such as embryonic development, the cell cycle, the nervous system and cellular metabolism, work in humans was established by studying their functioning in simpler organisms and then scaling up into more complex organisms(1). If you are going to study a human disease or process you can’t, for ethical reasons, perform the initial work in humans; you have to develop a model. In the last seminar we talked about breast cancer, specifically how human growth hormone affects gene expression in breast cancer cells. (See Breast Cancer and Biotechnology). As Dr. Perry said, this sort of experiment can’t be done in humans so the Liggins’ scientists used a model. The model they used was a cell model. Cell models can tell us a lot about processes such as gene expression, but at some point the research needs to be scaled up into a whole organism or animal model so that the effect of Why is Sarah using fruit flies? other tissues and cells on the biochemical Drosophila melanogaster is a small fly belonging to processes can be studied. the family Drosophilidae. It is often called fruit fly, or The main animals used as laboratory models for vinegar fly as it likes to hang around rotting fruit. The humans are rodents (rats and mice), zebrafish, fruit fly has a long history of use in science research. fruit flies and nematode worms. They are used It’s genome was sequenced in 2000 and there are because they are relatively easy to raise in the multiple online databases containing the Drosophila lab, and due to their short lifespans, genes. This means it is relatively easy to conduct experiments can be carried out quickly ‐ in days genetic analysis on them. What makes the fruit fly or months, rather than years. Compare this with particularly useful as a model for humans is that humans where our already long life span makes many of the Drosophila genes perform the same designing and carrying out aging experiments function as they do in humans(1) (3). cumbersome and largely impractical. 2 The Life History Trade‐Off. The Life History Trade‐Off is the key principle behind Sarah’s research. It refers to the idea that an increase in lifespan is achieved at the expense of successful reproduction – or conversely an increase in reproduction results in a sacrifice of life length. An individual can enjoy a long life or a large amount of offspring but not both. The life history trade‐off is seen across a diverse group of animals including nematodes, fruit fly, yeast, mice, rats and potentially humans. The goal of the Life History Trade‐Off is successful reproduction. The ’choice’ between the two options ‐ long life or more offspring is a response to environmental conditions. Organisms shorten their own expected lifespan by having a lot of offspring at a young age, in the hope that some of them survive; or to they live to an older age and only have a couple of offspring later in life in the hope that they have increased chances for survival then. Nutrient allocation is switched between growth and repair or reproduction. Both options have advantages in different environments (Fig. 3). In an environment where there is a lot of food it is in the best interests of the species to produce as many offspring as possible, as soon as possible, to take advantage of it. On the other hand, when there is little good food around, it is better for the species if the mother waits to produce her offspring until she finds lots of good food they can eat. She will have fewer children, but they will have a better chance of survival than if she had them back when there was no food to spare. A remarkable exception to this rule is the honey bee Fig. 3. Selection mechanisms maintaining alternate life queen. She is highly reproductively active yet far histories outlives all other bees in the hive. What causes aging? There are several different processes in the body that cause aging and most of these relate to the cell cycle, including shortening of telomere lengths, and the accumulation of DNA damage through the failure of repair mechanisms. (You learnt about the cell cycle in the last seminar Breast Cancer and Biotechnology) The other significant process that causes aging is oxidative stress. Oxidative stress is the damage to DNA and cells caused by a group of chemicals known as reactive oxygen species or free radicals. Free radicals are a by product of respiration in mitochondria where food is converted into energy. Aging is most noticeable in organisms where growth is completed before reproduction starts e.g. Insects, birds, and many mammal including humans. (See Life History Trade Off above) The main animals used for laboratory studies into aging all display this characteristic of reproduction occurring after growth is completed, which makes them good models for human aging. Scientists now know that there is no single process that results in aging, instead it is the accumulation of damage caused by a large number of independent processes with no common cause. This makes study of aging quite complicated but scientists are having considerable success with looking at the effects of single genes on aging. 3 The Evolutionary origins of aging. Aging is not an advantage to the organism so why does it exist? Given that aging is harmful to the organism it seems logical to expect natural selection to have selected against it. But remember natural selection only selects for features and processes that allow organisms to live long enough to reproduce. So the actual impact of natural selection weakens as organisms age because there a fewer individuals surviving for natural selection to act on. Genetics of Aging Aging is likely to be a polygenic trait i.e. many genes are involved and consequently many metabolic pathways. One of the key results from aging research over the last 20 years has been the finding that the genetic pathways that modulate aging are the same in multiple organisms, which provides encouragement for translating these results into humans(4). Genetic pathways and genes that carry out the same function in very different organisms are said to be evolutionary conserved. Evolutionary conserved genes are genes that have remained essentially unchanged throughout evolution. Conservation of a gene indicates that it is unique and essential for life , therefore natural selection acts to preserve it in the gene pool. The vast majority of genes in the human genome are also be found in other animals (Fig. 4.) 1%
1%
Prokaryotes
Humans only
21%
32%
Eukaryote and prokaryote
Vertebrate only
22%
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Animals and other eukaryotes
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Fig 4. Where do human genes come from? What research is increasingly finding out is that the genes that are essential for growth and development are highly conserved. A recent study identified 64 genes that extend lifespan in yeast. These genes are all highly conserved from yeast to humans making them possible candidates for regulating lifespan.(2) Scientists are identifying more and more genes with a role in longevity in non human animals and a corresponding function in humans, suggesting that mechanisms of aging are to some extent ‘shared’ between all animals. The evolutionary conservation of genes to do with aging further reinforces the usefulness of animal models in understanding processes involved in human aging. Interestingly many of the genes identified in longevity in the animal models are involved in pathways that contribute to matching the growth and reproductive rate of the animals to their nutritional status. 4 Dietary Restriction Environmental factors such as diet have a huge impact on aging. Reducing levels of food by 30—50% has been shown to significantly extend lifespan and reduce age–related diseases in mice and rats. This led to the theory of Caloric Restriction (CR) – that by manipulating the nutrition of the organism, and limiting the access to or availability of calories, we can induce a lifespan extension phenotype in a wide range of organisms. This includes but is not limited to nematodes, yeast, mice, rats, fruit fly and potentially humans. Restricting the calorie intake of these organisms results in not only lifespan extension but increased stress resistance and decreased fecundity (reproductive ability)(5) Scientists now realise, however, that it is not the restriction of calories that causes the extended lifespan but something else. Researchers recently discovered that in the case of the Fruit Fly, Drosophila melanogaster, it is the changing levels of protein and carbohydrate in the food which affects lifespan. In diets with identical calorie values, fruit fly eating lots of protein and little carbohydrate had really short lives, but laid a large amount of eggs. Equally, flies eating little protein and lots of carbohydrate lived till they were really old, but laid very few eggs throughout their lives. It was due to observations such as this that the name ‘Caloric Restriction’ was changed to the more accurate ‘Dietary Restriction’ (DR). The researchers think what is going on is that under poor nutrient conditions the organism reallocates nutrients from reproduction to looking after itself so it can survive the harsher conditions (the Life History Trade Off). Studies have shown that in fruit flies, reducing the intake of certain essential amino acids, extends lifespan but reduces reproduction(4). Scientists have found out that restricting intake of proteins, lipids or carbohydrates can have a major impact on aging and age‐related diseases. Dietary Restriction can increase lifespan in rodents, nematode worm , fruit fly and rhesus monkeys, and also protects against cancer, cataracts, diabetes, osteoporosis and kidney disease—all disease associated with aging. Calorie Restriction in humans Recent studies of people who consumed a calorie restricted diet for a number of years showed a lower body weight, body mass index (BMI) and total body fat as well as lower levels of insulin and higher sensitivity to insulin. They also had lower levels of inflammatory mediators. Other studies in humans suggest that Caloric Restriction may reduce risk factors for cardiovascular Reduced calorie diet disease and memory decline. These studies suggest that Caloric Restriction in humans reduces the risk factors associated with the diseases of aging.(4) Whether or not Dietary Restriction or Caloric Restriction has any widespread application for all of us is unclear because of the difficulties in getting people to stick to extremely low calorie diets for long enough to have any significant effects. So instead what the scientists are trying to do is to identify the genes that are affected by Dietary Restriction in order to develop pharmaceuticals that may achieve the same goal of longevity Normal balanced diet without the need to reduce nutrient intake. 5 Sarah’s Current Research Sarah’s current research is using fruit flies to find a genetic explanation for the phenomenon of dietary restriction. Scientists think that the different diets causes a change in gene expression which in turn alters how the organism uses its resources: either for reproduction or survival. The flies in the Sarah’s study were given one of four types of food to eat, each with decreasing amounts of protein and increasing amounts of carbohydrate, but the exact same number of calories. The flies eating the most protein lived for less than 26 days and those eating the least protein lived for up to 58 days. You can see this pattern in Fig. 4. Sarah wanted to find out which genes might be affected by the different diets. To do this samples of flies from populations feeding on each of the four different food types were taken at different ages and the mRNA extracted. Remember mRNA is Fig. 4. Survivorship of flies on 4 diets present only when a gene is turned ‘on’. (You learnt about how mRNA is used as an indicator of gene expression in the last seminar Breast Cancer and Biotechnology) Microarrays are used to measure the amount of mRNA. The microarrays Sarah used are covered with spots of all the genes in the fruit fly. If any of these genes have been turned up/down or on/off in the samples due to the different diets, the specific gene will light up when the sample mRNA touches it. The microarrays are used as a tool to identify any genes that are differentially expressed between two samples. Differentially expressed refers to genes that are turned on in one sample, for example the flies that die young, and off in the other sample e.g. the ’old’ flies. These genes could potentially be the ones that are responsible for causing this difference in lifespan. For example the genes in Table 1. show up as differentially expressed between the shortest lived flies and longest lived flies (in samples taken at the end of the fruit fly lifespan). Online genome databases are then used to identify what each gene does; this is the column headed ‘Molecular function’. The column headed ‘Biological process’ is a broader description of the ‘molecular function’. You may be able to work out what these processes are and how they promote longevity. When listed as ‘unknown’ the gene has yet to be studied in enough detail to tell us about its function—which is exciting as it may provide an as yet unknown clue about aging. Gene Symbol Gene name Molecular Function Biological process AttD Attacin‐D unknown Antibacterial response Alh Alhambra DNA binding, protein binding, zinc ion binding, transcription factor activity. Larval development, molting cycle, cutical development, haltere development. TotX TurandotX unknown Response to stress. 6 The data from the microarray results lists is put through another program that groups genes which appear on the list in functional groups; so if five genes are involved in the same process or pathway appear on the list, they are grouped together under their functional title ‐ for example ‘carbohydrate metabolic process’ (the breaking down of carbohydrate in the diet). The more genes showing on the lists—the more likely it is that that entire pathway or process is involved in this life history trade off phenomenon. From this analysis of the data we can identify interesting genes — for example genes involved in stress and immune response. This result is exciting because it supports our theories — the flies that are living for longer are investing more nutrients, effort and resources in responding to, dealing with and healing stressors on the body than they are to making and laying eggs. The next step is to go through these genes and mutate them (turn them on or off) in flies to see if we can force the flies to live longer even if they are not eating the special diet. These experiments will also help to prove that the mutated gene is responsible for the extension in lifespan, and not just a random light flash on the microarray. From this we know that the genes that are different between the samples are the ones that may be responsible for extending lifespan, and we can progress to asking deeper questions about these genes, what they do, how they work — and potentially their effect on aging and reproduction in other mammals and eventually humans. Because Drosophila melanogaster is a model organism, this research is important to you; Humans suffer from aging – what if we can find a gene which will let you live longer? Or be healthier in your old age? If you follow a specific diet (maybe even only in infancy) that will turn on/off important genes ‐ could you increase your chances for a longer life? And secondly; diet involved‐diseases that humans suffer from, such as diabetes and obesity, are becoming more common. Any genes that are changed by what you eat may be very important in these diseases. You could be your own genetic engineer by eating differently! References 1. Partridge, L. (2010). The new biology of aging. Philos Trans R Soc Lond B Biol Sci, 365(1537), 147–154. http://
www.ncbi.nlm.nih.gov/pmc/articles/PMC2842712/pdf/rstb20090222.pdf 2. Curran, S. P., & Ruvkun, G. (2007). Lifespan Regulation by Evolutionarily Conserved Genes Essential for Viability. PLoS Genet, 3(4), e56. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1847696/?
tool=pmcentrez&report=abstract 3. Partridge, L. (2009). Some highlights of research on aging with invertebrates, 2009. Aging Cell, 8(5), 509‐513. http://www3.interscience.wiley.com/journal/122467086/abstract 4. Katewa, S. D., & Kapahi, P. (2010). Dietary restriction and aging, 2009. Aging Cell, 9(2), 105‐112.http://
www3.interscience.wiley.com/journal/123247984/abstract 5. Partridge, L., Piper, M. D., & Mair, W. (2005). Dietary restriction in Drosophila. Mech Aging Dev, 126(9), 938‐950. 6. Paaby, A. B., & Schmidt, P. S. (2009). Dissecting the genetics of longevity in Drosophila melanogaster. Fly (Austin), 3(1), 29‐38. http://www.landesbioscience.com/journals/fly/article/7771/ For further information contact Michal Denny [email protected]; Sarah Morgan [email protected] Copyright © Liggins Institute 2010 http://LENS.auckland.ac.nz 7