Developing Technology and Changing Questions

The application of modern technologies to ageing research is proving invaluable in unravelling the complex genetic mechanisms behind longevity. As technology has progressed, the ageing questions that have been asked, and answered, have moved from the broad to the narrow.

33.1

Population Genetics

The question of why organisms age was the first to be tackled by modern ageing research. The competition between the various evolutionary theories of ageing, predominantly the mutation accumulation and antagonistic pleiotropy theories, drove not only the first longevity selection experiments (Rose and Charlesworth, 1981b), but also population genetics studies to explain the hereditary variation in rates of ageing (Rose and Charlesworth, 1981a). The heritability of longevity itself was broadly (although not universally) accepted as numerous studies of lab animals

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demonstrated genetic determination of lifespan (Goodrick, 1975; Deerberg et al., 1980) and a pilot longevity selection was able to extend lifespan in Drosophila (Rose and Charlesworth, 1981b). Experimental evolution was principally used to answer the question of why ageing evolved. It was postulated that mutation accumulation would lead to the additive genetic variation of fitness traits increasing with age, while antagonistic pleiotropy would lead to easily selectable changes in

longevity, by selecting on fitness traits at different ages. These ideas were tested and no increase in additive genetic variance of egg laying rate was found with age, while longevity was increased by selecting on late reproduction, supporting antagonistic pleiotropy as the dominant influence on longevity (Rose and Charlesworth, 1980). Another approach was sibling analysis of laboratory Drosophila populations, measuring life-history traits in sibling females and looking for correlations. Genetic variance for fecundity was found to be inversely correlated with lifespan, further suggesting an antagonistic pleiotropy relationship between the two traits (Rose and Charlesworth, 1981a). The UC Irvine, Wayne State University and Edinburgh lines were extensively studied to determine the evolutionary history of ageing. The UC Irving lines saw reduced fecundity in females during early life as a tradeoff for long life (Rose, 1984), as did the Wayne State lines supporting antagonistic pleiotropy (Luckinbill et al., 1984), while the Edinburgh lines saw no such difference, suggesting either mutation accumulation or a different pleiotropic trait (Partridge and Fowler, 1992).

These early hypotheses were supported by the study of quantitative trait loci (QTL). QTLs are simply locations in an organism’s DNA that correlate with a quantitative phenotype. Once identified, QTLs can be measured in populations to give an idea of how a phenotype is genetically determined. In the Wayne State selection lines, the lifespan extension effect was shown to be polygenic and a result of contributions from all chromosomes, although the third chromosome contributed the most

explaining 66-72% of lifespan variation in females (Luckinbill et al., 1988). Later, numerous QTLs were identified correlating with the selection regime, however not all of these correlated with longevity, showing how drift can lead to erroneous results in selection experiments (Curtsinger et al., 1998). The UC Irvine lines had the same response, showing that the lifespan extension was a result of epistatic control of chromosome 2 by genes on chromosome 3 (Arking, Dudas and Baker, 1993).

33.2

Microarray and RT-qPCR

The development of the polymerase chain reaction (PCR) in the early 80s was pivotal for genetic research, allowing any given DNA sequence to be replicated and produced in abundance (Mullis et al., 1986). Subsequently, the technique was improved by adding ethidium bromide, which fluoresces in the presence of double-stranded DNA, allowing the PCR reaction to be tracked in real-time

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(Higuchi et al., 1992). Concurrently, PCR was being developed for use in the quantification of mRNA (Rappolee et al., 1988), and combining the two led to reverse transcriptase qPCR (RT-qPCR)

protocols able to accurately measure relative mRNA levels, in real-time, all in the same tube (Gibson, Heid and Williams, 1996).

A related technology made possible by PCR is the expression microarray, allowing relative mRNA quantification of a huge number of genes simultaneously (Schena et al., 1995). Using microarrays allows large scale studies of genes for involvement in ageing (De Magalhães, Curado and Church, 2009), while RT-qPCR is routinely used in validation of microarray studies, or for focused

experiments where information on a few specific genes is required (Morey, Ryan and Dolah, 2006). When used with selection experiments, these transcriptomic techniques have allowed the

characterisation of some of the mechanisms behind lifespan extension. The selected flies of the University of Aarhus lines for instance showed that genes upregulated in the selected flies tended to be genes that were downregulated with age as whole. Additionally, genes of the "phototransduction and vision" functional group were found to be the most represented of the differentially expressed genes, mirroring a previous study selecting for increased heat resistance(J.G. Sørensen, Nielsen and Loeschcke, 2007; Sarup, Sørensen and Loeschcke, 2011). Candidate genes from this study were later confirmed using RT-qPCR and expression was also tested in the Groningen University directly

selected strains (Wit et al., 2013). From this study, the gene CG32638 was found to be expressed at a lower level in the long-lived lines from both selection regimes. This gene shares a conserved region with the human angiotensin II type 1 receptor protein, which is involved in the regulation of blood pressure and water salt balance.

Expression analysis of the UC Irvine lines found the standard expression profile of ageing, with both regimes seeing a downregulation of proteolysis, mitochondrial function intermediary metabolism and nucleic acid synthesis while protein synthesis, stress responses and immunity were upregulated. The long-lived lines were found to have increased expression proteolysis and xenobiotic

detoxification genes relative to the controls. Of the genes differentially expressed between the treatment groups, 27 were found to co-localize to QTLs on chromosome 3, found to be associated with the lifespan differences between the lines (Wilson et al., 2013).

Microarray transcriptomics has also been useful in investigating the link between longevity and stress resistance. A microarray comparison of the stress and longevity selected University of Aarhus lines found that the longevity selected lines shared many common expression changes with those selected for starvation and desiccation resistance (J. G. Sørensen, Nielsen and Loeschcke, 2007). Interestingly, it was found that lines selected for varying kinds of heat stress, heat knockdown,

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consistent high temperature or heat-shock, had dissimilar expression patterns from one another, suggesting the heat stress response is highly multifaceted and must be regulated carefully to protect against different sources of damage.

33.3

Genomics

With the completion of the Drosophila genome project (Adams et al., 2000), and the subsequent annotation of many of its genes and features (Tweedie et al., 2009), a powerful new tool was available in the analysis of long-lived flies. Using these annotations, smaller scale sequencing studies were able to sequence and characterise previously known genes of interest, for instance indy, shown to double average lifespan in some conditions when knocked out by p-element insertion (Rogina et al., 2000). Sequencing the indy gene, it was determined to be homologous to the mammalian sodium dicarboxylate cotransporter, which, coupled with its expression in the midgut, fat body and oenocytes, suggested that the knockout may lead to a reduced metabolic function mimicking dietary restriction. This success was controversial however when the result was found to be linked to the presence of the parasite Wolbachia (Toivonen et al., 2007), although this was later refuted (Helfand et al., 2009) and deletion of mammalian homologues of indy has since been suggested as a

promising target for the treatment of nutritional diseases of ageing such as obesity and type 2 diabetes (Birkenfeld et al., 2011).

On a broader scale, sequencing data has been used in Genome Wide Association Study (GWAS) approaches using Drosophila, to try and unpick the genomic differences between regular and unusually long-lived flies. Comparing the genomes of extremely long-lived flies to younger members of the same cohort, SNPs were mostly found in genes of the immune response and glutathione metabolic process. Older insects are extremely susceptible to pathogens, due to accumulated injuries providing numerous access routes, and such variation in immune response genes is unsurprising. Likewise, glutathione transferases are responsible for cleaning up various damageing metabolites that form as a result of oxidative stress, another process implicated in ageing-related mortality (Burke et al., 2014). Adding to this, a GWAS of the Drosophila melanogaster Genetic Reference Panel revealed genetic variants associated with ageing in 197 inbred strains with a wide distribution of mean lifespans. Several known ageing pathways were highlighted in this analysis, including proteolysis, carbohydrate metabolism, apoptosis and the TOR pathway, although individual SNPs contributing to longevity could not be effectively determined due to lack of power (Ivanov et al., 2015).

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