Tower lab research

    Changes in gene expression during aging as a guide to aging mechanisms

    We are using the laboratory fruit fly, Drosophila melanogaster, as a model to investigate basic mechanisms of aging.  Our approach is to identify the changes in gene expression that occur during normal aging and use this information as a guide to investigating underlying aging mechanisms. We have used a variety of approaches, including traditional molecular biology techniques, transgenic reporter constructs, micro-array analyses and RNAseq. The changes in gene expression during fly aging were found to be highly similar to the fly’s response to oxidative stress, and include increased expression of specific heat shock protein (Hsp) genes, induction of purine biosynthesis genes, dramatic induction of innate immune response genes, and the down-regulation of mitochondrial genes including electron transport chain (ETC); most or all of these responses appear to be conserved in mammalian aging.

    We have investigated the regulation of the Drosophila Hsp genes Hsp22 and Hsp70, and have found that conserved heat shock response elements in the promoters of these genes are required for their up-regulation during aging, and that the time-course is accelerated by mutations and environmental conditions that shorten life span and increase oxidative stress. Hsp22 localizes to the mitochondria and exhibits one of the largest aging-related increases known for a eukaryotic protein (>150-fold). We hypothesize that many of the gene expression changes during fly aging result from a failure to maintain normally functional mitochondria and a consequent oxidative stress and proteotoxic stress.  This idea is consistent with results from yeast, C. elegans and mammals that point to a central role for the mitochondria in modulating life span and aging phenotypes.

    Predictive biomarkers of aging & a novel video-tracking assay

    Several genes that are induced during aging and by oxidative stress (including hsp22hsp70, and the innate immune response genes Drosomycin and Metchnikowin) were analyzed using transgenic reporter constructs.  Fusing the regulatory regions of the genes to GFP (or DsRED) produced transgenic reporters that could be quantified longitudinally during fly aging. To facilitate these analyses, we have developed a video-based 3D tracking assay that allows fly movement, behavior, and transgenic reporter expression to be quantified simultaneously.  Strikingly, the expression of the immune reporters and the Hsp reporters was found to be predictive of remaining fly life span.  Hsps show promise as biomarkers of aging in C. elegans and humans as well.  In the future we plan to use these reporters to investigate the mechanisms that cause different individual animals to have different life spans.

    Identifying life span regulatory genes using conditional transgenic systems

    To identify genes that directly regulate life span we have focused on the development and application of conditional transgenic systems.  These include the FLP-out system based on yeast FLP recombinase (induced by a heat pulse), the Tet-on system (induced by doxycycline), and the Gene-Switch system (induced by RU486/mifepristone).  Using these systems we have over-expressed a series of logical candidate genes in the adult fly, and find that the redox-regulatory enzymes Cu/Zn-superoxide dismutase (Cu/ZnSOD) and mitochondrial Mn-superoxide dismutase (MnSOD) are both positive regulators of life span, and can yield life span increases ranging from 15-40%.  Our data, as well as that from other labs demonstrates that the life span increases caused by SOD are dependent upon the fly’s genotype, sex, and specific diet/environment. Transcriptional profiling and in vivo reporters indicate that Tet-on over-expression of MnSOD acts in young adult flies to create a mitochondrial unfolded protein response (UPRmt) in the oenocytes (liver-like cells).

    We have engineered a P-type transposable element with an outwardly-directed, Tet-on promoter (called “PdL”).  PdL enabled genetic screens for positive regulators of adult life span, and identified the genes Filamin, Fwd, Cct1, alpha-mannosidase II, and the regulatory subunit of the vacuolar H+-ATPase (V-ATPase) VhaSFD.

    Sexual antagonistic pleiotropy (SAP) of p53 and foxo

    We are investigating the regulation of Drosophila life span by sex and p53.  We have found that p53 has developmental stage-specific and sex-specific effects on adult life span indicative of sexual antagonistic pleiotropy (Waskar et al 2009 Aging 1:903-936).  Over-expression of wild-type p53 (isoform A) in adult flies can increase life span in a sex-specific and tissue-specific manner, and the foxo gene was found to act in males to create sexual dimorphism in the life span effects of p53 (Shen & Tower 2010 Exp Gerontol 45:97-105).  These results suggest that both p53 and foxo exhibit sexual antagonistic pleiotropy for adult life span.  Taken together the data integrate well with current evolutionary theories of aging, and suggest a working model in which antagonistic pleiotropy of gene function between developmental stages and sexes causes a failure in nuclear-mitochondrial signaling and mitochondrial maintenance, thereby leading to oxidative stress and mortality (Tower 2006 Mech Ageing Dev 127:705-718 TowerMAD2006.pdf).  In the future we plan to investigate how the sex-determination pathway interacts with life span regulatory genes to affect mitochondrial maintenance, hsp expression and life span.

    Genetics of aging working model

    Our working model for the genetics of aging involves the on/off state of a regulatory gene switch that is ON in females and OFF in males.  Males are therefore dependent upon maternal contribution to the egg of this genes activities.  This gene is proposed to be Sxl in Drosophila, sdc-2 in C. elegans, and Xist+Foxl2 in mammals (Tower 2006 Mech Ageing Dev 127:705-718 TowerMAD2006.pdf)(Tower 2017 Sex-Specific Gene Expression and Life Span Regulation 28:735-747).  The binary switch genes are proposed to regulate sexual differentiation, dosage compensation, mitochondrial transmission to offspring, and mitochondrial maintenance during aging (Tower 2014 Arch Biochem Biophys 576:17-31).  The mitochondria are hypothesized to promote sexual differentiation through production of hormones and ROS, and in turn sexual differentiation is hypothesized to promote mitochondrial maintenance failure and aging.  These models were suggested by the sexual antagonistic pleiotropy of p53 and foxo discussed above. Finally, we have recently reported that the progesterone and glucocorticoid antagonist mifepristone/RU486 can block a trade-off between reproductive metabolism and life span in Drosophila females, yielding life span increase of +100% (Tower et al Biogerontology 2017 18(3):413-427). Mifepristone effects include preventing midgut hypertrophy in mated females (Landis et al Front Genet. 2021 12:751647). Moreover, mifepristone increased life span of virgin females on both normal and high fat diets, without reducing food intake. In collaboration with Sean Curran’s lab, mifepristone was found to increase life span of mated C. elegans. The data suggest mifepristone mechanisms are conserved across species, and support the potential of mifepristone as an anti-aging intervention in humans.

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