Ageing Research Reviews 6 (2007) 6472 www.elsevier.

com/locate/arr

Review

Small laboratory sh as models for aging research

Glenn S. Gerhard *
Weis Center for Research, Geisinger Clinic, Danville, PA 17822, United States

Abstract Fish represent approximately half of all vertebrate species, yet have received little attention as models for aging research relative to invertebrate organisms or rodents. However, the basic gerontological characteristics of several sh species have been studied and provide compelling data for further investigation. In particular, guppies have proved to be an invaluable model for evolutionary analyses of aging, killish are short-lived and may be exploitable for life span manipulation studies, and zebrash come with a formidable armament of associated biological tools from their widespread use as a model of vertebrate development. These sh are well suited for the investigation of basic processes implicated in aging, such as insulin signaling, oxidative stress, and comparative studies of species with widely divergent longevities. Under-explored areas for which these sh may also provide unique research opportunities include their use as platforms for disease modeling, drug discovery, and regenerative medicine. # 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Vertebrate; Fish; Aging; Model; Review

1. Aging studies of sh The largest class of vertebrates are the shes (Liem, 1995), whose members include some of the most experimentally versatile and shortest and longest lived vertebrates known (Finch, 1990). While several species have been studied over the past several decades from a gerontological perspective, the amount of resources directed to aging research using sh has been essentially absent when compared with invertebrate models (Gerhard et al., 2004). This stands in stark contrast to other elds, especially developmental biology, where sh have ascended as a widely used vertebrate model bridging the economic and evolutionary gap between invertebrates and rodents. Despite the disproportionate allocation of research effort, signicant progress has been made with several small tropical sh species, which exhibit an age-related increase in mortality typical of gradual senescence and denite life span (Finch, 1990). In this group of sh, age-related degenerative changes and increases in various pathological lesions have been documented, as well as the characteristic senescent decline in reproductive capacity (Patnaik et al., 1994). In this review, the focus will be on the historical and contemporary uses of three actively investigated species; guppies, killish, and zebrash. 1.1. Guppy The guppy was perhaps the rst sh to be systematically studied from a gerontological perspective, largely due to the pioneering work of Comfort (Comfort, 1961a; Woodhead, 1998). Comfort conducted studies on caloric restriction
* Tel.: +1 570 271 8669; fax: +1 570 271 6701. E-mail address: gsgerhard@geisinger.edu. 1568-1637/$ see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.arr.2007.02.007

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(Comfort, 1960), n regeneration (Comfort and Doljanski, 1958), temperature manipulation (Comfort, 1969), and reproductive capacity (Comfort, 1961b). His work was followed by Woodhead, who conducted histological surveys of various tissues of guppies throughout their life span (Woodhead, 1978; Woodhead and Pond, 1984; Woodhead et al., 1983). For example, in the heart, no signicant changes were evident until the sh were 3 years old when a loss of muscle bers in the ventricles of old sh was noted, with deposition of collagen in the bulbus arteriosus. While histological analysis of the brains throughout the lifespan showed no overall loss of tissue, in both genders a loss of neurons from the stratum griseum periventriculare in the midbrain roof occurred with age. Brain and body size increased at a similar rate throughout adult life in male sh, although in old females brain growth appeared to cease after 2 years. The guppy continues to demonstrate its gerontological utility, driven mainly by the work of Reznick et al. (2001, 2006) and Reznick (1997), who has used the species to address the questions of why and how senescence evolves in natural populations. Guppy populations experience large differences in mortality rates depending upon their geographic location and co-existence of predators. High adult mortality rates due to predation select for individuals that develop more rapidly and reproduce earlier. Based upon these observations, evolutionary theory held that guppy populations that experience low mortality rates should experience delayed senescence and longer life spans relative to those that experience high mortality rates. However, guppies from high-predation environments actually live longer because they have a longer reproductive lifespan. Reproductive life span exerts a direct contribution to individual tness, thus post-reproductive lifespan in guppies appears to be unselected and randomly set at the end of the life span. Fish may also undergo reproductive senescence and extended post-reproductive life spans, despite the general observation that oocytes can be formed and produce viable offspring throughout the life span (Reznick et al., 2001). 1.2. Killish Another type of sh for which there is a rich gerontological pedigree are the killish. Over three decades ago, Liu and Walford conducted a variety of studies using a species of the Cynolebias genus of killish (Liu et al., 1975; Liu and Walford, 1966, 1969, 1970, 1972, 1975; Walford et al., 1969). One focus was upon the under-studied phenomenon of temperature modulated life span extension. They determined the effect of a modest reduction in temperature on life span and the primary pathological phenotypes of a species of killish in the laboratory. A decreased environmental temperature increased life span, as expected, but also increased growth rates and ultimate body size. The lowered temperature was also associated with favorable effects upon immune parameters and collagen cross-linking. They also reported the impact of reciprocal temperature transfers. Fish raised at a higher temperature until midlife, then transferred to a lower temperature lived longer than sh maintained at either the lower or higher temperature, or those initially raised at the lower temperature then transferred to higher temperature at midlife. Surprisingly, no further studies have followed upon these provocative observations. Gerontological studies on killish were also conducted by Markovsky (Markofsky, 1976; Markofsky and Matias, 1977; Markofsky and Milstoc, 1979a,b; Markofsky and Perlmutter, 1972, 1973), who documented degenerative histopathological changes in the liver and kidney with age as well as the relationship between growth sexual maturity and longevity. Killish re-emerged when Jagadeeswaran again began to study their potential for aging research as a genetic model (Herrera and Jagadeeswaran, 2004). A major attraction of killish species is their relatively short life span (less than 1 year), which if coupled to the types of genetic manipulations currently applied to other sh species, could be a major advantage for identifying genes associated with aging and longevity. Their short life span derives from their life history as annual sh, generally existing in seasonal ponds in which their life expectancy in the wild is limited by the duration of the wet season. Their corresponding life span in captivity is also short, although their maintenance is complicated by storage of their eggs under relatively dry conditions for an extended time period as part of their life history. Killish are also popular aquarium shes and many different species are maintained by aquarium enthusiasts. Cellerino has also extended the work on aging in killish (Genade et al., 2005; Terzibasi et al., 2007; Valdesalici and Cellerino, 2003; Valenzano and Cellerino, 2006; Valenzano et al., 2006a,b). One species manifests a maximum life span of about 3 months, although some controversy surrounds the interpretation of the longevity data for this very short lived organism (Cellerino, 2005; Jagadeeswaran, 2005). Indeed, the seeming race to shortevity in the search for temporally tractable short-lived organisms may seem to run counter to those researchers whose goals include the identication of aging mechanisms in notoriously long-lived species such as humans. Nevertheless, if such short-lived

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species can be shown to manifest public mechanisms of senescence and are not short-lived because of private early death mechanisms, they may be useful models. Indeed, the demonstration of histological and behavioral biomarkers related to aging, and the ability to clone by homology genes related to aging, supports the use of killish as a genetic model for aging research. The possibility therefore exists that such a species may be used to genetically manipulate life span over a time frame similar to that of Drosophila. Another possible use for such short-lived species is through comparative studies of similar organisms with disparate life spans (Gerhard et al., 2004). 1.3. Zebrash Zebrash are a small (12 in.) fresh water tropical cyprinid sh native to India. They were initially attractive to developmental biologists because of their transparent embryos, rapid ex utero development, prolic reproductive capacity (100200+ eggs per clutch), and the relatively modest amount of resources required to maintain large numbers of sh (Eisen, 1996). They are physically large enough to isolate signicant amounts of specic tissues, especially skeletal muscle, yet are sufciently small to allow for substantial economies of scale. The genetic resources for zebrash continue to expand with an increasingly dense genetic map and expressed sequence tag assemblage, the existence of hundreds of genetic mutants from large scale mutagenesis experiments (Driever et al., 1996), the availability of transgenic morpholino knock down techniques (Nasevicius and Ekker, 2000) and other genetic manipulations such as haploid embryos, homozygous diploid progeny (Postlethwait and Talbot, 1997), and the ongoing sequencing of its genome (Vogel, 2000). This author has detailed the case for zebrash as a model for studying basic mechanisms of aging elsewhere (Gerhard, 2003; Gerhard and Cheng, 2002), as have Keller and Murtha (Keller and Murtha, 2004; Murtha and Keller, 2003; Murtha et al., 2003) and Kishi (Kishi, 2004; Kishi et al., 2003; Yu et al., 2006). Each investigator has contributed important basic information validating the use of zebrash as a potentially valuable model for aging research. Perhaps the major reason why zebrash has remained outside the mainstream of gerontological research (Gerhard et al., 2004) is its relatively long average life span of about 3 years (Gerhard et al., 2002). The relative impracticality of a 3 years mean life span is balanced by the truly impressive array of biological tools for manipulating and analyzing zebrash, rivaling mice, worms, and ies, and leading some to speculate that zebrash may become the vertebrate system of choice for a wide spectrum of biological questions that need to be investigated in vivo at cellular and subcellular resolutions (Beis and Stainier, 2006). Questions related to aging have not yet become part of this wide spectrum. 2. Temperature reduction (TR) and caloric restriction (CR) Body temperature has been reported as a predictor of longevity in primates and humans (Roth et al., 2002) and, in multivariate analyses, was found to be one of four variables that accounted for most of the variance in maximum life spans across 85 species (Finch, 1990). Such evidence indirectly implicates body temperature as a signicant factor in aging. However, mechanistic studies on temperature reduction (TR) and life span have been almost exclusively performed in invertebrates, mainly insects (Sohal and Allen, 1986). Elegant studies have documented the inverse correlation between thermally regulated metabolic rate and life span in Dipteran species (Hollingsworth, 1969; McArthur and Sohal, 1982). Indeed, a signicant portion of the empiric framework of aging theories invoking metabolic rate, metabolic potential, and oxidative stress are based upon data obtained from insects. However, ndings derived from invertebrate models warrant replication in a vertebrate ectotherm before such results can be extrapolated to all poikilotherms (Sohal and Allen, 1990). In addition, potentially important variables are not accounted for in many insect TR studies, including caloric intake, metabolic rate, and histopathology prole, which are easily accommodated using sh species. Following on previous studies of TR on killish (Liu and Walford, 1972, 1975), the effects of temperature on longevity, behavior, and age-associated histological markers of senescence were determined in a short-lived killish species (Valenzano et al., 2006a). Lowering temperature from 25 to 22 8C increased both median and maximum lifespan due to a reduction in the slope of the age-dependent acceleration in death rate. TR also retarded the onset of age-related locomotor and learning decits and decreased the accumulation of the age-related marker lipofuscin. The effects of TR on gene expression in zebrash skeletal muscle have also been determined and indicated that an oxidative stress response was induced (Malek et al., 2004). The role of oxidative stress as a potential longevity promoting mechanism of TR has been investigated by relatively few laboratories and in relatively few models

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(McArthur and Sohal, 1982; Sohal et al., 1985; Farmer and Sohal, 1987). In general, oxidative damage to lipids and proteins and ROS generation are increased at higher temperatures, although exceptions are apparent (Farmer and Sohal, 1987). Even the relationship between metabolic rate and temperature is not simple and direct in certain insects (McArthur and Sohal, 1982). Few studies have been conducted in non-insect ectothermic organisms. Caloric restriction (CR) is a well-studied environmental manipulation that can extend the mean and maximum life spans of rodents and other organisms. The life span prolonging effects of CR have been documented for a number of diverse species, rendering it close to being a universal phenomenon (Rose, 1991). CR has been previously studied in the guppy, but the effect was modest and the regimen atypical (Comfort, 1960, 1963). Indeed, the author concluded that rather than a generic slowing of aging, the post-ponement of a particular cause of death may have occurred. Thus, whether CR delays aging in a small sh species may not yet be known. The effects of CR and TR may be related. While TR may result in a reduction in food intake, CR may exert its longevity promoting effects in homeotherms through a reduction of body temperature. CR dramatically lowers body temperature in mice (Duffy et al., 1990), which readily enter a torpor-like state while fully fed controls rarely exhibit such changes. Rats (Duffy et al., 1997) and non-human primates (Lane et al., 1996) on CR regimens also have lower mean body temperatures than AL controls, although the reduction is small compared to that attained in mice. Nevertheless, a reduction in body temperature appears to be an effect shared among diverse mammalian species subject to CR. Mounting evidence also implicates oxidative stress as a key mechanism in the effects of CR upon life span (Sohal and Weindruch, 1996; Merry, 2000; Wanagat et al., 1999). As vertebrates, sh generally exhibit similar toxicological and adaptive responses to oxidative stress as do mammals (Kelly et al., 1998; Winston, 1991). Fish species may therefore be an ideal model on which to further explore the mechanisms of CR and TR. 3. Age-related disease models Specic age-related diseases, such as Alzheimers, have also been studied in sh (Newman et al., 2007). For example, molecular studies on presenilins (Leimer et al., 1999) and beta amyloid (Musa et al., 2001), which play prominent roles in the molecular pathogenesis of Alzheimers disease, have been performed using zebrash. Zebrash lacking the presenilin enhancer 2 (Pen-2) protein demonstrate excessive p53-dependent apoptosis and neuronal loss (Campbell et al., 2006). Another age-related disorder, Parkinsons disease (PD) has been modeled in zebrash by treating with PD-inducing neurotoxins which causes behavioral alterations and decreased locomotor activity (Bretaud et al., 2004). Inactivation of DJ-1 in zebrash, a gene which when mutated in humans leads to early onset Parkinsons disease, resulted in increased p53 and Bax expression and loss of dopaminergic neurons after exposure to hydrogen peroxide (Bretaud et al., 2007). In addition to neurodegenerative diseases, other age-related disorders have been modeled in sh. For example, a zebrash model of osteoporosis has been reported. Exposure of zebrash larva to vitamin D3 analogs and intermittent parathyroid hormone administration resulted in dose-dependent increases in the formation of mineralized bone, whereas continuous exposure to PTH resulted in net bone loss (Barrett et al., 2006; Fleming et al., 2005). In addition to mechanistic studies, these models also represent potential platforms for drug discovery (vide infra). 4. Drug discovery The search for elixirs of youth is centuries old, although the application of modern pharmacological approaches has only recently begun (Geesaman, 2006). Chemical genetic studies to identify and characterize compounds that delay aging and extend lifespan using invertebrate models have been reported (Gill et al., 2003; Bauer et al., 2004). Several anti-aging drug candidates have been identied and characterized (Collins et al., 2006; Evason et al., 2005). For example, resveratrol, a natural phytoalexin found in grapes and red wine, has been implicated as a potential anti-aging compound (Baur and Sinclair, 2006). Exploiting the very short life span of a killish species, resveratrol was added to the food starting in early adulthood and caused a dose-dependent increase in median and maximum life span, as well as a delay in the age-dependent decline in activity and cognitive performances (Valenzano and Cellerino, 2006; Valenzano et al., 2006b). Resveratrol also reduced the expression of neurobrillary degeneration in the brain of this short-lived species. These studies highlight the potential utility of sh as models for characterizing new anti-aging compounds.

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Fig. 1. Microplate-based assays using zebrash. (A) Over 20 zebrash embryos easily t into a single well of a 96-well microplate. This allows for high signal strength using in vivo assays (e.g., uorescent probes) for high throughput compound screening. (B) Light microscopy of 1-day-old zebrash embryo. Embryo is curled within its chorion. (C) Same embryo as in (B) exposed to 20 ,70 -dichlorodihydrouorescein diacetate, a probe that uoresces green following oxidation by ROS. Such probes may be useful in a high-throughput compound screening protocol.

In addition to their use as species for the replication and characterization of candidate anti-aging compounds, largescale screens for the discovery of medically relevant compounds have now become feasible using zebrash, which has emerged as an increasingly used platform for high-throughput drug screening (Zon and Peterson, 2005). Screening for small molecules using live, multi-cellular organisms rather than cultured cells or cell-free solutions is an advantage for studying complex processes, such as aging, that cannot be recapitulated in simpler in vitro assays, and also allows for simultaneous assessment of toxicity. For the rst few weeks of life, zebrash can easily t into a well of a 96-well plate (Fig. 1), enabling high-throughput screening assays that require only small amounts of test compounds, which readily diffuse into young zebrash. A variety of assay end-points have been described (Rubinstein, 2006), including several with potential gerontological relevance. For example, apoptotic cells can also be visualized in live zebrash embryos using acridine orange staining, a metachromatic intercalator sensitive to DNA conformation (Brand et al., 1996; Furutani-Seiki et al., 1996), which has been proposed as a screen for therapeutic compounds affecting apoptosis (Parng et al., 2004). Other assays, including in vivo uorescence assays for reactive oxygen species are amenable to a microplate-based format (Fig. 1). The variety of experimental techniques available for manipulating zebrash may be incorporated into the drug discovery process. Among those techniques include the use of morpholino oligos (Nasevicius and Ekker, 2000), which when microinjected knockdown the expression of genes with potential relevance to aging in zebrash. Standard techniques for the microinjection of DNA or RNA can also be used to increase the expression of specic proteins. For example, reduction of IGF-I or IGF-I signaling has been correlated with increased longevity and retarded aging in several diverse species (Bartke, 2005). Over-expression of IGF-I in zebrash embryos through microinjection with IGF-I in vitro transcribed mRNA, results in dorsalized embryos with shortened tail and trunk regions (Eivers et al.,

Fig. 2. Microinjection of IGF-1 RNA into zebrash embryos. (A) Normal 1-day-old zebrash embryo. (B) Zebrash embryo 1 day after microinjection with in vitro transcribed IGF-1 RNA showing a shortened twisted tail with an apparently normal head region. (C) Phenotype of IGF-1 microinjected embryo at 2 days post-fertilization showing pericardial effusion and severe tail abnormality.

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2004), an easily identiable abnormal phenotype (Fig. 2). This phenotype may be amenable for use in screening for compounds that attenuate or block this phenotype, i.e., those compounds that inhibit the increased IGF-1 signaling. Yolk sac microinjection of morpholinos or mRNA can be accomplished quickly and efciently due to the relatively large size of the embryos facilitating high throughput screening. 5. Regenerative medicine Tissue engineering, cell replacement therapy, and regenerative medicine are promising approaches to aging and age-related disorders (Gurtner et al., 2006) and have employed comparative approaches (Alvarado and Tsonis, 2006). Fish have been used for studies on organ regeneration for many years, including early work by Comfort on the effect of aging and temperature on n regeneration in the guppy (Comfort and Doljanski, 1958; Comfort, 1969). Fin regeneration in sh has been studied for over a century but has recently been reinvigorated by the biological resources associated with the zebrash (Akimenko et al., 2003). Little is known about molecular mechanisms of n regeneration, and cellular mechanisms are incompletely dened, but the tools now available using zebrash may accelerate discovery (Poss et al., 2003). An organ upon which zebrash has shed signicant light is the heart. Cardiac injury in mammals results in scarring with minimal regeneration of heart muscle. In contrast, zebrash fully regenerate heart muscle within weeks of a onefth resection of the ventricule (Poss et al., 2002). Regeneration occurs through proliferation of cardiac myocytes at the leading edge of the regenerating muscle. In zebrash with mutations in the Mps1 mitotic checkpoint kinase, a critical cell cycle regulator, regeneration did not occur but instead scars formed. Further studies indicated that regeneration occurred by both myocardial and epicardial cells in concert in an Fgf-dependent manner (Lepilina et al., 2006). Further studies may help pinpoint the molecular targets for exploiting regeneration in mammalian species. 6. Summary Despite the dearth of attention paid to sh models in aging research, such species present tremendous scientic opportunities. The focus on three such sh in this review serves to highlight the potential of this class of organisms. References
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