Brad A. Rikke

Longevity genes in the nematode Caenorhabditis elegans also mediate increased resistance to stress and prevent disease.

More than 40 single-gene mutants in Caenorhabditis elegans have been demonstrated to lead to increased lifespan (a rigorous, operational test for being a gerontogene) of 20% or more; these are referred to collectively as ‘Age’ mutants. Age mutants must change key functions that are rate-limiting determinants of longevity; moreover, important genes can be identified independently of prior hypotheses as to actual mode of gene action in extending longevity and/or ‘slowing’ of ageing. These Age mutants define as many as nine (possibly) distinct pathways and/or modes of action, as defined by primary phenotype. Each of three well-studied mutants (age-1, clk-1, and spe-26) alters age-specific mortality rates in a fashion unique to itself. In age-1 mutants, the decreases in mortality rates are quite dramatic, with an almost tenfold drop in mortality throughout most of life. All Age mutants (so far without exception) increase the ability of the worm to respond to several (but not all) stresses, including heat, UV, and reactive oxidants. We have used directed strategies as well as random mutagenesis to identify novel genes that increase the worms ability to resist stress. Two genes (daf-16 and old-1) are epistatic to the long-life phenotype of most mutants and also yield over-expression strains that are stress-resistant and long-lived. We have also used a variety of approaches to determine what transcriptional alterations are associated with increased longevity (and with ageing itself), including whole-genome expression studies using microarrays and GFP reporter constructs. We suggest that the role of the Age genes in both longevity and stress resistance indicates that a major evolutionary determinant of longevity is the ability to respond to stress. In mammals, both dietary restriction and hormesis are phenomena in which the endogenous level of resistance to stress has been upregulated; both of these interventions extend longevity, suggesting possible evolutionary conservation.

Strain variation in the response of body temperature to dietary restriction

Dietary restriction (DR, also referred to as calorie restriction, energy restriction, and food restriction) retards senescence and increases longevity in mammals. DR also lowers mean body temperature (T(b)), and thus mean T(b) might be useful as a covariate of DR-induced life extension. Indeed, lower T(b) could itself underlie some of the beneficial life-extension effects that occur during DR. To assess the relationship between lower T(b) during DR and life extension, we asked whether significant strain variation exists in the T(b) response of mice being fed 60% ad libitum (AL). Individually-housed, female mice from 28 strains, representing a genealogically diverse sample of the classical inbred strains, were directly compared. The mean T(b)s in response to DR exhibited highly significant strain variation, ranging from 1.5 degrees C below normal to a phenomenal 5 degrees C below normal. This variation was not explained by differences in loss of thermoregulation, AL adiposity, sensitivity to a nonadaptive hypothermia, motor activity, thermal arousal, absolute food intake, or efficacy of nutrient extraction. The variation in strain mean T(b) was also present in the absence of torpor. This strain variation could be used to critically test whether lower T(b) is a covariate of life extension during DR.

Genetic structure of the LXS panel of recombinant inbred mouse strains: a powerful resource for complex trait analysis

The set of LXS recombinant inbred (RI) strains is a new and exceptionally large mapping panel that is suitable for the analysis of complex traits with comparatively high power. This panel consists of 77 strains—more than twice the size of other RI sets— and will typically provide sufficient statistical power (β = 0.8) to map quantitative trait loci (QTLs) that account for ∼25% of genetic variance with a genomewide p < 0.05. To characterize the genetic architecture of this new set of RI strains, we genotyped 330 MIT microsatellite markers distributed on all autosomes and the X Chromosome and assembled error-checked meiotic recombination maps that have an average F2-adjusted marker spacing of ∼4 cM. The LXS panel has a genetic structure consistent with random segregation and subsequent fixation of alleles, the expected 3–4 × map expansion, a low level of nonsyntenic association among loci, and complete independence among all 77 strains. Although the parental inbred strains—Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS)—were derived originally by selection from an 8-way heterogeneous stock selected for differential sensitivity to sedative effects of ethanol, the LXS panel is also segregating for many other traits. Thus, the LXS panel provides a powerful new resource for mapping complex traits across many systems and disciplines and should prove to be of great utility in modeling the genetics of complex diseases in human populations.

Fat maintenance is a predictor of the murine lifespan response to dietary restriction

Dietary restriction (DR), one of the most robust life‐extending manipulations, is usually associated with reduced adiposity. This reduction is hypothesized to be important in the life‐extending effect of DR, because excess adiposity is associated with metabolic and age‐related disease. Previously, we described remarkable variation in the lifespan response of 41 recombinant inbred strains of mice to DR, ranging from life extension to life shortening. Here, we used this variation to determine the relationship of lifespan modulation under DR to fat loss. Across strains, DR life extension correlated inversely with fat reduction, measured at midlife (males, r = −0.41, P < 0.05, n = 38 strains; females, r = −0.63, P < 0.001, n = 33 strains) and later ages. Thus, strains with the least reduction in fat were more likely to show life extension, and those with the greatest reduction were more likely to have shortened lifespan. We identified two significant quantitative trait loci (QTLs) affecting fat mass under DR in males but none for lifespan, precluding the confirmation of these loci as coordinate modulators of adiposity and longevity. Our data also provide evidence for a QTL previously shown to affect fuel efficiency under DR. In summary, the data do not support an important role for fat reduction in life extension by DR. They suggest instead that factors associated with maintaining adiposity are important for survival and life extension under DR.

Genetic dissection of dietary restriction in mice supports the metabolic efficiency model of life extension

Dietary restriction (DR) has been used for decades to retard aging in rodents, but its mechanism of action remains an enigma. A principal roadblock has been that DR affects many different processes, making it difficult to distinguish cause and effect. To address this problem, we applied a quantitative genetics approach utilizing the ILSXISS series of mouse recombinant inbred strains. Across 42 strains, mean female lifespan ranged from 380 to 1070days on DR (fed 60% of ad libitum [AL]) and from 490 to 1020days on an AL diet. Longevity under DR and AL is under genetic control, showing 34% and 36% heritability, respectively. There was no correlation between lifespans on DR and AL; thus different genes modulate longevity under the two regimens. DR lifespans are significantly correlated with female fertility after return to an AL diet after various periods of DR (R=0.44, P=0.006). We assessed fuel efficiency (FE, ability to maintain growth and body weight independent of absolute food intake) using a multivariate approach and found it to be correlated with longevity and female fertility, suggesting possible causality. We found several quantitative trait loci responsible for these traits, mapping to chromosomes 7, 9, and 15. We present a metabolic model in which the anti-aging effects of DR are consistent with the ability to efficiently utilize dietary resources.

Identification of a genetic region in mice that specifies sensitivity to propofol.

Background Long‐sleep (LS) and short‐sleep (SS) mice, initially selected for differential sensitivity to ethanol, also exhibit differential sensitivity to propofol. By interbreeding LS and SS mice to obtain progeny whose chromosomes are a patchwork of the LS and SS chromosomes, the authors determined whether differential propofol sensitivity cosegregates with any particular chromosomal region(s). Such cosegregation is the essence of genetic linkage mapping and a first step toward isolating a gene that can modulate propofol sensitivity in mammals. A gene underlying a quantitative trait such as anesthetic sensitivity is commonly called a quantitative trait locus (QTL). Methods The propofol dose was 20 mg/kg injected retroorbitally. Sensitivity was measured as the duration of the loss of righting reflex (LORR). The LORR and propofol brain levels at awakening were determined for 24 LSXSS recombinant‐inbred (RI) strains, derived by intercrossing LS and SS for two generations followed by > 20 generations of inbreeding. A genetic linkage between LORR and an albino mutation on chromosome 7 was investigated further using 164 second‐generation progeny (F2 s) from intercrossing inbred LS and inbred SS mice, similar to the LSXSS RIs except F2 s are not inbred. The linkage between propofol sensitivity and the albino locus also was investigated using additional genetic markers on chromosome 7. Statistical significance was assessed by interval mapping using a regression method for RIs and Mapmaker/QTL (Whitehead Institute, Cambridge, MA) for F2 s. Results Genetic mapping in the LSXSS RIs revealed a QTL tightly linked to the Tyr (albino) locus that accounts for nearly all of the genetic difference in propofol sensitivity between LS and SS mice. Analysis of propofol brain levels at awakening indicated that this QTL results from differential neurosensitivity. Mapping in F2 s confirmed the genetic linkage to Tyr. Mice (ISS c/c x C57BL/6 c2j /C) that differed only by an albino mutation at Tyr were not differentially sensitive to propofol. Conclusions A single QTL, called Lorp1, underlies most of the genetic difference in propofol neurosensitivity between LS and SS mice. Although this QTL is tightly linked to Tyr, propofol sensitivity is not modulated by albinism. For mapping this QTL, the LSXSS RIs proved to be an especially powerful resource, localizing the candidate‐gene region to a 99% confidence interval of only 2.5 centimorgans.

Lower body temperature as a potential mechanism of life extension in homeotherms.

Although best known for his studies on the anti-aging effects of dietary restriction, Dr Roy Walford began his career by studying the anti-aging effects of lowering body temperature. As a tribute to his long and productive career, we review these pioneering studies and the singular influence these have had on our own thinking about the potential for lower body temperature to extend the life span of homeotherms. We show our results from a study of six classical inbred strains of mice that depict marked strain variation in the body temperature response to dietary restriction. In addition, we show a genome scan from a recombinant inbred strain panel in which we identified a significant quantitative trait locus on murine chromosome 9 and a provisional locus on chromosome 17 that specify variation in the response of body temperature to dietary restriction. These discoveries suggest that we can now extend the studies of Dr Walford to critically test whether lower body temperature can prolong the life span of mammals.

A Microarray Analysis of Potential Genes Underlying the Neurosensitivity of Mice to Propofol

Establishing the mechanism of action of general anesthetics at the molecular level is difficult because of the multiple targets with which these drugs are associated. Inbred short sleep (ISS) and long sleep (ILS) mice are differentially sensitive in response to ethanol and other sedative hypnotics and contain a single quantitative trait locus (Lorp1) that accounts for the genetic variance of loss-of-righting reflex in response to propofol (LORP). In this study, we used high-density oligonucleotide microarrays to identify global gene expression and candidate genes differentially expressed within the Lorp1 region that may give insight into the molecular mechanism underlying LORP. Microarray analysis was performed using Affymetrix MG-U74Av2 Genechips® and a selection of differentially expressed genes was confirmed by semiquantitative reverse transcription-polymerase chain reaction. Global expression in the brains of ILS and ISS mice revealed 3423 genes that were significantly expressed, of which 139 (4%) were differentially expressed. Analysis of genes located within the Lorp1 region showed that 26 genes were significantly expressed and that just 2 genes (7%) were differentially expressed. These genes encoded for the proteins AWP1 (associated with protein kinase 1) and “BTB (POZ) domain containing 1,” whose functions are largely uncharacterized. Genes differentially expressed outside Lorp1 included seven genes with previously characterized neuronal functions and thus stand out as additional candidate genes that may be involved in mediating the neurosensitivity differences between ISS and ILS.

No evidence that competition for food underlies lifespan shortening by dietary restriction in multiply housed mice: response to commentary

Department of Integrative Physiology, University of Colorado,Boulder, CO 80309, USAOur recent report (Liao et al., 2010) that dietary restriction(DR) shortened lifespan in many strains of recombinant inbred(RI) mice was unexpected in the context of a long history ofresearch on the life-extending action of DR (Weindruch W Masoro, 2005). It is important to recognize,however,thatourreportisnotthe first(see Liaoet al.,2010,fora number of earlier reports in which DR had no effect onlifespan, or even led to shorter lifespan). Although we hadhypothesized that there would be strain variation in the extentof lifespan extension by DR, we too had overlooked dissonantdata, which in retrospect would have led us to expand ourhypothesis to include possible negative effects of DR. Thegenetic variation we have found in the response to DR, particu-larly given its magnitude, provides a valuable tool for elucidatingunderlying genes and pathways that mediate the lifespan-mod-ulatingactionofDR–theultimategoalofresearch inthisarea.We are pleased to respond to the commentary by Mattson(2010) and the opportunity it offers to provide additional infor-mation bearing on mechanisms by which DR can shorten as wellas lengthen lifespan. The commentary offers some interestingsuggestions about these mechanisms. Here, we provide datathat largely refute these suggestions. We also respond to othercomments and speculations raised in the commentary thatdeserveclarification.The author’s primary explanation for the lifespan shorteningby DR of some strains is based on postulated effects of multiplehousing. The author assumes that multiple housing results incompetition for limited food, thereby leading to dominancebehaviour and unequal distribution of food among the individu-als. The commentary speculates that the dominant mousewouldeat adisproportionate shareof the food,andthus receiveless than the normal 40% restriction, and that subordinate micein the cage would be restricted more than 40% leading to earlydeath, i.e., shortened lifespan in relation to ad libitum (AL) fedcontrols of the same strain. Our published data, as well as newdata presented here, do not support this idea. First, exclusionfrom the analysis of early deaths, which in Mattson’s modelwould represent subordinate mice with exceptionally low foodintake, has little impact on the frequency or distribution of RIstrains with shortened lifespan (supporting Fig. S3, Liao et al.,2010). Second, taking body weight as an indicator of feedingdominance, there is no evidence that such dominance led toshortened life of subordinates. Inmales, arguedby theauthor tobe more prone to feeding dominance, mean lifespan of themost subordinate (i.e. smallest) mouse was no shorter than thatof the most dominant (i.e., largest) mouse, and the same is truefor females, both for the strains that had significantly shortenedlifespans under DR (Fig. 1), as well as for strains with shortenedlifespans that did not reach statistical significance (data notshown). Finally, although we were unable to measure the foodconsumption of individual mice housed multiply, using bodyweight as a measure of food consumption provides little evi-dence formarkedinequality offood intakeamongcagemates.Iffood were unequally distributed among cagemates because ofcompetition, the range and variance of body weight in DR cageswould be greater than that in AL fed cages. Neither range (datanot shown) nor coefficient of variation of body weight was sig-nificantly greater in the DR than in the AL cages (Table 1). Thesefindings are consistent with our behavioural observations of themice during feeding in this study as well as previously (Ikenoet al., 2005). Although we are in agreement with the commen-tary that DR mice consume virtually all of their allotment within1–2 h of feeding, we see no fighting during this period. Whenfood is available, each DR mouse spends its time obtainingpellets of food from the hopper and consuming them. In somecases, a mouse will take the pellet from another mouse butthat mouse will obtain another pellet from the hopper andboth will have food to eat (C.-Y. Liao, V. Diaz, J. F. Nelson;unpublished observations). The food hoppers are large enoughtoallowallmicetoaccessfoodsimultaneously.The most compelling evidence that competition for food doesnot underlie the DR shortening of lifespan is the finding that DR

Genetic Models and Mapping Genes in the Study of Anaesthetic Action

The molecular action of anaesthetic agents is a problem well studied but not well understood. A novel approach to identifying molecular pathways involved in anaesthetic drug action involves isolating the genes mediating anaesthetic sensitivity in animal models. Several animal models have been derived using differential drug sensitivity as a screening phenotype. This method has produced both invertebrate (Drosophlla melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae) and vertebrate (rodent) animal lines that differ in their central nervous system (CNS) response to anaesthetic agents. Lines can be derived from spontaneous or induced mutagenic processes, or selective breeding schemes. Inbred and recombinant lines possessing inherent differential drug sensitivities also are available. All can be used in a method of gene mapping known as quantitative trait loci (QTL) mapping. Anaesthetic sensitivity is a quantitative measure and is likely influenced by multiple genes known as QTLs where each QTL corresponds to a single gene. In this review we describe several genetic models currently used in studying anaesthetic action. We focus primarily on a mouse model which has been used to identify a QTL mediating propofol neurosensitivity in a selectively bred mouse line known as Long Sleep (LS) and Short Sleep (SS) mice.