Identification of genetic markers of biological age and metabolism

A method of measuring the biological age of a multicellular organism is disclosed. In one embodiment this method comprises the steps of obtaining a sample of nucleic acid isolated from the organism's organ, tissue or cell and determining the expression pattern of a panel of sequences within the nucleic acid that have been predetermined by either increase or decrease in response to biological aging of the organ, tissue or cell. A method of obtaining biomarkers of aging is also disclosed. This method comprises the step of comparing a gene expression profile of a young multicellular organism subject's organ, tissue or cells; a gene expression profile from a chronologically aged subject's organ, tissue or cell; and a gene expression profile from a chronologically aged but biologically younger subject's organ, tissue or cell and identifying gene expression alterations that are observed when comparing the young subjects and the chronologically aged subjects and are not observed or reduced in magnitude when comparing the young subjects and the chronologically aged but biologically younger subjects.

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Description
CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to provisional application 60/148,540, filed Aug. 12, 1999, U.S. provisional application 60/178,232, filed Jan. 26, 2000 and 60/211,923 filed Jun. 16, 2000. These provisional applications are incorporated by reference as if fully set forth herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT BACKGROUND OF THE INVENTION

[0003] A common feature of most multicellular organisms is the progressive and irreversible physiological decline that characterizes senescence. Although genetic and environmental factors can influence the aging process, the molecular basis of senescence remains unknown. Postulated mechanisms include cumulative damage to DNA leading to genomic instability, epigenetic alterations that lead to altered gene expression patterns, telomere shortening in replicative cells, oxidative damage to critical macromolecules and nonenzymatic glycation of long-lived proteins (S. M. Jazwinski, Science 273:54, 1996; G. M. Martin, et al., Nature Gen. 13:25, 1996; F. B. Johnson, et al., Cell 96:291, 1996; K. B. Beckman and B. N. Ames, Physiol. Revs. 78:547, 1998). Factors which contribute to the difficulty of elucidating mechanisms and testing interventions include the complexity of organismal senescence and the lack of molecular markers of biological age (biomarkers). Aging is complex in that underlying mechanisms in tissues with limited regenerative capacities (e.g., skeletal and cardiac muscle, brain), which are composed mainly of postmitotic (non-dividing) cells, may differ markedly from those operative in proliferative tissues. Accordingly, approaches which provide a global assessment of senescence in specific tissues would greatly increase understanding of the aging process and the possibility of pharmaceutical, genetic or nutritional intervention.

[0004] Genetic manipulation of the aging process in multicellular organisms has been achieved in Drosophila, through the over-expression of catalase and Cu/Zn superoxide dismutase (W. C. Orr and R. S. Sohal, Science 263:1128, 1994; T. L. Parkes, et al., Nat. Genet. 19:171, 1998), in the nematode C. elegans, through alterations in the insulin receptor signaling pathway (S. Ogg, et al., Nature 389:994, 1997; S. Paradis and G. Ruvkun, Genes Dev. 12:2488-2498, 1998; H. A. Tissenbaum and G. Ruvkun, Genetics 148:703, 1998), and through the selection of stress-resistant mutants in either organism (T. E. Johnson, Science 249:908, 1990; S. Murakami and T. E. Johnson, Genetics 143:1207, 1996; Y. J. Lin, et al., Science 282:943, 1998). In mammals, there has been limited success in the identification of genes that control aging rates. Mutations in the Werner Syndrome locus (WRN) accelerate the onset of a subset of aging-related pathology in humans, but the role of the WRN gene product in the modulation of normal aging is unknown (C. E. Yu, et al., Science 272:258, 1996; D. B. Lombard and L. Guanrente, Trends Genet. 12:283, 1996).

[0005] In contrast to the current lack of genetic interventions to retard the aging process in mammals, caloric restriction (CR) appears to slow the intrinsic rate of aging (R. Weindruch and R. L. Walford, The Retardation of Aging and Disease by Dietary Restriction (CC. Thomas, Springfield, Ill., 1988; L. Fishbein, Ed., Biological Effects of Dietary Restriction (Springer-Verlag, New York, 1991; B. P. Yu, Ed., Modulation of Aging Processes by Dietary Restriction (CRC Press, Boca Raton, Fla. 1994). Most studies have involved laboratory rodents which, when subjected to a long-term, 25-50% reduction in calorie intake without essential nutrient deficiency, display delayed onset of age-associated pathological and physiological changes and extension of maximum lifespan.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention will allow the evaluation of aging interventions on a molecular and tissue-specific basis through the identification of aging biomarkers. In particular, the use of gene expression profiles allows the measurement of aging rates of target organs, tissues and cells, and to what extent aging is delayed by specific interventions, as determined by quantitative analysis of mRNA abundance. Because aging-related gene expression profiles can be classified in subgroups according to function, the invention also allows for the determination of how function-specific aspects of aging are affected. This particular feature will allow for determination of combination therapies that prevent or reverse most aging related changes in particular organs, tissues, and cells.

[0007] In one embodiment, the present invention is a method of measuring the biological age of a multicellular organism comprising the steps of (a) obtaining a sample of nucleic acid isolated from the organism's organ, tissue or cell, wherein the nucleic acid is RNA or a cDNA copy of RNA and (b) determining the expression pattern of a panel of sequences within the nucleic acid that have been predetermined to either increase or decrease in response to biological aging of the organ, tissue or cell. Preferably, the expression patterns of at least ten sequences are determined in step (b) and the organism is a mammal, most preferably a rodent.

[0008] In one preferred embodiment of the method described above, the nucleic acid is isolated from a mammalian tissue selected from the group consisting of brain tissue, heart tissue, muscle tissue, skin, liver tissue, blood, skeletal muscle, lymphocytes and mucosa.

[0009] In another embodiment the present invention is a method of obtaining biomarkers of aging comprising the steps of: (a) comparing a gene expression profile of a young multicellular organism subject's organ, tissue or cells; a gene expression profile from a chronologically aged (and therefore biologically aged) subject's organ, tissue or cell; and a gene expression profile from a chronologically aged but biologically younger subject's organ, tissue or cell, and (b) identifying gene expression alterations that are observed when comparing the young subjects and the chronologically aged subjects and are not observed or reduced in magnitude when comparing the young subjects and chronologically aged and biologically younger subjects. Preferably, one uses high density oligonucleotide arrays comprising at least 5-10% of the subject's gene expression product to compare the subject's gene expression profile, and caloric restriction to obtain a chronologically aged but biologically younger subject.

[0010] In a preferred embodiment of the method described above, the gene expression profile indicates a two-fold or greater increase or decrease in the expression of certain genes in biologically aged subjects. In a more preferred embodiment of the present invention, the gene expression profile indicates a three-fold or greater or, most preferably three-fold or greater, increase or decrease in the expression of certain genes in aged subjects.

[0011] In another embodiment, the present invention is a method of measuring biological age of muscle tissue comprising the step of quantifying the mRNA abundance of a panel of biomarkers selected from the group consisting of markers described in the Tables 1, 2, 15 and 16. A method of measuring biological age of brain tissue comprising the step of quantifying the mRNA abundance of a panel of biomarkers selected from the group consisting of markers described in Tables 5, 6, 9, 10, 11, 12, 13 and 14.

[0012] In another embodiment, the present invention is a method for screening a compound for the ability to inhibit or retard the aging process in a multicellular organism tissue, organ or cell, preferably mammalian tissue, organ or cell, comprising the steps of: (a) dividing test organisms into first and second samples; (b) administering a test compound to the organisms of the first sample; (c) analyzing tissues, organisms and cells of the first and second samples for the level of expression of a panel of sequences that have been predetermined to either increase or decrease in response to biological aging of the tissue, (d) comparing the analysis of the first and second samples and identifying test compounds that modify the expression of the sequences of step (c) in the first sample such that the expression pattern is indicative of tissue that has an inhibited or retarded biological age.

[0013] It is an object of the present invention to evaluate or screen compounds for the ability to inhibit or retard the aging process.

[0014] It is also an object of the present invention to measure the biological age of a multicellular organism, such as a mammal in a tissue or cell-specific basis.

[0015] It is also an object of the present invention to obtain biomarkers of aging.

[0016] Other objects, features and advantage of the present invention will become apparent to one of skill in the art after review of the specification and claims.

DETAILED DESCRIPTION OF THE INVENTION

[0017] One of the major impediments to the development of pharmaceutical, genetic or nutritional interventions aimed at retarding the aging process is the lack of a molecular method for measuring the aging process in humans or experimental animals. A suitable biomarker of the aging process should reflect biological age (physiological condition) as opposed to chronological age. Additionally, the biomarker should be amenable to quantitation, and reflect aging-related alterations at the molecular level in the tissue under study. Importantly, any such biomarker must be validated with the use of a model of retarded aging.

[0018] Caloric restriction, when started either early in life or in middle-age, represents the only established paradigm of aging retardation in mammals. (R. Weindruch and R. L. Walford, “The Retardation of Aging and Disease by Dietary Restriction” (C. C. Thomas, Springfield, Ill, 1988)) The effects of caloric restriction on age-related parameters are broad: caloric restriction increases mean and maximum lifespan, reduces and delays both spontaneous and induced carcinogenesis, almost completely suppresses autoimmunity associated with aging, and reduces the incidence of several age-induced diseases. (R. Weindruch and R. L. Walford, supra, 1988) Therefore, we expect that the rate of change of most proposed aging biomarkers should be retarded by caloric restriction.

[0019] By “biological age” we mean the physiological state of an animal or tissue relative to the physiological changes that occur throughout the animal's lifespan. By “chronological age” we mean the age of an animal as measured by a time scale such as month or years.

[0020] Because gene expression patterns are responsive to both intracellular and extracellular events, we reasoned that simultaneous monitoring of thousands of genes on a tissue-specific or organ-specific basis would reveal a set of genes that are altered in expression levels as a consequence of biological aging. Although alterations in gene expression with aging had been previously investigated for some genes, a global analysis of gene expression patterns during aging, and the validation of such patterns as a tool to measure biological age through the use of a model of retarded aging had not been previously performed. Such global analysis is required to identify genes that are expressed differentially as a consequence of aging on different cell types that compose the tissue under study and will allow a quantitative assessment of aging rates.

[0021] There exists a large and growing segment of the population in developed countries that is suffering from age-associated disorders, such as sarcopenia (loss of muscle mass), neurodegenerative conditions, and cardiac disease. Therefore, the market for compounds that prevent aging-associated disorders and improve quality of life for the elderly is likely to drive research and development of novel drugs by the pharmaceutical industry. As an example, many drugs, nutraceuticals and vitamins are thought to influence aging favorably, but their use remains limited due to the lack of FDA approval. The inability to assess biological aging in tissues at the molecular level precludes proper animal and human testing of such compounds.

[0022] In one embodiment, the invention is a method for measuring the biological aging process of a multicellular organism, such as a mammal, at the organ, tissue or cellular level through the characterization of the organism's gene expression patterns. This method preferably comprises obtaining a cDNA copy of the organism's RNA and determining the expression pattern of a panel of particular sequences (preferably at least 5 sequences, most preferably at least 10 sequences and more preferably at least 20, 30, 40, or 50 sequences) within the cDNA that have been predetermined to either increase or decrease in response to biological aging of the organ, tissue or cell. (We refer to nucleotide sequences with alterartions in expression patterns characteristic of biological age as “biomarkers.”) One may characterize the biological age of the organism by determining how many and at what level the biomarkers are altered.

[0023] Tables 1-4 and 15-16 describe a specific gene expression profiles determined in skeletal muscle of mice. Tables 1, 2, 15 and 16 describe aging-related increases and decreases in gene expression in gastrocnemius of mice. (Tables 1 and 2 were prepared using a high density oligonucleotide array of over 6,300 genes, while Tables 15 and 16 were prepared using a high density oligonucleotide array of 19,000 genes.) Tables 3 and 4 describe caloric restriction related decreases and increases in gene expression. Tables 1 and 2 contain a column (“CR reversal”) describing the influence of caloric restriction on the increased or decreased expression. Tables 5-8 describe a similar analysis of the gene expression profile determined neocortex tissue of mice and Tables 9 and 10 describe a gene expression profile determined on the cerebellum tissue in mice. Tables 11-14 describe gene expression profiles determined in mouse heart. (Tables 11 and 12 were prepared with the 19,000 high density oligonucleotide chip, while Tables 13 and 14 were prepared using the less dense gene chip.) From these gene expression profiles, one may select many biomarkers.

[0024] For example, in order to either measure or determine biological age in skeletal muscle, one would select markers in Tables 1 and 2 that reflect changes in gene expression that have been shown to be either partially or completely inhibited by caloric restriction in skeletal muscle such as AA0071777, L06444, AA14576, etc. Genes that were not affected by caloric restriction (such as W84988, Table 1) may represent chronological markers or aging, and therefore are less useful for the measurement of aging rates. One may determine which genes are or are not affected by caloric restriction by examination of the “CR reversal” lane of Tables 1 or 2.

[0025] If one wished to examine a tissue, organ or cell that is not represented in Tables 1-16, one would prepare samples and tabulate results from those samples as described below in the Examples. In this manner, one may examine any tissue, organ or cell for biological aging. Preferably, one would wish to examine a tissue selected from the group consisting of brain tissue, heart tissue, muscle tissue, skin, liver tissue, blood, lymphocytes, skeletal tissue and mucosa.

[0026] For example, choosing markers from Tables 1 and 2 to examine the efficacy of a test compound in aging prevention, one could design a PCR-based amplification strategy or a DNA microarray hybridization strategy to quantify the mRNA abundance for markers W08057, AA114576, 11071777, 11106112, D29016 and M16465 as a function of aging, using animals of several age groups, such as 6 months, 12 months, 18 months, 24 months and 30 months. (The marker designations refer to Gene Bank accession number entries.) A second set of animals would be given a test compound intended to slow the aging process at 10 months of age (middle age). Animals from the experimental group would be sacrificed or biopsied at the ages of 12 months, 18 months, 24 months and 30 months. If the test compound is successful, the normal aging-related alterations in expression of these particular markers will be prevented or attenuated.

[0027] One would follow the same protocol in using the other tables for marker selection. One would match the tissue to be analyzed with the appropriate table. For example, if one were analyzing muscle tissue, one might choose markers from Tables 1 and 2.

[0028] In another embodiment, the present invention is a method of obtaining and validating novel mammalian biomarkers of aging. Preferably, this method comprises the steps of comparing the gene expression profile from a young subject's organ, tissue or cells with samples from individuals that are both chronologically and biologically aged. This is followed by comparison of the gene expression profile of the chronologically and biologically aged individuals with that of individuals that display similar chronological ages, but a younger biological age, such as animals under caloric restriction. Gene expression alterations that are prevented or retarded by caloric restriction represent markers of biological age, as opposed to chronological age.

[0029] In one version of this embodiment, one would preferably use high density oligonucleotide arrays representing at least 5-10% of the subject's genes, as described in Lee, et al. at Science 285(5432):1390-1393, 1999 and Lee, et al., Nat. Genet. 25(3):294-297, 2000. (Both Lee, et al., supra, 1999 and Lee, et al., supra, 2000 are incorporated by reference as if fully set forth herein.)

[0030] For example, Lee, et al., supra, 1999 details the comparison between gastrocnemius muscle from 5 month (young) and 30 month (aged) mice, and 30 month mice under caloric restriction. Lee, et al., supra, 1999 disclose that of the 6500 genes surveyed in the oligonucleotide array, 58 (0.9%) displayed a greater than 2-fold increase in expression levels as a function of age and 55 (0.8%) displayed a greater than 2-fold decrease in expression. The most substantial expression change was for the mitochondrial sarcomeric creatine kinase (Mi-CK) gene (3.8-fold). Sequences that display a greater than three-fold alteration (increase or decrease) with aging, which are prevented or restricted by caloric restriction, such as W08057, AA114576, AA071777, AA106112, D29016, M16465, are likely to be particularly good aging biomarkers.

[0031] Lee, et al., supra, 2000 describes the comparison between cDNAs isolated from neocortex tissue for the same three groups of mice described above. Lee, et al., supra, 2000 disclose that of the 6347 genes surveyed, 63 (1%) displayed a greater than 1.7-fold increase in expression levels with aging in the neocortex, whereas 63 genes (1%) displayed a greater than 2.1-fold increase in expression in the cerebellum. Functional classes were assigned and regulatory mechanisms inferred for specific sets of alterations (see Tables 5-10). Of these, 20% (13/63), and 33% (17-51) could be assigned to an inflammatory response in the neocortex and cerebullum, respectively. Transcriptional alterations of several genes in this category were shared by the two brain regions, although fold-changes tended to be higher in the cerebellum, perhaps due to reduced tissue size and/or reduced heterogeneity at the cellular level. These transcriptional alterations include the microglial and macrophage migration factor Mps1 and the Cd40L receptor, which is a mediator of the microglial activation pathway. Also induced was Lysozyme C and beta(2) microglobulin which are markers of inflammation in the human CNS. Interestingly, a concerted induction of the complement cascade components C4, C1qA, C1qB and C1qC was observed, a part of the humoral immune system involved in inflammation and cytolysis.

[0032] In another embodiment, the present invention is a method of screening a test compound for the ability to inhibit or retard the aging process in mammalian tissue. In a typical example of this embodiment, one would first treat a test mammal with a test compound and then analyze a representative tissue of the mammal for the level of expression of a panel of biomarkers. Preferably, the tissue is selected from the group consisting of brain tissue, heart tissue, muscle tissue, blood, skeletal muscle, mucosa, skin and liver tissue. One then compares the analysis of the tissue with a control, untreated mammal and identifies test compounds that are capable of modifying the expression of the biomarker sequences in the mammalian samples such that the expression is indicative of tissue that has an inhibited or retarded biological age. This expression pattern would be more similar to an expression pattern found in biologically younger subjects.

[0033] As an example, a group of young rodents (mice) would be divided into a control and a test group. The test group would receive a test compound as a dietary supplement added to food from age 5 months to 30 months, whereas the control group would receive a standard diet during this time period. At age 30 months, several tissues would be collected from animals from each group, and a gene expression profile would be obtained. Each animal's gene expression profile would be compared to that of a 5 month (young) animals receiving the standard diet. One would then examine if, for any of the organs investigated, the gene expression pattern fo the animals receiving the test compound was more similar to that of young animals, compared to the experimental group that received a standard diet.

[0034] In another embodiment, the present invention is a method of detecting whether a test compound mimics the gene profile induced by caloric restriction. This method typically comprises the steps of exposing the mammal to a test compound and measuring the level of a panel of biomarkers. One then determines whether the expression pattern of the tissue mimics the expression pattern induced by caloric restriction.

[0035] For example, if one wished to examine skeletal muscle, the test compound would be analyzed for induction of genes observed to be induced by caloric restriction in Tables 3 and 4.

EXAMPLES

[0036] 1. In General

[0037] In order to test our hypothesis, we performed gene expression profiling of over 6300 genes in skeletal muscle, neocortex tissue, and cerebellum tissue and 19,000 genes in skeletal muscle and heart tissue of 5-month and 30-month old C57Bl6 mice, using high density oligonucleotide arrays. We found that a number of genes demonstrated alterations in gene expression profile as a function of chronological age and that these genes were broadly divided into a few classes listed in the Tables, such as stress response, energy metabolism, biosynthesis, protein metabolism and neuronal growth.

[0038] In order to validate the use of gene expression profiles as biomarkers of biological age, we investigated the role of caloric restriction, the only intervention known to retard the aging process in mammals, on gene expression profiles. Our analysis demonstrated that 30-month old calorically restricted animals display either complete or partial prevention of most aging associated alterations in gene expression, validating the use of gene expression profiles as a biomarkers of the aging process. In the process we have discovered a gene expression profile that is specifically associated with caloric restriction. We believe that this profile provides genetic markers for this metabolic state.

[0039] In like fashion, the present invention allows the determination of biological age in any organism through the determination of age-related variations in mRNA abundance. Such determination can be achieved through generation of cDNA from the mRNA of the organism and quantification of the cDNA product through hybridization to DNA microarrays, preferably as described here. Alternatively, any technique that allows for the quantitative determination of mRNA abundance may be used, such as quantitative PCR, Northern blotting and RNAse protection assays.

[0040] 2. Experimental Protocols

[0041] Details on the methods employed to house and feed male C57BL/6 mice, a commonly used model in aging research with an average lifespan of ˜30 months, were recently described (T. D. Pugh, et al., Cancer Res. 59:642, 1999). Briefly, mice were purchased from Charles River Laboratories (Wilmington, Mass.) at 1.5 months of age. After receipt in Madison, the mice were housed singly in the specific pathogen-free Shared Aging Rodent Facility at the Madison Veterans Administration Geriatric Research, Education and Clinical Center, and provided a non-purified diet (PLI5001 (Purina Labs, St. Louis, Mo.) and acidified water ad libitum for one week. The mice were then allocated into two groups and fed one of two nearly isocaloric (˜4.1 kcal/g), semi-purified diets. Each mouse in the control group was fed 84 kcal/week of the control diet (TD91349 (Teklad, Madison, Wis.)) which is ˜5-20% less than the range of individual ad libitum intakes. This dietary intake was used so that the control mice were not obese and retained motor activity up to the age of sacrifice. Each mouse subjected to CR was fed 62 kcal/week of the restricted diet (TD9351 (Teklad, Madison, Wis.)), resulting in a 26% reduction of caloric intake. The latter diet was enriched in protein, vitamins and minerals such that caloric restriction (CR) and control mice were fed nearly identical amounts of these components. The fat component, corn oil, was at the same level (13.5%) in both diets, leading to a 26% reduction in fat intake for the calorie-restricted mice. The adult body weights of the mice averaged ˜32 g for controls and ˜23 g for those on CR. Mice were euthanized by rapid cervical dislocation, autopsied to exclude animals showing overt disease, and the gastrocnemius muscle was removed from each limb, combined in a micocentrifuge tube, and immediately flash-frozen in liquid nitrogen and then stored at −80° C. All aspects of animal care were approved by the appropriate committees and conformed with institutional guidelines.

[0042] Total RNA was extracted from frozen tissue using TRIZOL reagent (Life Technologies) and a power homogenizer (Fisher Scientific) with the addition of chloroform for the phase separation before isopropyl alcohol precipitation of total RNA. Poly(A)+ RNA was purified from the total RNA with oligo-dT linked Oligotex resin (Qiagen). One microgram of poly(A)+ RNA was converted into double-stranded cDNA (ds-cDNA) using SuperScript Choice System (Life Technologies) with an oligo dT primer containing a T7 RNA polymerase promoter region (Genset). After second strand synthesis, the reaction mixture was extracted with phenol/chloroform/isoamyl alcohol. Phase Lock Gel (5 Prime→3 Prime, Inc.) was used to increase ds-cDNA recovery. The ds-cDNA was collected by ethanol precipitation. The pellet was resuspended in 3 &mgr;l of DEPC-treated water. In vitro transcription was performed using a T7 Megascript Kit (Ambion) with 1.5 &mgr;l of ds-cDNA template in the presence of a mixture of unlabeled ATP, CTP, GTP, and UTP and biotin-labeled CTP and UTP (bio-11 -CTP and bio-16-UTP (Enzo)). Biotin-labeled cRNA was purified using a RNeasy affinity column (Quiagen). The amount of biotin-labeled cRNA was determined by measuring absorbance at 260 nm. Biotin-labeled cRNA was fragmented randomly to sizes ranging from 35 to 200 bases by incubating at 94° C. for 35 minutes in 40 mM Tris-acetate pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate. The hybridization solutions contained 100 mM MES, 1 M (Na+), 20 mM EDTA, and 0.1% Tween 20. In addition, the hybridization solutions contained 50 pM oligonucleotide B2 (a biotin-labeled control oligonucleotide used for making grid alignments), 0.1 mg/mL herring sperm DNA, and 0.5 mg/mL acetylated BSA. The final concentration of fragmented cRNA was 0.05 &mgr;g/&mgr;l in the hybridization solutions. Hybridization solutions were heated to 99° C. for 5 minutes followed by 45° C. for 5 minutes before being placed in the gene chip. 10 &mgr;g of cRNA was placed in the gene chip. Hybridizations were carried out at 45° C. for 16 hours with mixing on a rotisserie at 60 rpm. Following hybridization, the hybridization solutions were removed, and the gene chips were installed in fluidics systems for wash and stain. The fluidics system (Affymetrix GeneChip Fluidics tation 400) performed two post-hybridization washes (a non-stringent wash and a stringent wash), staining with streptavidin-phycoerythrin, and one post-stain wash. The gene chips were read at a resolution of 6 &mgr;m using a Hewlett Packard Gene array scanner. Data collected from two scanned images were used for the analysis.

[0043] Detailed protocols for data analysis of Affymetrix microarrays and extensive documentation of the sensitivity and quantitative aspects of the method have been described (D. J. Lockhart, Nature Biotech. 14:1675, 1996). The Affymetrix GeneChip MU6500 set was derived from selected genes and ESTs from the Aug. 15, 1996 release of GeneBank. Briefly, each gene is represented by the use of ˜20 perfectly matched (PM) and mismatched (MM) control probes. The MM probes act as specificity controls that allow the direct subtraction of both background and cross-hybridization signals. The number of instances in which the PM hybridization signal is larger than the MM signal is computed along with the average of the logarithm of the PM:MM ratio (after background subtraction) for each probe set. These values are used to make a matrix-based decision concerning the presence or absence of an RNA molecule. All calculations are performed by Affymetrix software. To determine the quantitative RNA abundance, the average of the differences representing PM minus MM for each gene-specific probe family is calculated, after discarding the maximum, the minimum, and any outliers beyond three standard deviations. For example, to calculate fold changes (FC) between data sets obtained from young (y) vs. old (o) mice, the following formula was used: 1 FC = SI o - SI y the ⁢   ⁢ smallest ⁢   ⁢ of ⁢   ⁢ either ⁢   ⁢ SI y ⁢   ⁢   ⁢ or ⁢   ⁢ SI y + 1 ⁢   ⁢ if ⁢   ⁢ SI o ≥ SI o ⁢   ⁢ or - 1 ⁢   ⁢ if ⁢   ⁢ SI o < SI y

[0044] Where SIo is the average signal intensity from a gene-specific probe family from an old mouse and SIy is that from a young mouse.

[0045] Alternatively, if the Qfactor, a measure of the non-specific fluorescence intensity background, is larger the smallest of either SIy or SIo, the FC is calculated as: 2 FC = SI o - SI y Q factor

[0046] The Qfactor is automatically calculated for different regions of the microarray, and therefore minimizes the calculation of spurious fold changes. Average of pair-wise comparisons were made between study groups, each composed of three animals using Excel software. As an example, each 5-month-old mouse was compared to each 30-month-old mouse generating a total of nine pair-wise comparisons.

[0047] The murine 19K gene chip allows one to monitor more than 19,000 clustered murine EST transcripts selected from the TIGR (The Institute for Genome Research) database. This database is created by assembling ESTs into virtual transcripts called tentative mouse consensus sequences (Tcs). These sequence contigs are assigned a TC (tentative mouse consensus) number. Therefore, each TC number represents a unique transcript and allows one to check or obtain the sequence from the TIGR mouse gene index.

[0048] 3. Results

[0049] The results of our analysis are shown below in Tables 1-16. Tables 1-4 and 15-16 are the result of the analysis of mouse gastrocnemias muscle. Tables 1 and 15 describe aging-related increases in gene expression, Tables 2 and 16 describe aging-related decrease in gene expression, Table 3 describes caloric restriction related increases, and Table 4 describes caloric restriction related decreases in gene expression. Tables 5-10 describe results obtained using mouse brain tissue. Table 5 describes aging-related increases in gene expression in neocortex, Table 6 describes aging-related decreases in gene expression in neocortex, Table 7 describes caloric restriction related increases in gene expression in neocortex, Table 8 describes caloric restriction related decreases in gene expression in neocortex, Table 9 describes aging-related increases in gene expression in the cerebellum, and Table 10 describes aging-related decreases in gene expression in the cerebellum.

[0050] Tables 11-14 are the result of the analysis of mouse heart muscle. Tables 11 and 12, obtained by use of the Mu19K Gene Chip, disclose up-regulated and down-regulated aging-related genes. Tables 13 and 14, obtained from the Mu6500 Gene Chip, disclose up-regulated and down-regulated aging-related genes. 1 TABLE 1 Aging-related increases in gene expression in gastrocnemius muscle of C57BL/6 mice* &Dgr; Age CR ORF (fold) Gene Class/Function Reversal AA106112 3.8 Mitochondrial Sarcomeric Creatine Energy Metabolism/ATP generation C Kinase AA071777 3.8 Synaptic Vesicle Protein 2 Growth Factor/Neunte extension 51% Y00094 3.6 Ypt 1/ras-related GTP Binding Transport/Protein trafficking C Protein W10855 3.5 Methyl CpG Binding Protein DNA metabolism/gene silencing C W08057 3.5 Heat Shock 27 kDa Protein Stress Response/Chaperone C M17790 3.5 Serum Amyloid A Isoform 4 Stress Response/Unknown N L06444 3.5 GDF-9 Growth Factor/Unknown 50% AA114576 3.4 Heat Shock 71 kDa Protein Stress Response Chaperone C W84988 3.3 Transcription Regulatory Protein Transcriptional Factor/Unknown N SWI3 X64587 3.2 U2AF RNA Metabolism/Splicing Factor C D87902 3.2 ARF5 Transport/ADP-nbosylation 87% U19118 3.0 LRG-21 Transcriptional Factor/Macrophage activation 42% AA068057 2.9 RabB Signal Transduction/Unknown C U05837 2.9 Beta-Hexosaminidase Catabolism/Lysosomal enzyme C W85446 2.8 Protein Kinase C Inhibitor 1 Signal Transduction/Unknown 74% Homolog AA060167 2.8 Pre-B Cell Enhancing Factor Growth Factor/Cytokine C Precursor M37760 2.7 Serine-2 Ultrahigh Sulfur Protein Unknown 45% AA096992 2.7 G25K GTP-Binding Protein Signal Transduction/Unknown N AA008255 2.7 Adaptin Complex Small Chain Unknown 37% Homolog AA166502 2.6 EIF-4A-II RNA Metabolism/RNA helicase N X66602 2.6 POU-domain protein Transcriptional Factor/Unknown N X79828 2.6 NK 10 Transcriptional Factor/Unknown N V00719 2.6 Alpha-Amylase-1 Energy Metabolism/Starch metabolism N L28177 2.6 GADD45 Stress Response/Chaperone checkpoint 77% W50941 2.5 Nucleotide Pyrophosphatase Unknown N X53257 2.5 Neurotrophin-3 Growth Factor/Reinnervation of muscle 50% M74570 2.4 Aldehyde Dehydrogenase II Stress Response/Aldehyde detoxification 29% D49473 2.4 Sox17 Transcriptional Factor/Unknown 86% AA117284 2.3 Zinc Finger Protein 43 (HTF6) Transcriptional Factor/Unknown N W63835 2.3 Beta-centractin Structural/Contractility 60% AA089097 2.2 Phosphatidylcholine-transfer Transport/Lipid turnover C Protein AA059662 2.2 Protease Do Precursor Stress Response Protease C L22482 2.2 HIC-5 Stress Response Senescence and differentiation C X78197 2.2 AP-2 Beta Transcriptional Factor/Neurogenesis N AA059664 2.2 IGF Binding Protein Growth Factor/Cellular senescence C V00714 2.2 Alpha Globin Structural/Hemoglobin component C X99963 2.2 rhoB Stress Response/Unknown 87% AA014024 2.1 Dynactin Transport/Neuronal transport 55% X65627 2.1 TNZ2 Stress Response/RNA Metabolism 64% X95503 2.1 GTP-Binding Protein (IRG-47) Signal Transduction/Unknown 85% V00727 2.1 FBJ-MuSV Provirus/None C X12807 2.1 pp2.5 Unknown C W08049 2.1 MAGP Structural/Microfibnl glycoprotein N AA066425 2.1 CO-029 Structural/Cell surface glycoprotein N W82998 2.1 POLYA + RNA Export Protein RNA Metabolism/RNA export 44% X89749 2.1 mTGIF Transcriptional Factor/Neuronal differentiation C L07918 2.1 GDP-Dissociation Inhibitor Transport/membrane dynamics N X63190 2.1 PEA3 Transcriptional Factor/Response to muscle injury C *The influence of CR on the increased expression with age of specific ORFs is denoted as either C (complete, ≧90%), N (none) or partial (≧20%, percentage effect indicated).

[0051] 2 TABLE 2 Aging-related decreases in gene expression in gastrocnemius muscle of C57BL/6 mice* &Dgr; Age CR ORF (fold) Gene Class/Functlon Reversal D29016 −6.4 Squalene Synthase Biosynthesis/Cholesterol/fatty acid 52% synthesis AA106126 −4.9 Myosin Heavy Chain, Perinatal Structural Protein/Muscle contraction C D31898 −4.4 Protein Tyrosine Phosphatase, Signal Transduction/Unknown 79% PTPBR7 U29762 −4.3 Albumin Gene D-Box Binding Transcriptional Factor/Albumin synthesis 85% Protein AA061310 −4.1 Mitochondrial LON Protease Energy Metabolism/Mitochondrial biogenesis C AA162443 −3.6 Protein Phosphatase PP2a Signal Transduction/Unknown C M89797 −3.5 Wnt-4 Signal Transduction/Unknown 72% M16465 −3.4 Calpactin I Light Chain Signal Transduction/Calcium effector C X74134 −3.2 Ovalbumin Transcription Factor I Transcriptional Factor/Unknown N U08020 −3.2 Alpha 1 Type 1 Collagen Structural Protein/Extracellular matrix N X58251 −3.1 Pro-alpha-2(l) Collagen Structural Protein/Extracellular matrix N AA138226 −3.1 Clathrin Light Chain B Intracellular Transport/Vesicle transport C X85214 −3.0 Ox40 Signal Transduction/T Cell activation 50% D76440 −2.9 Necdin Growth Factor/neuronal growth 47% suppressor AA107752 −2.9 EF-1-Gamma Protein Metabolism/Protein synthesis 63% W55037 −2.9 Alpha Enolase Energy Metabolism/Glycolysis 68% X74134 −2.8 COUP-TFI Transcription Factor/Unknown 28% U06146 −2.8 Desintegrin-related Protein Unknown 28% U39545 −2.8 BMP8b Growth Factor/Unknown C X75014 −2.7 Phox2 Homeodomain Protein Transcriptional Factor/Neuronal 65% differentiation and survival U22031 −2.6 20S Proteasome Subunit Protein Metabolism/Protein turnover 44% U70210 −2.5 TR2L Transcriptional Factor/Apoptosis modulator N X76652 −2.5 3f8 Structural Protein/Neuronal adhesion N W54288 −2.5 PKCSH Signal Transduction/Unknown C M81475 −2.5 Phosphoprotein Phosphatase Energy Metabolism/Glycogen metabolism C U22394 −2.3 mSin3 Transcriptional Factor/Inhibitor of 46% cell proliferation M83336 −2.3 gp130 Signal Transduction/Unknown 77% L34611 −2.3 PTHR Signal Transduction/Ca homeostasis N X52046 −2.3 Pro-Alpha1 (III) Collagen Structural Protein//Extracellular matrix N L2450 −2.2 DNA Binding-protein Unknown 58% AA103356 −2.2 Calmodulin Signal Transduction/Calcium effector N L37092 −2.2 p130PITSL Cyclin-kinase DNA Metabolism/Cell cycle control N AA061604 −2.2 Ubiquitin Thiolesterase Protein Metobolism/Protein turnover C AA139680 −2.2 DNA Polymerase Alpha Primase DNA Metabolism/DNA replication N AA034842 −2.1 ERV1 DNA Metabolism/Maintenance of MtDNA 46% M21285 −2.1 Stearoyl-CoA Desaturase Biosynthesis/ synthesis C U11274 −2.1 PmuAUF1-3 RNA Metabolism/RNA degradation N U73744 −2.1 HSP70 Stress Response/Chaperone N J03398 −2.1 MDR Membrane Protein/Unknown N AA145829 −2.1 26S Proteasome Component TBP1 Protein Metabolism/Protein turnover C M32240 −2.1 GAS3 Growth Factor/Apoptosis and growth arrest 55% L00681 −2.1 Unp Ubiquitin Specific Protease Protein Metabolism/Protein turnover N U34277 −2.0 PAF Acelylhydrolase Unknown N U35741 −2.0 Rhodanese Protein Metabolism/Mitochondrial C protein folding W53731 −2.0 Signal Recognition Particle Intracellular Transport/Protein trafficking C Receptor AA044497 −2.0 Zinc Finger Protein 32 Transcriptional Factor/Unknown 40% L27842 −2.0 PMP35 Energy Metabolism/Poroxisome assembly 60% AA106406 −2.0 ATP Synthase A Chain Energy Metobolism/ATP synthesis N AA041826 −2.0 IPP-2 Energy Metabolism/Glycogen Metabolism C * The influence of CR on the increased expression with age of specific ORFs is denoted as either C (complete, ≧90%), N (none) or partial (≧20%, percentage effect indicated).

[0052] 3 TABLE 3 Caloric restriction-related increases in gene expression &Dgr; CR ORF (fold) Gene Class/Function U68267 9.6 Myosin Binding Protein H Structural/Myofibnl interactions (MyBP-H) X13135 4.7 Fatty Acid Synthase Biosynthesis/Fatty acid synthesis U05809 4.5 LAF1 Transketolase Energy Metabolism/Carbohydrate metabolism W53351 4.1 Fructose-bisphosphate Energy Metabolism/Glycolysis Aldotase M15501 3.5 Cardiac Muscle Alpha Actin Structural/Muscle contraction AA071776 3.5 Glucose-6-Phosphate Energy Metabolism/Glycolysis Isomerase AA073283 3.3 Cardiac Muscle Myosin Beta-Actin Structural/Contractile protein AA138226 2.9 Clathrin Light Chain B Transport/Axonal transport L42115 2.9 Insulin-Activated Amino Acid Transport/Aminoacid transport Transporter U37222 2.8 Adipocyte Complement- Growth Factor/Unknown Related Protein (Acrp30) W89939 2.7 FK506-Binding Protein Signal Transduction/Neuronal (FKBP-12) regeneration X16314 2.5 Glutamine Synthetase Biosynthesis/Glutamine synthesis AA080277 2.5 Sodium Potassium ATPase Membrane Protein/Ion pump Alpha-2 Chain W30250 2.5 Myosin Light Chain 1 Structural/Contractile protein AA137659 2.4 Cytochrome P450-IIC12 Biosynthesis/Steroid biosynthesis AA031112 2.4 ZFP-37 Transcriptional Factor/Unknown U34295 2.3 Glucose Dependent Energy Metabolism/Insulin sensitizer Insulinotropic Polypeptide W54288 2.3 Protein Kinase-C Substrate Signal Transduction/AGE receptor (80K-H) U01841 2.3 Peroxisome Proliferator Energy Metabolism/Insulin sensitizer Receptor Gamma (PPAR) AA109527 2.3 Actin 1 Structural/Contractile protein AA145829 2.3 26S Protease Subunit TBP-1 Protein Metabolism/26S proteasome component Y00137 2.3 Lymphotoxin-Beta Signal Transduction/Cytokine AA107752 2.2 Elongation Factor 1-gamma Protein Metabolism/Protein synthesis AA016431 2.2 Keratinocyte Lipid-binding Unknown/Fatty acid binding Protein M93275 2.1 Adipose Differentiation Unknown Related Protein (ADFP) W53731 2.1 Signal Recognition Particle Protein Metabolism/Protein synthesis Receptor Alpha Subunit U60328 2.1 Proteasome Activator PA28 Protein Metabolism/Protein turnover Alpha Subunit W78478 2.1 Gamma E-crystallin Unknown X67083 2.1 Chop-10 (gadd153) Stress-Response/Growth arrest U40189 2.1 Neuropeptide Y Unknown AA020281 2.1 Progesterone Reductase Metabolic/Progesterone metabolism AA022083 2.0 Huntingtin Unknown X59990 2.0 mCyP-S1 (Cyclophilin) Protein Metabolism/Protein folding X56548 2.0 Purine Nucleoside Biosynthesis/Purine turnover Phosphorylase L28116 2.0 PPAR Delta Energy Metabolism/Peroxisome induction U43319 2.0 Frizzled 6 Unknown X14432 2.0 Thrombomodulin Unknown L32973 2.0 Thymidylate Kinase Biosynthesis/dTTP sythesis D76440 1.9 Necdin Growth Factor/Neuronal growth suppressor L36860 1.9 GCAP Signal Transduction/Calcium-binding regulatory protein W08293 1.9 Translocon-Associated Protein Metabolism/Protein Protein Delta translocation AA041826 1.9 Protein Phosphatase Energy Metabolism/Inhibition Inhibitor 2 (IPP-2) of glycogen synthesis D42083 1.9 Fructose 1.6-bisphosphatase Energy Metabolism/Gluconeogenesis AA008737 1.9 Peroxisomal Protein PAS8 Transport/Peroxisome targeting W57495 1.8 60S Ribosomal Protein L23 Protein Metabolism/Protein synthesis D83585 1.8 Proteasome Z Subunit Protein Metabolism/Protein turnover M13366 1.8 Glycerophosphate Energy Metabolism/Electron Dehydrogenase transport to mitochondna U37091 1.8 Carbonic Anhydrase IV Energy Metabolism/CO2 disposal * The genes listed on this table were not influenced by age. Reversal of aging-associated changes are listed in Tables 1 and 2. Energy Metabolism and Biosynthetic classes are highlighted in blue.

[0053] 4 TABLE 4 Caloric restriction-related decreases in gene expression &Dgr; DR ORF (fold) Gene Class/Function AA062328 −3.4 DnaJ Homolog 2 Stress Response/Chaperone X03690 −2.5 Ig Heavy Chain Constant Immune Function/Antibody Region mu(b) U60453 −2.3 Ezh1 (Zeste Homolog 2) Transcriptional Factor/Gene silencing M83380 −2.3 relB Transcriptional Factor/Unknown D38613 −2.1 921-L Presynaptic Protein Unknown X82457 −2.0 es64 Unknown U35646 −2.0 Aminopeptidase Protein Metabolism/Protein turnover W13412 −1.9 ATP Synthase Coupling Energy Metabolism/ATP synthesis Factor B M92416 −1.9 FGF-6 Growth Factor/Muscle regeneration U58497 −1.9 mp86 (Mnb Protein Kinase) Signal Transduction/Unknown L29454 −1.9 Fbn-1 (Fibrillin) Structural/Microfibnl organization U56773 −1.9 Pelle-like Protein Kinase Signal Transduction/Unknown D49439 −1.9 TFIID Subunit p80 Transcriptional Factor/Unknown D31943 −1.9 Inducible SH2-Containing Growth Factor/Cytokine Protein U47737 −1.9 TSA-1 Signal Transduction/T cell function X63023 −1.9 Cytochrome P-450-IIIA Stress Response/Detoxification X53476 −1.8 HMG-14 DNA Metabolism/Chromalin remodeling L33768 −1.8 JAK3 Signal Transduction/T cell function U03283 −1.8 Cyp 1b1 Cytochrome P450 Stress Response/Detoxification U14390 −1.8 Aldehyde Dehydrogenase-3 Stress Response/Detoxification U75530 −1.8 PHAS-II Protein Metabolism/Translation inhibitor X13605 −1.8 Histone H3.3 DNA metabolism/Chromatin remodeling U65313 −1.8 G3BP DNA metabolism/Helicase AA062349 −1.8 P31 Protein Metabolism/Protein turnover X76850 −1.8 MAPKAP2 Stress Response/Unknown D43694 −1.8 Math-1 Transcription Factor/Neuronal differentiation U66887 −1.8 RAD50 DNA Metabolism/DNA repair M83219 −1.8 MRP14 Growth Factor/Inflammation Z14986 −1.8 SAMDC Biosynthesis/Polyamine synthesis W17516 −1.8 NEDD8 Unknown D78641 −1.7 Membrane Glycoprotein Unknown D26123 −1.7 Carbonyl Reductase Unknown U71205 −1.7 nt Signal Transduction/Unknown U31510 −1.7 ADP-ribosyltransferase Protein Metabolism/ADP-ribosylation L4406 −1.7 Hsp 105-beta Stress Response/Chaperone AA059718 −1.7 DNA Polymerase Beta DNA Metabolism/DNA repair D16464 −1.7 HES-1 Transcription Factor/Neuronal differentiation D87963 −1.7 ETFR-1 Transcriptional Factor/Unknown U12236 −1.7 Alpha M290 Integrin Signal Transduction/Cell and matrix adhesion X98848 −1.7 6-phosphofructo-2-kinase Energy Metabolism/glycolysis W41974 −1.7 ATP-Dependent RNA RNA Metabolism/Unknown Helicase-Homolog X75285 −1.6 Fibulin-2 Structural/Basement membrane M96265 −1.6 GALT Energy Metabolism/Glycolysis D67015 −1.6 97kDa Nuclear Pore Transport/Nuclear import Targeting Complex AA002750 −1.6 5-lypoxygenase Activating Biosynthesis/Leukotriene synthesis Protein (FLAP) X93357 −1.6 SYT Transcriptional Factor/Unknown W13191 −1.6 Thyroid Hormone Receptor Metabolic/Thyroid hormone receptor Alpha-2 U43206 −1.6 Phosphatidylethanolamine Signal Transduction/Unknown Binding Protein W11169 −1.6 SUI1ISO1 Protein Metabolism/Translation initiation factor W42234 −1.6 XPE DNA Metabolism/DNA repair W08897 −1.6 Seryl-tRNA Synthetase Protein Metabolism/Protein synthesis AA027739 −1.6 Heterogeneous Nuclear Transcriptional Factor/Unknown Ribonucleoprotein K * The genes listed on this table were not influenced by age. Reversal of aging-associated changes are listed in Tables 1 and 2. DNA Repair and Stress Response classes are highligted in green.

[0054] 5 TABLE 5 Aging-related increases in gene expression in neocortex of C57BL/6 mice* &Dgr; Age Signal Intensity CR ORF (fold) SE old Young Gene Class Prevention M88354 5.7 1.9 165 −109 Vasopressin-neurophysin II Osmotic stress  68% M17440 4.9 0.2 786 141 Complement C4 Immune/inflammatory  52% AA120109 4.1 0.8 278 65 Interferon-induced protein 6-16 homolog Immune/inflammatory 100% M88355 2.7 0.6 195 70 Oxytocin-neurophysin Osmotic stress  23% AA037945 2.5 0.2 254 73 Beta-SNAP homolog Transport N AA162093 2.5 0.2 145 21 Pre-mRNA splicing factor PRP22 RNA metabolism N AA137962 2.4 0.2 150 39 RAS-related protein RAB-14 Neurotransmitter release N K01347 2.3 0.4 420 178 Glial fibrillary acidic protein (GFAP) Stress response  38% AA027404 2.3 0.1 129 −43 Na/K-transporting ATPase beta-2 chain Ionic transport N U60593 2.3 0.4 279 131 Cap43 Stress response N AA137871 2.3 0.6 55 −35 Phosphatidylinositol-4-phosphate 5-kinase Signal transduction N U61751 2.3 0.2 299 128 VAMP-1 Transport N M21050 2.2 0.2 209 74 Lysozyme C Immune/inflammatory  54% AA153990 2.2 0.9 343 155 GTP: AMP phosphotransferase Energy metabolism 100% mitochondrial W29462 2.1 0.3 114 −49 Calpactin I light chain Structural N L39123 2.1 0.2 1887 768 Apolipoprotein D (apoD) Stress response N U16297 2.0 0.5 124 47 Cytochrome B561 Transport N M26251 2.0 0.3 484 260 Vimentin Stress response N AA163911 2.0 0.2 130 38 Casein kinase I, delta isoform Stress response N AA022006 2.0 0.2 115 −48 CD40L receptor precursor Immune/inflammatory N AA124859 2.0 0.2 17 −54 ICAM-2 Immune/inflammatory N Y00305 1.9 0.2 225 101 Potassium channel protein-1 Transport N AA116604 1.9 0.1 515 272 Cathepsin Z Stress response  70% M95200 1.9 0.3 168 92 Vascular endothelial growth factor Growth factor N L16894 1.9 0.4 123 −71 Cyclophilin C-AP Stress response 100% L20315 1.9 0.2 120 66 MPS1 gene Immune/inflammatory N AA028501 1.9 0.2 74 16 Cytochrome c oxidase subunit VIII-H Energy metabolism N X86569 1.9 0.2 24 −31 LIM-kinase Unknown N AA105716 1.9 0.2 107 14 Fructose-1,6-bisphosphatase homolog Energy metabolism  87% W13646 1.8 0.1 1278 705 Ti-225 (ubiqurtin) Stress response N J03236 1.8 0.3 681 362 JunB Stress response  46% X52886 1.8 0.1 1050 555 Cathepsin D Stress response  64% AA028273 1.8 0.3 331 153 Protein phosphatase inhibitor 2 (IPP-2) Unknown N X16995 1.8 0.1 757 375 N10 Steroid metabolism N X16995 1.8 0.1 624 363 Complement C1q B-chain Immune/inflammatory 100% X66295 1.8 0.1 823 467 Complement C1q C-chain Immune/inflammatory  75% U22445 1.8 0.5 201 160 Serine/threonine kinase (Akt2) Energy metabolism 100% U17297 1.8 0.2 6 −43 Integral membrane phosphoprotein 7.2b Unknown N AA059700 1.8 0.2 1467 797 MHC class I B(2)-microglobulin Immune/inflammatory  64% L29503 1.8 0.1 192 103 Myelin/oligodendrocyte glycoprotein (Omg) Unknown N AA168918 1.8 0.4 326 166 Na/K-transporting ATPase gamma chain Transport N M90364 1.8 0.1 326 202 Beta-caterun Stress response N AA061086 1.8 0.2 179 89 Hsp40 Stress response  52% W50891 1.8 0.3 41 −3 Creatine kinase Energy metabolism N W67046 1.8 0.2 105 71 Exodus-2 Immune/inflammatory N W13875 1.8 0.2 216 125 Myosin regulatory light chain 2-A Unknown N X67083 1.8 0.3 121 47 Chop-10 GADD153 Stress esponse N AA089110 1.8 0.2 23 −35 Dynein beta chain, ciliary Transport N V00727 1.7 0.3 404 236 c-fos(p55) Stress response 100% AA062328 1.7 0.2 113 23 DNAJ protein homolog 2 Stress response N AA122619 1.7 0.3 14 −43 Set protein (HLA-DR associated protein II) Unknown N M73741 1.7 0.2 1313 730 Alpha-B2-crystallin gene Stress response  67% X70393 1.7 0.4 146 65 Inter-alpha-inhibitor H3 chain lmmune/inflammatory  56% AA124698 1.7 0.7 100 42 Lethal(1)discs large-1 Unknown N W14434 1.7 0.2 401 240 Fructose-bisphosphate aldolase Energy metabolism N W89579 1.7 0.2 83 −3 RAS-related protein RAB-4 Signal transduction N AA089333 1.7 0.1 336 221 Cathepsin S precursor Stress response  56% U19521 1.7 0.2 70 31 Vesicle transport protein (munc-18c) Transport N AA107137 1.7 0.3 204 118 Casein kinase 1, gamma Unknown N AA106166 1.7 0.2 2312 1372 Elongation factor 2(EF-2) homolog RNA metabolism N M31811 1.7 0.1 748 457 Clathrin light chain B Transport 100% AA140487 1.7 0.3 23 −25 Cyclophilin A homolog Stress response 100% U37419 1.7 0.2 58 −29 G protein alpha subunit (GNA-15) Signal transduction N AA114781 1.7 0.2 52 26 Uridylate kinase DNA metabolism N X58861 1.6 0.1 1128 694 Complement C1Q alpha-chain Immune/inflammatory 100% AA048650 1.6 0.2 169 100 Estradiol 17 B-dehydrogenase 3 homolog Steroid metabolism N W46723 1.6 0.2 83 46 Creatine kinase, B chain homolog Energy metabolism N U16162 1.6 0.7 112 82 Prolyl 4-hydroxylase alpha(1)-subunit Structural N X68273 1.6 0.2 105 73 Macrosialin Immune-inflammatory N W48962 1.6 0.7 87 38 B-adrenergic receptor kinase 1 Signal transduction N AA063858 1.6 0.2 135 80 RHO-related GTP-binding protein RHOG Signal transduction 100% M15525 1.6 0.1 22 −58 Laminin B1 Neuronal outgrowth N AA068780 1.6 0.1 275 187 Phosphoserine aminotransferase homolog Unknown  76% U27462 1.6 0.3 133 79 BS4 peptide Unknown N AA106077 1.6 0.1 116 64 Glutathione peroxidase Stress response  76% AA119959 1.6 0.2 194 128 Protein transport protein SEC23 Transport N AA061170 1.6 0.2 39 −18 NEDD-4 protein Unknown N X16151 1.6 0.2 93 61 T-lymphocyte activation 1 protein (ETa-1) Immune/inflammatory N W29462 1.6 0.3 114 −49 Calpactin I light chain (p11) Unknown N AA097579 1.6 0.1 24 −20 Zinc finger protein 91 homolog Unknown  52% X64070 1.6 0.3 252 163 46kDa marinose 6-phosphate receptor Lysosomal N W48519 1.6 0.2 98 100 GRP94 homolog Stress response N X78682 1.6 0.2 408 269 B-cell receptor associated protein (BAP) 32 Unknown N AA106166 1.6 0.2 2312 1372 Elongation factor 2 homolog Protein metabolism N AA169054 1.6 0.2 279 184 GTP-binding protein GTR1 Signal transduction N W51181 1.6 0.3 42 25 DNA-directed RNA polymerase II RNA metabolism  75% AA036390 1.6 0.2 146 83 DNA-binding protein inhibitor ID-1 Transcriptional factor  75% L08115 1.5 0.2 309 236 Human CD9 antigen homolog Structural 100% U37353 1.5 0.2 191 121 Protein phosphatase 2A B'alpha3 Signal transduction N regulatory subunit L10244 1.5 0.2 316 206 Spermidine/spermine N1-acetyltransferase Polyamine metabolism N J05154 1.5 0.2 72 6 Cholesterol acyltransferase (LCAT) Steroid metabolism N D43643 1.5 0.2 62 36 YL-1 Unknown N M34141 1.5 0.1 39 5 COX-1 lmmune/inflammatory 100% L28177 1.5 0.1 35 −9 GADD 45 Stress response N X85992 1.5 0.1 51 10 Semaphorin C Neuronal remodelling N AA098307 1.5 0.2 85 47 Tubulin beta 5 Microtubule component N *The values presented for Signal Intensity are the averages of three mice per age group and are expressed as data for old/young mice. The prevention by CR is shown as being none (N) or the calculated percentage effect. The SE was calculated for the nine pairwise compansons and was obtained by dividing the standard deviation by the square root of 3. The method from which signal intensity is used to estimate fold changes is described in the Methods section of the manuscript

[0055] 6 TABLE 6 Aging-related increases in gene expression in neocortex of C57BL/6 mice* &Dgr; Age Signal Intensity CR ORF (fold) SE old Young Gene Class Prevention X74134 −3.0 1.1 157 387 Ovalbumin upstream promoter Transcriptional factor N L24430 −2.7 0.6 56 161 Osteocalcin precursor Unknown N AA124352 −2.5 0.5 19 274 Neuromedin B precursor homolog Neurotransmission  54% D31898 −2.2 0.5 116 253 Protein tyrosine phosphatase. PTPBR7 Unknown N W29468 −2.2 0.3 133 284 Myosin light chain 2 mRNA Unknown N AA065993 −2.2 0.3 16 115 GTP-binding nuclear protein RAN homolog Signal transduction N U35323 −2.1 0.3 11 135 H2-M Unknown N W98695 −2.1 0.2 3 120 Plasma retinol-binding protein precursor Steroid metabolism N AA062463 −2.1 0.2 63 168 Kidney androgen-regulated protein Steroid metabolism N U38196 −2.1 0.6 64 151 Palmytoylated protein p55 Signal transduction 100% L36135 −2.1 0.3 −42 32 T cell receptor delta chain, C region Immune/inflammatory N D32200 −2.1 0.3 38 101 Hes-3 Unknown N W98898 −2.1 0.4 −21 125 Transforming protein RFP Growth factor N U29762 −2.0 0.2 396 744 Albumin gene D-Box binding protein Circadian rhythm N AA138711 −2.0 0.5 222 321 Protein kinase C inhibitor protein Unknown N W13586 −2.0 0.3 135 548 Atnal/fetal isoform myosin alkali light chain Structural  49% X67812 −2.0 0.3 41 120 ret proto-oncogene Unknown N M97812 −2.0 0.2 12 85 REX-1 Steroid metabolism N W11011 −2.0 0.4 418 673 NEDD8 Protein metabolism N X13538 −2.0 0.2 66 176 Hox-1 4 gene Growth factor N X66405 −2.0 0.5 186 330 Collagen alpha 1 chain type VI Structural 100% AA050791 −2.0 0.5 194 355 Creatine kinase, M chain Energy metabolism N W55515 −1.9 0.4 132 243 Cyclic-AMP-dependent ATF-4 Transcriptional factor 100% L33416 −1.9 0.3 184 291 Clone p85 secreted protein Unknown 100% X70398 −1.9 0.9 186 325 PTZ-17 Growth factor N M84412 −1.8 0.1 46 128 Antigen (Ly-9) Immune/inflammatory  47% AA067927 −1.8 0.2 63 132 DNA-PK-catalytic subunit DNA metabolism N Y09585 −1.8 0.4 143 212 Serotonin 4L receptor Neurotransmission N X95255 −1.8 0.1 6 72 Gli3 protein Growth factor N U37459 −1.8 0.1 37 87 Glial-derived neurotrophic factor (GDNF) Growth factor N M99377 −1.8 0.3 121 270 Alpha-2 adrenergic receptor Neurotransmission N D83585 −1.8 0.5 916 1457 Proteasome Z subunit Protein metabolism N U52222 −1.8 0.2 61 160 Mel-1a melatonin receptor Neuropeptide N M13710 −1.7 0.3 120 219 Interferon alpha-7 gene Immune/inflammatory N D76446 −1.7 0.2 103 199 TAK1 Stress response N U64445 −1.7 0.2 12 56 Ubiquitin fusion-degradation protein (ufd1l) Protein metabolism 100% U39545 −1.7 0.3 144 235 Bone morphogenetic protein 8B (Bmp8b) Growth factor N W59776 −1.7 0.2 95 174 Vacuolar ATP synthase catalytic subunit A pH regulation N AA071792 −1.7 0.2 36 89 GSTP-1 Protein metabolism N AA052547 −1.7 0.3 −2 95 PA-FABP homolog Unknown 100% D63819 −1.7 0.2 61 143 Neuropeptide Y-YII receptor Neuropeptide N W08326 −1.7 0.2 173 265 51PK(L) homolog Unknown N AA000468 −1.7 0.2 113 195 p55CDC DNA metabolism 100% U66203 −1.7 0.2 111 181 FHF-3 Growth factor N AA051632 −1.7 0.2 112 167 MEK5 Signal transduction  61% AA051147 −1.7 0.2 114 264 Chemotaxis protein cheY homolog Unknown N X84692 −1.7 0.2 24 91 Spnr mRNA for RNA binding protein RNA metabolism N U53925 −1.7 0.3 100 169 HCF1 Unknown  33% AA038142 −1.7 0.3 251 376 RCC1 DNA metabolism N W54682 −1.7 0.1 87 188 Antithrombin-III precursor (ATIII) lmmune/inflammatory N U13705 −1.7 0.2 324 494 Plama glutathione peroxidase (MUSPGPX) Stress response  44% X75384 −1.7 0.2 91 158 SAX-1 Growth factor N Z32767 −1.7 0.3 117 205 RAD52 DNA metabolism  76% AA107752 −1.6 0.6 225 336 Elongation factor 1-gamma Protein metabolism N M12836 −1.6 0.6 56 116 T-cell receptor gamma chain gene C-region lmmune/inflammatory N AA060704 −1.6 0.2 975 1407 Glutathione S-transferase MU 5 Unknown N AA118294 −1.6 0.1 99 161 Vitronectin homolog Unknown N AA123026 −1.6 0.1 72 166 Pancreatitis-associated protein 3 homolog Unknown 100% AA065652 −1.6 0.1 39 99 Ubiquitin carboxyl-terminal hydrolase Protein metabolism N W46104 −1.6 0.2 19 58 DNA-repair protein XP-E DNA metabolism N M88694 −1.6 0.2 67 109 Thioether S-methyltransferase Unknown  57% AA117004 −1.6 0.1 6 61 Heat shock cognate 71 KD protein homolog Stress response N M15501 −1.6 0.1 229 325 Adult cardiac muscle alpha-actin Structural 100% U49430 −1.6 0.2 78 108 Ceruloplasmin Transport N X69019 −1.6 0.2 36 71 Hox 3.5 gene. complete cds Growth factor N M28666 −1.6 0.2 317 496 Porphobilinogen deaminase Biosynthesis  44% W368759 −1.6 0.1 49 112 CMP-N-acetylneuraminate-beta-1,4- Sialyltransferase N galactoside alpha-2,3-sialyltransferase W11666 −1.6 0.2 105 207 apolipoprotein H Lipid metabolism N W09925 −1.6 0.1 26 102 Endothelial actin-binding protein Growth factor  74% AA116282 −1.6 0.1 140 355 TNF alpha precursor Immune/inflammatory  56% D37791 −1.6 0.0 556 895 Beta-1,4,-galactosyltransferase Unknown N W12658 −1.6 0.2 143 216 FKBP-rapamycin associated protein (FRAP) Unknown N Z468454 −1.6 0.2 −16 39 Preproglucagon Energy metabolism N AA103045 −1.5 0.1 57 106 Cleavage stimulation factor. 64 Kd subunit RNA metabolism N AA108891 −1.5 0.2 4 62 Putative ATP-dependent RNA helicase RNA metabolism  55% AA153522 −1.5 0.3 80 159 Serine/threonine protein kinase sulu Unknown N M23501 −1.5 0.2 33 101 TCA3 Unknown  61% AA063762 −1.5 0.1 112 193 Zinc finger protein 36 homolog (KOX18) Unknown  63% AA098588 −1.5 0.1 84 137 Zinc finger protein HRX (ALL-1) Unknown  57% W15873 −1.5 0.2 161 258 tctex-1 mRNA Unknown  61% AA170748 −1.5 0.1 −14 48 40S Ribosomal protein S4 Unknown N W80326 −1.5 0.1 −11 86 Sex-determining protein FEM-1 Unknown N AA140159 −1.5 0.2 65 134 Thiol-specific antioxidant protein homolog Stress response N D16492 −1.5 0.1 19 58 RaRF Unknown  56% D85845 −1.5 0.2 48 88 Atonal homolog-3 Growth factor N L06451 −1.5 0.1 −55 87 Agouti switch protein mRNA Unknown 100% AA166500 −1.5 0.2 51 141 Transcriptional regulatory protein RPD3 Unknown N L28035 −1.5 0.1 377 578 Protein kinase C-gamma mRNA Unknown 100% U52197 −1.4 0.1 296 439 Poly(A) polymerase V RNA metabolism N D29763 −1.4 0.1 799 1130 Seizure-related, product 6 type 3 precursor Unknown/response  50% U22015 −1.4 0.1 89 130 Retinoid X receptor interacting protein Steroid metabolism 100% *The values presented for Signal Intensity are the averages of three mice per age group and are expressed as data for old/young mice. The prevention by CR is shown as being none (N) or the calculated percentage effect. The SE was calculated for the nine pairwise comparisons and was obtained by dividing the standard deviation by the square root of 3. The method from which signal intensity is used to estimate fold changes is described in the Methods section of the manuscript.

[0056] 7 TABLE 7 Caloric restriction-related increases in gene expression in neocortex of C57BL/6 mice* CR Signal Intensity ORF Increase SE CR Control Gene Class J04971 4.1 0.7 410 87 Slow/cardiac troponin C (cTnC) Unknown D13903 3.1 1.2 150 49 MPTPdelta (type A) Growth factor M36660 3.1 0.3 24 −114 NAD(P)H menadione oxidoreductase Stress response M55617 3.1 0.6 27 −48 MMCP-4 unknown W65178 3.0 0.3 39 −35 BMP-1 Growth factor AA118682 3.0 0.6 62 −12 Trithorax homolog 2 Transcriptional factor AA014816 3.0 0.7 257 38 Prolactin homolog Unknown U39904 2.9 1.4 100 −169 Citron, putative rho/rac effector Signal transduction AA061310 2.9 0.7 87 29 Mitochondnal LON protease Energy metabolism U02098 2.8 0.5 82 36 Pur-alpha DNA metabolism M29395 2.8 0.3 38 −20 Orotidine-5-monophosphate decarboxylase DNA metabolism M23236 2.8 0.5 16 −57 Retrovirus POL protein homolog Unknown M13019 2.8 0.4 −15 −130 Thymidylate synthase DNA metabolism X76858 2.6 0.4 58 −17 phi AP3 Unknown W56940 2.5 0.2 81 24 Neuronal-glial cell adhesion molecule homolog Unknown X59846 2.4 0.6 215 156 GAS 6 Growth factor U05247 2.4 0.3 666 250 c-Src kinase Signal transduction AA104316 2.3 0.3 25 −46 Type-I ER resident kinase PERK Stress response L04302 2.3 0.2 49 2 Thrombospondin 3 Structural W55507 2.3 0.3 31 −14 D(2) Dopamine receptor Neurotransmission AA014909 2.3 0.4 56 −39 Gastrula zinc finger protein XLCGF20.1 Unknown U46923 2.2 0.8 71 −13 G protein-coupled receptor GPR19 Unknown M34857 2.2 0.1 176 57 Hox-2.5 Growth factor M74227 2.2 0.3 162 48 Cyclophilin C (cyp C) Immune/inflammatory W12794 2.2 0.3 48 −59 Transforming protein MAF homolog Transcriptional factor X62940 2.2 0.1 2199 931 TSC-22 Unknown L06451 2.2 0.1 136 −55 Agouti switch protein Unknown AA052547 2.2 0.1 74 −2 Fatty acid-binding protein, epidermal (E-FABP) Transport W17956 2.2 0.4 108 −2 Zinc finger protein 42 homolog Unknown X95226 2.2 0.4 53 −1 Dystrobrevin Structural AA152808 2.2 0.2 141 24 Proteine kinase PASK Signal transduction AA014512 2.1 0.5 32 −3 Unknown Unknown W74811 2.1 0.4 17 −46 Apolipoprotein c-II precursor (APO-CII) Transport U69270 2.1 0.7 323 210 LIM domain binding protein 1 (Ldb1) Growth factor W54720 2.1 0.2 100 19 Ca**-transporting ATPase (brain isoform 1) Unknown X13460 2.1 0.1 313 151 Annexin VI Signal transduction U61362 2.1 0.3 57 −35 Groucho-related gene 1 protein (Grg 1) Unknown W09323 2.1 0.3 91 −11 Endothelin-2 precursor (ET-2) Unknown W70403 2.1 0.2 17 −19 mafF Unknown AA071685 2.0 0.4 93 47 Elongation factor 1-alpha chain homolog Protein metabolism W14673 2.0 0.4 133 8 BAT3 Unknown W53409 2.0 0.3 33 −28 Protein kinase C homolog, alpha type Signal transduction U19880 2.0 0.1 28 −6 D4 dopamine receptor gene Neurotransmission M75875 2.0 0.4 280 119 MHC H2-K homolog Unknown W62842 2.0 0.2 12 −24 ATP synthase lipid-binding protein P2 precursor Energy metabolism U48397 2.0 0.3 126 40 Aquaponn 4 Osmotic stress J00475 2.0 0.3 74 −34 Ig alpha chain region C Immune/inflammatory M57960 2.0 0.2 21 −18 Carboxylesterase Unknown X57800 2.0 0.1 560 274 PCNA DNA metabolism U36277 2.0 0.3 123 70 I-kappa B alpha chain Stress response AA015291 2.0 0.3 140 67 Probable E1-E2 ATPase unknown W82109 2.0 0.3 73 29 Kinesin light chain (KLC) Transport M83380 1.9 0.2 25 −26 RelB Immune/inflammatory U13174 1.9 0.2 36 2 Basolateral Na-K-2Cl cotransporter Transport M33960 1.9 0.2 19 1 Plasminogen activator inhibitor (PAI-1) Growth factor X72310 1.9 0.3 106 38 DRTF-polypeptide-1 (DP-1) Transcriptional factor AA059886 1.9 0.2 8 −52 Retinal degeneration C protein Apoptotic factor U02278 1.9 0.2 18 −32 Hox-B3 Growth factor AA072842 1.9 0.2 126 72 Na*- and Cl-dependent transporter NTT73 Transport M98339 1.9 0.2 113 −15 GATA-4 Transcriptional factor W13427 1.9 0.3 195 94 Platelet factor 4 precursor Unknown U44955 1.9 0.2 45 2 Alpha3 connexin gene Transport L24191 1.9 0.1 104 25 Intrinsic factor Transport W08109 1.9 0.3 142 99 Protein kinase C inhibitor 1 (PKCl-1) homolog Unknown W36570 1.9 0.3 146 67 DNA mismatch repair protein MSH2 DNA metabolism Z34524 1.8 0.2 42 −20 Protein kinase D Signal transduction AA105081 1.8 0.2 46 −1 Initiation factor IF-2, mitochondrial Protein metabolism U18797 1.8 0.2 95 −3 MHC class I antigen H-2M3 Unknown M11988 1.8 0.3 141 82 Hox-A6 Growth factor U17961 1.8 0.2 123 81 p62 ras-GAP associated phosphoprotein Signal transduction W85103 1.8 0.1 24 −17 IGF binding protein 4 precursor homolog Energy metabolism X07997 1.8 0.2 230 128 MHC class I T-cell antigen Lyt3.1 Immune/inflammatory W46723 1.8 0.3 164 83 Creatine kinase, B chain homolog Unknown W48464 1.8 0.4 18 −7 Protein-tyrosine phosphatase MEG2 homolog Unknown L06322 1.8 0.1 84 −4 Delta opioid receptor Neurotransmission W49178 1.8 0.1 605 508 Tubulin beta-1 chain homolog Structural W48477 1.8 0.2 106 61 Thyrotroph embryonic factor homolog Unknown W64225 1.8 0.3 80 44 G21 Unknown L28167 1.8 0.2 88 45 Zinc finger protein Unknown W97199 1.8 0.3 37 62 Negative regulator of transcription subunit 2 Transcriptional factor X01971 1.8 0.2 20 −35 Interferon alpha 5 (Mu IFN-alpha 5) Immune/inflammatory AA061266 1.8 0.3 164 125 Oxysterol-binding protein homolog Transport U21855 1.8 0.3 94 31 CAF1 Transcriptional factor W87078 1.8 0.1 182 90 Unknown Unknown W34687 1.8 0.3 188 105 Actin alpha skeletal muscle homolog Structural K01238 1.8 0.3 191 127 Interferon alpha 2 Immune/inflammatory U15635 1.8 0.2 70 9 IFN-gamma induced (Mg11) Unknown L13968 1.8 0.1 98 26 UCR-motif DNA-binding protein Transcriptional factor M86567 1.8 0.2 122 60 GABA-A receptor alpha-2 subunit Neurotransmission M87861 1.8 0.3 51 −22 Granule membrane protein 140 Structural W55350 1.8 0.3 14 −4 Phosphatidylinositol transfer protein B isoform Unknown L43567 1.8 0.1 35 −21 B-cell receptor gene lmmune/inflammatory AA153196 1.8 0.2 55 −19 Ubiqurlin-activating enzyme E1 homolog Protein metabolism M28312 1.8 0.1 109 41 Metalloprotease inhibitor TIMP1 Immune/inflammatory *The values presented for Signal Intensity are the averages of three mice per age group and are expressed as data for old CR/old control mice. The SE was calculated for the nine pairwise comparisons and was obtained by dividing the standard deviation by the square root of 3. The method from which signal intensity is used to estimate fold changes is described in the Methods section of the manuscript.

[0057] 8 TABLE 8 Caloric restriction-related decreases in gene expression in neocortex of C57BL/6 mice* CR De- Signal Intensity ORF crease SE CR Control Gene Class X76505 −7.2 1.0 −195 73 Tyro 10 Signal transduction U43088 −6.3 1.1 −109 164 IL-17 (CTLA-8) Immune/inflammatory W50186 −5.6 2.1 −38 129 Heavy chain homolog Unknown Y07711 −3.5 0.5 28 151 Zyxin Signal transduction Z47205 −3.1 0.8 45 200 PLZF Transcriptional factor AA000203 −2.8 0.7 −93 26 Corticosteroid-binding globulin precursor Transport W83658 −2.6 0.5 51 197 Guanine nucleotide-binding protein Signal transduction G(I)/G(S)/G(O) homolog L46815 −2.6 0.2 8 67 Ig kappa chain recombination and transcription DNA metabolism enhancer AA153484 −2.4 0.5 208 456 SERCA2 Ion transport W51466 −2.4 0.4 12 147 Chlorine channel protein P64 homolog Unknown U27398 −2.4 0.4 39 132 XPC DNA Metabolism X58069 −2.2 0.7 54 164 H2A.X DNA metabolism U50712 −2.2 0.4 54 156 MCP-5 Immune/inflammatory M61909 −2.1 0.3 39 125 NF-kappa-B p65 Stress response AA072643 −2.1 0.4 49 110 Midkine precursor homolog Stress response L01991 −2.1 0.3 48 132 PANG Unknown L04678 −2.1 0.2 −64 138 Integrin beta 4 subunit Structural W64628 −2.1 0.4 62 197 Guanine nucleotide-binding protein Signal transduction G(I)/G(S)/G(O) gamma-7 subunit X54098 −2.0 0.3 55 136 lamin B2 Structural AA023458 −2.0 0.3 20 107 Heat shock 27 KD protein homolog Stress response D63380 −2.0 0.2 −19 32 Alpha-1,3-fucosyltransferase Protein metabolism U15548 −2.0 0.3 −30 42 Beta 2 thyroid hormone receptor Energy metabolism AA123385 −2.0 0.2 57 117 Phosphorylase B kinase gamma catalytic chain Energy metabolism X57349 −2.0 0.4 −10 49 Transferrin receptor Transport D00659 −2.0 0.1 1 35 Aromatase P450 Biosynthesis AA028875 −2.0 0.2 −32 54 Glycine-rich cell wall structural homolog Lysosomal X76291 −2.0 0.1 11 79 Ihh (Indian Hedgehog) Signal transduction AA041982 −1.9 0.3 44 84 LARK Circadian regulation AA118758 −1.9 0.2 103 206 Multifunctional aminoacyl-tRNA synthetase Protein synthesis W75353 −1.9 0.3 90 162 Apolipoprotein C-IV Transport W55410 −1.9 0.2 30 111 Tubulin gamma chain homolog Unknown L20343 −1.9 0.2 22 102 L-type calcium channel beta 2a subunit isoform Transport W91095 −1.9 0.5 44 93 Valyl-tRNA synthetase Protein metabolism X81593 −1.9 0.1 53 119 Winged-helix domain Transcriptional factor M38248 −1.9 0.2 −6 25 BALB8N Unknown J04694 −1.8 0.3 48 134 Alpha-1 type IV collagen Structural L47650 −1.8 0.3 50 85 STAT6 R Immune/inflammatory AA023595 −1.8 0.1 38 133 Frizzled protein precursor Signal transduction AA015168 −1.8 0.2 42 97 Interferon-gamma receptor beta chain homolog Immune/inflammatory AA013951 −1.8 0.1 32 38 Creatine transporter homolog Energy metabolism W78443 −1.8 0.2 17 106 MKP-X Signal transduction D31842 −1.8 0.2 66 126 PTP36 Structural W50138 −1.8 0.2 1 162 Putative serine/threonine-protein kinase B0464.5 Unknown L35307 −1.8 0.2 33 104 c-Krox Transcriptional factor AA073154 −1.8 0.3 31 68 Alpha-catern homolog Structural W12720 −1.8 0.3 149 251 RAP-2B homolog Signal transduction AA170169 −1.8 0.2 −17 37 Elongation factor 1-gamma homolog Protein metabolism W48951 −1.8 0.3 8 30 Voltage-dependent anion-selective channel Unknown protein 2 homolog M35732 −1.8 0.3 −13 17 Seminal vesicle secretory protein IV Unknown AA145515 −1.8 0.3 68 187 Pre-MRNA splicing factor PRP6 RNA metabolism W13162 −1.8 0.1 −7 62 Cell division protein kinase 4 DNA metabolism J03482 −1.8 0.2 42 113 Histone H1 DNA metabolism W82793 −1.8 0.1 −4 59 Topoisomerase E III homolog DNA metabolism Z31360 −1.8 0.3 1 51 P/L01 Unknown Y09632 −1.8 0.1 16 37 Rabkinesin-6 Transport AA066621 −1.8 0.2 13 63 60S ribosomal protein L10 Protein metabolism U67874 −1.8 0.3 46 85 Ubiqurtin thiolesterase family Protein metabolism AA109714 −1.8 0.3 562 968 SKP1 RNA metabolism AA007957 −1.8 0.2 210 357 Threonyl-tRNA synthetase homolog Protein metabolism AA162633 −1.8 0.2 46 95 Isoleucyl-tRNA synthetase Protein metabolism M17299 −1.8 0.3 29 101 Phosphoglycerate kinase (pgk-2) Energy metabolism AA050102 −1.7 0.3 211 263 Elongation factor 2 (EF-2) Protein metabolism W54637 −1.7 0.2 72 137 Tubulin bets-2 chain class-II homolog Unknown D10028 −1.7 0.3 167 312 Glutamate receptor channel subunit zeta 1 Neurotransmission M28587 −1.7 0.2 −52 30 Alpha leukocyte interferon Immune/inflammatory AA023506 −1.7 0.2 60 144 Insulin receptor substrate-3 Energy metabolism W70629 −1.7 0.3 92 158 COPII Protein metabolism U33626 −1.7 0.3 66 125 PML isoform 1 (Pml) Unknown AA144746 −1.7 0.2 42 92 EF-1-delta Protein metabolism M19380 −1.7 0.3 1406 2303 Calmodulin (Cam III) Signal transduction AA144136 −1.7 0.2 43 100 Choline kinase R1 homolog Biosynthesis AA165847 −1.7 0.3 331 509 EF-1-alpha2 homolog Protein metabolism W33415 −1.7 0.2 90 136 ATP citrate-lyase Unknown U35233 −1.6 0.1 71 109 Endothelin-1 Vasoconstrictive peptide W57384 −1.9 0.3 6 15 ATP synthase A chain homolog Energy metabolism X60452 −1.6 0.3 124 200 Cytochrome P-450IIIA Stress response AA022127 −1.6 0.1 172 279 Vascular endothelial growth factor Unknown AA168841 −1.6 0.2 169 289 Serine/threonine-protein kinase PAK Unknown AA120586 −1.6 0.1 9 64 Apolipoprotein B-100 precursor Stress response AA104561 −1.6 0.2 104 166 EIF-4A homolog Protein metabolism X17071 −1.6 0.1 25 90 Trophoblast-specific protein Growth factor M96265 −1.6 0.1 153 250 Galactose-1-phosphate uridyl transferase Biosynthesis AA145160 −1.6 0.2 178 287 Translational initiation factor 2 alpha Protein metabolism X63473 −1.6 0.1 69 110 m4 muscannic acetylcholine receptor Neurotransmission AA002750 −1.5 0.2 176 290 5-lipoxygenase activating protein (FLAP) Immune/inflammatory W64698 −1.5 0.2 51 63 Protein kinase C inhibitor 1 Signal transduction U63841 −1.5 0.1 120 197 NeuroD3 Growth factors U04294 −1.5 0.1 99 150 Potassium channel subunit (m-eag) Transport M33227 −1.5 0.2 259 396 Cryptdin-related (CRS4C) Immune/ inflammatory U20532 −1.5 0.1 45 67 P45 NF-E2 related factor 2 (Nrf2) Transcriptional factor AA140026 −1.5 0.1 378 519 DNA directed RNA polymerase polypeptide G DNA metabolism W09025 −1.5 0.1 47 68 ATP synthase B chain homolog Energy metabolism W29163 −1.5 0.1 342 465 Leydig cell tumor 10kd protein homolog Unknown AA155191 −1.5 0.1 36 65 Kinesin heavy chain Transport M80360 −1.5 0.1 63 96 Rep-3 DNA metabolism AA044561 −1.4 0.2 93 132 PEP carboxykinase - mitochondnal Energy metabolism AA096843 −1.4 0.2 130 175 Unknown Unknown X57277 −1.4 0.1 908 1298 Rac 1 Signal transduction W82998 −1.4 0.1 256 363 BUB3 DNA metabolism *The values presented for Signal Intensity are the averages at three mice per age group and are expressed as data for old CR/old control mice. The SE was calculated for the nine pairwise comparisons and was obtained by dividing the standard deviation by the square root of 3. The method from which signal intensity is used to estimate fold changes is described in the Methods section of the manuscript.

[0058] 9 TABLE 9 Aging-related increases in gene expression in the cereum of C57BL/6 mice* Fold Signal Intensity CR ORF Change SE Old Young Gene Class Prevention AA120109 9.3 3.4 254 29 lnterferon-induced protein 6-16 precursor Immune/inflammatory N M21050 6.4 0.9 291 14 Lysozyme P (Lzp-s) Immune  88 X56824 5.7 1.9 160 89 Tumor-induced 32 kD protein (p32) Unknown 100 V00727 5.6 2.6 282 57 c-fos Stress  30 M13019 4.9 0.7 109 3 Thymidylale synthase DNA metabolism  87 L16894 4.7 1.0 192 5 Cyclophilin C (CyCAP) Immune/inflammatory N AA146437 4.7 0.3 841 169 Cathepsin S precursor Stress  62 X58861 4.4 0.2 719 160 C1Q alpha-chain Immune/inflammatory  80 W67046 4.3 0.8 50 1 C6 chemokine Immune/inflammatory N X66295 4.1 0.6 508 147 C1q C-chain Immune/inflammatory  56 W65899 4.1 1.8 152 58 Guanine nucleotide-binding protein Signal transduction  80 U00677 4.1 2.2 16 −10 Syntrophin-1 Neurotransmission 100 X68273 3.9 1.8 108 −37 Macrosialin Immune/inflammatory N U19854 3.9 0.5 35 −63 Ubiqurtinating enzyme E2-20K Protein metabolism 100 U63133 3.9 1.1 318 95 Emv-3 Viral N L20315 3.8 0.1 97 26 MPS1 Immune/inflammatory  56 K01347 3.8 0.7 337 109 Glial fibrillary acidic protein (GFAP) Stress  61 M17440 3.7 0.3 445 116 Sex-limited protein (SIpA) Immune/inflammatory N X91144 3.6 1.3 38 −2 P-selectin glycoprotein ligand 1 Immune/inflammatory 100 U43084 3.5 0.8 54 18 IFIT-2 Glucocorticoid-attenuated response Immune/inflammatory N AA089333 3.4 0.2 208 61 Cathepsin S precursor Stress  71 X83733 3.4 0.3 71 −7 SAP62-AMH RNA metabolism 100 W45750 3.3 1.3 197 257 Guanine nucleotide-binding protein G(T) Signal transduction 100 M22531 3.3 0.2 431 146 Clq B-chain Immune/inflammatory  65 AA031244 3.1 0.4 83 9 DNAJ protein homolog HSJ1 Stress 100 M60429 3.1 0.8 121 37 Ig-gamma 1 chain Immune/inflammatory 100 AA036067 3.0 0.4 815 311 Apolipoprotein E precursor (APO-E) Lipid transport  28 U06119 2.9 0.3 27 4 Cathepsin H prepropeptide (ctsH) Stress response  55 AA106347 2.9 0.3 243 57 Angiotensinogen precursor Osmoregulation  80 W98998 2.9 0.7 182 79 Neurogenic locus notch homolog protein 1 Immune/inflammatory  100 AA059700 2.8 0.3 2013 687 MHC class I B(2)-microglobulin Immune/inflammatory  45 U73037 2.8 0.8 69 41 Interferon regulatory factor 7 (7) Immune/inflammatory  50 Y00964 2.8 0.3 780 316 beta-hexosaminidase (Hexb) Unknown  47 X55315 2.8 0.6 63 15 Fetus cerebral cortex for 3UTR Transcription factor 100 U37465 2.8 0.1 15 −7 Protein tyrosine phosphatase phi (PTPphi) Unknown  63 L07803 2.7 1.2 24 −15 trombospondin 2 Structural N U19119 2.7 0.3 52 −5 G-proiein-like LRG-47 Immune/inflammatory N X52886 2.6 0.2 893 326 Cathepsin D Stress response  38 W70578 2.6 1.2 31 7 Antigen WC1 1 Immune/inflammatory  81 X16705 2.6 0.4 93 −4 Laminin B1 Structural  84 W57539 2.6 0.3 28 6 Oocyte zinc finger protein XLCOF8 Unknown N X52308 2.6 0.4 32 9 Thrombin Fibrinogen activation  91 U70859 2.6 0.7 109 46 Cationic amino acid transporter (CAT3) AA transport  49 U41497 2.6 1.1 160 40 Very-long chain acyl-CoA dehydrogenase Lipid metabolism 100 AA089339 2.6 0.5 76 31 Cystatin C precursor Immune/inflammatory 100 X16151 2.5 0.1 239 95 Early T-lymphocyte activation 1 protein Immune/inflammatory  49 U37419 2.5 0.5 111 −2 G protein alpha subunit (GNA-15) Unknown N K02785 2.5 0.5 15 −6 r-fos Stress response N M12289 2.5 0.5 39 25 Pennatal skeletal myosin heavy chain Structural 100 X58849 2.4 0.4 59 13 Murine Hox-4.7 Developmental 100 AA063858 2.4 0.2 89 32 Rho-related GTP-binding protein RHOG Signal transduction  74 D10632 2.4 0.2 33 −27 Zinc finger protein Transcription factor N U33005 2.3 0.4 35 −8 tbc1 Unknown N W85160 2.3 0.7 70 41 40S ribosomal protein S4, X isoform Unknown 100 U57331 2.3 1.0 42 15 Transcription factor Tbx6 (tbx6) Developmental  92 U44731 2.3 0.2 71 20 Putative purine nucleotide binding protein Immune/inflammatory N W87253 2.3 0.6 58 16 Integrin beta-5 Subunit precursor Cell adhesion 100 U53142 2.3 0.2 223 101 Endothelial constitutive nitric oxide Synthase Neurotranmission N AA087715 2.3 0.1 85 −6 GTPase-activating protein SPA-1 Unknown N D49429 2.3 0.3 554 251 Rad21 homolog DNA metabolism  73 AA155318 2.3 0.4 291 129 HNRP1 RNA metabolism N AA032593 2.3 0.1 99 17 Transducin beta chain 2 Signal transduction  83 X03690 2.3 0.2 45 −13 lg mu chain Immune/inflammatory  93 M26417 2.3 0.5 54 28 T cell receptor beta chain Immune/inflammatory 100 X86374 2.2 0.6 73 38 TAG7 Immune/inflammatory  38 W90894 2.2 0.3 27 −11 Cell division protein kinase 4 DNA metabolism 100 M84005 2.2 0.7 83 51 Olfactory receptor 15 Odor receptor  23 X55573 2.2 0.5 55 19 Brain-derived neurotrophic factor Growth factor N W30129 2.2 0.3 90 −16 Phosphatidylinositol glycan hmolog Structural 100 AA163771 2.2 0.3 153 67 EIF-28 epsilon subunit Protein metabolism N X72910 2.1 0.4 96 44 HSA-C Unknown N AA116604 2.1 0.2 303 181 Cathepsin Z Stress response  64 L16462 2.1 0.4 51 4 BCL2-related protein A1 Apoptosis  58 L13732 2.1 0.4 53 29 Natl. resistance-asstd. macrophage protein1 Immune/inflammatory  85 D37791 2.1 0.1 934 424 Beta-1,4-galactosyltransferase Protein metabolism  82 AA125097 2.0 0.1 618 313 Unknown Unknown  94 AA109998 2.0 0.2 40 12 Hexokinase D homolog Energy metabolism 100 M88127 2.0 0.2 33 −8 APC2 homolog Unknown  82 X13538 2.0 0.5 114 45 Hox-1,4 Growth/development 100 V01527 2.0 0.5 28 10 H2-IA-beta Immune/inflammatory 100 AA144411 2.0 0.1 86 79 Unknown Unknown 100 X63535 2.0 0.1 55 21 Tyrosine-protein kinase receptor UFO Signal transduction N M83348 2.0 0.1 42 22 Pregnancy specific glycoprotein homolog Unknown N W08211 2.0 0.2 62 26 TGF-beta receptor type III Signal transduction 100 W13136 2.0 0.4 266 87 Angiotenisinogen Osmoregulation  36 W46084 2.0 0.1 89 45 Unknown Unknown N U73744 2.0 0.1 3958 2909 Heat shock 70 Stress response 100 D29763 1.9 0.2 465 271 Seizure-related, product 6 type 3 Unknown  47 AA118121 1.9 1.0 51 37 lsoleucyl-tRNA synthetase Protein metabolism N M27034 1.9 0.2 258 163 MHC class 1 D-region Immune/inflammatory N U35249 1.9 0.1 68 36 CDK-activating kinase assembly factor DNA metabolism  61 J03776 1.9 0.4 37 22 Down regulatory protein (rpt-1r) of IL-2 receptor Immune/inflammatory N U28728 1.9 0.3 221 112 Els Signal transduction  66 AA124192 1.9 0.2 411 244 Unknown Unknown  44 W63809 1.8 0.4 136 80 Unknown Unknown  73 X16834 1.8 0.2 455 182 Galectin-3 Immune/inflammatory N X16995 1.8 0.2 351 221 N10 nuclear hormonal receptor homolog Unknown 100 J02870 1.8 0.2 848 380 40S ribosomal protein SA Protein metabolism 100 L21768 1.8 0.2 153 76 EGF15 Growth factor  68 AA117284 1.8 0.1 217 123 Zinc finger protein homolog Unknown N *The values presented for Signal Intensity are the averages of three mice per age group and are expressed as data for old/young mice. The prevention by CR is shown as being none (N) or the calculated percentage effect The SE was calculated for the nine pairwise compansons and was obtained by dividing the standard deviation by the square root of 3. The method from which signal intensity is used to estimate fold changes is described in the Methods section of the manuscript.

[0059] 10 TABLE 10 Aging-related increases in gene expression in the cereum of C57BL/6 mice* Fold Signal Intensity CR ORF Change SE Old Young Gene Class Prevention U00445 −4.3 1.4 39 132 Glucose-6-phosphatase Energy metabolism  79 W48504 −4.1 1.1 32 78 phosphoneuroprotein 14 homolog) Unknown N AA153337 −3.9 0.7 67 218 Myosin regulatory light chain 2 (MLC-2). Unknown  61 W51213 −3.9 0.5 14 57 NEDD-4 homolog Protein metabolism  55 X56304 −3.1 0.4 2 27 Tenascin Growth/development N W12681 −3.1 0.6 30 126 Hepatocyte growth factor Growth/development  37 Z68889 −2.9 1.0 30 70 Wnt-2 homolog Growth/development N W55684 −2.8 0.6 13 37 Brain protein i47 Unknown N U04827 −2.8 0.5 94 219 Brain fatty acid-binding protein (B-FABP) Growth/development N AA008066 −2.7 1.0 1 61 Pre-mRNA splicing factor PRP22 Unknown  74 W55300 −2.7 0.7 20 47 Fatty acid-binding protein, heart (H-FABP) Unknown  71 D13903 −2.7 0.5 7 37 MPTPdelta (type A) Growth/development N AA013976 −2.6 0.5 162 405 POL polyprotein; reverse transcriptase; Unknown N ribonuclease H W10865 −2.6 0.2 14 142 Myosin light chain 1, atnal/foetal isoform Unknown N AA020296 −2.5 0.2 −162 166 NG9 Growth/development 100 W64865 −2.5 1.1 10 31 Stat-3 Unknown N AA139694 −2.5 0.3 64 203 Beta-myosin heavy chain Transport 100 U29762 −2.5 0.3 304 657 Albumin gene D-Box binding protein Transcription Factor N M87276 −2.4 0.5 16 34 Thrombospondin Structural  52 X02677 −2.4 0.2 63 160 Anion exchange protein Anion exchanger 100 X04836 −2.4 0.2 22 68 T-cell antigen CD4 Immune/inflammatory 100 X87242 −2.4 0.3 48 111 unc-33 Growth/development  70 AA163021 −2.4 0.2 28 143 Annexin VIII Signal transduction  84 M31810 −2.4 0.3 29 113 P-protein membrane transporter Transport 100 M97900 −2.4 0.6 18 49 Unknown Unknown  20 M15008 −2.4 0.6 101 227 Steroid 21-hydroxylase B Steroid metabolism 100 M99377 −2.4 0.5 77 191 Alpha-2 adrenergic receptor Neurotransmission N M32490 −2.4 0.3 62 122 Cyr61 Growth/development  41 AA168350 −2.3 0.3 130 237 Cysteinyl-tRNA synthetase Protein metabolism  83 AA061206 −2.3 0.2 8 52 Unp (ubiquitin protease) Protein metabolism N W12794 −2.3 0.3 23 96 Unknown Unknown  78 AA050593 −2.3 0.1 5 69 Unknown Unknown  62 AA050715 −2.3 0.3 64 148 Smoothelin Structural  92 AA106463 −2.2 0.3 110 277 Phosphoenolpyruvate carboxykinase. Energy metabolism N X90829 −2.2 0.3 −16 9 Lbx1 Growth/development N X65588 −2.2 0.3 −1 24 mp41 Neurotransmission N J00475 −2.2 0.2 −23 58 lg alpha chain Immune/inflammatory N X03019 −2.2 0.3 4 71 GM-CSF Immune/inflammatory  26 W34687 −2.2 0.4 62 115 Alpha-actin Transport  78 W75614 −2.2 0.4 27 56 Alpha-synuclein Growth/development N AA068153 −2.2 0.3 14 39 Polyadenylate-binding protein RNA metabolism  55 U36842 −2.1 0.5 22 36 Riap 3-inhibitor of apoptosis Apoptosis 100 W09127 −2.1 0.3 3 85 60S ribosomal protein L22 Protein metabolism 100 D63819 −2.1 0.2 29 87 Neuropeptide Y-Y1 receptor Neurotransmission N M33884 −2.1 0.1 70 139 Env polyprotein Viral protein  55 AA144430 −2.1 0.3 64 156 NF-KB P100 inhibitory subunit Stress response  48 AA168554 −2.1 0.3 119 246 Unknown Unknown  85 U35730 −2.1 0.8 12 30 Jerky Unknown N M92649 −2.1 0.4 45 112 nitric oxide synthase Neurotransmission N D12907 −2.1 0.2 55 126 Serine protease inhibitor homologue Unknown  85 M17327 −2.1 0.2 234 566 Env polyprotein Viral protein  56 AA170444 −2.1 0.2 172 246 Ubiquitin-activating enzyme E1 Protein metabolism 100 W12658 −2.1 0.3 203 415 FKBP-rapamycin associated protein Unknown N AA123026 −2.1 0.3 60 116 REG 2 Unknown 100 W13125 −2.1 0.5 111 232 Phenylalanyl-tRNA synthetase beta chain Protein metabolism N AA103862 −2.1 0.4 53 143 Unknown Unknown N U21301 −2.1 0.6 30 62 c-mer tyrosine kinase receptor Signal transduction N W13586 −2.1 0.1 29 136 Myosin light chain 1 homolog Transport 100 W42217 −2.1 0.1 69 143 Ribosomal protein S20 Protein metabolism 100 AA153522 −2.1 0.4 95 191 Serine/threonine kinase Signal transduction  78 W30612 −2.0 0.1 70 160 Chloride intracellular channel 3 Transport 100 W11621 −2.0 0.4 78 138 Zinc finger protein 126 Unknown N X72805 −2.0 0.3 25 63 CD-1 histone H1t DNA metabolism N L08407 −2.0 0.3 38 117 Collagen type XVII Structural N AA145609 −2.0 0.2 55 134 cAMP responsive element modifier Transcriptional factor  34 W12756 −2.0 0.1 48 117 Unknown Unknown  92 W75523 −2.0 0.3 48 95 Vertebrate homolog of C. elegans Lin-7 type 2 Unknown N D85904 −1.9 0.3 69 129 Heat shock 70-related protein Apg-2 Stress response N AA138911 −1.8 0.2 176 311 RNA helicase PRP16 RNA metabolism 100 W42216 −1.8 0.1 183 361 SWI/SNF related homolog Transcriptional factor  74 W12395 −1.8 0.4 141 237 Transcription elongation factor A (SII) Transcriptional factor  88 K03235 −1.8 0.1 84 149 Prolifenn 2 Growth factor 100 AA145859 −1.8 0.1 4110 5250 Unknown Unknown 100 W57194 −1.8 0.2 61 108 Ubiquitin carboxyl terminal hydrolase 12 Protein metabolism N AA166440 −1.7 0.1 229 389 Phosphatidylserine decarboxylase Protein metabolism N L33726 −1.7 0.1 69 128 Fascin homolog 1 Structural 100 L35549 −1.7 0.4 30 38 Y-box binding protein homolog Unknown 100 AA154514 −1.7 0.1 7639 12878 ATP synthase A chain (protein 6) homolog Energy metabolism 100 AA143937 −1.7 0.1 384 697 Beta-centractin Transport  70 AA027387 −1.7 0.1 169 270 Rab-4B Transport  51 L38971 −1.7 0.2 205 334 Integral membrane protein 2 Unknown  43 W10526 −1.7 0.1 193 301 Ca** channel, voltage-dep., gamma subunit 1 Transport  90 W12204 −1.6 0.2 114 200 Ca2+/calmodulin-dependent protein kinase Signal transduction N isoform gamma B AA170173 −1.6 0.1 149 289 NTT-73 Transport 100 M64403 −1.6 0.1 126 208 Cyclin D1 homolog DNA metabolism 100 W13191 −1.6 0.1 288 347 Thyroid hormone receptor alpha 2 Energy metabolism  87 U47543 −1.6 0.1 121 205 NGF1-A binding protein 2 (NAB2) Growth factor N D70848 −1.6 0.2 154 246 Zic2 (cerebellar zinc finger protein) Neural development  77 X56518 −1.6 0.3 106 164 Acetylcholinesterase Neurotransmission N AA144588 −1.6 0.2 233 368 Beta-adrenergic receptor kinase 2 homolog Neurotransmission  33 AA139828 −1.6 0.1 224 351 gonadotropin inducible transcription repressor-1 Unknown 100 homolog AA061170 −1.6 0.2 43 65 WW-domain oxidoreductase homolog Unknown N X58287 −1.6 0.3 84 153 mR-PTPu Signal transduction N L13129 −1.6 0.1 162 220 Annexin A7 Exocytosis  90 D85037 −1.6 0.1 50 77 Doc2beta Neruotransmission N U30823 −1.6 0.2 55 102 Myocyte enhancer factor-2A Transcriptional factor  33 W64791 −1.6 0.1 92 143 Galactokinase Energy metabolism N X52622 −1.6 0.1 274 377 IN Viral protein 100 AA063914 −1.5 0.1 175 267 Alpha-tubulin Transport 64 *The values presented for Signal Intensity are the averages of three mice per age group and are expressed as data for old/young mice. The prevention by CR is shown as being none (N) or the calculated percentage effect. The SE was calculated for the nine pairwise comparisons and was obtained by dividing the standard deviation by the square root of 3. The method from which signal intensity is used to estimate fold changes is described in the Methods section of the manuscript.

[0060] 11 TABLE 11 Genes upregulated by aging in C57BL/6 mice heart from Mu19K GeneChip Fold Probe Set oc1 oc2 oc3 yc1 yc2 yc3 Change TC27774 396 218 490 −1328 −2197 −1280 25.8 TC35932 71 1391 355 −596 −507 −1500 17.2 TC39719 938 595 1380 529 −129 −562 14.6 TC24697 1510 2431 3697 173 −823 −537 13.9 TC17809 4141 4286 4415 224 369 921 11.0 TC28794 1358 1313 1445 349 −38 657 10.4 TC16257 439 867 471 −121 −528 166 10.3 TC34515 1687 1117 966 465 −1068 −1737 9.4 TC29214 102 154 188 −381 −122 −209 9.0 TC32857 733 915 524 200 82 90 8.3 TC37114 553 803 466 377 −99 59 8.2 TC17940 947 1889 1474 −54 160 −1487 8.1 TC39890 912 1658 1190 639 617 8 7.7 TC39498 1080 738 1754 −29 634 −462 7.3 TC25820 340 510 325 −353 −315 −575 6.1 TC24908 12482 8941 7330 1337 1838 1387 5.8 TC29305 1271 1020 827 841 382 606 5.5 TC16024 739 1570 995 603 312 123 4.8 TC33899 304 287 240 64 30 73 4.8 TC16184 1294 3064 3523 428 388 447 4.7 TC39399 338 421 286 −81 208 27 4.5 TC17839 1506 946 2315 248 512 146 4.5 TC18386 1822 1967 1585 281 566 477 4.4 TC27769 3796 5647 3986 1260 975 2286 4.4 TC37583 433 617 758 119 425 93 4.3 TC22269 6795 7593 8793 920 2322 5205 4.1 TC28239 2039 1359 881 227 495 604 4.1 TC34440 340 310 258 21 −437 −170 4.1 TC39301 803 1692 1539 27 710 778 4.1 TC29662 997 2372 1701 174 650 694 4.0 TC33757 339 323 257 49 76 231 3.9 TC29977 858 631 879 102 541 335 3.9 TC19997 419 358 384 84 67 266 3.8 TC27675 4002 5625 6693 1292 1580 1426 3.8 TC21921 677 779 864 339 43 229 3.8 TC41800 915 441 1157 −8 69 180 3.7 TC31694 2158 2467 2245 449 306 976 3.7 TC28855 282 194 355 67 127 62 3.6 TC31277 311 243 445 44 182 172 3.6 TC21628 176 422 304 124 76 68 3.5 TC36063 498 623 390 −80 346 −52 3.5 TC33608 514 449 479 140 165 124 3.4 TC38147 420 212 473 61 173 211 3.3 TC23622 112 328 186 −55 60 99 3.2 TC34697 549 450 752 89 356 370 3.2 TC22213 1892 2305 2099 655 730 644 3.1 TC31569 282 113 247 73 127 4 3.1 TC28942 517 1055 1020 301 364 224 3.0

[0061] 12 TABLE 12 Genes downregulated by aging in C57BL/6 mice heart from Mu19K GeneChip Fold Probe Set oc1 oc2 oc3 yc1 yc2 yc3 Change TC27282 20 −2020 −2141 5078 970 879 −86.2 TC32064 −217 −844 −511 2335 2211 2176 −58.6 TC24160 −1155 −3091 −2382 427 4103 4674 −56.2 TC14603 867 −2795 −2128 4729 2680 2255 −53.4 TC22507 −1155 −1599 −1409 1319 2177 2942 −50.4 TC15929 −1203 −1586 −1787 1348 1014 2026 −47.0 TC19943 −687 −669 −428 2880 2552 1067 −41.7 TC18736 −1142 787 −1647 2711 3654 4006 −33.0 TC19957 1242 −501 958 6796 6771 5343 −30.5 TC37452 175 −1172 −441 820 2013 1233 −27.3 TC33452 532 −740 −465 2021 880 719 −26.3 TC14870 −289 −1650 −2496 30 209 1249 −25.2 TC26312 −118 −73 −146 406 1251 1344 −24.3 TC25802 −688 −736 −1968 31 707 695 −23.7 TC14624 −227 −943 −758 1675 718 352 −22.6 TC41568 −684 −3089 −1954 7 711 129 −22.6 TC16488 −1548 −57 −1609 1055 1739 190 −22.5 TC18539 122 1114 −269 3415 2604 2614 −21.6 TC37617 −1738 −296 −2150 2156 2231 422 −20.6 TC39618 −56 −204 −168 769 1196 887 −19.5 TC37350 −1070 −657 −655 1944 1258 260 −19.5 TC36639 1496 −3251 −23 4489 2756 6211 −19.4 TC16420 48 −674 −17 1059 1053 1072 −18.6 TC37529 177 151 333 6190 3159 2499 −18.3 TC15736 −67 −1109 −1133 242 530 647 −18.2 TC36992 498 −2096 −450 2140 2451 1214 −17.9 TC28761 326 −105 847 4047 2990 1712 −17.9 TC25360 −1421 −2210 −2177 332 173 204 −17.2 TC16633 −66 −612 −638 626 240 496 −17.0 TC18250 145 −416 −464 2429 890 804 −16.3 TC35586 −337 −526 6 762 782 328 −16.2 TC37067 2006 137 2589 7334 6130 5348 −16.0 TC40509 176 −216 197 2219 724 1177 −15.9 TC37745 380 −1137 141 822 1566 1043 −15.8 TC24220 648 227 48 1916 1805 2138 −14.9 TC17700 159 −80 −657 565 810 690 −14.4 TC17256 −2800 −3715 −3550 629 2754 950 −13.4 TC37672 −117 427 247 1149 1712 1737 −13.0 TC18637 202 −208 −312 1012 907 794 −12.8 TC15863 −639 250 289 882 794 1198 −12.7 TC23647 −575 334 −1428 1821 2149 2101 −12.5 TC16841 375 −198 430 1177 1044 1257 −12.3 TC27576 −70 75 428 596 1326 857 −12.2 TC21963 −281 −437 −368 944 136 231 −12.2 TC36608 −527 −316 −140 343 254 7 −12.1 TC26887 60 188 −100 589 933 734 −11.9 TC24501 539 518 79 4279 1947 1811 −11.8 TC36239 902 −102 843 1587 1899 2152 −11.3 TC38050 −47 −81 115 324 633 645 −11.3 TC37660 −1 −617 −203 450 240 314 −11.1 TC34986 −1 −98 −28 726 315 235 −10.7 TC30885 402 −55 27 878 734 398 −10.4 TC16723 478 276 62 1703 1736 1138 −10.3 TC20671 −70 −827 −303 948 1087 410 −10.2 TC14753 −332 −265 −325 418 335 276 −10.1 TC16229 −156 515 107 1224 681 1077 −10.1 TC24641 −372 −382 −329 127 845 718 −10.0 TC35052 139 −86 −19 504 459 447 −9.9 TC20554 158 392 625 1255 896 1199 −9.8 TC25572 −470 −460 −871 472 1340 791 −9.5 TC21262 220 −336 1193 2061 1581 2928 −9.5 TC25416 48 −285 −104 487 554 460 −9.5 TC41297 373 −176 455 1093 976 991 −9.4 TC37701 −219 −338 −398 830 294 236 −9.4 TC34944 364 462 369 3507 3271 3393 −9.3 TC31449 −7 53 −51 300 252 217 −9.0 TC41997 167 −142 199 682 1057 893 −8.8 TC36033 −164 −295 −678 1048 194 241 −8.8 TC27468 584 492 560 1011 1031 929 −8.8 TC16039 603 −2181 −1612 2105 1544 1004 −8.6 TC19352 −918 −290 −600 1103 700 859 −8.5 TC25041 229 −697 −295 726 515 558 −8.4 TC35104 548 1 563 1294 1692 715 −8.3 TC25357 143 −277 −40 897 788 1407 −8.0 TC22194 119 −63 −176 477 440 633 −7.9 TC20469 284 −303 −850 1031 591 674 −7.7 TC41078 −35 −289 42 551 232 148 −7.7 TC39603 417 −253 300 813 952 586 −7.6 TC36846 64 −83 117 606 487 353 −7.2 TC24619 −11 −273 −224 212 483 418 −7.1 TC15831 1167 1269 87 3253 1942 1814 −7.1 TC25629 −4 −309 −341 387 106 167 −7.1 TC23144 −91 −175 −322 770 114 393 −7.0 TC29553 77 −27 −110 93 283 185 −7.0 TC36286 −312 −574 −44 702 929 668 −6.8 TC23964 1265 1225 276 6611 4409 5007 −6.8 TC37675 19 103 139 408 734 469 −6.6 TC41144 236 58 273 1095 734 708 −6.6 TC40883 −31 −251 88 201 473 370 −6.6 TC27606 −640 −765 −579 232 208 394 −6.5 TC14712 1140 643 −15 1661 1331 2644 −6.5 TC26859 803 95 985 3249 2325 2184 −6.4 TC33246 168 −216 −384 517 283 384 −6.4 TC37343 180 −27 34 459 508 346 −6.3 TC37275 1193 720 808 1722 1828 1992 −6.3 TC18134 685 695 488 145 57 96 −6.2 TC40210 166 −245 91 354 502 400 −6.1 TC17241 438 −110 756 1750 2691 2519 −6.1 TC21038 133 −138 −206 600 218 168 −6.1 TC22355 12 −396 −116 182 232 177 −6.1 TC38075 111 −40 11 533 588 613 −6.0 TC38184 −263 −107 58 293 235 92 −6.0 TC37491 239 166 349 1404 1500 1141 −5.9 TC33420 −132 −208 −114 388 128 88 −5.9 TC37318 1331 188 833 1241 3321 2861 −5.8 TC37916 −273 −62 −202 198 55 43 −5.8 TC17885 −178 169 −288 1591 1472 1445 −5.7 TC15884 390 −134 −109 734 431 493 −5.6 TC40452 −94 −141 107 291 339 359 −5.6 TC29330 512 370 140 2164 1174 930 −5.6 TC17616 101 46 57 531 853 808 −5.6 TC21414 −62 −2 −143 111 296 344 −5.5 TC17717 36 −83 −144 222 172 209 −5.4 TC31495 156 155 77 280 502 371 −5.3 TC18144 2048 819 1400 3236 3117 3190 −5.3 TC19650 −120 −282 −56 358 86 18 −5.2 TC25815 36 224 90 490 506 508 −5.2 TC37544 470 242 458 527 767 691 −5.1 TC38870 119 −35 187 1057 704 587 −5.1 TC26789 111 49 −68 240 243 270 −5.0 TC37493 103 250 396 993 982 795 −5.0 TC41579 465 120 253 959 557 669 −5.0 TC17620 326 452 303 721 565 788 −4.9 TC18572 29 −130 −51 208 264 348 −4.9 TC41021 217 84 43 611 329 306 −4.9 TC25021 61 95 69 471 440 235 −4.9 TC37829 −235 −243 92 142 292 771 −4.7 TC19783 35 −10 249 371 604 767 −4.6 TC24373 −111 −424 171 376 384 395 −4.6 TC41191 54 −407 −30 741 36 721 −4.6 TC30942 281 146 19 1772 1068 1025 −4.5 TC14554 28 −147 44 651 479 471 −4.5 TC32618 210 68 260 435 504 448 −4.5 TC35574 1063 295 1619 2598 3642 3046 −4.5 TC39584 1090 1014 538 2430 3908 4185 −4.4 TC37290 −26 −15 90 541 212 211 −4.3 TC14567 968 216 267 2605 1842 1044 −4.2 TC30986 66 −14 76 306 151 178 −4.2 TC35356 211 −3 224 474 598 338 −4.2 TC35554 91 −100 89 572 566 558 −4.2 TC22851 810 416 520 3098 1773 1661 −4.2 TC20860 316 118 498 1291 739 695 −4.1 TC41573 212 88 343 656 1162 931 −4.1 TC32333 471 489 542 2274 1696 1350 −4.1 TC20845 164 222 −12 508 438 361 −4.0 TC37484 192 −14 236 408 384 494 −4.0 TC33993 −342 −140 −253 161 567 752 −4.0 TC37769 670 107 485 2676 1219 1617 −3.9 TC31667 435 73 167 1141 556 585 −3.9 TC18679 1123 1055 1090 638 626 366 −3.9 TC21666 5 81 −153 203 351 195 −3.8 TC41350 213 83 206 680 403 479 −3.8 T021304 −109 −65 −63 243 38 61 −3.7 TC39507 −137 −208 −77 310 61 22 −3.7 TC19129 827 722 469 1364 1364 1142 −3.6 TC21197 −376 −1186 −1054 1746 1222 416 −3.6 TC38888 67 8 50 292 106 199 −3.6 TC32452 992 974 1165 2411 2887 2965 −3.5 TC14511 739 660 298 942 1924 2211 −3.5 TC29246 716 546 538 1125 991 1222 −3.4 TC15902 137 −4 55 350 211 209 −3.4 TC37774 378 234 424 1148 1146 952 −3.3 TC27288 377 394 816 1451 1663 1554 −3.3 TC31668 −76 −153 −46 170 103 10 −3.3 TC41983 252 −1 190 240 490 429 −3.3 TC14823 933 420 557 1168 2494 1983 −3.3 TC40714 416 939 354 1914 1744 1041 −3.3 TC20259 272 22 86 330 285 513 −3.3 TC23344 462 577 862 1602 2043 2131 −3.3 TC27282 1068 765 508 3300 1911 1689 −3.2 TC21501 500 1332 782 4505 3307 3468 −3.2 TC34693 −14 177 761 1242 1088 1137 −3.2 TC41186 231 120 272 1122 579 641 −3.1 TC26140 276 −43 141 279 541 452 −3.1 TC20981 −59 −53 −38 137 67 86 −3.1 TC39851 97 −176 80 457 204 169 −3.0 TC26095 283 532 336 1142 776 909 −3.0 TC16932 125 188 91 490 284 323 −3.0 TC22052 100 118 149 375 356 323 −3.0

[0062] 13 TABLE 13 Genes upregulated by aging in C57BL/6 mice heart from Mu6500 GeneChip ORF oc1 oc2 oc3 yc1 yc2 yc3 Fold Change X60103 242 223 238 13 −52 65 11.8 AA117446 273 512 453 155 118 66 6.8 M21829 82 83 141 24 45 52 5.4 L07297 69 103 101 −52 −30 −43 5.1 X94998 208 168 223 −8 −35 80 5.1 W36875 149 126 153 15 64 64 4.9 U00677 171 108 187 18 77 5 4.3 M17440 311 354 372 90 84 61 4.0 U08210 45 24 38 −10 4 −17 3.9 AA097087 326 628 684 140 181 143 3.5 X62622 180 134 235 81 112 27 3.5 U25844 702 607 584 186 204 191 3.3 D13664 218 202 130 40 75 75 3.3 U00674 55 48 15 −9 11 15 3.3 Z31663 0 63 55 −42 −100 −88 3.2 X91824 155 121 140 58 60 69 3.2 AA152695 38 42 26 8 8 14 3.2 AA014024 111 219 218 110 59 72 3.1 D16497 1888 1428 3023 664 996 517 3.1 AA036050 52 52 49 18 9 9 3.1 L41154 408 305 476 128 152 157 3.1 AA168633 585 654 733 167 253 246 3.1 L20276 1761 1059 1201 260 600 829 3.0

[0063] 14 TABLE 14 Genes downregulated by aging in C57BL/6 mice heart from Mu6500 GeneChip ORF oc4 oc5 oc6 yc1 yc2 yc3 Fold Change X54149 52 16 −69 106 139 84 −6.2 X98475 −7 37 38 202 136 79 −6.1 U25114 185 133 69 326 301 283 −5.4 U58885 −16 33 105 315 212 301 −5.3 X85169 −1 −32 −75 48 43 11 −5.0 AA028728 68 −19 17 90 99 116 −4.9 D14336 100 17 26 141 202 176 −4.8 W29790 72 91 13 259 196 195 −4.8 L11163 181 334 −18 401 820 512 −4.5 AA068712 18 −12 −15 61 69 70 −4.5 D43643 26 −12 −58 69 61 45 −4.3 Y08361 35 1 −35 88 54 84 −4.2 W57425 −6 −31 −61 36 9 13 −4.2 L17076 130 103 97 645 491 431 −4.1 U08215 45 27 −1 160 74 73 −3.8 AA068780 28 −5 −34 86 32 64 −3.8 AA072334 66 43 88 194 160 136 −3.7 AA060808 98 30 57 226 159 155 −3.7 W84060 15 36 6 56 91 63 −3.7 X97796 16 5 −24 72 53 37 −3.6 X60831 49 35 7 52 59 84 −3.6 AA003162 152 28 108 274 204 224 −3.6 W08293 174 130 106 508 356 342 −3.5 AA107999 47 6 −18 77 72 56 −3.5 Z47205 112 93 21 127 181 253 −3.3 AA107137 46 −19 −31 87 165 125 −3.2 U70017 34 0 3 126 63 48 −3.2 W34891 0 19 19 41 40 36 −3.2 M90364 141 94 103 394 273 326 −3.1 W20652 26 43 38 75 63 84 −3.1 W10926 48 −1 −5 99 34 82 −3.1 X53532 13 14 15 92 36 57 −3.0 W77701 167 90 68 369 347 251 −3.0 U53455 22 29 24 127 62 85 −3.0 U09218 17 22 2 57 71 29 −3.0 D78141 29 24 5 54 74 65 −3.0

[0064] 15 TABLE 15 Genes upregulated by aging in C57BL/6 mice gastrocnemius from Mu19K GeneChip Probe Set oc1 oc2 oc3 yc1 yc2 yc3 Fold Change TC22507 1496 5100 4680 −861 −868 2232 12.3 TC41260 2271 2776 1202 345 337 214 7.1 TC15427 3952 6832 4863 392 2541 1658 6.2 TC17528 309 830 202 −401 −87 58 4.8 TC39719 467 1194 956 −96 −68 639 4.6 TC30023 3484 1557 2722 −471 784 −100 4.2 TC15105 2869 2887 744 424 221 −401 4.2 TC22814 9874 12120 6784 1463 3030 4227 4.2 TC32898 3770 1780 2282 1470 299 598 4.0 TC17624 932 1910 1154 96 704 295 3.9 TC38243 3651 2564 2668 2227 1427 370 3.3 TC32537 2652 2455 3025 723 614 1165 3.3 TC16833 1263 1056 635 427 417 −26 3.1 TC37853 655 965 895 237 151 275 3.1 TC35747 768 1198 1174 477 809 145 3.0 TC36248 3727 6677 4613 2357 2860 1045 2.9 TC16809 2167 1306 1781 648 1219 566 2.8 TC37410 1198 1044 612 564 545 38 2.8 TC29110 1462 775 696 −808 −441 −1038 2.7 TC41340 615 744 603 435 182 403 2.7 TC20762 1280 839 1046 582 553 149 2.7 TC41486 2628 3390 2900 754 2234 1251 2.7 TC30327 3780 2597 2167 628 1606 1354 2.6 TC41030 402 383 450 125 −70 −187 2.6 TC37927 1283 1988 419 −684 −704 −690 2.5 TC35232 206 291 846 −414 −154 −217 2.5 TC40552 676 624 566 180 272 −14 2.5 TC35879 761 606 643 217 248 316 2.5 TC36106 553 81 381 35 −28 −309 2.4 TC14958 431 569 687 37 86 338 2.4 TC15563 1782 2034 1615 779 1031 423 2.4 TC37009 5627 4674 6716 3156 3535 2177 2.4 TC38613 14275 16183 14699 6963 8380 4717 2.4 TC17122 5461 6072 4547 2524 2633 1687 2.4 TC27769 44054 58886 54326 31194 27436 14076 2.4 TC33822 6543 3341 4435 1353 2737 2536 2.4 TC20391 102 324 227 −201 −286 −15 2.4 TC38653 687 826 298 244 59 122 2.4 TC40473 533 539 263 57 118 124 2.3 TC17622 1714 1541 1071 926 397 609 2.3 TC18112 756 793 703 610 211 251 2.3 TC19062 2563 4000 2391 1565 2019 1229 2.3 TC16585 4312 3985 4720 2520 2316 1346 2.3 TC37317 726 1068 673 494 398 258 2.3 TC40165 817 869 775 448 588 182 2.2 TC21714 1174 1390 1120 808 475 702 2.2 TC17422 31965 35070 40903 13173 19477 14605 2.2 TC37018 592 437 367 217 172 79 2.2 TC16885 2486 2538 923 −830 765 −522 2.2 TC34291 13707 19389 10341 8383 5255 6989 2.2 TC37463 1444 1417 1078 922 520 513 2.2 TC24549 8515 9554 5391 4618 4038 3446 2.2 TC35324 321 607 357 140 137 156 2.1 TC31058 1436 1266 1773 514 303 159 2.1 TC15920 2072 2001 1360 477 1197 809 2.1 TC29793 1532 1993 2224 458 1173 801 2.1 TC37926 2769 2562 1750 865 1108 1169 2.1 TC40454 1344 2480 2437 590 1123 786 2.1 TC17515 3386 4354 3900 2340 2892 1179 2.1 TC35819 2072 2558 2188 1248 1174 959 2.1 TC39079 1639 1879 1394 538 1352 726 2.1 TC35125 1031 714 880 300 652 40 2.0 TC40951 11 565 108 −204 −192 −530 2.0 TC37262 680 922 706 269 530 3 2.0 TC31287 2040 2088 2058 336 1232 1246 2.0 TC40137 334 303 464 69 135 144 2.0 TC31251 1652 1328 1412 654 696 592 2.0 TC31522 6212 5990 6621 3005 3336 4224 2.0 TC37833 1464 1782 872 587 766 423 2.0 TC23026 462 265 318 105 88 74 2.0 TC33710 5381 4005 5984 1782 3214 2638 2.0 TC14237 978 1638 1423 877 412 747 2.0 TC32046 2438 2103 1415 898 512 1318 2.0 TC15245 2305 2606 4096 1771 1589 503 2.0 TC30375 15067 24645 27999 11194 14149 9870 2.0 TC24289 383 454 679 143 283 −134 2.0 TC30683 1269 622 565 −320 97 122 2.0

[0065] 16 TABLE 16 Genes downregulated by aging in C57BL/6 mice gastrocnemius from Mu19K GeneChip Probe Set oc1 oc2 oc3 yc1 yc2 yc3 Fold Change TC39172 282 384 1189 1388 1492 1767 −8.6 TC24050 −1117 −243 252 388 1315 2392 −6.8 TC34953 3835 5266 6073 35656 21430 31766 −6.3 TC34306 1324 565 −353 1427 2241 3278 −5.6 TC26537 3726 2008 378 6454 4146 9861 −5.2 TC35355 245 −492 187 765 951 1217 −4.9 TC40742 −394 229 395 1281 1132 1041 −4.7 TC24501 152 253 −108 981 536 1084 −4.6 TC14421 419 1398 344 2366 1833 2615 −4.5 TC21687 −959 88 1433 2686 2066 2732 −4.5 TC25229 369 −201 79 1383 638 1283 −4.2 TC34953 379 2950 2267 5359 3465 5921 −3.9 TC24344 473 528 359 1189 1506 2141 −3.7 TC33957 4504 2776 5281 12197 14665 15262 −3.6 TC40061 4693 1355 4866 7669 10158 7310 −3.5 TC36858 −65 113 276 904 449 854 −3.3 TC15621 3342 3801 2088 5802 5651 7667 −3.1 TC22866 2973 2064 3961 6385 9965 9570 −3.1 TC36347 1077 2585 1662 4287 6166 4493 −3.0 TC26944 13744 8497 7171 26871 31183 24244 −3.0 TC36854 −679 139 −105 2255 4600 2220 −2.9 TC32868 −194 501 −963 1491 1485 569 −2.9 TC33934 −2432 4016 2471 8604 6093 6420 −2.9 TC34857 819 360 −165 2160 2933 3161 −2.9 TC37125 1946 486 1276 2675 2376 2256 −2.7 TC34321 1133 1989 1051 2901 3233 3270 −2.6 TC35099 1565 3225 2314 3774 5816 7280 −2.6 TC22794 420 153 343 1106 1654 1016 −2.6 TC28206 −519 −812 −715 778 784 816 −2.5 TC17374 44879 40619 41419 95128 124767 111416 −2.5 TC19536 38 165 264 626 476 617 −2.5 TC39309 708 927 1767 2405 2161 1651 −2.5 TC14511 2772 859 1861 2932 4587 3089 −2.4 TC25977 −125 907 −393 1714 939 1724 −2.4 TC34555 713 2541 2642 3098 3608 4297 −2.4 TC40318 2484 2040 3012 5440 5650 5710 −2.4 TC22050 721 421 545 944 1092 1638 −2.4 TC23531 264 555 298 677 1076 612 −2.4 TC35434 1150 743 1300 2736 2496 1833 −2.4 TC37551 −265 73 −169 118 422 232 −2.4 TC34651 792 2193 2064 3432 3751 4517 −2.3 TC40365 −286 −312 −315 176 172 252 −2.3 TC26535 4580 11925 9572 12361 20086 21438 −2.2 TC25372 12 141 −161 348 276 386 −2.2 TC28752 816 1567 2442 3958 2783 2378 −2.2 TC21901 1491 754 1326 2284 2539 2382 −2.2 TC41250 628 279 660 782 1093 1096 −2.2 TC20836 102 182 514 781 452 820 −2.2 TC39607 1263 1289 765 1277 1861 1895 −2.2 TC33236 1991 2588 3851 5152 4945 5421 −2.1 TC41556 1138 1047 1367 2263 1972 1988 −2.1 TC41884 475 55 193 650 406 693 −2.1 TC31627 606 494 1343 1839 1123 2105 −2.1 TC35120 1298 1479 752 2993 2032 1705 −2.1 TC37978 664 425 875 1444 1620 1546 −2.1 TC32191 329 1419 700 2118 1560 2187 −2.0 TC39472 5773 5966 4650 9742 11750 11019 −2.0 TC36773 2894 3313 4085 5414 7595 6159 −2.0 TC38302 459 289 306 621 809 568 −2.0 TC28179 11576 8026 7030 16063 14643 19203 −2.0

Claims

1. A method of measuring the biological age of a multicellular organism comprising the steps of:

(a) obtaining a sample of nucleic acid isolated from the organism's organ, tissue or cell, wherein the nucleic acid is RNA or a cDNA copy of RNA and
(b) determining the gene expression pattern of a panel of specific sequences within the nucleic acid pool described in (a) that have been predetermined to either increase or decrease in response to biological aging of the organ, tissue or cell, where the gene expression pattern comprises the relative level of mRNA or cDNA abundance for the panel of specific sequences.

2. The method of claim 1 wherein the expression patterns of at least ten sequences are determined in step (b).

3. The method of claim 2 wherein the expression patterns of at least 20 sequences are determined in step (b).

4. The method of claim 3 wherein the expression levels of at least 30 sequences are determined in step (b).

5. The method of claim 4 wherein the expression levels of at least 40 sequences are determined in step (b).

6. The method of claim 5 wherein the expression levels of at least 50 sequences are determined in step (b).

7. The method of claim 1 wherein the organism is a mammal.

8. The method of claim 7 wherein the mammal is slected from the group consisting of humans, rats and mice.

9. The method of claim 1 wherein the nucleic acid is isolated from a tissue selected from the group consisting of brain tissue, heart tissue, muscle tissue, skin, liver tissue, blood, skeletal muscle, lymphocytes and mucosa.

10. The method of obtaining biomarkers of aging comprising the steps of:

(a) comparing a gene expression profile of a young multicellular organism subject's organ, tissue or cells; a gene expression profile from a biologically and chronologically aged subject's organ, tissue or cell; and a gene expression profile from a chronologically aged but biologically younger subject's organ, tissue or cell, and
(b) identifying gene expression alterations that are observed when comparing the young subjects and the chronologically and biologically aged subjects and are not observed or reduced in magnitude when comparing the young subjects and chronologically aged but biologically younger subjects.

11. The method of claim 10 wherein one uses high density oligonucleotide arrays comprising at least 5-10% of the subject's genes to compare the subjects gene expression profile.

12. The method of claim 10 wherein the gene expression profile indicates a two-fold or greater increase or decrease in the expression of certain genes in chronologically aged subjects.

13. The method of claim 10 wherein the gene expression profile indicated a 3-fold or greater increase or decrease in the expression of certain genes in chronologically aged subjects.

14. The method of claim 10 wherein the gene expression profile indicates a 4-fold or greater increase or decrease in the expression of certain genes in chronologically aged subjects.

15. A method of measuring biological age of muscle tissue comprising the step of quantifying the mRNA abundance of a panel of biomarkers selected from the group consisting of markers W08057, AA114576, 11071777, 11106112, D29016, and M16465.

16. A method of measuring biological age of muscle tissue comprising the step of quantifying the mRNA abundance of a panel of biomarkers selected from the group consisting of markers described in Tables 1, 2, 15, and 16.

17. A method of measuring biological age of brain tissue comprising the step of quantifying the mRNA abundance of a panel of biomarkers selected from the group consisting of markers M17440, K01347, AA116604 and X16995.

18. The method of claim 10 wherein the subject is a mammal.

19. The method of claim 18 wherein the mammal is selected from the group consisting of humans, mice and rats.

20. A method of measuring biological age of brain tissue comprising the step of quantifying the mRNA abundance of a panel of biomarkers selected from the group consisting of markers described in Tables 5, 6, 9, and 10.

21. A method of measuring biological age of heart tissue comprising the step of quantifying the mRNA abundance of a panel of biomarkers selected from the group consisting of markers described in Tables 11, 12, 13 and 14.

22. A method for screening a compound for the ability to inhibit or retard the aging process in multicellular organisms tissue, organ or cell comprising the steps of:

(a) dividing test organisms into first and second mammalian samples;
(b) exposing the organisms of the first sample to a test compound;
(c) analyzing tissues, organs or cells of the first and second samples for the level of expression of a panel of sequences that have been predetermined to either increase or decrease in response to biological aging of the tissue;
(d) comparing the analysis of the first and second samples and identifying test compounds that modify the expression of the sequences of step (c) in the first sample such that the expression pattern is indicative of tissue, organ or cell that has an inhibited or retarded biological age.

23. A method as in claim 22, wherein the organism is a mammal.

24. The method of claim 23, wherein the mammal is selected from the group consisting of humans, rats and mice.

25. A method as in claim 23, wherein the tissue is selected from the group consisting of brain tissue, heart tissue, muscle tissue, blood, skeletal muscle, mucosa, skin, lymphocytes and liver tissue.

26. A method of detecting whether a test compound mimics the gene profile induced by caloric restriction, comprising the steps of:

(a) exposing a multicellular organism to the test compound, and
(b) measuring the expression level of a panel of sequences predetermined to either increase or decrease in response to biological aging in a tissue, organ or cell of the organism and comparing the measurement to a measurement obtained in the same tissue, organ or cell in calorically restricted subjects.

27. The method of claim 26 wherein the multicellular organism is a mammal.

28. The method of claim 27 wherein the mammal is selected from the group consisting of humans, rodents and mice.

Patent History
Publication number: 20030157526
Type: Application
Filed: Dec 2, 2002
Publication Date: Aug 21, 2003
Inventors: Richard H. Weindruch (Madison, WI), Tomas A. Prolla (Madison, WI), Cheol-Koo Lee (Madison, WI)
Application Number: 10307706
Classifications
Current U.S. Class: 435/6
International Classification: C12Q001/68;