WO2001012851A2 - Identification of genetic markers of biological age and metabolism - Google Patents

Abstract

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 subject and the chronologically aged but biologically younger subjects.

Description

IDENTIFICATION OF GENETIC MARKERS OF BIOLOGICAL AGE AND METABOLISM

CROSS-REFERENCE TO RELATED APPLICATION

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

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with United States government support awarded by the following agencies: NIH Grant No: AG11915. The United States has certain rights in this invention.

BACKGROUND OF THE INVENTION

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 rephcative 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, Phvsiol. 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 nutπtional intervention

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).

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, IL, 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, FL 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

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.

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. 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 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.

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. 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.

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.

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

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.

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

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

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.

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" (CC Thomas, Springfield, IL, 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.

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

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. 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 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. 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 descπbe 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.

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, AA114576, 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 c are not affected by caloric restriction by examination of the "CR reversal" lane of Tables 1 or 2.

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. 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 M 16465 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.

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.

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 caloπc restriction represent markers of biological age, as opposed to chronological age. 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 li- at Sαence 285(5432): 1390-1393, 1999 and Lee, et al., Nat Genet. 25(3).294-297, 2000. (Both Lee, et aj., supra. 1999 and Lee, et aj., supra. 2000 are incorporated by reference as if fully set forth herein.) For example, Lee, et aj., 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 mitochondπal sarcomeπc creatine kinase (Mi-CK) gene (3.8-fold) Sequences that display a greater than threefold 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

Lee, et aj., 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) microglobuiin 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.

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.

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.

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. 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

1. In General 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 C57BI6 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. 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.

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.

2. Experimental Protocols 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, MA) 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 isocaloπc (-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, WI)) 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, WI)), 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. 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 oiigo 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 μl of DEPC-treated water. In vitro transcription was performed using a T7 Megascript Kit (Ambion) with 1.5 μl 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-labeied 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 μg/μl 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 μg 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 fiuidics systems for wash and stain. The fiuidics system (Affymetrix GeneChip Fiuidics 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 μm using a Hewlett Packard Gene array scanner. Data collected from two scanned images were used for the analysis.

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 August 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: FC = SL - SL + 1 if SI₀≥SI₀ or -1 if Sl₀ < Sl_y the smallest of either Sl_y or Sl₀

Where Sl₀ is the average signal intensity from a gene-specific probe family from an old mouse and Sl_y is that from a young mouse. Alternatively, if the Q_facIor, a measure of the non-specific fluorescence intensity background, is larger the smallest of either Sl_y or Sl₀, the FC is calculated as:

FC = SL - SL Qfactor

The Q_factor 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.

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.

3. Results

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.

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.

Table 1. Aging-related increases in gene expression in gastrocnemius muscle of C57BL76 mice

OHF Δ Age Gene Class Function CR (fold) Reversal

AA106112 3.8 Mitochondnal Sarcomenc Creatine Energy Metabolism/ATP generation Kinase

AA071777 3.8 Synaptic Vesicle Protein 2 Growth Facfor/Neuπte extension 51 %

Y00094 3.6 Ypt 1/ras-related GTP Binding Transport Protein trafficking C

Protein W108S5 3.5 Methyl CpG Binding Protein DNA metabolism/gene silencing C

W08057 3.5 Heat Shock 27 kDa Protein Stress Response Chaoerone C

M 17790 3.5 Serum Amyloid A Isoform 4 Stress Pespoπse/Uπknovvn N

L06444 3.5 GDF-9 Growth Factor/Unknown 50%

AA114576 3.4 Heat Shock 71 kDa Protein Stress Response-Chaoerone C

W84988 3.3 Transcnption Regulatory Protein Transcnptional Factor/Unknown N

SWI3 X64587 3 2 U2AF RNA Metabolism/Splicing Factor C

D87902 3.2 ARF5 TranspoπVADP-nbosylation 87%

U19118 3.0 LRG-21 Transcnptional Factor/Macrophage activation 42%

AA068057 2.9 RabB Signal TransductiorVUnknown C

U05837 2.9 Beta-Hexosaminidase Catabolism/Lysosomal enzyme C

W85446 2.8 Protein Kinase C Inhibitor 1 Signal TransductiorVUnknown 74%

Homolog

AA060167 2.8 Pre-B Cell Enhancing Factor Growth Factor/Cytokine

Precursor

M37760 2.7 Seπne-2 Ultrahign Sulfur Protein Unknown 45%

AA096992 2.7 G25K GTP-Binding Protein Signal TransductiorVUnknown N

AA0082S5 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 Transcnptional Factor/Unknown N

X79828 2.6 NK 10 Transcnptional Factor/Unknown N

V0O719 2.6 Alpha- Amylase-1 Energy Metabolism Starch metabolism N

128177 2.6 GADD45 Stress Response/Cell cvcle cneckpoint 77%

W50941 2.5 Nucleotide Pyrophosphatase Unknown N

X53257 2.5 Neurotrophιn-3 Growth Factor/Reinπervation of muscle 50%

M74570 2.4 Aldehyde Dehydrogeπase II Stress Response Aldeτ-de detoxification 29%

D49473 2.4 Sox 17 Transcnptional Factor/Unknown 86%

AA117284 2.3 Zinc Finger Protein 43 (HTF6) Transcnptional 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 Transcnptional Factor/Neurogenesis N

AA059664 2.2 IGF Binding Protein Growth Factor/Cellular senescence C

V00714 2.2 Alpha Globm Structural/Hemoglobin component C

X99963 2.2 rhoB Stress Response<Unkι -wn 87%

AA014024 2.1 Dynactin Transport Neuronal transport 55%

X65627 2.1 TMZ2 Stress Pesponse-RNA 'n<-τ?.bolιsm 64%

X95503 2.1 GTP-Binding Protein (IRG-47) Signal TransductiorVUnknown 85%

V00727 2 1 FBJ-MuSV Provirus None C

X 12807 2 1 PP2.5 Unknown C

W08049 2.1 MAGP Structurat 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 Transcnptional Factor/Neuronat differentiation C

L07918 2 1 GDP-Dissociation Inhibitor Transport membrane dynamics N

X63190 2.1 PEA3 Transcnptional Factor/Response to muscle iniury 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). Table 2. Aging-related decreases in gene expression in gastrocnemius muscle of C57BL/6 mice^*

ORF Δ Age Gene Class/Function CR (fold) Reversal

D29016 -6 4 Squalene Synthase Biosvnlhesis/ChcfP cro] fattv acid 52% synthesis

AA106126 -4 9 Myosm Heavy Chain. Peππatal Structural ProteirVMuscle contraction C

D31898 -4 4 Protein Tyrosine Phosphatase. Signal TransductiorVUnknown 79% PTPBR7

U29762 -4 3 Albumin Gene D-Box Binding Transcnptional Factor/Albumin synthesis 85% Protein

AAO61310 -4 1 Mitochondnal LON Protease Energy Metabolisr- Mitochondnal biogenesis C

AA162443 -3 6 Protein Phosphatase PP2a Signal TransductiorVUnknown C

M89797 -3.5 Wnt-4 Signal TransductiorVUnknown 72%

M16465 -3 4 Calpactin I Light Cham Signal Transduction/Calcium effector C

X74134 -3 2 Ovalbumin Transcπption Factor I Transcnptional Factor/Unknown N

U08020 -3.2 Alpha 1 Type 1 Collagen Structural Protein Extracellular matnx N

X58251 -3 1 Pro-alpha-2(l) Collagen Structural Protein Extracellular matnx N

AA138226 -3 1 Clathπn Light Chain B Intracellular Transport Vesicle transport C

X85214 -3.0 Ox40 Signal Traπsduction T Cell activation 50%

D76440 -2 9 Necdin Growth Factor/neuronal growth 47% suppressor

AA107752 -2 9 EF-1 -Gamma ^proteιn Meta olic protein synthesis 63% 55037 -2.9 Alpha Enolase Energy Metabolisn ι Glvcolvsis 68%

X74134 -2 8 COUP-TFI Transcπption Factor/Unknown 28%

U06146 -2 8 Desintegnn-related Protein Unknown 28%

U39545 -2 8 BMPβb Growth Factor/Unknown C

X75014 -2.7 Phox2 Homeodomain Protein Transcnptional Factor/Neuroπal 65% differentiation and survival

U22031 -2.6 20S Proteasome Subunit Protein Metabolism rroleiπ turnover 44%

U70210 -2.5 TR2L Transcnptional Factor/Apoptosis modulator N

X76652 -2.5 3f8 Structural Protein/Neuronal adhesion N

W54288 -2.5 PKCSH Signal TransductiorVUnknown C

M81475 -2.5 Phosphoprotem Phosphatase Energy Metabolisn llvcogon metabolism C

U22394 -2.3 mSιn3 Transcnptional Factor/Inhibitor of 46% cell proliferation

M83336 -2.3 gp130 Signal TransductiorVUnknown 77%

L34611 -2.3 PTHR Signal TraπsductiorVCa homeostasis N

X52046 -2 3 Pro-Alpha1 (III) Collagen Structural Proteirv/Extracellular matnx 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 Metabolisr^{1 D}r tcιn turnover C

AA139680 -2.2 DNA Polymerase Alpha Pπmase DNA Metabolism/DNA replication N

AA034842 -2 1 ERV1 DNA Metabolism/Maintenance of MtDNA 46%

M21285 -2 1 Stearoyl-CoA Desaturase Biosynthesis/PU FA 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 Metabolic-

turnover C

M32240 -2 1 GAS3 Growth Factor/Apoptosis and growth arrest 55%

L00681 -2.1 Unp Ubiquitin Specific Protease Protein Meiaoonsr- ^ )tcιn turnover N

U34277 -2 0 PAF Acetylhydrolase Unknown N

U35741 -2.0 Rhodanese Protein Metaooif rπ Mitochondnal C protein foldinπ

W53731 -2 0 Signal Recognition Particle Intracellular Transport/Protein trafficking C

Receptor AA044497 -2 0 Zinc Finger Protein 32 Transcnptional Factor/Unknown 40%

L27842 -2 0 PMP35 Energy Metaboiisr- °πroxιsome assembly 60%

AA106406 -2 0 ATP Synthase A Chain Energy Metabonsπ- ATP synthesis N

AA041826 -20 IPP-2 Energy Metaool'sr- '.ilvcogeπ 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)

D76440

W57495

Table Λ Caloπc restπction-related decreases in gene expression

'The genes listed on this table were not influenced bv age Reversal of aging- associated changes are listed in Tables I and 2 DNA Repair and Stress Response classes are high gted in green Table 5. Aging-related increases in gene expression in neocortex of C57BU6 mice^*

ORF A Age SE Signal Intensity Gene Class CR (fold) Prevention Old Young

M88354 5 7 1 9 1 65 1 09 Vasooressm-neuropnysin II Osmotic stress 68% 17440 4 9 0 2 786 141 Complement C4 Immune inllammatory 52",.

AA120109 4 1 0 8 278 65 Inteneron-induced protein 6-16 homolog Immune/inflammatory 100°. 883S5 2 7 0 6 1 95 70 Oxviocin neurophysin Osmotic stress 23°,.

AAQ37945 2 5 0 2 254 73 Beta SNAP homolog Transport N

AA 162093 2 5 0 2 1 45 2 1 Pre-mRNA splicing factor PRP22 RNA metabolism N

AA137962 2 4 0 2 1 50 39 RAS-relatea protein RAB-14 Neurotransmmer release N

K01347 2 3 0 4 420 1 78 G at fibπiiary acidic protein (GFAP) Stress response 38°/.

AA027404 2 3 0 1 1 29 -43 Na K-transpomng ATPase beta-2 chain Ionic transport N

U60593 2 3 0 4 279 1 31 Cap43 Stress response N

AA137871 2 3 0 6 55 3-> Phospnatιoylιnosιtol-4-pnosphate 5-kιnase Signal transduction N

U61751 2 3 0 2 299 1 28 VAMP-1 Transport N

M21050 2 2 0 2 209 74 Lvsozyme C Immune inflammatory 54%

AA 1S3990 2 2 0 9 343 1 55 GTP AMP onospnotransferase Energy metabolism 100% mitocnoπαπal W29462 2 1 0 3 1 1 4 -49 Calpactiπ 1 light chain Structural N

L39123 2 1 0 2 1 887 768 Apolipopratein D (apoD) Stress response N

U 16297 2 0 0 5 1 24 47 Cyiocπrome B561 Transport N

M26251 2 0 0 3 484 260 Vimentm Stress response N

AA16391 1 2 0 0 2 1 30 38 Casein kinase 1 delta isoform Stress response N

AA022006 2 0 0 2 1 1 5 -48 CD40L receptor precursor Immune inflammatory N

AA124859 2 0 0 2 1 7 -54 ICAM-2 Immune/inflammatory N

Y00305 1 9 0 2 225 1 01 Potassium channel proteιn-1 Transport N

AA116604 1 9 0 1 515 272 Catnepsin Z Stress response 70%

M95200 1 9 0 3 1 68 92 Vascular endothelial growth factor Growth factor N

L 16894 1 9 0 4 1 23 -71 Cvclophiliπ C-AP Stress response 100%

L20315 1 9 0 2 1 20 66 MPS1 gene Immune inllammatory N

AA02B501 1 9 0 2 74 1 6 Cytochrome c oxidase subunit VIH-H Energy metabolism N

X86569 1 9 0 2 24 -31 LIM-kinase Unknown N

AA105716 1 9 0 2 1 07 1 4 Fructose- 1 6-bιsphosphatase homolog Energy metabolism 87%

W13646 1 8 0 1 1278 705 Tl 225 (uDiαumn) Stress response N

J03236 1 8 0 3 6B 1 362 JuπB Stress response 46%

X528B6 1 8 0 1 1050 555 Catneosin D Stress response 64%

AA028273 1 8 0 3 331 1 53 Protein pπosphatase inhibitor 2 (IPP 2) Unknown N

X 16995 1 8 0 1 757 375 N10 Steroid metabolism N

X 16995 1 8 0 1 624 363 Complement Clq B chain Immune/inflammatory 100%

X66295 1 8 0 1 823 467 Complement C1q C-chain Immune inllammatory 75%

U22445 1 8 0 5 201 1 60 Senne/threonine kinase (Akt2) Energy metabolism 100%

U 17297 1 8 0 2 6 -43 Integral memorane phosonoprotein 7 2b Unknown N

AA0S9700 1 8 0 2 1467 797 MHC class I B(2)-mιcroglobulιn Immune inllammatory 64%

L29503 1 8 0 1 1 92 1 03 Myelin/oligooendrocyte 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-catenin Stress response N

AA061086 1 8 0 2 1 79 89 Hsp40 Stress response 52%

W50891 1 8 0 3 4 1 -3 Creatine kinase Energy metabolism N

W67046 1 8 0 2 105 7 1 Exodus 2 Immune inllammatory N

W13875 1 8 0 2 21 6 1 25 Myosm regulatory light chain 2 A Unknown N

X67083 1 8 0 3 1 21 47 Chop-10 GADD153 Stress response 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 1 13 23 ONAJ protein homolog 2 Stress response N AA122619 1.7 0.3 1 4 -43 Set protein (HLA-DR assoαateα protein II) Unknown N

M73741 1 7 0.2 1313 730 Alpna-B2-cfystaMιn gene Stress response 67°,

X70393 1.7 0 4 146 65 Inter-alpha-inhibrtor H3 chain Immune inltammatory 561

AA124698 1.7 0 7 1 00 42 etnaK 1 (discs large- 1 Unknown N

W14434 1.7 0.2 401 240 Fruαose-busphosohaie a joiase Energy metabolism N

W89579 1 7 0.2 83 -3 RAS-relaied protein RAB-4 Signal transαuction N

AA089333 1.7 0.1 336 221 Cathepsin S precursor Stress response 56',.

U19521 1.7 0.2 70 3 1 Veside transport protein (munc-lBe) Transport N

AA107137 1 7 0.3 204 1 1 8 Casein kinase I. gamma Unknown N

AA106166 1.7 0.2 2312 1372 Elongation factor 2 (EF-2) homolog RNA metabolism N

M3181 1 1.7 0 1 748 457 Clathnn light chain B Transport 100%

AA 140487 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 transαuction N

AA114781 1.7 0.2 52 26 Undylate kinase DNA metabolism N

X58851 1.6 0.1 1 1 28 694 Complement C1Q alpha-chain Immune inttammatdry ιoc«.

AA048650 1.6 0.2 1 69 1 00 Estradiol 17 β-dehyorogenase 3 homolog Stemid metabolism N

W46723 1.6 0.2 83 46 Creatine kinase. B chain homolog Energy metabolism N

U16162 1.6 0.7 1 1 2 82 Protyl 4-hydroxylase alpha(l)-subunιt Structural N

X68273 1.6 0.2 1 05 73 Macrosialin Immune inflammatory N

W48952 1 6 0.7 87 38 rj-adrenergic receptor kinase 1 Signal transαuction N

AA063858 1.6 0.2 1 35 80 RHO-related GTP-btnding protein RHOG Signal transduction 100%

Ml 5525 1.6 0.1 22 -58 Laminm B1 Neuronal outgrowth N

AA068780 1.6 0.1 275 1 87 Phosphoseπne aminotransterase homolog Unknown 76%

U27462 1.6 0.3 1 33 79 BS4 peptide Unknown N

AA106077 1.6 0.1 1 1 6 64 Glutathione perc αdase Stress response 76%

AA119959 1.6 0.2 1 94 128 Protein transport protein SEC23 Transport N

AA061170 1.6 0.2 39 -1 8 NEOD-4 protein Unknown N

X16151 1.6 0.2 93 61 T-h/mphocyte activation 1 protein (ETa-1 ) Immune inflammatory N

W29462 1.6 0.3 1 1 4 -49 Calpactin I light chain <p11 ) Unknown N

AA097579 1.6 0.1 24 -20 Zinc linger protein 91 homolog Unknown 52%

X64070 1.6 0.3 252 1 63 46kDa mannose 6-pnospnate receptor Lysosomal N

W48519 1.6 0.2 98 1 00 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 1 84 GTP-btnding protein GTR1 Signal transduction N

W51181 1.6 0.3 42 25 DNA-directed RNA potvmerase II RNA metabolism 75%

AA036390 1.6 0.2 1 46 83 DNA-binding protein inhibitor ID-1 Transcnptional factor 75%

L08115 1.5 0.2 309 236 Human C09 antigen homolog Structural 100%

U37353 1.5 0.2 1 91 1 21 Protein phosphatase 2A B'alpha3 Signal transduction N regulatory suPunit

L 10244 1.5 0.2 31 6 206 Spertnidine spermine 1 -acetyltransterase Polyamme metabolism N

J05154 1.5 0.2 72 6 Cholesterol acyltransterase (LCAT) Steroid metabolism N

D43643 1.5 0.2 62 36 YL-1 Unknown N

M3 141 1.5 0.1 39 5 COX-1 Immune/inllammatory 100%

128177 1.5 0.1 35 -9 GADD 5 Stress response N

X85992 1.5 0.1 5 1 ι o Semaphonn C Neuronal remodelling N

AA09B307 1.5 0.2 85 47 Tubulm beta 5 Microtubule component N

'The values presented for Signal Intensity are the averages of three mice per age group ana are expressed as data for old/young mice. The prevention Dy CR is shown as oeing none (N) or the calculated percentage etlect. The SE was calculated for the nine pairwise compansons and was obtained by dividing tne standard deviation by the square root of 3. The method from which signal intensity is used to estimate told changes is described in the

Memoes section of tne manuscnpt. Table 6. Agiπg-reiated decreases in gene expression in neocortex of C57BU6 mice^*

ORF Λ Age SE Signal Intensity Gene C as CR (fold) Prevention Old Younα

X7 134 -3.0 1 1 157 387 Ovalbumin uostream promoter Transcnptional tacto' N

L2 430 -2.7 0.6 56 161 Osieocaiciπ precursor Unknown N

AA124352 -2.5 0.5 19 274 Neuromeαin B precursor homolog Neurotransmssion 54",.

D31B98 -2.2 0.5 116 253 Protein tyrosine phosonatase. PTPBR7 Unknown r.

W29468 -2.2 0.3 133 284 Myosm light chain 2 mRNA Unknown N

AA065993 -2.2 0.3 16 115 GTP-bind g nuclear protein RAN homolog Signal transduction N

U3S323 -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 anσrogen-regulated protein Steroid metabolism N

U38196 -2.1 0.6 64 151 Palmytoviated protein p55 Signal transαuction 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 04 -21 125 Transforming protein RFP Growth factor N

U29762 -2.0 0.2 396 744 Albumin gene D-Box binding protein Orcadian rhythm N

AA13871 1 -2.0 0.5 222 321 Protein kinase C inhibitor protein Unknown N

W13586 -2.0 0.3 135 548 Atnal tetal isotorm yosm alkali light chain Structural 49%

X67S12 -2.0 0.3 41 120 ret proto-oncogene Unknown N

M37312 -2.0 0.2 12 85 REX-1 Steroid metabolism N

-2.0 0 4 418 673 NEDD8 Protein metabolism N

X 13Ξ26 -2.0 0.2 66 176 Hox-1 4 gene Growth factor N

X66405 -2.0 0.5 186 330 Collagen aipna 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-depeπdent ATF-4 Transcnptional factor ιoo^«/.

L33 16 -1.9 0.3 184 291 Clone p85 secreted protein Unknown 100%

X7039B -1.9 0.9 186 325 PTZ-17 Growth factor N

M84 12 -1.8 0.1 46 128 Antigen (Ly-9) Immune inflammatory 47%

AA067g27 -1.8 0.2 63 132 DNA-PK-catalylic subunit DNA metabolism N

Y09585 -1.8 0 4 143 212 Serotonin 4L receptor Neuratransmission N

X95255 -1.8 0 1 6 72 GIΪ3 protein Growth factor N

U37459 -1.6 o.i 37 87 Glial-denved neurotroohic lactor (GDNF) Growth lactor N

M99377 -1 8 0.3 121 270 Alpha-2 aorenergic receptor Neurotransmission N

D83595 -1.8 0.5 916 1457 Proteasome Z subunit Protein metabolism N

U52222 -1.8 0.2 61 160 Mel- la meiatonm receptor Neuropeptide N

M13710 -1 7 0.3 120 219 Interteron aιpna-7 gene Immune/inllammatory N

D76446 •1.7 0.2 103 199 TAK1 Stress response N

U64445 •1 7 0.2 12 56 Ubiquitin lusion-αegradation protein (ufdll) Protein metabolism 100%

U39545 ■1.7 0.3 144 235 Bone morohogenetic protein 8B (BmpBb) 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 recepior Neuropeptide N

W08326 -1.7 0.2 173 265 51 PK(L) homolog Unknown N

AA00O466 •1 7 0.2 1 T3 195 P55CDC DNA metabolism 100%

U6G203 -1.7 0.2 111 181 FHF-3 Growth factor N

AA051632 •1.7 0.2 112 167 MEK5 Signal transduction 61%

AA051 147 -1.7 0.2 114 264 Chemotaxis proiein cheY homolog Unknown N

X84692 -1.7 0.2 24 91 Spnr mRNA lor 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 Antrthrompin-lll precursor (AT1II) Immune inflammatory N

U 13705 -1 7 02 324 494 Piama giutathione peroxioase (MUSPGPX) Stress response 44%

X75384 -1 7 0 2 91 158 SAX-i Growth factor

Z32767 -1 7 0 3 117 205 RAD52 DNA metabolism 76

AA 107752 -1 6 0 6 225 336 Elongation factor 1 -gamma Protein metabolism N

M 12836 •1 6 06 56 1 16 T-eell receptor gamma cnain gene C-region immune/inflammatory N

AA060704 -1 6 0 2 975 1407 Giutathione S-transterase MU 5 Unknown N

AA 118294 •1 6 0 1 99 161 Vitronectin homolog Unknown N

AA123026 •1 6 0 1 72 166 PancreaDtis-assoαated protein 3 homolog Unknown 100%

AA065652 -1 6 0 1 39 99 Ubiαurtm carooxyl-terminal nyorolase Protein metabolism N

W46104 -1 6 0 2 19 58 DNA-repair protein XP-E DNA metabolism N

MS8694 -1 6 0 2 67 109 Thioetner S-methyltransferase Unknown 57%

AA117004 -1 6 0 1 6 61 Heat snock cognate 71 KD proiein 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 Cerulcplasmin Transport N

X69019 -1 6 0 2 36 71 Hox 3.5 gene, complete cos Growth lactor N

M28666 -1 6 0 2 317 496 Porphobilinogen deamtnase Biosynthesis 44%

W36B759 -1 6 0 1 49 112 CMP-N-acetyιnβurammate-beta-1.4- Sialylranslerase N galactoside alpha-2.3- siaryrtransterasβ

W11666 -1 6 02 105 207 apolipoprotem H ϋpid metabolism N

W09925 -1 6 0 1 26 102 Endothelial actin-binding protein Growth factor 74%

AA 116282 -1 6 0 1 140 355 TNF alpha precursor Immune/inflammatory 56%

D37791 -1 6 0 0 556 895 Beta-1 4.-gala osvttransterase Unknown N

W 12658 -1 6 0 2 143 216 FKBP-rapamyαn associated protein (FRAP) Unknown N

Z468454 -1 6 0 2 -16 39 Preproglucagon Energy metabolism N

AA 103045 -1 5 0 1 57 106 Cleavage stimulation taαor. 64 Kd subunit RNA metabolism N

AA108891 -1 5 0 2 4 62 Putative ATP-dependent RNA helicase RNA metabolism 55%

A A153522 -1 5 0 3 80 159 Senne 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%

AA09858B -1 5 0 1 84 137 Zinc finger protein HRX (ALL-1 ) Unknown 57%

W 15873 -1 5 0 2 161 258 ιcιex-1 mRNA Unknown 61%

AA170748 -1 5 0 1 -14 48 40S Ribosomal protein S4 Unknown N

W80326 -1 5 0 1 -1 1 86 Sex-determinmg protein FEM-i Unknown N

AA140159 -1 5 0 2 65 134 Thiol-specific antioxidant protein homolog Stress response N

D16492 •1 5 0 1 19 5B RaRF Unknown 56%

D85B45 -1 5 0 2 48 88 Atonal homolog-3 Growth taαor N

L06451 -1 5 0 1 -55 87 Agouti switch protein mRNA Unknown 100%

AA 166500 ^■1 5 0 2 51 141 Transcnptional regulatory protein RPD3 Unknown N

L28035 -1 5 0 ' 377 57B Protein kinase C-gamma mRNA Unknown 100%

U52197 -1 4 0 1 296 439 Poly(A) poiymerase V RNA metabolism N

D29763 -1 4 0 1 799 1 130 Seizure-related, product 6 type 3 precursor Unknown response 50%

U22015 -1 4 0 1 89 130 Retmoid X receptor interacting protein Steroid metabolism 100%

"The values presenied 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 tor the nine pairwise ccmpansons and was obtaⁱned bv dⁱviding the standard deviation by the square root of 3 The method from which signal intensity is used lo estimate fold changes is descnoed in the Methods section of tne manuscript Table 7. Caloric restriction-related increases in gene expression in neocortex of C57BL 6 mice"

ORF CR SE Signal intensity Gene Class

Increase

CR Control

J04971 0 7 87 Siow/carαiac troponin C (cTnC) Unit no wn

D 13903 3 1 1.2 150 49 MPTPdefta (type A) Growth tacioπ

M36660 3 1 0 2 2i NAD(P)H menadione oxiαoreduc ase Stress response

MS5617 3 l 0 6 27 -18 MMCP-4 unknown

W65178 3 0 0.3 39 -35 BMP-1 Growth tactor

AA118682 3.0 0.6 62 -12 Tπtnorax homolog 2 Transcnptional taαor

AA014B16 3.0 0 7 257 38 Prolactin homolog Unknown

U39904 2.9 100 -169 Citron, putative mo rac effector Signal transαuction

AA061310 2.9 0 7 87 29 Mitochondnal LON protease Energy metabolism

U0209B 2.8 0.5 82 36 Pur-alpha DNA metabolism

M29395 2.8 0.3 36 -20 Orptιdιne-5-monpphosphate 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 tactor

U05247 2 4 0.2 686 250 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 D(2) Dopamme receptor Neurolransmission

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 lactor

M74227 2.2 0.3 162 48 Cyclophilin C (cyp C) Immune/inflammatory

W12794 2.2 0.3 48 -59 Transforming protein MAF homolog Transcnptional tactor

X62940 2.2 0.1 2199 931 TSC-22 Unknown

L.06 51 2.2 0.1 136 -55 Agouti switch protein Unknown

AA052547 2.2 0.1 74 -2 Fatty acid-binding protein, epidermal (E-FABP1 Transport

W17956 2.2 0 4 108 -2 Zinc finger protein 42 homolog Unknown

X95226 2.2 0.4 53 Dvsirobrevin Structural

A A152808 2.2 0.2 24 Proteine kinase PASK Signal transduction

AA014512 2.1 0.5 32 .3 Unknown Unknown

W7481 1 2 l 04 -16 Aoolipoprotein c-ll precursor (APO-CII) Transport

U69270 2 1 0.7 323 210 LIM domain binding protein 1 (Ldb1 ) Growth taαor

W54720 2 1 0.2 100 19 Ca"'-transpoπιng ATPase (brain isotorm 1) Unknown 13460 2 1 0.1 313 151 Annexin VI Signal transduction

U61362 2.1 0.3 57 ^■35 Groucho-related gene 1 protein (Grg1) Unknown

W09323 2.1 0.3 91 Enoothelιn-2 precursor (ET-2) Unknown

W70403 2.1 0.2 17 mafF Unknown

AA071685 2.0 04 93 Elongation factor 1 -alpha chain homolog Protein metabolism

W14673 2.0 0.4 133 8 BAT3 Unknown

W53409 2.0 0.3 33 -28 Protem kinase C homolog. alpha type Signal transαuction

U 19880 2.0 0.1 ^■ 2B -6 D4 dopamme receptor gene Neurolransmission

M75875 2.0 0 4 280 1 19 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

J0O475 2.0 0.3 74 •34 Ig alpha chain region C Immune/inllammatory

M57960 2.0 0.2 21 -18 Carboxylesterase Unknown

X57B00 2.0 0 1 560 274 PCNA DNA metabolism

U36277 2.0 0.3 123 70 l-kappa B alpha chain Stress response AA015291 Probable E1-E2 ATPase Unknown

WB2109 -; Kinesm light cnain (KLC Transpor-

M83380 1 9 RelB Immune/inllammatory

U13174 1 9 Basoiateral Na-K-2CI coiranspoπer Transport

M33960 1 9 Plasmmogen activator inhioitor (PAI-1) Growth taαor

X72310 1 9 DRTF-poιypeptιde-1 (DP 1 ) Transcnptional taαor

AA059886 1 9 Retinal degeneration C protein Apoptotic taαor

U02278 1 9 Hox-B3 Growin taαor

AA072B42 1 9 Na - and Cl -αeoendeπt transporter NTT73 Transpor

M98339 1 9 GATA-4 Transcnptional tactor

W13427 1 9 Platelet taαor 4 precursor Unknown

U44955 1 9 Alpha3 connexin gene Transport

L24191 1 9 0 1 Intrinsic taαor Transport

W08109 Protein kinase C inhibitor 1 (PKCI-1) homolog Unknown

W36570 DNA mismatch repair protein MSH2 DNA metaoolism

Z34524 Protein kinase D Signal transduction

AA 105081 Initiation taαor IF-2. mrtocnondnal Protein metabolism

U 18797 MHC class I antigen H-2M3 Unknown

M1 1988 Hox-A6 Growth taαor

U17961 p62 ras-GAP associated pnosDhoprotein Signal transduction 85103 0 1 IGF binding protein 4 precursor homolog Energy metaoolism

X07997 MHC class I T-cell antigen Lyl3 1 Immune inflammatory

W46723 Creatine kinase B chain nomolog Unknown

W48464 Protem-tyrosine phospnatase MEG2 homolog Unknown

L06322 1 0 1 Delta opoid receptor Neurotransmission

W49178 1 0 1 Tubulm beta-1 chain homolog Struαurai

W48477 1 Thyrotroph embryonic faαor nomolog Unknown

W64225 1 G21 Unknown

L28167 1 Zinc finger protein Unknown

W97199 1 Negative regulator of transcπption subunit 2 Transcnptional taαor

X01971 1 Inteneron alpha 5 (Mu IFN-alpha 5) Immune inllammatory

AA061266 1 Oxysterol-binding protein nomolog Transport

U21855 1 CAF1 Transcnptional taαor

W8707B 1 0 1 Unknown Unknown

W34687 1 Aαtn alpha skeletal muscle nomolog Structural

K01238 1 Interteron alpha 2 Immune/inflammatory

U 15635 1 IFN-gamma induced (Mg11 ) Unknown

L1396B 1 0 1 UCR-motif DNA-bindmg protein Transcnptional faαor

M86567 1 GABA-A receptor alpha-2 subunit Neurotransmission

M87B61 1 Granule membrane protein 140 Structural

W55350 1 Phospnatidylmositol transfer protein 13 isotorm Unknown

L43567 1 B-cell receptor gene Immune/inflammatory

AA153196 1 Ubiquitin-aαrvating enzyme E1 homolog Protein mβtapolism

M28312 1 Metalloprotease inhibitor TIMP1 Immune inllammatory

^■The values presenied tor Signal Intensity are the averages of three mice per age group and are expressed as data tor old CR/old control mice The SE was calculated tor the nine pairwise comoaπsons and was obtained by dividing the standard deviation bv tne square root of 3 The method from which signal intensity is used to estimate told cnanges is descnbed m the Methods section of the manuscπpi Table 8. Caloric restriction-related decreases in gene expression in neocortex of C57BL76 mice^'

ORF CR SE Signal Intensity Class Decrease CR Control

1.0 -195 73 Tyro 10 Signal transquαion

X76505 -7.2 09 164 IL-1 (CTLA-8) Immune/intiammatory

U430BB -6.3 1.1 -1

Unknown

W501B6 -5.6 2.1 -38 129 Heavy chain homolog

Signal transαuαion

Y07711 -3.5 0.5 28 151 Zyxm 1 0.8 45 200 PLZF Transcnptional taαor

Z47205 -3.

0.7 -93 26 Cortieosteroid-binding globulin precursor Transport

AA0OO203 -2.B

W83658 -2.6 0.5 51 197 Guanine nudeotide-bmdmg protein Signal transαuαion

G{IVG(SVG(0) nomolog

-2.6 0.2 8 67 Ig kappa chain recombination and transcnpuon DNA metabolism

L46815 enhancer

AA153484 -2.4 0.5 208 456 SERCA2 Ion transport channel protein P64 homolog Unknown 51466 -2.4 0.4 12 147 Chlorine .4 0.4 39 132 XPC DNA Metabolism

U27398 -2

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 04 49 110 Midkine precursor homolog Stress response

L01991 -2.1 0.3 48 132 PANG Unknown

L04678 -2.1 0.2 -64 138 Integnn beta 4 subunit Struαura!

W6462B -2.1 0.4 62 197 Guanine nucleotide-binding protein Signal transduαion G(IVG(S)/G(0) gamma-7 subunit

X54098 -2.0 0.3 55 136 lamm B2 Structural

AA023458 -2.0 0.3 20 107 Heat shock 27 KD protein nomolog Stress response

D633B0 -2.0 0.2 -19 32 Alpha-1.3-fucosyπransferase Protein metabolism

U 15548 -2.0 0.3 -30 42 Beta 2 thyroid hormone receptor Energy metabolism

AA123385 -2.0 0.2 57 117 Phosohoiylase B kinase gamma catalytic chain Energy metabolism

X57349 -2.0 0.4 -10 49 Transfemn receptor Transport

D00659 -2.0 0.1 1 35 Aromatase P450 Biosynthesis

AA028875 -2.0 0.2 -32 54 Glyciπe-πch 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 Orcadian regulation

AA118758 -1.9 0.2 103 206 Multriunαional aminoacyl-lRNA synthetase Protein synthesis

W75353 -1.9 0.3 90 162 Apolipoprotein C-IV Transport

W55410 -1.9 0.2 30 11 1 Tubulin gamma chain homolog Unknown

L20343 -1.9 0.2 22 102 L-type calcium channel beta 2a suPunit isotorm Transport

W91095 -1.9 0.5 44 93 Valyl-tRNA synthetase Protein metabolism

X81593 -1.9 0.1 53 119 Winged-helix domain Transcnptional taαor

M38248 -1.9 0.2 -6 25 BALB8N Unknown

J04694 -1.8 0.3 48 134 Alpha- 1 type IV collagen Structural

147650 -1.8 0.3 50 85 ΞTAT6 R Immune /inflammatory

AA023595 -1.8 0.1 38 133 Frizzled protein precursor Signal transduαion

AA015168 -1.8 0.2 42 97 Interteron-gamma receptor beta chain homolog Immune /inflammatory

AAO 13951 -1.8 0.1 32 38 Creatine transporter homolog Energy metabolism

W78443 -1.B 0.2 17 106 MKP-X Signal transduαion

D31842 -1.8 0.2 66 126 PTP36 Structural

W50138 -1.8 0.2 1 162 Putative serme threonine-proiein kinase B0464.5 Unknown

135307 -1.8 0.2 33 104 c-Krox Transcnptional taαor

AA073154 -1.8 0.3 31 68 Alpha-catenin homolog Structural

W 12720 -1.8 0.3 149 251 RAP-2B homolog Signal transduction

AA170169 -1.8 0.2 -17 37 Elongation taαor 1 -gamma homolog Protein metabolism

W4B951 -1.8 0.3 8 30 Voltage-dependent amon-seleαive channel Unknown protein 2 homolog

M35732 0.3 -13 Seminal vesicle secretory protein IV Unknown RNA metaoolism

Pre-MRNA splicing taαor PRP6

AA145515 ^■1 B DNA metaDOiism

Cell division protein kinase 4

C -

W13162 -1 E DNA metabolism

Histone HI

J03482 -1 8 DNA metabolism

Topoisomerase E III homolog

W82793 • 1 8 0 1 Unknown

P L01

Z31360 • 1 8 Transport

Rabkιnesιn-6

Y09632 -1 8 0 ' Protein metaDOiism

60S πposomai protein L10

AA066621 ■1 8 Protein metabolism

Ubiouitm mioiesierase tamⁱly

U67874 -1 8 RNA metabolism

SKP1

AA109714 ■1 8 Protein metabolism

Tnreonyl-tRNA syπmetase nomolog

AA007957 ■1 8 Protein metabolism

Isoleucyl-tRNA syπtnetase

AA162633 ■1 8

Phosphogtyceraie kinase (pgk-2) Energy metabolism

M 17299 -1 8 Protein metabolism

Elongation taαor 2 (EF-2)

AA050102 -1 7 Unknown

Tubulin βeta-2 chain class-ll homolog

W54637 -1 7 Neurotransmission

Glutamate receptor channel subunit i^βta 1

D 10028 -1 - Immune /iπllammatory

Alpha leukpcyte interteron

M2B587 -1 7 Energy metabolism

Insulin receptor suβstraιe-3

AA023506 -1 7 Protein metabolism

COPII 70629 -1 7 Unknown

PML isotorm 1 (Pml)

U33626 -1 7 Protein metabolism

EF-1 -delta

AA144746 -1 7 Signal transαuction

Calmodulin (Cam III) 19380 -1 7 1406 2303

Biosynthesis

Cholme kinase Rl homolog

AA144136 •1 7

EF-1 -alpha2 homolog Protein metabolism

AA165847 -1 7

Unknown

ATP crtraie-iyase

W33415 -1 7

Vasoconstπctive peptide

Endothβlιn-1

U35233 -1 6

ATP syntnase A chain nomolog Energy metabolism

W57384 -1 9

Cytochrome P-450IMA Stress response

X60 52 -1 6

Vascular endothelial growth factor Unknown

AA022127 -1 6

SenneΛhreonine-protein kinase PAK Unknown

AA168841 -1 6

Apolipoprotein B-100 precursor Stress response

AA120586 -1 6 Protein metabolism

EIF-4A nomolog

AA104561 -1 6

Tropnoblast-specific protein Growth taαor

X17071 -1 6 0 1

Galaαose-1 -phosphate uπdyl transt^βrase Biosynthesis

M96265 -1 6 0 1

Translational initiation tactor 2 alpha Protein metabolism

AA145160 -1 6 m4 muscannic acerylcholinβ receptor Neurotransmission

X63473 -1 6 0 1

5-lιooxygenase aαrvating protein (FLAP) Immune /inflammatory

AA002750 ^■1 5

Protein kinase C inhibitor 1 Signal transduction

W6469B •1 5

Growth factors

NeuroDS

U63841 -1 5 0 1

Potassium cnannel subunit (m-eag) Transport

U04294 ■1 5

Crypioin-related (CRS4C) Immune /inflammatory

M33227 •1 5

P45 NF-E2 related tactor 2 (Nrt2) Transcnptional tactor

U20532 •1 5 0 1

DNA direαed RNA poiymerase polypeptide G DNA metabolism

AA140026 -1 5 0 1

ATP syntnase B chain nomolog Energy metabolism

W09025 -1 5 0 1

Leydig cell tumor 10kd protein homolog Unknown

W29163 -1 5 0 1

Kinesm heavy chain Transport

AA155191 -1 5

DNA metabolism

Rep-3

M80360 -1 5 0 1

PEP caiboxykmase - mitochondnal Energy metabolism

AA044561 -1 4

Unknown Unknown

AA096843 -1 4

Signal transduction

X57277 -1 4 1298 Rad

DNA metabolism

BUB3

W82998 -1 4

'The values presented lor S_ignal Intensity are the averages of three mice per age group and are expressed as data tor old CR old control m_ice The SE was calculated tor the n_ine pairwise compansons and was obtained by dividⁱng the standard deviation by the square root of 3 The method from wh_ich s_ignal intensity is used to estimate fold changes is descnbed in the Methods section of the manuscnpL Table 9. Aging-related increases in gene expression in the cerebellum of C57BL76 mice^*

ORF Fold Change SE Signal Intensity Gene Class CR Old Young Prevention

AA120109 9.3 3 4 254 29 Interferon-mduced protein 6-16 precursor Immune/inflammatory N

M21050 6 4 0 9 291 14 Lysozyme P (Lzp-s) Immune 68

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 T ymidyiate synthase DNA metabolism 87 16894 4 7 1.0 192 5 Cycloohilin 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-cham Immune/inflammatory 56 65899 4.1 1.8 152 58 Guanine nucleotide-bmdmg protein Signal transduction 80

U00677 4.1 2.2 16 -10 Syntrophιn-ι Neurotransmission 100

X68273 3.9 1.8 108 -37 acrosiann Immune/inflammatory N

U19854 3.9 0.5 35 -63 Ubiquitmatiπg 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 inllammatory 56

K01347 3 8 07 337 109 Glial fibπllarv acidic protein (GFAP) Stress 61

Ml 7440 3.7 0 3 445 1 16 Sex-limited protein (SlpA) Immune/inflammatory N

X91144 3.6 1 3 38 P-selectm glycoprotein ligand 1 Immune/inflammatory 100

U43084 3.5 0.8 54 18 IFIT-2 Glucocorticoid-atlenuateα resoonse Immune/inflammatory N

AA089333 3.4 0.2 208 61 Cathepsin S precursor Stress 71

X83733 3 4 0 3 71 SAP62-AMH RNA metaoolism 100

W45750 3.3 1.3 197 257 Guanine nucleotide-binding protein G(T) Signal transduαion 100

M22531 3.3 02 431 146 Clq B-chaiπ 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

U061 19 2.9 0.3 27 4 Cathepsin H prepropeptide (ctsH) Stress response 55

AA106347 2.9 0.3 243 57 Aπgiotensinogen precursor Osmo regulation 80

W98998 2.9 0 7 182 79 Neurogenic locus notch homolog protein 1 Immune/inflammatory 100

AA059700 28 0.3 2013 687 MHC class 1 B(2)-mιcroglobulιn Immune/inflammatory 45

U73037 2.8 0 8 69 41 Interferon regulatory tactor 7 (mιrf7) Immune/inflammatory 50

Y00964 2.8 0 3 780 316 beta-hexosaminidase (Hexb) Unknown 47

X55315 2.8 0.6 63 15 Fetus cereDral cortex for 3UTR Transcπption faαor 100

U37465 2.B 0 1 15 -7 Protein tyrosine phosphatase phi (PTPphi) Unknown 63

L07803 2.7 1.2 24 -15 trombospondin 2 Structural N

U191 19 2.7 0.3 52 -5 G-protein-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

X 16705 2.6 0 4 93 -4 Lammin B1 Structural 84 57539 2.6 0 3 28 6 Oocyte zinc finger protein XLCOF8 Unknown N

X52308 2.6 0 4 32 9 Thrombin Fibπnogen activation 91

U70859 2.6 0 7 109 46 Canonic ammo acid transporter (CAT3) AA transport 49

U41497 2.6 1 1 160 40 Very-long chain acyl-CoA dehydrogenase ϋpid 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 11 1 G protein alpha subunit (G A-15) Unknown N

K027B5 2.5 0.5 15 -6 r-fos Stress response N 12289 2.5 0.5 39 25 Peπnatal skeletal myosm heavy chain Slructural 100

X58849 2 4 04 59 13 Munne Hox-4.7 Developmental 100

AA063858 2 4 0.2 B9 32 Rho-related GTP-biπding protein RHOG Signal transduction 74

D 10632 2.4 0.2 33 ^■27 Zinc finger protein Transcπption lactor N

U33005 2.3 04 35 -8 tbd Unknown N

W85160 2.3 0 7 70 41 40S πbosomal protein S4. X isotorm Unknown 100

U57331 2.3 1 0 42 15 Transcription factor Tbx6 (tbxβ) Developmental 92 U44731 2 3 02 71 20 Putative punπe nucieotide binding protein .une inflammatory N

W87253 2 3 06 58 16 Iniegnn beta-5 subunit precursor Cell adhesion 100

U53142 2 3 02 223 101 Endothelial constitutive nitπc oxioe synthase Neurotransmission N

AA087715 2 3 0 1 85 -61 GTPase-aαrvating protein SPA-1 Unknown N

D49429 2 3 0 3 554 251 Rad2l homolog DNA metaoolism 73

AA155318 2 3 0 4 291 129 HNRP1 RNA metabolism N

AA032593 2 3 0 1 99 17 Transducin beta chain 2 Signal transduαion 83

X03690 2 3 02 45 -13 Ig mu chain Immune/inflammatory 93

M26417 2 3 0 5 54 28 T cell receptor beta chain Immune/inflammatory 100

X86374 2 2 06 73 38 TAG7 Immune/inflammatory 38

W90894 22 0 3 27 -1 1 Cell division protein kinase 4 DNA metabolism 100

M84005 22 0 7 83 51 Olfaαory receptor 15 Odor receptor 23

X55573 2 2 0 5 55 19 Braiπ-deπved πeurotrophic faαor Growth taαor N

W30129 2 2 0 3 90 16 Phosphatidylinositol glycan hmolog Struαural 100

AA163771 2 2 0 3 153 67 EIF-2B epsiloπ subunit Protein metabolism N

X72910 2 1 0 4 96 44 HSA-C Unknown N

AA 116604 2 1 0 2 303 181 Cathepsin Z Stress response 64 16462 2 1 0 4 51 4 BCL2-related protein A1 Apoptosis 58 13732 2 1 04 53 29 Natl resistance-asstd macrpphage protein 1 Immune inflammatory 85

D37791 2 1 0 1 934 424 Beta-1 4-gaιacιosyltransιerase 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

X 13538 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 20 0 1 B6 79 Unknown Unknown 100

X63535 2 0 0 1 55 21 Tvrosine-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 transduαion 100 13136 2 0 0 4 266 87 Angiotenisinogen Os oregulation 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

AA1 18121 1 9 1 0 51 37 Isoleucyl-tRNA synthetase Protein metabolism N

M27034 1 9 02 258 163 MHC class 1 D-region Immune/inflammatory N

U35249 1 9 0 1 68 36 CDK-activating kinase assemply factor DNA metabolism 61

J03776 1 9 04 37 22 Down regulatory protein (rpt-1r) of IL-2 receptor Immune/inflammatory N

U28728 1 9 0 3 221 112 Efs Signal transαuction 66

AA124192 1 9 0 2 411 244 Unknown Unknown 44

W63809 1 8 0 4 136 80 Unknown Unknown 73

X 16834 1 8 0 2 455 182 Galectιn-3 Immune/inflammatory N

X16995 1 8 0 2 351 221 10 nuclear hormonal receptor homolog Unknown 100

J02870 1 8 0 2 848 380 40S πbosomal protein SA Protein metabolism 100

L21768 1 8 02 153 76 EGF15 Growth faαor 68

AA117284 1 B 0 1 217 123 Zinc finger protein homolog Unknown N

'The values presented for Signal Intensity are tne averages of three mice per age group and are expressed as data for old/young mice The prevention bv CR is snown as being none (N) or the calculateα percentage effeα The SE was calculted for the nine pairwise compansons and was obtained bv dividing the standard deviation by tne square root of 3 The method from which signal intensity is used to estimate told changes is descnbed in the Methods section of the manuscπpt Table 10. Aging-related decreases in gene expression in the cerebellum of C57BL/6 mice^* e Class CR

ORF Fold Change SE Signal Intensity Gen Old Young Prevention

39 132 Glucose-6-phosphatase Energy metabolism

U00445 43 1 4 79

W48504 -4 1 1 1 32 78 phosphoneuroprotein 14 homolog) Unknown N

AA153337 -3 9 07 67 218 Mvosin regulatory light chain 2 (MLC-2) Unknown 61 -3 9 05 14 57 NEDD-4 homolog Protein metabolism

W51213 55

XS6304 -3 1 04 2 27 Teπascin Growth/development N

W12681 3 1 0 6 30 126 Hepatocyle growth taαor Growth/development 37

Z6B889 -2 9 1 0 30 70 Wnt-2 homolog Growth/development N

W55684 -2 8 0 6 13 37 Brain protein ι47 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 soliαng taαor PRP22 Unknown 74

W55300 -2 7 0 7 20 47 Fatty acid-binding protein, heart (H-FABP) Unknown 71

D 13903 -2 7 0 5 7 37 MPTPdelta (type A) Growth development N

AA013976 -2 6 0 5 162 405 POL potyprotein, reverse transcπptase. Unknown N πbonuclease H

W10865 -2 6 0 2 14 142 Myosm light chain 1. amal foetal isoform Unknown N

AA020296 -2 5 0 2 -162 166 NG9 Growth/development 100

W64865 -2 5 1 1 ιo 31 Stat-3 Unknown N

AA139694 -2 5 03 64 203 Beta-myosin heavy chain Transport 100

U29762 ^■2 5 03 304 657 Albumin gene D-Box binding protein Transcription Faαor N

M8727S ■2 4 05 16 34 Thrombospondin Structural 52

X02677 ■2 4 02 63 160 Anion exchange protein Anion exchanger 100

X04836 -2 4 02 22 68 T-cell antigen CD4 Immune/inflammatory 100

X87242 -2 4 03 48 111 unc-33 Growth/development 70

AA 163021 -2 4 02 28 143 Annexin VIII Signal transduαion 84

M31810 -2 4 03 29 113 P-prptem membrane transporter Transport 100

M97900 -2 4 06 18 49 Unknown Unknown 20

M 15008 -2 4 06 101 227 Steroid 21-hydroxylase B Steroid metabolism 100

M99377 -2 4 05 77 191 Alpha-2 adrenergic receptor Neurotransmission N

M32490 -2 4 03 62 122 Cyr€1 Growth/development 41

AA168350 -2 3 03 130 237 Cystemyl-tRNA synthetase Protein metabolism 83

AA061206 -2 3 02 52 Unp (ubiquitin protease) Protein metabolism N

W12794 -2 3 03 23 96 Unknown Unknown 78

AA050593 -2 3 01 5 69 Unknown Unknown 62

AA050715 -2 3 03 64 148 Smoothelin Structural 92

AA106463 -2 2 03 110 277 Phosptioenolpyruvate carboxykinase Energy metabolism N

X90829 -2 2 03 -16 9 Lbx1 Growth/development N

X65588 -2 2 03 -1 24 mp41 Neurotransmission N

J00475 -2 2 02 -23 58 Ig alpha chain Immune/inflammatory N

X03019 -2 2 03 4 71 GM-CSF Immune/inflammatory 26

W34687 •2 2 04 62 115 Alpha-aαin Transport 78

W75614 ^■2 2 04 27 56 Alpha-synuclein Growth development N

AA068153 -2 2 03 14 39 Polyadenylate-bmding protein RNA metabolism 55

U36842 -2 1 05 22 36 Riap 3-ιnhιbιtor of apoptosis Apoptosis 100

W09127 -2 1 03 3 85 60S nbosomal protein L22 Protein metabolism 100

D63819 -2 1 02 29 87 Neuropeptide Y-Y1 receptor Neurotransmission N

M33884 ^■2 1 01 70 139 Env polyprotein Viral protein 55

AA144430 -2 1 03 64 156 NF-KB P100 inhibitory subunit Stress response 48

AA168554 ^■2 1 03 119 246 Unknown Unknown 85

U35730 -2 1 08 12 30 Jerky Unknown N

M92649 -2 1 04 45 112 mtπc oxide synthase Neurotransmission N

D 12907 -2 1 02 55 126 Seπne protease inhibitor homologue Unknown 85 l 7327 -2 1 02 234 566 Env polyprotein Viral protein 56

AA170444 -2 1 02 172 246 Ubiquitin-actrvaling enzyme E1 Protein metabolism 100

W12658 •2 1 03 203 415 FKBP-rapamycin associated protein 'Jnknown N

AA123026 ^■2 1 03 60 116 REG 2 Unknown 100 W13125 -2.1 0 5 11 1 432 Phenylalanyl-tRNA synthetase beta chain P^ .i 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 transαuαion N

W 13586 -2.1 0 1 29 136 Mvosin light chain 1 homolog Transport 100

W42217 -2 1 0 1 69 143 Ribosomal protein S20 Protein metabolism 100

AA 153522 -2.1 04 95 191 Senne/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 Hit DNA metabolism N

L08407 -2.0 0.3 38 117 Collagen type XVII Struαural N

AA145609 -2.0 0.2 55 134 cAMP responsive element modifier Transcnptional faαor 34

W12756 -2.0 0.1 48 117 Unknown Unknown 92

W75523 -2.0 0.3 48 95 Vertebrate homolog of C. elegans ϋn-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 31 1 RNA helicase PRP16 RNA metabolism 100

W42216 -1 8 0 1 183 361 SWI/SNF related hdmolog Transcnptional faαor 74

W12395 -1.8 04 141 237 Transcπption elongation taαor A (Sll) Transcnptional faαor 88

K03235 -1.8 0.1 84 149 Prolifeπn 2 Growth faαor 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 Phosphatidylsenne decarboxylase Protein metabolism N

L33726 -1 7 0.1 69 128 Fascm homolog 1 Struαural 100

L35549 -1 7 04 30 38 Y-box binding protein homolog Unknown ιoo

AA154514 -1 7 0.1 7639 12878 ATP synthase A chain (protein 6) homolog Energy metabolism 100

AA 143937 -1 7 0.1 384 697 Beta -centra clin Transport 70

AA027387 -1.7 0.1 169 270 Rab-4B Transport 51

L3B971 -1.7 0.2 205 334 Integral membrane protein 2 Unknown 43

W 10526 -1.7 0.1 193 301 Ca- channel, vottage-dep., gamma subunit 1 Transport 90

W12204 -1 6 0.2 114 200 Ca2+/calmodulιn-dependeπt protein kinase Signal transduction N isotorm gamma B AA170173 -1.6 0.1 149 289 NTT-73 Transport 100

M64403 -1.6 0 1 126 208 Cyc n D1 homolog DNA metabolism 100

W13191 -1.6 0 1 2B8 347 Thyroid hormone receptor alpha 2 Energy metabolism 87

U47543 -1 6 0 1 121 205 NGF1-A binding protein 2 (NAB2) Growth faαor N

D70848 -1 6 0.2 154 246 Zιc2 (cerebellar zinc finger protein) Neural development 77

X56518 -1 6 0 3 106 164 Acetylcholmesterase Neurotransmission N

AA144588 -1.6 0.2 233 368 Beta-adrenergic receptor kinase 2 homolog Neurotransmission 33

AA139B2B -1 6 0 1 224 351 gonadotropiπ inducible transcπption repressor-1 Unknown 100 homolog AA061170 -1.6 0.2 43 65 W -doma oxidoreductase homolog Unknown N

X58287 -1.6 0.3 84 153 mR-PTPu Signal transduαion N

L13129 -1.6 0.1 162 220 Annexin A7 Exocytosis 90

D85037 -1.6 0.1 50 77 Doc2beta Nenjotransmission N

U30823 -1 6 0.2 55 102 Myocyte enhancer taαor-2A Transcnptional taαor 33

W64791 -1 6 0.1 92 143 Galacfokinase Energy metabolism N

X52622 -1 6 0 1 274 377 IN Viral protein 100

AA063914 -1.5 0.1 175 267 Aloha-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 bv CR is shown as being none (N) or the calculated percentage effect. The SE was calculated for the nine pairwise ccmpansons 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 descnbed in the Methods seαion of the manuscnpt. Table 11 (3enes uoregulated by aging in C57BL6 mice heart from u19K GeneChip

Probe Set OC1 oc2 oc3 ! yd yc2 yc3 Fold Change

TC27774 396 218 490 I -1328 -2197 -1280 25.8

TC35932 71 1391 355 -596 -507 -1500 17.2

TC39719 938 595 1380 I 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 ¹ -121 -528 166 10.3

TC34515 1687 1117 966 465 -1068 -1737 9.4

TC2921 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 73R 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 47

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 34

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 Table 12 Genes downreguiated py aging in C57BL76 mice heart from Mu19K GeneChip

Probe Set od oc2 oc3 yd yc2 yc3 I Fold 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 ¹ -534

TC22507 -1155 -1599 -1409 1319 2177 2942 j -504

TC15929 -1203 -1586 -1787 1348 1014 2026 -47.0

TC19943 -687 -669 -428 2880 2552 1067 -417

TC18736 -1142 787 -1647 2711 3654 4006 -33.0

TC19957 1242 -501 958 6796 6771 5343 -30.5

TC37452 175 -1172 -441 I 820 2013 1233 -27.3

TC33452 , 532 -740 -465 i 2021 880 719 -26.3

TC14870 -289 -1650 -2496 I 30 209 1249 -25.2

TC26312 -118 -73 -146 I 406 1251 1344 -24.3

TC25802 -688 -736 -1968 31 707 695 -23.7

TC14624 -227 -943 -758 1675 718 352 -22.6

TC41568 J -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 ¹ 769 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 j 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 -149

TC17700 159 -80 -657 565 810 690 -144

TC17256 -2800 -3715 -3550 629 2754 950 -134

TC37672 -117 427 247 1149 1712 1737 -13.0

TC18637 202 -208 -312 1012 907 794 -12.8

TC15863 ¹ -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 -118

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 i 478 276 62 1703 1736 1138 -10.3

TC20671 | -70 -827 -303 948 1087 410 -10.2

TC-,4753 -332 -265 -325 418 335 276

-10.1

Probe Set ^[ OC1 oc2 oc3 i yd yc2 yc3 Fold Change

TC20259 272 22 86 _! 330 285 513 -3.3

TC23344 462 577 862 1602 2043 2131 -3.3

TC27282 1068 765 508 ¹ 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 ¹ -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

Table13. Genes upregulated bv aging in C57BL76 mice heart from Mu6500 GeneChip

ORF oc1 oc2 oc3 yd yc2 yc3 Fold Chanαe

X60103 242 223 238 13 -52 65 11.8

AA117446 273 512 453 155 5 118 66 6.8 21829 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

M 17440 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

D 13664 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

AAO 14024 111 219 218 110 59 72 3.1

D 16497 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

Table 14 Genes downregulated by aging in C57BL 6 mice heart from Mu6500 GeneChip

ORF oc4 oc5 oc6 ' yd yc2 yc3 I Fold Change

X54149 I ⁵² 16 -69 > 106 139 84 -6.2

X98475 37 38 202 136 79 -6.1

-⁷

U25114 i 185 133 69 326 301 283 -5.4

U58885 I -16 33 105 315 212 301 -5.3

X85169 ¹ -1 -32 -75 48 43 11 -5.0

AA028728 j 68 -19 17 90 99 116 -4.9

D14336 i 100 17 26 141 202 176 -48

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 I 69 61 45 ! -4.3

Y08361 35 1 -35 , 88 54 84 -4.2

W57425 -6 -31 -61 I 36 9 13 -4.2

L17076 130 103 97 i 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 j 17 22 2 57 71 29 -3.0

D78141 j 29 24 ⁵ I 54 74 65 | -3.0

Probe Set i OC1 oc2 oc3 I vc1 yc2 vc3 Fold Change

TC29793 1532 1993 2224 458 1173 801 2.1

TC37926 i 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 I 2072 2558 2188 1248 1174 959 2.1

TC39079 ^' 1639 1879 1394 538 1352 726 2.1

TC35125 ¹ 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

Table 16. Genes downregulated by aging in C57BL/6 mice gastrocnemius from Mu19K GeneChip

Probe Set oc1 oc2 oc3 yd 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 ¹ -63

TC34306 1324 565 -353 . 1427 2241 3278 -56

TC26537 3726 2008 378 6454 4146 9861 -52

TC35355 ¹ 245 -492 187 765 951 1217 -49

TC40742 -394 229 395 1281 1132 1041 -47

TC24501 , 152 253 -108 981 536 1084 -46

TC14421 419 1398 344 2366 1833 2615 i ^"45

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 ¹ -3.5

TC36858 -65 113 276 904 449 854 i -3.3

TC15621 3342 3801 2088 5802 5651 7667 J -31

TC22866 2973 2064 3961 6385 9965 9570 I -3.1

TC36347 1077 2585 1662 4287 6166 4493 -3.0

TC26944 13744 8497 7171 26871 31183 24244 I -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 -24

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 -24

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 Probe Set od oc2 oc3 yd yc2 yc3 Fold Change

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 j -2.0

Claims

CLAIMSWe claim:

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 0 sequences are determined in step (b).

6. The method of claim 5 wherein the expression levels of at ieast 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 sleeted 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-foid 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, and10.

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.