LONGEVITY SIGNATURES AND THEIR APPLICATIONS
20220249504 · 2022-08-11
Inventors
- Vadim GLADYSHEV (Boston, MA, US)
- Alexander TYSHKOVSKIY (Boston, MA, US)
- Anastasia SHINDYAPINA (Boston, MA, US)
Cpc classification
A61K31/519
HUMAN NECESSITIES
A61K31/519
HUMAN NECESSITIES
A61K31/5377
HUMAN NECESSITIES
A61K31/4184
HUMAN NECESSITIES
A61K31/416
HUMAN NECESSITIES
A61K31/4745
HUMAN NECESSITIES
A61K31/192
HUMAN NECESSITIES
A61K31/416
HUMAN NECESSITIES
A61K31/4422
HUMAN NECESSITIES
A61K31/5377
HUMAN NECESSITIES
A61K31/192
HUMAN NECESSITIES
A61K31/4745
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
C12Q1/6883
CHEMISTRY; METALLURGY
International classification
A61K31/5377
HUMAN NECESSITIES
A61K31/4184
HUMAN NECESSITIES
Abstract
The present disclosure relates to compositions and methods useful for elongating the lifespan of a subject (e.g., a mammalian subject, such as a human). Additionally or alternatively, the compositions and methods of the disclosure can be used to treat, prevent, and/or delay the onset of various geriatric syndromes in such a subject. The disclosure also provides compositions and methods that can be used to identify new interventions, such as chemical agents, lifestyle changes, or diets, that can be used to increase lifespan and to treat, prevent, and/or delay the onset of geriatric syndromes.
Claims
1. A method of increasing the lifespan of a mammalian subject, the method comprising administering to the subject a therapeutically effective amount of Selumetinib (6-(4-Bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide), LY294002 (2-Morpholin-4-yl-8-phenylchromen-4-one), AZD-8055 (5-[2,4-bis[(3S)-3-methyl-4-morpholinyl]pyrido[2,3-d]pyrimidin-7-yl]-2-methoxy-benzenemethanol), KU-0063794 (rel-5-[2-[(2R,6S)-2,6-dimethyl-4-morpholinyl]-4-(4-morpholinyl)pyrido[2,3-d]pyrimidin-7-yl]-2-methoxybenzenemethanol), Celastrol (3-Hydroxy-9β,13α-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic acid), Ascorbyl Palmitate ([(2S)-2-[(2R)-4,5-Dihydroxy-3-oxo-2-furyl]-2-hydroxy-ethyl] hexadecanoate), Oligomycin-a ((1R,4E,5'S,6S,6'S,7R,8S,10R,11R,12S,14R,15S,16R,18E,20E,22R,25S,27R,28S,29R)-22-ethyl-7,11,14,15-tetrahydroxy-6′-[(2R)-2-hydroxypropyl]-5′,6,8,10,12,14,16,28,29-nonamethyl-3′,4′,5′,6′-tetrahydro-3H,9H,13H-spiro[2,26-dioxabicyclo[23.3.1]nonacosa-4,18,20-triene-27,2′-pyran]-3,9,13-trione), NVP-BEZ235 (2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile), Importazole (N-(1-Phenylethyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine), Ryuvidine (2-methyl-5-[(4-methylphenyl)amino]-4,7-benzothiazoledione), NSC-663284 (6-Chloro-7-[[2-(4-morpholinyl)ethyl]amino]-5,8-quinolinedione), P1-828 (2-(4-Morpholinyl)-8-(4-aminopheny)l-4H-1-benzopyran-4-one), Pyrvinium pamoate (6-(Dimethylamino)-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)ethenyl]-1-methyl-4,4′-methylenebis[3-hydroxy-2-naphthalenecarboxylate] (2:1)-quinolinium), P1-103 (3-[4-(4-morpholinyl)pyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]-phenol), YM-155 (4,9-dihydro-1-(2-methoxyethyl)2-methyl-4,9-dioxo-3-(2-pyrazinylmethyl)-1H-naphth[2,3-d]imidazolium, bromide), Prostratin ((1aR,1 bS,4aR,7aS,7bR,8R,9aS)-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropa[3,4]benzo[1,2-e]azulen-9a-yl acetate), BCI hydrochloride (3-(cyclohexylamino)-2,3-dihydro-2-(phenylmethylene)-1H-inden-1-one, monohydrochloride), Dorsomorphin-Compound C (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)pyrazolo[1,5-a]pyrimidine), VU-0418947-2 (6-Phenyl-N-[(3-phenylphenyl)methyl]-3-pyridin-2-yl-1,2,4-triazin-5-amine), JNK-9L (4-[3-fluoro-5-(4-morpholinyl)phenyl]-N-[4-[3-(4-morpholinyl)-1,2,4-triazol-1-yl]phenyl]-2-pyrimidinamine), Phloretin (3-(4-Hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one), ZG-10 ((E)-4-(4-(dimethylamino)but-2-enamido)-N-(3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide), Proscillaridin (5-[(3S,8R,9S,10R,13R,14S,17R)-14-Hydroxy-10,13-dimethyl-3-((2R,3R,4R,5R,6R)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-2H-pyran-2-one), YC-1 (3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole), IKK-2-inhibitor-V (N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide), Anisomycin ((2R,3S,4S)-4-hydroxy-2-(4-methoxybenzyl)-pyrrolid in-3-yl acetate), Colforsin ([(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-5-acetyloxy-3-ethenyl-10,10b-dihydroxy-3,4a,7,7,10a-Pentamethyl-1-oxo-5,6,6a,8,9,10-hexahydro-2H-benzo[f]chromen-6-yl] 3-d imethylaminopropanoate), Rilmenidine (N-(Dicyclopropylmethyl)-4,5-dihydro-1,3-oxazol-2-amine), GDC-0941 (Pictilisib, 4-(2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine), Valdecoxib (4-(5-methyl-3-phenylisoxazol-4-yl)benzenesulfonamide), Myricetin (3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), Cyproheptadine (4-(5H-Dibenzo[a,d]cyclohepten-5-ylidene)-1-methylpiperidine), Vorinostat (N-Hydroxy-N′-phenyloctanediamide), Nifedipine (3,5-Dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate), Phylloquinone (2-Methyl-3-[(E)-3,7,11,15-tetramethylhexadec-2-enyl]naphthalene-1,4-dione), Withaferin-A ((4β,5β,6β,22R)-4,27-Dihydroxy-5,6:22,26-diepoxyergosta-2,24-diene-1,26-dione), Temsirolimus ((1R,2R,4S)-4-{(2R)-2-[(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1,4]oxazacyclohentriacontin-3-yl]propyl}-2-methoxycyclohexyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate), SN-38 (4,11-diethyl-4,9-dihydroxy-(4S)-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione), GSK-1059615 (5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidenedione), Tipifarnib (6-[(R)-amino-(4-chlorophenyl)-(3-methylimidazol-4-yl)methyl]-4-(3-chlorophenyl)-1-methylquinolin-2-one), Linifanib (1-[4-(3-amino-1H-indazol-4-yl)phenyl]-3-(2-fluoro-5-methylphenyl)urea), WYE-354 (4-[6-[4-[(methoxycarbonyl)amino]phenyl]-4-(4-morpholinyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-methyl ester-1-piperidinecarboxylic acid), MK-212 (6-Chloro-2-(1-piperazinyl)pyrazine hydrochloride), and/or Enzastaurin (3-(1-Methylindol-3-yl)-4-[1-[1-(pyridin-2-ylmethyl)piperidin-4-yl]indol-3-yl]pyrrole-2,5-dione), thereby increasing the lifespan of the subject.
2. A method of reducing the frailty index of a mammalian subject, the method comprising administering to the subject a therapeutically effective amount of Selumetinib, LY294002, AZD-8055, KU-0063794, Celastrol, Ascorbyl Palmitate, Oligomycin-a, NVP-BEZ235, Importazole, Ryuvidine, NSC-663284, PI-828, Pyrvinium pamoate, PI-103, YM-155, Prostratin, BCI hydrochloride, Dorsomorphin-Compound C, VU-0418947-2, JNK-9L, Phloretin, ZG-10, Proscillaridin, YC-1, IKK-2-inhibitor-V, Anisomycin, Colforsin, Rilmenidine, GDC-0941, Valdecoxib, Myricetin, Cyproheptadine, Vorinostat, Nifedipine, Phylloquinone, Withaferin-A, Temsirolimus, SN-38, GSK-1059615, Tipifarnib, Linifanib, WYE-354, MK-212, and/or Enzastaurin, thereby reducing the frailty index of the subject.
3. A method of improving learning ability in a mammalian subject, the method comprising administering to the subject a therapeutically effective amount of Selumetinib, LY294002, AZD-8055, KU-0063794, Celastrol, Ascorbyl Palmitate, Oligomycin-a, NVP-BEZ235, Importazole, Ryuvidine, NSC-663284, PI-828, Pyrvinium pamoate, PI-103, YM-155, Prostratin, BCI hydrochloride, Dorsomorphin-Compound C, VU-0418947-2, JNK-9L, Phloretin, ZG-10, Proscillaridin, YC-1, IKK-2-inhibitor-V, Anisomycin, Colforsin, Rilmenidine, GDC-0941, Valdecoxib, Myricetin, Cyproheptadine, Vorinostat, Nifedipine, Phylloquinone, Withaferin-A, Temsirolimus, SN-38, GSK-1059615, Tipifarnib, Linifanib, WYE-354, MK-212, and/or Enzastaurin, thereby improving the learning ability of the subject.
4. A method of delaying onset of a geriatric syndrome in a mammalian subject, the method comprising administering to the subject a therapeutically effective amount of Selumetinib, LY294002, AZD-8055, Celastrol, KU-0063794, Ascorbyl Palmitate, Oligomycin-a, NVP-BEZ235, Importazole, Ryuvidine, NSC-663284, PI-828, Pyrvinium pamoate, PI-103, YM-155, Prostratin, BCI hydrochloride, Dorsomorphin-Compound C, VU-0418947-2, JNK-9L, Phloretin, ZG-10, Proscillaridin, YC-1, IKK-2-inhibitor-V, Anisomycin, Colforsin, Rilmenidine, GDC-0941, Valdecoxib, Myricetin, Cyproheptadine, Vorinostat, Nifedipine, Phylloquinone, Withaferin-A, Temsirolimus, SN-38, GSK-1059615, Tipifarnib, Linifanib, WYE-354, MK-212, and/or Enzastaurin, thereby delaying the onset of a geriatric syndrome in the subject.
5. The method of any one of claims 1-4, wherein the subject is a human.
6. A pharmaceutical composition comprising Selumetinib, LY294002, AZD-8055, Celastrol, or ascorbyl palmitate, and one or more pharmaceutically acceptable excipients, wherein the composition is formulated for administration to a human in combination with a meal.
7. A pharmaceutical composition comprising Selumetinib, LY294002, AZD-8055, KU-0063794, Celastrol, Ascorbyl Palmitate, Oligomycin-a, NVP-BEZ235, Importazole, Ryuvidine, NSC-663284, PI-828, Pyrvinium pamoate, PI-103, YM-155, Prostratin, BCI hydrochloride, Dorsomorphin-Compound C, VU-0418947-2, JNK-9L, Phloretin, ZG-10, Proscillaridin, YC-1, IKK-2-inhibitor-V, Anisomycin, Colforsin, Rilmenidine, GDC-0941, Valdecoxib, Myricetin, Cyproheptadine, Vorinostat, Nifedipine, Phylloquinone, Withaferin-A, Temsirolimus, SN-38, GSK-1059615, Tipifarnib, Linifanib, WYE-354, MK-212, and/or Enzastaurin, and one or more pharmaceutically acceptable excipients, wherein the composition is formulated for administration to a human in combination with a meal.
8. A dietary supplement comprising Selumetinib, LY294002, AZD-8055, KU-0063794, Celastrol, Ascorbyl Palmitate, Oligomycin-a, NVP-BEZ235, Importazole, Ryuvidine, NSC-663284, PI-828, Pyrvinium pamoate, PI-103, YM-155, Prostratin, BCI hydrochloride, Dorsomorphin-Compound C, VU-0418947-2, JNK-9L, Phloretin, ZG-10, Proscillaridin, YC-1, IKK-2-inhibitor-V, Anisomycin, Colforsin, Rilmenidine, GDC-0941, Valdecoxib, Myricetin, Cyproheptadine, Vorinostat, Nifedipine, Phylloquinone, Withaferin-A, Temsirolimus, SN-38, GSK-1059615, Tipifarnib, Linifanib, WYE-354, MK-212, or Enzastaurin, or a combination thereof.
9. The dietary supplement of claim 8, wherein the dietary supplement is formulated for administration to a human in combination with a meal.
10. A method of increasing the lifespan of a mammalian subject, the method comprising providing the subject with a treatment that (i) increases expression of one or more genes set forth in any of Tables 1-10 and/or (ii) decreases expression of one or more genes set forth in any of Tables 11-20.
11. A method of reducing the frailty index in a mammalian subject, the method comprising providing the subject with a treatment that (i) increases expression of one or more genes set forth in any of Tables 1-10 and/or (ii) decreases expression of one or more genes set forth in any of Tables 11-20.
12. A method of improving learning ability in a mammalian subject, the method comprising providing the subject with a treatment that (i) increases expression of one or more genes set forth in any of Tables 1-10 and/or (ii) decreases expression of one or more genes set forth in any of Tables 11-20.
13. A method of delaying onset of a geriatric syndrome in a mammalian subject, the method comprising providing the subject with a treatment that (i) increases expression of one or more genes set forth in any of Tables 1-10 and/or (ii) decreases expression of one or more genes set forth in any of Tables 11-20.
14. The method of any one of claims 10-13, wherein the treatment comprises administration of an agent, a lifestyle change, a change in disease status, or a combination thereof.
15. The method of claim 14, wherein the treatment comprises administration of an agent.
16. The method of claim 15, wherein the agent comprises a small molecule, a peptide, a peptidomimetic, an interfering ribonucleic acid (RNA), an antibody, an aptamer, or a gene therapy.
17. The method of claim 16, wherein the agent comprises a small molecule.
18. The method of claim 17, wherein the agent comprises a compound represented by formula (I) ##STR00009## wherein one or two of X.sup.5, X.sup.6 and X.sup.8 is N, and the other(s) is/are CH; R.sup.7 is selected from halo, OR.sup.01, SR.sup.S1, NR.sup.N1R.sup.N2, NR.sup.N7aC(═O)R.sup.C1, NR.sup.N7bSO.sub.2R.sup.S2a, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group; R.sup.01 and R.sup.S1 are selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.1-7 alkyl group; R.sup.N1 and R.sup.N2 are independently selected from H, an optionally substituted C.sub.1-7 alkyl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, or R.sup.N1 and R.sup.N2, together with the nitrogen to which they are bound, form a heterocyclic ring comprising from 3 to 8 ring atoms; R.sup.C1 is selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, an optionally substituted C.sub.1-7 alkyl group; NR.sup.N8R.sup.N9, wherein R.sup.N8 and R.sup.N9 are independently selected from H, an optionally substituted C.sub.1-7 alkyl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, or R.sup.N8 and R.sup.N9, together with the nitrogen to which they are bound, form a heterocyclic ring comprising from 3 to 8 ring atoms; R.sup.S2a is selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.1-7 alkyl group; R.sup.N7a and R.sup.N7b are selected from H and a C.sub.1-4 alkyl group; R.sup.N3 and R.sup.N4, together with the nitrogen to which they are bound, form an optionally substituted heterocyclic ring comprising from 3 to 8 ring atoms; R.sup.2 is selected from H, halo, OR.sup.02, SR.sup.S2b, NR.sup.N5R.sup.N6, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, wherein R.sup.02 and R.sup.S2b are selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.1-7 alkyl group; and R.sup.N5 and R.sup.N6 are independently selected from H, an optionally substituted C.sub.1-7 alkyl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, or R.sup.N5 and R.sup.N6, together with the nitrogen to which they are bound, form an optionally substituted heterocyclic ring comprising from 3 to 8 ring atoms, or a pharmaceutically acceptable salt thereof.
19. The method of claim 18, wherein the agent comprises KU-0063794, represented by formula (1) ##STR00010##
20. The method of any one of claims 17-19, wherein the agent comprises Selumetinib, LY294002, AZD-8055, Celastrol, or ascorbyl palmitate.
21. The method of any one of claims 14-20, wherein the treatment comprises a lifestyle change.
22. The method of claim 21, wherein the lifestyle change comprises a dietary change.
23. The method of any one of claims 14-22, wherein the agent is administered to the subject orally, intraarticularly, intravenously, intramuscularly, rectally, cutaneously, subcutaneously, topically, transdermally, sublingually, nasally, intravesicularly, intrathecally, epidurally, or transmucosally.
24. The method of claim 23, wherein the agent is administered to the subject orally.
25. The method of any one of claims 14-24, wherein the agent is formulated as a tablet, capsule, gel cap, powder, liquid solution, or liquid suspension.
26. The method of any one of claims 10-25, further comprising monitoring the subject for (i) an increase in expression of one or more genes set forth in Tables 1-10 and/or (ii) a decrease in expression of one or more genes set forth in Tables 11-20 following treatment.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0107]
[0112]
[0121]
[0129]
[0135]
[0141]
[0146]
[0151]
[0154]
[0158]
[0161]
[0164]
At the level of gene expression change, rapamycin shows significant positive correlation only with itself (median Spearman correlation coefficient=0.088; BH adjusted Mann-Whitney test p-value=2.8.Math.10.sup.−3). Although thought to be CR mimetic, rapamycin shows slight (median Spearman correlation coefficient=−0.049) but significant (BH adjusted Mann-Whitney test p-value=2.Math.10.sup.−3) negative correlation with CR at the level of gene expression. For every intervention, violinplot shows the distribution of Spearman correlation coefficient between gene expression changes of every dataset of rapamycin and the corresponding intervention. 250 genes consisting of 125 genes with the lowest p-value in each pair of datasets were used for calculation.
Estradiol: 17-α-estradiol; Snell: Snell dwarf mice; Ames: Ames dwarf mice; Little: Little mice; CR: Caloric restriction; MR: Methionine Restriction; EOD: Every-other-day feeding; FGF21 over: FGF21 overexpression; GHRKO: Growth Hormone Receptor Knockout.
[0165]
[0169]
[0172]
Gene expression changes in response to interventions are scored against longevity signatures to identify candidate compounds with lifespan-extending effects. Statistical significance of association with longevity signatures is calculated using permutation test and adjusted with Benjamini-Hochberg procedure.
[0173]
The latter include gene signatures of individual interventions (CR, rapamycin and GH deficiency), common signatures (Interventions common) and signatures associated with the effect on lifespan (Maximum and median lifespan). Cells are colored based on significance score, calculated as log.sub.10(adjusted p-value) corrected by sign of regulation. Sirt6 Over: Sirt6 Overexpression.
[0174]
CMap is used for prediction of perspective compounds. Mouse and human primary hepatocytes and mouse in vivo models are used for validation.
[0175]
Four-month old UM-HET3 mice were subjected to diets for 1 month, followed by gene expression analyses. The figure shows the significance of associations between longevity signatures and gene expression changes in response to predicted compounds.
[0176]
[0177]
AZD-8055 extends lifespan of C57BL/6 male mice when given late in life. Arrow indicates the treatment onset. n=15 in the AZD-8055 group, and n=14 in the Control group.
[0178]
Left panel: AZD-8055 was given to 31-month-old C57BL/6 mice, n=10 for males and n=4 for females the AZD-8055 group, and n=18 for males and n=8 for females for the Control group. Right panel: same as in left panel, but gait speed was assessed in males. AZD-8055 was given to 31-month-old C57BL/6 mice, n=6 for the AZD-8055 group, and n=17 for the Control group.
[0179]
The treatment does not lead to glucose intolerance in old C57BL/6 mice. 23-month-old mice were treated for 2.5 months prior to analyses.
[0180]
Selumetinib extends lifespan of C57BL/6 mice when given late in life. Left: survival of males and females combined (n=20 per group). Right: an independent cohort of female mice (n=15 per group), until they were sacrificed for biochemical experiments. Arrows indicate the onset of treatment.
[0181]
Left: Selumetinib improves frailty index of 31-month-old C57BL/6 female mice (n=8 for Selumetinib, n=10 for Control). Right: Gait speed.
[0182]
Population of immune cells in the spleen is not altered by Selumetinib (n=7 for Control, n=12 for Selumetinib). 27-month-old C57BI/6 females were used. Cells were analyzed by FACS: B-cells are CD45+CD19+, T-cells are CD45+CD3+, and myeloid cells are CD45+CD11b+.
[0183]
Arrow indicates the onset of treatment. n=19 for the Celastrol group, and n=20 for the Control group.
[0184]
Celastrol does not affect frailty index or gait speed. n=10 per sex per treatment.
[0185]
Arrow indicates the onset of treatment. n=14 for the LY294002 group, and n=14 for the Control group.
[0186]
Both frailty index and gait speed are improved by this compound in 31-month-old C57BL/6 male mice. Left: frailty index: n=9 for males and n=3 for females for the LY294002 group, and n=18 for males and n=8 for females for the Control group. Right: gait speed: n=9 for the LY294002 group, and n=17 for the Control group.
[0187]
No effect was observed. ns: not significant.
[0188]
Arrow indicates the onset of treatment. n=14 for the KU-0063794, and n=14 for the Control group.
[0189]
Both frailty index and gait speed are improved by this compound in 31-month-old C57BL/6 male mice. Left: frailty index: n=13 for males and n=2 for females for the KU-0063794 group, and n=18 for males and n=8 for females for the Control group. Right: gait speed: n=7 for the KU-0063794 group, and n=17 for the Control group.
[0190]
No effect of this compound was observed. ns: not significant.
DETAILED DESCRIPTION
[0191] The potential to live shorter or longer life is defined by the metabolic state of cells, and, in turn, is reflected in their gene expression patterns. The transition from a shorter- to a longer-lived state is observed when comparing the transcriptomes of (i) particular organs of mice subjected to interventions known to extend lifespan; (ii) cell types widely differing in lifespan, a parameter referred to as “cell turnover;” and (iii) particular organs between shorter- and longer-lived mammals.
[0192] Based on gene expression analyses of these models, transcriptomic patterns associated with lifespan have been identified, and an approach for identification of new lifespan-extending interventions has been developed. This approach was then applied to predict candidate longevity interventions. The present disclosure describes this approach and the validation of candidate prediction using different biological models.
Identification of Gene Expression Longevity Signatures
[0193] The gene expression patterns that reflect the transition from shorter to longer lived states are designated throughout the present disclosure as “longevity signatures.” A total of 10 longevity signatures have been developed based on the transcriptomes of (i) mice treated with 17 different lifespan-extending interventions (6 “intervention-based signatures”); (ii) 20 organs and cell types differing in cell turnover (1 “turnover-based signature”); and (iii) liver, kidney, and brain of 41 species of mammals differing 30-fold in lifespan (3 “organ-specific signatures”). Each of these gene signatures contains a set of genes that is up-regulated in longer-living cells, as well as a set of genes that is down-regulated in longer-living cells. The 6 intervention-based signatures are shown in Tables 1-6 (up-regulated genes) and in Tables 11-16 (down-regulated genes), below. The 1 turnover-based signature is shown in Table 7 (up-regulated genes) and Table 17 (down-regulated genes), below. The 3 organ-specific signatures are shown in Tables 8-10 (up-regulated genes) and Tables 18-20 (down-regulated genes), below.
[0194] As described in further detail in the working examples, below, the genes within the foregoing signatures were identified as having an expression pattern associated with lifespan by various metrics. For example, the intervention-based signatures were identified by analyzing gene expression patterns that are observed in mammals upon treatment with agents known to have a lengthening effect on lifespan. The intervention-based signatures include 3 signatures corresponding to the genes perturbed in response to individual longevity interventions (calorie restriction, rapamycin and growth hormone deficient mutants), 1 signature corresponding to the genes commonly perturbed by all interventions and 2 signatures corresponding to the genes, which expression change in response to interventions is associated with the effect on median or maximum lifespan. The turnover-based signature was identified by analyzing gene expression patterns across different cell types and tissues in humans and correlating genes that are up-regulated or down-regulated with cell lifespan. The organ-specific signatures were identified by analyzing the gene expression patterns in particular organs (liver, kidney, and brain) across 41 species of mammals and correlating genes that are up-regulated or down-regulated with the lifespan of the corresponding mammal.
[0195] In sum, the above procedures enabled the identification of 10 longevity signatures, captured by Tables 1-10 (up-regulated genes) and Tables 11-20 (down-regulated genes), that are characteristic of elevated lifespan. The sections that follow describe the procedures used to identify these signatures in further detail. The following sections also describe methods that can be used to screen for interventions (e.g., chemical agents and/or lifestyle changes, among others) capable of up-regulating one or more genes in Tables 1-10 and/or down-regulating one or more genes in Tables 11-20. Such interventions can be used to increase lifespan of a subject (e.g., a mammalian subject, such as a human), as well as to reduce the risk of frailty in a subject, improve the learning ability of the subject, and treat, prevent, and/or delay the onset of geriatric syndromes in a subject.
Identification of Candidate Lifespan-Extending Interventions Based on Longevity Signatures
[0196] This section provides an example of how the gene signatures described above can be used to screen for lifespan-extending interventions. Briefly, the gene signatures described above were screened for candidate longevity interventions across 3,300 compounds using the Connectivity Map (CMap) database. CMap aggregates gene expression data related to the response of several human cell lines to different drugs. This platform was utilized to identify compounds with the most significant positive association with the above longevity signatures. To account for possible differences in mechanisms behind different signatures, each of these signatures was analyzed separately. To search for other interventions potentially effecting lifespan, including genetic, pharmacological, and environmental interventions, the GEO database was also utilized, which contains gene expression datasets corresponding to the effect of many interventions on different biological models.
[0197] To statistically estimate the association of certain gene expression profiles with the above signatures, a gene set enrichment analysis (GSEA)-based approach was also developed, which examines whether a certain gene set is enriched among up- or down-regulated genes (
Validation of Predicted Interventions
[0198] To validate the above approach and predictions, specific gene expression datasets were chosen from the GEO database that correspond to the effect of certain interventions considered to be health- and lifespan-extending or shortening on mouse liver (
[0199] A significant positive association was detected with the majority of longevity signatures for all compounds predicted via CMap. Additionally, significant associations were detected for datasets from GEO, consistent with the predictions described above. For example, as described in the working examples below, mild hypoxia and Keap1 knockout perturbed gene expression in the same way as longevity interventions, whereas interleukin-6 injection and Mat1a knockout led to the opposite changes.
[0200] This approach was then expanded and compounds with the most significant positive association with different longevity associations using CMap were selected. These hits were verified using various biological models (
[0201] First, the identified hits were applied to human and mouse primary hepatocytes, and ensuing gene expression profiles were obtained. To treat human cells, 3 different doses of each agent were used, whereas for mice, a single dose of each agent was used. Using the GSEA-based approach, statistically significant (permutation test adjusted p-value <0.1) associations were identified with at least one longevity signature for 10 (25% of tested compounds) and 31 (44.3% of tested compounds) drugs in human and mouse hepatocytes, respectively.
[0202] Second, diets were prepared for 24 of the identified compounds. These diets were administered to mice for 1 month, and ensuing gene expression changes were monitored. Using the GSEA-based association test, 18 drugs (69% of tested compounds) were identified as having a statistically significant association with at least one longevity signature. Moreover, on average, every compound had significant associations with 2.8 different signatures, supporting the robustness of this approach (
[0203] Taken together, the above findings substantiate a method for unbiased identification of candidate longevity interventions and show how this method was validated in both cell culture and in vivo models. These findings are described in further detail in the working examples below.
Methods of Measuring Gene Expression
[0204] The expression level of a gene described herein (e.g., a gene set forth in one or more of the longevity signatures recited in Tables 1-20) can be ascertained, for example, by evaluating the concentration or relative abundance of mRNA transcripts derived from transcription of the gene. Additionally or alternatively, gene expression can be determined by evaluating the concentration or relative abundance of encoded protein produced by transcription and translation of the corresponding gene. Protein concentrations can also be assessed using functional assays. The sections that follow describe exemplary techniques that can be used to measure the expression level of a gene of interest. Gene expression can be evaluated by a number of methodologies known in the art, including, but not limited to, nucleic acid sequencing, microarray analysis, proteomics, in-situ hybridization (e.g., fluorescence in-situ hybridization (FISH)), amplification-based assays, in situ hybridization, fluorescence activated cell sorting (FACS), northern analysis and/or PCR analysis of mRNAs.
Nucleic Acid Detection
[0205] Nucleic acid-based methods for determining gene expression include imaging-based techniques (e.g., Northern blotting or Southern blotting). Northern blot analysis is a conventional technique well known in the art and is described, for example, in Molecular Cloning, a Laboratory Manual, second edition, 1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainview, N.Y. 11803-2500. Typical protocols for evaluating the status of genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis).
[0206] Gene detection techniques that may be used in conjunction with the compositions and methods described herein further include microarray sequencing experiments (e.g., Sanger sequencing and next-generation sequencing methods, also known as high-throughput sequencing or deep sequencing). Exemplary next generation sequencing technologies include, without limitation, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing platforms. Additional methods of sequencing known in the art can also be used. For instance, gene expression at the mRNA level may be determined using RNA-Seq (e.g., as described in Mortazavi et al., Nat. Methods 5:621-628 (2008) the disclosure of which is incorporated herein by reference in their entirety). RNA-Seq is a robust technology for monitoring expression by direct sequencing the RNA molecules in a sample. Briefly, this methodology may involve fragmentation of RNA to an average length of 200 nucleotides, conversion to cDNA by random priming, and synthesis of double-stranded cDNA (e.g., using the Just cDNA DoubleStranded cDNA Synthesis Kit from Agilent Technology). Then, the cDNA is converted into a molecular library for sequencing by addition of sequence adapters for each library (e.g., from Illumina®/Solexa), and the resulting 50-100 nucleotide reads are mapped onto the genome.
[0207] Gene expression levels may be determined using microarray-based platforms (e.g., single-nucleotide polymorphism arrays), as microarray technology offers high resolution. Details of various microarray methods can be found in the literature. See, for example, U.S. Pat. No. 6,232,068 and Pollack et al., Nat. Genet. 23:41-46 (1999), the disclosures of each of which are incorporated herein by reference in their entirety. Using nucleic acid microarrays, mRNA samples are reverse transcribed and labeled to generate cDNA. The probes can then hybridize to one or more complementary nucleic acids arrayed and immobilized on a solid support. The array can be configured, for example, such that the sequence and position of each member of the array is known. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. Expression level may be quantified according to the amount of signal detected from hybridized probe-sample complexes. A typical microarray experiment involves the following steps: 1) preparation of fluorescently labeled target from RNA isolated from the sample, 2) hybridization of the labeled target to the microarray, 3) washing, staining, and scanning of the array, 4) analysis of the scanned image and 5) generation of gene expression profiles. One example of a microarray processor is the Affymetrix GENECHIP® system, which is commercially available and comprises arrays fabricated by direct synthesis of oligonucleotides on a glass surface. Other systems may be used as known to one skilled in the art.
[0208] Amplification-based assays also can be used to measure the expression level of a gene described herein. In such assays, the nucleic acid sequences of the gene act as a template in an amplification reaction (for example, PCR, such as qPCR). In a quantitative amplification, the amount of amplification product is proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the expression level of the gene, corresponding to the specific probe used, according to the principles described herein. Methods of real-time qPCR using TaqMan probes are well known in the art. Detailed protocols for real-time qPCR are provided, for example, in Gibson et al., Genome Res. 6:995-1001 (1996), and in Heid et al., Genome Res. 6:986-994 (1996), the disclosures of each of which are incorporated herein by reference in their entirety. Levels of gene expression as described herein can be determined by RT-PCR technology. Probes used for PCR may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme.
Protein Detection
[0209] Gene expression can additionally be determined by measuring the concentration or relative abundance of a corresponding protein product. Protein levels can be assessed using standard detection techniques known in the art. Protein expression assays suitable for use with the compositions and methods described herein include proteomics approaches, immunohistochemical and/or western blot analysis, immunoprecipitation, molecular binding assays, ELISA, enzyme-linked immunofiltration assay (ELIFA), mass spectrometry, mass spectrometric immunoassay, and biochemical enzymatic activity assays. In particular, proteomics methods can be used to generate large-scale protein expression datasets in multiplex. Proteomics methods may utilize mass spectrometry to detect and quantify polypeptides (e.g., proteins) and/or peptide microarrays utilizing capture reagents (e.g., antibodies) specific to a panel of target proteins to identify and measure expression levels of proteins expressed in a sample (e.g., a single cell sample or a multi-cell population).
[0210] Exemplary peptide microarrays have a substrate-bound plurality of polypeptides, the binding of an oligonucleotide, a peptide, or a protein to each of the plurality of bound polypeptides being separately detectable. Alternatively, the peptide microarray may include a plurality of binders, including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display binders, yeast two-hybrid binders, aptamers, which can specifically detect the binding of specific oligonucleotides, peptides, or proteins. Examples of peptide arrays may be found in U.S. Pat. Nos. 6,268,210, 5,766,960, and 5,143,854, the disclosures of each of which are incorporated herein by reference in their entirety.
[0211] Mass spectrometry (MS) may be used in conjunction with the methods described herein to identify and characterize gene expression. Any method of MS known in the art may be used to determine, detect, and/or measure a protein or peptide fragment of interest, e.g., LC-MS, ESI-MS, ESI-MS/MS, MALDI-TOF-MS, MALDI-TOF/TOF-MS, tandem MS, and the like. Mass spectrometers generally contain an ion source and optics, mass analyzer, and data processing electronics. Mass analyzers include scanning and ion-beam mass spectrometers, such as time-of-flight (TOF) and quadruple (Q), and trapping mass spectrometers, such as ion trap (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), may be used in the methods described herein. Details of various MS methods can be found in the literature. See, for example, Yates et al., Annu. Rev. Biomed. Eng. 11:49-79, 2009, the disclosure of which is incorporated herein by reference in its entirety.
[0212] Prior to MS analysis, proteins in a sample obtained from the patient can be first digested into smaller peptides by chemical (e.g., via cyanogen bromide cleavage) or enzymatic (e.g., trypsin) digestion. Complex peptide samples also benefit from the use of front-end separation techniques, e.g., 2D-PAGE, HPLC, RPLC, and affinity chromatography. The digested, and optionally separated, sample is then ionized using an ion source to create charged molecules for further analysis. Ionization of the sample may be performed, e.g., by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. Additional information relating to the choice of ionization method is known to those of skill in the art.
[0213] After ionization, digested peptides may then be fragmented to generate signature MS/MS spectra. Tandem MS, also known as MS/MS, may be particularly useful for analyzing complex mixtures. Tandem MS involves multiple steps of MS selection, with some form of ion fragmentation occurring in between the stages, which may be accomplished with individual mass spectrometer elements separated in space or using a single mass spectrometer with the MS steps separated in time. In spatially separated tandem MS, the elements are physically separated and distinct, with a physical connection between the elements to maintain high vacuum. In temporally separated tandem MS, separation is accomplished with ions trapped in the same place, with multiple separation steps taking place over time. Signature MS/MS spectra may then be compared against a peptide sequence database (e.g., SEQUEST). Post-translational modifications to peptides may also be determined, for example, by searching spectra against a database while allowing for specific peptide modifications.
Pharmaceutical Compositions
[0214] Using the compositions and methods of the disclosure, one can screen for interventions (e.g., chemical agents, dietary supplements, diets, and/or lifestyle changes, among others) that are capable of effectuating a change in gene expression consistent with the longevity signatures set forth in one or more of Tables 1-20. For example, one may screen for an intervention that is capable of (i) up-regulating one or more of the genes set forth in Tables 1-10 and/or (ii) down-regulating one or more of the genes set forth in Tables 11-20. Such interventions are expected to enhance lifespan and promote the overall wellbeing of the subject, e.g., by reducing the risk of frailty in the subject, improving the learning ability of the subject, and/or preventing or delaying the onset of a geriatric syndrome in the subject.
[0215] Examples of agents that up-regulate one or more genes set forth in the longevity signatures shown in Tables 1-10 and/or down-regulate one or more gens set forth in the longevity signatures shown in Tables 11-20 include the following compounds. As described herein, such compounds may be used to enhance lifespan and promote the overall wellbeing of the subject, e.g., by reducing the risk of frailty in the subject, improving the learning ability of the subject, and/or preventing or delaying the onset of a geriatric syndrome in the subject. Examples of these compounds are KU-0063794 (rel-5-[2-[(2R,6S)-2,6-dimethyl-4-morpholinyl]-4-(4-morpholinyl)pyrido[2,3-d]pyrimidin-7-yl]-2-methoxybenzenemethanol), Ascorbyl Palmitate ([(2S)-2-[(2R)-4,5-Dihydroxy-3-oxo-2-furyl]-2-hydroxy-ethyl] hexadecanoate), Celastrol (3-Hydroxy-9β,13α-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic acid), Oligomycin-a ((1R,4E,5'S,6S,6'S,7R,8S,10R,11R,12S,14R,15S,16R,18E,20E,22R,25S,27R,28S,29R)-22-ethyl-7,11,14,15-tetrahydroxy-6′-[(2R)-2-hydroxypropyl]-5′,6,8,10,12,14,16,28,29-nonamethyl-3′,4′,5′,6′-tetrahydro-3H,9H,13H-spiro[2,26-dioxabicyclo[23.3.1]nonacosa-4,18,20-triene-27,2′-pyran]-3,9,13-trione), NVP-BEZ235 (2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile), AZD-8055 (5-[2,4-bis[(3S)-3-methyl-4-morpholinyl]pyrido[2,3-d]pyrimidin-7-yl]-2-methoxy-benzenemethanol), Importazole (N-(1-Phenylethyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine), Ryuvidine (2-methyl-5-[(4-methylphenyl)amino]-4,7-benzothiazoledione), NSC-663284 (6-Chloro-7-[[2-(4-morpholinyl)ethyl]amino]-5,8-quinolinedione), PI-828 (2-(4-Morpholinyl)-8-(4-aminopheny)l-4H-1-benzopyran-4-one), Pyrvinium pamoate (6-(Dimethylamino)-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)ethenyl]-1-methyl-4,4′-methylenebis[3-hydroxy-2-naphthalenecarboxylate] (2:1)-quinolinium), PI-103 (3-[4-(4-morpholinyl)pyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]-phenol), YM-155 (4,9-dihydro-1-(2-methoxyethyl)2-methyl-4,9-dioxo-3-(2-pyrazinylmethyl)-1H-naphth[2,3-d]imidazolium, bromide), Prostratin ((1aR,1bS,4aR,7aS,7bR,8R,9aS)-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropa[3,4]benzo[1,2-e]azulen-9a-yl acetate), BCI hydrochloride (3-(cyclohexylamino)-2,3-dihydro-2-(phenylmethylene)-1H-inden-1-one, monohydrochloride), Dorsomorphin-Compound C (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)pyrazolo[1,5-a]pyrimidine), VU-0418947-2 (6-Phenyl-N-[(3-phenylphenyl)methyl]-3-pyridin-2-yl-1,2,4-triazin-5-amine), JNK-9L (4-[3-fluoro-5-(4-morpholinyl)phenyl]-N-[4-[3-(4-morpholinyl)-1,2,4-triazol-1-yl]phenyl]-2-pyrimidinamine), Phloretin (3-(4-Hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one), ZG-10 ((E)-4-(4-(dimethylamino)but-2-enamido)-N-(3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide), Proscillaridin (5-[(3S,8R,9S,10R,13R,14S,17R)-14-Hydroxy-10,13-dimethyl-3-((2R,3R,4R,5R,6R)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-2H-pyran-2-one), YC-1 (3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole), IKK-2-inhibitor-V (N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide), Anisomycin ((2R,3S,4S)-4-hydroxy-2-(4-methoxybenzyl)-pyrrolidin-3-yl acetate), LY294002 (2-Morpholin-4-yl-8-phenylchromen-4-one), Colforsin ([(3R,4aR,5S,6S,6aS,10S,10aR,10b5)-5-acetyloxy-3-ethenyl-10,10b-dihydroxy-3,4a,7,7,10a-Pentamethyl-1-oxo-5,6,6a,8,9,10-hexahydro-2H-benzo[f]chromen-6-yl] 3-d imethylaminopropanoate), Rilmenidine (N-(Dicyclopropylmethyl)-4,5-dihydro-1,3-oxazol-2-amine), Selumetinib (6-(4-Bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide), GDC-0941 (Pictilisib, 4-(2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine), Valdecoxib (4-(5-methyl-3-phenylisoxazol-4-yl)benzenesulfonamide), Myricetin (3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), Cyproheptadine (4-(5H-Dibenzo[a,d]cyclohepten-5-ylidene)-1-methylpiperidine), Vorinostat (N-Hydroxy-N′-phenyloctanediamide), Nifedipine (3,5-Dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate), Phylloquinone (2-Methyl-3-[(E)-3,7,11,15-tetramethylhexadec-2-enyl]naphthalene-1,4-dione), Withaferin-A ((4β,5β,6β,22R)-4,27-Dihydroxy-5,6:22,26-diepoxyergosta-2,24-diene-1,26-dione), Temsirolimus ((1R,2R,4S)-4-{(2R)-2-[(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21 S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1,4]oxazacyclohentriacontin-3-yl]propyl}-2-methoxycyclohexyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate), SN-38 (4,11-diethyl-4,9-dihydroxy-(4S)-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione), GSK-1059615 (5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidenedione), Tipifarnib (6-[(R)-amino-(4-chlorophenyl)-(3-methylimidazol-4-yl)methyl]-4-(3-chlorophenyl)-1-methylquinolin-2-one), Linifanib (1-[4-(3-amino-1H-indazol-4-yl)phenyl]-3-(2-fluoro-5-methylphenyl)urea), WYE-354 (4-[6-[4-[(methoxycarbonyl)amino]phenyl]-4-(4-morpholinyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl-]methyl ester-1-piperidinecarboxylic acid), MK-212 (6-Chloro-2-(1-piperazinyl)pyrazine hydrochloride), and Enzastaurin (3-(1-Methylindol-3-yl)-4-[1-[1-(pyridin-2-ylmethyl)piperidin-4-yl]indol-3-yl]pyrrole-2,5-dione).
Formulations
[0216] The therapeutic or prophylactic agents described herein may be incorporated into a vehicle for administration into a patient (e.g., a mammal, such as a human). Pharmaceutical compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.
EXAMPLES
[0217] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regards as their invention.
Experimental Procedures
Example 1
Animals and Diets
[0218] Mice were subjected for methionine restriction (MR) as described in (Ables et al., 2012, 2015). Seven-weeks old male C57BL/6J mice were purchased from The Jackson Laboratory (Stock #000664, Bar Harbor, Me., USA) and housed in a conventional animal facility maintained at 20±2° C. and 50±10% relative humidity with a 12 h light: 12 h dark photoperiod. During a 1-week acclimatization, mice were fed Purina Lab Chow #5001 (St. Louis, Mo., USA). Mice were then weight matched and fed either a control (CF; 0.86% methionine w/w) or MR (0.12% methionine w/w) diet consisting of 14% kcal protein, 76% kcal carbohydrate, and 10% kcal fat (Research Diets, New Brunswick, N.J., USA) for 52 weeks. Body weight and food consumption were monitored twice weekly. Young mice were 8 weeks old (2 months) at the initiation of the experiments and 60 weeks old (14 months) upon termination. On the day of sacrifice, animals were fasted for 4 hours at the beginning of the light cycle. After mice were sacrificed by CO.sub.2 asphyxiation, liver samples were collected, flash frozen, and stored at −80° C. until analyzed.
[0219] Other mice used in this study were obtained from the colonies at University of Michigan Medical School and were subjected to interventions as described in (Harrison et al., 2014; Miller et al., 2011, 2014; Strong et al., 2016). Liver samples corresponding to lifespan-extending interventions for RNA-seq and metabolome analysis were taken at 6 and 12 months of age from male and female mice treated by drugs or exposed to caloric restriction (CR) diet from 4 months of age along with control mice, which were untreated littermate mice matched by age and sex. The design of experiment was, therefore, in accordance with intervention testing program (ITP) studies, which confirmed the lifespan-extending effect of these interventions. Interventions analyzed at 6 months of age included 40% CR, Protandim™ (1,200 ppm, as in (Strong et al., 2016)), rapamycin (42 ppm, as in (Miller et al., 2014)), 17-α-estradiol (14.4 ppm, as in (Strong et al., 2016)) and acarbose (1000 ppm, as in (Harrison et al., 2014)), while interventions analyzed at 12 months of age included 40% CR, acarbose (1000 ppm, as in (Harrison et al., 2014)) and rapamycin (14 ppm, as in (Miller et al., 2011, 2014)). All organisms received the same diet (Purina 5LG6) made in the same commercial diet kitchen (TestDiet, Richmond, Ind., USA). All mice, except for those subjected to CR, were fed ad libitum. Genetically heterogenous UM-HET3 strain, in which each mouse had unique genetic background but shared the same set of inbred grandparents (C57BL/6J, BALB/cByJ, C3H/HeJ, and DBA/2J), was used in this setting. This cross produces a set of genetically diverse animals, which minimizes the possibility that the identified signatures represent gene expression patterns specific to inbred lines. Moreover, this strain was used by ITP to test the lifespan extension potential of the compounds analyzed in this study.
[0220] Liver samples from Snell dwarf (Flurkey et al., 2001) and GHRKO (Coschigano et al., 2003) males, and their sex- and age-matched littermate controls, were taken from mice at 5 months of age belonging to (PW/J×C3H/HeJ)/F2 and (C57BL/6J×BALB/cByJ)/F2 strains, respectively.
[0221] Liver samples corresponding to tested compounds predicted with the longevity gene expression signatures via Connectivity Map (CMap) were taken at 4 months of age from UM-HET3 males given diets containing KU-0063794 (10 ppm, as in (Yongxi et al., 2015)), AZD-8055 (20 ppm, as in (García-Martínez et al., 2011)), ascorbyl-palmitate (6.3 ppm, as in (Veurink et al., 2003)) and rilmenidine (10 ppm, as in (Jackson et al., 2014)) for 1 month along with untreated littermate control mice of the same age and sex, which were fed ad libitum.
[0222] In all cases, interventions continued until the animals were sacrificed. For RNA-seq analysis corresponding to lifespan-extending interventions, 3 biological replicates were used for each experimental group, including both treated and control mice, resulting in the total of 78 samples. For metabolome analysis, we utilized at least 5 and 8 biological replicates per experimental group for treated and control mice, respectively, resulting in the total of 39 samples. For RNA-seq analysis corresponding to drugs predicted with longevity signatures, we used 4 and 8 biological replicates per experimental group for treated and control mice, respectively, resulting in the total of 24 samples. RNA was extracted from liver tissues with PureLink RNA Mini Kit as described in the protocol and passed to sequencing.
Example 2
RNAseq Data Processing and Analysis
[0223] For samples corresponding to lifespan-extending interventions, paired-end sequencing with 100 bp read length was performed on illumine HiSeq2000 platform. For samples corresponding to predicted compounds, libraries were prepared as described in (Hashimshony et al., 2016) and sequenced with 100 bp read length option on the Illumina HiSeq2500. Quality filtering and adapter removal were performed using Trimmomatic version 0.32. Processed/cleaned reads were then mapped with STAR (version 2.5.2b) (Dobin et al., 2013) and counted via featureCounts (Liao et al., 2014). To filter out genes with low number of reads, we left only genes with at least 6 reads in at least 66.6% of samples, which resulted in 12,861 and 9,352 detected genes according to Entrez annotation for RNAseq corresponding to lifespan-extending interventions and compounds predicted by CMap, respectively. Filtered data was then passed to RLE normalization (Anders and Huber, 2010).
[0224] Differential expression analysis was performed with R package edgeR (Robinson et al., 2009). For individual interventions, we declared gene expression to be significantly changed, if p-value, adjusted by Benjamini-Hochberg procedure (Benjamini and Hochberg, 1995), was smaller than 0.05 and fold change (FC) was bigger than 1.5 in any direction. When several doses and age groups were presented, we added separate factors accounting for that to the model and looked for genes significantly changed across these settings. As dose and age groups experiments were run separately and had their own controls, such factors allowed us to adjust for possible batch effect. The effects of certain interventions on different sexes were investigated separately. To determine the statistical significance of overlap between differentially expressed genes corresponding to certain interventions, we performed Fisher exact test separately for up- and downregulated genes, considering 12,861 detected genes as a background.
[0225] When performing analysis of the feminizing effect, gene expression differences were identified between control males and females from UM-HET3 strains for each age group. Gene was declared significant if p-value, adjusted by Benjamini-Hochberg procedure, was smaller than 0.05 and FC was bigger than 1.5 in any direction. The intersection of these gene sets was used for subsequent calculation of the feminizing effect and distances between sexes. The statistical significance of correlation between sex-associated differences and response to certain intervention (“feminizing effect”) was calculated using Spearman correlation test and adjusted for multiple comparisons with Benjamini-Hochberg procedure. When calculating correlation between response to certain intervention in specific age group (6 or 12 months) and female-associated differences, the latter were calculated using gene expression data for control males and females from the other age group (12 or 6 months, respectively). This approach provided us with unbiased correlations, based on different control samples and, therefore, free of regression to the mean effect. In case of MR, GHRKO and Snell dwarf mice, which possess their own controls, the feminizing effect was calculated using both age groups.
[0226] Differences in the feminizing effect of interventions in certain age groups between males and females was tested by Spearman correlation test, applied to the difference in log.sub.2FC of gender-associated genes in response to the specified conditions between males and females, and female-associated differences based on the other age group, with the following Benjamini-Hochberg adjustment. Manhattan distance between male and female gene expression profiles was calculated for individual samples in a pairwise manner using intersection of sex-specific gene sets across age groups. Unpaired Mann-Whitney test and Benjamini-Hochberg adjustment were used to assess statistical significance of difference between gender gene expression distances of control mice and animals subjected to interventions. Overlap between statistically significant sex-associated genes and genes differentially expressed in response to interventions was estimated by Fisher exact test similarly to comparison of individual interventions.
[0227] Heatmap of feminizing genes was created based on feminizing changes, aggregated across age groups, and log.sub.2FC of corresponding genes in response to individual interventions, aggregated across age groups as well (using edgeR). Only genes differentially expressed between control males and females (637 genes) were used to build the heatmap. Clustering was performed with average hierarchical approach and Spearman correlation distance.
[0228] To investigate genes responsible for the feminizing effect, we used single edgeR model to identify genes with changes associated with the feminizing effect, calculated within unbiased correlation analysis. We declared a gene to be significantly changed, if its Benjamini-Hochberg adjusted p-value was smaller than 0.05. We then took an intersection of sex-associated genes, aggregated across age groups, and genes associated with the feminizing effect, separately for up- and downregulated genes, to obtain the final list of common genes. This resulted in 164 upregulated and 153 downregulated genes.
Example 3
Metabolome Data Processing and Analysis
[0229] Metabolite profiling using four complimentary liquid chromatography-mass spectrometry (LC-MS) methods (Paynter et al., 2018) was applied to characterize metabolites and lipids of male and female UMHET-3 mice subjected to control diet, acarbose and rapamycin (Data S1A). The samples were homogenates of freshly frozen tissues of sacrificed animals, matched by age and sex. To filter out metabolites with low coverage, only metabolites detected in at least 66.6% of the samples were remained. Afterwards, filtered data were log10-transformed and scaled (Data S1B). The data were further aggregated with our previous metabolome dataset on acarbose, rapamycin, CR, GHRKO and Snell dwarf mice models together with the corresponding controls, obtained using similar experimental procedure (Ma et al., 2015). The second dataset was preprocessed in the same way as the first one. Genetic background, age groups and treatment doses in both datasets were consistent with those used for gene expression analysis.
[0230] Analysis of the feminizing effect was performed similarly to that described for gene expression. First, metabolites that differ between control males and females were identified for each dataset using limma. Metabolite was declared significant if p-value, adjusted by Benjamini-Hochberg procedure, was less than 0.1. Then, statistical significance of the feminizing effect was calculated using Spearman correlation test and adjusted for multiple comparisons with Benjamini-Hochberg. For unbiased analysis, when calculating correlation between the response to certain interventions in specific datasets (new or published one) and female-associated differences, the latter were used from the metabolite data corresponding to the other dataset (the published or the new one, respectively) together with the set of metabolites identified for that dataset. In the case of GHRKO and Snell dwarf mice, which had their own controls, the feminizing effect was calculated using both datasets.
[0231] Differences in the feminizing effect of certain interventions in certain datasets between males and females was tested by Spearman correlation test, applied to the difference in log.sub.2FC of gender-associated metabolites (identified based on the other dataset) in response to the specified conditions between males and females, and female-associated differences from the other dataset, with the following Benjamini-Hochberg adjustment. Manhattan distance between male and female metabolite profiles was calculated for individual samples in a pairwise manner using intersection of sex-specific metabolite sets across datasets. Unpaired Mann-Whitney test and Benjamini-Hochberg adjustment were used to assess statistical significance of difference between gender-associated metabolite profile distances of control mice and animals subjected to interventions.
Example 4
Functional Enrichment Analysis
[0232] For identification of functions enriched by genes differentially expressed in response to individual interventions within our RNAseq data and aggregated across datasets (CR, rapamycin and GH deficiency interventions), commonly changed across interventions (common signatures) as well as associated with the effect on lifespan, we performed GSEA (Subramanian et al., 2005) on a pre-ranked list of genes based on log.sub.10(p-value) corrected by the sign of regulation, calculated as:
log.sub.10(pv)×sgn(lfc),
where pv and lfc are p-value and logFC of certain gene, respectively, obtained from edgeR output, and sgn is signum function (is equal to 1, −1 and 0 if value is positive, negative and equal to 0, respectively). REACTOME, BIOCARTA, KEGG and GO biological process and molecular function from Molecular Signature Database (MSigDB) have been used as gene sets for GSEA (Subramanian et al., 2005). q-value cutoff of 0.1 was used to select statistically significant functions. Significance scores of enriched functions were calculated as:
significance score=−log.sub.10(qv)×sgn(NES),
where NES and qv are normalized enrichment score and q-value, respectively.
[0233] Horizontal and vertical barplots were shown for manually chosen statistically significant functions with size of barplot being dependent on value of significance score. For functions associated with the lifespan effect and common signatures across tissues, heatmap colored based on significance scores was used. Clustering of functions enriched by individual interventions within RNAseq data was performed based on NES of functions with statistically significant enrichment (q-value <0.1) by at least one intervention. Clustering has been performed with hierarchical average approach and Spearman correlation distance.
[0234] To identify functions enriched by genes shared by differences between males and females along with changes in response to lifespan-extending interventions in males, we performed Fisher exact test using Database for Annotation, Visualization and Integrated Discovery (DAVID) (Huang et al., 2009a, 2009b). INTERPRO, KEGG and GO BP and MF databases were used. We declared functions to be enriched if their Benjamini-Hochberg adjusted Fisher exact test p-value was smaller than 0.1.
[0235] To perform further functional enrichment analysis of molecular pathways by CR and GH deficiency, we applied iPANDA method (Ozerov et al., 2016) to every individual dataset related to these interventions and obtained corresponding pathway activation scores (PAS) for each of them. PAS is based on both statistical significance and the strength of activation of the certain pathway. As some of the individual datasets measure response to certain intervention using the same control sampling, to calculate the aggregated PAS together with its p-value for the certain intervention, we used mixed-effect model, based on all single PAS values obtained from individual datasets with random term corresponding to the use of the same control sampling for calculation of gene expression change. Mixed-effect model was built with R package metafor (Viechtbauer, 2010). Obtained p-values were adjusted for multiple comparisons with Benjamini-Hochberg procedure. Functions were considered to be significantly enriched if their adjusted p-value was smaller than 0.1. Barplots with manually chosen enriched functions were built with the size of bars corresponding to the value of significance score, calculated as:
significance score=−log.sub.10(adj.pv)×sgn(agPAS),
where adj. pv and agPAS are BH adjusted p-value and aggregated PAS obtained from mixed-effect model output, respectively.
Example 5
Aggregation of RNAseq and Microarray Datasets for Meta-Analysis
[0236] To identify signatures associated with lifespan extension and the effect of certain interventions, we expanded our data with publicly available datasets from Gene Expression Omnibus (GEO) (Edgar, 2002) and ArrayExpress (Kolesnikov et al., 2015) databases. For the analysis of signatures associated with certain interventions (CR, rapamycin, GH deficiency), we integrated available gene expression data obtained from liver of mice from healthy genetic strains on standard diets subjected to CR, rapamycin and mutations associated with GH deficiency (Ames dwarf mice, GHRKO, Little mice, Snell dwarf mice). For the analysis of signatures shared across lifespan-extending interventions, we included only the data with the experimental design statistically confirmed to extend lifespan. Finally, for the analysis of signatures associated with the lifespan extension effect, we integrated datasets on interventions with available and reliable survival data corresponding to the same experimental design (sex, strain, dose, age when the intervention started). In total, our hepatic meta-analysis covered 17 different interventions presented in 77 control-intervention datasets from 22 different sources (including ours) (
[0237] To aggregate data across different platforms and studies, we developed the following method. First, data within each study was preprocessed independently and log-transformed to conform to normal distribution if needed. Then, filtering of low-covered genes was performed with soft threshold. Then, all identifiers were mapped to Entrez ID gene format, and genes not detected in our RNAseq data were filtered out. This resulted in the coverage of 12,861 genes or less if some of these genes were filtered out because of the low coverage. Afterwards, samples within every study were normalized by quantile normalization and scaling, followed by multiplication by the certain value to make it on the same scale as RNAseq data with more natural interpretation. Finally, mean and standard error of logFC of every gene for every response to intervention was calculated together with p-value (along with Benjamini-Hochberg adjusted p-value) estimated by edgeR (Robinson et al., 2009) and limma (Ritchie et al., 2015) for RNAseq and microarrays datasets, respectively. This resulted in 2 values representing every gene from every dataset. Importantly, one study may include several datasets if several interventions or settings have been analyzed there, and sometimes, different interventions or doses share the same control samples. This may be a source of batch effect, which we removed during subsequent steps of the analysis.
Scaling of genes within every sample, performed before calculation of logFC, results in similar and comparable distribution of gene changes across different studies and platforms. Importantly, scaling is not performed after calculation of logFC as different interventions may lead to different size of gene expression profile perturbation. Indeed, lifespan-extending genetic manipulations generally lead to bigger perturbation of transcriptome compared to diets and compounds (
Example 6
[0238] Identification of genes associated with individual longevity interventions logFC calculated for every dataset were further used as inputs to the statistical tests for meta-analysis. To account for standard error of logFC and remove batch effect related to the belonging of several datasets to the same study or same control sampling within the study, we applied mixed-effect model using R package metafor (Viechtbauer, 2010). As an input, we used both mean and standard error of logFC. Such approach allowed us to account for the size of the effect and variance of estimated gene expression change within each individual dataset, which provides a more sensitive and accurate analysis compared to previous studies focused on the comparison of lists of differentially expressed genes.
[0239] When calculating gene expression changes of individual interventions across different sources (such as CR and rapamycin), to remove batch effect, belonging to the same study or control group was considered as a random term. When calculating such changes for GH deficiency interventions, we also included type of intervention as a random term. Using this procedure, we obtained aggregated logFC and corresponding p-value for every gene. Besides standard p-value, we also calculated leave-one-out (LOO) and robust p-value. ‘LOO p-value’ is defined as the highest p-value after removal of every study one by one. On the other hand, ‘robust p-value’ is the lowest p-value after the same procedure. Benjamini-Hochberg procedure was used to adjust every type of p-value for multiple comparisons. We declared genes to be differentially expressed in response to CR, rapamycin and GH deficiency across datasets if adjusted p-value was smaller than 0.01 and their LOO p-value was smaller than 0.01. The significance of overlap between the lists of differentially expressed genes obtained from meta-analysis was estimated by Fisher exact test separately for up- and downregulated genes, considering 12,861 detected genes as background.
[0240] Similarly, aggregated logFC together with p-values were calculated for all interventions presented in our data by multiple sources. For interventions presented as a single dataset, logFC and p-values were obtained from individual datasets as described previously. For interventions measured in several datasets from the same source, single edgeR or limma model was used depending on the origin of the data (RNAseq or microarray). This resulted in the matrix containing aggregated log.sub.2FC values of every gene in response to different interventions. To visualize change of each gene within each individual intervention, we built barplots representing aggregated log2FC of a certain gene in response to all intervention where it has been detected. Statistically significant changes were defined based on Benjamini-Hochberg adjusted p-value.
[0241] To identify upstream regulators of the detected gene expression response to CR, rapamycin and GH deficiency, we applied the Biobase Transfac platform (Matys, 2006). First, for every individual dataset, we identified transcription factor binding to sequences enriched in the promoters of differentially expressed genes using the platform. This resulted in a matrix, where every transcription factor was either enriched (1) or not (0) for the certain dataset. At this step, we excluded redundant IDs corresponding to different binding patterns of the same factor by considering factor to be enriched if at least one of its patterns is enriched. This resulted in 1,466 different upstream regulators. To identify factors overrepresented across different datasets of the same intervention, we applied permutation version of binomial statistical test as described in (Plank et al., 2012). Briefly, to identify the p-value threshold corresponding to the desired FDR (equal to 0.01), permutation test is performed, where 1 and 0 (corresponding to enrichment of different transcription factors) are shuffled within each dataset and number of false positives for different binomial test p-value thresholds are calculated. Based on the obtained numbers, p-value threshold ensuring FDR threshold of 0.01 is determined. The significance of overlap between enriched upstream regulators of different interventions was estimated by Fisher exact test, considering 1,466 non-redundant transcription factors as background.
Example 7
Analysis of Mutual Organization of Interventions
[0242] To assess similarity of gene expression response across interventions, we built a heatmap of aggregated log.sub.2FC of genes significantly changed in response to CR, rapamycin and GH deficiency interventions (2507 genes in total). Complete hierarchical clustering was employed for the heatmap. Correlation matrix representing similarity between aggregated logFC of different interventions was calculated based on Spearman correlation coefficient.
[0243] To calculate correlations between interventions in unbiased way, we applied the following approach. For every pair of interventions, including comparison of intervention with itself, we examined all pairs of datasets from different sources. For each such pair we selected 250 genes consisting of 125 genes with the most significant expression change (with the lowest p-values) from each dataset and calculated Spearman correlation coefficient within the pair. We reiterated this algorithm and, as a result, for every pair of interventions obtained distribution of Spearman correlation coefficients, calculated between datasets from different sources. For CR and rapamycin, we visualized these distributions using violinplot. One-sample Mann-Whitney test and Benjamini-Hochberg adjustment were used to check if means of correlation coefficients are different from 0 with statistical significance. We declared correlation coefficient to be significant if adjusted p-value was smaller than 0.1.
[0244] For correlation matrix we employed median values of Spearman correlation coefficients. By filtering out comparisons of datasets from the same source, we removed possible batch effect and ended up with independent and unbiased comparison of interventions. However, as some interventions were presented only within the same source, we couldn't estimate unbiased correlation for such cases. This missing data was visualized by grey boxes. The same was sometimes true for comparison of intervention with itself, as in this case we also employed only datasets from different sources. For this reason, correlation coefficient of intervention with itself was not equal to 1 in resulted unbiased correlation matrix. Complete hierarchical clustering approach was employed for visualization of correlation matrix.
[0245] To demonstrate similarities between different interventions in network mode, we employed Cytoscape (Shannon et al., 2003). Only edges between interventions with significant positive correlation coefficients (median Spearman correlation coefficient >0 and adjusted Mann-Whitney p-value <0.1) were shown. The width of edge was defined by the log.sub.10(adjusted p-value). Smaller p-value led to wider edge.
Example 8
[0246] Identification of Common Signatures and Genes Associated with the Lifespan Effect
[0247] To identify hepatic genes, whose expression change is shared across lifespan-extending interventions, we filtered out all interventions and settings with unproven lifespan extension effects. To account for possible differences in the intervention effect on lifespan across different sexes, ages, strains and doses, we only considered the datasets, whose experimental settings were shown to produce a statistically significant extension of lifespan. Therefore, for example, 40% CR in C57BL/6 females was excluded from the analysis as this setting doesn't lead to a statistically significant lifespan extension, contrary to 20% CR applied to the same mouse strain (Mitchell et al., 2016). In the case of drugs, we also filtered out the datasets containing the response to compounds, which had not been confirmed by ITP studies (such as metformin and resveratrol).
[0248] First, for every single gene we calculated number of interventions, where it is differentially expressed based on adjusted aggregated p-value estimated as described previously. We considered gene to be differentially expressed if its adjusted aggregated p-value was smaller than 0.1. However, this approach overfits genes changed in response to similar interventions (such as GH deficiency interventions) and doesn't take into account possible consistent changes, which may be, however, not significant due to low sampling size or high variance. To overcome this problem, we applied single mixed-effect model to every gene as described previously and looked for genes, whose aggregated logFC across lifespan-extending interventions is significantly different from 0. Here, however, we also included the type of intervention as a random term together with correlation matrix specifying similarities between general response of the interventions. This correlation matrix was taken from unbiased mutual organization analysis described previously. We declared genes to be significantly shared across interventions if Benjamini-Hochberg adjusted robust p-value, obtained after removal of every type of intervention one by one, was smaller than 0.05. The same approach was used to identify genes shared across lifespan-extending interventions in the skeletal muscle and WAT. Heatmap with expression changes of significant genes across individual datasets was clustered using a complete hierarchical approach.
[0249] To identify genes associated with the lifespan effect, first, we estimated three main metrics of lifespan for every available setting, including median lifespan ratio (in logarithmic scale), maximum lifespan ratio (in logarithmic scale), defined as ratio of average lifespan of 10% most survived individuals, and median hazard ratio, defined as ratio of slopes of survival curves at the median point (timepoint where 50% of cohort is remained survived). These metrics were obtained from published survival data for the corresponding interventions. To account for heterogeneity of our data, we integrated gene expression and longevity studies only if they were associated with the same experimental design (sex, dose, strain, age when intervention started). We then calculated average metric values across the studies to obtain most consistent and reliable estimates. Interventions or settings, for which no appropriate longevity study was available, were excluded.
[0250] Afterwards, we applied mixed-effect model as described previously to identify genes associated with each of the 3 numeric metrics of the lifespan effect. Control group and type of intervention were considered as random term, and correlation matrix between interventions was used to define covariance matrix. We declared genes to be significantly associated with the lifespan effect if Benjamini-Hochberg adjusted p-value and LOO p-value, obtained after removal of every intervention one by one, were both smaller than 0.05. The significance of overlap between lists of genes associated with different metrics of the lifespan effect was estimated by Fisher exact test separately for genes with positive and negative association, considering 12,861 detected genes as a background. Complete hierarchical clustering was used to sort genes on heatmap, representing logFC of genes with significant association across individual datasets. Individual datasets were sorted there based on their effect on maximum lifespan.
[0251] Overlap between gene signatures associated with lifespan extension and genes, whose manipulation was demonstrated to extend or shorten mouse lifespan, was estimated by Fisher exact test, as described previously. The latter set was obtained from GenAge database and included 84 and 44 genes with pro- and anti-longevity effects, respectively (De Magalhães and Toussaint, 2004).
Example 9
[0252] Test for Association with Longevity Signatures
[0253] To test association of interventions with longevity signatures related to individual interventions (CR, rapamycin and GH deficiency), common changes and lifespan effect association, we employed GSEA-based approach. First, for every signature we specified 250 genes with the lowest p-values and divided them into up- and downregulated genes. These lists were considered as gene sets. Then we ranked genes related to interventions of interest based on their p-values, calculated as described in functional enrichment section. When running association test for lifespan-extending interventions (
[0254] For interventions from publicly available sources (
[0255] For compounds predicted with the longevity signatures via CMap, we calculated p-values of gene expression changes compared to control independently for every drug using edgeR. We then converted them to log.sub.10(p-value) corrected by the sign of regulation as described earlier and proceeded to GSEA-based analysis.
[0256] We calculated GSEA scores separately for up- and downregulated lists of gene set as described in (Lamb et al., 2006) and defined final GSEA score as a mean of the two. To calculate statistical significance of obtained GSEA score, we performed permutation test where we randomly assigned genes to the lists of gene set maintaining their size. To get p-value of association between certain intervention and longevity signature, we calculated the frequency of real final GSEA score being bigger by absolute value than random final GSEA scores obtained as results of 3,000 permutations. To adjust for multiple comparisons, we performed Benjamini-Hochberg procedure. Resulted adjusted p-values were converted into significance scores as:
significance score=−log.sub.10(adj.pv)×sgn(GSEA score),
where adj. pv and GSEA score are BH adjusted p-value and final GSEA score, respectively. Heatmaps were colored based on values of significance scores.
Example 10
RNAseq Analysis Across Lifespan-Extending Interventions
[0257] We subjected 78 young adult mice to 8 interventions previously established to extend lifespan, including acarbose, 17-α-estradiol, rapamycin, Protandim, CR (40%), MR (0.12% methionine w/w), GHRKO and Pit1 knockout (Snell dwarf mice) (3 biological replicates were used in each experimental group;
[0258] Differentially expressed genes associated with each intervention were initially examined separately for males and females. Many differentially expressed genes were found to be common to interventions. For example, almost half of MR genes (44.3% upregulated and 41.8% downregulated genes) were altered significantly and in the same direction in Snell dwarf males and CR males and females (
[0259] Analysis of enriched functions using gene set enrichment analysis (GSEA) (Subramanian et al., 2005) revealed many similarities among the interventions (
[0260] In addition to common strategies, we detected some distinct signatures. For example, 17-α-estradiol in females and MR resulted in downregulation of oxidative phosphorylation. Although ribosomal protein genes, in general, represented the most common upregulated pattern across the interventions, this was not the case for mitochondrial ribosomal protein genes. Some interventions, including CR, GHRKO, Snell dwarf mice and acarbose in males, showed significant upregulation of these genes, whereas 17-α-estradiol in both sexes and MR showed their downregulation. Finally, fatty acid oxidation, which is known to be positively associated with the lifespan extension effect of several interventions (Amador-Noguez et al., 2004; Plank et al., 2012; Tsuchiya et al., 2004), was significantly downregulated when applied to females (
[0261] Interestingly, although MR mice resemble CR mice in stress resistance and endocrine changes, and MR mice share many differentially expressed genes with CR and growth hormone (GH) deficiency-associated interventions (i.e. GHRKO and Snell dwarf mice), MR displayed a quite distinct pattern at the level of functional enrichment (
[0262] To get a more global view on the similarities among interventions in terms of regulation of cellular pathways, we built a heatmap of normalized enrichment scores (NES) of all functions enriched by at least one intervention and clustered the data using an average hierarchical approach (
Example 11
Feminizing Effect of Lifespan-Extending Interventions
[0263] The finding of sex-specific gene expression changes in response to longevity interventions allowed us to examine this question in more detail. Several previous studies noted a feminizing effect of CR and GH deficiency on gene expression in males (Buckley and Klaassen, 2009; Estep et al., 2009; Fu and Klaassen, 2014; Li et al., 2013). To test if this effect is reproduced across different interventions, we first identified genes whose expression significantly differs between control males and females from UM-HET3 strains in both 6- and 12-month-old age groups. We then examined how lifespan-extending interventions affect these sex-associated differences. To analyze it in an unbiased way free of regression to the mean effect, for every intervention of a certain sex and age, we calculated the Spearman correlation of its gene expression response with the differences between males and females, calculated for another age group. In the case of Snell dwarf mice, GHRKO and MR, which had their own controls, we used both age groups for the calculation.
[0264] In males, we detected statistically significant feminizing-like patterns for genetic (GHRKO and Snell dwarf mice) and dietary (CR and MR) interventions at the gene expression level (
[0265] In females, the effect of interventions on sex-associated expression differences was mostly similar to that in males. For example, CR (Spearman correlation=0.12 and 0.2 and BH adjusted p-value=0.07 and 3.7.Math.10.sup.−3 for 12- and 6-month age groups, respectively) and 12-month old acarbose (Spearman correlation=0.19 and BH adjusted p-value=4.7.Math.10.sup.−3) females also exhibited a significant feminizing-like pattern (
[0266] Although various interventions had a different effect on feminizing genes across sexes, we observed a consistently stronger feminizing effect in males compared to females for every individual intervention and age group (Spearman correlation test BH adjusted p-value <2.6.Math.10.sup.−6), except for Protandim, which showed the opposite trend (
[0267] To validate our findings at the level of metabolome, we performed metabolite profiling of 39 12-month-old male and female mice subjected to control diet, acarbose and rapamycin (at least 5 biological replicates in each experimental group). We further aggregated this data with our previous dataset, which included female and male mice of the same age subjected to control diet, CR, acarbose and rapamycin as well as male GHRKO and Snell dwarf mice (Ma et al., 2015). Using a similar procedure, we identified metabolites that significantly differ between control males and females in each of the datasets and then used them to calculate the feminizing effect at the metabolome level. In agreement with the gene expression results, we observed a significant feminizing effect of genetic interventions (GHRKO and Snell dwarf mice), CR, and acarbose in males (
[0268] To better understand the nature of the feminizing pattern, we identified sex-associated genes which change in response to interventions is, at the same time, associated with the feminizing effect. With the FDR threshold of 0.05 and FC threshold of 1.5, we detected 355 sex-associated genes differentially expressed at a higher level in females and 282 genes expressed at a lower level (
[0269] Among downregulated sex-associated genes, we detected enrichment of complement and coagulation cascades (Fisher exact test BH adjusted p-value=9.8.Math.10.sup.−3) and major urinary proteins (MUP) genes (Fisher exact test BH adjusted p-value=0.021) (
[0270] Overall, the data show that the feminizing effect is shared by genetic and dietary lifespan-extending interventions in males at both gene expression and metabolome levels, and that this effect is achieved through perturbations of common genes and molecular pathways including those regulated by GH. The feminizing effect does not explain lifespan extension but is consistently higher in males compared to females subjected to the same intervention, regardless of its direction and size. It also appears to reduce gender-associated differences at the gene expression and metabolite levels, pointing to the converging effect of lifespan-extending interventions on hepatic transcriptome and metabolome across sexes.
Example 12
Signatures of CR, Rapamycin and Growth Hormone Deficiency
[0271] To obtain a comprehensive picture of gene expression responses to interventions, we collected all publicly available microarray datasets for mouse liver and conducted a meta-analysis across aggregated data. We first focused on 3 interventions: CR, rapamycin and interventions related to GH deficiency (GHRKO, Little mice, Snell and Ames dwarf mice). The latter group was combined, because these interventions, although targeting different genes involved in GH production and sensing, result in a similar effect on liver due to inability to activate GHR. In addition to this mechanistic notion, similarity among these interventions could also be seen at the level of hepatic gene expression as demonstrated by other groups (Amador-Noguez et al., 2004) and our results (
[0272] To overcome issues associated with differences in platforms across different studies, along with batch effects, we developed an integrative method, based on independent preprocessing and normalization of individual datasets and following aggregation of means and standard deviations of logFC for all genes detected in our RNAseq data (resulting in 12,861 genes). Importantly, to account for possible differences in the general effect of interventions on mouse transcriptome, we did not normalize distributions of logFC across datasets. To include information about standard deviations of logFC and account for possible batch effects due to the use of several datasets sharing the same control (e.g., if several doses were tested), we applied a mixed-effect model, considering shared control as a random term. We used this method to identify genes up- or downregulated across datasets associated with the same type of intervention. Our approach, contrary to the comparison of lists of differentially expressed genes used in previous meta-analyses (Plank et al., 2012; Swindell, 2008), accounts for the size of the effect and variance of gene expression change within each individual dataset and, therefore, provides a more accurate and sensitive analysis. Besides standard p-value, obtained from the mixed-effect model test, we calculated “leave-one-out” (LOO) p-value as the largest (least significant) p-value after removal of every study one by one.
[0273] In this procedure, genes were designated statistically significant if their BH adjusted p-value was <0.01 and LOO p-value was <0.01. With these thresholds, we identified 419 up- and 370 downregulated genes for CR, 894 up- and 879 downregulated genes for GH deficiency, and 127 up- and 100 downregulated genes for rapamycin (
[0274] By applying GSEA, we further identified several pathways shared by 2 or all 3 analyzed interventions (
[0275] To obtain further details on the regulation of molecular pathways by CR and GH deficiency, we used the iPANDA algorithm (Ozerov et al., 2016), which is another method of functional enrichment analysis that utilizes the sign of the effect of each specific gene on pathway activation or inhibition. We applied it to every individual dataset included in our meta-analysis and calculated an aggregated pathway activation score (PAS) along with its statistical significance using the mixed effect model described previously. In agreement with the GSEA output, we observed activation of TCA cycle, respiratory electron transport chain, urea cycle and PPAR pathways along with inhibition of alternative complement, interferon and insulin processing pathways in both CR (
[0276] To identify upstream regulators of observed gene expression changes, we analyzed enrichment of transcription factors associated with differentially expressed genes using the Biobase Transfac platform (Matys, 2006). First, for each individual dataset we identified transcription factors binding to sequences enriched in promoters of genes differentially expressed in the corresponding dataset. We then applied a binomial statistical test to identify factors whose enrichment was overrepresented across datasets within the same type of intervention. A permutation FDR threshold of 0.01 resulted in the identification of 161 transcription factor IDs enriched for CR, 213 IDs enriched for GH-deficient interventions and 17 IDs enriched for rapamycin (
Example 13
Mutual Organization of Gene Expression Profiles of Lifespan-Extending Interventions
[0277] We next performed a meta-analysis of the dataset that included, in addition to the gene expression data we generated, all publicly available microarray data on lifespan-extending interventions in mouse liver. We also included resveratrol and metformin, which are interventions that did not increase lifespan in the ITP mouse cohort at the concentrations used (Miller et al., 2011; Strong et al., 2013, 2016), but are known to share some molecular mechanisms with lifespan-extending CR (Barger et al., 2008; Dhahbi et al., 2005; Martin-Montalvo et al., 2013; Pearson et al., 2008), increase healthspan of mammals, including improvement of cardiovascular function and physical performance along with inhibition of inflammation (Baur and Sinclair, 2006; Martin-Montalvo et al., 2013; Pearson et al., 2008), and lead to increased longevity of the nematode Caenorhabditis elegans (De Haes et al., 2014; Viswanathan et al., 2005; Wood et al., 2004), short-lived fish Nothobranchius furzeri in case of resveratrol (Valenzano et al., 2006), and mice under certain conditions (Baur et al., 2006; Martin-Montalvo et al., 2013; Pearson et al., 2008). After integration of all available data, our dataset included 17 different interventions and 77 control-intervention comparisons across 22 different sources (
[0278] Aggregation of data was performed using the approach discussed above. Interestingly, comparison of standard deviations of gene expression fold change distributions in response to different interventions showed that genetic manipulations had the largest effects on gene expression profile (Mann-Whitney test p-value=0.003 between dietary and genetic intervention groups), whereas pharmacological interventions had the smallest effect (Mann-Whitney test p-value=1.71.Math.10.sup.−6 between pharmacological and dietary intervention groups) and dietary interventions were in the middle (
[0279] To examine how similar various interventions are in terms of gene expression signatures identified for CR, GH deficiency and rapamycin, we created a heatmap representing aggregated gene expression data across interventions for the identified genes (
[0280] To overcome the batch effect and investigate mutual organization of gene expression profiles of different interventions at the level of whole transcriptomes, we compared interventions pairwise, considering, for every pair of interventions, only pairs of control-intervention comparisons from different sources. For each of them, we calculated the Spearman correlation coefficient using the 250 most statistically significant differentially expressed genes. We then examined the distribution of these correlation coefficients among all pairs of control-intervention comparisons. Using this approach, we could get rid of the batch effect in that datasets from the same study were not compared when calculating the correlation coefficient. We also used the same unbiased procedure to obtain the distribution of correlation coefficients between different datasets of the same intervention. This let us investigate how consistent gene expression response to certain intervention is across different studies and experimental design settings.
[0281] For CR, this method resulted in statistically significant positive correlations with the majority of interventions, including all GH deficiency interventions (BH adjusted Mann-Whitney p-value <6.1.Math.10.sup.−10 for all of them), dietary interventions, such as CR itself (BH adjusted Mann-Whitney p-value=1.2.Math.10.sup.−95), MR and EOD (BH adjusted Mann-Whitney p-values <1.95.Math.10.sup.−5), as well as FGF21 overexpression, acarbose, 17-α-estradiol, metformin and resveratrol (BH adjusted Mann-Whitney p-values <3.2.Math.10.sup.−3) (
[0282] Using the same approach, we prepared a matrix with median Spearman correlation coefficients for every pair of interventions aggregated across all control-intervention comparisons from different sources (
[0283] Overall, most lifespan-extending interventions showed similar gene expression patterns both at the level of whole transcriptomes and particular genes. However, some interventions, such as rapamycin, Protandim, S6K1 −/− and MYC +/−, showed quite distinct transcriptional patterns in liver, and did not demonstrate statistically significant positive correlation with any other intervention (
Example 14
Common Signatures Across Lifespan-Extending Interventions
[0284] To identify gene signatures commonly up- or downregulated by lifespan-extending interventions, which could serve as an approximation of ‘necessary’ features and qualitative predictors of lifespan extension, we first identified statistically significant genes regulated by each individual intervention using the same approach as in case of CR, rapamycin and GH deficiency interventions, where datasets from several independent sources were present. To account for possible differences of the intervention effect on lifespan across doses, ages, strains and sexes, introduced by heterogeneity of our data, here we only considered the datasets, whose experimental conditions were shown to produce statistically significant extension of lifespan.
[0285] Using the intervention-wise approach, for every gene we calculated the number of interventions, where it was up- or downregulated (
[0286] To overcome this problem, we searched for genes shared by different interventions using a single mixed-effect model with an additional random term corresponding to intervention type and correlation matrix for this term composed from means of correlation coefficients of gene expression changes between the corresponding interventions across all possible pairs of datasets (
[0287] To detect genes commonly shared by most interventions, we weakened the criteria by letting one intervention to be an outlier. We accomplished this by removing each intervention one by one and taking the best remaining p-value (“robust p-value” approach). Using the BH adjusted robust p-value threshold of 0.05, we identified 166 upregulated and 134 downregulated genes (
[0288] Another interesting example of a gene commonly upregulated across lifespan-extending interventions is Brca1 (BH adjusted p-value=0.04) (
[0289] Several glutathione S-transferase genes were also significantly upregulated across lifespan-extending interventions, including Gstt2 (BH adjusted robust p-value=0.014), Gsto1 (BH adjusted robust p-value=0.037) and Gsta4 (BH adjusted robust p-value=0.013) (
[0290] To identify pathways associated with common up- and downregulated gene signatures, we performed functional GSEA (
[0291] To generalize our findings across tissues, we aggregated publicly available data on gene expression responses to lifespan-extending interventions in two additional tissues, skeletal muscle and white adipose tissue (WAD. Using the same methods and threshold criteria, we examined this dataset for common longevity signatures in each tissue. We identified 160 and 390 upregulated along with 123 and 325 downregulated genes for the muscle and WAT, respectively. Interestingly, there was almost no overlap between common gene expression signatures across different tissues (
Example 15
[0292] Signatures Associated with the Degree of Lifespan Extension
[0293] To identify genes positively and negatively associated with the degree of lifespan extension, potentially serving as quantitative predictors of longevity, we integrated a previously described mixed-effect regression model with 3 commonly used metrics of lifespan extension obtained from published survival data on corresponding interventions: median lifespan ratio, maximum lifespan ratio, calculated as the ratio of average lifespan of 10% longest-surviving individuals, and median hazard ratio, calculated as the ratio of slopes of survival curves at the timepoint where 50% of cohort is alive. We used these metrics as they seem to be the most consistent and robust to the effects of sampling size (Moorad et al., 2012). To account for heterogeneity of the data, we integrated gene expression and the longevity data only if they were associated with the same experimental design in terms of sex, strain, dose and the age at which the intervention started. As in the case of common signatures, we considered source and type of intervention as random terms and used the correlation matrix of interventions to account for similarity between them.
[0294] We designated genes as statistically significant if their BH adjusted p-value and LOO p-value, obtained after removal of every intervention one by one, were both <0.05. With these thresholds, we detected 351, 258 and 183 genes with positive and 264, 191 and 108 genes with negative association with maximum lifespan ratio, median lifespan ratio and median hazard ratio, respectively (
[0295] Other genes positively associated with changes in both maximum and median lifespan included members of fatty acid metabolism, including acyl-coenzyme A dehydrogenase Acadm (BH adjusted p-value=0.001 and 0.005 for maximum and median lifespan, respectively) and enoyl-coenzyme A delta isomerase Eci1 (BH adjusted p-value=2.2.Math.10.sup.−6 and 6.4.Math.10.sup.−6) (
[0296] Interestingly, the fat synthesis enzyme Dgat1, those knockout is associated with extension of mean and maximum lifespan in female mice by 23% and 8%, respectively (Streeper et al., 2012), was found to be slightly positively associated with median and maximum lifespan effects across interventions (slope coefficient=0.38 and 0.29 and BH adjusted p-value=0.007 and 0.04 for maximum and median lifespan, respectively) (
[0297] To check if such pattern is universal for different genes, we compared the identified genes shared across signatures and associated with the degree of lifespan effect with the genes whose perturbation was demonstrated to extend mouse lifespan, obtained from GenAge (18 pro- and 38 anti-longevity genes) (De Magalhães and Toussaint, 2004). Indeed, we observed almost no overlap between these gene sets (Fisher exact test p-value >0.33 for all pairwise comparisons) (
[0298] To identify pathways enriched by genes positively and negatively associated with the lifespan extension effect, we ran GSEA for all 3 metrics of lifespan extension and observed general consistency among them in terms of functional enrichment (
[0299] Interestingly, some of the hepatic genes and pathways could be used for the prediction of both lifespan extension per se (qualitative estimate) as well as degree of this effect (quantitative estimate), being both common signatures and signatures associated with the lifespan extension effect. We identified 26 genes being both commonly changed across interventions and associated with either median or maximum lifespan extension effect in the same direction. 17 of them were upregulated and positively associated with lifespan extension, while 9 were downregulated and negatively associated. The identified genes are involved in regulation of apoptosis (Aatk, Net1, Rb1, Sgms1), immune response (C4 bp, P2ry14, Slc15a4, Tap2, Rb1), transcription (Pir, Sall1), stress response (Net1, Nqo1, Pck2, Rb1), glucose metabolism (Pck2, Pgm1) and cellular transport (Ldirad3, Slc15a4, Slc25a30 and Tap2).
[0300] For example, Nqo1, encoding NAD(P)H-dependent quinone oxidoreductase involved in oxidative stress response, showed a significant positive association with maximum and median lifespan (BH adjusted p-value=0.002 and 7.74.Math.10.sup.−5, respectively) and was also commonly upregulated across lifespan-extending interventions (BH adjusted robust p-value=0.011) (
[0301] Another interesting example is Slc15a4, which codes for lysosome-based proton-coupled amino-acid transporter of histidine and oligopeptides from lysosome to cytosol. In dendritic cells, this protein regulates the immune response by transporting bacterial muramyl dipeptide (MDP) to cytosol and, therefore, activating the NOD2-dependent innate immune response (Nakamura et al., 2014). In addition, its activity affects endolysosomal pH regulation and probably v-ATPase integrity, required for mTOR activation (Kobayashi et al., 2014). Our data show that Slc15a4 is a common signature of lifespan-extending interventions (BH adjusted robust p-value=0.008) along with some other transporters (
[0302] As for the pathways, oxidative phosphorylation showed positive association with both common and lifespan effect associated signatures, and some functions involved in liver regulation of immune response showed negative association (
[0303] To make our data and tools available to the research community, we developed a web application, GENtervention, based on the R package shiny (Chang et al., 2016). It allows interrogation of gene expression data and, for every gene, it offers (i) expression change across different datasets related to every individual intervention (e.g.
Example 16
Application of Longevity Signatures for the Identification of New Candidates for Lifespan Extension
[0304] In this work, we obtained gene expression patterns (signatures) associated with the response to particular well-studied interventions (CR, rapamycin and GH deficiency interventions), as well as signatures based on gene sets commonly regulated across different interventions and associated with the degree of lifespan extension. We considered the possibility that these ‘longevity signatures’ could be used as predictors of new lifespan-extending interventions at the gene expression level. We examined this possibility with two approaches. First, we checked if the signatures can be used to predict potential association of interventions of interest with the longevity gene expression response using publicly available datasets. Second, we tested their capability to predict new candidates for lifespan extension using the Connectivity Map (CMap) platform (Lamb et al., 2006; Subramanian et al., 2017).
[0305] For the first study, we preprocessed 6 publicly available datasets on hepatic gene expression in response to certain in vivo interventions in mouse models, including injection of interleukin 6 (IL-6) (Ramadoss et al., 2010), knockout of methionine adenosyltransferase gene (Mat1a) (Alonso et al., 2017), hypoxia conditions (Baze et al., 2010), knockout of Keap1 coding for an inhibitor of acute stress regulator NRF2 (Osburn et al., 2008), supplementation of SIRT1 activator SRT2104 (Mercken et al., 2014b) and overexpression of the sirtuin gene Sirt6 (Kanfi et al., 2012). We then ran a GSEA-based association test using longevity signatures as input subsets (
[0306] Interleukin-6 (IL-6) is one of the best studied pro-inflammatory cytokines secreted by T cells and macrophages to support the immune response. It was shown to stimulate the inflammatory and auto-immune response during progression of diseases, including diabetes (Kristiansen and Mandrup-Poulsen, 2005), Alzheimer's disease (Swardfager et al., 2010), multiple myeloma (Gadó et al., 2000) and others. Moreover, IL-6 was shown to induce insulin resistance directly by inhibiting insulin receptor signal transduction (Senn et al., 2002). Finally, functions related to liver regulation of the immune response stimulated by IL-6 were enriched for genes both commonly downregulated and negatively associated with the lifespan extension effect of longevity interventions. We tested if the intraperitoneal injection of interleukin-6 into mouse bloodstream leads to hepatic gene expression changes associated with longevity signatures and detected a significant negative association with all longevity signatures (BH adjusted p-value <0.025) (
[0307] Methionine adenosyltransferase 1A (Matta) is an enzyme that catalyzes conversion of methionine to S-adenosylmethionine. This gene plays a crucial role in methionine and glutathione metabolism. Its activity in liver is increased 205% in Ames dwarf mice compared to wild-type animals (Uthus and Brown-Borg, 2003), and the introduction of GH to these mice led to ˜40% decrease in MAT activity in liver (Brown-Borg et al., 2005). Moreover, due to the role of MAT in methionine metabolism, MAT deficiency in liver leads to persistent hypermethioninemia (Ubagai et al., 1995), which can be thought of as the opposite of MR. Therefore, we expected that knockout of Mat1a could be negatively associated with longevity signatures. Indeed, the test for longevity association revealed a negative association of this intervention with 4 out of 6 longevity signatures, the exceptions being GH deficiency and median lifespan effect signatures (BH adjusted p-value <0.02) (
[0308] Hypoxia, a reduction in oxygen levels, has suggestive associations with longevity that are not yet well understood. First, aging is associated with hypoxia, e.g. showing 38% reduction in oxygen levels in adipose tissue (Zhang et al., 2011). Second, studies investigating the effect of hypoxia on longevity show contrasting results. Thus, one group showed that, in C. elegans, growth in low oxygen and mutation of VHL-1, a negative regulator of the main modulator of hypoxia HIF-1, extended worm lifespan up to 40% (Mehta et al., 2009). However, another group reported an increased lifespan in C. elegans following the deletion of HIF-1 gene under slightly different conditions (Chen et al., 2009). Also, by generating reactive oxygen species (ROS), hypoxia leads to activation of NRF2, one of the upstream regulators associated with the response to lifespan-extending interventions (
[0309] NRF2 is one of the key acute stress regulators, which, among others, activates XMEs (Baird and Dinkova-Kostova, 2011) commonly upregulated at the level of hepatic gene expression across different lifespan-extending interventions (
[0310] We also analyzed the association of sirtuin activation with longevity signatures using two mouse models, SIRT1 activator SRT2104 in males (Mercken et al., 2014b) and Sirt6 overexpression in both sexes (Kanfi et al., 2012). Both of these models were shown to extend lifespan of males, but the effect was modest (˜10% increase in median and maximum lifespan). Accordingly, we detected significant positive associations of these models in males with CR and signatures shared by lifespan-extending interventions. However, there was no consistent positive association with longevity signatures associated with the quantitative effect of lifespan extension, and we even observed a weak negative association for one of them (
[0311] To test if the longevity gene expression signatures may be translated across species, we analyzed their association with the hepatic response to CR in rhesus monkey (Macaca mulatta) males (Rhoads et al., 2018). We observed a strong significant association with the CR signature, pointing to the occurrence of the shared gene expression response to this intervention in mammals (
[0312] Finally, we tested if longevity signatures could be used to predict the difference in lifespan between different mouse strains, which may also be considered as genetic interventions. The GSE10421 dataset includes gene expression of for livers of male mice of 2 mouse strains tested at the same chronological age (7 weeks old): C57BL/6 and DBA/2 (Kautz et al., 2008). We ran a statistical model testing for genes with significant difference between these strains and subjected them to the longevity association test. All longevity signatures except for rapamycin showed a significant positive association with C57BL/6 gene expression profile compared to that of DBA/2 (BH adjusted p-value <5.3.Math.10.sup.−4) (
[0313] For the second study, to test if such approach may be used for the identification of new lifespan-extending drugs, we utilized the CMap platform developed by the Broad Institute (Lamb et al., 2006; Subramanian et al., 2017). This platform contains gene expression profiles of different human cell lines, subjected to more than 1,500 chemical compounds, and allows searching for perturbagens producing gene expression changes similar to the genetic signature of interest. To identify drugs with significant longevity effects, we ranked them based on their association with the maximum lifespan signature. We then chose four compounds from the top of the ranking, prepared diets with them and applied these diets to UM-HET3 male mice for 1 month. These drugs included two mTOR inhibitors KU-0063794 (García-Martínez et al., 2009) and AZD-8055 (Chresta et al., 2010), antioxidant ascorbyl-palmitate (Cort, 1974) and antihypertensive agent rilmenidine (Mpoy et al., 1988).
[0314] We performed RNAseq on the liver samples of mice subjected to the drugs, together with the corresponding controls. To check if the hits predicted based on human cell lines are reproduced in mouse tissues, we calculated a gene expression response to each of these drugs and ran an association test as described earlier (
Example 17
Identification of a Turnover-Based Longevity Signature
[0315] We identified genes whose expression correlated with cell and tissue turnover. Available turnover times fora number of tissues and cell types (in days) were supplemented with estimates from the literature and used as a bona fide measure of lifespan (‘lifespan trait’). We applied generalized least squares regression, tested different evolutionary models and selected the best fit model by maximum likelihood.
[0316] Two hundred eight out of 12,044 genes showed significant correlation with turnover at a false discovery rate (Q-value) of 0.05, with 75% (155 genes, including those shown in Table 17) in negative correlation and 25% (53 genes, including those shown in Table 7) in positive correlation. Notable genes with a positive correlation included the complex SNRPN-SNURF locus, which gives rise to a number of proteins and short non-coding RNAs. We visualized the protein—protein interaction network represented by these 208 genes, revealing significant enrichment for genes involved in cell cycle, immune signaling (NF-κB) and p53 signaling. In our data set, hematopoietic tissues (bone marrow and spleen) and monocytes constituted the samples with the shortest turnover. Removal of these data points in the regression analysis retained the ‘turnover signature’, with the overlapping gene set comprising critical cell cycle and apoptosis associated genes, such as CHEK1, CHEK2, MKI67, FOXM1, TP53 and BCL10, while a correlation with immune signaling-associated genes was lost.
[0317] The procedures used to determine the turnover-based longevity signatures are described in Seim et al., Aging and Mechanisms of Disease 2:16014 (2016), the disclosure of which is incorporated herein by reference in its entirety.
Example 18
Identification of Organ-Specific Longevity Signatures by Analysis of Gene Expression Profiles Across Various Species
[0318] An analysis of gene expression divergence was carried out on 41 species of mammals having different lifespans, including terrestrial mammals of young adult age belonging to Euungulata (n=4), Carnivora (n=4), Chiroptera (n=2), Didelphimorphia (n=1), Diprotodoncia (n=1), Erinaceomorpha (n=1), Lagomorpha (n=1), Monotremata (n=1), Primate (n=8), Rodentia (n=9) and Soricomorpha (n=1) lineages. The total divergence of examined lineages corresponded to a period of about 160 million years. Evolution of these mammals yielded widespread variation in life histories, such as time to maturity, maximum lifespan and oxygen consumption (as a measure of basal metabolic rate, BMR). The relationship between these life histories defines a set of lineage-specific functional tradeoffs and adaptive investments developed during environmental specialization. For example, most primates are characterized by longevity, slow growth and reduced BMR, whereas muroid species (Eumuroida) often use opportunistic-type strategies characterized by rapid development and growth, low body mass and short lifespan. Moreover, some organisms such as representatives of Chiroptera and Histriocognathi, feature Eumuroida-sized species, but possess life history attributes of larger, longer-lived mammals.
[0319] Gene expression in three organs (i.e., liver (Tables 10 and 20), kidney (Tables 9 and 19) and brain (Tables 8 and 18)) was analyzed because of their easier availability, dominance of one cell type (e.g., liver), difference in metabolic functions, size of organs (which is a limitation for smaller animals) and compatibility with previous data from other labs. The majority of the examined species was represented by duplicated (52-60% of species) or triplicated (30-42% of species) biological replicates to account for within species gene expression variation. 25-60 million of 51-bp paired-and RNA-seq reads for each biological replicate were generated (data not shown).
[0320] Reads were then mapped to genomic sequences of organisms from Ensembl and NCBI databases. Database gene model annotations were used and 1-1 orthologous sequence relationships for these organisms were precomputed to calculate gene expression values defined as fragments per kilobase of transcript per million RNA-seq reads mapped (FPKM). Depending on species, RNA-seq read alignment efficiency varied from 55-99% (data not shown). For 12 species with no available genome sequences, full-length transcriptomic contigs using RNA-seq reads were de novo assembled (data not shown), encoded peptides were ab initio predicted (data not shown), and orthologous relationships with database proteins were inferred. Analyses on the expression of protein coding genes with a 1:1 orthologous relationship were further focused, derived from the dataset of 19,643 unique groups of sequences (data not shown).
[0321] In arriving at the gene signatures set forth in Tables 8-10 (up-regulated genes in long-living mammals) and Tables 18-20 (down-regulated genes in long-living mammals), the most relevant genes and biological pathways associated with life histories were examined. Gene set enrichment analysis revealed statistically significant label overrepresentation in the central energy metabolism combining numerous associated pathways such as pyruvate metabolism, carbohydrate degradation pathways, catabolism of tryptophan, lysine and valine oxidation and biosynthesis of fatty acids, Ppar, peroxisome, Ampk, growth hormone signaling and others. Interestingly, divergent evolution of marine vertebrates led to adaptive variation in growth and lifespan (St-Cyr et al., 2008) associated with expression signatures closely related to those observed in the studied mammals, indicating fundamental relatedness of strategies governing parallel life history and transcriptome evolution in vertebrates.
[0322] The full set of procedures used to determine the organ-specific longevity signatures set forth in Tables 8-10 and 18-20 are described in US 2016/0333407, the disclosure of which is incorporated herein by reference in its entirety.
Example 19
Listing of Intervention-Based Longevity Signatures, Turnover-Based Longevity Signature, and Organ-Specific Longevity Signatures
[0323] The various intervention-based longevity signatures, turnover-based longevity signature, and organ-specific longevity signatures described herein are listed in Tables 1-20, below.
TABLE-US-00001 TABLE 1 Intervention-based gene signature 1 (Calorie restriction, up-regulated genes) Entry No. Gene 1 Fmo3 2 Gm4477 3 Slc22a27 4 Gm14420 5 Acmsd 6 Slc22a29 7 Eif4ebp3 8 Nrg4 9 Ctgf 10 Cyp39a1 11 Orm2 12 Per1 13 Fmo2 14 Por 15 Slc51b 16 Slco1a4 17 Etnppl 18 Coq10b 19 Pde6c 20 Cyp2c39 21 Akr1c19 22 Abcc4 23 Mthfd1l 24 Per2 25 Rdh9 26 Zbtb16 27 Tef 28 Cyp2a4 29 Cox7a1 30 Rorc 31 Gstt2 32 Cbr1 33 Igfbp1 34 Gde1 35 Mgst3 36 Txnip 37 Igfbp2 38 Tbc1d8 39 Akr1b7 40 Cdkn1c 41 Aldoc 42 Idh2 43 Gas2l3 44 Gsta4 45 Steap2 46 Gstt3 47 E130012A19Rik 48 Rnf145 49 Nampt 50 Fam84b 51 Tsc22d3 52 Mdh2 53 Slc37a4 54 Map2k6 55 Cnst 56 Irs2 57 Ces1b 58 Hspa2 59 Gm5621 60 Lonrf1 61 Zpr1 62 Fmo5 63 Enpp1 64 Dynll1 65 D630033O11Rik 66 Lrp4 67 Crym 68 Mknk2 69 Tat 70 Lpin2 71 Otud1 72 Ppl 73 Morc3 74 Rbm3 75 BC023829 76 Sall1 77 Ccbl2 78 Bmper 79 Ripk4 80 Stard5 81 Pls1 82 Glrx 83 Ldhb 84 Nudt19 85 Fmo4 86 Ndel1 87 Nhlrc2 88 Cry2 89 Acy1 90 Lmo4 91 Itpr1 92 Adamts7 93 Esrra 94 Vldlr 95 Etnk2 96 Asl 97 Marveld3 98 Klhl3 99 Hspa9 100 Pdk1 101 0610031O16Rik 102 Baiap2l1 103 Tk1 104 Gmnn 105 Dnajb6 106 Car2 107 Pck1 108 Gab1 109 Sestd1 110 Cnn3 111 Dctpp1 112 Tm4sf4 113 Scarf1 114 Rev1 115 Chchd7 116 Slc15a4 117 Hmgn5 118 Cd163 119 Man2a1 120 Sult1a1 121 Rpl22l1 122 Rps9 123 Slc6a8 124 Timm8a1 125 Fam73a 126 2410131K14Rik
TABLE-US-00002 TABLE 2 Intervention-based gene signature 2 (Growth hormone deficient mutants, up-regulated genes) Entry No. Gene 1 Sult1e1 2 Cyp2b13 3 Spink1 4 Hao2 5 Krt23 6 Lrtm2 7 Cyp4a14 8 Cyp39a1 9 Igfbp1 10 Cyp2b9 11 Atp6v0d2 12 Ppp1r3g 13 Pcp4l1 14 Serpina7 15 Chil1 16 Scd2 17 Vldlr 18 Abcc4 19 Lpl 20 Abcd2 21 Pde6c 22 5330417C22Rik 23 BC089597 24 Rcan2 25 Robo1 26 Slc16a5 27 Cyp2b10 28 Fam126a 29 Pls1 30 Defb1 31 Abcb1a 32 Gstt3 33 Nr4a1 34 Col4a5 35 Tceal8 36 8430408G22Rik 37 Lgals1 38 Slco1a4 39 Cyp4a31 40 Slc16a7 41 Il1m 42 Parp16 43 Pparg 44 Aldh1b1 45 Adora1 46 Orm2 47 Igfbp2 48 Gsta2 49 Usp18 50 Cables1 51 Adssl1 52 Serpina6 53 Ppargc1a 54 Tmem237 55 Rnf145 56 Cdkn1c 57 Cth 58 Crym 59 Tstd1 60 Cxcl1 61 Hexb 62 Tmtc2 63 Cd83 64 Idh2 65 Gstm3 66 Gsta4 67 Card10 68 1810046K07Rik 69 Sh2d4a 70 Cpe 71 Dclre1a 72 Tcea3 73 Cdpf1 74 Ldhb 75 Mfsd7c 76 Rdh9 77 Vnn3 78 Pla2g12a 79 Tenm3 80 As3mt 81 Gbp2 82 Arrdc4 83 Gadd45b 84 Mycl 85 Nqo1 86 Arhgap18 87 Ldlrad3 88 Wee1 89 Dqx1 90 Rab30 91 Smpd3 92 Rbp1 93 Enpp2 94 Tmem98 95 Slc25a48 96 Rhbg 97 Slc15a4 98 Mtmr11 99 Dusp6 100 Pigp 101 Sult1a1 102 Btg2 103 Meis1 104 Agt 105 Slc7a2 106 Cox7a1 107 Nhlrc2 108 Afp 109 Echdc3 110 Nudt19 111 Rassf3 112 Cers6 113 Btg1 114 Acad10 115 Ugp2 116 Lcn2 117 Fam134b 118 Ropn1l
TABLE-US-00003 TABLE 3 Intervention-based gene signature 3 (Rapamycin, up-regulated genes) Entry No. Gene 1 2700060E02Rik 2 Acsf3 3 Adh1 4 Alg2 5 Alyref 6 Apopt1 7 Arl14ep 8 Arpc3 9 Arpp19 10 Atp5c1 11 Atp5j 12 Atp5s 13 Bola3 14 Btbd1 15 Btbd3 16 Btf3l4 17 Car14 18 Cdc14b 19 Ces1f 20 Chtop 21 Churc1 22 Cnbp 23 Ctnnd1 24 Ctps 25 D630033O11Rik 26 Dctn6 27 Ddhd1 28 Dnttip1 29 Dtymk 30 Echdc3 31 Eif3l 32 Elac1 33 Erlin1 34 Erp44 35 Extl2 36 Fez2 37 Frat2 38 G6pc3 39 Gm5621 40 Gnai3 41 Gnpnat1 42 Hdac2 43 Hdgfrp2 44 Helb 45 Hnrnph3 46 Hprt 47 Ier3ip1 48 Ifrd1 49 Ift52 50 Igf1 51 Igsf5 52 Iscu 53 Isy1 54 Kctd5 55 Klkb1 56 Lamc1 57 Lasp1 58 Lrrfip1 59 Lum 60 Maob 61 Mapk1ip1 62 Med6 63 Med9 64 Mgat4b 65 Mien1 66 Mks1 67 Mphosph6 68 Mpp6 69 Mrpl30 70 Mrpl42 71 Mrpl57 72 Mrps16 73 Mrps25 74 Mrps27 75 Mterf4 76 Ndufa4 77 Ndufaf7 78 Ndufb3 79 Nsun4 80 Nucks1 81 Nudt7 82 Nudt9 83 Nxt1 84 Ola1 85 Oma1 86 Oxnad1 87 Pdzd11 88 Pkp2 89 Polr2h 90 Ppp3cb 91 Ppp3r1 92 Psmb7 93 Rab4a 94 Rad23a 95 Rbm22 96 Rdh7 97 Rhoa 98 Rnaseh1 99 Rnf7 100 Rpl27 101 Rpl36al 102 Rpl9 103 Rps17 104 Rrbp1 105 Slc12a6 106 Smim11 107 Snrpg 108 Sod2 109 Spop 110 Stxbp3 111 Taf11 112 Tmed10 113 Tmem125 114 Tmem216 115 Trabd 116 Ttf2 117 Txnl4a 118 Ube2d2a 119 Ufc1 120 Ugt2b36 121 Ugt3a1 122 Utp11l 123 Vamp4 124 Wwc1 125 Xkr9 126 Yipf4 127 Zfp938
TABLE-US-00004 TABLE 4 Intervention-based gene signature 4 (Common to all interventions, up-regulated genes) Entry No. Gene 1 1600020E01Rik 2 1810030O07Rik 3 2310010J17Rik 4 Aass 5 Acmsd 6 Actr6 7 Adcy3 8 Adrb2 9 Agmo 10 Agpat9 11 Akr1b7 12 Arpc3 13 B4galt6 14 Bambi 15 Bbs2 16 Bckdhb 17 Brap 18 Brca1 19 Cblb 20 Ccdc152 21 Cep78 22 Cep97 23 Cggbp1 24 Chrac1 25 Cluap1 26 Cmklr1 27 Cnn3 28 Cnst 29 Cps1 30 Crebrf 31 Cth 32 Cul4b 33 Cyp3a59 34 Cyp4a14 35 Dab2 36 Dbt 37 Ddr1 38 Dhrs11 39 Dynlt1b 40 Ecscr 41 Efcab2 42 Ehhadh 43 Epor 44 Erf 45 Exosc2 46 F13b 47 Fam105a 48 Fam167b 49 Fbxw9 50 Fhit 51 Fmo4 52 Gab1 53 Gch1 54 Ggta1 55 Gm10639 56 Gm16124 57 Gm29376 58 Gm5621 59 Gpc6 60 Grb7 61 Gsta4 62 Gsto1 63 Gstt2 64 Gtf2a2 65 Hacd2 66 Hmgn5 67 Hspa9 68 Inpp5a 69 Kcnn2 70 Kctd12b 71 Klf15 72 Las1l 73 Ldlrad3 74 Lrrc8c 75 Maoa 76 Maob 77 Map2k6 78 Map3k15 79 Map4k2 80 Mat2b 81 Mdh2 82 Med14 83 Megf9 84 Mertk 85 Mier1 86 Mospd2 87 Msr1 88 Mtmr7 89 ND4 90 Ndc1 91 Ndel1 92 Ndufa12 93 Net1 94 Neurl3 95 Nmnat1 96 Npc1 97 Npr3 98 Nqo1 99 Nsmce2 100 Nsmce4a 101 Ocln 102 Orc6 103 P2ry14 104 Pck2 105 Pde7a 106 Peli1 107 Pgm1 108 Phtf2 109 Pigu 110 Pir 111 Plekhb2 112 Plekhg3 113 Plk3 114 Postn 115 Ppic 116 Ppt1 117 Prkag1 118 Psmd9 119 Qk 120 Qpct 121 Rab4a 122 Rab9 123 Rbm22 124 Rbm3 125 Rdh16 126 Rdh9 127 Rilpl1 128 Rnase4 129 Rnaseh2b 130 Rpl10a 131 Rpusd1 132 Rragc 133 Rsph3a 134 Sall1 135 Sept8 136 Sertad2 137 Sestd1 138 Sfxn2 139 Sgk1 140 Sgms1 141 Slc15a4 142 Slc25a36 143 Slc2a2 144 Slc35a5 145 Slc51b 146 Smc4 147 Snhg20 148 Snx16 149 Snx6 150 Sorl1 151 Sos2 152 Stat3 153 Susd2 154 Tax1bp3 155 Tfap4 156 Timm8a1 157 Tmem50a 158 Tob2 159 Tpd52l1 160 Trim24 161 Txnrd1 162 Xpot 163 Zfp429 164 Zfp764 165 Zfp938 166 Zzz3
TABLE-US-00005 TABLE 5 Intervention-based gene signature 5 (Association with maximum lifespan change, up-regulated genes) Entry No. Gene 1 Fam19a2 2 Sult2a7 3 Ppp1r3g 4 AA465934 5 Serpina7 6 Slc16a5 7 Lpl 8 Nipal1 9 Robo1 10 Sybu 11 Col4a5 12 Gm26684 13 Ildr2 14 Clec4a1 15 Fam126a 16 ND3 17 Ldhb 18 Utp14b 19 Tstd1 20 Ndrg1 21 Cox7a1 22 Mfsd7c 23 Rassf3 24 2810013P06Rik 25 Nqo1 26 Igfbp2 27 Gys2 28 Il17rb 29 Hexa 30 Ldlrad3 31 Pla2g16 32 Chkb 33 Arrdc4 34 5730508B09Rik 35 Agpat9 36 Tnfrsf12a 37 Ropn1l 38 Hsd17b1l 39 Hunk 40 Wee1 41 Rcn2 42 Gabarapl1 43 Rpa2 44 Fads6 45 Cbln3 46 Zfp820 47 Sptssa 48 Slc16a10 49 Gskip 50 Ppm1k 51 Mettl10 52 Parp16 53 Sardh 54 Tcea3 55 Ddah2 56 Tmem25 57 Eci1 58 Acsm3 59 Dcxr 60 Dnajb2 61 Polr3gl 62 ND6 63 Galk1 64 Ttc38 65 Agxt2 66 Nudt16 67 Aldh3a2 68 Neurl2 69 Pgap3 70 Slc25a4 71 Megf9 72 Haus1 73 Macrod1 74 Lgals4 75 Spg21 76 Clk1 77 Zfp960 78 Cpt2 79 5031425E22Rik 80 Siva1 81 Pdk2 82 Hook2 83 Vegfb 84 Ppat 85 Immp2l 86 Postn 87 Acot7 88 Apoc2 89 Tceal8 90 Osgep 91 Mcee 92 Dhtkd1 93 Kctd12 94 Hpd 95 Cyp4f15 96 Ppp3cc 97 Mnat1 98 P2ry14 99 Fam167b 100 Adck3 101 Gldc 102 Gpr108 103 Fam195a 104 Spopl 105 Cbr4 106 Hint3 107 Cep85 108 Acadm 109 Dtnbp1 110 Aldh4a1 111 Kansl3 112 Mrpl42 113 Ddit3 114 Aaed1 115 Cdip1 116 Clasp2 117 Akr1b10 118 Lhfpl2 119 Emc8 120 Bfar 121 Mapkapk2 122 Fam118a 123 Abcb6 124 Ndufb3 125 Cmtm8 126 Rbks 127 Cul9 128 Il17ra 129 1190005I06Rik 130 Rbm15 131 Slc25a34 132 Tmem106b 133 Med20 134 Catsper2 135 Zfp7 136 Fh1 137 Ntan1 138 Sbds 139 Gm5621 140 Rab9 141 Nmnat1 142 Mospd2 143 Orc6 144 Cblb 145 Cnst 146 Crebrf 147 Maob 148 Ehhadh 149 Pir 150 Brca1 151 Map2k6 152 Cth 153 Ecscr 154 Adcy3 155 Exosc2 156 Cmklr1
TABLE-US-00006 TABLE 6 Intervention-based gene signature 6 (Association with median lifespan change, up-regulated genes) Entry No. Gene 1 Sult2a7 2 Fam19a2 3 AA465934 4 Serpinb1a 5 Serpina7 6 Col4a5 7 Fst 8 Nipal1 9 Cyp2b10 10 Pls1 11 Fam126a 12 Ldhb 13 Mfsd7c 14 Ndrg1 15 Tstd1 16 Nqo1 17 Igfbp2 18 Il17rb 19 As3mt 20 Ldlrad3 21 C330018D20Rik 22 Gys2 23 Arrdc4 24 Parp16 25 Rdh9 26 Rassf3 27 2810013P06Rik 28 Rnf186 29 Agpat9 30 Hsd17b11 31 Pla2g16 32 Wwtr1 33 Hexa 34 Chkb 35 Grtp1 36 Sptssa 37 Slc1a4 38 Fam151b 39 Ppm1k 40 ND6 41 Vnn3 42 Aldh1b1 43 Slc25a4 44 Aig1 45 Ttc38 46 Tsc22d4 47 Postn 48 Cbln3 49 Sult1a1 50 Kctd12 51 Tcea3 52 Mettl10 53 Ropn1l 54 Zfp820 55 Megf9 56 Rcn2 57 Ugcg 58 9330175E14Rik 59 H2-M3 60 Aaed1 61 Polr3gl 62 Nudt16 63 Clk1 64 Rpa2 65 Agxt2 66 Ppat 67 Macrod1 68 Gls2 69 Eci1 70 Gpr108 71 Haus1 72 Rell1 73 Mcee 74 Tmem106b 75 Tmem25 76 Vldlr 77 Spopl 78 Rnf170 79 Acadm 80 Cyp4f15 81 Adcy6 82 Ppp3cc 83 Spg21 84 Zfp960 85 Ntan1 86 P2ry14 87 Pgap3 88 Acot7 89 Mnat1 90 Aldh4a1 91 Tnnc1 92 Cbr4 93 Spred1 94 Vbp1 95 Cdip1 96 Adck3 97 Khdrbs3 98 Zbtb44 99 Acsm3 100 Dcxr 101 Crcp 102 Immp2l 103 Tmem123 104 Kansl3 105 Nrros 106 Ube2m 107 Anpep 108 Abcg1 109 Dnajb2 110 Fam118a 111 Plekhm3 112 Vamp2 113 Il16 114 Ndufab1 115 Sgms1 116 Crym 117 Far1 118 Ptpn6 119 Dcbld1 120 H2afv 121 Hint3 122 Sh3bp5l 123 Lyrm5 124 Gnpnat1 125 Ctnnd1 126 Pitrm1 127 Akr1b10 128 Marcks 129 Ppa1 130 Kdsr 131 Slc22a18 132 Spg7 133 Mtap 134 Tmem53 135 Polk 136 Mmachc 137 Ctsf 138 Nhlrc2 139 Gins1 140 Rbm26 141 Pgm1 142 Zfp36l2 143 Zfp961 144 Gm5621 145 Rab9 146 Nmnat1 147 Mospd2 148 Orc6 149 Cblb 150 Cnst 151 Crebrf 152 Maob 153 Ehhadh 154 Pir 155 Brca1 156 Map2k6 157 Cth 158 Ecscr 159 Adcy3 160 Exosc2 161 Cmklr1
TABLE-US-00007 TABLE 7 Cell turnover-based gene signature (up-regulated genes) Entry No. Gene 1 Ankrd34a 2 Bcl6b 3 Ccdc92 4 Celf2 5 Creld1 6 Cry2 7 Eif5b 8 Hey1 9 Pcdh9 10 Plekhg1 11 Shank3 12 Snrpn 13 Stoml1 14 Ttyh2 15 Zbtb46
TABLE-US-00008 TABLE 8 Organ-specific gene signature 1 (Brain, up-regulated genes) Entry No. Gene 1 Arl2 2 Arrb2 3 Atf3 4 AU022252 5 Bahcc1 6 Bcap31 7 Cdk5rap1 8 Chrne 9 Ciita 10 Cndp2 11 Creb5 12 Cxcr4 13 Cyr61 14 Ddx41 15 Dennd3 16 Dysf 17 Efna1 18 Eif2b5 19 Erh 20 Esyt1 21 Fam178b 22 Fam195b 23 Fam229a 24 Fer1l4 25 Fignl1 26 Fxyd6 27 Gchfr 28 Gpr179 29 Gsap 30 Hagh 31 Higd1a 32 Hnrnpdl 33 Lamtor5 34 Lgals3 35 Litaf 36 Mapk12 37 Mbd4 38 Metrnl 39 Morn2 40 Mrps11 41 Mst1 42 Myom2 43 Narfl 44 Nfkbia 45 Nlrc5 46 Npm2 47 Parp1 48 Pcsk7 49 Pex10 50 Pfkfb3 51 Phf1 52 Plac8l1 53 Plcb2 54 Prdx3 55 Prx 56 Psmf1 57 Psmg3 58 Qrich2 59 Rabl6 60 Rhbdl2 61 Slain1 62 Slc44a3 63 Srp14 64 Stag3 65 Syngr2 66 Tagln 67 Tap1 68 Timm50 69 Tmem14c 70 Top3a 71 Tssk3 72 Txnip 73 Ung 74 Zfp36
TABLE-US-00009 TABLE 9 Organ-specific gene signature 2 (Kidney, up-regulated genes) Entry No. Gene 1 2510039O18Rik 2 Ackr1 3 Alkbh7 4 Apex1 5 Apitd1 6 Arl2 7 B4galt7 8 Bcap31 9 Capg 10 Ccdc50 11 Cct7 12 Cel 13 Ciz1 14 Clip3 15 Cln3 16 Cnp 17 Cpsf31 18 Csrp1 19 Cyr61 20 Dbndd1 21 Dgcr14 22 Eef1g 23 Efna1 24 Eif3k 25 Eif4b 26 Fam195b 27 Fis1 28 Finc 29 Flot1 30 Fosl2 31 Fus 32 Gltscr2 33 Gnptg 34 Gypc 35 Hdac10 36 Hmg20b 37 Hnrnpd 38 Hnrnpdl 39 Hoxa5 40 Iqck 41 Itga5 42 Krt18 43 Larp6 44 Lgi2 45 LOC100862468 46 Lsm3 47 Map6d1 48 Meiob 49 Mrps15 50 Naca 51 Necab3 52 Nol4l 53 Nop56 54 Nr2f1 55 Nsl1 56 P4htm 57 Pcbp2 58 Pcgf2 59 Pdlim1 60 Plcb2 61 Psmf1 62 Rnase1 63 Rpa2 64 Rpl22 65 Rpl28 66 Rpl30 67 Rpl37 68 Rps9 69 Rtfdc1 70 Sdc1 71 Sh3kbp1 72 Skap1 73 Slc41a3 74 Slx1b 75 Smarcb1 76 Smyd3 77 Spata20 78 Ssbp3 79 Styxl1 80 Tbc1d8 81 Tppp3 82 Trub2 83 Ttc21a 84 Tub 85 Tubgcp6 86 Vps28 87 Wash1 88 Wdr13 89 Yipf3
TABLE-US-00010 TABLE 10 Organ-specific gene signature 3 (Liver, up-regulated genes) Entry No. Gene 1 2510039O18Rik 2 6820408C15Rik 3 Afap1l2 4 Agbl2 5 Akap12 6 Arhgap15 7 Arhgdib 8 Basp1 9 Blvrb 10 Bok 11 Cdh23 12 Cep112 13 Cited2 14 Col6a2 15 Crispld2 16 Ctgf 17 Cxcr4 18 Cyba 19 Cytip 20 Ddx41 21 Dkk3 22 Eef1d 23 Eef1g 24 Egr1 25 Eif3l 26 Erbb2 27 Evc2 28 Fam129b 29 Fam149a 30 Fanca 31 Fkbp11 32 Flna 33 Fos 34 Fosl2 35 Fus 36 Glipr2 37 Gltscr2 38 Gm7102 39 Gnptg 40 H2afv 41 Hebp2 42 Hmgb2 43 Hnrnpd 44 Hnrnpdl 45 Jun 46 Junb 47 Krtcap2 48 Larp6 49 Ldb2 50 Lgals1 51 Lmod1 52 Mbd4 53 Mbp 54 Med4 55 Mrps15 56 Myc 57 Naa20 58 Ncapd2 59 Ncf2 60 Nfkbia 61 Nr2f1 62 Nsmce1 63 Pdlim3 64 Pfkp 65 Plekho2 66 Ptprc 67 Ramp2 68 Rasl11a 69 Relt 70 Rpa2 71 Rpl10a 72 Rpl11 73 Rpl22 74 Rpl24 75 Rpl28 76 Rpl30 77 Rpl35a 78 Rpl37 79 Rpl38 80 Rpl5 81 Rpl6 82 Rps15a 83 Rps17 84 Rps19 85 Slc27a3 86 Slx1b 87 Stx11 88 Styxl1 89 Sumo2 90 Tagln 91 Tagln2 92 Thbs1 93 Tm6sf2 94 Trpv2 95 Tubgcp6 96 U2af1 97 Wbscr22 98 Wdr13
TABLE-US-00011 TABLE 11 Intervention-based gene signature 1 (Calorie restriction, down-regulated genes) Entry No. Gene 1 Mup14 2 Ifit1 3 Elovl3 4 Serpina12 5 Saa1 6 Adh6-ps1 7 Spon2 8 C6 9 Csrp3 10 Hsd3b7 11 Saa2 12 C9 13 Ifi47 14 Irgm2 15 Rsad2 16 Igtp 17 Ugt3a1 18 Paqr9 19 Nudt7 20 Fitm1 21 Pdilt 22 Cyp7b1 23 A230050P20Rik 24 Slc30a10 25 Ifit3 26 Slc22a7 27 Cyp2u1 28 Slc25a30 29 Isg15 30 Trim12c 31 Alas2 32 Car3 33 Klhdc7a 34 Insig2 35 Arsg 36 Insc 37 Sult5a1 38 Iigp1 39 C8b 40 Gna14 41 Oasl1 42 Ly6a 43 3010026O09Rik 44 Ifit3b 45 Sdr42e1 46 Cmpk2 47 Aox3 48 Psmb9 49 Cyp2d40 50 Tlcd2 51 Gdf15 52 Chn1os3 53 Adck5 54 Irgm1 55 Dhx58 56 Srd5a1 57 Mikl 58 Tsc22d1 59 Hsd17b2 60 Apon 61 Pctp 62 Dct 63 Tgtp1 64 Plcxd2 65 Eps8l2 66 MbH 67 Exoc4 68 Nsmf 69 Stat1 70 Cyp8b1 71 Irf9 72 Ldhd 73 S100a10 74 St3gal3 75 Lgals3bp 76 Fam89a 77 Apol9a 78 Plin2 79 Mx2 80 Nudt1 81 Lonp2 82 Tmem19 83 Parp14 84 Necab1 85 Slc15a3 86 LOC102640772 87 Smagp 88 Serpina10 89 Kynu 90 Parp9 91 Scamp5 92 Lasp1 93 Mapk15 94 Gsdmd 95 Cyp2f2 96 Zc3h12d 97 Nrp1 98 Irf5 99 St6gal1 100 Dpp7 101 Cldn2 102 Acy3 103 Cox19 104 Bcl3 105 Mocos 106 Fabp2 107 Trim34a 108 C4bp 109 Fpgs 110 Cxcl10 111 Acsf2 112 Fam114a1 113 Gbp7 114 Glo1 115 Ifi35 116 Rab29 117 Cmtm6 118 Pdcd4 119 Dock8 120 Aox1 121 3830406C13Rik 122 Csf2rb 123 Sept9 124 Tap1
TABLE-US-00012 TABLE 12 Intervention-based gene signature 2 (Growth hormone deficient mutants, down-regulated genes) Entry No. Gene 1 Hsd3b5 2 Slco1a1 3 Wfdc21 4 Elovl3 5 Igf1 6 Serpina3k 7 C8a 8 Igfals 9 Det 10 Keg1 11 Mup3 12 Susd4 13 Ppp1r14a 14 Serpina12 15 Cyp7b1 16 C6 17 Scara5 18 Dpy19l3 19 Nudt7 20 Csrp3 21 Egfr 22 Ces3a 23 Srd5a1 24 Crygn 25 Csad 26 C8b 27 Sdr9c7 28 Dmrta1 29 Ugt2b38 30 Onecut1 31 Socs2 32 Nat8 33 Cyp2u1 34 Slc30a10 35 F11 36 Gna14 37 Nrep 38 Pdilt 39 Slc10a2 40 Npr2 41 Cmah 42 Cyp4f14 43 Fabp5 44 Serpina11 45 Neb 46 Zap70 47 Cela1 48 Sult2a8 49 Fabp2 50 Ablim3 51 Derl3 52 Apcs 53 Sdf2l1 54 Gpc1 55 Phlda1 56 Celsr1 57 C9 58 Hsd17b2 59 Cadm4 60 Aox3 61 Slc25a30 62 Irf6 63 E2f8 64 Trp53inp2 65 Lift 66 Alas2 67 Pnpla7 68 Bmyc 69 Sntg2 70 Ugt2b1 71 Hspb1 72 Gadd45g 73 Ttc39c 74 Rapgef4 75 Arhgap44 76 Hsd3b2 77 Me1 78 Apoa4 79 Iigp1 80 A230050P20Rik 81 Cadps2 82 Creld2 83 Aacs 84 Ero1lb 85 Omd 86 Cish 87 Tmem19 88 Tars 89 Hes6 90 Ifi47 91 Slc25a33 92 Slc41a2 93 Cfh 94 Lrg1 95 Manf 96 Syvn1 97 Enho 98 Inhbe 99 Rdh11 100 Dpp7 101 Prlr 102 Sipa1l3 103 Slc22a30 104 Ifi47 105 Pdia6 106 Errfi1 107 Ccnf 108 Tnfaip8l1 109 Ifi35 110 Cyp2f2 111 Plekhb1 112 Zfand4 113 Orm1 114 D16Ertd472e 115 Mkx 116 Insc 117 Tspan33 118 Al661453 119 Mcm10 120 Sdr42e1 121 Sec11c 122 Hao1 123 Acsm1 124 Dnajb11 125 Tnk2 126 Slc34a2 127 Ctsc 128 Aatk 129 Zfp445 130 Dpy19l1 131 Pms1 132 Ldhd
TABLE-US-00013 TABLE 13 Intervention-based gene signature 3 (Rapamycin, down-regulated genes) Entry No. Gene 1 2310033P09Rik 2 Abcb4 3 Abcc3 4 Abhd13 5 Acnat2 6 Adcy9 7 Aqp1 8 Arhgap23 9 Arhgap30 10 Atff7p 11 Atg2a 12 Atp13a1 13 Baz1b 14 Cactin 15 Casp3 16 Ccdc97 17 Cdk13 18 Col4a3bp 19 Coro1c 20 Cpn2 21 Crim1 22 Cyb561d2 23 Cyp2b10 24 Ddhd2 25 Dkk3 26 Dnajc14 27 Dpy19l1 28 Egln2 29 Elmo3 30 Emp2 31 Endod1 32 Esyt1 33 Fam160a2 34 Fbxo18 35 Foxk2 36 Frk 37 Gga2 38 Hiatl1 39 Hif1an 40 Irs2 41 Iws1 42 Kank3 43 Keap1 44 Klhl18 45 Lrrk1 46 Man2b2 47 Mapk4 48 Mb21d2 49 Mybbp1a 50 Myo6 51 Naa25 52 Naa30 53 Naip2 54 Nek4 55 Neurl4 56 Nfic 57 Nhlrc3 58 Nipal3 59 Os9 60 P2rx4 61 Pear1 62 Phactr4 63 Pi4ka 64 Plekhm1 65 Pofut2 66 Prpf4 67 Psmd2 68 Ptpra 69 Rab3gap1 70 Rapgef5 71 Sart3 72 Scarb2 73 Selo 74 Serpinb6b 75 Sf3b3 76 Slc1a2 77 Slc35g1 78 Slc6a8 79 Slc7a5 80 Snx8 81 Stim1 82 Tcf25 83 Tfpi 84 Thsd1 85 Tle4 86 Tmem44 87 Tnfaip1 88 Tor1b 89 Tpp1 90 Traf7 91 Tstd2 92 Tubgcp6 93 Ubac1 94 Vgll4 95 Vstm4 96 Wfdc2 97 Wnt2 98 Zbtb11 99 Zfp58 100 Zfyve27
TABLE-US-00014 TABLE 14 Intervention-based gene signature 4 (Common to all interventions, down-regulated genes) Entry No. Gene 1 1110037F02Rik 2 1600012H06Rik 3 1700049G17Rik 4 2610015P09Rik 5 4933421O10Rik 6 9130023H24Rik 7 Aasdh 8 Aatk 9 Acp5 10 Adam17 11 Adh6-ps1 12 Alkbh8 13 Apol9b 14 Arhgef12 15 Bbx 16 BC017158 17 Bcdin3d 18 Braf 19 C4bp 20 Ccnj 21 Ccpg1os 22 Cdk17 23 Cenpj 24 Cfi 25 Cldn3 26 Cmtr1 27 Commd7 28 Cpn2 29 Cyb561 30 Cyb561a3 31 Cyb5d2 32 Cyp4f17 33 Cyp4v3 34 Ddost 35 E2f8 36 Ercc6 37 Erp29 38 Exoc1 39 Exoc2 40 Ext2 41 Fabp1 42 Fadd 43 Fam219b 44 Fem1a 45 Gatc 46 Gbp7 47 Ghr 48 Gorasp1 49 Gpat2 50 Gpc4 51 Gpr107 52 Gtf3c1 53 H2-T23 54 Hc 55 Hipk2 56 Hps4 57 Ikbkap 58 Il6st 59 Irf3 60 Itsn1 61 Kbtbd3 62 Kctd17 63 Klhl18 64 Larp1 65 Litaf 66 Lmbr1 67 Lmf1 68 LOC102631757 69 Lrrc14 70 Lysmd4 71 Malat1 72 Mfsd3 73 Mgmt 74 Mllt4 75 Mrps18a 76 Mrs2 77 Nbas 78 Nupl2 79 Oasl1 80 Onecut1 81 Osbpl9 82 Otud4 83 Oxr1 84 P4hb 85 Parp9 86 Pdcd4 87 Pdia6 88 Pgpep1 89 Pik3r4 90 Pnn 91 Polb 92 Prelp 93 Proz 94 Psmb9 95 Qprt 96 Rai14 97 Rb1 98 Rec114 99 Rfwd2 100 Rplp0 101 Saa4 102 Serpina10 103 Slc25a30 104 Smarcal1 105 Snap47 106 Snapc5 107 Snx17 108 Sox5 109 Spsb4 110 St3gal3 111 Stk16 112 Stk19 113 Tap2 114 Tbc1d5 115 Tfr2 116 Tlr5 117 Tmem261 118 Tmem8b 119 Trmt5 120 Ttc30b 121 Ttc41 122 Tuft1 123 Ube3a 124 Vcpip1 125 Vps18 126 Vwa5a 127 Wbp1 128 Wfdc2 129 Zfp507 130 Zfp595 131 Zfp729b 132 Zkscan7 133 Zmym2 134 Znfx1
TABLE-US-00015 TABLE 15 Intervention-based gene signature 5 (Association with maximum lifespan change, down-regulated genes) Entry No. Gene 1 Mup16 2 Mup19 3 Mup15 4 Mup14 5 Mup11 6 Nuggc 7 Ugt2b37 8 Tnik 9 Mfhas1 10 Npr2 11 B4galnt3 12 Srd5a1 13 Apcs 14 Slc3a1 15 Gm17296 16 Lrp2bp 17 Cmah 18 Sort1 19 Piezo1 20 Ablim3 21 Cml2 22 Errfi1 23 Ston1 24 Nup210 25 Zbtb20 26 Slc25a30 27 Cfh 28 C8a 29 Wdr91 30 Gm15446 31 Socs3 32 Hsph1 33 Mug2 34 Prex1 35 Whsc1 36 Tspan33 37 Fkbp5 38 Zdhhc14 39 Crp 40 Fan1 41 Ccbl1 42 Proca1 43 Tnk2 44 Fam135a 45 Orm1 46 Irf2 47 Stard4 48 Tbc1d4 49 Dock8 50 6430548M08Rik 51 Tmem45b 52 Slc35b1 53 Scfd2 54 Bhlhe40 55 Slc40a1 56 Crlf2 57 Ubr2 58 Fam89a 59 Coro1c 60 Slc25a23 61 Slc16a2 62 Kif13b 63 Dirc2 64 Ahsa2 65 Lrg1 66 Surf4 67 Itih3 68 Mgat5 69 Cdc42bpa 70 Frmd8 71 Simc1 72 Cipc 73 Bahcc1 74 Tango6 75 Kng1 76 Slco2b1 77 C1ra 78 Kdm4a 79 Adi1 80 Wipi1 81 Sccpdh 82 Lpgat1 83 Apof 84 Itih1 85 C4bp 86 Fn1 87 Gorasp1 88 Tap2 89 Cux1 90 Kynu 91 Ptpre 92 Xbp1 93 Ywhab 94 Sox13 95 Slc10a1 96 Brpf3 97 Lman1 98 Fbxl20 99 Ginm1 100 Acad9 101 Aars 102 4930453N24Rik 103 Tmed9 104 Tpst2 105 Aldh2 106 Tiam1 107 Dhx36 108 Ppib 109 Gucd1 110 Rps6ka1 111 Golgb1 112 Trim26 113 Adh6-ps1 114 Itsn1 115 1600012H06Rik 116 Exoc2 117 Ercc6 118 Fam219b 119 Mrps18a 120 Zfp729b 121 St3gal3 122 Vps18 123 Apol9b
TABLE-US-00016 TABLE 16 Intervention-based gene signature 6 Association with median lifespan change, down-regulated genes) Entry No. Gene 1 Mup16 2 Mup19 3 Mup14 4 Tnik 5 Mfhas1 6 Cfb 7 Srd5a1 8 Rpl35a 9 Serpina1d 10 Apcs 11 B4galnt3 12 Slc6a9 13 Npr2 14 C8a 15 Bhlhe40 16 Neb 17 Gm17296 18 1810064F22Rik 19 Gnai1 20 Coq2 21 Aatk 22 Gadd45g 23 Tspan33 24 Homer2 25 Gm15446 26 Cadm4 27 Wdr91 28 Sort1 29 Zdhhc14 30 F11 31 Prex1 32 Zbtb20 33 Stard4 34 Rap2a 35 Irf2 36 6430548M08Rik 37 2200002D01Rik 38 Crp 39 Crlf2 40 Slc17a2 41 Tnk2 42 Tmem45b 43 Simc1 44 Scfd2 45 Ubr2 46 Surf4 47 Slc35b1 48 Zfp324 49 Phlpp2 50 C1ra 51 Dirc2 52 Syt1 53 Tigar 54 Dpp7 55 Txndc11 56 D3Ertd254e 57 Slc25a23 58 Kynu 59 Sox13 60 Epb41l4b 61 Mgat5 62 Gorasp1 63 Kif13b 64 Ahsa2 65 Tmem19 66 Tango6 67 Slc38a2 68 Tbcel 69 Bahcc1 70 Slc6a13 71 Adamts5 72 Dhx36 73 Cdc42bpa 74 Serpinc1 75 Gne 76 Aldh2 77 Abtb1 78 Fbxl20 79 Trim26 80 Ppib 81 Coro1c 82 Slco2b1 83 Fyco1 84 Nr1h4 85 Ciz1 86 Myo6 87 Thada 88 Zfp335 89 Kng1 90 Edrf1 91 Spop 92 Gbf1 93 Cys1 94 Gucd1 95 Cdk5rap3 96 Capn1 97 Ctnnb1 98 Acad9 99 Adarb1 100 Ttc7b 101 Brpf3 102 Fgd6 103 Rabggta 104 Eif4ebp2 105 Lgals8 106 Tmed9 107 Cpn2 108 Adh6-ps1 109 Itsn1 110 1600012H06Rik 111 Exoc2 112 Ercc6 113 Fam219b 114 Mrps18a 115 Zfp729b 116 St3gal3 117 Vps18 118 Apol9b
TABLE-US-00017 TABLE 17 Cell turnover-based gene signature (down-regulated genes) Entry No. Gene 1 Ano10 2 Arfip1 3 Bcl10 4 Brca2 5 Bub1b 6 Ccnb2 7 Cdca3 8 Cdca8 9 Cenpf 10 Cenpw 11 Chek1 12 Chek2 13 Cnot1 14 Ddb2 15 Ddx52 16 Ect2 17 Eif6 18 Exo1 19 Fancd2 20 Foxm1 21 Gdi2 22 Hnrnpf 23 Kif11 24 Kif23 25 Mki67 26 Msh5 27 Nadsyn1 28 Ncapg 29 Ncaph 30 Net1 31 Nuf2 32 Orc1 33 Parpbp 34 Pdcd6ip 35 Plk4 36 Rars 37 Rcc1 38 Rccd1 39 Samd9l 40 Scyl2 41 Slc25a43 42 Spata18 43 Stk38 44 Trp53 45 Zwint
TABLE-US-00018 TABLE 18 Organ-specific gene signature 1 (Brain, down-regulated genes) Entry No. Gene 1 1810030O07Rik 2 A830018L16Rik 3 Actr2 4 Adarb1 5 Adprh 6 Agap2 7 Ankrd55 8 Ankrd63 9 Arfgef1 10 Atad1 11 Atp11b 12 Atp2b1 13 Atp6ap1l 14 Atp6v1c1 15 Atxn7l3 16 Cacnb3 17 Ccdc39 18 Cdadc1 19 Cds1 20 Cep120 21 Col1a1 22 Col4a1 23 Ctps2 24 Cyb5r4 25 Dclk3 26 Dgkb 27 Dlst 28 Dnajb5 29 Dnajc27 30 Dnal1 31 Dnm1l 32 Dtl 33 Dync2li1 34 Egflam 35 Fam13b 36 Fam83f 37 Fbxw2 38 Fmnl1 39 Fmo1 40 Gad1 41 Gapvd1 42 Gdap2 43 Gfra1 44 Gnal 45 Gng12 46 Gpcpd1 47 Gpld1 48 Gria3 49 Hapln1 50 Htr1f 51 Il1rap 52 Inpp5j 53 Ipmk 54 Jak2 55 Kcnj6 56 Kcnk2 57 Kcnt2 58 Klhdc7a 59 Lclat1 60 Mas1 61 Mb21d2 62 Mtmr3 63 Nckap1 64 Ndrg4 65 Opa1 66 Oxsr1 67 Palmd 68 Paqr9 69 Pcsk2 70 Pde1b 71 Pdyn 72 Pik3ca 73 Pitpna 74 Plxdc2 75 Pold3 76 Ppfia3 77 Ppm1e 78 Ppme1 79 Ppp1r9a 80 Ppp2r5a 81 Ppp3r1 82 Prkci 83 Purb 84 Rala 85 Rap2c 86 Rbm46 87 Ric1 88 Rock2 89 Rragc 90 Senp7 91 Sfmbt1 92 Slc22a8 93 Slc30a5 94 Slc35b4 95 Snx13 96 Sppl3 94 Src 98 Stk38 99 Strbp 100 Stx6 101 Sugt1 102 Susd2 103 Tbata 104 Tbc1d8b 105 Tenm4 106 Tgds 107 Tmem229a 108 Tomm70a 109 Trpc4 110 Tspan2 111 Ube3b 112 Ubr5 113 Vps54 114 Vti1a 115 Wdr36 116 Wnt6 117 Xkr4 118 Xpr1 119 Zfc3h1 120 Zfp106
TABLE-US-00019 TABLE 19 Organ-specific gene signature 2 (Kidney, down-regulated genes) Entry No. Gene 1 1110059E24Rik 2 1700067K01Rik 3 4930402H24Rik 4 4930453N24Rik 5 Abcd3 6 Abce1 7 Aco1 8 Aco2 9 Actl6a 10 Adprh 11 Adra2b 12 Agpat3 13 Arfgef2 14 Arhgap11a 15 Arid2 16 Arih1 17 Atad2b 18 Atg7 19 Atl2 20 Atp11a 21 Atp2b1 22 Atp5a1 23 Atp5b 24 Avpr2 25 Birc6 26 Brdt 27 Brwd1 28 Cab39l 29 Cacul1 30 Camk2n2 31 Camsap2 32 Casd1 33 Cep19 34 Cgrrf1 35 Chac2 36 Cisd1 37 Clock 38 Cmip 39 Cnot6l 40 Col4a3 41 Cul4b 42 Cyp2e1 43 D15Ertd621e 44 Dbt 45 Ddb1 46 Dgkq 47 Dlat 48 Dnajc13 49 Dpp9 50 Dynd1li1 51 Efr3a 52 Eif4g3 53 Etfa 54 Fam135a 55 Fam20b 56 Fam210a 57 Fam8a1 58 Fbxo33 59 Fetub 60 Fkbpl 61 Fmo2 62 Fmo5 63 Ghitm 64 Gxylt1 65 Hectd2 66 Igf1 67 Immt 68 Ipmk 69 Kbtbd8 70 Kcnj16 71 Kidins220 72 Kif20a 73 Klhl24 74 Kmt2a 75 Kras 76 Lace1 77 Lekr1 78 Lin7c 79 Lmod2 80 Lpgat1 81 Ltn1 82 Mapt 83 Mars2 84 Mgat3 85 Mier3 86 Mon2 87 Mvb12a 88 Ncbp1 89 Nckap1 90 Ndufa9 91 Ndufab1 92 Ndufs1 93 Ndufs2 94 Nek2 95 Neurl1b 96 Nsf 97 Nuak2 98 Odf3 99 Opa1 100 Oplah 101 Pank1 102 Papd5 103 Paqr9 104 Parg 105 Pcdh17 106 Pcsk6 107 Pcyt1a 108 Pigu 109 Pik3ca 110 Pitpnb 111 Pkn2 112 Pkp3 113 Ppp2ca 114 Ppp4r1 115 Ppp4r4 116 Rab18 117 Rbm46 118 Rfx7 119 Rhebl1 120 Rock2 121 Sacm1l 122 Sbk1 123 Sclt1 124 Sdhb 125 Sel1l 126 Slc16a13 127 Slc16a7 128 Slc34a1 129 Slc39a10 130 Slc5a1 131 Slc5a8 132 Slc6a6 133 Smagp 134 Snx13 135 Socs7 136 Srp54b 137 Stxbp5 138 Synj1 139 Taok1 140 Tcp11l2 141 Tert 142 Tgfbr1 143 Tm9sf3 144 Tmppe 145 Tmx3 146 Tnfaip8 147 Tnks2 148 Tns1 149 Top2a 150 Trpc3 151 Trpm7 152 Tulp4 153 Ubr5 154 Uhrf1 155 Wdr26 156 Wdr35 157 Wdr36 158 Xylb 159 Zbtb43 160 Zc3h12c 161 Zfp706
TABLE-US-00020 TABLE 20 Organ-specific gene signature 3 (Liver, down-regulated genes) Entry No. Gene 1 1110059E24Rik 2 1700006E09Rik 3 1700066M21Rik 4 Abcd3 5 Abce1 6 Abcf2 7 Acbd5 8 Acox1 9 Acpp 10 Ado 11 Agpat3 12 Ankrd52 13 Arid2 14 Arl5a 15 Arl5b 16 Armc1 17 Asb8 18 Asun 19 Atad2b 20 Atl2 21 Atp11b 22 Atp5a1 23 Atp5b 24 BC004004 25 Bmf 26 Bpnt1 27 Brpf1 28 Btbd8 29 Cacul1 30 Carnmt1 31 Cdip1 32 Cep152 33 Cgrrf1 34 Chac2 35 Chmp7 36 Chuk 37 Cisd1 38 Clock 39 Clpx 40 Cluap1 41 Cluh 42 Cps1 43 Cul4b 44 Cyb5r4 45 Dbt 46 Derl1 47 Dnajc3 48 Dtnbp1 49 Ehmt2 50 Erap1 51 Evi5 52 Fam175b 53 Fam214a 54 Fbxo45 55 Gan 56 Gmfb 57 Gnpnat1 58 Gpc4 59 Hectd1 60 Hsdl1 61 Igf1 62 Immt 63 Ipmk 64 Kbtbd8 65 Kif21a 66 Klhl11 67 Klhl24 68 Lace1 69 Larp4b 70 Lpar3 71 Lppr5 72 Lyrm2 73 Mafg 74 Map2k4 75 Mapt 76 Minpp1 77 Mtf1 78 Naa15 79 Nanp 80 Ndufa10 81 Nmt1 82 Nr3c1 83 Ogdh 84 Opa1 85 Opcml 86 Oprm1 87 Pafah1b1 88 Papss2 89 Parp16 90 Pcyt1a 91 Pitpnb 92 Pkp3 93 Plaa 94 Ppm1a 95 Ppp2r5e 96 Psmd1 97 Psmd11 98 Pvrl1 99 Rmnd1 100 Rnf4 101 Rragc 102 Sacm1l 103 Sbno1 104 Scai 105 Slc25a13 106 Slc25a15 107 Slc25a20 108 Slc25a23 109 Slc25a44 110 Slc25a46 111 Slc33a1 112 Slc38a7 113 Slc4a4 114 Slmap 115 Smim8 116 Smurf2 117 Snx13 118 Socs4 119 Sox6 120 Spef1 121 Sppl3 122 Stk35 123 Stx17 124 Sucla2 125 Suds3 126 Suv420h2 127 Synj2bp 128 Taok1 129 Tcp11l2 130 Tex2 131 Tm9sf3 132 Tmem106b 133 Tmem170b 134 Tmem63b 135 Tmppe 136 Trappc13 137 Ttc7 138 Tulp4 139 Uba3 140 Ube2a 141 Ube2w 142 Ubr5 143 Ufm1 144 Umad1 145 Usp14 146 Usp47 147 Vwa8 148 Wdr36 149 Wdtc1 150 Zbtb41 151 Zbtb44 152 Zfp740
Example 20
[0324] Procedure for Identifying Candidate Interventions Based on Association with Longevity Signatures
[0325] There are a variety of protocols that can be implemented in order to use the longevity gene signatures described herein to identify new interventions capable of extending lifespan, reducing frailty, improving learning ability, and/or preventing/delaying the onset of a geriatric syndrome. An exemplary protocol that can be used for this purpose is described below:
1. Download longevity signatures. Every signature contains two sets of genes. One of them includes genes positively associated with a certain longevity metric, and the other includes genes with the negative association.
2. Prepare dataset of interest. For every gene in the gene expression data of interest, calculate fold changes and corresponding p-values between intervention and control groups. For every gene, calculate significance score, defined as −log.sub.10(p. value)×sgn(logFC). Sort genes based on the significance score (from the highest value to the lowest).
3. Filter out excess genes. From particular longevity signature gene sets, filter out all genes that are not represented in the sorted list corresponding to gene expression dataset of interest.
4. Calculate connectivity score (metric of the effect size). Calculate connectivity scores separately for gene sets positively and negatively associated with longevity metric as described in (Lamb et al., 2006). First, calculate Kolmogorov-Smirnov enrichment statistics (ES) separately for positively and negatively associated genes. Then, calculate the final connectivity score as an average between the two:
5. Calculate p-value (metric of statistical significance). To calculate statistical significance of obtained connectivity score, apply permutation test. Randomly choose genes from the sorted list so that they form gene sets of the same size as longevity gene sets. Then calculate the connectivity score for these randomized signatures using the same algorithm as described above. Repeat this algorithm (e.g., 3,000 times). Then calculate p-value as the proportion of cases when the absolute value of random connectivity score is bigger than the absolute value of the real connectivity score:
6. Adjust p-values for multiple hypotheses. Adjust obtained p-values corresponding to different longevity signatures using multiple hypothesis correction techniques (e.g., Benjamini-Hochberg method). The resulting connectivity scores and adjusted p-values may be used as a metric of association between longevity signatures and gene expression response to the intervention of interest.
Example 21
Testing Longevity Interventions in Mice: Effects of Various Agents on Lifespan, Gait Speed, Frailty Index, and Muscle Function
Materials and Methods
[0326] Interventions were predicted in a screen based on the gene expression longevity signatures that we developed. The predicted interventions were then verified for gene expression responses in human and mouse primary cell culture (hepatocytes) and in live mice (after mice were fed for 1 month with the diets containing these interventions). The interventions that passed these tests were further assessed for the effect on lifespan of 2-year-old C57BI/6 mice. Older mice (2-year-old) were chosen for this experiment in order to mimic the effect of giving interventions to human subjects in their second half of life.
[0327] The basic scheme of the experiment is shown in
Results: AZD-8055
[0328] AZD-8055 was given to mice ad libitum in the amount of 20 mg/kg of food. This agent extends the lifespan of male mice (
Results: Selumetinib
[0329] Selumetinib was given ad libitum at the concentration of 100 mg/kg of diet. We found that it extends lifespan of C57BI/6 mice (
Results: Celastrol
[0330] Celastrol was given ad libitum at the concentration of 8 mg/kg of food. We found that it has a lifespan-extending effect (p=0.052) (
Results: LY294002
[0331] LY294002 was given ad libitum at the level of 600 mg/kg of diet. We found that it extends lifespan of male mice (
Results: KU-0063794
[0332] KU-0063794 was given ad libitum at the concentration of 10 mg/kg of diet. This agent was found to extend lifespan of male mice (p=0.052) (
REFERENCES
[0333] Aaron, E. A., and Powell, F. L. (1993). Effect of chronic hypoxia on hypoxic ventilatory response in awake rats. J. Appl. Physiol. 74, 1635-1640. [0334] Ables, G. P., Perrone, C. E., Orentreich, D., and Orentreich, N. (2012). Methionine-Restricted C57BL/6J Mice Are Resistant to Diet-Induced Obesity and Insulin Resistance but Have Low Bone Density. PLoS One 7, 1-12. [0335] Ables, G. P., Ouattara, A., Hampton, T. G., Cooke, D., Perodin, F., Augie, I., and Orentreich, D. S. (2015). Dietary methionine restriction in mice elicits an adaptive cardiovascular response to hyperhomocysteinemia. Sci. Rep. 5, 1-10.
al-Shawi, R., Wallace, H., Harrison, S., Jones, C., Johnson, D., and Bishop, J. O. (1992). Sexual dimorphism and growth hormone regulation of a hybrid gene in transgenic mice. Mol. Endocrinol. 6, 181-190. [0336] Alonso, C., Fernández-Ramos, D., Varela-Rey, M., Martinez-Arranz, I., Navasa, N., Van Liempd, S. M., Lavin Trueba, J. L., Mayo, R., Ilisso, C. P., de Juan, V. G., et al. (2017). Metabolomic Identification of Subtypes of Nonalcoholic Steatohepatitis. Gastroenterology 152, 1449-1461.e7. [0337] Amador-Noguez, D., Yagi, K., Venable, S., and Darlington, G. (2004). Gene expression profile of long-lived Ames dwarf mice and Little mice. Aging Cell 3, 423-441. [0338] Baird, L., and Dinkova-Kostova, A. T. (2011). The cytoprotective role of the Keap1-Nrf2 pathway. Arch. Toxicol. 85, 241-272. [0339] Balaban, R. S., Nemoto, S., and Finkel, T. (2005). Mitochondria, oxidants, and aging. Cell 120, 483-495. [0340] Barger, J. L., Kayo, T., Vann, J. M., Arias, E. B., Wang, J., Hacker, T. A., Wang, Y., Raederstorff, D., Morrow, J. D., Leeuwenburgh, C., et al. (2008). A low dose of dietary resveratrol partially mimics caloric restriction and retards aging parameters in mice. PLoS One 3. [0341] Baur, J. A., and Sinclair, D. A. (2006). Therapeutic potential of resveratrol: The in vivo evidence. Nat. Rev. Drug Discov. 5, 493-506. [0342] Baur, J. A., Pearson, K. J., Price, N. L., Jamieson, H. A., Lerin, C., Kalra, A., Prabhu, V. V, Allard, J. S., Lopez-Lluch, G., Lewis, K., et al. (2006). Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444, 337-342. [0343] Baze, M. M., Schlauch, K., and Hayes, J. P. (2010). Gene expression of the liver in response to chronic hypoxia. 275-288. [0344] Boylston, W. H., DeFord, J. H., and Papaconstantinou, J. (2006). Identification of longevity-associated genes in long-lived Snell and Ames dwarf mice. Age (Omaha). 28, 125-144. [0345] Brown-borg, H. M. (2007). Hormonal regulation of longevity in mammals. Ageing Res. Rev. 6, 28-45. [0346] Brown-Borg, H. M., Rakoczy, S. G., and Uthus, E. O. (2005). Growth hormone alters methionine and glutathione metabolism in Ames dwarf mice. Mech. Ageing Dev. 126, 389-398. [0347] Buckley, D. B., and Klaassen, C. D. (2009). Mechanism of Gender-Divergent UDP-Glucuronosyltransferase mRNA Expression in Mouse Liver and Kidney. 37, 834-840. [0348] Cao, L., Li, W., Kim, S., Brodie, S. G., and Deng, C. X. (2003). Senescence, aging, and malignant transformation mediated by p53 in mice lacking the brca1 full-length isoform. Genes Dev. 17, 201-213. [0349] Chang, W., Cheng, J., Allaire, J., Xie, Y., and McPherson, J. (2016). shiny: Web Application Framework for R. R Packag. Version 0.14.2. Https//CRAN.R-Project.Org/Package=shiny. [0350] Chen, D., Thomas, E. L., and Kapahi, P. (2009). HIF-1 modulates dietary restriction-mediated lifespan extension via IRE-1 in Caenorhabditis elegans. PLoS Genet. 5. [0351] Chresta, C. M., Davies, B. R., Hickson, I., Harding, T., Cosulich, S., Critchlow, S. E., Vincent, J. P., Ellston, R., Jones, D., Sini, P., et al. (2010). AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. Cancer Res. 70, 288-298. [0352] Cort, W. M. (1974). Antioxidant activity of tocopherols, ascorbyl palmitate, and ascorbic acid and their mode of action. J. Am. Oil Chem. Soc. 51, 321-325. [0353] Coschigano, K. T., Clemmons, D., Bellush, L. L., and Kopchick, J. J. (2000). Assessment of growth parameters and lifespan of GHR/BP gene-disrupted mice. Endocrinology 141, 2608-26β. [0354] Coschigano, K. T., Holland, A. N., Riders, M. E., List, E. O., Flyvbjerg, A., and Kopchick, J. J. (2003). Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin, and insulin-like growth factor I levels and increased life span. Endocrinology 144, 3799-3810. [0355] David, J., Van Herrewege, J., and Fouillet, P. (1971). Quantitative under-feeding of drosophila: Effects on adult longevity and fecundity. Exp. Gerontol. 6, 249-257. [0356] Dhahbi, J. M., Mote, P. L., Fahy, G. M., and Spindler, S. R. (2005). Identification of potential caloric restriction mimetics by microarray profiling. Am. Physiol. Soc. 23, 343-350. [0357] Estep, P. W., Warner, J. B., and Bulyk, M. L. (2009). Short-term calorie restriction in male mice feminizes gene expression and alters key regulators of conserved aging regulatory pathways. PLoS One 4. [0358] Fok, W. C., Chen, Y., Bokov, A., Zhang, Y., Salmon, A. B., Diaz, V., Javors, M., Wood, W. H., Zhang, Y., Becker, K. G., et al. (2014a). Mice fed rapamycin have an increase in lifespan associated with major changes in the liver transcriptome. PLoS One 9. [0359] Fok, W. C., Bokov, A., Gelfond, J., Yu, Z., Zhang, Y., Doderer, M., Chen, Y., Javors, M., Wood, W. H., Zhang, Y., et al. (2014b). Combined treatment of rapamycin and dietary restriction has a larger effect on the transcriptome and metabolome of liver. Aging Cell 13, 311-319. [0360] Fontana, L., Partridge, L., and Longo, V. D. (2010). Extending Healthy Life Span-From Yeast to Humans. Science (80-.). 328, 321-326. [0361] Fu, Z. D., and Klaassen, C. D. (2014). Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice. Toxicol. Appl. Pharmacol. 274, 137-146. [0362] Gadó, K., Domján, G., Hegyesi, H., and Falus, A. (2000). Role of interleukin-6 in the pathogenesis of multiple myeloma. Cell Biol. Int. 24, 195-209. [0363] García-Martínez, J. M., Alessi, D. R., Moran, J., Cosulich, S. C., Clarke, R. G., Gray, A., and Chresta, C. M. (2009). Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). Biochem. J. 421, 29-42. [0364] Garratt, M., Stockley, P., Armstrong, S. D., Beynon, R. J., and Hurst, J. L. (2011). The scent of senescence: Sexual signalling and female preference in house mice. J. Evol. Biol. 24, 2398-2409. [0365] Gautier, H. (1996). Interactions and control among metabolic of breathing rate, hypoxia, and control of breathing. 521-527. [0366] Gertz, M., Nguyen, G. T. T., Fischer, F., Suenkel, B., Schlicker, C., Fränzel, B., Tomaschewski, J., Aladini, F., Becker, C., Wolters, D., et al. (2012). A Molecular Mechanism for Direct Sirtuin Activation by Resveratrol. PLoS One 7, 1-12. [0367] Gokarn, R., Solon-Biet, S. M., Cogger, V. C., Cooney, G. J., Wahl, D., McMahon, A. C., Mitchell, J. R., Mitchell, S. J., Hine, C., De Cabo, R., et al. (2018). Long-term Dietary Macronutrients and Hepatic Gene Expression in Aging Mice. J Gerontol A Biol Sci Med Sci 00, 1-8. [0368] Gorrini, C., Baniasadi, P. S., Harris, I. S., Silvester, J., Inoue, S., Snow, B., Joshi, P. A., Wakeham, A., Molyneux, S. D., Martin, B., et al. (2013). BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival. J. Exp. Med. 210, 1529-1544. [0369] Grandison, R. C., Piper, M. D. W., and Partridge, L. (2009). Amino acid imbalance explains extension of lifespan by dietary restriction in Drosophila. Nature 462, 1061-1064. [0370] De Haes, W., Frooninckx, L., Van Assche, R., Smolders, A., Depuydt, G., Billen, J., Braeckman, B. P., Schoofs, L., and Temmerman, L. (2014). Metformin promotes lifespan through mitohormesis via the peroxiredoxin PRDX-2. Proc. Natl. Acad. Sci. 111, E2501-E2509. [0371] Harrison, D. E., Strong, R., Sharp, Z. D., Nelson, J. F., Astle, C. M., Flurkey, K., Nadon, N. L., Wilkinson, J. E., Frenkel, K., Carter, C. S., et al. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460, 392-395. [0372] Harrison, D. E., Strong, R., Allison, D. B., Ames, B. N., Astle, C. M., Atamna, H., Fernandez, E., Flurkey, K., [0373] Javors, M. A., Nadon, N. L., et al. (2014). Acarbose, 17-α-estradiol, and nordihydroguaiaretic acid extend mouse lifespan preferentially in males. Aging Cell 13, 273-282. [0374] Hine, C., Harputlugil, E., Zhang, Y., Ruckenstuhl, C., Lee, B. C., Brace, L., Longchamp, A., Trevino-Villarreal, J. H., Mejia, P., Ozaki, C. K., et al. (2015). Endogenous hydrogen sulfide production is essential for dietary restriction benefits. Cell 160, 132-144. [0375] Hofmann, J. W., Zhao, X., De Cecco, M., Peterson, A. L., Pagliaroli, L., Manivannan, J., Hubbard, G. B., Ikeno, Y., Zhang, Y., Feng, B., et al. (2015). Reduced expression of MYC increases longevity and enhances healthspan. Cell 160, 477-488. [0376] Houthoofd, K., and Vanfleteren, J. R. (2006). The longevity effect of dietary restriction in Caenorhabditis elegans. Exp. Gerontol. 41, 1026-1031. [0377] Huang, D. W., Sherman, B. T., and Lempicki, R. A. (2009a). Bioinformatics enrichment tools: Paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1-13. [0378] Huang, D. W., Lempicki, R. a, and Sherman, B. T. (2009b). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44-57. [0379] Imaoka, S., Fujita, S., and Funae, Y. (1991). Age-dependent exression of cytochrome P-450s in rat liver. BBA—Mol. Basis Dis. 1097, 187-192. [0380] Jain, I. H., Zazzeron, L., Goli, R., Alexa, K., Schatzman-Bone, S., Dhillon, H., Goldberger, O., Peng, J., Shalem, O., Sanjana, N. E., et al. (2016). Hypoxia as a therapy for mitochondrial disease. Science (80-.). 352, 54-61. [0381] Kabil, O., Vitvitsky, V., Xie, P., and Banerjee, R. (2011). The quantitative significance of the transsulfuration enzymes for H2S production in murine tissues. Antioxid. Redox Signal. 15, 363-372. [0382] Kamataki, T., Maeda, K., Shimada, M., Kitani, K., Nagai, T., and Kato, R. (1985). Age-Related Alteration in the Activities of Drug-Metabolizing Enzymes and Contents of Sex-Specific Forms of Cytochrome P-450 in Liver Microsomes from Male and Female Rats1. J. Pharmacol. Exp. Ther. 233, 222-228. [0383] Kanfi, Y., Naiman, S., Amir, G., Peshti, V., Zinman, G., Nahum, L., Bar-Joseph, Z., and Cohen, H. Y. (2012). The sirtuin SIRT6 regulates lifespan in male mice. Nature 483, 218-221. [0384] Kapahi, P., Zid, B. M., and Harper, T. (2004). Regulation of Lifespan in Drosophila by Modulation of Genes in the TOR Signaling Pathway. Curr. Biol. 14, 885-890. [0385] Kautz, L., Meynard, D., Monnier, A., Darnaud, V., Bouvet, R., Wang, R. H., Deng, C., Vaulont, S., Mosser, J., Coppin, H., et al. (2008). Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1, and Atoh8 in the mouse liver. Blood 112, 1503-1509. [0386] Knopf, J. L., Gallagher, J. F., and Held, W. A. (1983). Differential, multihormonal regulation of the mouse major urinary protein gene family in the liver. Mol. Cell. Biol. 3, 2232-2240. [0387] Kobayashi, T., Shimabukuro-Demoto, S., Yoshida-Sugitani, R., Furuyama-Tanaka, K., Karyu, H., Sugiura, Y., Shimizu, Y., Hosaka, T., Goto, M., Kato, N., et al. (2014). The histidine transporter SLC15A4 coordinates mTOR-dependent inflammatory responses and pathogenic antibody production. Immunity 41, 375-388. [0388] Kristiansen, O. P., and Mandrup-Poulsen, T. (2005). Interleukin-6 and Diabetes. Diabetes 54, S114 LP-S124. [0389] Lakowski, B., and Hekimi, S. (1998). The genetics of caloric restriction in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 95, 13091-13096. [0390] Lamb, J., Crawford, E. D., Peck, D., Modell, J. W., Blat, I. C., Wrobel, M. J., Lerner, J., Brunet, J., Subramanian, A., Ross, K. N., et al. (2006). The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease. Science (80-.). 313, 1929-1935. [0391] Leiser, S. F., and Miller, R. A. (2010). Nrf2 Signaling, a Mechanism for Cellular Stress Resistance in Long-Lived Mice. Mol. Cell. Biol. 30, 871-884. [0392] Li, X., Bartke, A., Berryman, D. E., Funk, K., Kopchick, J. J., List, E. O., Sun, L., and Miller, R. A. (2013). Direct and indirect effects of growth hormone receptor ablation on liver expression of xenobiotic metabolizing genes. AJP Endocrinol. Metab. 305, E942-E950. [0393] Lin, A. S., Defossez, P., Guarente, L., Lin, S., Defossez, P., and Guarentet, L. (2000). Requirement of NAD and SIR2 for Life-Span Extension by Calorie Restriction in Saccharomyces cerevisiae. Science (80-.). 289, 2126-2128. [0394] Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M., and Kroemer, G. (2013). The hallmarks of aging. Cell 153. [0395] Ma, S., Yim, S. H., Lee, S.-G., Kim, E. B., Lee, S.-R., Chang, K.-T., Buffenstein, R., Lewis, K. N., Park, T. J., Miller, R. A., et al. (2015). Organization of the Mammalian Metabolome according to Organ Function, Lineage Specialization, and Longevity. Cell Metab. 22, 332-343. [0396] De Magalhães, J. P., and Toussaint, 0. (2004). GenAge: A genomic and proteomic network map of human ageing. FEBS Lett. 571, 243-247. [0397] Martin-Montalvo, A., Mercken, E. M., Mitchell, S. J., Palacios, H. H., Mote, P. L., Scheibye-Knudsen, M., Gomes, A. P., Ward, T. M., Minor, R. K., Blouin, M.-J., et al. (2013). Metformin improves healthspan and lifespan in mice. Nat Commun. 4, 2192. [0398] Matys, V. (2006). TRANSFAC® and its module TRANSCompel®: transcriptional gene regulation in eukaryotes. Nucleic Acids Res. 34, D108-D110. [0399] Mehta, R., Steinkraus, K. A., Sutphin, G. L., Ramos, F. J., S, L., Huh, A., Davis, C., Chandler-brown, D., and Kaeberlein, M. (2009). Proteasomal Regulation of the Hypoxic Response Modulates Aging in C. elegans.Science (80-.). 324, 1196-1198. [0400] Mercken, E. M., Hu, J., Krzysik-Walker, S., Wei, M., Li, Y., Mcburney, M. W., de Cabo, R., and Longo, V. D. (2014a). SIRT1 but not its increased expression is essential for lifespan extension in caloric-restricted mice. Aging Cell 13, 193-196. [0401] Mercken, E. M., Mitchell, S. J., Martin-, A., Minor, R. K., Almeida, M., Gomes, A. P., Scheibye-knudsen, M., Hector, H., Licata, J. J., Zhang, Y., et al. (2014b). SRT2104 extends survival of male mice on a standard diet and preserves bone and muscle mass. 787-796. [0402] Miller, D. L., and Roth, M. B. (2007). Hydrogen sulfide increases thermotolerance and lifespan in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 104, 20618-20622. [0403] Miller, R. A., Harrison, D. E., Astle, C. M., Floyd, R. A., Flurkey, K., Hensley, K. L., Javors, M. A., Leeuwenburgh, C., Nelson, J. F., Ongini, E., et al. (2007). An aging Interventions Testing Program: Study design and interim report. Aging Cell 6, 565-575. [0404] Miller, R. A., Harrison, D. E., Astle, C. M., Baur, J. A., Boyd, A. R., De Cabo, R., Fernandez, E., Flurkey, K., Javors, M. A., Nelson, J. F., et al. (2011). Rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogeneous mice. Journals Gerontol.—Ser. A Biol. Sci. Med. Sci. 66 A, 191-201. [0405] Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., Javors, M. A., Li, X., Nadon, N. L., Nelson, J. F., et al. (2014). Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. Aging Cell 13, 468-477. [0406] Mitchell, S. J., Madrigal-Matute, J., Scheibye-Knudsen, M., Fang, E., Aon, M., Gonzalez-Reyes, J. A., Cortassa, S., Kaushik, S., Gonzalez-Freire, M., Patel, B., et al. (2016). Effects of Sex, Strain, and Energy Intake on Hallmarks of Aging in Mice. Cell Metab. 23, 1093-1112. [0407] Moorad, J. A., Promislow, D. E. L., Nate, F., and Miller Richard A. (2012). A comparative assessment of univariate longevity measures using zoological animal records. Aging Cell 11, 940-948. [0408] Mpoy, M., Vandeleene, B., Ketelslegers, J. M., and Lambert, A. E. (1988). Treatment of systemic hypertension in insulin-treated diabetes mellitus with rilmenidine. Am. J. Cardiol. 61, 5-8. [0409] Mutter, F. E., Park, B. K., and Copple, I. M. (2015). Value of monitoring Nrf2 activity for the detection of chemical and oxidative stress. Biochem. Soc. Trans. 43, 657-662. [0410] Nakamura, N., Lill, J. R., Phung, Q., Jiang, Z., Bakalarski, C., De Mazière, A., Klumperman, J., Schlatter, M., Delamarre, L., and Mellman, I. (2014). Endosomes are specialized platforms for bacterial sensing and NOD2 signalling. Nature 509, 240-244. [0411] Narod, S. A., and Foulkes, W. D. (2004). BRCA1 and BRCA2: 1994 and beyond. Nat. Rev. Cancer 4, 665-676. [0412] Osburn, W. O., Yates, M. S., Dolan, P. D., Chen, S., Liby, K. T., Sporn, M. B., Taguchi, K., Yamamoto, M., and Kensler, T. W. (2008). Genetic or pharmacologic amplification of Nrf2 signaling inhibits acute inflammatory liver injury in mice. Toxicol. Sci. 104, 218-227. [0413] Ozerov, I. V., Lezhnina, K. V., lzumchenko, E., Artemov, A. V., Medintsev, S., Vanhaelen, Q., Aliper, A., Vijg, J., Osipov, A. N., Labat, I., et al. (2016). In silico Pathway Activation Network Decomposition Analysis (iPANDA) as a method for biomarker development. Nat. Commun. 7, 1-11. [0414] Pearson, K. J., Baur, J. A., Lewis, K. N., Peshkin, L., Price, N. L., Labinskyy, N., Swindell, W. R., Kamara, D., Minor, R. K., Perez, E., et al. (2008). Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span. Cell Metab. 8, 157-168. [0415] Plank, M., Wuttke, D., van Dam, S., Clarke, S. A., and de Magalhães, J. P. (2012). A meta-analysis of caloric restriction gene expression profiles to infer common signatures and regulatory mechanisms. Mol. Biosyst. 8, 1339. [0416] Ramadoss, P., Chiappini, F., Bilban, M., and Hollenberg, A. N. (2010). Regulation of hepatic six transmembrane epithelial antigen of prostate 4 (STEAP4) expression by STAT3 and CCAAT/enhancer-binding protein α. J. Biol. Chem. 285, 16453-16466. [0417] Rhoads, T. W., Burhans, M. S., Chen, V. B., Coon, J. J., Colman, R. J., Anderson, R. M., Rhoads, T. W., Burhans, M. S., Chen, V. B., Hutchins, P. D., et al. (2018). Caloric Restriction Engages Hepatic RNA Processing Mechanisms in Rhesus Monkeys Resource Caloric Restriction Engages Hepatic RNA Processing Mechanisms in Rhesus Monkeys. Cell Metab. 27, 677-688.e5. [0418] Richie, J. P., Leutzinger, Y., Parthasarathy, S., Malloy, V., Orentreich, N., and Zimmerman, J. a (1994). Methionine restriction increases blood glutathione and longevity in F344 rats. FASEB J. 8, 1302-1307. Roberts, S. A., Simpson, D. M., Armstrong, S. D., Davidson, A. J., Robertson, D. H., McLean, L., Beynon, R. J., and Hurst, J. L. (2010). Darcin: A male pheromone that stimulates female memory and sexual attraction to an individual male's odour. BMC Biol. 8. [0419] Rowland, J. E., Lichanska, A. M., Linda, M., White, M., Aniello, E. M., Maher, S. L., Brown, R., Teasdale, R. D., Noakes, P. G., Waters, M. J., et al. (2005). In Vivo Analysis of Growth Hormone Receptor Signaling Domains and Their Associated Transcripts In Vivo Analysis of Growth Hormone Receptor Signaling Domains and Their Associated Transcripts. Mol. Cell. Biol. 25, 66-77. [0420] Rusli, F., Boekschoten, M. V., Zubia, A. A., Lute, C., Müller, M., and Steegenga, W. T. (2015). A weekly alternating diet between caloric restriction and medium fat protects the liver from fatty liver development in middle-aged C57BL/6J mice. Mol. Nutr. Food Res. 59, 533-543. [0421] Senn, J. J., Klover, P. J., Nowak, I. A., and Mooney, R. A. (2002). Interleukin-6 induces cellular insulin resistance in hepatocytes. Diabetes 51, 3391-3399. [0422] Steinbaugh, M. J., Sun, L. Y., Bartke, A., and Miller, R. A. (2012). Activation of genes involved in xenobiotic metabolism is a shared signature of mouse models with extended lifespan. Am. J. Physiol. Endocrinol. Metab. 303, E488-95. [0423] Steiner, A. A., and Branco, L. G. S. (2002). Hypoxia-Induced Anapyrexia: Implications and Putative Mediators. Annu. Rev. Physiol. 64, 263-288. [0424] Streeper, R. S., Grueter, C. A., Salomonis, N., Cases, S., Levin, M. C., Koliwad, S. K., Zhou, P., Hirschey, M. D., Verdin, E., and Farese, R. V. (2012). Deficiency of the lipid synthesis enzyme, DGAT1, extends longevity in mice. Aging (Albany. N.Y.). 4, 13-27. [0425] Strong, R., Miller, R. A., Astle, C. M., Floyd, R. A., Flurkey, K., Hensley, K. L., Javors, M. A., Leeuwenburgh, C., Nelson, J. F., Ongini, E., et al. (2008). Nordihydroguaiaretic acid and aspirin increase lifespan of genetically heterogeneous male mice. Aging Cell 7, 641-650. [0426] Strong, R., Miller, R. A., Astle, C. M., Baur, J. A., De Cabo, R., Fernandez, E., Guo, W., Javors, M., Kirkland, J. L., Nelson, J. F., et al. (2013). Evaluation of resveratrol, green tea extract, curcumin, oxaloacetic acid, and medium-chain triglyceride oil on life span of genetically heterogeneous mice. Journals Gerontol.—Ser. A Biol. Sci. Med. Sci. 68, 6-16. [0427] Strong, R., Miller, R. A., Antebi, A., Astle, C. M., Bogue, M., Denzel, M. S., Fernandez, E., Flurkey, K., Hamilton, K. L., Lamming, D. W., et al. (2016). Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α-glucosidase inhibitor or a Nrf2-inducer. Aging Cell 15, 872-884.
[0428] Subramanian, A., Tamayo, P., Mootha, V. K., Mukherjee, S., Ebert, B. L., Gillette, M. a, Paulovich, A., Pomeroy, S. L., Golub, T. R., Lander, E. S., et al. (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U.S.A 102, 15545-15550. [0429] Subramanian, A., Narayan, R., Corsello, S. M., Peck, D. D., Natoli, T. E., Lu, X. L., Gould, J., Doench, J. G., Bittker, J. A., Root, D. E., et al. (2017). A Next Generation Connectivity Map: L1000 platform and the first 1,000,000 profiles. Cell 171, 1437-1452. [0430] Sun, L. Y., Spong, A., Swindell, W. R., Fang, Y., Hill, C., Huber, J. A., Boehm, J. D., Westbrook, R., Salvatori, R., and Bartke, A. (2013). Growth hormone-releasing hormone disruption extends lifespan and regulates response to caloric restriction in mice. Elife 2, e01098. [0431] Swardfager, W., Lanctt, K., Rothenburg, L., Wong, A., Cappell, J., and Herrmann, N. (2010). A meta-analysis of cytokines in Alzheimer's disease. Biol. Psychiatry 68, 930-941. [0432] Swindell, W. R. (2008). Comparative analysis of microarray data identifies common responses to caloric restriction among mouse tissues. Mech. Ageing Dev. 129, 138-153. [0433] Sykiotis, G. P., and Bohmann, D. (2008). Keap1/Nrf2 Signaling Regulates Oxidative Stress Tolerance and Lifespan in Drosophila. Dev. Cell 14, 76-85. [0434] Sziráki, A., Tyshkovskiy, A., and Gladyshev, V. N. (2018). Global remodeling of the mouse DNA methylome during aging and in response to calorie restriction. Aging Cell e12738. [0435] Tissenbaum, H. a, and Guarente, L. (2001). Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410, 227-230. [0436] Tsuchiya, T., Dhahbi, J. M., Cui, X., Mote, P. L., Bartke, A., and Spindler, S. R. (2004). Additive regulation of hepatic gene expression by dwarfism and caloric restriction. Physiol. Genomics 17, 307-315. [0437] Tullet, J. M. A., Hertweck, M., An, J. H., Baker, J., Hwang, J. Y., Liu, S., Oliveira, R. P., Baumeister, R., and Blackwell, T. K. (2008). Direct Inhibition of the Longevity-Promoting Factor SKN-1 by Insulin-like Signaling in C. elegans. Cell 132, 1025-1038. [0438] Ubagai, T., Lei, K. J., Huang, S., Mudd, S. H., Levy, H. L., and Chou, J. Y. (1995). Molecular mechanisms of an inborn error of methionine pathway. Methionine adenosyltransferase deficiency. J. Clin. Invest. 96, 1943-1947. [0439] Uthus, E. O., and Brown-Borg, H. M. (2003). Altered methionine metabolism in long living Ames dwarf mice. Exp. Gerontol. 38, 491-498. [0440] Valenzano, D. R., Terzibasi, E., Genade, T., Cattaneo, A., Domenici, L., and Cellerino, A. (2006). Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr. Biol. 16, 296-300. [0441] Vellai, T., Takacs-Vellai, K., Zhang, Y., Kovacs, A. L., Orosz, L., and Müller, F. (2003). Genetics: influence of TOR kinase on lifespan in C. elegans. Nature 426, 620. [0442] Viswanathan, M., Kim, S. K., Berdichevsky, A., and Guarente, L. (2005). A role for SIR-2.1 regulation of ER stress response genes in determining C. elegans life span. Dev. Cell 9, 605-615. [0443] Wauthier, V., Verbeeck, R., and Buc Calderon, P. (2007). The Effect of Ageing on Cytochrome P450 Enzymes: Consequences for Drug Biotransformation in the Elderly. Curr. Med. Chem. 14, 745-757. [0444] Waxman, D. J., and Holloway, M. G. (2009). Sex Differences in the Expression of Hepatic Drug Metabolizing Enzymes. Mol Pharmacol 76, 215-228. [0445] Weindruch, R., Walford, R. L., Fligiel, S., and Guthrie, D. (1986). The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. J. Nutr. 116, 641-654. [0446] Wood, J. G., Regina, B., Lavu, S., Hewitz, K., Helfand, S. L., Tatar, M., and Sinclair, D. (2004). Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature 430, 686-689. [0447] Yuan, R., Tsaih, S., Petkova, S. B., Evsikova, C. M. De, Marion, M. A., Bogue, M. A., Mills, K. D., Peters, L. L., Bult, C. J., Rosen, C. J., et al. (2009). Aging in inbred strains of mice: Study design and interim report on median lifespan and circulating IGF1 levels. Aging Cell 8, 277-287. [0448] Zahn, J. M., Sonu, R., Vogel, H., Crane, E., Mazan-Mamczarz, K., Rabkin, R., Davis, R. W., Becker, K. G., Owen, A. B., and Kim, S. K. (2006). Transcriptional profiling of aging in human muscle reveals a common aging signature. PLoS Genet. 2, 1058-1069. [0449] Zhang, L., Ebenezer, P. J., Dasuri, K., Fernandez-Kim, S. O., Francis, J., Mariappan, N., Gao, Z., Ye, J., Bruce-Keller, A. J., and Keller, J. N. (2011). Aging is associated with hypoxia and oxidative stress in adipose tissue: implications for adipose function. Am. J. Physiol. Endocrinol. Metab. 301, E599-607. [0450] Zhang, Y., Xie, Y., Berglund, E. D., Colbert Coate, K., He, T. T., Katafuchi, T., Xiao, G., Potthoff, M. J., Wei, W., Wan, Y., et al. (2012). The starvation hormone, fibroblast growth factor-21, extends lifespan in mice. Elife 2012, 1-14. [0451] Zhao, Y., Tyshkovskiy, A., Muñoz-Espin, D., Tian, X., Serrano, M., de Magalhaes, J. P., Nevo, E., Gladyshev, V. N., Seluanov, A., and Gorbunova, V. (2018). Naked mole rats can undergo developmental, oncogene-induced and DNA damage-induced cellular senescence. Proc. Natl. Acad. Sci. 115, 1801-1806. [0452] Zhou, Y., Xu, B. C., Maheshwari, H. G., He, L., Reed, M., Lozykowski, M., Okada, S., Cataldo, L., Coschigamo, K., Wagner, T. E., et al. (1997). A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc. Natl. Acad. Sci. U.S.A 94, 13215-13220. [0453] Ables, G. P., Perrone, C. E., Orentreich, D., and Orentreich, N. (2012). Methionine-Restricted C57BL/6J Mice Are Resistant to Diet-Induced Obesity and Insulin Resistance but Have Low Bone Density. PLoS One 7, 1-12. [0454] Ables, G. P., Ouattara, A., Hampton, T. G., Cooke, D., Perodin, F., Augie, I., and Orentreich, D. S. (2015). Dietary methionine restriction in mice elicits an adaptive cardiovascular response to hyperhomocysteinemia. Sci. Rep. 5, 1-10. [0455] Alonso, C., Fernández-Ramos, D., Varela-Rey, M., Martinez-Arranz, I., Navasa, N., Van Liempd, S. M., Lavin Trueba, J. L., Mayo, R., Ilisso, C. P., de Juan, V. G., et al. (2017). Metabolomic Identification of Subtypes of Nonalcoholic Steatohepatitis. Gastroenterology 152, 1449-1461.e7. [0456] Anders, S., and Huber, W. (2010). Differential expression analysis for sequence count data. Genome Biol. 11. [0457] Baze, M. M., Schlauch, K., and Hayes, J. P. (2010). Gene expression of the liver in response to chronic hypoxia. Physiol Genomics 41, 275-288. [0458] Benjamini, Y., and Hochberg, Y. (1995). Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. R. Stat. Soc. Ser. B 57, 289-300. [0459] Coschigano, K. T., Holland, A. N., Riders, M. E., List, E. O., Flyvbjerg, A., and Kopchick, J. J. (2003). Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin, and insulin-like growth factor I levels and increased life span. Endocrinology 144, 3799-3810. [0460] Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T. R. (2013). STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21. [0461] Edgar, R. (2002). Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207-210. [0462] Flurkey, K., Papaconstantinou, J., Miller, R. A., and Harrison, D. E. (2001). Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production. Proc. Natl. Acad. Sci. 98, 6736-6741. [0463] García-Martínez, J. M., Wullschleger, S., Preston, G., Guichard, S., Fleming, S., Alessi, D. R., and Duce, S. L. (2011). Effect of PI3K- and mTOR-specific inhibitors on spontaneous B-cell follicular lymphomas in PTEN/LKB1-deficient mice. Br. J. Cancer 104, 1116-1125. [0464] Harrison, D. E., Strong, R., Allison, D. B., Ames, B. N., Astle, C. M., Atamna, H., Fernandez, E., Flurkey, K., Javors, M. A., Nadon, N. L., et al. (2014). Acarbose, 17-α-estradiol, and nordihydroguaiaretic acid extend mouse lifespan preferentially in males. Aging Cell 13, 273-282. [0465] Hashimshony, T., Senderovich, N., Avital, G., Klochendler, A., de Leeuw, Y., Anavy, L., Gennert, D., Li, S., Livak, K. J., Rozenblatt-Rosen, O., et al. (2016). CEL-Seq2: Sensitive highly-multiplexed single-cell RNA-Seq. Genome Biol. 17, 1-7. [0466] Huang, D. W., Sherman, B. T., and Lempicki, R. A. (2009a). Bioinformatics enrichment tools: Paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1-13. [0467] Huang, D. W., Lempicki, R. a, and Sherman, B. T. (2009b). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44-57. [0468] Jackson, K. L., Palma-Rigo, K., Nguyen-Huu, T. P., Davern, P. J., and Head, G. A. (2014). Actions of rilmenidine on neurogenic hypertension in BPH/2J genetically hypertensive mice. J. Hypertens. 32, 575-586. [0469] Kanfi, Y., Naiman, S., Amir, G., Peshti, V., Zinman, G., Nahum, L., Bar-Joseph, Z., and Cohen, H. Y. (2012). The sirtuin SIRT6 regulates lifespan in male mice. Nature 483, 218-221. [0470] Kautz, L., Meynard, D., Monnier, A., Darnaud, V., Bouvet, R., Wang, R. H., Deng, C., Vaulont, S., Mosser, J., Coppin, H., et al. (2008). Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1, and Atoh8 in the mouse liver. Blood 112, 1503-1509. [0471] Kolesnikov, N., Hastings, E., Keays, M., Melnichuk, O., Tang, Y. A., Williams, E., Dylag, M., Kurbatova, N., Brandizi, M., Burdett, T., et al. (2015). ArrayExpress update-simplifying data submissions. Nucleic Acids Res. 43, D1113-D1116. [0472] Lamb, J., Crawford, E. D., Peck, D., Modell, J. W., Blat, I. C., Wrobel, M. J., Lerner, J., Brunet, J., Subramanian, A., Ross, K. N., et al. (2006). The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease. Science (80-.). 313, 1929-1935. [0473] Liao, Y., Smyth, G. K., and Shi, W. (2014). FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923-930. [0474] Ma, S., Yim, S. H., Lee, S.-G., Kim, E. B., Lee, S.-R., Chang, K.-T., Buffenstein, R., Lewis, K. N., Park, T. J., Miller, R. A., et al. (2015). Organization of the Mammalian Metabolome according to Organ Function, Lineage Specialization, and Longevity. Cell Metab. 22, 332-343. [0475] De Magalhães, J. P., and Toussaint, 0. (2004). GenAge: A genomic and proteomic network map of human ageing. FEBS Lett. 571, 243-247. [0476] Matys, V. (2006). TRANSFAC® and its module TRANSCompel®: transcriptional gene regulation in eukaryotes. Nucleic Acids Res. 34, D108-D110. [0477] Mercken, E. M., Mitchell, S. J., Martin-, A., Minor, R. K., Almeida, M., Gomes, A. P., Scheibye-knudsen, M., Hector, H., Licata, J. J., Zhang, Y., et al. (2014). SRT2104 extends survival of male mice on a standard diet and preserves bone and muscle mass. 787-796. [0478] Miller, R. A., Harrison, D. E., Astle, C. M., Baur, J. A., Boyd, A. R., De Cabo, R., Fernandez, E., Flurkey, K., Javors, M. A., Nelson, J. F., et al. (2011). Rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogeneous mice. Journals Gerontol.—Ser. A Biol. Sci. Med. Sci. 66 A, 191-201. [0479] Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., Javors, M. A., Li, X., Nadon, N. L., Nelson, J. F., et al. (2014). Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. Aging Cell 13, 468-477. [0480] Mitchell, S. J., Madrigal-Matute, J., Scheibye-Knudsen, M., Fang, E., Aon, M., Gonzalez-Reyes, J. A., Cortassa, S., Kaushik, S., Gonzalez-Freire, M., Patel, B., et al. (2016). Effects of Sex, Strain, and Energy Intake on Hallmarks of Aging in Mice. Cell Metab. 23, 1093-1112. [0481] Osburn, W. O., Yates, M. S., Dolan, P. D., Chen, S., Liby, K. T., Sporn, M. B., Taguchi, K., Yamamoto, M., and Kensler, T. W. (2008). Genetic or pharmacologic amplification of Nrf2 signaling inhibits acute inflammatory liver injury in mice. Toxicol. Sci. 104, 218-227. [0482] Ozerov, I. V., Lezhnina, K. V., lzumchenko, E., Artemov, A. V., Medintsev, S., Vanhaelen, Q., Aliper, A., Vijg, J., Osipov, A. N., Labat, I., et al. (2016). In silico Pathway Activation Network Decomposition Analysis (iPANDA) as a method for biomarker development. Nat. Commun. 7, 1-11. [0483] Paynter, N. P., Balasubramanian, R., Giulianini, F., Wang, D. D., Tinker, L. F., Gopal, S., Deik, A. A., Albert, C. M., Clish, C. B., and Rexrode, K. M. (2018). Metabolic Predictors of Incident Coronary Heart Disease in Women. Circulation 137, 841-853. [0484] Plank, M., Wuttke, D., van Dam, S., Clarke, S. A., and de Magalhaes, J. P. (2012). A meta-analysis of caloric restriction gene expression profiles to infer common signatures and regulatory mechanisms. Mol. Biosyst. 8, 1339. [0485] Ramadoss, P., Chiappini, F., Bilban, M., and Hollenberg, A. N. (2010). Regulation of hepatic six transmembrane epithelial antigen of prostate 4 (STEAP4) expression by STAT3 and CCAAT/enhancer-binding protein α. J. Biol. Chem. 285, 16453-16466. [0486] Rhoads, T. W., Burhans, M. S., Chen, V. B., Coon, J. J., Colman, R. J., Anderson, R. M., Rhoads, T. W., Burhans, M. S., Chen, V. B., Hutchins, P. D., et al. (2018). Caloric Restriction Engages Hepatic RNA Processing Mechanisms in Rhesus Monkeys. Cell Metab. 27, 677-688.e5. [0487] Ritchie, M. E., Phipson, B., Wu, D., Hu, Y., Law, C. W., Shi, W., and Smyth, G. K. (2015). Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47. [0488] Robinson, M. D., McCarthy, D. J., and Smyth, G. K. (2009). edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140. [0489] Shannon, P., Markiel, A., Owen Ozier, 2, Baliga, N. S., Wang, J. T., Ramage, D., Amin, N., Schwikowski, B., and Ideker, T. (2003). Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2498-2504. [0490] St-Cyr, J., Derome, N. & Bernatchez, L. (2008) The transcriptomics of life-history trade-offs in whitefish species pairs (Coregonus sp.). Mol. Ecol. 17, 1850-1870. [0491] Strong, R., Miller, R. A., Antebi, A., Astle, C. M., Bogue, M., Denzel, M. S., Fernandez, E., Flurkey, K., Hamilton, K. L., Lamming, D. W., et al. (2016). Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α-glucosidase inhibitor or a Nrf2-inducer. Aging Cell 15, 872-884. [0492] Subramanian, A., Tamayo, P., Mootha, V. K., Mukherjee, S., Ebert, B. L., Gillette, M. a, Paulovich, A., Pomeroy, S. L., Golub, T. R., Lander, E. S., et al. (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U.S.A 102, 15545-15550. [0493] Veurink, G., Liu, D., Taddei, K., Perry, G., Smith, M. A., Robertson, T. A., Hone, E., Groth, D. M., Atwood, C. S., and Martins, R. N. (2003). Reduction of inclusion body pathology in ApoE-deficient mice fed a combination of antioxidants. Free Radic. Biol. Med. 34, 1070-1077. [0494] Viechtbauer, W. (2010). Conducting Meta-Analyses in R with the metafor Package. J. Stat. Softw. 36, 1-48. [0495] Yongxi, T., Haijun, H., Jiaping, Z., Guoliang, S., and Hongying, P. (2015). Autophagy inhibition sensitizes KU-0063794-mediated anti-HepG2 hepatocellular carcinoma cell activity in vitro and in vivo. Biochem. Biophys. Res. Commun. 465, 494-500.
Enumerated Embodiments of the Invention
[0496] The invention is also characterized by the following enumerated embodiments:
[0497] 1. A method of identifying an agent capable of increasing the lifespan of a mammalian subject, the method comprising contacting the agent with a cell comprising one or more genes set forth in any of Tables 1-20, wherein a finding that the agent (i) increases expression of one or more genes in any of Tables 1-10 and/or (ii) decreases expression of one or more genes in any of Tables 11-20 identifies the agent as being capable of increasing the lifespan of a mammalian subject.
[0498] 2. The method of embodiment 1, wherein the subject is a human.
[0499] 3. The method of embodiment 1 or 2, wherein the cell comprises one or more genes set forth in any of Tables 1-6 or Tables 11-16, wherein a finding that the agent (i) increases expression of one or more genes in any of Tables 1-6 and/or (ii) decreases expression of one or more genes in any of Tables 11-16 identifies the agent as being capable of increasing the lifespan of the mammalian subject.
[0500] 4. The method of embodiment 3, wherein the cell comprises two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 1 and/or Table 11.
[0501] 5. The method of embodiment 3 or 4, wherein the cell comprises two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 2 and/or Table 12.
[0502] 6. The method of any one of embodiments 3-5, wherein the cell comprises two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 3 and/or Table 13.
[0503] 7. The method of any one of embodiments 3-6, wherein the cell comprises two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 4 and/or Table 14.
[0504] 8. The method of any one of embodiments 3-7, wherein the cell comprises two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 5 and/or Table 15.
[0505] 9. The method of any one of embodiments 3-8, wherein the cell comprises two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 6 and/or Table 16.
[0506] 10. The method of any one of embodiments 1-9, wherein the cell comprises one or more genes set forth in Table 7 or Table 17, wherein a finding that the agent (i) increases expression of one or more genes in Table 7 and/or (ii) decreases expression of one or more genes in Table 17 identifies the agent as being capable of increasing the lifespan of the mammalian subject.
[0507] 11. The method of embodiment 10, wherein the cell comprises two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 7 and/or Table 17.
[0508] 12. The method of any one of embodiments 1-11, wherein the cell comprises one or more genes set forth in any of Tables 8-10 or Tables 18-20, wherein a finding that the agent (i) increases expression of one or more genes in any of Tables 8-10 and/or (ii) decreases expression of one or more genes in any of Tables 18-20 identifies the agent as being capable of increasing the lifespan of the mammalian subject.
[0509] 13. The method of embodiment 12, wherein the cell comprises two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 8 and/or Table 18.
[0510] 14. The method of embodiment 12 or 13, wherein the cell comprises two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 9 and/or Table 19.
[0511] 15. The method of any one of embodiments 12-14, wherein the cell comprises two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 10 and/or Table 20.
[0512] 16. The method of any one of embodiments 1-15, wherein the agent is contacted with the cell by administering the agent to a test subject comprising the cell.
[0513] 17. The method of embodiment 16, wherein the test subject is a mammal.
[0514] 18. The method of embodiment 17, wherein the test subject is a mouse.
[0515] 19. The method of any one of embodiments 1-18, wherein expression of the one or more genes in the cell is determined by RNA-seq.
[0516] 20. The method of any one of embodiments 1-19, the method further comprising administering the identified agent to a mammalian subject to increase the lifespan of the subject and/or to treat an age-related disease.
[0517] 21. A collection of (i) gene expression signatures as set forth in any of Tables 1-10, or a combination thereof, that are upregulated, and (ii) gene expression signatures as set forth in any of Tables 11-20, or a combination thereof, that are downregulated.
[0518] 22. A composition comprising a biological sample and a plurality of nucleic acid primers suitable for amplification of one or more genes set forth in any of Tables 1-10 and/or Tables 11-20.
[0519] 23. The composition of embodiment 22, wherein the nucleic acid primers are at least 85% complementary to a portion of one or more of the genes set forth in any of Tables 1-10 and/or Tables 11-20.
[0520] 24. The composition of embodiment 23, wherein the nucleic acid primers are at least 90% complementary to a portion of one or more of the genes set forth in any of Tables 1-10 and/or Tables 11-20.
[0521] 25. The composition of embodiment 24, wherein the nucleic acid primers are at least 95% complementary to a portion of one or more of the genes set forth in any of Tables 1-10 and/or Tables 11-20.
[0522] 26. The composition of embodiment 25, wherein the nucleic acid primers are 100% complementary to a portion of one or more of the genes set forth in any of Tables 1-10 and/or Tables 11-20.
[0523] 27. The composition of any one of embodiments 22-26, wherein the nucleic acid primers are suitable for amplification of one or more genes set forth in any of Tables 1-6 or Tables 11-16.
[0524] 28. The composition of embodiment 27, wherein the nucleic acid primers are suitable for amplification of two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 1 and/or Table 11.
[0525] 29. The composition of embodiment 27 or 28, wherein the nucleic acid primers are suitable for amplification of two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 2 and/or Table 12.
[0526] 30. The composition of any one of embodiments 27-29, wherein the nucleic acid primers are suitable for amplification of two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 3 and/or Table 13.
[0527] 31. The composition of any one of embodiments 27-30, wherein the nucleic acid primers are suitable for amplification of two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 4 and/or Table 14.
[0528] 32. The composition of any one of embodiments 27-31, wherein the nucleic acid primers are suitable for amplification of two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 5 and/or Table 15.
[0529] 33. The composition of any one of embodiments 27-32, wherein the nucleic acid primers are suitable for amplification of two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 6 and/or Table 16.
[0530] 34. The composition of any one of embodiments 22-33, wherein the nucleic acid primers are suitable for amplification of one or more genes set forth in Table 7 or Table 17.
[0531] 35. The composition of embodiment 34, wherein the nucleic acid primers are suitable for amplification of two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 7 and/or Table 17.
[0532] 36. The composition of any one of embodiments 22-35, wherein the nucleic acid primers are suitable for amplification of one or more genes set forth in any of Tables 8-10 or Tables 18-20.
[0533] 37. The composition of embodiment 36, wherein the nucleic acid primers are suitable for amplification of two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 8 and/or Table 18.
[0534] 38. The composition of embodiment 36 or 37, wherein the nucleic acid primers are suitable for amplification of two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 9 and/or Table 19.
[0535] 39. The composition of any one of embodiments 36-38, wherein the nucleic acid primers are suitable for amplification of two, three, four, five, six, seven, eight, nine, ten, or more genes set forth in Table 10 and/or Table 20.
[0536] 40. A method of increasing the lifespan of a mammalian subject, the method comprising providing the subject with a treatment that (i) increases expression of one or more genes set forth in any of Tables 1-10 and/or (ii) decreases expression of one or more genes set forth in any of Tables 11-20.
[0537] 41. A method of reducing the frailty index in a mammalian subject, the method comprising providing the subject with a treatment that (i) increases expression of one or more genes set forth in any of Tables 1-10 and/or (ii) decreases expression of one or more genes set forth in any of Tables 11-20.
[0538] 42. A method of improving learning ability in a mammalian subject, the method comprising providing the subject with a treatment that (i) increases expression of one or more genes set forth in any of Tables 1-10 and/or (ii) decreases expression of one or more genes set forth in any of Tables 11-20.
[0539] 43. A method of delaying onset of a geriatric syndrome in a mammalian subject, the method comprising providing the subject with a treatment that (i) increases expression of one or more genes set forth in any of Tables 1-10 and/or (ii) decreases expression of one or more genes set forth in any of Tables 11-20.
[0540] 44. A method of increasing the lifespan of a mammalian subject, the method comprising administering to the subject a therapeutically effective amount of Selumetinib (6-(4-Bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide), LY294002 (2-Morpholin-4-yl-8-phenylchromen-4-one), AZD-8055 (5-[2,4-bis[(3S)-3-methyl-4-morpholinyl]pyrido[2,3-d]pyrimidin-7-yl]-2-methoxy-benzenemethanol), KU-0063794 (rel-5-[2-[(2R,6S)-2,6-dimethyl-4-morpholinyl]-4-(4-morpholinyl)pyrido[2,3-d]pyrimidin-7-yl]-2-methoxybenzenemethanol), Celastrol (3-Hydroxy-9β,13α-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic acid), Ascorbyl Palmitate ([(2S)-2-[(2R)-4,5-Dihydroxy-3-oxo-2-furyl]-2-hydroxy-ethyl] hexadecanoate), Oligomycin-a ((1R,4E,5'S,6S,6'S,7R,8S,10R,11R,12S,14R,15S,16R,18E,20E,22R,25S,27R,28S,29R)-22-ethyl-7,11,14,15-tetrahydroxy-6′-[(2R)-2-hydroxypropyl]-5′,6,8,10,12,14,16,28,29-nonamethyl-3′,4′,5′,6′-tetrahydro-3H,9H,13H-spiro[2,26-dioxabicyclo[23.3.1]nonacosa-4,18,20-triene-27,2′-pyran]-3,9,13-trione), NVP-BEZ235 (2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile), Importazole (N-(1-Phenylethyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine), Ryuvidine (2-methyl-5-[(4-methylphenyl)amino]-4,7-benzothiazoledione), NSC-663284 (6-Chloro-7-[[2-(4-morpholinyl)ethyl]amino]-5,8-quinolinedione), P1-828 (2-(4-Morpholinyl)-8-(4-aminopheny)l-4H-1-benzopyran-4-one), Pyrvinium pamoate (6-(Dimethylamino)-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)ethenyl]-1-methyl-4,4′-methylenebis[3-hydroxy-2-naphthalenecarboxylate] (2:1)-quinolinium), P1-103 (3-[4-(4-morpholinyl)pyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]-phenol), YM-155 (4,9-dihydro-1-(2-methoxyethyl)2-methyl-4,9-dioxo-3-(2-pyrazinylmethyl)-1H-naphth[2,3-d]imidazolium, bromide), Prostratin ((1aR,1bS,4aR,7aS,7bR,8R,9aS)-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropa[3,4]benzo[1,2-e]azulen-9a-yl acetate), BCI hydrochloride (3-(cyclohexylamino)-2,3-dihydro-2-(phenylmethylene)-1H-inden-1-one, monohydrochloride), Dorsomorphin-Compound C (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)pyrazolo[1,5-a]pyrimidine), VU-0418947-2 (6-Phenyl-N-[(3-phenylphenyl)methyl]-3-pyridin-2-yl-1,2,4-triazin-5-amine), JNK-9L (4-[3-fluoro-5-(4-morpholinyl)phenyl]-N-[4-[3-(4-morpholinyl)-1,2,4-triazol-1-yl]phenyl]-2-pyrimidinamine), Phloretin (3-(4-Hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one), ZG-10 ((E)-4-(4-(dimethylamino)but-2-enamido)-N-(3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide), Proscillaridin (5-[(3S,8R,9S,10R,13R,14S,17R)-14-Hydroxy-10,13-dimethyl-3-((2R,3R,4R,5R,6R)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-2H-pyran-2-one), YC-1 (3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole), IKK-2-inhibitor-V (N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide), Anisomycin ((2R,3S,4S)-4-hydroxy-2-(4-methoxybenzyl)-pyrrolidin-3-yl acetate), Colforsin ([(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-5-acetyloxy-3-ethenyl-10,10b-dihydroxy-3,4a,7,7,10a-Pentamethyl-1-oxo-5,6,6a,8,9,10-hexahydro-2H-benzo[f]chromen-6-yl] 3-d imethylaminopropanoate), Rilmenidine (N-(Dicyclopropylmethyl)-4,5-dihydro-1,3-oxazol-2-amine), GDC-0941 (Pictilisib, 4-(2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine), Valdecoxib (4-(5-methyl-3-phenylisoxazol-4-yl)benzenesulfonamide), Myricetin (3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), Cyproheptadine (4-(5H-Dibenzo[a,d]cyclohepten-5-ylidene)-1-methylpiperidine), Vorinostat (N-Hydroxy-N′-phenyloctanediamide), Nifedipine (3,5-Dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate), Phylloquinone (2-Methyl-3-[(E)-3,7,11,15-tetramethylhexadec-2-enyl]naphthalene-1,4-dione), Withaferin-A ((4β,5β,6β,22R)-4,27-Dihydroxy-5,6:22,26-diepoxyergosta-2,24-diene-1,26-dione), Temsirolimus ((1R,2R,4S)-4-{(2R)-2-[(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1,4]oxazacyclohentriacontin-3-yl]propyl}-2-methoxycyclohexyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate), SN-38 (4,11-diethyl-4,9-dihydroxy-(4S)-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione), GSK-1059615 (5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidenedione), Tipifarnib (6-[(R)-amino-(4-chlorophenyl)-(3-methylimidazol-4-yl)methyl]-4-(3-chlorophenyl)-1-methylquinolin-2-one), Linifanib (1-[4-(3-amino-1H-indazol-4-yl)phenyl]-3-(2-fluoro-5-methylphenyl)urea), WYE-354 (4-[6-[4-[(methoxycarbonyl)amino]phenyl]-4-(4-morpholinyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]methyl ester-1-piperidinecarboxylic acid), MK-212 (6-Chloro-2-(1-piperazinyl)pyrazine hydrochloride), and/or Enzastaurin (3-(1-Methylindol-3-yl)-4-[1-[1-(pyridin-2-ylmethyl)piperidin-4-yl]indol-3-yl]pyrrole-2,5-dione), thereby increasing the lifespan of the subject.
[0541] 45. A method of reducing the frailty index of a mammalian subject, the method comprising administering to the subject a therapeutically effective amount of Selumetinib, LY294002, AZD-8055, KU-0063794, Celastrol, Ascorbyl Palmitate, Oligomycin-a, NVP-BEZ235, Importazole, Ryuvidine, NSC-663284, P1-828, Pyrvinium pamoate, P1-103, YM-155, Prostratin, BCI hydrochloride, Dorsomorphin-Compound C, VU-0418947-2, JNK-9L, Phloretin, ZG-10, Proscillaridin, YC-1, IKK-2-inhibitor-V, Anisomycin, Colforsin, Rilmenidine, GDC-0941, Valdecoxib, Myricetin, Cyproheptadine, Vorinostat, Nifedipine, Phylloquinone, Withaferin-A, Temsirolimus, SN-38, GSK-1059615, Tipifarnib, Linifanib, WYE-354, MK-212, and/or Enzastaurin, thereby reducing the frailty index of the subject.
[0542] 46. A method of improving learning ability in a mammalian subject, the method comprising administering to the subject a therapeutically effective amount of Selumetinib, LY294002, AZD-8055, KU-0063794, Celastrol, Ascorbyl Palmitate, Oligomycin-a, NVP-BEZ235, Importazole, Ryuvidine, NSC-663284, P1-828, Pyrvinium pamoate, P1-103, YM-155, Prostratin, BCI hydrochloride, Dorsomorphin-Compound C, VU-0418947-2, JNK-9L, Phloretin, ZG-10, Proscillaridin, YC-1, IKK-2-inhibitor-V, Anisomycin, Colforsin, Rilmenidine, GDC-0941, Valdecoxib, Myricetin, Cyproheptadine, Vorinostat, Nifedipine, Phylloquinone, Withaferin-A, Temsirolimus, SN-38, GSK-1059615, Tipifarnib, Linifanib, WYE-354, MK-212, and/or Enzastaurin, thereby improving the learning ability of the subject.
[0543] 47. A method of delaying onset of a geriatric syndrome in a mammalian subject, the method comprising administering to the subject a therapeutically effective amount of Selumetinib, LY294002, AZD-8055, Celastrol, KU-0063794, Ascorbyl Palmitate, Oligomycin-a, NVP-BEZ235, Importazole, Ryuvidine, NSC-663284, PI-828, Pyrvinium pamoate, PI-103, YM-155, Prostratin, BCI hydrochloride, Dorsomorphin-Compound C, VU-0418947-2, JNK-9L, Phloretin, ZG-10, Proscillaridin, YC-1, IKK-2-inhibitor-V, Anisomycin, Colforsin, Rilmenidine, GDC-0941, Valdecoxib, Myricetin, Cyproheptadine, Vorinostat, Nifedipine, Phylloquinone, Withaferin-A, Temsirolimus, SN-38, GSK-1059615, Tipifarnib, Linifanib, WYE-354, MK-212, and/or Enzastaurin, thereby delaying the onset of a geriatric syndrome in the subject.
[0544] 48. The method of any one of embodiments 40-47, wherein the subject is a human.
[0545] 49. The method of any one of embodiments 40-43, wherein the treatment comprises administration of an agent, a lifestyle change, a change in disease status, or a combination thereof.
[0546] 50. The method of embodiment 49, wherein the treatment comprises administration of an agent.
[0547] 51. The method of embodiment 50, wherein the agent comprises a small molecule, a peptide, a peptidomimetic, an interfering ribonucleic acid (RNA), an antibody, an aptamer, or a gene therapy.
[0548] 52. The method of embodiment 51, wherein the agent comprises a small molecule.
[0549] 53. The method of embodiment 52, wherein the agent comprises a compound represented by formula (I)
##STR00005##
[0550] wherein one or two of X.sup.5, X.sup.6 and k is N, and the other(s) is/are CH;
[0551] R.sup.7 is selected from halo, OR.sup.01, SR.sup.S1 NR.sup.N1R.sup.N2, NR.sup.N7aC(═O)R.sup.C1, NR.sup.N7bSO.sub.2R.sup.s2a, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group;
[0552] R.sup.01 and R.sup.S1 are selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.1-7 alkyl group;
[0553] R.sup.N1 and R.sup.N2 are independently selected from H, an optionally substituted C.sub.1-7 alkyl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, or R.sup.N1 and R.sup.N2, together with the nitrogen to which they are bound, form a heterocyclic ring comprising from 3 to 8 ring atoms;
[0554] R.sup.C1 is selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, an optionally substituted C.sub.1-7 alkyl group;
[0555] NR.sup.N8R.sup.N9, wherein R.sup.N8 and R.sup.N9 are independently selected from H, an optionally substituted C.sub.1-7 alkyl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, or R.sup.N8 and R.sup.N9, together with the nitrogen to which they are bound, form a heterocyclic ring comprising from 3 to 8 ring atoms; R.sup.S2a is selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.1-7 alkyl group;
[0556] R.sup.N7a and R.sup.N7b are selected from H and a C.sub.1-4 alkyl group;
[0557] R.sup.N3 and R.sup.N4, together with the nitrogen to which they are bound, form an optionally substituted heterocyclic ring comprising from 3 to 8 ring atoms;
[0558] R.sup.2 is selected from H, halo, OR.sup.02, SR.sup.S2b, NR.sup.N5R.sup.N6, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, wherein R.sup.02 and R.sup.S2b are selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.1-7 alkyl group; and
[0559] R.sup.N5 and R.sup.N6 are independently selected from H, an optionally substituted C.sub.1-7 alkyl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, or R.sup.N5 and R.sup.N6, together with the nitrogen to which they are bound, form an optionally substituted heterocyclic ring comprising from 3 to 8 ring atoms,
[0560] or a pharmaceutically acceptable salt thereof.
[0561] 54. The method of embodiment 53, wherein the agent comprises KU-0063794, represented by formula (1)
##STR00006##
[0562] 55. The method of any one of embodiments 52-54, wherein the agent comprises Selumetinib, LY294002, AZD-8055, Celastrol, or ascorbyl palmitate.
[0563] 56. The method of any one of embodiments 49-55, wherein the treatment comprises a lifestyle change.
[0564] 57. The method of embodiment 56, wherein the lifestyle change comprises a dietary change.
[0565] 58. The method of any one of embodiments 49-57, wherein the agent is administered to the subject orally, intraarticularly, intravenously, intramuscularly, rectally, cutaneously, subcutaneously, topically, transdermally, sublingually, nasally, intravesicularly, intrathecally, epidurally, or transmucosally.
[0566] 59. The method of embodiment 58, wherein the agent is administered to the subject orally.
[0567] 60. The method of any one of embodiments 49-59, wherein the agent is formulated as a tablet, capsule, gel cap, powder, liquid solution, or liquid suspension.
[0568] 61. The method of any one of embodiments 40-60, further comprising monitoring the subject for (i) an increase in expression of one or more genes set forth in Tables 1-10 and/or (ii) a decrease in expression of one or more genes set forth in Tables 11-20 following the treatment.
[0569] 62. A pharmaceutical composition comprising a compound represented by formula (I)
##STR00007##
[0570] wherein one or two of X.sup.5, X.sup.6 and X.sup.8 is N, and the other(s) is/are CH;
[0571] R.sup.7 is selected from halo, OR.sup.01, SR.sup.S1, NR.sup.N1R.sup.N2, NR.sup.N7aC(═O)R.sup.C1, NR.sup.N7bSO.sub.2R.sup.S2a, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group;
[0572] R.sup.01 and R.sup.S1 are selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.1-7 alkyl group;
[0573] R.sup.N1 and R.sup.N2 are independently selected from H, an optionally substituted C.sub.1-7 alkyl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, or R.sup.N1 and R.sup.N2, together with the nitrogen to which they are bound, form a heterocyclic ring comprising from 3 to 8 ring atoms;
[0574] R.sup.C1 is selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, an optionally substituted C.sub.1-7 alkyl group;
[0575] NR.sup.N8R.sup.N9, wherein R.sup.N8 and R.sup.N9 are independently selected from H, an optionally substituted C.sub.1-7 alkyl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, or R.sup.N8 and R.sup.N9, together with the nitrogen to which they are bound, form a heterocyclic ring comprising from 3 to 8 ring atoms;
[0576] R.sup.S2a is selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.1-7 alkyl group;
[0577] R.sup.N7a and R.sup.N7b are selected from H and a C.sub.1-4 alkyl group; R.sup.N3 and R.sup.N4, together with the nitrogen to which they are bound, form an optionally substituted heterocyclic ring comprising from 3 to 8 ring atoms;
[0578] R.sup.2 is selected from H, halo, OR.sup.02, SR.sup.S2b, NR.sup.N5R.sup.N6, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, wherein R.sup.02 and R.sup.S2b are selected from H, an optionally substituted C.sub.5-20 aryl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.1-7 alkyl group; and
[0579] R.sup.N5 and R.sup.N6 are independently selected from H, an optionally substituted C.sub.1-7 alkyl group, an optionally substituted C.sub.5-20 heteroaryl group, and an optionally substituted C.sub.5-20 aryl group, or R.sup.N5 and R.sup.N6, together with the nitrogen to which they are bound, form an optionally substituted heterocyclic ring comprising from 3 to 8 ring atoms,
[0580] or a pharmaceutically acceptable salt thereof,
[0581] wherein the composition comprises one or more pharmaceutically acceptable excipients and is formulated for administration to a subject in combination with a meal.
[0582] 63. The pharmaceutical composition of embodiment 62, wherein the compound is KU-0063794, represented by formula (1)
##STR00008##
[0583] 64. A pharmaceutical composition comprising Selumetinib, LY294002, AZD-8055, Celastrol, or ascorbyl palmitate, and one or more pharmaceutically acceptable excipients, wherein the composition is formulated for administration to a subject in combination with a meal.
[0584] 65. A pharmaceutical composition comprising Selumetinib, LY294002, AZD-8055, KU-0063794, Celastrol, Ascorbyl Palmitate, Oligomycin-a, NVP-BEZ235, Importazole, Ryuvidine, NSC-663284, PI-828, Pyrvinium pamoate, PI-103, YM-155, Prostratin, BCI hydrochloride, Dorsomorphin-Compound C, VU-0418947-2, JNK-9L, Phloretin, ZG-10, Proscillaridin, YC-1, IKK-2-inhibitor-V, Anisomycin, Colforsin, Rilmenidine, GDC-0941, Valdecoxib, Myricetin, Cyproheptadine, Vorinostat, Nifedipine, Phylloquinone, Withaferin-A, Temsirolimus, SN-38, GSK-1059615, Tipifarnib, Linifanib, WYE-354, MK-212, and/or Enzastaurin, and one or more pharmaceutically acceptable excipients, wherein the composition is formulated for administration to a subject in combination with a meal.
[0585] 66. The pharmaceutical composition of any one of embodiments 62-65, wherein the composition is a tablet, capsule, gel cap, powder, liquid solution, or liquid suspension.
[0586] 67. The pharmaceutical composition of any one of embodiments 62-66, wherein the subject is a mammal.
[0587] 68. The pharmaceutical composition of embodiment 67, wherein the mammal is a human. 69. A dietary supplement comprising Selumetinib, LY294002, AZD-8055, KU-0063794, Celastrol, Ascorbyl Palmitate, Oligomycin-a, NVP-BEZ235, Importazole, Ryuvidine, NSC-663284, PI-828, Pyrvinium pamoate, PI-103, YM-155, Prostratin, BCI hydrochloride, Dorsomorphin-Compound C, VU-0418947-2, JNK-9L, Phloretin, ZG-10, Proscillaridin, YC-1, IKK-2-inhibitor-V, Anisomycin, Colforsin, Rilmenidine, GDC-0941, Valdecoxib, Myricetin, Cyproheptadine, Vorinostat, Nifedipine, Phylloquinone, Withaferin-A, Temsirolimus, SN-38, GSK-1059615, Tipifarnib, Linifanib, WYE-354, MK-212, or Enzastaurin, or a combination thereof.
[0588] 70. The dietary supplement of embodiment 69, wherein the dietary supplement is a tablet, capsule, gel cap, powder, liquid solution, or liquid suspension.
[0589] 71. The dietary supplement of embodiment 69 or 70, wherein the dietary supplement is formulated for administration to a subject in combination with a meal.
[0590] 72. The dietary supplement of embodiment 71, wherein the subject is a mammal.
[0591] 73. The dietary supplement of embodiment 72, wherein the mammal is a human.
OTHER EMBODIMENTS
[0592] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
[0593] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
[0594] Other embodiments are within the claims.