Neuronal SIRT1 Activation as a Novel Mechanism Underlying the Prevention of Alzheimer Disease Amyloid Neuropathology by Calorie Restriction
2006; Elsevier BV; Volume: 281; Issue: 31 Linguagem: Inglês
10.1074/jbc.m602909200
ISSN1083-351X
AutoresWeiping Qin, Tianle Yang, Lap Ho, Zhong Zhao, Jun Wang, Linghong Chen, Wei Zhao, Meenakshisundaram Thiyagarajan, Donal MacGrogan, Joseph T. Rodgers, Pere Puigserver, Junichi Sadoshima, Haiteng Deng, Steve Pedrini, Sam Gandy, Anthony A. Sauve, Giulio Maria Pasinetti,
Tópico(s)Tryptophan and brain disorders
ResumoNicotinamide adenine dinucleotide (NAD)+-dependent sirtuins have been identified to be key regulators in the lifespan extending effects of calorie restriction (CR) in a number of species. In this study we report for the first time that promotion of the NAD+-dependent sirtuin, SIRT1-mediated deacetylase activity, may be a mechanism by which CR influences Alzheimer disease (AD)-type amyloid neuropathology. Most importantly, we report that the predicted attenuation ofβ-amyloid content in the brain during CR can be reproduced in mouse neurons in vitro by manipulating cellular SIRT1 expression/activity through mechanisms involving the regulation of the serine/threonine Rho kinase ROCK1, known in part for its role in the inhibition of the non-amyloidogenic α-secretase processing of the amyloid precursor protein. Conversely, we found that the expression of constitutively active ROCK1 in vitro cultures significantly prevented SIRT1-mediated response, suggesting that α-secretase activity is required for SIRT1-mediated prevention of AD-type amyloid neuropathology. Consistently we found that the expression of exogenous human (h) SIRT1 in the brain of hSIRT1 transgenics also resulted in decreased ROCK1 expression and elevatedα-secretase activity in vivo. These results demonstrate for the first time a role for SIRT1 activation in the brain as a novel mechanism through which CR may influence AD amyloid neuropathology. The study provides a potentially novel pharmacological strategy for AD prevention and/or treatment. Nicotinamide adenine dinucleotide (NAD)+-dependent sirtuins have been identified to be key regulators in the lifespan extending effects of calorie restriction (CR) in a number of species. In this study we report for the first time that promotion of the NAD+-dependent sirtuin, SIRT1-mediated deacetylase activity, may be a mechanism by which CR influences Alzheimer disease (AD)-type amyloid neuropathology. Most importantly, we report that the predicted attenuation ofβ-amyloid content in the brain during CR can be reproduced in mouse neurons in vitro by manipulating cellular SIRT1 expression/activity through mechanisms involving the regulation of the serine/threonine Rho kinase ROCK1, known in part for its role in the inhibition of the non-amyloidogenic α-secretase processing of the amyloid precursor protein. Conversely, we found that the expression of constitutively active ROCK1 in vitro cultures significantly prevented SIRT1-mediated response, suggesting that α-secretase activity is required for SIRT1-mediated prevention of AD-type amyloid neuropathology. Consistently we found that the expression of exogenous human (h) SIRT1 in the brain of hSIRT1 transgenics also resulted in decreased ROCK1 expression and elevatedα-secretase activity in vivo. These results demonstrate for the first time a role for SIRT1 activation in the brain as a novel mechanism through which CR may influence AD amyloid neuropathology. The study provides a potentially novel pharmacological strategy for AD prevention and/or treatment. Sirtuins are a family of NAD+-dependent histone/protein deacetylases that are highly conserved in their catalytic domains and are distributed across all kingdoms of life (1Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1306) Google Scholar, 2Gray S.G. Ekstrom T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (495) Google Scholar, 3Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Crossref PubMed Scopus (618) Google Scholar, 4Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (497) Google Scholar). These enzymes utilize NAD+ as a substrate to catalyze deacetylation of specific acetylated-protein substrates (1Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1306) Google Scholar, 5Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2788) Google Scholar). Sirtuins can deacetylate a variety of substrates and are, therefore, involved in a broad range of physiological functions, including control of gene expression, metabolism, and aging (6Bordone L. Guarente L. Nat. Rev. Mol. Cell Biol. 2005; 6: 298-305Crossref PubMed Scopus (848) Google Scholar). Accumulating evidence implicates sirtuins in calorie restriction (CR)-mediated health effects including increased organism longevity in yeast, worms, flies, and mammals (1Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1306) Google Scholar, 2Gray S.G. Ekstrom T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (495) Google Scholar, 3Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Crossref PubMed Scopus (618) Google Scholar, 4Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (497) Google Scholar, 6Bordone L. Guarente L. Nat. Rev. Mol. Cell Biol. 2005; 6: 298-305Crossref PubMed Scopus (848) Google Scholar). Mammalian genomes encode seven distinct sirtuins (SIRT1-SIRT7). SIRT1 is induced by CR 4The abbreviations used are: CR, calorie restriction; AD, Alzheimer disease; AL, ad libitum; Aβ, β-amyloid; NAD+, nicotinamide adenine dinucleotide; NAM, nicotinamide; ROCK, serine/threonine Rho kinase; APP, amyloid precursor protein; WT, wild-type; sAPPα, soluble APP; DN, dominant negative; CA, constitutively active; H4-k16Ac, acetylated histone 4 lysine 16 residue; Ad, adenovirus; CHO, Chinese hamster ovary; m.o.i., multiplicity of infection; h, human; NeuN, neuronal nuclear-specific. in several tissues and has been implicated in various effects such as stress resistance, reduced apoptosis, and metabolic changes associated with CR (1Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1306) Google Scholar). SIRT1 is also expressed in the developing and the adult mammalian brain (7Hisahara S. Chiba S. Matsumoto H. Horio Y. J. Pharmacol. Sci. 2005; 98: 200-204Crossref PubMed Scopus (48) Google Scholar). Based on these considerations and on the evidence that CR prevents AD-type amyloid neuropathology in animal models (8Wang J. Ho L. Qin W. Rocher A.B. Seror I. Humala N. Maniar K. Dolios G. Wang R. Hof P.R. Pasinetti G.M. FASEB J. 2005; 19: 659-661Crossref PubMed Scopus (223) Google Scholar, 9Patel N.V. Gordon M.N. Connor K.E. Good R.A. Engelman R.W. Mason J. Morgan D.G. Morgan T.E. Finch C.E. Neurobiol. Aging. 2005; 26: 995-1000Crossref PubMed Scopus (281) Google Scholar), we sought to test the hypothesis that CR may reduce AD-type amyloid neuropathology through mechanisms involving promotion of SIRT1. The relevance of CR treatment in experimental models of AD to human pathology is supported by recent epidemiological evidence suggesting that humans who maintain a low calorie diet have a reduced risk of developing AD (10Luchsinger J.A. Tang M.X. Shea S. Mayeux R. Arch. Neurol. 2002; 59: 1258-1263Crossref PubMed Scopus (373) Google Scholar, 11Hendrie H.C. Ogunniyi A. Hall K.S. Baiyewu O. Unverzagt F.W. Gureje O. Gao S. Evans R.M. Ogunseyinde A.O. Adeyinka A.O. Musick B. Hui S.L. J. Am. Med. Assoc. 2001; 285: 739-747Crossref PubMed Scopus (321) Google Scholar, 12Mattson M.P. Neurology. 2003; 60: 690-695Crossref PubMed Scopus (111) Google Scholar). Abnormal Aβ deposition within the brain is a hallmark of AD neuropathology. Accumulation of aggregated Aβ is hypothesized to initiate a pathological cascade resulting in onset and progression of AD (13Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar). Aβ species with different amino and carboxyl termini are generated from amyloid precursor protein (APP) through sequential proteolysis by β- and γ-secretases in amyloidogenic processing pathways (14Gandy S. Neurobiol. Aging. 2002; 23: 1009-1016Crossref PubMed Scopus (29) Google Scholar). A 1–40 amino acid form of Aβ (Aβ1–40) is the major secreted product of these cleavages, whereas the minor 1–42 amino acid form of Aβ (Aβ1–42) that contains two additional residues at its carboxyl terminus has been suggested as the initiating molecule in the pathogenesis of AD (13Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar). In the non-amyloidogenic pathway, APP is cleaved within the Aβ domain by a third α-secretase, precluding generation and deposition of intact amyloidogenic Aβ peptides in the brain (14Gandy S. Neurobiol. Aging. 2002; 23: 1009-1016Crossref PubMed Scopus (29) Google Scholar). CR extends the life span in a wide variety of animals and has for decades been the only lifestyle-related regimen known to promote longevity and prevent morbidity in mammals (1Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1306) Google Scholar, 2Gray S.G. Ekstrom T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (495) Google Scholar, 3Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Crossref PubMed Scopus (618) Google Scholar, 4Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (497) Google Scholar, 6Bordone L. Guarente L. Nat. Rev. Mol. Cell Biol. 2005; 6: 298-305Crossref PubMed Scopus (848) Google Scholar). The recent determination that the yeast sirtuin Sir2 is required for CR-induced increases in lifespan in this organism suggests that sirtuins may play key roles in mediating CR-related health effects in other organisms (1Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1306) Google Scholar, 2Gray S.G. Ekstrom T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (495) Google Scholar, 3Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Crossref PubMed Scopus (618) Google Scholar, 4Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (497) Google Scholar, 6Bordone L. Guarente L. Nat. Rev. Mol. Cell Biol. 2005; 6: 298-305Crossref PubMed Scopus (848) Google Scholar). The latter hypothesis is supported by a number of recent studies showing that increasing sirtuin gene dosage promotes lifespan increases in worms and flies (1Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1306) Google Scholar, 6Bordone L. Guarente L. Nat. Rev. Mol. Cell Biol. 2005; 6: 298-305Crossref PubMed Scopus (848) Google Scholar). In addition, studies in mammals show that sirtuins (and SIRT1 in particular) regulate stress resistance, adipogenesis/adipolysis, and metabolic responses to fasting (6Bordone L. Guarente L. Nat. Rev. Mol. Cell Biol. 2005; 6: 298-305Crossref PubMed Scopus (848) Google Scholar). The sirtuin deacetylation reaction consumes NAD+, releases NAM, and forms a novel metabolite 2′-O-acetyl-(ADP) ribose along with a deacetylated lysine product (1Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1306) Google Scholar). Furthermore, NAM inhibits yeast Sir2 and SIRT1 at 50–150 μm concentrations (15Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar). The recycling of NAM back to NAD+ by the enzyme nicotinamide phosphoribosyltransferase may be crucial for maintenance of cellular NAD+ and for maintaining SIRT1 functions (1Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1306) Google Scholar, 6Bordone L. Guarente L. Nat. Rev. Mol. Cell Biol. 2005; 6: 298-305Crossref PubMed Scopus (848) Google Scholar). Indeed, NAD+ levels in liver were recently shown to increase with fasting (16Rodgers J.T. Lerin C. Haas W. Gygi S.P. Spiegelman B.M. Puigserver P. Nature. 2005; 434: 113-118Crossref PubMed Scopus (2584) Google Scholar), and changes in the NAD+/ NAM ratio in vivo may critically influence cellular responses to CR and modulate SIRT1 activities in mammals (17Lamming D.W. Wood J.G. Sinclair D.A. Mol. Microbiol. 2004; 53: 1003-1009Crossref PubMed Scopus (200) Google Scholar). In this study we first examined if CR treatment influences SIRT1 expression/activity and the NAD+/NAM ratio in the brain and then systematically explored the causal link between SIRT1-mediated deacetylase activity and Aβ peptide generation in vitro. We report for the first time that promotion of NAD+-dependent SIRT1 deacetylase activity may be a mechanism by which a CR dietary regimen may prevent experimental AD amyloid neuropathology. Most importantly, we report that a mechanism through which SIRT1 in part prevents Aβ peptide generation is through promotion of the non-amyloidogenic processing of APP by means of inhibition of ROCK1 expression. These findings point to SIRT1 activation and enhancement of the NAD+/nicotinamide (NAM) ratio in the brain as potentially attractive pharmacological strategies for AD prevention and/or treatment. Tg2576 Mice and Diets—In this study 4-month-old female Tg2576 mice (18Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3700) Google Scholar) (Taconic, Inc., Germantown, NY) were randomly assigned to CR or ad libitum (AL) dietary regimens. CR was achieved by feeding Tg2576 mice 70% of the calories consumed by the pair-controlled AL animals, as previously reported (8Wang J. Ho L. Qin W. Rocher A.B. Seror I. Humala N. Maniar K. Dolios G. Wang R. Hof P.R. Pasinetti G.M. FASEB J. 2005; 19: 659-661Crossref PubMed Scopus (223) Google Scholar). At 10 months of age, the mice were sacrificed, and brain specimens were harvested as previously reported (8Wang J. Ho L. Qin W. Rocher A.B. Seror I. Humala N. Maniar K. Dolios G. Wang R. Hof P.R. Pasinetti G.M. FASEB J. 2005; 19: 659-661Crossref PubMed Scopus (223) Google Scholar). All animal studies were conducted following protocols approved by the Mount Sinai School of Medicine Institutional Animal Use Committee. Measurement of NAD+ and NAM—A weighed aliquot of frozen pulverized tissue allocated for biochemical studies (cerebellum) was extracted in 800 μl of HClO4 (1.1 m) containing 6 nmol of [18O]NAD+ (91% isotopic enrichment) and 2.5 nmol of [18O]NAM (96% isotopic enrichment). After centrifugation for 3 min at room temperature, supernatants were neutralized with KOH and injected onto a high performance liquid chromatography for separation of NAM and NAD+ from other cell components. NAD+ and NAM peaks were collected as determined by retention time of authentic standards. Samples were lyophilized, and matrix-assisted laser desorption ionization-mass spectroscopy (MS) (positive mode) was used to detect NAD+, and ESI-MA (positive mode) was used to measure the NAM signal (19Sauve A.A. Moir R.D. Schramm V.L. Willis I.M. Mol. Cell. 2005; 17: 595-601Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). A peak ratio of 664/666 provided quantitation of the nmol of NAD+ in a sample as determined from the 6 nmol of [18O]NAD+ added to the original sample. Similarly, NAM levels were determined using the ratio 123/125 multiplied by 2.5 nmol of NAM to obtain total nmol of NAM per sample. Both NAD+ and NAM mole contents were then divided by the tissue volume (density, 1.0 μl/mg of tissue) to obtain the metabolite concentration in tissue. The methodology employed is based upon a previous approach that measured NAM contents in yeast (19Sauve A.A. Moir R.D. Schramm V.L. Willis I.M. Mol. Cell. 2005; 17: 595-601Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Corrections were applied for isotopic abundances, and relevant blanks and controls were also run. Each sample group contained at least four animals, and each sample was run in duplicate. Neocortical SIRT1 Immunocytochemical Localization—Neocortical tissue sections (50 μm) adjacent to those used for assessment of amyloid plaque pathology in CR Tg2576 mice were washed with Tris-buffered saline (20 mm, pH 7.4) followed by incubation with 0.2% Triton X-100 for 30 min for permeabilization. Endogenous peroxidase activity was quenched by incubating the sections with 0.3% hydrogen peroxide for 5 min. After a rinse with Tris-buffered saline (TBS), the sections were incubated with blocking buffer (5% goat serum in TBS) for 120 min. The sections were then incubated with primary antibodies SMI-32 (1:2000 dilution, Sternberger Monoclonals Inc., Lutherville, MD) and anti-SIRT1 IgG (1:100 dilution, Upstate Biotechnology, Inc., Lake Placid, NY) in blocking buffer at 4 °C overnight. The sections were washed 3 times with Tris-buffered saline and then incubated with a mixture of anti-mouse IgG Texas Red-conjugated and anti-rabbit IgG peroxidase-conjugated (1:200 dilution) antibodies in blocking buffer for 2 h at 37 °C. The specific labeling of SIRT was detected using Tyramide Signal Amplification kit (Molecular Probs™) according to the manufacturer's instructions. The sections were mounted with Vectasheald, the sections were observed under either a fluorescent microscope (Zeiss Axioplan 2 Imaging system) or Leica TCS-SP (UV) confocal system, and images were acquired. Cell Culture and Treatments—Embryonic (E14) neocortical primary neuronal cultures derived from Tg2576 transgenic mice (Tg2576 neurons) were prepared as previously described (20Kelley K.A. Ho L. Winger D. Freire-Moar J. Borelli C.B. Aisen P.S. Pasinetti G.M. Am. J. Pathol. 1999; 155: 995-1004Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Chinese hamster ovary (CHO) cells expressing human APP carrying the K670N, M671L Swedish mutation (APPswe)(a gift from Dr. Robakis) as in Tg2576 neurons were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum, 1% streptomycin/penicillin (Invitrogen), and 400 μg/ml G418 (Invitrogen). In a cell culture study, CHO-APPswe cells or Tg2576 neurons were seeded at 4 × 104 cells/cm2 and cultured at 37 °C in the presence of 5% CO2. For adenoviral infection or pharmacological studies, Tg2576 neuronal (5 days old) or CHO-APPswe (24 h after plating), cultures were infected with wild-type (WT) SIRT1 (16Rodgers J.T. Lerin C. Haas W. Gygi S.P. Spiegelman B.M. Puigserver P. Nature. 2005; 434: 113-118Crossref PubMed Scopus (2584) Google Scholar), dominant negative (DN) SIRT1 (21Alcendor R.R. Kirshenbaum L.A. Imai S. Vatner S.F. Sadoshima J. Circ. Res. 2004; 95: 971-980Crossref PubMed Scopus (281) Google Scholar), or LacZ control adenoviruses at doses defined as multiplicities of infection (m.o.i.) or co-treatment with NAD+. NAD+ (75 mm, Sigma) was freshly prepared and dissolved in water before use. For the constitutively active (CA) ROCK1 study, CHO-APPswe cells were transfected with pCAG empty vector or CA ROCK1 cDNA followed by infection with Ad-WT SIRT1 of Ad-lacZ 6 h later. The transfection was carried out using Lipofectamine 2000 reagent (Invitrogen) according to the manufacture's instructions and as previously described (22Pedrini S. Carter T.L. Prendergast G. Petanceska S. Ehrlich M.E. Gandy S. PLoS Med. 2005; 2: e18Crossref PubMed Scopus (133) Google Scholar). Conditioned medium was collected at indicated time points for Aβ detection. Cell viability was assessed by LDH assay. Aβ Enzyme-linked Immunosorbent Assay—The quantitative assessment of Aβ peptides in brain and cultured cells was performed as previously descried (23Qin W. Ho L. Pompl P.N. Peng Y. Zhao Z. Xiang Z. Robakis N.K. Shioi J. Suh J. Pasinetti G.M. J. Biol. Chem. 2003; 278: 50970-50977Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Western Blot Analysis—Aliquots of frozen pulverized neocortical tissue (cingular and parietal cortex) were lysed in radioimmune precipitation assay buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mm Tris, pH 8.0) containing protease inhibitors. Lysates were then subjected to Western blot analysis with the indicated antibody as previously reported (8Wang J. Ho L. Qin W. Rocher A.B. Seror I. Humala N. Maniar K. Dolios G. Wang R. Hof P.R. Pasinetti G.M. FASEB J. 2005; 19: 659-661Crossref PubMed Scopus (223) Google Scholar). The following antibodies were used in this study: polyclonal anti-SIRT1 antibody (1:3000), polyclonal anti-histone H4 (Ac16) (1:2000, Serotec, Raleigh, NC), polyclonal antiamyloid precursor protein carboxyl-terminal (751–770) antibody (anti-O443, 1:5000 dilution, Calbiochem), monoclonal 22C11 antibody (1:1000, Chemicon International, Temecula, CA), monoclonal 6E10 antibody (1:1000, Senetek, St. Louis, MO), rabbit polyclonal antibody against ROCK1 (1:5000; Chemicon International), and polyclonal β-actin antibody (1:3000, Sigma). Secretase Activity Assays—α-, β-, and γ-secretase activities were assessed using commercially available kits (R&D Systems, Minneapolis, MN) as previously described (24Rezai-Zadeh K. Shytle D. Sun N. Mori T. Hou H. Jeanniton D. Ehrhart J. Townsend K. Zeng J. Morgan D. Hardy J. Town T. Tan J. J. Neurosci. 2005; 25: 8807-8814Crossref PubMed Scopus (586) Google Scholar, 25Burns M. Gaynor K. Olm V. Mercken M. LaFrancois J. Wang L. Mathews P.M. Noble W. Matsuoka Y. Duff K. J. Neurosci. 2003; 23: 5645-5649Crossref PubMed Google Scholar). SIRT1 Transgenic Mice—For the generation of the hSIRT1 transgenic mice, a 2.3-kilobase cDNA fragment containing the entire coding region of the hSIRT1 (a gift from Dr. Sauve) was inserted into a unique HindIII-NotI restriction site located within the second exon of the rat neuron-specific enolase gene, as previously described in this laboratory (20Kelley K.A. Ho L. Winger D. Freire-Moar J. Borelli C.B. Aisen P.S. Pasinetti G.M. Am. J. Pathol. 1999; 155: 995-1004Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). A 7.3-kilobase SalI fragment containing the hSIRT1 was purified and micro-injected into 1-cell mouse eggs as previously described (20Kelley K.A. Ho L. Winger D. Freire-Moar J. Borelli C.B. Aisen P.S. Pasinetti G.M. Am. J. Pathol. 1999; 155: 995-1004Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Founders were identified by PCR-based genotyping, and generation of individual F1 lines was obtained by mating founders with strain-matched C57B6-SJL WT mice for analysis of SIRT1 and ROCK1 expression and α-secretase activity. Statistical Analysis—All values are expressed as the means ± S.E. Differences between means were analyzed using a two-tailed Student t test. In all analyses the null hypothesis was rejected at the 0.05 level. All statistical analyses were performed using the Prism Stat program (GraphPad Software, Inc., San Diego, CA). CR Increases SIRT1 Expression and NAD+ Levels in the Brains of ∼10-Month-old Tg2576 Mice—For the in vivo studies, beginning at 4 months of age, Tg2576 transgenic mice, which model AD-type amyloid neuropathology (18Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3700) Google Scholar), were enrolled in a CR diet regimen providing ∼30% fewer daily calories relative to average caloric consumption of age- and gender-matched control AL-fed Tg2576 mice as previously described (8Wang J. Ho L. Qin W. Rocher A.B. Seror I. Humala N. Maniar K. Dolios G. Wang R. Hof P.R. Pasinetti G.M. FASEB J. 2005; 19: 659-661Crossref PubMed Scopus (223) Google Scholar). Consistent with previous evidence, CR treatment resulted in body weight stabilization over the ∼6 month study period among CR Tg2576 mice relative to the AL-fed group (supplemental Fig. 1). When mice were sacrificed at ∼10 months of age, we found a significant elevation of SIRT1 protein content (p < 0.05) (Fig. 1A) and NAD+ content (p < 0.001) (Fig. 1B) as well as a significant reduction in NAM content (p < 0.01) (Fig. 1B) in the brain of CR Tg2576 mice relative to the age- and gender-matched AL-fed control Tg2576 mice (see supplemental Fig. 2 for more information on NAD+/NAM assay). The increased SIRT1 protein content and the ∼4-fold increased NAD+/NAM ratio (p < 0.001) in the brain of CR Tg2576 mice (Fig. 1B) coincided with an apparent marked elevation of SIRT1 deacetylase activity in the brain, as reflected by significantly reduced acetylation levels of the SIRT1 substrate H4-k16Ac (p < 0.05), assessed by Western blot assay using an antibody specific for H4-k16ac residue (Fig. 1C) (26Vaquero A. Scher M. Lee D. Erdjument-Bromage H. Tempst P. Reinberg D. Mol. Cell. 2004; 16: 93-105Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar), relative to AL-fed Tg2576 control mice. The observed difference in immunoreactive H4-k16Ac content was not attributable to different H4 contents in the brain of CR relative to AL Tg2576 mice (data not shown). In parallel control studies we found that CR-mediated SIRT1 activation in the brain of Tg2576 coincided with reduced acetylated H3-k9Ac levels in the absence of significant changes in H4-k8Ac relative to control AL-fed-treated Tg2576 mice (data not shown). Our evidence is consistent with a previous report that SIRT1 deacetylates histone polypeptides with a preference for H4-k16Ac and H3-K9Ac (26Vaquero A. Scher M. Lee D. Erdjument-Bromage H. Tempst P. Reinberg D. Mol. Cell. 2004; 16: 93-105Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar). Based on this evidence and the fact that t H4-k16Ac is selectively influenced in the brain of Tg2576 in response to CR (Fig. 1C), we decided to monitor H4-k16Ac signaling as the specific index for SIRT1-mediated deacetylase activity in vitro (see below). The elevation in SIRT1 deacetylase activity in this study coincided with significantly decreased Aβ1–40 and Aβ1–42 peptide contents (Fig. 1D) in the same brain tissue and was accompanied by near complete prevention of AD-type amyloid neuropathology (supplemental Fig. 3, A and B). The evidence revealing CR-mediated attenuation in AD-type neuropathology in 10-month-old CR Tg2576 mice in this study, is consistent with our previous independent study showing similar responses in 12-month-old Tg2576 mice after 9 months of CR treatment (8Wang J. Ho L. Qin W. Rocher A.B. Seror I. Humala N. Maniar K. Dolios G. Wang R. Hof P.R. Pasinetti G.M. FASEB J. 2005; 19: 659-661Crossref PubMed Scopus (223) Google Scholar). To identify the cellular localization of SIRT1 expression in CR Tg2576 mice, we immunocytochemically surveyed SIRT1 in adjacent contralateral tissue from neocortical sections used to evaluate AD-type amyloid neuropathology. We found that SIRT1 immunoreactivity in CR Tg2576 mice (green immunostaining) was primarily localized within the boundaries of both the outer (OG) and inner (IG) granular layers of the neocortex (Fig. 1E, panel A); sparse SIRT1 immunolabeling was also detected in both outer (OPL) and inner (IPL) pyramidal layers of the parietal neocortex as reflected by co-localization of SIRT1 and SMI-32 immunoreactive signal (red immunostaining), which is a specific marker for neocortical pyramidal neurons (Fig. 1E; panel A). Similar neuronal SIRT1 immunoreactive distribution was found in the neocortex of AL-fed Tg2576 (data not shown). Further examination of cellular localization of SIRT1 in the neocortical granular layer in adjacent tissue sections revealed that the SIRT1 immunoreactive signal (Fig. 1E, panel b, green staining) selectively co-localized with neuronal nuclear-specific (NeuN) immunoreactivity (Fig. 1E, panel c, red staining) as determined by overlapping SIRT1 and NeuN staining (Fig. 1E, panel d). The selective neuronal distribution of SIRT1 protein in the neocortex and other brain regions of CR Tg2576 mice 5G. M. Pasinetti, unpublished observation. suggests that neuronal SIRT1 deacetylase may selectively influence APP processing independently from glial inflammatory-associated Aβ processing and amyloid plaque generation (27Mrak R.E. Griffin W.S. Neurobiol. Aging. 2005; 26: 349-354Crossref PubMed Scopus (531) Google Scholar). Thus, despite the availability of evidence that exogenous SIRT1 expression in glia may attenuate inflammatory neurotoxicity in vitro (28Chen J. Zhou Y. Mueller-Steiner S. Chen L.F. Kwon H. Yi S. Mucke L. Gan L. J. Biol. Chem. 2005; 280: 40364-40374Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar), CR-mediated SIRT1 responses with respect to AD amyloid neuropathology in the brain could be primarily neuronal based upon the extent to which SIRT1 localizes to these cell types in the brain. Based on this evidence we conducted a systematic series of in vitro studies to evaluate the role of neuronal SIRT1 in CR-mediated attenuation of AD-type Aβ neuropathology in the brain of CR Tg2576 mice. In particular, we investigated if the predicted attenuation in Aβ peptide content by CR in the brain can be achieved by manipulating the expression/activity of SIRT1 in primary embryonic Tg2576 cortico-hippocampal neurons (Tg2576 neurons; E16) in vitro. SIRT1 Expression Causally Promotes α-Secretase Activity and Attenuates Aβ Peptides Generation in Primary Tg2576 Neuron Cultures and CHO-APPswe Cells—We found that viral expression of WT (16Rodgers J.T. Lerin C. Haas W. Gygi S.P. Spiegelman B.M. Puigserver P. Nature. 2005; 434: 113-118Crossref PubMed Scopus (2584) Google Scholar) SIRT1 (10 m.o.i.), leading to an ∼3-fold elevation in SIRT1 protein content in the Tg2576 neurons (Fig. 2A and ins
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