Sirt-1 promotes CREB-dependent gene expression. ( A ) Forskolin-in- ducible physical association of CREB with Sirt-1 in PC12 cells. mycCREB or mycCREB- Δ LZ were transfected in PC12 cells and immunoprecipitated from protein lysates untreated or stimulated with Fsk for 30 min; the presence of Sirt-1 in immunocomplexes was veri fi ed by immunoblotting ( Upper ); anti- Ser133 CREB and anti-myc immunoblotting were used to con fi rm CREB phosphorylation by Fsk and assess the expression level of transfected CREB isoforms, respectively ( Lower ). ( Ba ) ChIP assays showing parallel interaction of CREB and Sirt-1 with the CRE-containing promoter regions of nNOS and PGC- 1 α in hippocampal neurons treated with NGF. Promoter ampli fi cation from total chromatin input is also reported as control. ( b ) CREB mediates Sirt-1 interaction with CRE-containing promoter regions. NGF-inducible binding of Sirt-1 to nNOS ( Upper ) and Sirt-1 promoter ( Lower ) are drastically reduced in hippocampal neurons lacking CREB. Binding of CREB to the same promoter regions con fi rms severe reduction of CREB binding in Cre-infected cells. rIgG (rabbit IgG) is a negative control for ChIP. Total chromatin input was equal throughout the lanes. ( C ) RT-PCR analysis showing reduced induction of nNOS and PGC-1 α mRNA by NGF in cortical neurons treated with the Sirt-1 inhibitor Nicotinamide (NAM). ( D ) ( a and b ) Representative RT-PCR analysis of PGC-1 α and nNOS mRNA expression in whole brains from WT and Sirt-1-de- fi cient mice under both AL and CR (6 mo) feeding. Each lane represents a pool of two mice. Actin was used as loading control. ( c ) Western blot analysis of whole brain protein homogenates from WT and Sirt-1 KO mice (fed AL) indicating reduced expression of nNOS but normal levels of immunoreactive CREB and total histone H4. Anti-AcH4K16 and antiactin immunostaining con fi rm, respectively, reduced deacetylase activity in the SirtKO sample and equal protein loading in the two lanes. 

Contexts in source publication

Context 1

... tion of CREB protein, and the expression level of a number of mRNAs known to be regulated by CREB (23). CREB1 protein expression in control mice was not changed by the dietary regimen; instead, an increase in CREB phosphorylation on Serine 133 in the hippocampi of the CR group was revealed by phospho-speci fi c immunoblotting (Fig. 2 A ), suggesting that CR activates CREB in this brain area. Accordingly, mRNAs of “ canonical ” CREB targets peroxisome proliferator-activated receptor- γ coactivator-1 α ( PGC- 1 α ), nNOS , and phosphoenolpyruvate carboxykinase ( PEPCK ) were induced by CR in the cortex and hippocampus of CREB- pro fi cient mice (Fig. 2 B ). Of note, both PGC-1 α and NO, the product of NOS enzymes, are known to promote mitochondrial biogenesis and to participate in organismal response to CR (26). The three mRNAs were overall down-regulated in BCKO mice fed AL compared with controls, and their induction by CR, observed in control mice, was nearly abolished (Fig. 2 B ). Interestingly, the expression of other putative CREB targets like bcl-2 , NGF , and c-fos were unaffected by CR and CREB deletion (Fig. S2). Thus, collectively, these fi ndings demonstrate that nutrient availability selectively regulates CREB-dependent gene expression in the forebrain. We next asked how CR may affect CREB activity. Because Sirt- 1 is a metabolic sensor involved in several biological consequences of nutrient deprivation (27, 6), and animals lacking Sirt-1 in the brain show defective behavioral and hormonal responses to CR (7), we investigated the sirtuin as a potential CREB interactor. Sirt-1 mRNA was drastically reduced both in the cortex and hippocampus of BCKO mice, irrespective of the dietary regimen. Moreover, the moderate induction by CR that we observed in control brains was completely lost in the corresponding CREB- de fi cient tissues (Fig. 2 C ). Finally, acetylation of Histones H3 (AcK9) and H4 (AcK16), an inverse correlate of Sirt-1 activity (28), was abnormally high in BCKO hippocampal homogenates (Fig. 2 D ), con fi rming an overall reduction of Sirt-1 activity in this area of the brain. Transcriptional Regulation of Sirt-1 by CREB in Neurons. These observations suggested that Sirt-1 may represent a direct transcriptional target of CREB. Accordingly, CREB activators NGF and Forskolin (Fsk) (29) raised the level of immunoreactive Sirt-1 and of the corresponding mRNA in cultured primary cortical and hippocampal neurons (Fig. 3 A ). Bioinformatic analysis of the mouse Sirt-1 locus (NC_000076) revealed the presence of several putative cAMP Responsive Elements (CRE) both upstream and downstream of the transcription start site (TSS) (30), and an ∼ 300- bp segment encompassing two of those elements (TGACG at +1,998 and CGTCA at +2,012 from the annotated TSS) drove transcriptional response to Fsk and PKA in a standard luciferase reporter assay performed in PC12 pheochromocytoma cells (Fig. 3 B , a and b ). Moreover, ChIP from hippocampal neurons revealed that CREB binds the same genomic region in a fashion inducible by NGF (Fig. 3 Bc ). Finally, in primary neurons isolated from CREB loxP/loxP mice, deletion of CREB by adenoviral delivery of the Cre recombinase (Fig. 3 C , a and b ) led to a reduction of Sirt-1 immunoreactivity (Fig. 3 Cc ), and inhibited Sirt-1 mRNA induction by NGF and Fsk (Fig. 3 Cb ). Thus, we conclude that CREB directly regulates Sirt-1 mRNA and protein expression in neurons. Known transcriptional regulators of Sirt-1, such as p53 and Foxo3a (31), also interact with, and are either positively or negatively modulated by the deacetylase (32, 33); similarly, a myc-tagged form of human CREB1 and Sirt-1 could be easily coimmunopre- cipated in naive PC12 cells (Fig. 4 A ), indicating physical association between the two proteins; the association was rapidly induced by the PKA agonist Fsk, in parallel with phosphorylation of CREB on Serine 133. Short-time (30 min) stimulation with Fsk did not affect the total amount of cellular Sirt-1, but rather increased the stoichiometry of the binding to CREB, suggesting either an increased af fi nity between the two proteins, or the enhanced interaction with intermediate molecular partners co-recruited at target gene promoters. In keeping with the latter possibility, a mutant CREB that retains the phosphorylation site but is unable to bind DNA because of the deletion of the C-terminal transactivation domain ( Δ LZ CREB), displayed marginal physical interaction with Sirt-1, nor was the binding induced by Fsk (Fig. 4 A ). To con fi rm that SIRT1 and CREB colocalize on CREB- responsive chromatin regions in cortical neurons, cross-linked chromatin was immunoprecipitated with an anti-Sirt-1 antiserum and Sirt-1 binding to the regions of nNOS and PGC-1 α promoters surrounding the CRE elements determined by semiquantitative PCR. Sirt-1 was found to interact with both genes, in a fashion inducible by NGF and with a binding kinetic similar to that of CREB (Fig. 4 Ba ). Sirt-1 also bound the same Sirt-1 chromatin region surrounding the +1,998/+2,012 half-CRE sites that copre- cipitates with CREB, suggesting that Sirt-1 may self-regulate its CRE-dependent transactivation (Fig. 4 Bb ). Of note, Sirt-1 interaction with nNOS and Sirt-1 chromatin was indeed mediated by CREB, as revealed by its drastic reduction in CREB loxP/loxP neurons following the recombinase-mediated deletion of the factor (Fig. 4 Bb ) and by the failure of forced Sirt-1 overexpression to restore Sirt-1 chromatin binding in CREB-de fi cient cells (Fig. S3). To determine whether Sirt-1 modulates CREB transcriptional activity, cortical neurons were stimulated with NGF in the presence of sirtuin inhibitor Nicotinamide (34); induction of nNOS and PGC-1 α mRNA by NGF was blunted upon inhibition of Sirt-1 deacetylase activity (Fig. 4 C ). A similar result was obtained in naive PC12 cells by siRNA-mediated knock-down of Sirt-1 (Fig. S4 A ). In addition, Sirt-1 – de fi cient PC12 displayed impaired differentiation by NGF (as assessed by quanti fi cation of neurite outgrowth), a response largely dependent on CREB and nNOS in this cell model (35, 36) (Fig. S4 B ). In a complementary set of experiments, lentivirus-mediated overexpression of Sirt-1 increased the expression of nNOS and PGC-1 α mRNA in hippocampal neurons treated with NGF. However, Sirt-1 did not fully restore the expression of these genes in CREB-de fi cient cells, nor NGF protection from hydrogen peroxide-dependent cell death, which is largely lost in these cells (Fig. S5), was recovered upon transduction of the Sirt-1 cDNA. Collectively, the above data suggest that Sirt-1 and CREB are, at least to some extent, re- ciprocally dependent, and act in concert in the context of neurotrophin signaling. Unlike previously reported fi ndings (16), manipulations aimed at modulating Sirt-1 expression and activity in the above contexts did not affect the level of immunoreactive CREB1 (Fig. S4 Aa ). KO Mice. Finally, to verify whether Sirt-1 actually regulates CREB- dependent transcription in vivo and this is relevant for response to CR, we analyzed the expression of CREB targets nNOS and PGC- 1 α mRNAs in whole brains of Sirt-1 KO mice fed AL or CR for 25 – 28 wk (37). This analysis revealed that both genes are markedly hypoexpressed in Sirt-de fi cient mice compared with littermate controls under both dietary regimens (Fig. 4 D ), and their induction by CR, that was more pronounced for PGC-1 α , was also attenuated (Fig. 4 D , a and b ). The amount of immunoreactive CREB was comparable in brain homogenates of the two strains (Fig. 4 Dc ), nor CREB acetylation on lysine residues was affected by either Sirt-1 deletion or CR (Fig. S6), indicating that, at least in these experimental settings, Sirt-1 regulates CREB transcriptional activity independent of the acetylation and expression level of the factor. Taken together, the above fi ndings strongly suggest that Sirt-1 modulates the expression of CREB-dependent genes in mouse brain, and by extension, identify in the decrease of CREB- dependent transcription a molecular signature for a defective brain response to CR, shared by CREB and Sirt-1 mutant mice (7). The CREB transcription factor has been widely investigated as a metabolic sensor and regulator of glucose homeostasis in liver and fat tissue (15), and as a master switch of calcium and neuro- trophin-triggered transcriptional programs regulating neuronal differentiation, survival, and plasticity in the brain and peripheral nervous system (10, 11, 18). Evidence also exist for CREB roles in the control of appetite and food intake in the hypothalamus (38), but whether neuronal plasticity and high-order cognitive functions may be in fl uenced by nutrient cues and energy metabolism through CREB, an issue relevant to major diseases like AD and type 2 diabetes (3, 4), remains to be established. Our demonstra- tion of impaired electrophysiological, cognitive, and emotional response to CR in brain CREB KO mice, clearly suggests that this may indeed be the case. Interestingly, these differences emerge in the context of an overall comparable feeding behavior and metabolic response to CR between control and BCKO mice (Fig. 1 Ab ), indicating that potential effects of hypothalamic CREB signaling on energy balance and appetite regulation unlikely account for the observed phenotypes (38). Data reported in Fig. 2, showing CREB phosphorylation and transcriptional activation by CR, indicate that CREB is metaboli- cally regulated in the cortex and hippocampus, although the mechanism of such regulation needs to be further investigated. Because plasma from CR rodents has been previously shown to induce Sirt-1 expression in several organs/tissues, a humoral/hormonal mechanism for CREB activation could be envisaged (39); as an alternative, NO (40) or oxygen species (41) may mediate, cell-autonomously, this effect. Further research in this direction is warranted. Our ...

Context 2

... phosphorylation on Serine 133 in the hippocampi of the CR group was revealed by phospho-speci fi c immunoblotting (Fig. 2 A ), suggesting that CR activates CREB in this brain area. Accordingly, mRNAs of “ canonical ” CREB targets peroxisome proliferator-activated receptor- γ coactivator-1 α ( PGC- 1 α ), nNOS , and phosphoenolpyruvate carboxykinase ( PEPCK ) were induced by CR in the cortex and hippocampus of CREB- pro fi cient mice (Fig. 2 B ). Of note, both PGC-1 α and NO, the product of NOS enzymes, are known to promote mitochondrial biogenesis and to participate in organismal response to CR (26). The three mRNAs were overall down-regulated in BCKO mice fed AL compared with controls, and their induction by CR, observed in control mice, was nearly abolished (Fig. 2 B ). Interestingly, the expression of other putative CREB targets like bcl-2 , NGF , and c-fos were unaffected by CR and CREB deletion (Fig. S2). Thus, collectively, these fi ndings demonstrate that nutrient availability selectively regulates CREB-dependent gene expression in the forebrain. We next asked how CR may affect CREB activity. Because Sirt- 1 is a metabolic sensor involved in several biological consequences of nutrient deprivation (27, 6), and animals lacking Sirt-1 in the brain show defective behavioral and hormonal responses to CR (7), we investigated the sirtuin as a potential CREB interactor. Sirt-1 mRNA was drastically reduced both in the cortex and hippocampus of BCKO mice, irrespective of the dietary regimen. Moreover, the moderate induction by CR that we observed in control brains was completely lost in the corresponding CREB- de fi cient tissues (Fig. 2 C ). Finally, acetylation of Histones H3 (AcK9) and H4 (AcK16), an inverse correlate of Sirt-1 activity (28), was abnormally high in BCKO hippocampal homogenates (Fig. 2 D ), con fi rming an overall reduction of Sirt-1 activity in this area of the brain. Transcriptional Regulation of Sirt-1 by CREB in Neurons. These observations suggested that Sirt-1 may represent a direct transcriptional target of CREB. Accordingly, CREB activators NGF and Forskolin (Fsk) (29) raised the level of immunoreactive Sirt-1 and of the corresponding mRNA in cultured primary cortical and hippocampal neurons (Fig. 3 A ). Bioinformatic analysis of the mouse Sirt-1 locus (NC_000076) revealed the presence of several putative cAMP Responsive Elements (CRE) both upstream and downstream of the transcription start site (TSS) (30), and an ∼ 300- bp segment encompassing two of those elements (TGACG at +1,998 and CGTCA at +2,012 from the annotated TSS) drove transcriptional response to Fsk and PKA in a standard luciferase reporter assay performed in PC12 pheochromocytoma cells (Fig. 3 B , a and b ). Moreover, ChIP from hippocampal neurons revealed that CREB binds the same genomic region in a fashion inducible by NGF (Fig. 3 Bc ). Finally, in primary neurons isolated from CREB loxP/loxP mice, deletion of CREB by adenoviral delivery of the Cre recombinase (Fig. 3 C , a and b ) led to a reduction of Sirt-1 immunoreactivity (Fig. 3 Cc ), and inhibited Sirt-1 mRNA induction by NGF and Fsk (Fig. 3 Cb ). Thus, we conclude that CREB directly regulates Sirt-1 mRNA and protein expression in neurons. Known transcriptional regulators of Sirt-1, such as p53 and Foxo3a (31), also interact with, and are either positively or negatively modulated by the deacetylase (32, 33); similarly, a myc-tagged form of human CREB1 and Sirt-1 could be easily coimmunopre- cipated in naive PC12 cells (Fig. 4 A ), indicating physical association between the two proteins; the association was rapidly induced by the PKA agonist Fsk, in parallel with phosphorylation of CREB on Serine 133. Short-time (30 min) stimulation with Fsk did not affect the total amount of cellular Sirt-1, but rather increased the stoichiometry of the binding to CREB, suggesting either an increased af fi nity between the two proteins, or the enhanced interaction with intermediate molecular partners co-recruited at target gene promoters. In keeping with the latter possibility, a mutant CREB that retains the phosphorylation site but is unable to bind DNA because of the deletion of the C-terminal transactivation domain ( Δ LZ CREB), displayed marginal physical interaction with Sirt-1, nor was the binding induced by Fsk (Fig. 4 A ). To con fi rm that SIRT1 and CREB colocalize on CREB- responsive chromatin regions in cortical neurons, cross-linked chromatin was immunoprecipitated with an anti-Sirt-1 antiserum and Sirt-1 binding to the regions of nNOS and PGC-1 α promoters surrounding the CRE elements determined by semiquantitative PCR. Sirt-1 was found to interact with both genes, in a fashion inducible by NGF and with a binding kinetic similar to that of CREB (Fig. 4 Ba ). Sirt-1 also bound the same Sirt-1 chromatin region surrounding the +1,998/+2,012 half-CRE sites that copre- cipitates with CREB, suggesting that Sirt-1 may self-regulate its CRE-dependent transactivation (Fig. 4 Bb ). Of note, Sirt-1 interaction with nNOS and Sirt-1 chromatin was indeed mediated by CREB, as revealed by its drastic reduction in CREB loxP/loxP neurons following the recombinase-mediated deletion of the factor (Fig. 4 Bb ) and by the failure of forced Sirt-1 overexpression to restore Sirt-1 chromatin binding in CREB-de fi cient cells (Fig. S3). To determine whether Sirt-1 modulates CREB transcriptional activity, cortical neurons were stimulated with NGF in the presence of sirtuin inhibitor Nicotinamide (34); induction of nNOS and PGC-1 α mRNA by NGF was blunted upon inhibition of Sirt-1 deacetylase activity (Fig. 4 C ). A similar result was obtained in naive PC12 cells by siRNA-mediated knock-down of Sirt-1 (Fig. S4 A ). In addition, Sirt-1 – de fi cient PC12 displayed impaired differentiation by NGF (as assessed by quanti fi cation of neurite outgrowth), a response largely dependent on CREB and nNOS in this cell model (35, 36) (Fig. S4 B ). In a complementary set of experiments, lentivirus-mediated overexpression of Sirt-1 increased the expression of nNOS and PGC-1 α mRNA in hippocampal neurons treated with NGF. However, Sirt-1 did not fully restore the expression of these genes in CREB-de fi cient cells, nor NGF protection from hydrogen peroxide-dependent cell death, which is largely lost in these cells (Fig. S5), was recovered upon transduction of the Sirt-1 cDNA. Collectively, the above data suggest that Sirt-1 and CREB are, at least to some extent, re- ciprocally dependent, and act in concert in the context of neurotrophin signaling. Unlike previously reported fi ndings (16), manipulations aimed at modulating Sirt-1 expression and activity in the above contexts did not affect the level of immunoreactive CREB1 (Fig. S4 Aa ). KO Mice. Finally, to verify whether Sirt-1 actually regulates CREB- dependent transcription in vivo and this is relevant for response to CR, we analyzed the expression of CREB targets nNOS and PGC- 1 α mRNAs in whole brains of Sirt-1 KO mice fed AL or CR for 25 – 28 wk (37). This analysis revealed that both genes are markedly hypoexpressed in Sirt-de fi cient mice compared with littermate controls under both dietary regimens (Fig. 4 D ), and their induction by CR, that was more pronounced for PGC-1 α , was also attenuated (Fig. 4 D , a and b ). The amount of immunoreactive CREB was comparable in brain homogenates of the two strains (Fig. 4 Dc ), nor CREB acetylation on lysine residues was affected by either Sirt-1 deletion or CR (Fig. S6), indicating that, at least in these experimental settings, Sirt-1 regulates CREB transcriptional activity independent of the acetylation and expression level of the factor. Taken together, the above fi ndings strongly suggest that Sirt-1 modulates the expression of CREB-dependent genes in mouse brain, and by extension, identify in the decrease of CREB- dependent transcription a molecular signature for a defective brain response to CR, shared by CREB and Sirt-1 mutant mice (7). The CREB transcription factor has been widely investigated as a metabolic sensor and regulator of glucose homeostasis in liver and fat tissue (15), and as a master switch of calcium and neuro- trophin-triggered transcriptional programs regulating neuronal differentiation, survival, and plasticity in the brain and peripheral nervous system (10, 11, 18). Evidence also exist for CREB roles in the control of appetite and food intake in the hypothalamus (38), but whether neuronal plasticity and high-order cognitive functions may be in fl uenced by nutrient cues and energy metabolism through CREB, an issue relevant to major diseases like AD and type 2 diabetes (3, 4), remains to be established. Our demonstra- tion of impaired electrophysiological, cognitive, and emotional response to CR in brain CREB KO mice, clearly suggests that this may indeed be the case. Interestingly, these differences emerge in the context of an overall comparable feeding behavior and metabolic response to CR between control and BCKO mice (Fig. 1 Ab ), indicating that potential effects of hypothalamic CREB signaling on energy balance and appetite regulation unlikely account for the observed phenotypes (38). Data reported in Fig. 2, showing CREB phosphorylation and transcriptional activation by CR, indicate that CREB is metaboli- cally regulated in the cortex and hippocampus, although the mechanism of such regulation needs to be further investigated. Because plasma from CR rodents has been previously shown to induce Sirt-1 expression in several organs/tissues, a humoral/hormonal mechanism for CREB activation could be envisaged (39); as an alternative, NO (40) or oxygen species (41) may mediate, cell-autonomously, this effect. Further research in this direction is warranted. Our data identify in the complex interplay with the nutrient- sensitive histone deacetylase Sirt-1 a molecular connection between CREB and neuronal response to CR. Previous work from others has compellingly involved ...

Context 3

... mouse Sirt-1 locus (NC_000076) revealed the presence of several putative cAMP Responsive Elements (CRE) both upstream and downstream of the transcription start site (TSS) (30), and an ∼ 300- bp segment encompassing two of those elements (TGACG at +1,998 and CGTCA at +2,012 from the annotated TSS) drove transcriptional response to Fsk and PKA in a standard luciferase reporter assay performed in PC12 pheochromocytoma cells (Fig. 3 B , a and b ). Moreover, ChIP from hippocampal neurons revealed that CREB binds the same genomic region in a fashion inducible by NGF (Fig. 3 Bc ). Finally, in primary neurons isolated from CREB loxP/loxP mice, deletion of CREB by adenoviral delivery of the Cre recombinase (Fig. 3 C , a and b ) led to a reduction of Sirt-1 immunoreactivity (Fig. 3 Cc ), and inhibited Sirt-1 mRNA induction by NGF and Fsk (Fig. 3 Cb ). Thus, we conclude that CREB directly regulates Sirt-1 mRNA and protein expression in neurons. Known transcriptional regulators of Sirt-1, such as p53 and Foxo3a (31), also interact with, and are either positively or negatively modulated by the deacetylase (32, 33); similarly, a myc-tagged form of human CREB1 and Sirt-1 could be easily coimmunopre- cipated in naive PC12 cells (Fig. 4 A ), indicating physical association between the two proteins; the association was rapidly induced by the PKA agonist Fsk, in parallel with phosphorylation of CREB on Serine 133. Short-time (30 min) stimulation with Fsk did not affect the total amount of cellular Sirt-1, but rather increased the stoichiometry of the binding to CREB, suggesting either an increased af fi nity between the two proteins, or the enhanced interaction with intermediate molecular partners co-recruited at target gene promoters. In keeping with the latter possibility, a mutant CREB that retains the phosphorylation site but is unable to bind DNA because of the deletion of the C-terminal transactivation domain ( Δ LZ CREB), displayed marginal physical interaction with Sirt-1, nor was the binding induced by Fsk (Fig. 4 A ). To con fi rm that SIRT1 and CREB colocalize on CREB- responsive chromatin regions in cortical neurons, cross-linked chromatin was immunoprecipitated with an anti-Sirt-1 antiserum and Sirt-1 binding to the regions of nNOS and PGC-1 α promoters surrounding the CRE elements determined by semiquantitative PCR. Sirt-1 was found to interact with both genes, in a fashion inducible by NGF and with a binding kinetic similar to that of CREB (Fig. 4 Ba ). Sirt-1 also bound the same Sirt-1 chromatin region surrounding the +1,998/+2,012 half-CRE sites that copre- cipitates with CREB, suggesting that Sirt-1 may self-regulate its CRE-dependent transactivation (Fig. 4 Bb ). Of note, Sirt-1 interaction with nNOS and Sirt-1 chromatin was indeed mediated by CREB, as revealed by its drastic reduction in CREB loxP/loxP neurons following the recombinase-mediated deletion of the factor (Fig. 4 Bb ) and by the failure of forced Sirt-1 overexpression to restore Sirt-1 chromatin binding in CREB-de fi cient cells (Fig. S3). To determine whether Sirt-1 modulates CREB transcriptional activity, cortical neurons were stimulated with NGF in the presence of sirtuin inhibitor Nicotinamide (34); induction of nNOS and PGC-1 α mRNA by NGF was blunted upon inhibition of Sirt-1 deacetylase activity (Fig. 4 C ). A similar result was obtained in naive PC12 cells by siRNA-mediated knock-down of Sirt-1 (Fig. S4 A ). In addition, Sirt-1 – de fi cient PC12 displayed impaired differentiation by NGF (as assessed by quanti fi cation of neurite outgrowth), a response largely dependent on CREB and nNOS in this cell model (35, 36) (Fig. S4 B ). In a complementary set of experiments, lentivirus-mediated overexpression of Sirt-1 increased the expression of nNOS and PGC-1 α mRNA in hippocampal neurons treated with NGF. However, Sirt-1 did not fully restore the expression of these genes in CREB-de fi cient cells, nor NGF protection from hydrogen peroxide-dependent cell death, which is largely lost in these cells (Fig. S5), was recovered upon transduction of the Sirt-1 cDNA. Collectively, the above data suggest that Sirt-1 and CREB are, at least to some extent, re- ciprocally dependent, and act in concert in the context of neurotrophin signaling. Unlike previously reported fi ndings (16), manipulations aimed at modulating Sirt-1 expression and activity in the above contexts did not affect the level of immunoreactive CREB1 (Fig. S4 Aa ). KO Mice. Finally, to verify whether Sirt-1 actually regulates CREB- dependent transcription in vivo and this is relevant for response to CR, we analyzed the expression of CREB targets nNOS and PGC- 1 α mRNAs in whole brains of Sirt-1 KO mice fed AL or CR for 25 – 28 wk (37). This analysis revealed that both genes are markedly hypoexpressed in Sirt-de fi cient mice compared with littermate controls under both dietary regimens (Fig. 4 D ), and their induction by CR, that was more pronounced for PGC-1 α , was also attenuated (Fig. 4 D , a and b ). The amount of immunoreactive CREB was comparable in brain homogenates of the two strains (Fig. 4 Dc ), nor CREB acetylation on lysine residues was affected by either Sirt-1 deletion or CR (Fig. S6), indicating that, at least in these experimental settings, Sirt-1 regulates CREB transcriptional activity independent of the acetylation and expression level of the factor. Taken together, the above fi ndings strongly suggest that Sirt-1 modulates the expression of CREB-dependent genes in mouse brain, and by extension, identify in the decrease of CREB- dependent transcription a molecular signature for a defective brain response to CR, shared by CREB and Sirt-1 mutant mice (7). The CREB transcription factor has been widely investigated as a metabolic sensor and regulator of glucose homeostasis in liver and fat tissue (15), and as a master switch of calcium and neuro- trophin-triggered transcriptional programs regulating neuronal differentiation, survival, and plasticity in the brain and peripheral nervous system (10, 11, 18). Evidence also exist for CREB roles in the control of appetite and food intake in the hypothalamus (38), but whether neuronal plasticity and high-order cognitive functions may be in fl uenced by nutrient cues and energy metabolism through CREB, an issue relevant to major diseases like AD and type 2 diabetes (3, 4), remains to be established. Our demonstra- tion of impaired electrophysiological, cognitive, and emotional response to CR in brain CREB KO mice, clearly suggests that this may indeed be the case. Interestingly, these differences emerge in the context of an overall comparable feeding behavior and metabolic response to CR between control and BCKO mice (Fig. 1 Ab ), indicating that potential effects of hypothalamic CREB signaling on energy balance and appetite regulation unlikely account for the observed phenotypes (38). Data reported in Fig. 2, showing CREB phosphorylation and transcriptional activation by CR, indicate that CREB is metaboli- cally regulated in the cortex and hippocampus, although the mechanism of such regulation needs to be further investigated. Because plasma from CR rodents has been previously shown to induce Sirt-1 expression in several organs/tissues, a humoral/hormonal mechanism for CREB activation could be envisaged (39); as an alternative, NO (40) or oxygen species (41) may mediate, cell-autonomously, this effect. Further research in this direction is warranted. Our data identify in the complex interplay with the nutrient- sensitive histone deacetylase Sirt-1 a molecular connection between CREB and neuronal response to CR. Previous work from others has compellingly involved neuronal Sirt-1 in hormonal and metabolic adaptation to dietary restriction in mice (7). Results presented here clearly suggest that the two molecules are part of the same CR-sensitive signaling cascade. However, other genes potentially relevant to mitochondrial biogenesis and neuronal response to calorie restriction, namely nNOS and PGC-1 α (40), and some PGC-1 α targets — including CPT1 , CoxIV , and UCP-2 — are also critically down-regulated, together with Sirt-1, in the cortex and hippocampus of BCKO mice (Fig. 2 B and Fig. S2 b ). This fi nding, and evidence from Fig. S3 that overexpression of Sirt-1 does not rescue NGF signaling in CREB-de fi cient hippocampal neurons in vitro, suggests that up-regulation of Sirt-1 is not the only mechanism whereby CREB participates in neuronal response to nutrients. Instead, cooperation with CREB is likely critical for the action of neuronal Sirt-1 in CR. This view is supported by the unexpected fi nding that CREB-dependent genes involved in neuronal plasticity, survival, and stress resistance (42, 43), and induced by calorie restriction (Fig. 2), are markedly down-regulated in Sirt-1 – de fi cient cultured neurons (PC12) and in the brain of Sirt-1 KO mice. Because the latter strain is, like BCKO mice, impaired in brain response to reduced food intake (7), the above evidence further support the notion that CREB-dependent transcription has a pivotal role in the neuronal effects of calorie restriction, and identify in the CREB – Sirt-1 axis a major component of the nutrient sensitive molecular network that connects caloric intake and energy metabolism to brain health. Biochemical details of how Sirt-1 affects CREB activity remains to be clari fi ed. Unlike other transcription factors, CREB does not appear to be deacetylated by Sirt-1 (Fig. S6); moreover, we could not detect consistent effects of Sirt-1 on the expression of CREB (Fig. 4 Dc , and Figs. S4 Aa and S6), unlike that described by Gao et al. (16). Those authors, however, made their important observations on Sirt-1 Δ ex4 mice that, unlike Sirt-1KO mice used in our experiments, do express an inactive form of Sirt-1 potentially acting in a dominant negative fashion against other molecules (deacetylases?) capable of regulating CREB expression. On the other hand, Sirt-1 ...

Context 4

... and downstream of the transcription start site (TSS) (30), and an ∼ 300- bp segment encompassing two of those elements (TGACG at +1,998 and CGTCA at +2,012 from the annotated TSS) drove transcriptional response to Fsk and PKA in a standard luciferase reporter assay performed in PC12 pheochromocytoma cells (Fig. 3 B , a and b ). Moreover, ChIP from hippocampal neurons revealed that CREB binds the same genomic region in a fashion inducible by NGF (Fig. 3 Bc ). Finally, in primary neurons isolated from CREB loxP/loxP mice, deletion of CREB by adenoviral delivery of the Cre recombinase (Fig. 3 C , a and b ) led to a reduction of Sirt-1 immunoreactivity (Fig. 3 Cc ), and inhibited Sirt-1 mRNA induction by NGF and Fsk (Fig. 3 Cb ). Thus, we conclude that CREB directly regulates Sirt-1 mRNA and protein expression in neurons. Known transcriptional regulators of Sirt-1, such as p53 and Foxo3a (31), also interact with, and are either positively or negatively modulated by the deacetylase (32, 33); similarly, a myc-tagged form of human CREB1 and Sirt-1 could be easily coimmunopre- cipated in naive PC12 cells (Fig. 4 A ), indicating physical association between the two proteins; the association was rapidly induced by the PKA agonist Fsk, in parallel with phosphorylation of CREB on Serine 133. Short-time (30 min) stimulation with Fsk did not affect the total amount of cellular Sirt-1, but rather increased the stoichiometry of the binding to CREB, suggesting either an increased af fi nity between the two proteins, or the enhanced interaction with intermediate molecular partners co-recruited at target gene promoters. In keeping with the latter possibility, a mutant CREB that retains the phosphorylation site but is unable to bind DNA because of the deletion of the C-terminal transactivation domain ( Δ LZ CREB), displayed marginal physical interaction with Sirt-1, nor was the binding induced by Fsk (Fig. 4 A ). To con fi rm that SIRT1 and CREB colocalize on CREB- responsive chromatin regions in cortical neurons, cross-linked chromatin was immunoprecipitated with an anti-Sirt-1 antiserum and Sirt-1 binding to the regions of nNOS and PGC-1 α promoters surrounding the CRE elements determined by semiquantitative PCR. Sirt-1 was found to interact with both genes, in a fashion inducible by NGF and with a binding kinetic similar to that of CREB (Fig. 4 Ba ). Sirt-1 also bound the same Sirt-1 chromatin region surrounding the +1,998/+2,012 half-CRE sites that copre- cipitates with CREB, suggesting that Sirt-1 may self-regulate its CRE-dependent transactivation (Fig. 4 Bb ). Of note, Sirt-1 interaction with nNOS and Sirt-1 chromatin was indeed mediated by CREB, as revealed by its drastic reduction in CREB loxP/loxP neurons following the recombinase-mediated deletion of the factor (Fig. 4 Bb ) and by the failure of forced Sirt-1 overexpression to restore Sirt-1 chromatin binding in CREB-de fi cient cells (Fig. S3). To determine whether Sirt-1 modulates CREB transcriptional activity, cortical neurons were stimulated with NGF in the presence of sirtuin inhibitor Nicotinamide (34); induction of nNOS and PGC-1 α mRNA by NGF was blunted upon inhibition of Sirt-1 deacetylase activity (Fig. 4 C ). A similar result was obtained in naive PC12 cells by siRNA-mediated knock-down of Sirt-1 (Fig. S4 A ). In addition, Sirt-1 – de fi cient PC12 displayed impaired differentiation by NGF (as assessed by quanti fi cation of neurite outgrowth), a response largely dependent on CREB and nNOS in this cell model (35, 36) (Fig. S4 B ). In a complementary set of experiments, lentivirus-mediated overexpression of Sirt-1 increased the expression of nNOS and PGC-1 α mRNA in hippocampal neurons treated with NGF. However, Sirt-1 did not fully restore the expression of these genes in CREB-de fi cient cells, nor NGF protection from hydrogen peroxide-dependent cell death, which is largely lost in these cells (Fig. S5), was recovered upon transduction of the Sirt-1 cDNA. Collectively, the above data suggest that Sirt-1 and CREB are, at least to some extent, re- ciprocally dependent, and act in concert in the context of neurotrophin signaling. Unlike previously reported fi ndings (16), manipulations aimed at modulating Sirt-1 expression and activity in the above contexts did not affect the level of immunoreactive CREB1 (Fig. S4 Aa ). KO Mice. Finally, to verify whether Sirt-1 actually regulates CREB- dependent transcription in vivo and this is relevant for response to CR, we analyzed the expression of CREB targets nNOS and PGC- 1 α mRNAs in whole brains of Sirt-1 KO mice fed AL or CR for 25 – 28 wk (37). This analysis revealed that both genes are markedly hypoexpressed in Sirt-de fi cient mice compared with littermate controls under both dietary regimens (Fig. 4 D ), and their induction by CR, that was more pronounced for PGC-1 α , was also attenuated (Fig. 4 D , a and b ). The amount of immunoreactive CREB was comparable in brain homogenates of the two strains (Fig. 4 Dc ), nor CREB acetylation on lysine residues was affected by either Sirt-1 deletion or CR (Fig. S6), indicating that, at least in these experimental settings, Sirt-1 regulates CREB transcriptional activity independent of the acetylation and expression level of the factor. Taken together, the above fi ndings strongly suggest that Sirt-1 modulates the expression of CREB-dependent genes in mouse brain, and by extension, identify in the decrease of CREB- dependent transcription a molecular signature for a defective brain response to CR, shared by CREB and Sirt-1 mutant mice (7). The CREB transcription factor has been widely investigated as a metabolic sensor and regulator of glucose homeostasis in liver and fat tissue (15), and as a master switch of calcium and neuro- trophin-triggered transcriptional programs regulating neuronal differentiation, survival, and plasticity in the brain and peripheral nervous system (10, 11, 18). Evidence also exist for CREB roles in the control of appetite and food intake in the hypothalamus (38), but whether neuronal plasticity and high-order cognitive functions may be in fl uenced by nutrient cues and energy metabolism through CREB, an issue relevant to major diseases like AD and type 2 diabetes (3, 4), remains to be established. Our demonstra- tion of impaired electrophysiological, cognitive, and emotional response to CR in brain CREB KO mice, clearly suggests that this may indeed be the case. Interestingly, these differences emerge in the context of an overall comparable feeding behavior and metabolic response to CR between control and BCKO mice (Fig. 1 Ab ), indicating that potential effects of hypothalamic CREB signaling on energy balance and appetite regulation unlikely account for the observed phenotypes (38). Data reported in Fig. 2, showing CREB phosphorylation and transcriptional activation by CR, indicate that CREB is metaboli- cally regulated in the cortex and hippocampus, although the mechanism of such regulation needs to be further investigated. Because plasma from CR rodents has been previously shown to induce Sirt-1 expression in several organs/tissues, a humoral/hormonal mechanism for CREB activation could be envisaged (39); as an alternative, NO (40) or oxygen species (41) may mediate, cell-autonomously, this effect. Further research in this direction is warranted. Our data identify in the complex interplay with the nutrient- sensitive histone deacetylase Sirt-1 a molecular connection between CREB and neuronal response to CR. Previous work from others has compellingly involved neuronal Sirt-1 in hormonal and metabolic adaptation to dietary restriction in mice (7). Results presented here clearly suggest that the two molecules are part of the same CR-sensitive signaling cascade. However, other genes potentially relevant to mitochondrial biogenesis and neuronal response to calorie restriction, namely nNOS and PGC-1 α (40), and some PGC-1 α targets — including CPT1 , CoxIV , and UCP-2 — are also critically down-regulated, together with Sirt-1, in the cortex and hippocampus of BCKO mice (Fig. 2 B and Fig. S2 b ). This fi nding, and evidence from Fig. S3 that overexpression of Sirt-1 does not rescue NGF signaling in CREB-de fi cient hippocampal neurons in vitro, suggests that up-regulation of Sirt-1 is not the only mechanism whereby CREB participates in neuronal response to nutrients. Instead, cooperation with CREB is likely critical for the action of neuronal Sirt-1 in CR. This view is supported by the unexpected fi nding that CREB-dependent genes involved in neuronal plasticity, survival, and stress resistance (42, 43), and induced by calorie restriction (Fig. 2), are markedly down-regulated in Sirt-1 – de fi cient cultured neurons (PC12) and in the brain of Sirt-1 KO mice. Because the latter strain is, like BCKO mice, impaired in brain response to reduced food intake (7), the above evidence further support the notion that CREB-dependent transcription has a pivotal role in the neuronal effects of calorie restriction, and identify in the CREB – Sirt-1 axis a major component of the nutrient sensitive molecular network that connects caloric intake and energy metabolism to brain health. Biochemical details of how Sirt-1 affects CREB activity remains to be clari fi ed. Unlike other transcription factors, CREB does not appear to be deacetylated by Sirt-1 (Fig. S6); moreover, we could not detect consistent effects of Sirt-1 on the expression of CREB (Fig. 4 Dc , and Figs. S4 Aa and S6), unlike that described by Gao et al. (16). Those authors, however, made their important observations on Sirt-1 Δ ex4 mice that, unlike Sirt-1KO mice used in our experiments, do express an inactive form of Sirt-1 potentially acting in a dominant negative fashion against other molecules (deacetylases?) capable of regulating CREB expression. On the other hand, Sirt-1 dependent modulation of nutrient sensitive cofactors of CREB, like TORC/Crtc (38), has been described in the liver ...

Context 5

... for response to CR, we analyzed the expression of CREB targets nNOS and PGC- 1 α mRNAs in whole brains of Sirt-1 KO mice fed AL or CR for 25 – 28 wk (37). This analysis revealed that both genes are markedly hypoexpressed in Sirt-de fi cient mice compared with littermate controls under both dietary regimens (Fig. 4 D ), and their induction by CR, that was more pronounced for PGC-1 α , was also attenuated (Fig. 4 D , a and b ). The amount of immunoreactive CREB was comparable in brain homogenates of the two strains (Fig. 4 Dc ), nor CREB acetylation on lysine residues was affected by either Sirt-1 deletion or CR (Fig. S6), indicating that, at least in these experimental settings, Sirt-1 regulates CREB transcriptional activity independent of the acetylation and expression level of the factor. Taken together, the above fi ndings strongly suggest that Sirt-1 modulates the expression of CREB-dependent genes in mouse brain, and by extension, identify in the decrease of CREB- dependent transcription a molecular signature for a defective brain response to CR, shared by CREB and Sirt-1 mutant mice (7). The CREB transcription factor has been widely investigated as a metabolic sensor and regulator of glucose homeostasis in liver and fat tissue (15), and as a master switch of calcium and neuro- trophin-triggered transcriptional programs regulating neuronal differentiation, survival, and plasticity in the brain and peripheral nervous system (10, 11, 18). Evidence also exist for CREB roles in the control of appetite and food intake in the hypothalamus (38), but whether neuronal plasticity and high-order cognitive functions may be in fl uenced by nutrient cues and energy metabolism through CREB, an issue relevant to major diseases like AD and type 2 diabetes (3, 4), remains to be established. Our demonstra- tion of impaired electrophysiological, cognitive, and emotional response to CR in brain CREB KO mice, clearly suggests that this may indeed be the case. Interestingly, these differences emerge in the context of an overall comparable feeding behavior and metabolic response to CR between control and BCKO mice (Fig. 1 Ab ), indicating that potential effects of hypothalamic CREB signaling on energy balance and appetite regulation unlikely account for the observed phenotypes (38). Data reported in Fig. 2, showing CREB phosphorylation and transcriptional activation by CR, indicate that CREB is metaboli- cally regulated in the cortex and hippocampus, although the mechanism of such regulation needs to be further investigated. Because plasma from CR rodents has been previously shown to induce Sirt-1 expression in several organs/tissues, a humoral/hormonal mechanism for CREB activation could be envisaged (39); as an alternative, NO (40) or oxygen species (41) may mediate, cell-autonomously, this effect. Further research in this direction is warranted. Our data identify in the complex interplay with the nutrient- sensitive histone deacetylase Sirt-1 a molecular connection between CREB and neuronal response to CR. Previous work from others has compellingly involved neuronal Sirt-1 in hormonal and metabolic adaptation to dietary restriction in mice (7). Results presented here clearly suggest that the two molecules are part of the same CR-sensitive signaling cascade. However, other genes potentially relevant to mitochondrial biogenesis and neuronal response to calorie restriction, namely nNOS and PGC-1 α (40), and some PGC-1 α targets — including CPT1 , CoxIV , and UCP-2 — are also critically down-regulated, together with Sirt-1, in the cortex and hippocampus of BCKO mice (Fig. 2 B and Fig. S2 b ). This fi nding, and evidence from Fig. S3 that overexpression of Sirt-1 does not rescue NGF signaling in CREB-de fi cient hippocampal neurons in vitro, suggests that up-regulation of Sirt-1 is not the only mechanism whereby CREB participates in neuronal response to nutrients. Instead, cooperation with CREB is likely critical for the action of neuronal Sirt-1 in CR. This view is supported by the unexpected fi nding that CREB-dependent genes involved in neuronal plasticity, survival, and stress resistance (42, 43), and induced by calorie restriction (Fig. 2), are markedly down-regulated in Sirt-1 – de fi cient cultured neurons (PC12) and in the brain of Sirt-1 KO mice. Because the latter strain is, like BCKO mice, impaired in brain response to reduced food intake (7), the above evidence further support the notion that CREB-dependent transcription has a pivotal role in the neuronal effects of calorie restriction, and identify in the CREB – Sirt-1 axis a major component of the nutrient sensitive molecular network that connects caloric intake and energy metabolism to brain health. Biochemical details of how Sirt-1 affects CREB activity remains to be clari fi ed. Unlike other transcription factors, CREB does not appear to be deacetylated by Sirt-1 (Fig. S6); moreover, we could not detect consistent effects of Sirt-1 on the expression of CREB (Fig. 4 Dc , and Figs. S4 Aa and S6), unlike that described by Gao et al. (16). Those authors, however, made their important observations on Sirt-1 Δ ex4 mice that, unlike Sirt-1KO mice used in our experiments, do express an inactive form of Sirt-1 potentially acting in a dominant negative fashion against other molecules (deacetylases?) capable of regulating CREB expression. On the other hand, Sirt-1 dependent modulation of nutrient sensitive cofactors of CREB, like TORC/Crtc (38), has been described in the liver and may occur in neuronal cells. Alternatively, Sirt-1 may be recruited by CREB on target promoters and act either on histones (Fig. 2 D ) or, in trans , on other (maybe com- peting) transcription factors. In conclusion, CREB1, an effector of neurotrophins involved in several age-associated neurodegenerative diseases, mediates at least some brain responses to dietary restriction. This action involves the up-regulation of Sirt-1 that in turn bolsters the CREB- dependent expression of genes involved in neuronal metabolism, survival and plasticity (Fig. S7). Although molecular details on how Sirt-1 regulates gene transcription by CREB need to be further investigated, the CREB-Sirt-1 axis outlines a unique molecular network at the crossroad of energy metabolism, metabolic diseases, and brain ...

Context 6

... object and spatial memory. This indicator of neuronal plasticity declines in rodents with age, in a fashion that is attenuated by calorie restriction (24). To extend our analysis of CR effects on the brain of CREB-de fi cient mice, we tested LTP at CA3-CA1 synapses in hippocampal brain slices obtained from animals euthanized after 5 wk of either CR or AL feeding regimen. As previously shown (25), LTP was not affected by CREB status in the two AL-fed groups. Strikingly, however, LTP increased in control mice under CR [AL 136.6 ± 8.0% ( n = 10 slices) vs. CR 161.3 ± 8.2% ( n = 9 slices); P < 0.05] but not in the BCKO strain [AL 130.3 ± 8.6% ( n = 9 slices) vs. CR 121.0 ± 8.5% ( n = 10 slices), n.s.] (Fig. 1 C ), indicating that bene fi cial effect of CR on LTP is also abrogated by brain-speci fi c CREB deletion. The increased LTP induced by CR in control mice was independent on changes in basal synaptic transmission, because input/output curves obtained plotting fi eld excitatory postsynaptic potential (fEPSP) amplitudes vs. stimulus intensity in hippocampal brain slices of CR and AL animals were superimposable. Because CREB appeared to participate in brain response to CR, we assessed whether diet affected the total amount/phosphoryla- tion of CREB protein, and the expression level of a number of mRNAs known to be regulated by CREB (23). CREB1 protein expression in control mice was not changed by the dietary regimen; instead, an increase in CREB phosphorylation on Serine 133 in the hippocampi of the CR group was revealed by phospho-speci fi c immunoblotting (Fig. 2 A ), suggesting that CR activates CREB in this brain area. Accordingly, mRNAs of “ canonical ” CREB targets peroxisome proliferator-activated receptor- γ coactivator-1 α ( PGC- 1 α ), nNOS , and phosphoenolpyruvate carboxykinase ( PEPCK ) were induced by CR in the cortex and hippocampus of CREB- pro fi cient mice (Fig. 2 B ). Of note, both PGC-1 α and NO, the product of NOS enzymes, are known to promote mitochondrial biogenesis and to participate in organismal response to CR (26). The three mRNAs were overall down-regulated in BCKO mice fed AL compared with controls, and their induction by CR, observed in control mice, was nearly abolished (Fig. 2 B ). Interestingly, the expression of other putative CREB targets like bcl-2 , NGF , and c-fos were unaffected by CR and CREB deletion (Fig. S2). Thus, collectively, these fi ndings demonstrate that nutrient availability selectively regulates CREB-dependent gene expression in the forebrain. We next asked how CR may affect CREB activity. Because Sirt- 1 is a metabolic sensor involved in several biological consequences of nutrient deprivation (27, 6), and animals lacking Sirt-1 in the brain show defective behavioral and hormonal responses to CR (7), we investigated the sirtuin as a potential CREB interactor. Sirt-1 mRNA was drastically reduced both in the cortex and hippocampus of BCKO mice, irrespective of the dietary regimen. Moreover, the moderate induction by CR that we observed in control brains was completely lost in the corresponding CREB- de fi cient tissues (Fig. 2 C ). Finally, acetylation of Histones H3 (AcK9) and H4 (AcK16), an inverse correlate of Sirt-1 activity (28), was abnormally high in BCKO hippocampal homogenates (Fig. 2 D ), con fi rming an overall reduction of Sirt-1 activity in this area of the brain. Transcriptional Regulation of Sirt-1 by CREB in Neurons. These observations suggested that Sirt-1 may represent a direct transcriptional target of CREB. Accordingly, CREB activators NGF and Forskolin (Fsk) (29) raised the level of immunoreactive Sirt-1 and of the corresponding mRNA in cultured primary cortical and hippocampal neurons (Fig. 3 A ). Bioinformatic analysis of the mouse Sirt-1 locus (NC_000076) revealed the presence of several putative cAMP Responsive Elements (CRE) both upstream and downstream of the transcription start site (TSS) (30), and an ∼ 300- bp segment encompassing two of those elements (TGACG at +1,998 and CGTCA at +2,012 from the annotated TSS) drove transcriptional response to Fsk and PKA in a standard luciferase reporter assay performed in PC12 pheochromocytoma cells (Fig. 3 B , a and b ). Moreover, ChIP from hippocampal neurons revealed that CREB binds the same genomic region in a fashion inducible by NGF (Fig. 3 Bc ). Finally, in primary neurons isolated from CREB loxP/loxP mice, deletion of CREB by adenoviral delivery of the Cre recombinase (Fig. 3 C , a and b ) led to a reduction of Sirt-1 immunoreactivity (Fig. 3 Cc ), and inhibited Sirt-1 mRNA induction by NGF and Fsk (Fig. 3 Cb ). Thus, we conclude that CREB directly regulates Sirt-1 mRNA and protein expression in neurons. Known transcriptional regulators of Sirt-1, such as p53 and Foxo3a (31), also interact with, and are either positively or negatively modulated by the deacetylase (32, 33); similarly, a myc-tagged form of human CREB1 and Sirt-1 could be easily coimmunopre- cipated in naive PC12 cells (Fig. 4 A ), indicating physical association between the two proteins; the association was rapidly induced by the PKA agonist Fsk, in parallel with phosphorylation of CREB on Serine 133. Short-time (30 min) stimulation with Fsk did not affect the total amount of cellular Sirt-1, but rather increased the stoichiometry of the binding to CREB, suggesting either an increased af fi nity between the two proteins, or the enhanced interaction with intermediate molecular partners co-recruited at target gene promoters. In keeping with the latter possibility, a mutant CREB that retains the phosphorylation site but is unable to bind DNA because of the deletion of the C-terminal transactivation domain ( Δ LZ CREB), displayed marginal physical interaction with Sirt-1, nor was the binding induced by Fsk (Fig. 4 A ). To con fi rm that SIRT1 and CREB colocalize on CREB- responsive chromatin regions in cortical neurons, cross-linked chromatin was immunoprecipitated with an anti-Sirt-1 antiserum and Sirt-1 binding to the regions of nNOS and PGC-1 α promoters surrounding the CRE elements determined by semiquantitative PCR. Sirt-1 was found to interact with both genes, in a fashion inducible by NGF and with a binding kinetic similar to that of CREB (Fig. 4 Ba ). Sirt-1 also bound the same Sirt-1 chromatin region surrounding the +1,998/+2,012 half-CRE sites that copre- cipitates with CREB, suggesting that Sirt-1 may self-regulate its CRE-dependent transactivation (Fig. 4 Bb ). Of note, Sirt-1 interaction with nNOS and Sirt-1 chromatin was indeed mediated by CREB, as revealed by its drastic reduction in CREB loxP/loxP neurons following the recombinase-mediated deletion of the factor (Fig. 4 Bb ) and by the failure of forced Sirt-1 overexpression to restore Sirt-1 chromatin binding in CREB-de fi cient cells (Fig. S3). To determine whether Sirt-1 modulates CREB transcriptional activity, cortical neurons were stimulated with NGF in the presence of sirtuin inhibitor Nicotinamide (34); induction of nNOS and PGC-1 α mRNA by NGF was blunted upon inhibition of Sirt-1 deacetylase activity (Fig. 4 C ). A similar result was obtained in naive PC12 cells by siRNA-mediated knock-down of Sirt-1 (Fig. S4 A ). In addition, Sirt-1 – de fi cient PC12 displayed impaired differentiation by NGF (as assessed by quanti fi cation of neurite outgrowth), a response largely dependent on CREB and nNOS in this cell model (35, 36) (Fig. S4 B ). In a complementary set of experiments, lentivirus-mediated overexpression of Sirt-1 increased the expression of nNOS and PGC-1 α mRNA in hippocampal neurons treated with NGF. However, Sirt-1 did not fully restore the expression of these genes in CREB-de fi cient cells, nor NGF protection from hydrogen peroxide-dependent cell death, which is largely lost in these cells (Fig. S5), was recovered upon transduction of the Sirt-1 cDNA. Collectively, the above data suggest that Sirt-1 and CREB are, at least to some extent, re- ciprocally dependent, and act in concert in the context of neurotrophin signaling. Unlike previously reported fi ndings (16), manipulations aimed at modulating Sirt-1 expression and activity in the above contexts did not affect the level of immunoreactive CREB1 (Fig. S4 Aa ). KO Mice. Finally, to verify whether Sirt-1 actually regulates CREB- dependent transcription in vivo and this is relevant for response to CR, we analyzed the expression of CREB targets nNOS and PGC- 1 α mRNAs in whole brains of Sirt-1 KO mice fed AL or CR for 25 – 28 wk (37). This analysis revealed that both genes are markedly hypoexpressed in Sirt-de fi cient mice compared with littermate controls under both dietary regimens (Fig. 4 D ), and their induction by CR, that was more pronounced for PGC-1 α , was also attenuated (Fig. 4 D , a and b ). The amount of immunoreactive CREB was comparable in brain homogenates of the two strains (Fig. 4 Dc ), nor CREB acetylation on lysine residues was affected by either Sirt-1 deletion or CR (Fig. S6), indicating that, at least in these experimental settings, Sirt-1 regulates CREB transcriptional activity independent of the acetylation and expression level of the factor. Taken together, the above fi ndings strongly suggest that Sirt-1 modulates the expression of CREB-dependent genes in mouse brain, and by extension, identify in the decrease of CREB- dependent transcription a molecular signature for a defective brain response to CR, shared by CREB and Sirt-1 mutant mice (7). The CREB transcription factor has been widely investigated as a metabolic sensor and regulator of glucose homeostasis in liver and fat tissue (15), and as a master switch of calcium and neuro- trophin-triggered transcriptional programs regulating neuronal differentiation, survival, and plasticity in the brain and peripheral nervous system (10, 11, 18). Evidence also exist for CREB roles in the control of appetite and food intake in the hypothalamus (38), but whether neuronal plasticity and high-order cognitive functions ...

Context 7

... targets peroxisome proliferator-activated receptor- γ coactivator-1 α ( PGC- 1 α ), nNOS , and phosphoenolpyruvate carboxykinase ( PEPCK ) were induced by CR in the cortex and hippocampus of CREB- pro fi cient mice (Fig. 2 B ). Of note, both PGC-1 α and NO, the product of NOS enzymes, are known to promote mitochondrial biogenesis and to participate in organismal response to CR (26). The three mRNAs were overall down-regulated in BCKO mice fed AL compared with controls, and their induction by CR, observed in control mice, was nearly abolished (Fig. 2 B ). Interestingly, the expression of other putative CREB targets like bcl-2 , NGF , and c-fos were unaffected by CR and CREB deletion (Fig. S2). Thus, collectively, these fi ndings demonstrate that nutrient availability selectively regulates CREB-dependent gene expression in the forebrain. We next asked how CR may affect CREB activity. Because Sirt- 1 is a metabolic sensor involved in several biological consequences of nutrient deprivation (27, 6), and animals lacking Sirt-1 in the brain show defective behavioral and hormonal responses to CR (7), we investigated the sirtuin as a potential CREB interactor. Sirt-1 mRNA was drastically reduced both in the cortex and hippocampus of BCKO mice, irrespective of the dietary regimen. Moreover, the moderate induction by CR that we observed in control brains was completely lost in the corresponding CREB- de fi cient tissues (Fig. 2 C ). Finally, acetylation of Histones H3 (AcK9) and H4 (AcK16), an inverse correlate of Sirt-1 activity (28), was abnormally high in BCKO hippocampal homogenates (Fig. 2 D ), con fi rming an overall reduction of Sirt-1 activity in this area of the brain. Transcriptional Regulation of Sirt-1 by CREB in Neurons. These observations suggested that Sirt-1 may represent a direct transcriptional target of CREB. Accordingly, CREB activators NGF and Forskolin (Fsk) (29) raised the level of immunoreactive Sirt-1 and of the corresponding mRNA in cultured primary cortical and hippocampal neurons (Fig. 3 A ). Bioinformatic analysis of the mouse Sirt-1 locus (NC_000076) revealed the presence of several putative cAMP Responsive Elements (CRE) both upstream and downstream of the transcription start site (TSS) (30), and an ∼ 300- bp segment encompassing two of those elements (TGACG at +1,998 and CGTCA at +2,012 from the annotated TSS) drove transcriptional response to Fsk and PKA in a standard luciferase reporter assay performed in PC12 pheochromocytoma cells (Fig. 3 B , a and b ). Moreover, ChIP from hippocampal neurons revealed that CREB binds the same genomic region in a fashion inducible by NGF (Fig. 3 Bc ). Finally, in primary neurons isolated from CREB loxP/loxP mice, deletion of CREB by adenoviral delivery of the Cre recombinase (Fig. 3 C , a and b ) led to a reduction of Sirt-1 immunoreactivity (Fig. 3 Cc ), and inhibited Sirt-1 mRNA induction by NGF and Fsk (Fig. 3 Cb ). Thus, we conclude that CREB directly regulates Sirt-1 mRNA and protein expression in neurons. Known transcriptional regulators of Sirt-1, such as p53 and Foxo3a (31), also interact with, and are either positively or negatively modulated by the deacetylase (32, 33); similarly, a myc-tagged form of human CREB1 and Sirt-1 could be easily coimmunopre- cipated in naive PC12 cells (Fig. 4 A ), indicating physical association between the two proteins; the association was rapidly induced by the PKA agonist Fsk, in parallel with phosphorylation of CREB on Serine 133. Short-time (30 min) stimulation with Fsk did not affect the total amount of cellular Sirt-1, but rather increased the stoichiometry of the binding to CREB, suggesting either an increased af fi nity between the two proteins, or the enhanced interaction with intermediate molecular partners co-recruited at target gene promoters. In keeping with the latter possibility, a mutant CREB that retains the phosphorylation site but is unable to bind DNA because of the deletion of the C-terminal transactivation domain ( Δ LZ CREB), displayed marginal physical interaction with Sirt-1, nor was the binding induced by Fsk (Fig. 4 A ). To con fi rm that SIRT1 and CREB colocalize on CREB- responsive chromatin regions in cortical neurons, cross-linked chromatin was immunoprecipitated with an anti-Sirt-1 antiserum and Sirt-1 binding to the regions of nNOS and PGC-1 α promoters surrounding the CRE elements determined by semiquantitative PCR. Sirt-1 was found to interact with both genes, in a fashion inducible by NGF and with a binding kinetic similar to that of CREB (Fig. 4 Ba ). Sirt-1 also bound the same Sirt-1 chromatin region surrounding the +1,998/+2,012 half-CRE sites that copre- cipitates with CREB, suggesting that Sirt-1 may self-regulate its CRE-dependent transactivation (Fig. 4 Bb ). Of note, Sirt-1 interaction with nNOS and Sirt-1 chromatin was indeed mediated by CREB, as revealed by its drastic reduction in CREB loxP/loxP neurons following the recombinase-mediated deletion of the factor (Fig. 4 Bb ) and by the failure of forced Sirt-1 overexpression to restore Sirt-1 chromatin binding in CREB-de fi cient cells (Fig. S3). To determine whether Sirt-1 modulates CREB transcriptional activity, cortical neurons were stimulated with NGF in the presence of sirtuin inhibitor Nicotinamide (34); induction of nNOS and PGC-1 α mRNA by NGF was blunted upon inhibition of Sirt-1 deacetylase activity (Fig. 4 C ). A similar result was obtained in naive PC12 cells by siRNA-mediated knock-down of Sirt-1 (Fig. S4 A ). In addition, Sirt-1 – de fi cient PC12 displayed impaired differentiation by NGF (as assessed by quanti fi cation of neurite outgrowth), a response largely dependent on CREB and nNOS in this cell model (35, 36) (Fig. S4 B ). In a complementary set of experiments, lentivirus-mediated overexpression of Sirt-1 increased the expression of nNOS and PGC-1 α mRNA in hippocampal neurons treated with NGF. However, Sirt-1 did not fully restore the expression of these genes in CREB-de fi cient cells, nor NGF protection from hydrogen peroxide-dependent cell death, which is largely lost in these cells (Fig. S5), was recovered upon transduction of the Sirt-1 cDNA. Collectively, the above data suggest that Sirt-1 and CREB are, at least to some extent, re- ciprocally dependent, and act in concert in the context of neurotrophin signaling. Unlike previously reported fi ndings (16), manipulations aimed at modulating Sirt-1 expression and activity in the above contexts did not affect the level of immunoreactive CREB1 (Fig. S4 Aa ). KO Mice. Finally, to verify whether Sirt-1 actually regulates CREB- dependent transcription in vivo and this is relevant for response to CR, we analyzed the expression of CREB targets nNOS and PGC- 1 α mRNAs in whole brains of Sirt-1 KO mice fed AL or CR for 25 – 28 wk (37). This analysis revealed that both genes are markedly hypoexpressed in Sirt-de fi cient mice compared with littermate controls under both dietary regimens (Fig. 4 D ), and their induction by CR, that was more pronounced for PGC-1 α , was also attenuated (Fig. 4 D , a and b ). The amount of immunoreactive CREB was comparable in brain homogenates of the two strains (Fig. 4 Dc ), nor CREB acetylation on lysine residues was affected by either Sirt-1 deletion or CR (Fig. S6), indicating that, at least in these experimental settings, Sirt-1 regulates CREB transcriptional activity independent of the acetylation and expression level of the factor. Taken together, the above fi ndings strongly suggest that Sirt-1 modulates the expression of CREB-dependent genes in mouse brain, and by extension, identify in the decrease of CREB- dependent transcription a molecular signature for a defective brain response to CR, shared by CREB and Sirt-1 mutant mice (7). The CREB transcription factor has been widely investigated as a metabolic sensor and regulator of glucose homeostasis in liver and fat tissue (15), and as a master switch of calcium and neuro- trophin-triggered transcriptional programs regulating neuronal differentiation, survival, and plasticity in the brain and peripheral nervous system (10, 11, 18). Evidence also exist for CREB roles in the control of appetite and food intake in the hypothalamus (38), but whether neuronal plasticity and high-order cognitive functions may be in fl uenced by nutrient cues and energy metabolism through CREB, an issue relevant to major diseases like AD and type 2 diabetes (3, 4), remains to be established. Our demonstra- tion of impaired electrophysiological, cognitive, and emotional response to CR in brain CREB KO mice, clearly suggests that this may indeed be the case. Interestingly, these differences emerge in the context of an overall comparable feeding behavior and metabolic response to CR between control and BCKO mice (Fig. 1 Ab ), indicating that potential effects of hypothalamic CREB signaling on energy balance and appetite regulation unlikely account for the observed phenotypes (38). Data reported in Fig. 2, showing CREB phosphorylation and transcriptional activation by CR, indicate that CREB is metaboli- cally regulated in the cortex and hippocampus, although the mechanism of such regulation needs to be further investigated. Because plasma from CR rodents has been previously shown to induce Sirt-1 expression in several organs/tissues, a humoral/hormonal mechanism for CREB activation could be envisaged (39); as an alternative, NO (40) or oxygen species (41) may mediate, cell-autonomously, this effect. Further research in this direction is warranted. Our data identify in the complex interplay with the nutrient- sensitive histone deacetylase Sirt-1 a molecular connection between CREB and neuronal response to CR. Previous work from others has compellingly involved neuronal Sirt-1 in hormonal and metabolic adaptation to dietary restriction in mice (7). Results presented here clearly suggest that the two molecules are part of the same CR-sensitive signaling cascade. However, other genes ...

Context 8

... overall down-regulated in BCKO mice fed AL compared with controls, and their induction by CR, observed in control mice, was nearly abolished (Fig. 2 B ). Interestingly, the expression of other putative CREB targets like bcl-2 , NGF , and c-fos were unaffected by CR and CREB deletion (Fig. S2). Thus, collectively, these fi ndings demonstrate that nutrient availability selectively regulates CREB-dependent gene expression in the forebrain. We next asked how CR may affect CREB activity. Because Sirt- 1 is a metabolic sensor involved in several biological consequences of nutrient deprivation (27, 6), and animals lacking Sirt-1 in the brain show defective behavioral and hormonal responses to CR (7), we investigated the sirtuin as a potential CREB interactor. Sirt-1 mRNA was drastically reduced both in the cortex and hippocampus of BCKO mice, irrespective of the dietary regimen. Moreover, the moderate induction by CR that we observed in control brains was completely lost in the corresponding CREB- de fi cient tissues (Fig. 2 C ). Finally, acetylation of Histones H3 (AcK9) and H4 (AcK16), an inverse correlate of Sirt-1 activity (28), was abnormally high in BCKO hippocampal homogenates (Fig. 2 D ), con fi rming an overall reduction of Sirt-1 activity in this area of the brain. Transcriptional Regulation of Sirt-1 by CREB in Neurons. These observations suggested that Sirt-1 may represent a direct transcriptional target of CREB. Accordingly, CREB activators NGF and Forskolin (Fsk) (29) raised the level of immunoreactive Sirt-1 and of the corresponding mRNA in cultured primary cortical and hippocampal neurons (Fig. 3 A ). Bioinformatic analysis of the mouse Sirt-1 locus (NC_000076) revealed the presence of several putative cAMP Responsive Elements (CRE) both upstream and downstream of the transcription start site (TSS) (30), and an ∼ 300- bp segment encompassing two of those elements (TGACG at +1,998 and CGTCA at +2,012 from the annotated TSS) drove transcriptional response to Fsk and PKA in a standard luciferase reporter assay performed in PC12 pheochromocytoma cells (Fig. 3 B , a and b ). Moreover, ChIP from hippocampal neurons revealed that CREB binds the same genomic region in a fashion inducible by NGF (Fig. 3 Bc ). Finally, in primary neurons isolated from CREB loxP/loxP mice, deletion of CREB by adenoviral delivery of the Cre recombinase (Fig. 3 C , a and b ) led to a reduction of Sirt-1 immunoreactivity (Fig. 3 Cc ), and inhibited Sirt-1 mRNA induction by NGF and Fsk (Fig. 3 Cb ). Thus, we conclude that CREB directly regulates Sirt-1 mRNA and protein expression in neurons. Known transcriptional regulators of Sirt-1, such as p53 and Foxo3a (31), also interact with, and are either positively or negatively modulated by the deacetylase (32, 33); similarly, a myc-tagged form of human CREB1 and Sirt-1 could be easily coimmunopre- cipated in naive PC12 cells (Fig. 4 A ), indicating physical association between the two proteins; the association was rapidly induced by the PKA agonist Fsk, in parallel with phosphorylation of CREB on Serine 133. Short-time (30 min) stimulation with Fsk did not affect the total amount of cellular Sirt-1, but rather increased the stoichiometry of the binding to CREB, suggesting either an increased af fi nity between the two proteins, or the enhanced interaction with intermediate molecular partners co-recruited at target gene promoters. In keeping with the latter possibility, a mutant CREB that retains the phosphorylation site but is unable to bind DNA because of the deletion of the C-terminal transactivation domain ( Δ LZ CREB), displayed marginal physical interaction with Sirt-1, nor was the binding induced by Fsk (Fig. 4 A ). To con fi rm that SIRT1 and CREB colocalize on CREB- responsive chromatin regions in cortical neurons, cross-linked chromatin was immunoprecipitated with an anti-Sirt-1 antiserum and Sirt-1 binding to the regions of nNOS and PGC-1 α promoters surrounding the CRE elements determined by semiquantitative PCR. Sirt-1 was found to interact with both genes, in a fashion inducible by NGF and with a binding kinetic similar to that of CREB (Fig. 4 Ba ). Sirt-1 also bound the same Sirt-1 chromatin region surrounding the +1,998/+2,012 half-CRE sites that copre- cipitates with CREB, suggesting that Sirt-1 may self-regulate its CRE-dependent transactivation (Fig. 4 Bb ). Of note, Sirt-1 interaction with nNOS and Sirt-1 chromatin was indeed mediated by CREB, as revealed by its drastic reduction in CREB loxP/loxP neurons following the recombinase-mediated deletion of the factor (Fig. 4 Bb ) and by the failure of forced Sirt-1 overexpression to restore Sirt-1 chromatin binding in CREB-de fi cient cells (Fig. S3). To determine whether Sirt-1 modulates CREB transcriptional activity, cortical neurons were stimulated with NGF in the presence of sirtuin inhibitor Nicotinamide (34); induction of nNOS and PGC-1 α mRNA by NGF was blunted upon inhibition of Sirt-1 deacetylase activity (Fig. 4 C ). A similar result was obtained in naive PC12 cells by siRNA-mediated knock-down of Sirt-1 (Fig. S4 A ). In addition, Sirt-1 – de fi cient PC12 displayed impaired differentiation by NGF (as assessed by quanti fi cation of neurite outgrowth), a response largely dependent on CREB and nNOS in this cell model (35, 36) (Fig. S4 B ). In a complementary set of experiments, lentivirus-mediated overexpression of Sirt-1 increased the expression of nNOS and PGC-1 α mRNA in hippocampal neurons treated with NGF. However, Sirt-1 did not fully restore the expression of these genes in CREB-de fi cient cells, nor NGF protection from hydrogen peroxide-dependent cell death, which is largely lost in these cells (Fig. S5), was recovered upon transduction of the Sirt-1 cDNA. Collectively, the above data suggest that Sirt-1 and CREB are, at least to some extent, re- ciprocally dependent, and act in concert in the context of neurotrophin signaling. Unlike previously reported fi ndings (16), manipulations aimed at modulating Sirt-1 expression and activity in the above contexts did not affect the level of immunoreactive CREB1 (Fig. S4 Aa ). KO Mice. Finally, to verify whether Sirt-1 actually regulates CREB- dependent transcription in vivo and this is relevant for response to CR, we analyzed the expression of CREB targets nNOS and PGC- 1 α mRNAs in whole brains of Sirt-1 KO mice fed AL or CR for 25 – 28 wk (37). This analysis revealed that both genes are markedly hypoexpressed in Sirt-de fi cient mice compared with littermate controls under both dietary regimens (Fig. 4 D ), and their induction by CR, that was more pronounced for PGC-1 α , was also attenuated (Fig. 4 D , a and b ). The amount of immunoreactive CREB was comparable in brain homogenates of the two strains (Fig. 4 Dc ), nor CREB acetylation on lysine residues was affected by either Sirt-1 deletion or CR (Fig. S6), indicating that, at least in these experimental settings, Sirt-1 regulates CREB transcriptional activity independent of the acetylation and expression level of the factor. Taken together, the above fi ndings strongly suggest that Sirt-1 modulates the expression of CREB-dependent genes in mouse brain, and by extension, identify in the decrease of CREB- dependent transcription a molecular signature for a defective brain response to CR, shared by CREB and Sirt-1 mutant mice (7). The CREB transcription factor has been widely investigated as a metabolic sensor and regulator of glucose homeostasis in liver and fat tissue (15), and as a master switch of calcium and neuro- trophin-triggered transcriptional programs regulating neuronal differentiation, survival, and plasticity in the brain and peripheral nervous system (10, 11, 18). Evidence also exist for CREB roles in the control of appetite and food intake in the hypothalamus (38), but whether neuronal plasticity and high-order cognitive functions may be in fl uenced by nutrient cues and energy metabolism through CREB, an issue relevant to major diseases like AD and type 2 diabetes (3, 4), remains to be established. Our demonstra- tion of impaired electrophysiological, cognitive, and emotional response to CR in brain CREB KO mice, clearly suggests that this may indeed be the case. Interestingly, these differences emerge in the context of an overall comparable feeding behavior and metabolic response to CR between control and BCKO mice (Fig. 1 Ab ), indicating that potential effects of hypothalamic CREB signaling on energy balance and appetite regulation unlikely account for the observed phenotypes (38). Data reported in Fig. 2, showing CREB phosphorylation and transcriptional activation by CR, indicate that CREB is metaboli- cally regulated in the cortex and hippocampus, although the mechanism of such regulation needs to be further investigated. Because plasma from CR rodents has been previously shown to induce Sirt-1 expression in several organs/tissues, a humoral/hormonal mechanism for CREB activation could be envisaged (39); as an alternative, NO (40) or oxygen species (41) may mediate, cell-autonomously, this effect. Further research in this direction is warranted. Our data identify in the complex interplay with the nutrient- sensitive histone deacetylase Sirt-1 a molecular connection between CREB and neuronal response to CR. Previous work from others has compellingly involved neuronal Sirt-1 in hormonal and metabolic adaptation to dietary restriction in mice (7). Results presented here clearly suggest that the two molecules are part of the same CR-sensitive signaling cascade. However, other genes potentially relevant to mitochondrial biogenesis and neuronal response to calorie restriction, namely nNOS and PGC-1 α (40), and some PGC-1 α targets — including CPT1 , CoxIV , and UCP-2 — are also critically down-regulated, together with Sirt-1, in the cortex and hippocampus of BCKO mice (Fig. 2 B and Fig. S2 b ). This fi nding, and evidence from Fig. S3 that overexpression of Sirt-1 does not rescue NGF ...

Context 9

... in cultured primary cortical and hippocampal neurons (Fig. 3 A ). Bioinformatic analysis of the mouse Sirt-1 locus (NC_000076) revealed the presence of several putative cAMP Responsive Elements (CRE) both upstream and downstream of the transcription start site (TSS) (30), and an ∼ 300- bp segment encompassing two of those elements (TGACG at +1,998 and CGTCA at +2,012 from the annotated TSS) drove transcriptional response to Fsk and PKA in a standard luciferase reporter assay performed in PC12 pheochromocytoma cells (Fig. 3 B , a and b ). Moreover, ChIP from hippocampal neurons revealed that CREB binds the same genomic region in a fashion inducible by NGF (Fig. 3 Bc ). Finally, in primary neurons isolated from CREB loxP/loxP mice, deletion of CREB by adenoviral delivery of the Cre recombinase (Fig. 3 C , a and b ) led to a reduction of Sirt-1 immunoreactivity (Fig. 3 Cc ), and inhibited Sirt-1 mRNA induction by NGF and Fsk (Fig. 3 Cb ). Thus, we conclude that CREB directly regulates Sirt-1 mRNA and protein expression in neurons. Known transcriptional regulators of Sirt-1, such as p53 and Foxo3a (31), also interact with, and are either positively or negatively modulated by the deacetylase (32, 33); similarly, a myc-tagged form of human CREB1 and Sirt-1 could be easily coimmunopre- cipated in naive PC12 cells (Fig. 4 A ), indicating physical association between the two proteins; the association was rapidly induced by the PKA agonist Fsk, in parallel with phosphorylation of CREB on Serine 133. Short-time (30 min) stimulation with Fsk did not affect the total amount of cellular Sirt-1, but rather increased the stoichiometry of the binding to CREB, suggesting either an increased af fi nity between the two proteins, or the enhanced interaction with intermediate molecular partners co-recruited at target gene promoters. In keeping with the latter possibility, a mutant CREB that retains the phosphorylation site but is unable to bind DNA because of the deletion of the C-terminal transactivation domain ( Δ LZ CREB), displayed marginal physical interaction with Sirt-1, nor was the binding induced by Fsk (Fig. 4 A ). To con fi rm that SIRT1 and CREB colocalize on CREB- responsive chromatin regions in cortical neurons, cross-linked chromatin was immunoprecipitated with an anti-Sirt-1 antiserum and Sirt-1 binding to the regions of nNOS and PGC-1 α promoters surrounding the CRE elements determined by semiquantitative PCR. Sirt-1 was found to interact with both genes, in a fashion inducible by NGF and with a binding kinetic similar to that of CREB (Fig. 4 Ba ). Sirt-1 also bound the same Sirt-1 chromatin region surrounding the +1,998/+2,012 half-CRE sites that copre- cipitates with CREB, suggesting that Sirt-1 may self-regulate its CRE-dependent transactivation (Fig. 4 Bb ). Of note, Sirt-1 interaction with nNOS and Sirt-1 chromatin was indeed mediated by CREB, as revealed by its drastic reduction in CREB loxP/loxP neurons following the recombinase-mediated deletion of the factor (Fig. 4 Bb ) and by the failure of forced Sirt-1 overexpression to restore Sirt-1 chromatin binding in CREB-de fi cient cells (Fig. S3). To determine whether Sirt-1 modulates CREB transcriptional activity, cortical neurons were stimulated with NGF in the presence of sirtuin inhibitor Nicotinamide (34); induction of nNOS and PGC-1 α mRNA by NGF was blunted upon inhibition of Sirt-1 deacetylase activity (Fig. 4 C ). A similar result was obtained in naive PC12 cells by siRNA-mediated knock-down of Sirt-1 (Fig. S4 A ). In addition, Sirt-1 – de fi cient PC12 displayed impaired differentiation by NGF (as assessed by quanti fi cation of neurite outgrowth), a response largely dependent on CREB and nNOS in this cell model (35, 36) (Fig. S4 B ). In a complementary set of experiments, lentivirus-mediated overexpression of Sirt-1 increased the expression of nNOS and PGC-1 α mRNA in hippocampal neurons treated with NGF. However, Sirt-1 did not fully restore the expression of these genes in CREB-de fi cient cells, nor NGF protection from hydrogen peroxide-dependent cell death, which is largely lost in these cells (Fig. S5), was recovered upon transduction of the Sirt-1 cDNA. Collectively, the above data suggest that Sirt-1 and CREB are, at least to some extent, re- ciprocally dependent, and act in concert in the context of neurotrophin signaling. Unlike previously reported fi ndings (16), manipulations aimed at modulating Sirt-1 expression and activity in the above contexts did not affect the level of immunoreactive CREB1 (Fig. S4 Aa ). KO Mice. Finally, to verify whether Sirt-1 actually regulates CREB- dependent transcription in vivo and this is relevant for response to CR, we analyzed the expression of CREB targets nNOS and PGC- 1 α mRNAs in whole brains of Sirt-1 KO mice fed AL or CR for 25 – 28 wk (37). This analysis revealed that both genes are markedly hypoexpressed in Sirt-de fi cient mice compared with littermate controls under both dietary regimens (Fig. 4 D ), and their induction by CR, that was more pronounced for PGC-1 α , was also attenuated (Fig. 4 D , a and b ). The amount of immunoreactive CREB was comparable in brain homogenates of the two strains (Fig. 4 Dc ), nor CREB acetylation on lysine residues was affected by either Sirt-1 deletion or CR (Fig. S6), indicating that, at least in these experimental settings, Sirt-1 regulates CREB transcriptional activity independent of the acetylation and expression level of the factor. Taken together, the above fi ndings strongly suggest that Sirt-1 modulates the expression of CREB-dependent genes in mouse brain, and by extension, identify in the decrease of CREB- dependent transcription a molecular signature for a defective brain response to CR, shared by CREB and Sirt-1 mutant mice (7). The CREB transcription factor has been widely investigated as a metabolic sensor and regulator of glucose homeostasis in liver and fat tissue (15), and as a master switch of calcium and neuro- trophin-triggered transcriptional programs regulating neuronal differentiation, survival, and plasticity in the brain and peripheral nervous system (10, 11, 18). Evidence also exist for CREB roles in the control of appetite and food intake in the hypothalamus (38), but whether neuronal plasticity and high-order cognitive functions may be in fl uenced by nutrient cues and energy metabolism through CREB, an issue relevant to major diseases like AD and type 2 diabetes (3, 4), remains to be established. Our demonstra- tion of impaired electrophysiological, cognitive, and emotional response to CR in brain CREB KO mice, clearly suggests that this may indeed be the case. Interestingly, these differences emerge in the context of an overall comparable feeding behavior and metabolic response to CR between control and BCKO mice (Fig. 1 Ab ), indicating that potential effects of hypothalamic CREB signaling on energy balance and appetite regulation unlikely account for the observed phenotypes (38). Data reported in Fig. 2, showing CREB phosphorylation and transcriptional activation by CR, indicate that CREB is metaboli- cally regulated in the cortex and hippocampus, although the mechanism of such regulation needs to be further investigated. Because plasma from CR rodents has been previously shown to induce Sirt-1 expression in several organs/tissues, a humoral/hormonal mechanism for CREB activation could be envisaged (39); as an alternative, NO (40) or oxygen species (41) may mediate, cell-autonomously, this effect. Further research in this direction is warranted. Our data identify in the complex interplay with the nutrient- sensitive histone deacetylase Sirt-1 a molecular connection between CREB and neuronal response to CR. Previous work from others has compellingly involved neuronal Sirt-1 in hormonal and metabolic adaptation to dietary restriction in mice (7). Results presented here clearly suggest that the two molecules are part of the same CR-sensitive signaling cascade. However, other genes potentially relevant to mitochondrial biogenesis and neuronal response to calorie restriction, namely nNOS and PGC-1 α (40), and some PGC-1 α targets — including CPT1 , CoxIV , and UCP-2 — are also critically down-regulated, together with Sirt-1, in the cortex and hippocampus of BCKO mice (Fig. 2 B and Fig. S2 b ). This fi nding, and evidence from Fig. S3 that overexpression of Sirt-1 does not rescue NGF signaling in CREB-de fi cient hippocampal neurons in vitro, suggests that up-regulation of Sirt-1 is not the only mechanism whereby CREB participates in neuronal response to nutrients. Instead, cooperation with CREB is likely critical for the action of neuronal Sirt-1 in CR. This view is supported by the unexpected fi nding that CREB-dependent genes involved in neuronal plasticity, survival, and stress resistance (42, 43), and induced by calorie restriction (Fig. 2), are markedly down-regulated in Sirt-1 – de fi cient cultured neurons (PC12) and in the brain of Sirt-1 KO mice. Because the latter strain is, like BCKO mice, impaired in brain response to reduced food intake (7), the above evidence further support the notion that CREB-dependent transcription has a pivotal role in the neuronal effects of calorie restriction, and identify in the CREB – Sirt-1 axis a major component of the nutrient sensitive molecular network that connects caloric intake and energy metabolism to brain health. Biochemical details of how Sirt-1 affects CREB activity remains to be clari fi ed. Unlike other transcription factors, CREB does not appear to be deacetylated by Sirt-1 (Fig. S6); moreover, we could not detect consistent effects of Sirt-1 on the expression of CREB (Fig. 4 Dc , and Figs. S4 Aa and S6), unlike that described by Gao et al. (16). Those authors, however, made their important observations on Sirt-1 Δ ex4 mice that, unlike Sirt-1KO mice used in our experiments, do express an inactive form of Sirt-1 potentially acting in a dominant negative fashion against ...

Context 10

... fi c CREB deletion. The increased LTP induced by CR in control mice was independent on changes in basal synaptic transmission, because input/output curves obtained plotting fi eld excitatory postsynaptic potential (fEPSP) amplitudes vs. stimulus intensity in hippocampal brain slices of CR and AL animals were superimposable. Because CREB appeared to participate in brain response to CR, we assessed whether diet affected the total amount/phosphoryla- tion of CREB protein, and the expression level of a number of mRNAs known to be regulated by CREB (23). CREB1 protein expression in control mice was not changed by the dietary regimen; instead, an increase in CREB phosphorylation on Serine 133 in the hippocampi of the CR group was revealed by phospho-speci fi c immunoblotting (Fig. 2 A ), suggesting that CR activates CREB in this brain area. Accordingly, mRNAs of “ canonical ” CREB targets peroxisome proliferator-activated receptor- γ coactivator-1 α ( PGC- 1 α ), nNOS , and phosphoenolpyruvate carboxykinase ( PEPCK ) were induced by CR in the cortex and hippocampus of CREB- pro fi cient mice (Fig. 2 B ). Of note, both PGC-1 α and NO, the product of NOS enzymes, are known to promote mitochondrial biogenesis and to participate in organismal response to CR (26). The three mRNAs were overall down-regulated in BCKO mice fed AL compared with controls, and their induction by CR, observed in control mice, was nearly abolished (Fig. 2 B ). Interestingly, the expression of other putative CREB targets like bcl-2 , NGF , and c-fos were unaffected by CR and CREB deletion (Fig. S2). Thus, collectively, these fi ndings demonstrate that nutrient availability selectively regulates CREB-dependent gene expression in the forebrain. We next asked how CR may affect CREB activity. Because Sirt- 1 is a metabolic sensor involved in several biological consequences of nutrient deprivation (27, 6), and animals lacking Sirt-1 in the brain show defective behavioral and hormonal responses to CR (7), we investigated the sirtuin as a potential CREB interactor. Sirt-1 mRNA was drastically reduced both in the cortex and hippocampus of BCKO mice, irrespective of the dietary regimen. Moreover, the moderate induction by CR that we observed in control brains was completely lost in the corresponding CREB- de fi cient tissues (Fig. 2 C ). Finally, acetylation of Histones H3 (AcK9) and H4 (AcK16), an inverse correlate of Sirt-1 activity (28), was abnormally high in BCKO hippocampal homogenates (Fig. 2 D ), con fi rming an overall reduction of Sirt-1 activity in this area of the brain. Transcriptional Regulation of Sirt-1 by CREB in Neurons. These observations suggested that Sirt-1 may represent a direct transcriptional target of CREB. Accordingly, CREB activators NGF and Forskolin (Fsk) (29) raised the level of immunoreactive Sirt-1 and of the corresponding mRNA in cultured primary cortical and hippocampal neurons (Fig. 3 A ). Bioinformatic analysis of the mouse Sirt-1 locus (NC_000076) revealed the presence of several putative cAMP Responsive Elements (CRE) both upstream and downstream of the transcription start site (TSS) (30), and an ∼ 300- bp segment encompassing two of those elements (TGACG at +1,998 and CGTCA at +2,012 from the annotated TSS) drove transcriptional response to Fsk and PKA in a standard luciferase reporter assay performed in PC12 pheochromocytoma cells (Fig. 3 B , a and b ). Moreover, ChIP from hippocampal neurons revealed that CREB binds the same genomic region in a fashion inducible by NGF (Fig. 3 Bc ). Finally, in primary neurons isolated from CREB loxP/loxP mice, deletion of CREB by adenoviral delivery of the Cre recombinase (Fig. 3 C , a and b ) led to a reduction of Sirt-1 immunoreactivity (Fig. 3 Cc ), and inhibited Sirt-1 mRNA induction by NGF and Fsk (Fig. 3 Cb ). Thus, we conclude that CREB directly regulates Sirt-1 mRNA and protein expression in neurons. Known transcriptional regulators of Sirt-1, such as p53 and Foxo3a (31), also interact with, and are either positively or negatively modulated by the deacetylase (32, 33); similarly, a myc-tagged form of human CREB1 and Sirt-1 could be easily coimmunopre- cipated in naive PC12 cells (Fig. 4 A ), indicating physical association between the two proteins; the association was rapidly induced by the PKA agonist Fsk, in parallel with phosphorylation of CREB on Serine 133. Short-time (30 min) stimulation with Fsk did not affect the total amount of cellular Sirt-1, but rather increased the stoichiometry of the binding to CREB, suggesting either an increased af fi nity between the two proteins, or the enhanced interaction with intermediate molecular partners co-recruited at target gene promoters. In keeping with the latter possibility, a mutant CREB that retains the phosphorylation site but is unable to bind DNA because of the deletion of the C-terminal transactivation domain ( Δ LZ CREB), displayed marginal physical interaction with Sirt-1, nor was the binding induced by Fsk (Fig. 4 A ). To con fi rm that SIRT1 and CREB colocalize on CREB- responsive chromatin regions in cortical neurons, cross-linked chromatin was immunoprecipitated with an anti-Sirt-1 antiserum and Sirt-1 binding to the regions of nNOS and PGC-1 α promoters surrounding the CRE elements determined by semiquantitative PCR. Sirt-1 was found to interact with both genes, in a fashion inducible by NGF and with a binding kinetic similar to that of CREB (Fig. 4 Ba ). Sirt-1 also bound the same Sirt-1 chromatin region surrounding the +1,998/+2,012 half-CRE sites that copre- cipitates with CREB, suggesting that Sirt-1 may self-regulate its CRE-dependent transactivation (Fig. 4 Bb ). Of note, Sirt-1 interaction with nNOS and Sirt-1 chromatin was indeed mediated by CREB, as revealed by its drastic reduction in CREB loxP/loxP neurons following the recombinase-mediated deletion of the factor (Fig. 4 Bb ) and by the failure of forced Sirt-1 overexpression to restore Sirt-1 chromatin binding in CREB-de fi cient cells (Fig. S3). To determine whether Sirt-1 modulates CREB transcriptional activity, cortical neurons were stimulated with NGF in the presence of sirtuin inhibitor Nicotinamide (34); induction of nNOS and PGC-1 α mRNA by NGF was blunted upon inhibition of Sirt-1 deacetylase activity (Fig. 4 C ). A similar result was obtained in naive PC12 cells by siRNA-mediated knock-down of Sirt-1 (Fig. S4 A ). In addition, Sirt-1 – de fi cient PC12 displayed impaired differentiation by NGF (as assessed by quanti fi cation of neurite outgrowth), a response largely dependent on CREB and nNOS in this cell model (35, 36) (Fig. S4 B ). In a complementary set of experiments, lentivirus-mediated overexpression of Sirt-1 increased the expression of nNOS and PGC-1 α mRNA in hippocampal neurons treated with NGF. However, Sirt-1 did not fully restore the expression of these genes in CREB-de fi cient cells, nor NGF protection from hydrogen peroxide-dependent cell death, which is largely lost in these cells (Fig. S5), was recovered upon transduction of the Sirt-1 cDNA. Collectively, the above data suggest that Sirt-1 and CREB are, at least to some extent, re- ciprocally dependent, and act in concert in the context of neurotrophin signaling. Unlike previously reported fi ndings (16), manipulations aimed at modulating Sirt-1 expression and activity in the above contexts did not affect the level of immunoreactive CREB1 (Fig. S4 Aa ). KO Mice. Finally, to verify whether Sirt-1 actually regulates CREB- dependent transcription in vivo and this is relevant for response to CR, we analyzed the expression of CREB targets nNOS and PGC- 1 α mRNAs in whole brains of Sirt-1 KO mice fed AL or CR for 25 – 28 wk (37). This analysis revealed that both genes are markedly hypoexpressed in Sirt-de fi cient mice compared with littermate controls under both dietary regimens (Fig. 4 D ), and their induction by CR, that was more pronounced for PGC-1 α , was also attenuated (Fig. 4 D , a and b ). The amount of immunoreactive CREB was comparable in brain homogenates of the two strains (Fig. 4 Dc ), nor CREB acetylation on lysine residues was affected by either Sirt-1 deletion or CR (Fig. S6), indicating that, at least in these experimental settings, Sirt-1 regulates CREB transcriptional activity independent of the acetylation and expression level of the factor. Taken together, the above fi ndings strongly suggest that Sirt-1 modulates the expression of CREB-dependent genes in mouse brain, and by extension, identify in the decrease of CREB- dependent transcription a molecular signature for a defective brain response to CR, shared by CREB and Sirt-1 mutant mice (7). The CREB transcription factor has been widely investigated as a metabolic sensor and regulator of glucose homeostasis in liver and fat tissue (15), and as a master switch of calcium and neuro- trophin-triggered transcriptional programs regulating neuronal differentiation, survival, and plasticity in the brain and peripheral nervous system (10, 11, 18). Evidence also exist for CREB roles in the control of appetite and food intake in the hypothalamus (38), but whether neuronal plasticity and high-order cognitive functions may be in fl uenced by nutrient cues and energy metabolism through CREB, an issue relevant to major diseases like AD and type 2 diabetes (3, 4), remains to be established. Our demonstra- tion of impaired electrophysiological, cognitive, and emotional response to CR in brain CREB KO mice, clearly suggests that this may indeed be the case. Interestingly, these differences emerge in the context of an overall comparable feeding behavior and metabolic response to CR between control and BCKO mice (Fig. 1 Ab ), indicating that potential effects of hypothalamic CREB signaling on energy balance and appetite regulation unlikely account for the observed phenotypes (38). Data reported in Fig. 2, showing CREB phosphorylation and transcriptional activation by CR, indicate that CREB is metaboli- cally ...