Journal: Journal of Biological Chemistry

Article Title: Neuronal SIRT1 Activation as a Novel Mechanism Underlying the Prevention of Alzheimer Disease Amyloid Neuropathology by Calorie Restriction

doi: 10.1074/jbc.m602909200

Figure Lengend Snippet: FIGURE 1. Calorie restriction increases SIRT1 expression and NAD levels in the brain of 10-month-old Tg2576 mice. A, in this study 4-month-old female Tg2576 mice were fed AL or caloric CR diets for 6 months. Neocortex tissue lysates (cingulated and parietal cortex) from AL- and CR-treated mice were separated by SDS-PAGE and probed with a rabbit polyclonal antibody against SIRT1; 3T3 cell-nuclear extract controlled for specificity of SIRT1 signal in the brain, whereas -actin immunoreactivity served as a loading control. B, assess- ment of NAD and NAM contents in the brain of CR- and AL-fed control Tg2576 mice. C, assessment of neocortical SIRT1 activity by Western blots using an antibody specific for acetylated histone 4 lysine 16 residue (H4-k16Ac). D, assessment of neocortical A1–40 and A1–42 peptide concentrations in CR- and AL-fed control Tg2576 mice. E, cellular and regional distribution of neocortical SIRT1 immunoreactivity in the contralateral neocortex used for SIRT1 Western blot analysis; panel a, green and red reflect SIRT1 and SMI-32 immunoreac- tivities respectively; panels b–c, green and red immunostaining reflect SIRT1 and NeuN immunoreactivities, respectively; panel d, yellow reflects overlapping SIRT1 and NeuN immunoreactivities in CR Tg2576 mouse neocortex. In panel a, the arrows point to SMI-32/SIRT1-positive neurons by double-labeled immunocyto- chemistry. In panels b–d, arrows point to the same labeled neurons. Bar graphs demonstrate group means S.E.; n 4 animals/group; *, p 0.05; **, p 0.01, ***, p 0.001; 2-tailed t test, AL-fed versus CR-treated group. OG, outer granular layer; IG, inner granular layer; OPL, outer pyramidal layer; IPL, inner pyramidal layer.

Article Snippet: The following antibodies were used in this study: polyclonal anti-SIRT1 antibody (1:3000), polyclonal anti-histone H4 (Ac16) (1:2000, Serotec, Raleigh, NC), polyclonal antiamyloid precursor protein carboxyl-terminal (751–770) antibody (anti-O443, 1:5000 dilution, Calbiochem), monoclonal 22C11 antibody (1:1000, Chemicon International, Temecula, CA), monoclonal 6E10 antibody (1:1000, Senetek, St. Louis, MO), rabbit polyclonal antibody against ROCK1 (1:5000; Chemicon International), and polyclonal -actin antibody (1:3000, Sigma).

Techniques: Expressing, SDS Page, Control, Activity Assay, Western Blot, Residue, Immunostaining, Labeling

Journal: Journal of Biological Chemistry

Article Title: Neuronal SIRT1 Activation as a Novel Mechanism Underlying the Prevention of Alzheimer Disease Amyloid Neuropathology by Calorie Restriction

doi: 10.1074/jbc.m602909200

Figure Lengend Snippet: FIGURE 4. ROCK1 is involved in SIRT1-mediated activation of -secretase activity. A, brain ROCK1 expression is decreased in Tg2576 mice in response to CR treatment. In this study 4-month-old female Tg2576 mice were fed AL or CR for 6 months. Neocortex tissue lysates from AL and CR mice were separated by SDS-PAGE and probed with a rabbit polyclonal antibody against ROCK1; -actin immunoreactivity served as a loading control. B and C, viral WT SIRT1 in CHO-APPswe cells or DN SIRT1 in Tg2576 neuron cultures results in reduced or increased levels of ROCK1, respectively, as determined by Western blot analysis as shown in A. D, CHO-APPswe cells were transfected with pCAG (empty) vector or CA ROCK1 cDNA in combination with Ad-lacZ or Ad-WT SIRT1 viral infection. The resulting conditioned medium and cell lysates 48 h post-transfection/infection were assessed for sAPP concentra- tion (expressed as % of total sAPP immunoreactivity) and full-length APP level (expressed as % of total actin immunoreactivity), respectively. Inset, Western blot analysis of SIRT1, ROCK1 expression, and sAPP concentration and full-length APP level. E, quantification of SIRT1 expression in the brain of 3-month-old human hSIRT1 transgenic mice. Inset, Western blot analysis of SIRT1, ROCK1 expression, and -actin. Neocortex tissue lysates from hSIRT1 transgenics and WT mice were separated by SDS-PAGE and probed with rabbit polyclonal antibodies against SIRT1 and ROCK1; -actin immunoreactivity served as a loading control. F, quantification of ROCK1 expression in the brain of 3-month-old hSIRT1 transgenic mice. G, fluori- metric assessment of -secretase activity in the brain of 3-month-old hSIRT1 transgenic mice. In A–G, results are expressed as a percentage of treatment in the control group; in A–D, values represent the means S.E. of determinations made in three separate culture preparations; n 3 per culture preparation. In E–G, values represent the means S.E. of determinations made in two individual hSIRT1 transgenic F1 lines. *, p 0.05 versus control group. SIRT1 Tg, (h) SIRT1 transgenic mice.

Article Snippet: The following antibodies were used in this study: polyclonal anti-SIRT1 antibody (1:3000), polyclonal anti-histone H4 (Ac16) (1:2000, Serotec, Raleigh, NC), polyclonal antiamyloid precursor protein carboxyl-terminal (751–770) antibody (anti-O443, 1:5000 dilution, Calbiochem), monoclonal 22C11 antibody (1:1000, Chemicon International, Temecula, CA), monoclonal 6E10 antibody (1:1000, Senetek, St. Louis, MO), rabbit polyclonal antibody against ROCK1 (1:5000; Chemicon International), and polyclonal -actin antibody (1:3000, Sigma).

Techniques: Activation Assay, Activity Assay, Expressing, SDS Page, Control, Western Blot, Transfection, Plasmid Preparation, Infection, Concentration Assay, Transgenic Assay

Journal: Journal of Biological Chemistry

Article Title: Neuronal SIRT1 Activation as a Novel Mechanism Underlying the Prevention of Alzheimer Disease Amyloid Neuropathology by Calorie Restriction

doi: 10.1074/jbc.m602909200

Figure Lengend Snippet: FIGURE 1. Calorie restriction increases SIRT1 expression and NAD levels in the brain of 10-month-old Tg2576 mice. A, in this study 4-month-old female Tg2576 mice were fed AL or caloric CR diets for 6 months. Neocortex tissue lysates (cingulated and parietal cortex) from AL- and CR-treated mice were separated by SDS-PAGE and probed with a rabbit polyclonal antibody against SIRT1; 3T3 cell-nuclear extract controlled for specificity of SIRT1 signal in the brain, whereas -actin immunoreactivity served as a loading control. B, assess- ment of NAD and NAM contents in the brain of CR- and AL-fed control Tg2576 mice. C, assessment of neocortical SIRT1 activity by Western blots using an antibody specific for acetylated histone 4 lysine 16 residue (H4-k16Ac). D, assessment of neocortical A1–40 and A1–42 peptide concentrations in CR- and AL-fed control Tg2576 mice. E, cellular and regional distribution of neocortical SIRT1 immunoreactivity in the contralateral neocortex used for SIRT1 Western blot analysis; panel a, green and red reflect SIRT1 and SMI-32 immunoreac- tivities respectively; panels b–c, green and red immunostaining reflect SIRT1 and NeuN immunoreactivities, respectively; panel d, yellow reflects overlapping SIRT1 and NeuN immunoreactivities in CR Tg2576 mouse neocortex. In panel a, the arrows point to SMI-32/SIRT1-positive neurons by double-labeled immunocyto- chemistry. In panels b–d, arrows point to the same labeled neurons. Bar graphs demonstrate group means S.E.; n 4 animals/group; *, p 0.05; **, p 0.01, ***, p 0.001; 2-tailed t test, AL-fed versus CR-treated group. OG, outer granular layer; IG, inner granular layer; OPL, outer pyramidal layer; IPL, inner pyramidal layer.

Article Snippet: The following antibodies were used in this study: polyclonal anti-SIRT1 antibody (1:3000), polyclonal anti-histone H4 (Ac16) (1:2000, Serotec, Raleigh, NC), polyclonal antiamyloid precursor protein carboxyl-terminal (751–770) antibody (anti-O443, 1:5000 dilution, Calbiochem), monoclonal 22C11 antibody (1:1000, Chemicon International, Temecula, CA), monoclonal 6E10 antibody (1:1000, Senetek, St. Louis, MO), rabbit polyclonal antibody against ROCK1 (1:5000; Chemicon International), and polyclonal -actin antibody (1:3000, Sigma).

Techniques: Expressing, SDS Page, Control, Activity Assay, Western Blot, Residue, Immunostaining, Labeling

Journal: Journal of Biological Chemistry

Article Title: Neuronal SIRT1 Activation as a Novel Mechanism Underlying the Prevention of Alzheimer Disease Amyloid Neuropathology by Calorie Restriction

doi: 10.1074/jbc.m602909200

Figure Lengend Snippet: FIGURE 2. SIRT1 expression causally promotes -secretase activity and attenuates A peptides generation in primary Tg2576 neuron cultures and CHO-APPswecells.A,Adenovirus-mediatedoverexpressionofWTSIRT1inprimaryneuronculturesorCHO-APPswecellleadstoreducedcontentsofA1–40and A1–42 released into the culture media. D, adenovirus-mediated overexpression of a dominant-negative DN SIRT1 construct in Tg2576 mouse neurons led to increased A1–40 and A1–42 contents in the conditioned medium. In A and D, A contents were assessed by enzyme-linked immunosorbent assay 48 h post-infection (10 m.o.i.). Insets, Western blot analysis of SIRT1 expression and SIRT1 activity using anti-SIRT1 antibody and H4-k16Ac antibody, respectively. B and E, fluorimetric assessment of -, -, and -secretase activities in CHO-APPswe cell in response to adenoviral-SIRT1 (B) or in Tg2576 neurons in response to adenoviral-DN-SIRT1 expression (E). C and F, assessment of changes in sAPP concentration and full-length APP (expressed as % of total sAPP and actin immunoreactivity, respectively) in the same CHO-APPswe cultures (C) or Tg2576 neurons (F) in response to adenoviral -SIRT1 or adenoviral DN SIRT1 expression. Results are expressed as % of control (adenoviral-LacZ) infection; values represent the means S.E. of determinations made in three separate culture preparations; n 3 per culture preparation; *, p 0.05; **, p 0.01; ***, p 0.001 versus control group.

Article Snippet: The following antibodies were used in this study: polyclonal anti-SIRT1 antibody (1:3000), polyclonal anti-histone H4 (Ac16) (1:2000, Serotec, Raleigh, NC), polyclonal antiamyloid precursor protein carboxyl-terminal (751–770) antibody (anti-O443, 1:5000 dilution, Calbiochem), monoclonal 22C11 antibody (1:1000, Chemicon International, Temecula, CA), monoclonal 6E10 antibody (1:1000, Senetek, St. Louis, MO), rabbit polyclonal antibody against ROCK1 (1:5000; Chemicon International), and polyclonal -actin antibody (1:3000, Sigma).

Techniques: Expressing, Activity Assay, Over Expression, Dominant Negative Mutation, Construct, Enzyme-linked Immunosorbent Assay, Infection, Western Blot, Concentration Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Neuronal SIRT1 Activation as a Novel Mechanism Underlying the Prevention of Alzheimer Disease Amyloid Neuropathology by Calorie Restriction

doi: 10.1074/jbc.m602909200

Figure Lengend Snippet: FIGURE 3. NAD treatment attenuates A generation in Tg2576 mouse cortico-hippocampal neuron cultures in a dose-dependent fashion. A, NAD treatments for 24 h resulted in dose-dependent inhibition of A concentration in Tg2576 mouse neurons. B, fluorimetric assessment of -, -, and -secretase activities. C, assessment of changes in sAPP concentration expressed as percent of total sAPP immunoreactivity and full-length APP concentrations relative to -actin immunoreactive signal in the same specimen as in A. In A–C, results are expressed as the percent of vehicle treatment in the control group; values represent the means S.E. of determinations made in three separate culture preparations; n 3 per culture, *, p 0.05; **, p 0.01 versus vehicle control group. D, role of SIRT1 in NAD-mediated attenuation of A generation. Tg2576 mouse neurons were infected with DN SIRT1 adenovirus and co-cultured with 1 mM NAD. A1– 40 and A1– 42 concentration in the conditioned medium were assessed by enzyme-linked immunosorbent assay 72 h post-infection (20 m.o.i.). In D, results are expressed as a percentage of vehicle treatment in the control group; values represent means S.E. of determinations made in three separate culture preparations; n 3 per culture. *, p 0.05 versus vehicle control group. **, p 0.05 versus NAD treatment-only group.

Article Snippet: The following antibodies were used in this study: polyclonal anti-SIRT1 antibody (1:3000), polyclonal anti-histone H4 (Ac16) (1:2000, Serotec, Raleigh, NC), polyclonal antiamyloid precursor protein carboxyl-terminal (751–770) antibody (anti-O443, 1:5000 dilution, Calbiochem), monoclonal 22C11 antibody (1:1000, Chemicon International, Temecula, CA), monoclonal 6E10 antibody (1:1000, Senetek, St. Louis, MO), rabbit polyclonal antibody against ROCK1 (1:5000; Chemicon International), and polyclonal -actin antibody (1:3000, Sigma).

Techniques: Inhibition, Concentration Assay, Control, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Neuronal SIRT1 Activation as a Novel Mechanism Underlying the Prevention of Alzheimer Disease Amyloid Neuropathology by Calorie Restriction

doi: 10.1074/jbc.m602909200

Figure Lengend Snippet: FIGURE 4. ROCK1 is involved in SIRT1-mediated activation of -secretase activity. A, brain ROCK1 expression is decreased in Tg2576 mice in response to CR treatment. In this study 4-month-old female Tg2576 mice were fed AL or CR for 6 months. Neocortex tissue lysates from AL and CR mice were separated by SDS-PAGE and probed with a rabbit polyclonal antibody against ROCK1; -actin immunoreactivity served as a loading control. B and C, viral WT SIRT1 in CHO-APPswe cells or DN SIRT1 in Tg2576 neuron cultures results in reduced or increased levels of ROCK1, respectively, as determined by Western blot analysis as shown in A. D, CHO-APPswe cells were transfected with pCAG (empty) vector or CA ROCK1 cDNA in combination with Ad-lacZ or Ad-WT SIRT1 viral infection. The resulting conditioned medium and cell lysates 48 h post-transfection/infection were assessed for sAPP concentra- tion (expressed as % of total sAPP immunoreactivity) and full-length APP level (expressed as % of total actin immunoreactivity), respectively. Inset, Western blot analysis of SIRT1, ROCK1 expression, and sAPP concentration and full-length APP level. E, quantification of SIRT1 expression in the brain of 3-month-old human hSIRT1 transgenic mice. Inset, Western blot analysis of SIRT1, ROCK1 expression, and -actin. Neocortex tissue lysates from hSIRT1 transgenics and WT mice were separated by SDS-PAGE and probed with rabbit polyclonal antibodies against SIRT1 and ROCK1; -actin immunoreactivity served as a loading control. F, quantification of ROCK1 expression in the brain of 3-month-old hSIRT1 transgenic mice. G, fluori- metric assessment of -secretase activity in the brain of 3-month-old hSIRT1 transgenic mice. In A–G, results are expressed as a percentage of treatment in the control group; in A–D, values represent the means S.E. of determinations made in three separate culture preparations; n 3 per culture preparation. In E–G, values represent the means S.E. of determinations made in two individual hSIRT1 transgenic F1 lines. *, p 0.05 versus control group. SIRT1 Tg, (h) SIRT1 transgenic mice.

Article Snippet: The following antibodies were used in this study: polyclonal anti-SIRT1 antibody (1:3000), polyclonal anti-histone H4 (Ac16) (1:2000, Serotec, Raleigh, NC), polyclonal antiamyloid precursor protein carboxyl-terminal (751–770) antibody (anti-O443, 1:5000 dilution, Calbiochem), monoclonal 22C11 antibody (1:1000, Chemicon International, Temecula, CA), monoclonal 6E10 antibody (1:1000, Senetek, St. Louis, MO), rabbit polyclonal antibody against ROCK1 (1:5000; Chemicon International), and polyclonal -actin antibody (1:3000, Sigma).

Techniques: Activation Assay, Activity Assay, Expressing, SDS Page, Control, Western Blot, Transfection, Plasmid Preparation, Infection, Concentration Assay, Transgenic Assay