Figure 1.
Effect of modulators of SIRT1 in neuronal dendritic branching.
Hippocampal neurons at 6 days in vitro (DIV) were treated for 24 h with 10 µM resveratrol (Res), 1 mM NAD+ or 1 mM nicotinamide (NC). Neurons were fixed and MAP-2 immunostained for dendrite counting at 7 DIV. (A) Representative images of MAP-2 immunostaining in control neurons (a), and neurons treated with Res (b), NAD+ (c), NC (d). Scale bar: 10 µm. (B, C) Quantification of total dendritic branch tip number (TDBTN) (B), and covertures area of neurons (C) in all conditions normalized to control neurons. (D) Sholl analysis control neurons (black circles), and neurons treated with RES (white circle), NAD+ (red triangle) or nicotinamide (blue triangle). Resveratrol and NAD+ treatment significantly increased the number of intersection at 23–45 µm from the some compared to control neurons (p<0.01 at 23–45 µm). Neurons treated with nicotinamide did not change the intersection number compared to control. Statistical analysis was performed by Kruskal-Wallis/Dunn. n = 50 neurons. Error bars indicate S.E.M. *p<0.05; **p<0.01.
Table 1.
Effect of resveratrol, NAD+ or nicotinamide in the number of primary and secondary dendrites.
Figure 2.
SIRT1 deacetylase activity is present in Hippocampal neurons.
Hippocampal neurons were transfected at 3 DIV with GFP, SIRT1/GFP or SIRT1H363Y/GFP. Representative images are shown of the immunodetection of SIRT1 in control neurons transfected only with GFP (A’), transfected with SIRT1/GFP (B’) or SIRT1H363Y/GFP (C’). SIRT1 deacetylase activity was detected with a specific antibody to detect acetylated histone H3 in control neurons (A’’, white arrow), SIRT1 positive neurons (B’’, acetylated histone H3: white arrow and desacetylated histone H3 :white circle with yellow arrow) and neurons transfected with SIRT1H363Y (C’’, acetylated histone H3: white arrow). GFP positive cells (A, B and C) and merged images are also shown for the three experimental conditions (A’’’, B’’’ and C’’’). Scale bar, 10 µm.
Table 2.
Effect of SIRT1 in primary and secondary dendrite number.
Figure 3.
SIRT1 deacetylase activity regulates dendritic arbor complexity in hippocampal neurons.
Neurons at 3 DIV were transfected with EGFP, SIRT1/EGFP or SIRT1H363Y/EGFP, then were treated at 4 DIV with Res (1, 10 or 50 µM), fixed at 6 DIV and immunostained against MAP-2 for dendrite analysis. (A) Representative images of control neurons transfected with EGFP (left column), SIRT1/EGFP (middle column) or SIRT1H363Y/EGFP (right column) and treated with different concentration of Res. Scale bar, 10 µm. (B, C) Quantification of TDBTN (B), and coverage area (C) in the presence or absence of different concentrations of Res (1, 10 or 50 µM). Ten fields were randomly selected photography. Values were normalized against control neuron transfected with EGFP. (D) Sholl analysis of neurons tansfected with GFP (black circles), GFP + Res 10 µM (white circle), SIRT1/GFP (red triangle) or SIRT1/GFP + Res 10µM (blue triangle). Both, Res treatment and SIRT1 transfection significantly increases the number of intersection at 25–80 µm from the soma (p<0.01 at 25–80 µm) and a higher increase in the number of intersection at 40–90 µm from the some (p<0.001 at 40–90 µm). (E) Sholl analysis of neurons transfected with the dominant negative of SIRT1. EGFP (black circles), SIRT1H363Y/EGFP (white circle), SIRT1H363Y/EGFP + Res (red triangle). SIRT1H363Y/EGFP transfection significantly decreased the number of intersection at 28–45 µm from the soma compared to control EGFP trasfected neurons (p<0,01 at 28–45 µm), and it prevented the effect of Res. Experiments were made triplicate and p values were determined by Kruskal-Wallis/Dunn. Error bars indicate S.E.M. n = 50–60 neurons. *p<0.01; **p<0.001.
Figure 4.
ROCK activity is involved in the regulatory effect of RES on dendrite development.
Hippocampal neurons were treated at 3 DIV with 5 µM TAT-TI-JIP, 1µM SP600125 or 100 µM Y-27632 in the presence or absence of 50 µM RES. Neurons were fixed at 6 DIV and immunostained against MAP-2. (A) Representative images of MAP-2 immunostaining in all experimental conditions. Scale bar, 10 µm. (B) Quantification of TDBTN and coverage area of neurons in all conditions shown in (A). Values were normalized against control neurons. (C) Sholl analysis of control neurons (black circles), and neurons treated with TAT-TI-JIP (cian circle), TAT-TI-JIP + RES (red triangle), SP600125 (blue triangle), SP600125+RES (green square), RES treatment reverts the JIP and SP600125 effect significantly increasing the number of intersection at 26–50 µm from the soma compared to non treated neurons (p<0,01 at 26–50 µm). Error bars indicate S.E.M. n = 50–60 neurons. (D) Sholl analysis of control neurons (black circles), and neurons treated with Y−27632 (red circle), Y−27632+ RES (blue triangle). Treatment with Y−27632 in the presence or absence of RES significantly increases the number of intersection at 24–70 µm from the some compared with non treated neurons (p<0,01 at 24–70 µm). (E) Quantification of TDBTN in neurons treated with different doses of Y−27632 in the presence or absence of Res. (F) Representative immunoblot against total Myosin Phosphatase Target 1 (MYPT1) and MYPT 1 phosphorylated at T853 (pMYPT), a specific substrate of ROCK kinase. Densitometric values of pMYPT1 bands were normalized against total MYPT1, and then each condition expressed as relative to the control condition without treatment. Experiments were carried out in triplicate. Students t-test, *p<0.01.
Figure 5.
SIRT1 regulates the maintenance of the dendritic tree.
Hippocampal neurons at 10 DIV were transfected with GFP, SIRT1/EGFP or SIRT1H363Y/EGFP. Neurons were fixed at 14 DIV and immunostained against Tau protein. (A) Representative images of Tau immunodetection in trasfected GFP positive neurons. Scale bar, 10 µm. (B) Quantification of TDBTN and coverage area in transfected neurons. (C) Sholl analysis of neurons tansfected with GFP (black circles), SIRT1/GFP (white circle), or SIRT1H363Y/GFP (black triangle). The number of intersections does not change in neurons transfected with SIRT1 compared to control GFP neurons. SIRT1H363Y/GFP transfection significantly decrease the number of intersection at 18–45 µm compared to control neurons (p<0.01 at 18–45 µm). Error bars indicate S.E.M. Experiments were made in triplicate n = 50 neurons. p values were determined by Kruskal-Wallis/Dunn. *p<0.05.
Figure 6.
The effect of SIRT1 expression on dendritic spine density.
(A) Hippocampal Neurons were transfected at 10 DIV and analyzed at 21 DIV. Representative images of dendritic spines in neurons expressing GFP, SIRT1/GFP or SIRT1H363Y/GFP. (B) Dendritic spine density (per 10 µm) in transfected neurons at 21 DIV. (C) Density of the different kinds of dendritic spines (per 10 µm) in transfected neurons. p values were determined by Kruskal-Wallis/Dunn. Error bars indicate S.E.M. n = 40 neurons. *p<0.01; **p<0.001.
Figure 7.
NAD+ and RES protect neurons from toxicity produced by Aβ aggregates.
Hippocampal neurons at 21 DIV were treated with 5 and 10 µM Aβ oligomers for 12 h in the presence or absence of 50 µM RES or 2 mM NAD+. (A) Representative images of control and treated neurons inmunostained against Tau (red) and Hoeschst (blue). Scale bar 10 µM. (B) Percentage of apoptotic nuclei in neurons in the different experimental conditions. (C) MTT assay of hippocampal neurons treated with different concentration of Aβ fibrils with or without RES or NAD+ and *p<0.01.
Figure 8.
SIRT1 preserves dendritic integrity in hippocampal neurons challenged with Aβ fibrils.
Hippocampal neurons at 10 DIV were transfected with EGFP, SIRT1/EGFP or SIRT1H363Y/GFP and then treated at 14 DIV with 5 µM Aβ for 12 h and then fixed and immunostained against Tau to analyze dendrites. (A) Representative images of EGFP transfected neurons with or without treatment with Aβ 5 µM, and neurons transfected with SIRT1/GFP or SIRT1H363Y/GFP challenged with Aβ. Scale bar, 10 µm. (B,C) Normalized TDBTN (B) and coverage area (C) of experimental conditions shown in (A). (D) Sholl analysis of transfected neurons: GFP (black circles), GFP + Aβ 5 µM (with circle), SIRT1/GFP + Aβ 5 µM (red triangle), SIRT1H363Y/GFP + Aβ 5 µM (blue triangle). Treatment with Aβ fibrils significantly decrease the number of intersection at 28–110 µm from the soma compared with GFP transfected neurons (p<0,01 at 28–100 µm). (E) Percentage apoptotic nuclei under the different condition assayed. p values were determined by Kruskal-Wallis/Dunn (*p<0.01). Error bars indicate S.E.M. n = 40 transfected neurons.