Nicotinamide Restores Cognition in Alzheimer's Disease Transgenic Mice via a Mechanism Involving Sirtuin Inhibition and Selective Reduction of Thr231-Phosphotau

Nicotinamide prevents memory impairments in a hippocampal-dependent task in the 3xTg-AD mice. 3xTg-AD mice were treated with nicotinamide or vehicle for 4 months in their drinking water. Mice were trained and tested on the spatial memory version of the Morris water maze (MWM; n = 8 per group). A, Acquisition curves shown for the 7 d of training on the MWM. Nicotinamide prevents spatial memory deficits during training in the 3xTg-AD mice. All mice were trained to criterion in the MWM task (as indicated by dashed line at 25 s escape latency). B–D, Mice were given a memory probe with the platform removed at 1.5 h or 24 h after the last training trial. B, 3xTg-AD mice treated with nicotinamide made significantly more platform crosses at both short- and long-term probes than vehicle-treated 3xTg-AD mice. NonTg mice treated with nicotinamide performed better at the 1.5 h probe than vehicle treated NonTg mice. C, 3xTg-AD mice treated with nicotinamide exhibited significantly decreased latencies to cross the platform location compared with vehicle-treated 3xTg-AD mice, at both the 1.5 and 24 h probes. NonTg mice treated with nicotinamide had decreased latencies to cross the platform location compared with vehicle treated nontransgenic mice at only the 1.5 h probe. D, No significant differences in the time spent in the opposite quadrant were seen with nicotinamide treatment compared with vehicle for either 3xTg-AD or NonTg mice. E, Nicotinamide prevents contextual fear memory deficits in a mainly amygdala-dependent task. Mice were tested for retention of memory for fear-associated environments 1.5 and 24 h after training. Mice were taken out after 180 s if they did not cross over. F, Nicotinamide treatment does not affect cortex-dependent novel object recognition. No significant differences were seen between 3xTg-AD mice treated with nicotinamide or vehicle on their ability to remember a prior object, either 1.5 or 24 h after habituation with the object. Error bars indicate SEM. (*p < 0.05) for control 3xTg-AD mice vs nicotinamide treated 3xTg-AD mice, (**p < 0.05) for control nonTg mice vs control 3xTg-AD mice, and (^#p < 0.05) for control nonTg mice vs nicotinamide treated nonTg mice.

Nicotinamide treatment does not affect Aβ load or production. Soluble (A) and insoluble (B) Aβ₄₀ and Aβ₄₂ levels were measured from 3xTg-AD whole-brain homogenates from animals treated for 4 months with nicotinamide or vehicle. No significant differences were seen between treatments. C–H, DAB staining with 6E10 shows Aβ-like immunoreactivity in 40 μm sections from nicotinamide- and vehicle-treated mice. Staining was apparent in the hippocampal region (C, F), amygdala (D, G), and cortex (E, H; original magnification, 5×), but no differences were seen with treatment. I, Western blot analyses of protein extracts from whole-brain homogenates of 3xTg-AD mice treated for 4 months with either nicotinamide (N; n = 8) or vehicle (C; n = 8) shown as alternating lanes. Steady-state levels of APP and APP CTF's C83 and C99 were unaffected by nicotinamide treatment. J, Quantification of I normalized to β-actin levels as a loading control. Error bars indicate SEM.

Tau pathology is decreased after nicotinamide treatment. Western blot analyses of protein extracts from whole-brain homogenates of 3xTg-AD mice treated for 4 months with either nicotinamide (N; n = 8) or vehicle (C; n = 8) shown as alternating lanes. Steady-state levels of total human tau (HT7) and tau phosphorylated at ser199/202 (AT8), thr231 (AT180), and thr181 (AT270) are shown. B, Quantification of A normalized to β-actin levels as a loading control. Error bars indicate SEM, and * indicates significance versus vehicle treatment (p < 0.05). Nicotinamide treatment dramatically reduces Thr213-phosphotau immunoreactivity. C–H, DAB staining with HT7 shows human tau immunoreactivity in 40 μm sections from nicotinamide- and vehicle-treated mice. Staining was apparent in the hippocampal region (C, F), amygdala (D, G) but not cortex (E, I; original magnification, 5×). I, J, Confocal microscopy images from 40 μm brain sections from nicotinamide- and vehicle-treated 3xTg-AD mice. CA1 pyramidal neurons shown (magnification 60×). Ubiquitin immunoreactivity shown in red for all panels. Total human tau (HT7) reactivity shown in green for I and human tau phosphorylated at thr231 (AT180) reactivity shown in green for J. Merge image shown for both total human tau and ubiquitin and for AT180-tau and ubiquitin.

Nicotinamide treatment increases p25 and reduces monoubiquitinated-tau. Western blot analyses of protein extracts from whole-brain homogenates of 3xTg-AD mice treated for 4 months with either nicotinamide (N; n = 8) or vehicle (C; n = 8) shown as alternating lanes. Steady-state levels of GSK3α/β, inactive GSK3β (phosphorylated at ser9), cdk5 and p35/p25 shown. B, Quantification of A normalized to β-actin levels as a loading control. Error bars indicate SEM, and * indicates significance versus vehicle treatment (p < 0.05). C, One hundred micrograms of brain homogenate immunoprecipitated with HT7 to isolate total human tau and then probed with anti-ubiquitin. Monoubiquitinated tau is identified by the molecular weight, which is 9 kDa heavier than human tau. Mouse heavy IgG chains also shown (55 kDa). No other ubiquitin-positive bands were seen. D, Overexpression of 5 μg of either wild-type human tau or Thr231 phospho-mimic human tau (T231E) in 3T3 cells with coexpression of myc-actin as a transcriptional and loading control (n = 10 per condition). Steady-state levels of the T231E phospho-mimic are significantly reduced, whereas myc-actin levels are consistent with wild-type tau myc-actin levels showing that transcriptional production of the proteins are not affected, but rather tau stability. E, Filter retardation assay of insoluble tau shows that wild-type human tau accumulates into high molecular weight aggregates, and that this is reduced with Thr231 phosphorylation mimic T231E tau. Memcode protein staining shown as a loading control. F, Quantification of D and E. Error bars indicate SEM, and * indicates significance versus wild-type tau (p < 0.05).

Nicotinamide treatment inhibits brain sirtuins and increase acetylated α-tubulin and MAP2c. Western blot analyses of protein extracts from whole-brain homogenates of 3xTg-AD mice treated for 4 months with either nicotinamide (N; n = 8) or vehicle (C; n = 8) shown as alternating lanes. A, Steady-state levels of acetylated α-tubulin showing an increase with nicotinamide treatment, and steady-state levels of a band corresponding to acetyl-α-tubulin dimer, which shows a large increase with nicotinamide treatment. Total steady-state levels of monomeric and dimeric α-tubulin are also shown, although no differences were evident between nicotinamide and vehicle treatment. B, Quantification of A. C, Steady-state levels of PSD-95, synaptophysin, MAP2a, MAP2b, and MAP2c shown. Increases were seen in MAP2c with nicotinamide treatment. D, Quantification of C normalized to actin as a loading control. Error bars indicate SEM, and * indicates significance versus vehicle treatment (p < 0.05).