Cyclooxygenase-1 inhibition reduces amyloid pathology and improves memory deficits in a mouse model of Alzheimer's disease

Abstract

Several epidemiological and preclinical studies suggest that non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit cyclooxygenase (COX), reduce the risk of Alzheimer's disease (AD) and can lower β-amyloid (Aβ) production and inhibit neuroinflammation. However, follow-up clinical trials, mostly using selective cyclooxygenase (COX)-2 inhibitors, failed to show any beneficial effect in AD patients with mild to severe cognitive deficits. Recent data indicated that COX-1, classically viewed as the homeostatic isoform, is localized in microglia and is actively involved in brain injury induced by pro-inflammatory stimuli including Aβ, lipopolysaccharide, and interleukins. We hypothesized that neuroinflammation is critical for disease progression and selective COX-1 inhibition, rather than COX-2 inhibition, can reduce neuroinflammation and AD pathology. Here, we show that treatment of 20-month-old triple transgenic AD (3 × Tg-AD) mice with the COX-1 selective inhibitor SC-560 improved spatial learning and memory, and reduced amyloid deposits and tau hyperphosphorylation. SC-560 also reduced glial activation and brain expression of inflammatory markers in 3 × Tg-AD mice, and switched the activated microglia phenotype promoting their phagocytic ability. The present findings are the first to demonstrate that selective COX-1 inhibition reduces neuroinflammation, neuropathology, and improves cognitive function in 3 × Tg-AD mice. Thus, selective COX-1 inhibition should be further investigated as a potential therapeutic approach for AD.

Fig. 1

SC-560 treatment modulates microglial activation state. Representative immunostaining of microglial cells in the hippocampal subiculum of Non-Tg and 3 × Tg-AD mice treated with vehicle or SC-560. Double-staining of CD11b (a), Iba-1 (b), FcγRIII/II (c), CD68 (e), Ym1 (h), and amyloid deposits (Congo red or 6E10) showing reactive microglial cells surrounding amyloid plaques. Scale bar: a, e, h, 50 μm; b, c, 100 μm. (d) Expression of mRNA for pro-inflammatory factors and alternative activation markers in the brains of 3 × Tg-AD mice measured by quantitative real-time PCR. (f) Representative western blot of Ym1 in the brain homogenates 3 × Tg-AD mice treated with vehicle or SC-560. β-actin was used as loading control. (g) Quantification of Ym1 expression. Data shown are the means ± SEM (n = 6 mice per group). *p < 0.05 versus vehicle-treated 3 × Tg-AD mice; **p < 0.01 versus vehicle-treated 3 × Tg AD mice.

Fig. 2

Effect of SC-560 treatment on astrocyte activation. (a) Representative immunostaining of astrocytes in the hippocampal subiculum of 3 × Tg-AD mice treated with vehicle (left) or SC-560 (right). Double-staining of glial fibrillary acidic protein (GFAP) and amyloid deposits (Congo red) showing reactive astrocyte surrounding amyloid plaques. Scale bar, 100 μm. (b) Representative western blot of GFAP in the brain homogenates of 3 × Tg-AD mice treated with vehicle or SC-560. β-actin was used as loading control. (c) Quantification of GFAP expression. Data shown are the means ± SEM (n = 6 mice per group).

Fig. 3

SC-560 treatment reduces amyloid deposits. (a, b) Representative images of Congo red (a) and thioflavin S staining (b) in hippocampal subiculum in 3 × Tg-AD mice treated with vehicle or SC-560. Scale bar, 100 μm. Boxed regions (left) indicate the areas magnified (right), respectively. (c) Quantification of Congophilic amyloid deposits in the subiculum. Data are means ± SEM (n = 6 per group). *p < 0.05 versus vehicle-treated 3 × Tg-AD mice. (d) Representative western blot of full-length APP (APP-FL) and APP C-terminal fragments (CTFs) in the brain homogenates of 3 × Tg-AD mice treated with vehicle or SC-560. (e) Quantification of APP-FL expres-sion. Data shown are the means ± SEM (n = 6 mice per group).

Fig. 4

SC-560 treatment reduces phosphorylated tau. (a) Representative images of AT8 staining (a) in hippocampal subiculum in 3 × Tg-AD mice treated with vehicleorSC-560. Scale bar, 100 μm. Boxed regions (left) indicate the areas magnified (right), respectively. (b) Quantification of AT8-positive cells in the subiculum. Data are means ± SEM (n = 6 per group). *p < 0.05 versus vehicle-treated 3 × Tg-AD mice. (c) Representative western blot of phosphorylated tau (AT8) and total tau (TAU-5) in the brain homogenates of 3 × Tg-AD mice treated with vehicle or SC-560. β-actin was used as loading control. (d) Quantification of AT8 and TAU-5 expression. Data shown are the means ± SEM (n = 6 mice per group). *p < 0.05 versus vehicle-treated 3 × Tg-AD mice.

Fig. 5

SC-560 treatment increases phosphorylated glycogen syn-thase kinase-3β (GSK-3β). (a) Representative western blot of GSK-3β phosphorylated at Ser 9, total GSK-3β, cyclin-dependent kinase 5 (CDK5), and protein phosphatase 2A (PP2A) in the brain homogenates of 3 × Tg-AD mice treated with vehicle or SC-560. (b) Quantification of phosphorylated GSK-3β (Ser 9) expression. Data shown are the means ± SEM (n = 6 mice per group). *p < 0.05 versus vehicle-treated 3 × Tg-AD mice.

Fig. 6

SC-560-treated aged 3 × Tg-AD mice show improved spatial learning and memory. (a) Schematic representation of experimental design. The arrowheads represent the injections for the SC-560 or vehicle. (b) Acquisition of spatial learning in the Morris Water Maze hidden-platform task. Escape latency represents time taken to escape to the hidden platform. (c and d) Probe trial without platform. Probe analysis was performed on day 5 (1.5 h after training day 5) and day 6 (24 h after training day 5). Latency is measured as the time to cross the exact platform location (c). Platform crossing is calculated as the number of crosses over the exact location of the hidden platform (d). Data are means ± SEM (n = 6 per group). *p < 0.05 versus vehicle-treated 3 × Tg-AD mice.