Cyclooxygenase inhibition targets neurons to prevent early behavioural decline in Alzheimer's disease model mice

Abstract

Identifying preventive targets for Alzheimer's disease is a central challenge of modern medicine. Non-steroidal anti-inflammatory drugs, which inhibit the cyclooxygenase enzymes COX-1 and COX-2, reduce the risk of developing Alzheimer's disease in normal ageing populations. This preventive effect coincides with an extended preclinical phase that spans years to decades before onset of cognitive decline. In the brain, COX-2 is induced in neurons in response to excitatory synaptic activity and in glial cells in response to inflammation. To identify mechanisms underlying prevention of cognitive decline by anti-inflammatory drugs, we first identified an early object memory deficit in APPSwe-PS1ΔE9 mice that preceded previously identified spatial memory deficits in this model. We modelled prevention of this memory deficit with ibuprofen, and found that ibuprofen prevented memory impairment without producing any measurable changes in amyloid-β accumulation or glial inflammation. Instead, ibuprofen modulated hippocampal gene expression in pathways involved in neuronal plasticity and increased levels of norepinephrine and dopamine. The gene most highly downregulated by ibuprofen was neuronal tryptophan 2,3-dioxygenase (Tdo2), which encodes an enzyme that metabolizes tryptophan to kynurenine. TDO2 expression was increased by neuronal COX-2 activity, and overexpression of hippocampal TDO2 produced behavioural deficits. Moreover, pharmacological TDO2 inhibition prevented behavioural deficits in APPSwe-PS1ΔE9 mice. Taken together, these data demonstrate broad effects of cyclooxygenase inhibition on multiple neuronal pathways that counteract the neurotoxic effects of early accumulating amyloid-β oligomers.

Keywords: Alzheimer’s disease; cyclooxgenases; hippocampus; ibuprofen; kynurenine pathway.

Anti-inflammatory drugs like ibuprofen reduce Alzheimer’s disease risk when taken before cognitive decline begins, but the mechanism behind this protection has remained elusive. Woodling, Colas et al. report that ibuprofen changes neuronal gene expression and prevents memory decline in a mouse model of Alzheimer’s disease, identifying new targets for research.

Figure 1

Amyloid-β oligomers induce COX-2 expression in hippocampal neurons in a COX-2 dependent manner. Primary rat hippocampal neurons were stimulated with amyloid-β ₄₂ oligomers and/or SC-236 for 8 h and expression was assayed by quantitative PCR. ( A ) COX-2 mRNA is induced by amyloid-β ₄₂ oligomers in a dose dependent manner ( ^**P < 0.01 by Tukey’s multiple comparison; n = 8 independent samples per group). ( B ) Co-treatment with the COX-2 inhibitor SC-236 (100 nM) prevents amyloid-β ₄₂ oligomer (1 µM) induced increase in COX-2 expression ( ^****P < 0.0001 versus control vehicle, ^##P < 0.01 versus amyloid-β vehicle by Bonferroni multiple comparison; n = 8 independent samples per group). ( C ) SC-236 (100 nM) prevents amyloid-β ₄₂ oligomer (5 µM) induced increase in COX-2 expression ( ^****P < 0.0001 versus control vehicle, ^###P < 0.001 versus amyloid-β vehicle by Bonferroni multiple comparison; n = 8 independent samples per group).

Figure 2

Behavioural deficits precede increases in oxidative stress and amyloid-β plaque deposition in APP-PS1 mice and are prevented by ibuprofen. ( A ) γ-Ketoaldehydes (γ-KAs) are highly reactive products of lipid peroxidation that form permanent adducts on proteins; levels of lysine γ-KA-adducts reflect cumulative oxidative injury to proteins. β-lactam lysyl-adducts were quantified by LC-ESI/MS/MS and increase in posterior cortex samples after 4 months of age (red arrow; n = 9–14 mice/group; ANOVA effects of genotype and age, P < 0.01; ^***P < 0.001 by Bonferroni multiple comparison). ( B ) Quantification of hippocampal area covered by Congo Red indicates that amyloid plaque deposition begins after 4 months of age (red arrow; n = 5–14 mice per group; ANOVA P < 0.0001; ^***P < 0.001 by Bonferroni multiple comparison). Images show Congo Red staining in the dentate gyrus (DG, dotted line), where multiple plaques are first seen at 6 months of age. Scale bar = 100 μm. ( C ) APP-PS1 mice show object discrimination in the 24-h NOR task at 3 months, but are impaired at 4 months (red arrow; n = 7–13 mice/group; ^**P < 0.01; * P < 0.05 by paired t -test to identify significant object memory versus 0 h discrimination). ( D ) Ibuprofen administration beginning at 3 months of age prevents NOR deficits at 24 h in APP-PS1 mice ( n = 8–10 mice/group treated with either vehicle or ibuprofen from 3–6 months; ^**P < 0.01; * P < 0.05 by paired t -test versus 0 h discrimination). ( E ) Ibuprofen prevents high-speed locomotor activity during NOR testing in APP-PS1 mice (time with speed > 25 m/min; ^**P < 0.01 by Bonferroni multiple comparison). ( F ) Total object exploration time is not changed in ibuprofen-treated mice in the 24-h NOR task. ( G ) EEG recordings demonstrate cortical epileptiform activity in the form of brief high-intensity discharges, or spikes (red arrow), in 6 month APP-PS1 mice. ( H ) Quantification of spike frequency and duration demonstrates no effect of ibuprofen treatment in APP-PS1 mice ( n = 7–9 mice/group; P -values from Mann-Whitney U-tests). ( I ) Levels of COX-derived PGE ₂ are increased in hippocampus of APP-PS1 mice and reduced by treatment with ibuprofen for 3 months ( n = 5–20 mice per group aged 6–8 months; * P < 0.05 versus wild-type vehicle by Bonferroni multiple comparison).

Figure 3

Ibuprofen regulates expression of neuronal genes associated with LTP and LTD neuronal plasticity pathways. Hippocampi from wild-type and APP-PS1 mice treated with either vehicle or ibuprofen from 3–6 months of age were examined by microarray analysis. ( A ) KEGG pathway analysis for significantly regulated genes ( P < 0.05) is shown for each comparison. KEGG pathways for the wild-type vehicle versus APP-PS vehicle comparison shows enrichment of biological pathways involved in lysosome function, protein and RNA degradation, and translation. Ibuprofen treatment selectively enriches for KEGG pathways involved in synaptic plasticity in both APP-PS and wild-type mice (highlighted in red). Comparison between wild-type ibuprofen and APP-PS ibuprofen shows additional neuronal enrichment for axon guidance and neurotrophin signaling pathways. ( B and C ) Hierarchical clustering was performed on KEGG pathway neuronal genes that are regulated by ibuprofen in wild-type and APP-PS1 mice. ( B ) In wild-type hippocampus, ibuprofen induces an overall increase in gene expression of LTP genes. ( C ) In APP-PS1 hippocampus, ibuprofen induces a converse suppression of plasticity genes involved in both LTP and LTD. Shown in bold italics are genes shared in the LTP KEGG pathways in wild-type and APP-PS1 mice. wt = wild-type.

Figure 4

Ibuprofen broadly regulates neuronal gene expression and neurotransmitter levels in APP-PS1 mice. ( A ) Hierarchical clustering of hippocampal genes significantly regulated ( P < 0.01) in the APP-PS1 + vehicle versus APP-PS1 + ibuprofen comparison shows neuronal genes (highlighted in red) that are regulated by ibuprofen. ( B ) Penk and Tdo2 are the two neuronal genes most highly regulated by ibuprofen in APP-PS1 mice, as confirmed by quantitative PCR analysis ( n = 8–10 mice per group, * P < 0.05, ^**P < 0.01 by Bonferroni multiple comparison test). ( C–E ) Hippocampi from wild-type and APP-PS1 mice ± vehicle or ibuprofen for 3 months were analysed by HPLC-MS/MS at 6 months ( n = 6–14 mice per group). ( C ) Norepinephrine levels are significantly reduced in APP-PS1 hippocampus and significantly increased with ibuprofen treatment ( ^****P < 0.0001 and ^**P < 0.01 by Bonferroni multiple comparison). ( D ) Dopamine levels increase with ibuprofen treatment (effect of treatment P < 0.05). ( E ) Glutamate levels show an interaction effect between genotype and treatment, ( P < 0.05). WT = wild-type.

Figure 5

Ibuprofen reduces hippocampal TDO2 levels in APP-PS1 mice. ( A ) TDO2 metabolizes the essential amino acid tryptophan, the precursor of serotonin, to kynurenine; kynurenine is further metabolized to multiple neuroactive compounds either down the kynurenic acid arm (KYNA; in green) or down the 3-hydroxykynurenine (3-HK) and anthranillic acid (AA) arms (in blue) to quinolinic acid (QA) and NAD+. ( B ) TDO2 is enriched in hippocampal neurons, as shown in Gene Expression Nervous System Atlas of Tdo2 promoter-GFP reporter line (GENSAT; BAC BX2805, mouse line QE60). ( C ) Immunostaining shows enrichment of TDO2 protein in the hippocampal dentate-CA3 mossy fibre tract, with lower expression in CA3-1 regions (TDO2 immunofluorescence is white, nuclear DAPI is blue). ( D ) Western blot and ( E ) quantification of hippocampal TDO2 shows effects of ibuprofen in wild-type and APP-PS1 mice ( n = 4–5 per group treated from 4 to 7 months, ANOVA P = 0.019 for effect of ibuprofen and P = 0.052 for effect of genotype). ( F ) HPLC shows effect of ibuprofen on hippocampal kynurenine levels in wild-type and APP-PS1 mice ( n = 8–11 per group treated from 3 to 6 months; ANOVA P = 0.014 for effect of ibuprofen; * P < 0.05 by Bonferroni multiple comparison). WT = wild-type.

Figure 6

Hippocampal TDO2 is regulated by amyloid-β ₄₂ oligomers and neuronal COX-2. ( A–E ) Primary rat hippocampal neurons were co-treated with amyloid-β ₄₂ oligomers and/or the indicated compounds and assayed for expression of Tdo2 by quantitative PCR. ( A ) Amyloid-β ₄₂ oligomer treatment increases Tdo2 expression at 8 h ( n = 11–12 per group; * P < 0.05 and ^***P < 0.001 by Tukey’s multiple comparison). ( B ) Stimulation of hippocampal neurons with 100 nM PGE ₂ increases Tdo2 expression at 6 h ( n = 4–7 per group; * P < 0.05 by unpaired t -test). ( C ) Induction of Tdo2 by amyloid-β ₄₂ oligomers (5 μM) at 8 h is reduced by ibuprofen (10 µM) ( n = 16–18 per group; ^#P < 0.0001 versus control vehicle, ^***P < 0.001 versus control vehicle, and ^****P < 0.0001 versus amyloid-β vehicle by Bonferroni multiple comparisons). ( D ) Induction of Tdo2 by amyloid-β ₄₂ oligomers (5 μM) at 8 h is reduced by the COX-2 inhibitor SC-236 (100 nM) ( n = 5–7 per group; ^#P < 0.01 versus control vehicle and ^***P < 0.001 versus amyloid-β vehicle by Bonferroni multiple comparisons). ( E ) In neurons treated with amyloid-β ₄₂ oligomers (5 μM) for 8 h, both SC-236 (100 nM) and ibuprofen (10 μM) reduce Tdo2 expression with no additional effect of ibuprofen in SC-236 treated neurons ( n = 5–8 per group; * P < 0.05 by Bonferroni multiple comparisons). ( F ) Immunostaining for human COX-2 (hCOX-2) in Thy1-hCOX-2 hippocampi from lines 303 and 316 shows hCOX-2 expressed in dentate gyrus and CA subregions of hippocampus ( upper panel ); higher magnification of hCOX-2 staining is shown in lower panels in CA1 pyramidal neurons. ( G ) Tdo2 mRNA expression is increased in hippocampus of 3-month-old line 303 and line 316 mice ( n = 7–11 mice per group; * P < 0.05 and ^**P < 0.01 by unpaired t -test). ( H ) LC-MS/MS of hippocampi from 3-month-old line 303 mice shows increases in kynurenine pathway metabolites 3-OH kynurenine and AA ( n = 4–7 mice per group; * P < 0.05 by unpaired t -test). WT = wild-type.

Figure 7

Hippocampal TDO2 activity regulates NOR performance. ( A ) Representative image of AAV-mCherry fluorescence 3 weeks after injection of hippocampus. Expression (in red) is seen in the molecular layer and in CA3 (DAPI nuclear stain in blue). ( B ) HPLC of tryptophan (Trp) and kynurenine (Kyn) levels in hippocampi of AAV-mCherry and AAV-mCherry-TDO2 mice shows elevated kynurenine levels 3 weeks after injection of AAV-mCherry-TDO2 ( n = 10 mice per group, * P < 0.05 and ^**P < 0.01 by unpaired t -test). ( C ) NOR memory at 24 h was tested 3 weeks after hippocampal AAV-mCherry or AAV-mCherry-TDO2. AAV-mCherry-TDO2 injected mice are unable to perform the 24 h NOR task. ( n = 9 mice per group; * P < 0.05 paired t -test). ( D ) Wild-type and APP-PS1 mice were administered vehicle or 680C91 (7.5 mg/kg/day) from 3 months to 4 months of age and tested for NOR at 24 h. 680C91 treatment prevented the impaired NOR behaviour of APP-PS1 mice while impairing the NOR behaviour of wild-type mice (wild-type + vehicle, n = 15; wild-type + 680C91, n = 23; APP-PS1 + vehicle, n = 11; APP-PS1 + 680C91, n = 14; * P < 0.05 paired t -test). WT = wild-type.

Figure 8

Ibuprofen does not alter amyloid-β metabolism in 6 month APP _Swe -PS1 _ΔE9 mice. APP-PS1 mice were administered vehicle (Veh) or ibuprofen (Ibu) from 3 months to 6 months ( n = 8–10 mice per group; P -values from unpaired t -tests). ( A ) Soluble amyloid-β levels from ELISA of RIPA-soluble cortex extracts are unchanged by ibuprofen. ( B ) Total levels of amyloid-β ₄₂ or amyloid-β ₄₀ from guanidine-extracted cortex samples demonstrate no effect of ibuprofen on amyloid-β levels. ( C ) 6E10 (anti-amyloid-β) immunostaining and ( D ) quantification demonstrate no effect of ibuprofen on plaque coverage in the hippocampus. Arrows indicate plaques in representative images. Scale bar = 200 μm. ( E ) Congo Red (CR) quantification demonstrates no effect of ibuprofen on amyloid plaque density in the hippocampus. ( F ) Quantitative western blot of cortical lysates and ( G ) densitometric quantification demonstrate no effect of ibuprofen on β- and α-secretase cleavage products (C-terminal fragments, CTF) of APP. ( H ) Lipid peroxidation, as measured by quantification of LG-lysine adducts, is reduced to wild-type levels in posterior cortex of ibuprofen-treated APP-PS1 mice ( n = 7–10 mice/group; * P < 0.05 by Bonferroni multiple comparison test). ( I ) Model illustrating mechanisms associated with prevention of amyloid-β oligomer-induced behavioural deficits by NSAIDs. Amyloid-β oligomers are generated at the synapse and elicit excitatory synaptic activity that drives expression of the neuronal immediate early gene COX-2. Increased COX-2 activity leads to synaptic injury ( Andreasson et al. , 2001 ; Dore et al. , 2003 ). NSAIDs reduce COX-2 activity, and restore neuronal function by modulating expression of neuronal plasticity genes, neurotransmitter levels, and the first committed step in the kynurenine pathway. WT = wild-type.