Aspirin Up-Regulates Synaptic Function.

Calcium influx through NMDA- and AMPA-type glutamate receptors regulates diverse processes, including kinase and phosphatase activities, protein trafficking, structural and functional synaptic plasticity, cell growth, cell survival, and apoptosis (11⇓–13). Therefore, we investigated whether aspirin could evoke the calcium influx in cultured hippocampal neurons. Interestingly, following aspirin treatment, both AMPA and NMDA elicited a stronger calcium influx (Fig. 1 A and B). Dendritic spines are the major sites of excitatory synaptic transmission in the central nervous system, and their size and density influence the functioning of neuronal circuits (14, 15). Therefore, we examined the effect of aspirin on spine density in cultured hippocampal neurons and found that aspirin treatment significantly increased the density (Fig. 1 C and D). We validated these observations by measuring spine size. Consistent with the up-regulation of spine density, aspirin treatment significantly increased spine size in the hippocampal neurons (Fig. 1E). Among the neurotrophins (NTs), BDNF stands out for its ability to regulate the formation of plasticity and neuronal networks in the hippocampus (16⇓⇓⇓–20). Interestingly, aspirin treatment stimulated the expression of BDNF mRNA in the hippocampal neurons (Fig. 1 F and G). Immunoblot (Fig. 1 H and I) and immunocytochemical analyses (Fig. 1J) further corroborated that aspirin can up-regulate the expression of BDNF protein in E18 hippocampal neurons.

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Fig. 1.

Aspirin up-regulates synaptic plasticity in hippocampal neurons. Hippocampal neurons were treated with 5 µM aspirin for 18 h, followed by monitoring AMPA-induced (A) and NMDA-induced (B) calcium influx in a PerkinElmer VICTOR X2 luminescence spectrometer. To nullify the secondary involvement of AMPA receptor in NMDA-dependent calcium currents, hippocampal neurons were treated with NMDA together with NASPM, followed by the recording of calcium influx. Similarly, AMPA-dependent calcium influx was measured in the presence of N20C. Results are presented as the mean of three independent experiments. (C) Hippocampal neurons were treated with 5 µM aspirin for 18 h, followed by immunostaining with neuronal marker MAP2 (white) and Alexa Fluor 647-conjugated phalloidin (red) for spines. (Scale bars: 10 μm.) (D) Spine density was measured from phalloidin-stained hippocampal neurons in response to aspirin treatment and plotted as a function of 10-μm-long dendrites using boxplot analysis. Results are the mean ± SD of spines measured from 13 different neurons. Significance of the mean was tested with one-way ANOVA (effector: treatment), described as ^aF_{(1, 26)} = 2.948; P < 0.05 (= 0.01113). (E) Spine size was calculated from 13 different aspirin-treated hippocampal neurons using the strategy depicted in the cartoon. Results are the mean ± SD of spines measured from 13 different neurons. One-way ANOVA (effector: treatment) with ^aF_{(1, 26)} = 3.846; P < 0.05 (= 0.0213) was applied to test the significance of the mean between groups. (F and G) Hippocampal neurons were treated with aspirin for 6 h, followed by monitoring the mRNA expression of Bdnf by (F) RT- PCR (F) and real-time PCR (G). Results are the mean ± SD of three independent experiments. ^aP < 0.01 vs. control; ^bP < 0.001 vs. control. (H) Following dose-dependent aspirin treatment for 18 h, the expression of BDNF protein was investigated in hippocampal neurons by immunoblot analysis. (I) Relative density of BDNF protein expression compared with actin was calculated using ImageJ software. Results are the mean ± SD of three independent experiments. ^aP < 0.001 vs. control. (J) Hippocampal neurons were immunostained with MAP2 (green) and BDNF (red). (Scale bars: 20 μm.)

In addition to BDNF, other members of the NT family, NT3 and NT4, also participate in the regulation of synaptic plasticity (21, 22). BDNF and NT4 mediate their synaptic modulatory effect through receptor tropomyosin-related kinase B (TrkB) (23). NTs also can mediate programmed cell death by activating another NT receptor, p75^NTR (21). Therefore, we next examined the effect of aspirin on these NTs and their receptors in cultured hippocampal neurons. Aspirin treatment increased the mRNA expression (SI Appendix, Fig. S1 A and B) and protein expression (SI Appendix, Fig. S1C) of NT3 in hippocampal neurons. Similarly, aspirin also up-regulated the mRNA expression of NT4 and TrkB (SI Appendix, Fig. S1 A and B). However, under similar treatment conditions, p75^NTR mRNA expression remained unaltered (SI Appendix, Fig. S1 A and B). Taken together, these results suggest that aspirin is capable of stimulating the plasticity-associated functions in hippocampal neurons.

Aspirin Induces the Activation of PPARα.

We next investigated the mechanism by which aspirin up-regulates hippocampal plasticity. We previously showed that ligand-dependent activation of PPARα can promote plasticity-associated function in hippocampal neurons (24). This prompted us to investigate if aspirin can activate PPARα in hippocampal neurons. Since PPARs bind to a consensus sequence, PPRE (25), to study the activation of PPAR, we transfected mouse hippocampal neurons with tk-PPREx3-Luc, a PPRE-dependent luciferase construct, and measured luciferase activity. Aspirin treatment induced PPRE-driven luciferase activity in a dose-dependent manner, with maximum induction observed at 5 μM (SI Appendix, Fig. S2A). Similarly, aspirin also induced PPRE-driven luciferase activity in WT mouse astrocytes (SI Appendix, Fig. S2B). The inability of aspirin to induce PPRE-driven luciferase activity in Ppara-null astrocytes (SI Appendix, Fig. S2B) suggests that aspirin requires PPARα to increase PPRE reporter activity.

Aspirin Serves as a Ligand of PPARα.

Since aspirin induced the activation of PPARα, we examined whether aspirin can serve as a ligand of PPARα. We first constructed a 3D structure of mouse PPARα using homology modeling, performed with the swissmodel software module using human PPARα (Protein Data Bank ID code 1KKQ) as a template. We then used swissdock, a rigid body protein-ligand docking tool, to predict the interaction between aspirin and the LBD of PPARα at the molecular level. Based on electrostatic (E_tot) and desolvation (E_sol) energies, swissdock in conjugation with Chimera software resolved and displayed the most stable docked structure. According to this analysis, we found that aspirin docked very well in the interface of the LBD of PPARα (Fig. 2 A and B). We also observed that aspirin engaged with the Tyr314 residue of the PPARα LBD with a strong hydrogen bond interaction (3.51 A°). We further corroborated these observations by a mutation analysis, in which Tyr314 residue was mutated to aspartate (Y314DPPARα). The docked structures of aspirin with Y314DPPARα clearly indicated that the mutation indeed impaired the binding affinity of aspirin to PPARα with a very weak electrostatic interaction at a distance of 7.8 A° (Fig. 2C).

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Fig. 2.

Characterization of interaction of aspirin with PPARα at molecular level. (A) A rigid-body in silico docked pose of the PPARα LBD with aspirin. The interaction was evaluated with free energy (ΔG) = −6.46 kcal/mol, desolvation energy (E_sol) = −1,823.1 kcal/mol, and total energy (E_tot) = −1,611.88 kcal/mol). (B) Electrostatic potential surface of interaction between the LBD and aspirin. Red indicates a negatively charged surface; blue, a positively charged surface; white, a neutral surface. Black circle highlights the docked pose of aspirin. (C) While analyzing the interaction between aspirin and PPARα, the most stable docked structure was generated by similar rigid-body analysis with free energy (ΔG) = −5.49 kcal/mol, desolvation energy (E_sol) = −2,866.05 kcal/mol, and total energy (E_tot) = −2,531.20 kcal/mol. (D) Thermal shift assay of flPPARα analyzed with two doses of aspirin (5 and 10 μM). The melting of PPARα was monitored using a SYBR Green real-time melting strategy. (E) Thermal shift analyses of Y314DPPARα performed with a 5 μM concentration of aspirin. (F) Increasing doses of aspirin altered the maximum fluorescence derived from the melting of PPARα and Y314D PPARα proteins, as plotted in this dose–response curve. (G) TR-FRET analysis confirming the interaction between aspirin and PPARα. The curve was plotted as 520-nm/490-nm ratio of response with increasing doses of aspirin. Curve-fitting was done using GraphPad Prism. The analysis generated EC₅₀ (4.194 μM) and Hill slope (9.267) values based on the sigmoidal curve-fitting equation: Y = bottom + (X_{Hill slope}) × (top − bottom)/(X_{Hill slope} + EC_{50Hill slope}). Nuclear fractions of GFP- transduced (H), flPPARα- transduced (I), and Y314DPPARα- transduced (J) Ppara-null astrocytes treated with 5 μM aspirin were extracted using a chloroform:methanol extraction procedure, followed by GC-MS analysis. Results were analyzed and confirmed after three independent experiments.

Similarly, we also performed rigid body docking analyses to evaluate the affinities of aspirin-like molecules and other nonsteroidal anti-inflammatory drugs (NSAIDs) with PPARα. Five different compounds with structural similarities to aspirin, including ibuprofen, celecoxib, methyl salicylate, methyl-4-hydroxy benzoate, and naproxen, were docked with the LBD of PPARα (SI Appendix, Fig. S3). Interestingly, our in silico analyses revealed that these structural homologs of aspirin displayed very weak interactions with PPARα (SI Appendix, Fig. S3), suggesting that compared with other structural homologs, only aspirin displays a stronger affinity toward the PPARα LBD.

To experimentally confirm the binding and activation of PPARα by aspirin, thermal shift assays were carried out. In brief, full-length recombinant PPARα (flPPARα) was synthesized with lentivirus (24, 26) and its melting profile was monitored with the aid of a SYBR Green reaction at 27–94 °C. A typical sigmoidal melting curve with a melting temperature of 50.4 °C clearly indicated that the recombinant PPARα protein is conformationally stable. Interestingly, in the melting assay revealed, 5 μM of aspirin strongly shifted the melting curve of PPARα to 53.6 °C (Fig. 2D). While analyzing the extent of shift of PPARα protein in response to 5 and 10 μM aspirin, we found that 10 μM aspirin was unable to generate a larger shift (54.2 °C) in the thermal curve of PPARα suggesting that 5 μM aspirin shows optimal binding with PPARα. In contrast, aspirin exhibited a thermal shift of only 0.98 °C with mutated Y314DPPARα protein (Fig. 2E), indicating an interaction of aspirin with the Y314 residue of PPARα. Consequently, our kinetic plot of maximum thermal response vs. increasing doses of aspirin ranging from 1 to 10 μM (Fig. 2F) clearly revealed that 5 μM aspirin displayed maximum binding affinity with flPPARα. Again, aspirin exhibited very little binding affinity with mutated Y314DPPARα protein (Fig. 2F).

The physiological interaction between aspirin and PPARα can be influenced by recruitment of the coactivator PGC-1α. Therefore, to validate the interaction of aspirin with PPARα physiologically, we performed a time-resolved FRET coactivator assay. FRET analyses confirmed that aspirin indeed displayed a strong interaction with PPARα (Fig. 2G). The binding curve resulted an EC₅₀ value as low as 4.19 μM, with a Hill slope of 9.27. To further characterize the interaction between aspirin and PPARα at the molecular level, GC-MS analysis was performed in astroglial cell lysate. In brief, Ppara-null astrocytes were transduced with flPPARα or Y314D-PPARα followed by treatment with 5 μM aspirin for 1 h. Cells were homogenized, and nuclear chloroform extracts were prepared, passed through a GFP-affinity column, eluted, fractionated with chloroform and methanol, and then analyzed by GC-MS. Interestingly, aspirin displayed a strong interaction with PPARα, as detected in affinity-purified nuclear extracts of GFP-flPPARα-transduced (Fig. 2I), but not GFP-transduced (Fig. 2H), Ppara-null astroglial cells. The interaction was identified by the appearance of a sharp peak of acetyl salicylate at an m/z ratio of 180 (Fig. 2F). In contrast, Y314D mutation completely ablated the interaction of aspirin with PPARα, as we did not observe any peak at an m/z ratio of 180, suggesting that Tyr314 is required for the interaction of PPARα with aspirin (Fig. 2J).

Under physiological conditions, aspirin is known to be metabolized to other derivatives that also may exhibit an interaction with PPARα. Accordingly, the affinity-purified chloroform fraction of GFP-flPPARα–transduced Ppara-null astrocytes also identified the peak for 2-ethyl hexyl salicylate (SI Appendix, Fig. S4 A and B). However, we detected a smaller peak of 2-ethyl hexyl salicylate in the nuclear extracts of Ppara-null astrocytes transduced with Y314DPPARα (SI Appendix, Fig. S4C). Collectively, these data suggest that along with aspirin, its derivative 2-ethyl hexyl salicylate also displays a strong ligand-binding affinity with PPARα at its Y314 residue.

Aspirin-Induced Activation of PPARα Is Dependent on Its Interaction with the Y314 Residue of PPARα.

First, to confirm that aspirin-mediated induction of PPRE luciferase activity is due to activation of PPARα, we overexpressed PPARα in Ppara-null astrocytes. Interestingly, lentivirus-mediated insertion of flPPARα followed by stimulation with 5 μM aspirin significantly up-regulated PPRE luciferase activity in Ppara-null astrocytes (SI Appendix, Fig. S5). However, aspirin failed to induce PPRE luciferase activity in Y314DPPARα-transduced Ppara-null astrocytes (SI Appendix, Fig. S5). Taken together, these results clearly demonstrate that aspirin interacts with the Y314 residue of PPARα, and that this interaction is crucial for aspirin-induced transcriptional activation of PPARα.

Aspirin-Induced Stimulation of Synaptic Function Is Dependent on Its Interaction with the Y314 Residue of PPARα.

We next wanted to determine whether aspirin stimulates synaptic function through PPARα. Assessment of calcium influx through ionotropic receptors, including NMDA and AMPA receptors, has been considered the most reliable procedure for analyzing hippocampal function (11⇓–13). Interestingly, aspirin was unable to induce calcium influx in cultured hippocampal neurons isolated from Ppara-null mice compared with WT mice (Fig. 3 A and B), suggesting that aspirin requires PPARα to stimulate calcium influx in hippocampal neurons.

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Fig. 3.

Aspirin stimulates synaptic plasticity in hippocampal neurons via PPARα. WT and Ppara-null E18 hippocampal neurons were treated with 5 μM aspirin for 18 h, followed by analyzing AMPA-driven (A) and NMDA-driven (B) calcium influx. Ppara-null hippocampal neurons were transduced with either empty vector (blue) or flPPARα (green) via a lentivirus strategy for 48 h, followed by stimulation with aspirin (dark green) or gemfibrozil (red). After 18 h of stimulation, neurons were analyzed for AMPA-driven (C) and NMDA-driven (D) calcium influx. Similarly, Ppara-null (αKO) hippocampal neurons were transduced with lenti-Y314DPPARα (purple), followed by stimulation with aspirin (dark purple) or gemfibrozil red). After 18 h, AMPA-stimulated (E) and NMDA-stimulated (F) calcium influx was measured. AMPA-driven (G) and NMDA-driven (H) calcium influx was measured in Y314DPPARα-transduced WT E18 hippocampal neurons after treatment with aspirin and gemfibrozil. Calcium influx was monitored for 300 repeats in a PerkinElmer VICTOR X2 fluorimeter. Results represent three independent experiments. (I) WT and Ppara-null mouse hippocampal neurons transduced with lentivector containing flPPARα were treated with aspirin for 18 h, followed by immunostaining with neuronal marker MAP2 (green) and Alexa Fluor 647-conjugated phalloidin (red) to stain dendritic spines. (Insets) Enlarged views of dendrites, boxed within white rectangles in the respective lower-magnification images. (Scale bars: 20 μm.) (J) Spine density was measured from phalloidin-stained hippocampal neurons and plotted as a function of 10-μm-long dendrites. Statistical analyses of spine density were performed in 13–21 dendrites per group using two-way ANOVA considering genotype [F_{2, 105} = 135; P < 0.0001 (=0.000000)] and treatment [*F_{1, 105} = 3.04; P < 0.05 (=0.03107)] as two independent variables. Interaction statistics between two independent variables were calculated as well [F_{2, 105} = 6.14; P < 0.01 (=0.0030)]. Tukey’s HSD post hoc test was applied to assess the significance of the mean. (K) Spine size was quantified in seven to nine dendrites per group and analyzed with two-way ANOVA. The effects of genotype [F_{2, 31} = 19.1; P < 0.0001 (= 0.0000000)], treatment [F_{1, 31} = 14.8; P < 0.001 (= 0.0006)], and their interaction [F_{2, 31} = 2.91; P > 0.05 (= 0.0692)] were compared between groups. Tukey’s HSD post hoc test was applied to measure significance.

Next, to establish a direct role of Y314 residue of PPARα in aspirin-induced calcium influx, we overexpressed flPPARα and mutated Y314DPPARα in Ppara-null hippocampal neurons, followed by stimulation with 5 μM of aspirin. Interestingly, aspirin significantly stimulated both AMPA- and NMDA-mediated calcium currents in flPPARα-transduced (Fig. 3 C and D), but not Y314DPPARα-transduced (Fig. 3 E and F), Ppara-null hippocampal neurons.

To further confirm a direct role for PPARα in the regulation of ionotropic calcium conduction in aspirin-stimulated neurons, we also overexpressed the Y314DPPARα construct in WT hippocampal neurons, followed by stimulation with aspirin. Interestingly, infusion of Y314DPPARα attenuated both NMDA- and AMPA-driven ionotropic calcium influxes (Fig. 3 G and H) in WT hippocampal neurons. In all cases, we also validated the effect of aspirin with gemfibrozil, a classical PPARα agonist. Taken together, these results suggest that the interaction of aspirin with PPARα Tyr314 is essential for aspirin-induced up-regulation of calcium influx through NMDA- and AMPA-sensitive receptors.

Along with the estimation of calcium influx, quantification of dendritic spine density is an important measure to assess hippocampal function. Therefore, we adopted a phalloidin-based quantification analysis of dendritic spines in aspirin-treated hippocampal neurons. In brief, Ppara-null hippocampal neurons were transduced with flPPARα for 3–4 d and then treated overnight with aspirin. Then these cells and WT neurons were labeled with phalloidin and the dendritic marker MAP2. Accordingly, aspirin significantly increased spine density in WT, but not Ppara-null, hippocampal neurons. Interestingly, the introduction of flPPARα, but not with empty vector, significantly increased spine counts in aspirin-stimulated hippocampal neurons (Fig. 3 I and J). We further confirmed these observations by measuring spine size (Fig. 3K) under the different treatment conditions. Collectively, these results suggest that aspirin up-regulates morphological plasticity in hippocampal neurons via PPARα.

Aspirin Stimulates the Transcription of CREB via Its Interaction with the Y314 Residue of PPARα.

CREB plays a central role in regulating hippocampal plasticity and memory (27, 28). Recently we found that PPARα is not directly involved in the transcription of plasticity-related molecules and that PPARα controls hippocampal plasticity via transcriptional up-regulation of CREB (24). Therefore, we investigated whether aspirin also controls Creb transcription via PPARα. Accordingly, we observed that aspirin rapidly up-regulated the expression of Creb mRNA in primary mouse astrocytes (Fig. 4 A and B). As clearly shown in Fig. 4 C and D, aspirin increased the expression of CREB mRNA in a dose-dependent manner in WT and Pparb-null, but not Ppara-null, astrocytes. Immunoblot (Fig. 4 E and F) and immunofluorescence analyses (Fig. 4G) again confirmed that aspirin indeed up-regulated the expression of CREB protein in the WT and Pparb-null, but not Ppara-null, astrocytes. However, overexpression of PPARα restored the expression of Creb mRNA (SI Appendix, Fig. S6 A and B) and CREB protein (SI Appendix, Fig. S6 C–E) in Ppara-null astrocytes as well as hippocampal neurons (SI Appendix, Fig. S6 F and G), indicating the involvement of PPARα in aspirin-induced up-regulation of CREB. Moreover, pretreatment with GW9662, a PPARγ antagonist, was unable to suppress the expression of Creb mRNA in aspirin-treated WT astrocytes, thus nullifying the potential involvement of PPARγ (SI Appendix, Fig. S7 A and B). However, under similar treatment conditions, GW6471, a PPARα antagonist, inhibited the aspirin-mediated expression of Creb mRNA (SI Appendix, Fig. S7 C and D). Accordingly, aspirin also induced the recruitment of PPARα, but not PPARβ or PPARγ, to the Creb promoter (Fig. 4 H–J), suggesting that aspirin stimulates the transcription of Creb via PPARα.

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Fig. 4.

Aspirin increases the transcription of CREB via PPARα. (A and B) WT mouse astrocytes were treated with aspirin (5 μM) for various durations, followed by measurement of the mRNA expression of CREB by RT-PCR (A) and real-time PCR (B). Results are mean ± SD of three independent experiments. ^aP < 0.05 vs. control; ^bP < 0.01 vs. control; ^cP < 0.001 vs. control. (C and D) RT-PCR (C) and real-time PCR (D) analyses were performed to analyze the mRNA expression of CREB in the WT, Ppara-null, and Pparb-null primary mice astrocytes treated with increasing doses (0, 2, and 5 μM) of aspirin. Results are the mean ± SD of three independent experiments. ^aP < 0.001 vs. control. (E and F) WT (Left), Ppara-null (Middle), and Pparb-null (Right) mouse primary astrocytes were treated with increasing doses of aspirin and then analyzed by immunoblot analysis (E), followed by relative densitometric analysis (F) of the CREB protein. Results are mean ± SD of three independent experiments. ^aP < 0.001 vs. control. (G) Immunofluorescence analyses of GFAP (green) and CREB (red) were performed in WT, Ppara-null, and Pparb-null primary astrocytes after treatment with 5 μM aspirin. (Scale bars: 20 μm.) (H) Schematic representation of the Creb gene promoter with PPRE. (I and J) ChIP assays were performed using antibodies against PPARα, PPARβ, and PPARγ in aspirin-treated WT primary astrocytes. In brief, the promoter of the Creb gene was pulled down with PPARα, PPARβ, and PPARγ antibodies, followed by RT-PCR (I) and real-time PCR (J). Results are mean ± SD of three independent experiments. ^aP < 0.001 vs. control. (K) WT astrocytes were transfected with either WT-pCREB-Luc (blue) or mut-pCREB-Luc (red) constructs, followed by treatment with increasing doses of aspirin. After 2 h, luciferase activity was measured in a Promega GloMax luminometer. Results are mean ± SD of three independent experiments. ^aP < 0.05 vs. control; ^bP < 0.01 vs. control; ^cP < 0.001 vs. control.

To further verify the role PPARα in aspirin-mediated transcription of the Creb gene, a PPRE containing the Creb promoter upstream of a luciferase reporter gene (pCreb-luc) construct was transfected into astrocytes, followed by stimulation with aspirin. As shown in Fig. 4K, 5 μM of aspirin significantly stimulated Creb promoter-driven luciferase activity in astrocytes; however, aspirin failed to induce luciferase activity driven by mutated Creb promoter in which the PPRE was mutated, suggesting that aspirin-mediated transcription of Creb is dependent on PPARα. To understand this specificity, we also monitored the effect of gemfibrozil, a prototype activator of PPARα, on activation of the Creb promoter. Consistent with a role for PPARα in this process, gemfibrozil also induced reporter activity driven by the wt-Creb promoter, but not by the mutated Creb promoter (SI Appendix, Fig. S8).

Since aspirin binds to the Y314 residue of PPARα, to determine whether this interaction is crucial for aspirin-mediated transcription of Creb, Ppara-null astrocytes were transduced with lenti-GFP, lenti-fl-Ppara, and lenti-Y314DPpara. As expected, aspirin remained unable to induce the Creb promoter-driven luciferase activity in lenti-GFP–transduced Ppara-null astrocytes (SI Appendix, Fig. S9A); however, aspirin markedly induced Creb promoter-driven luciferase activity in lenti-fl-Ppara–transduced Ppara-null astrocytes (SI Appendix, Fig. S9B). In contrast, aspirin did not induce Creb promoter-driven reporter activity in lenti-Y314DPpara–transduced Ppara-null astrocytes (SI Appendix, Fig. S9C), confirming the crucial role of PPARα Y314 in aspirin-mediated up-regulation of Creb promoter activity.

To understand the functional significance of aspirin-mediated up-regulation of CREB via PPARα, we examined whether aspirin requires PPARα to up-regulate BDNF, one of the downstream targets of CREB. As evident from the immunofluorescence analyses, we noted that following aspirin treatment, BDNF expression was significantly higher in WT, but not Ppara-null, hippocampal (Fig. 5A) and cortical neurons (Fig. 5B). Similarly, aspirin was also observed to up-regulate the expression of Bdnf mRNA (Fig. 5 C and D) in WT and Pparb-null, but not Ppara-null, astrocytes. These results were further corroborated by immunoblot (Fig. 5 E and F) and immunocytochemical (Fig. 5G) analyses demonstrating induction of BDNF protein by aspirin in WT and Pparb-null, but not Ppara-null, astrocytes.

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Fig. 5.

Aspirin increases the expression of BDNF via PPARα. (A) WT and Ppara-null hippocampal neurons were treated with 5 μM aspirin for 18 h, followed by double-labeling of MAP2 (green) and BDNF (red). (Scale bars: 20 μm.) (B) Similar immunofluorescence analyses of BDNF were performed in WT and Ppara-null cortical neurons. (C and D) Primary astrocytes isolated from WT (Left), Ppara-null (Middle), and Pparb-null (Right) mice were treated with increasing doses of aspirin, and Bdnf mRNA was analyzed by RT-PCR (C) and real-time PCR (D). Results are mean ± SD of three independent experiments. ^aP < 0.05 vs. control; ^bP < 0.01 vs. control. (E and F) Similarly, protein expression of BDNF was determined by immunoblot analysis (E) followed by densitometric analysis (F) in WT (Left), Ppara-null (Middle), and Pparb-null (Right) mouse astrocytes treated with increasing dose of aspirin. The relative density of BDNF protein compared with actin was calculated using ImageJ software. Results are mean ± SD of three independent experiments. ^aP < 0.05 vs. control; ^bP < 0.01 vs. control. (G) Dual immunofluorescence analyses of GFAP (green) and BDNF (red) were performed in WT, Ppara-null, and Pparb-null mouse astrocytes in response to 5 μM of aspirin. (Scale bars: 20 μm.)

Aspirin Enhances the Expression of Plasticity-Associated Hippocampal Proteins and Protects Memory in the FAD5X Mouse Model of AD via PPARα.

We next analyzed whether aspirin uses PPARα to protect hippocampal properties in vivo in mouse brain. For this, we generated Ppara-null mice on the FAD5X transgenic background (26). These bigenic animals are reliable tools for studying the role of PPARα in AD-related pathologies in mice (26, 29). In this experiment, 6-mo-old FAD5X and FAD5X/Ppara-null mice were fed with aspirin (2 mg/kg body weight/d) for 4 wk, and then the hippocampi of these animals were tested for expression of CREB and CREB-dependent plasticity-associated proteins. Aspirin increased the expression of CREB (Fig. 6 C and D) and BDNF (Fig. 6 A and B) in the hippocampi of FAD5X, but not FAD5X/Ppara-null mice. Similarly, chronic administration of aspirin also increased the expression of other CREB-dependent proteins, including PSD95 (Fig. 6 E and F and SI Appendix, Fig. S10), and NR2A (Fig. 6 G and H), in the hippocampi of FAD5X mice but not FAD5X/Ppara-null mice, suggesting that PPARα plays an essential role in aspirin-induced expression of plasticity-associated proteins in the hippocampus.

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Fig. 6.

Aspirin enhances plasticity-associated molecules and promotes calcium influx in the hippocampus of FAD5X mice via PPARα. The 6- to 7-mo-old FAD5X and FAD5X/Ppara-null transgenic mice (n = 5 or 6 per group) were gavaged with aspirin (2 mg/kg body weight) for 30 d and the hippocampi were analyzed for the expression of different plasticity-associated proteins. Immunoblot analyses were performed for CREB (A), BDNF (C), PSD95 (E), and NR2A (G) in hippocampal extracts of NTG, FAD5X, FAD5X + aspirin, FAD5X/Ppara-null, and FAD5X/Ppara-null + aspirin mice. The relative densities of CREB (B), BDNF (D), PSD95 (F), and NR2A (H) proteins were measured using ImageJ. Results are presented as mean ± SEM of five or six mice per group. The significance of the mean was assessed using one-way ANOVA followed by Bonferroni’s post hoc test. ^aP < 0.001 vs. control-CREB; ^aP < 0.001 vs. control-BDNF; ^aP < 0.001 vs. control-PSD95; ^aP < 0.001 vs. control-NR2A. (I and J) AMPA-dependent (I) and NMDA-dependent (J) calcium currents were measured in the hippocampal slices of NTG, FAD5X, FAD5X + aspirin, FAD5X/Ppara-null, and FAD5X/Ppara-null + aspirin mice. The arrow indicates the application of AMPA and NMDA in the assay. Results are representative of three independent experiments.

We next analyzed the effect of aspirin on the ionotropic calcium influx through NMDA and AMPA receptors in hippocampal slices of FAD5X and FAD5X/Ppara-null mice. Consistent with the increased expression of plasticity-associated molecules, aspirin feeding up-regulated AMPA-dependent (Fig. 6I) and NMDA-dependent (Fig. 6J) calcium influx, as measured in organotypic hippocampal slices from FAD5X animals, but not from FAD5X/Ppara-null bigenic mice. Taken together, these results suggest that aspirin requires PPARα for controlling the expression of plasticity-associated molecules and their function in vivo in the hippocampus.

We then explored the effect of aspirin in improving hippocampus-dependent behaviors, including learning and memory, in FAD5X and FAD5X/Ppara-null mice. Hippocampus-dependent spatial learning and behavior can be reliably monitored using the Barnes maze test (24, 26) (Fig. 7A). As expected, and as reported in previous studies (26, 30), FAD5X mice displayed decreased spatial behaviors, as indicated by latency (Fig. 7B) and errors (Fig. 7C), compared with age-matched nontransgenic (NTG) mice. However, aspirin feeding significantly improved latency (Fig. 7B); [F_{1, 18} = 6.22; P < 0.05 (= 0.0226)] and decreased errors (Fig. 7C) [F_{1, 18} = 5.48; P < 0.05 (= 0.0310)] in FAD5X mice. In contrast, aspirin treatment was unable to improve spatial learning in FAD5X/Ppara-null mice, indicating an essential role for PPARα in aspirin-mediated improvement in hippocampus-dependent behaviors (Fig. 7B). The T-maze test assesses context-dependent hippocampal behavior, which may be a better index for testing cognition in mice. Consistently, our T-maze test also exhibited significant improvement in the performance of FAD5X mice, as demonstrated by the increased number of positive turns and reduction of errors, whereas FAD5X/Ppara-null mice did not show any improvement in T-maze test results following aspirin treatment (Fig. 7 D and E), indicating a pivotal role of PPARα in aspirin-mediated improvement in memory and learning in FAD5X mice.

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Fig. 7.

Aspirin protects memory and alleviates stress-related behaviors in the FAD5X mice via PPARα. The 6- to 7-mo-old FAD5X and FAD5X/Ppara-null transgenic mice were fed orally with aspirin (2 mg/kg/d) for 30 d, followed by assessment of hippocampus-dependent spatial behavior using the Barnes maze test. (A) Representative heat maps summarizing the overall activity of mice on the apparatus recorded with a Noldus camera and visualized using EthoVision XT software. (B and C) Latency (B) and number of errors made (C) by NTG, FAD5X, FAD5X + aspirin, FAD5X/Ppara-null, and FAD5X/Ppara-null + aspirin mice. Results are presented as mean ± SEM of 5–6 mice per group, and the significance of mean was assessed using two-way ANOVA followed by Bonferroni’s post hoc test. Context-dependent hippocampal behavior was analyzed using the T-maze test. (D and E) Number of positive turns (D) and number of errors (E) made by NTG, control and aspirin-treated FAD5X and FAD5X/Ppara-null transgenic mice on an appetitive T-maze conditioning task were manually recorded. Results are presented as mean ± SEM of five or six mice per group, and the significance of the mean was monitored assessed using two-way ANOVA followed by Bonferroni’s post hoc test. Stress-like behavior was investigated in an open-field apparatus. (F–H) Representative track plots (F) and graphs of center zone frequency (G) and total corner time duration (H) of NTG, FAD5X, FAD5X + aspirin, FAD5X/Ppara-null, and FAD5X/Ppara-null + aspirin mice. Results are presented as mean ± SEM of 5 or 6 mice per group, and the significance of the mean was monitored by two-way ANOVA followed by Bonferroni’s post hoc test.

To test the significance between groups, we performed two-way ANOVA with treatment [^aF_{1, 18} = 14.68; P < 0.01 (= 0.0012)] and genotype [^bF_{1, 18} = 6.893; P < 0.05 (= 0.0172)] as two effectors. Since the decreased latency in either the Barnes maze or T-maze test could be confounded with the increased locomotion of animals, we also monitored the speed of these animals in an open-field arena after 1 mo of aspirin feeding (SI Appendix, Fig. S11A). We did not observe any significant difference in total distance moved (SI Appendix, Fig. S11B) or velocity (SI Appendix, Fig. S11C) across the different groups of animals, nullifying the possibility of interference by increased locomotion in the hippocampus-dependent behaviors.

Since hippocampal function is connected to stress and depression (31⇓–33), we investigated the effect of aspirin on stress-related behaviors in the FAD5X and FAD5X/Ppara-null bigenic mice by analyzing their locomotive performance in the open-field arena (Fig. 7F). The FAD5X mice exhibited greater stress-related behaviors than seen in the NTG mice, as indicated by decreased center zone frequency and increased latency in corners (Fig. 7 G and H). However, aspirin treatment significantly enhanced center zone frequency and reduced latency in corners (Fig. 7 G and H) in the FAD5X mice. On the other hand, aspirin treatment was unable to reduce stress-related behaviors in the FAD5X/Ppara-null mice, further indicating that PPARα is required for the amelioration of stress-related behavior on aspirin treatment. Collectively, these results demonstrate that aspirin up-regulates hippocampal plasticity, improves memory, and alleviates stress-like behaviors in a mouse model of AD via PPARα.