Activation of extrasynaptic, but not synaptic, NMDA receptors modifies amyloid precursor protein expression pattern and increases amyloid-ß production

Abstract

Calcium is a key mediator controlling essential neuronal functions depending on electrical activity. Altered neuronal calcium homeostasis affects metabolism of amyloid precursor protein (APP), leading to increased production of β-amyloid (Aβ), and contributing to the initiation of Alzheimer's disease (AD). A linkage between excessive glutamate receptor activation and neuronal Aβ release was established, and recent reports suggest that synaptic and extrasynaptic NMDA receptor (NMDAR) activation may have distinct consequences in plasticity, gene regulation, and neuronal death. Here, we report for the first time that prolonged activation of extrasynaptic NMDAR, but not synaptic NMDAR, dramatically increased the neuronal production of Aβ. This effect was preceded by a shift from APP695 to Kunitz protease inhibitory domain (KPI) containing APPs (KPI-APPs), isoforms exhibiting an important amyloidogenic potential. Conversely, after synaptic NMDAR activation, we failed to detect any KPI-APP expression and neuronal Aβ production was not modified. Calcium imaging data showed that intracellular calcium concentration after extrasynaptic NMDAR stimulation was lower than after synaptic activation. This suggests distinct signaling pathways for each pool of receptors. We found that modification of neuronal APP expression pattern triggered by extrasynaptic NMDAR activation was regulated at an alternative splicing level involving calcium-/calmodulin-dependent protein kinase IV, but overall APP expression remained identical. Finally, memantine dose-dependently inhibited extrasynaptic NMDAR-induced KPI-APPs expression as well as neuronal Aβ release. Altogether, these data suggest that a chronic activation of extrasynaptic NMDAR promotes amyloidogenic KPI-APP expression leading to neuronal Aβ release, representing a causal risk factor for developing AD.

Figure 1.

Characterization of prolonged synaptic and extrasynaptic NMDAR activation in cortical neuron cultures at DIV12. Intracellular Ca²⁺ concentrations were monitored by Fura-2 calcium imaging. A, Cortical neurons were exposed for 2 min to Bic/4-AP and consecutively to Bic/4-AP with MK-801 (10 μm), an open channel blocker, for 3 min to irreversibly block activated synaptic NMDAR. Efficiency of NMDAR blockade was controlled by the absence of Ca²⁺ response under Bic/4-AP treatment in the presence of CNQX (10 μm), to avoid any AMPA receptor-dependent response. After washing, a dose ranging of NMDA (from 15 to 200 μm for 2 min each) was applied to the culture, and Ca²⁺ entry in neurons was monitored. Blockade of synaptic NMDAR was finally checked by applying Bic/4-AP + CNQX. B, Calcium responses after Bic/4-AP or increasing doses of NMDA application were quantified by measuring area under curve and represented in histograms (n = 3; n = 137; **p < 0.01). C, Cortical neuron cultures were exposed for 1 h to 50 μm Bic/2.5 mm 4-AP, and cellular Ca²⁺ entry was monitored using Fura-2. D, After blockade of synaptic response as described in A, cortical neuron cultures were treated for 1 h with 30 μm NMDA to selectively activate extrasynaptic NMDAR. Calcium response was monitored using Fura-2. E, Intracellular calcium concentration was quantified by measuring the area under the curve during the whole treatment duration (1 h for both treatments) (C, D). Statistical analysis was realized by ANOVA followed by Bonferroni–Dunn's test (Bic/4-AP: n = 3; n = 99; 30 μm NMDA after MK801 blockade: n = 3; n = 135; **p < 0.01).

Figure 2.

Effect of selective and prolonged activation of NMDAR on cellular morphology and neuronal death. A, Primary cultured cortical neurons at DIV 12 were subjected to synaptic (50 μm Bic + 2.5 mm 4-AP) or extrasynaptic (50 μm Bic + 2.5 mm 4-AP + 10 μm MK801 followed by 30 μm NMDA application) NMDAR activation for 12 and 24 h. The level of LDH released in the neuronal cell culture medium was determined by measuring the decrease in absorbance at 340 nm resulting from the oxidation of NADH. Results are expressed as mean ± SD from five independent experiments. Histograms represent neuronal cell death normalized to maximum neuronal death (0.1% Triton X-100 for 10 min). Statistical analysis was performed by ANOVA followed by Bonferroni-Dunn's test. n = 5; **p < 0.01 vs corresponding control; ##p < 0.01 vs extrasynaptic NMDAR stimulation. B, Morphological analysis of eGFP-transfected neuronal cultures. eGFP-labeled cortical neurons (DIV 12; transfection efficiency of 3 to 6%) were treated according to the synaptic protocol or the extrasynaptic protocol described in A for 12 or 24 h. Images represent the projection of z-stack images acquired by confocal microscopy at the indicated times and are representative of the cultures. Scale bar, 20 μm. Arrows are pointing to typical dendritic varicosities. C, Quantification of living cells, dead cells, and varicosity-containing cells in cultures exposed or not to synaptic or extrasynaptic treatment during the indicated times (12 or 24 h). Quantification of the three cell populations was achieved on four independent cultures by counting eGFP-transfected neurons on the overall coverslip under confocal microscope. Histograms represent means ± SD, and statistical analysis was performed by ANOVA followed by Bonferroni–Dunn's test (n = 4; *p < 0.05). D, Quantification of living cells, necrotic cells, and apoptotic cells in cultures exposed or not to synaptic or extrasynaptic protocol during the indicated times (12 or 24 h). Quantification of the three cell populations was achieved on six independent cultures using a double-stain apoptosis detection kit containing the blue-fluorescent Hoechst 33342 dye and the red-fluorescent PI dye, which is permeant only to dead cells. Counting of living, apoptotic, and necrotic cell populations was performed under a fluorescence microscope. Histograms represent means ± SD and statistical analysis was performed by ANOVA followed by Bonferroni–Dunn's test (n = 6; *p < 0.05). E, The photo is an illustration of apoptotic and living cells stained with Hoechst 33342. Arrows are pointing to typical apoptotic cells exhibiting apoptotic bodies. Scale bar, 20 μm.

Figure 3.

Synaptic, but not extrasynaptic, NMDAR activation induces BDNF mRNA expression in cortical neuron cultures. A, Primary cultured cortical neurons at 12 DIV were treated with 50 μm Bic + 2.5 mm 4-AP for 1, 3, or 6 h (synaptic protocol). Specificity of NMDAR in the effects of Bic/4-AP application was checked using AP-5 and nimodipine (to block VSCC), and CNQX (to block AMPA receptors). At the end of each time point, total RNA was extracted from neuron cultures and reverse transcribed in cDNA. Real-time PCR analysis was performed to quantify relative expression of BDNF mRNA in the different samples. The expression level of interest gene was analyzed according to the ΔΔCt method (comparative Ct method), where Ct is the threshold cycle value and cyclophilin the housekeeping gene. Histograms represent means ± SD, and statistical analysis was performed by ANOVA followed by Bonferroni-Dunn's test (n = 4; *p < 0.05, **p < 0.01 vs control; ##p < 0.05 vs Bic/4-AP treatment). B, Extrasynaptic NMDAR activation was performed by blocking activated NMDAR with 10 μm MK-801 for 3 min. After three extensive washings with culture medium, neurons were incubated at 37°C for 1 h before the addition of 30 μm NMDA for 1, 3, and 6 h. BDNF mRNA expression was measured as described above. Results are expressed as mean ± SD from five independent treatments.

Figure 4.

Synaptic and extrasynaptic NMDAR activity differently modulates APP expression in primary cultured neurons. A, Primary cultured cortical neurons at 12DIV were treated with 50 μm Bic + 2.5 mm 4-AP (synaptic protocol) for 1 h, 3, or 6 h. Real-time PCR analysis was performed to quantify relative expression of APP695 mRNA in the different samples. The expression level of interest gene was analyzed according to the ΔΔCt method (comparative Ct method) where Ct is the threshold cycle value and cyclophilin the housekeeping gene. Results are expressed as mean ± SD from five independent treatments. APP mRNA expression in control samples has been arbitrarily set at 100%. Statistical analysis was realized by ANOVA followed by Bonferroni–Dunn's test (n = 5; **p < 0.01 to control). B, Neurons at DIV 12 were treated as before (in A), and real-time PCR analysis was performed to quantify relative expression of KPI-APP mRNA in the different samples with expression in control set at 100%. Results are expressed as mean ± SD from five independent treatments. Statistical analysis was realized by ANOVA followed by Bonferroni-Dunn's test (n = 5). C, Neurons at DIV 12 were exposed to 50 μm Bic/2.5 mm 4-AP for 2 min, and consecutively to Bic/4-AP with 10 μm MK801 for 3 min. After three extensive washings, cultures were incubated at 37°C for 1 h before the addition of 30 μm NMDA for 1, 3, and 6 h (extrasynaptic protocol). Total RNA was extracted, and real-time PCR analysis was performed to quantify relative expression of APP695 mRNA with 100% level arbitrarily set in the control. Statistical analysis was realized by ANOVA followed by Bonferroni-Dunn's test (n = 5; **p < 0.01 to control). D, KPI-APP mRNA expression was measured in cultured neurons exposed to extrasynaptic protocol, and results were analyzed as described in C (n = 5; **p < 0.01 to control). E, Representative immunoblot of KPI-APP after selective activation of synaptic NMDAR. Primary cultured cortical neurons at DIV12 were washed two times with serum-free DMEM before being treated with 50 μm Bic + 2.5 mm 4-AP for 12, 18, and 24 h (synaptic protocol). At each time point, cells were lysed in RIPA buffer, and immunoblots were performed with anti KPI-APP antibody. Blots were rehybridized with an anti-actin antibody to estimate the total amount of proteins loaded. F, Top, Representative immunoblot of KPI-APP after selective activation of extrasynaptic NMDAR. Primary cultured cortical neurons at DIV 12 were exposed to 50 μm Bic/2.5 mm 4-AP for 2 min, and consecutively to Bic/4-AP with 10 μm MK801 for 3 min. After three extensive washings, cultures were incubated at 37°C for 1 h before the addition of 30 μm NMDA for 12 and 24 h (extrasynaptic protocol). Cell cultures were lysed in RIPA buffer, and immunoblots were performed with anti KPI-APP antibody. Absolute controls (Abs Con) were only subjected to washings with DMEM. Controls at 12 and 24 h (C) were exposed to Bic/4-AP for 2 min, and consecutively to Bic/4-AP with MK801 for 3 min without further NMDA application. Bottom, Relative expression of KPI-APP protein compared with actin from immunoblot presented in the top. Densitometric analysis of the protein bands was performed with ImageJ software. Each column is the mean ± SD from three immunoblots (n = 3). Statistical analysis was realized by ANOVA followed by Bonferroni–Dunn's test (n = 3; **p < 0.01 vs respective control). G, Representative immunoblot of KPI-APPs versus APP695 protein expression in neurons subjected to synaptic or extrasynaptic protocol for 24 h. APP isoforms were revealed using the 22C11 monoclonal antibody. Protein loading was normalized using β-actin immunodetection.

Figure 5.

KPI-APP protein expression in cortical neurons exposed to extrasynaptic NMDAR activation is mediated by calcium signaling pathway. A, Immunoblotting analysis of KPI-APP protein in cortical neuron cultures 24 h after synaptic NMDAR activation with or without MK-801 blocking, or after extrasynaptic NMDAR activation (extrasynaptic protocol) in the presence or not of 10 μm BAPTA-AM, an intracellular calcium chelator, or 10 μm KN-93, an inhibitor of CaM kinases. Blots were rehybridized with an anti-actin antibody to estimate the total amount of proteins loaded. B, Relative expression of KPI-APP compared with actin from experiments presented in A. Densitometric analysis of the protein bands was performed with ImageJ software. Each column is the mean ± SD from three immunoblots (n = 3). Statistical analysis was realized by ANOVA followed by Bonferroni-Dunn's test (n = 3; **p < 0.01 vs control; ##p < 0.01 vs extrasynaptic NMDAR activation). C, Immunoblotting analysis of KPI-APP protein in cortical neuron cultures 12 and 24 h after extrasynaptic NMDAR activation in the presence or not of 1 μm STO-609, a selective Ca²⁺/calmodulin-dependent protein kinase kinase. Blots were rehybridized with an anti-actin antibody to estimate the total amount of proteins loaded. D, Relative expression of KPI-APP compared with actin from experiments presented in C. Densitometric analysis of the protein bands was performed with ImageJ software. Each column is the mean ± SD from three immunoblots (n = 3). Statistical analysis was realized by ANOVA followed by Bonferroni–Dunn's test (n = 3; **p < 0.01 vs respective control; ##p < 0.01 vs extrasynaptic NMDAR activation).

Figure 6.

Synaptic NMDAR activation downregulates overall APP mRNA expression, whereas extrasynaptic NMDAR activation acts on APP isoforms mRNA ratio via an alternative splicing pathway. A, Real-time PCR analysis of total APP mRNA expression in cortical neurons exposed to Bic/4AP for 1, 3, or 6 h (synaptic protocol). A new pair of primers directed against the common part of the different APP isoforms was designed. At each time, APP mRNA expression in treated neurons was compared with its control. The expression level of APP was analyzed according to the ΔΔCt method (comparative Ct method) where Ct is the threshold cycle value and cyclophilin is the housekeeping gene. Results are representative of three independent experiments performed in triplicate. Statistical analysis was realized by ANOVA followed by Bonferroni-Dunn's test (n = 9; **p < 0.01 to respective control). B, Real-time PCR analysis of total APP mRNA expression in cortical neurons exposed to extrasynaptic NMDAR activation for 1, 3, or 6 h (extrasynaptic protocol). At each time, APP mRNA expression in treated neurons was compared with its control. Results are representative of three independent experiments performed in triplicate and expression level of APP was analyzed according to the ΔΔCt method. C, APP695/KPI-APP mRNA ratio in cortical neuron cultures treated with 50 μm Bic/2.5 mm 4-AP for 1, 3, and 6 h (synaptic protocol). Absolute quantification of APP695 and KPI-APP mRNA in each sample was performed by real-time PCR using known concentrations of DNA standard molecules (PCR products). Histograms represent the relative part of the two mRNA populations with the total of both mRNA arbitrarily set at 100%. D, APP695/KPI-APP mRNA ratio in cortical neuron cultures exposed to 50 μm Bic/2.5 mm 4-AP for 2 min, and consecutively to Bic/4-AP with 10 μm MK801 for 3 min. After three extensive washings, cultures were incubated at 37°C for 1 h before the addition of 30 μm NMDA for 1, 3, and 6 h (extrasynaptic protocol). Absolute quantification of APP695 and KPI-APP mRNA in each sample was performed by real-time PCR using known concentrations of DNA standard molecules (PCR products) as described in Materials and Methods. Histograms represent the relative part of the two mRNA populations with the total of both mRNA arbitrarily set at 100%. Statistical analysis was performed by ANOVA followed by Bonferroni-Dunn's test (n = 3; **p < 0.01 vs respective control). Abs Con, Absolute control; C, control (Bic/4-AP and MK-801 blocking); N, NMDA treatment after MK-801 blocking. E, Expression of KPI-APP isoforms is induced in neurons exposed to extrasynaptic but not to synaptic NMDAR activation. Immunocytochemical analysis of KPI-APP expression in cultured cortical neurons subjected to synaptic or extrasynaptic NMDAR activation for 24 h. Neurons were stained with an antibody raised against the neuronal marker MAP-2 (red), and KPI-APP protein expression was determined by using the monoclonal antibody directed against the KPI domain of APP (green). KPI-APP appeared colocalized (yellow) to neuronal cytosol and plasma membrane when images were overlaid. No expression of KPI-APP was detected in neurons exposed to Bic/4-AP for 24 h (synaptic protocol). Scale bars, 20 μm.

Figure 7.

Knockdown of CaMKIV reduces extrasynaptic NMDAR activation-induced KPI-APP expression in cortical neuron cultures. A, Cortical neuron cultures at 9 DIV were transfected with a mixture of two siRNA directed against CaMK IV using INTERFERin as transfection reagent. Two concentrations of siRNA (5 and 10 nm each) and two incubation times with siRNA/INTERFERin complex (4 and 24 h) were tested. For the scramble siRNA control, a single concentration (20 nm) was used. CaMK IV mRNA expression was measured by real-time PCR 48 h after transfection. Each column is the mean ± SD of six independent transfections (n = 6), and statistical analysis was performed by ANOVA followed by Bonferroni–Dunn's test (*p < 0.05, **p < 0.01 vs control). B, Cortical neuron cultures at 9 DIV were transfected with a mixture of two siRNAs (10 nm each) directed against CaMK IV. Different conditions of transfection were tested as described in A. Forty-eight hours after transfection, CaMK IV protein expression was evaluated by Western blot analysis. Each column is the mean ± SD of three independent transfections (n = 3), and statistical analysis was performed by ANOVA followed by Bonferroni-Dunn's test (*p < 0.05, **p < 0.01 vs control). C, Following preliminary experiments (A, B), neurons at 9 DIV were transfected with two siRNAs directed against CaMKIV (10 nm for each siRNA, 4 h of incubation). After three washings, neuron cultures were incubated for 48 h at 37°C and exposed to extrasynaptic NMDAR activation for 1, 3, and 6 h (extrasynaptic protocol). For each time, extrasynaptic treatment was compared with its respective control (non-siRNA-transfected neuron cultures). For each end-point, KPI-APP mRNA expression was measured by real-time PCR as described before. Each column is the mean ± SD of six independent experiment (n = 6). Statistical analysis was performed by ANOVA followed by Bonferroni–Dunn's test (*p < 0.05 vs control). D, Transfection of 20 μm nonsilencing FAM-labeled siRNA (green) in cultured cortical neurons at DIV12. Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 25 μm. E, Neuron cultures at DIV9 were transfected with two siRNAs directed against CaMKIV (10 nm for each siRNA, 4 h of incubation) or with a scrambled siRNA (20 nm). Forty-eight hours later, the cultures were exposed to extrasynaptic NMDAR activation for 12 and 24 h. Proteins were extracted in RIPA buffer in the presence of protease inhibitors for Western blot analysis (top). Quantification of band intensities was performed with ImageJ software (bottom). Each column is the mean ± SD from three immunoblots (n = 3). Statistical analysis was realized by ANOVA followed by Bonferroni–Dunn's test (n = 3; **p < 0.01 vs control).

Figure 8.

A, B, Extrasynaptic, but not synaptic, NMDAR activation downregulates the expression of two splicing factors, hnRNPA1 (A) and SC35 (B), at the mRNA level in cortical neuron cultures. Primary cultured cortical neurons at 12 DIV were treated according to the previously described synaptic protocol or extrasynaptic protocol for 1, 3, and 6 h. Extrasynaptic NMDAR activation was performed in neurons, transfected or not with siRNA directed against CaMKIV. At the end of each time point, total RNA was extracted from neurons and real-time PCR analysis was performed using primers specific for hnRNPA1 or SC35. Results are expressed as mean ± SD from three independent treatments with mRNA expression level in controls (Con) arbitrarily set at 100%. Statistical analysis was realized by ANOVA followed by Bonferroni–Dunn's test (n = 3; **p < 0.01 to control; #p < 0.05 to respective time of extrasynaptic treatment).

Figure 9.

Memantine dose-dependently inhibits extrasynaptic NMDAR-induced KPI-APP protein expression and Aβ production from cortical neuron cultures. A, Immunoblotting analysis of KPI-APP protein in cortical neuron cultures exposed for 24 h to Bic/4-AP (synaptic protocol), to Bic/4AP + MK-801 followed by three washings, to 7.5 μm NMDA, and to extrasynaptic NMDAR activation for 12 or 24 h. For extrasynaptic activation, neuron cultures were incubated in the presence or not of increasing doses of memantine (0.1, 1, and 10 μm). Blots were rehybridized with an anti-actin antibody to estimate the total amount of proteins loaded. Con, Control. B, Relative quantification of KPI-APP protein expression compared with actin from experiments presented in A. Densitometric analysis of the protein bands was performed with ImageJ software. Each column is the mean ± SD from three immunoblots (n = 3). Statistical analysis was realized by ANOVA followed by Bonferroni–Dunn's test (n = 3; **p < 0.01 vs control; ##p < 0.01 vs extrasynaptic NMDAR activation). C, Primary cultured cortical neurons at 12 DIV were treated for 24 h with DMEM (control), with 50 μm Bic + 2.5 mm 4-AP (synaptic protocol) or according to extrasynaptic protocol in the presence of increasing concentrations of memantine. After 24 h, conditioned culture media were harvested in the presence of a protease inhibitor mixture and finally desalted and concentrated 25-fold using Microcon columns (Amicon; Millipore) with a nominal molecular weight limit of 3 kDa (YM-3). Media were processed for measurement of Aβ(1-42) levels using a mouse/rat Aβ(1-42) ELISA kit, as detailed in Materials and Methods. Each column is the mean ± SD of nine independent treatments (n = 9), and statistical analysis was performed by ANOVA followed by Bonferroni-Dunn's post-test (**p < 0.01 vs control; ##p < 0.01 vs extrasynaptic NMDAR activation).

Figure 10.

Memantine at low or high doses inhibits the increase in KPI-APP expression in cortex of mice injected intraperitoneally with NMDA. Swiss mice received an intraperitoneal injection of saline vehicle or memantine (1 or 30 mg/kg). Thirty minutes after this first injection, animals received a second intraperitoneal injection of PBS, pH 7.4, or NMDA in a dose of 120 mg/kg. Mice were exposed to this treatment for 6 h (for RNA extraction) or 24 h (for protein extraction). A, Real-time PCR analysis of KPI-APP mRNA expression in cortical tissue of mice injected with 120 mg/kg NMDA. Total RNA was isolated from cortices by acidic phenol/chloroform extraction before to be reverse transcribed in cDNA. The expression level of APP was analyzed according to the ΔΔCt method with cyclophilin as the housekeeping gene. Results are representative of six independent experiments. Statistical analysis was realized by ANOVA followed by Bonferroni-Dunn's test (n = 6; **p < 0.01 vs control). B, Immunoblotting analysis of KPI-APP protein expression in cortical tissue of mice injected or not with 120 mg/kg NMDA. Blots were rehybridized with an anti-actin antibody to estimate the total amount of proteins loaded. Relative expression of KPI-APP compared with actin is presented in histogram below the blot. Each column is the mean ± SD from six immunoblots (n = 6). Statistical analysis was realized by ANOVA followed by Bonferroni–Dunn's test (n = 6; *p < 0.05 vs control). C, Real-time PCR analysis of KPI-APP mRNA expression in cortical tissue of mice 6 h after 120 mg/kg NMDA injection, with or without memantine (1 or 30 mg/kg). (n = 6; **p < 0.01 vs control; # p < 0.05 vs NMDA alone). D, Immunoblotting analysis of KPI-APP protein expression in cortical tissue of NMDA-injected mice with or without memantine (1 or 30 mg/kg). Relative expression of KPI-APP normalized with actin is presented in histogram below the blot. Each column is the mean ± SD from six immunoblots (n = 6). Statistical analysis was realized by ANOVA followed by Bonferroni–Dunn's test (n = 6; *p < 0.05 vs control; #p < 0.05 vs NMDA alone). E, Real-time PCR analysis of KPI-APP mRNA expression in cortical tissue of mice 6 h after injection of saline or memantine alone (1 or 30 mg/kg). F, Top, Immunoblotting analysis of KPI-APP protein expression in cortical tissue of mice 24 h after injection of saline or memantine alone (1 or 30 mg/kg). Blots were rehybridized with an anti-actin antibody to estimate the total amount of proteins loaded. Bottom, Relative quantification of KPI-APP protein expression compared with actin from experiments presented above. Densitometric analysis of the protein bands was performed with ImageJ software. Each column is the mean ± SD from three immunoblots (n = 3).