Journal: bioRxiv

Article Title: Age-dependent NMDA receptor function is regulated by the Amyloid Precursor Protein

doi: 10.1101/2022.07.20.500736

Figure Lengend Snippet: (a) Schematic diagram showing the locations of stimulating and recording electrodes in the hippocampus and in a CA1 pyramidal neuron for whole cell patch-clamp experiments. (b) Comparison of representative whole-cell patch-clamp recordings of pharmacologically isolated NMDAR EPSCs, normalized to the peak amplitude (in %), from infant, adult and aged mice, illustrating differences in deactivation kinetics. (c) The weighted time constant (τ weighted ) was calculated using the relative contribution of both slow/fast components of NMDAR EPSCs and reflects the overall deactivation kinetics. Results are expressed as the mean ± SEM (Kruskal Wallis followed by an Uncorrected Dunn’s multiple comparisons test using the adult group as reference, **p<0.01, n=28-44). (d) Time course of ifenprodil (5µM) effect on pharmacologically isolated NMDAR EPSC amplitude in CA1 pyramidal neurons, measured by whole-cell patch clamp in infant, adult and aged C57BL/6 wild-type mice. Results are expressed as the mean ± SEM (n=9-12). (e) Traces show NMDAR EPSCs recorded before (CTR) and after 30 min of Ifenprodil 5 µM perfusion (Ifen). (f) GluN2B contribution was calculated as the percentage of change in NMDAR EPSC amplitude after 30 min of ifenprodil perfusion. Results are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, *p<0.05, ****p<0.0001, n=9-12). (g) Schematic representation and immunoblotting analysis of PSD-enriched fractions from the hippocampal tissue of adult C57BL/6 wild-type mice. Membranes were immunoblotted with antibodies for GluN2B, PSD-95 and synaptophysin. PSD-fractions are enriched in PSD-95, whereas non-PSD fractions contain high levels of synaptophysin. (h) Representative western blot of hippocampal lysates subjected to biochemical fractionation to obtain PSD-enriched fractions from infant, adult and aged C57BL/6 mice. Membranes were immunoblotted with antibodies for GluN2A, GluN2B and PSD-95. (i, j) Results from PSD-enriched fractions were normalized with PSD-95 and are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, **p<0.01, n=5). (k) Results show the relative GluN2B/GluN2A in PSD-enriched fractions and are expressed as the mean ± SEM (Kruskal Wallis followed by an Uncorrected Dunn’s multiple comparisons test using the adult group as reference, *p<0.05, n=5).

Article Snippet: Incubation with primary antibodies was performed overnight at 4°C in a humidified chamber, with antibodies diluted in 3% BSA PBS: APP Y188 (1:100, ab32136, Abcam), GluN2B (1:100, AGC-003, Alomone), GluN2A (1:100, AGC-002, Alomone), PSD-95 (1:50, ADI-VAM-PS002-E, Enzo).

Techniques: Patch Clamp, Isolation, Western Blot, Fractionation

Journal: bioRxiv

Article Title: Age-dependent NMDA receptor function is regulated by the Amyloid Precursor Protein

doi: 10.1101/2022.07.20.500736

Figure Lengend Snippet: (a) A slow was calculated as the amplitude of the slow component of NMDAR EPSCs normalized to the total amplitude (%), measured by whole-cell patch-clamp recordings in CA1 hippocampal neurons from infant, adult and aged C57BL/6 mice. Results are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, **p<0.01,****p<0.0001, n=28-44). (b) Representative western of hippocampal lysates from infant, adult and aged C57BL/6 wild-type mice. Membranes were immunoblotted with antibodies for GluN2A, GluN2B and β-actin. (c) Results from blots as shown in (b) from hippocampal lysates were normalized with β-actin and are expressed as the mean ± SEM (Kruskal Wallis test followed by Uncorrected Dunn’s test using the adult group as reference, ****p<0.0001, n=8). (d) Results from blots as shown in (b) from hippocampal lysates were normalized with β-actin and are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, **p<0.01, *p<0.05, n=8). (e) Results from blots as shown in (b) from hippocampal lysates show the GluN2B/GluN2A relative ratio and are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, ****p<0.0001, n=8). (f) Representative western blot of hippocampal lysates from infant, adult and aged C57BL/6 wild-type mice immunoprecipitated for PSD-95. Membranes were immunoblotted with antibodies for GluN2A, GluN2B and PSD-95. (g, h) Results from blots as shown in (f) from PSD-95 immunoprecipitated samples were normalized with PSD-95 and are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, ****p<0.001, n=4). (i) Results from PSD-95 immunoprecipitated samples show the GluN2B/GluN2A relative ratio expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test using the adult group as reference, ****p<0.0001, n=4). (j) Representative western blot of prefrontal cortex human samples (21 to 89 years old). Membranes were immunoblotted with antibodies for GluN2B and GAPDH. (k) Linear regression graph calculated from blots as shown in (j) shows the variation in GluN2B relative levels (normalized with GAPDH) depending on the age of human subjects (n=12). Statistical analysis was performed using Pearson’s correlation (two-tailed p value). Dotted lines represent the 95% confidence intervals. The values obtained for 20–25-year-old subjects were used as reference.

Article Snippet: Incubation with primary antibodies was performed overnight at 4°C in a humidified chamber, with antibodies diluted in 3% BSA PBS: APP Y188 (1:100, ab32136, Abcam), GluN2B (1:100, AGC-003, Alomone), GluN2A (1:100, AGC-002, Alomone), PSD-95 (1:50, ADI-VAM-PS002-E, Enzo).

Techniques: Patch Clamp, Western Blot, Immunoprecipitation, Two Tailed Test

Journal: bioRxiv

Article Title: Age-dependent NMDA receptor function is regulated by the Amyloid Precursor Protein

doi: 10.1101/2022.07.20.500736

Figure Lengend Snippet: (a) Representative western blot of hippocampal PSD-enriched fractions from infant, adult and aged wild-type C57BL/6 mice. Membranes were immunoblotted with antibodies for APP and PSD-95. (b) APP levels were normalized with PSD-95 and are expressed as the mean ± SEM (Kruskal Wallis test followed by Uncorrected Dunn’s test for multiple comparisons using the adult group as reference, ***p<0.001, n=8). (c) Representative western blot of synaptosome fractions from the hippocampi of infant, adult and aged wild-type C57BL/6 mice immunoprecipitated for APP. Membranes were immunoblotted with antibodies for GluN2A, GluN2B and APP. (d) Representative western blot of postmortem brain tissue (prefrontal cortex) from human subjects (adult=22 and aged=89 years old) immunoprecipitated for APP. Membranes were immunoblotted with antibodies for GluN2B and APP. (e) Time course of NMDAR EPSC amplitude measured by whole-cell patch clamp in CA1 pyramidal neurons of infant C57BL/6 wild-type mice during 60 min of incubation with an antibody against the APP C-terminal (Y188). In the control condition, the antibody was heat inactivated (boiled Y188). Results are expressed as the mean ± SEM (n=6-10). The schematic diagram shows the strategy used to mask the APP C-terminal domain - the antibody was added to the intracellular solution in the patch pipette to diffuse into the intracellular space. (f) Traces show NMDAR EPSCs recorded at 20min (baseline) and 60min. The Y188 antibody was inside the patch pipette during the whole course of the experiment (60min). (g) The percentage of Y188-sensitive NMDAR EPSCs was determined for infant, adult and aged C57BL/6 wild-type mice. The effect was calculated comparing the baseline amplitude (15-20 min) with the final amplitude (60 min) and normalized with the control condition (boiled Y188) for each age. Results are expressed as the mean ± SEM (One-way ANOVA followed by an Uncorrected Fisher’s LSD’s multiple comparisons test, ***p<0.001, n=8-14). (h) Time course of NMDAR EPSC amplitude measured by whole-cell patch clamp in CA1 pyramidal neurons of infant C57BL/6 wild-type mice during 90 min of incubation with the Y188 antibody and perfusion with ifenprodil (5µM) at 60-90 min. In the control condition, the antibody was heat inactivated (boiled Y188) (n=1). The schematic diagram shows the strategy used to block APP (Y188 antibody in the patch pipette) and to inhibit GluN2B-NMDAR (ifenprodil perfusion). (i) Traces show NMDAR EPSCs recorded at 20min (baseline), 60min and 90min. The Y188 antibody was inside the patch pipette during the whole course of the experiment (60min), whereas Ifenprodil perfusion occurred from 60 to 90min. (j) The percentage of ifenprodil-sensitive NMDAR EPSCs in infant mice was calculated comparing the amplitude at 60 min with the final amplitude (90 min). The effect of ifenprodil on NMDAR EPSCs was calculated in neurons without antibody incubation (No antibody, used as reference), incubated with the Y188 antibody (Y188) or the heat-inactivated antibody (boiled Y188) for 90 min. Results are expressed as the mean ± SEM (One-way ANOVA followed by Tukey’s multiple comparisons test, **p<0.01, *p<0.05, n= 4-6).

Article Snippet: Incubation with primary antibodies was performed overnight at 4°C in a humidified chamber, with antibodies diluted in 3% BSA PBS: APP Y188 (1:100, ab32136, Abcam), GluN2B (1:100, AGC-003, Alomone), GluN2A (1:100, AGC-002, Alomone), PSD-95 (1:50, ADI-VAM-PS002-E, Enzo).

Techniques: Western Blot, Immunoprecipitation, Patch Clamp, Incubation, Transferring, Blocking Assay

Journal: bioRxiv

Article Title: Age-dependent NMDA receptor function is regulated by the Amyloid Precursor Protein

doi: 10.1101/2022.07.20.500736

Figure Lengend Snippet: (a) Representative immunocytochemistry analysis of APP immunofluorescence in rat primary neuronal cultures (14 days in vitro (DIV)) transfected with shAPP or the respective control (shCTR) at DIV7, as indicated in the timeline. mCherry (reporter plasmid) is shown in red, APP is labelled in green and cell nuclei are stained with Hoechst in blue. Transfected neurons are indicated by arrows. (b) APP immunoreactivity (%) in transfected neurons is expressed as the mean ± SEM, using the control condition as reference (Mann-Whitney test, ****p<0.0001, n=20-21 cells, 3 independent cultures). (c) Representative immunocytochemistry analysis of rat primary neuronal cultures (DIV14) transfected with shAPP or the respective control (shCTR) at DIV7. mCherry (reporter plasmid), labelled in red, was used to identify dendrites of transfected neurons. GluN2B is shown in magenta and PSD-95 is labelled in green. Higher magnification images are shown at the bottom, with arrows indicating GluN2B/PSD95 co-localization. (d, e) Results of analysis of GluN2B synaptic content (GluN2B – PSD 95 co-localization) and area from dendrites of transfected neurons are expressed as the mean ± SEM, using the control condition as reference (%) (Mann-Whitney test, *p<0.05, n= 39, 3 independent cultures) (f) Results of analysis of PSD95 area from dendrites of transfected neurons are expressed as the mean ± SEM, using the control condition as reference (%) (Unpaired t-test, *p<0.05, n= 39, 3 independent cultures).

Article Snippet: Incubation with primary antibodies was performed overnight at 4°C in a humidified chamber, with antibodies diluted in 3% BSA PBS: APP Y188 (1:100, ab32136, Abcam), GluN2B (1:100, AGC-003, Alomone), GluN2A (1:100, AGC-002, Alomone), PSD-95 (1:50, ADI-VAM-PS002-E, Enzo).

Techniques: Immunocytochemistry, Immunofluorescence, In Vitro, Transfection, Plasmid Preparation, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Age-dependent NMDA receptor function is regulated by the Amyloid Precursor Protein

doi: 10.1101/2022.07.20.500736

Figure Lengend Snippet: (a, b) Quantification of GluN2B average cluster size and fluorescence density in primary neuronal cultures (DIV14) transfected with shAPP or the respective control (shCTR) at DIV7. Results are expressed as the mean ± SEM, using the control condition as reference (%) (Mann-Whitney test, n=39 dendrites, 3 independent cultures). (c) Quantification of PSD-95 average cluster size in primary neuronal cultures transfected with shAPP or the respective control (shCTR). Results are expressed as the mean ± SEM, using the control condition as reference (%) (Unpaired t-test, n=39 dendrites, 3 independent cultures, **p<0.01). (d) Quantification of the relative PSD-95 fluorescence density in primary neuronal cultures transfected with shAPP or the respective control (shCTR). Results are expressed as the mean ± SEM, using the control condition as reference (%) (Mann-Whitney test, n=39 dendrites, 3 independent cultures).

Article Snippet: Incubation with primary antibodies was performed overnight at 4°C in a humidified chamber, with antibodies diluted in 3% BSA PBS: APP Y188 (1:100, ab32136, Abcam), GluN2B (1:100, AGC-003, Alomone), GluN2A (1:100, AGC-002, Alomone), PSD-95 (1:50, ADI-VAM-PS002-E, Enzo).

Techniques: Fluorescence, Transfection, MANN-WHITNEY