Journal: Nature Communications
Article Title: Lupus autoantibodies act as positive allosteric modulators at GluN2A-containing NMDA receptors and impair spatial memory
Figure Lengend Snippet: a Bar graphs (mean ± SEM) (left to right, n = 6, 6, 8, 8, 6, 6 cells recorded) showing normalized whole-cell peak current amplitudes for diheteromeric (N1/N2A-N2A or N1/N2B-N2B) or triheteromeric (N1/N2A-N2B) receptors at 10 µg/mL of B1 or G11 antibody (* *p < 0.01, two-sided Mann–Whitney U test) (left to right, p = 0.00507, 0.00865, 0.298; for ns, p = 0.581). b Representative images from immunocytochemistry of HEK293T cells not transfected (no DNA) or transfected with GluN1/GluN2A or GluN1/GluN2A (D285K), stained with B1 or G11 (10 μg/mL) (green, Alexa-488) and DAPI (blue) counterstain. Scale (white bar): 40 µm. c Mean fluorescence intensity in (B) (mean ± SEM) ( n = 5 coverslips of cells, all conditions) (* **p < 0.001, one-way ANOVA with post-hoc Tukey’s test) (Tukey’s: no DNA vs hN2A, p = 0.000156; hN2A vs hN2A(D285K), p = 0.000348). d Single charge reversal prevents DNRAb (10 μg/mL) potentiation of whole-cell currents (mean ± SEM, n = 5 recorded cells, all conditions). e Left, current records of triheteromeric (N1/N2A-N2A(D285K)) or diheteromeric (N1/N2A(D285K)-N2A(D285K)) NMDARs. Currents displayed as in Fig. . Right, bar graphs (mean ± SEM) (left to right, n = 5, 6, 5, 7, 5, 6 recorded cells) showing normalized current amplitude for diheteromeric and triheteromeric receptors at 10 µg/mL of B1 or G11 ( *p < 0.05, ** p < 0.01, two-sided Mann–Whitney U test) (left to right, p = 0.0358, 0.00577, 0.194). f Immunocytochemistry of DIV14 primary hippocampal cultures incubated either in control (B1 + vehicle) or in DNRAb (G11) at 10 µg/mL. G11 was incubated either alone (+vehicle), with TCN-201 (not shown), MPX-004, or ifenprodil at 3 µM. Upper panels, representative images of neurons stained with antibodies against neuronal β-tubulin, Tubb3 (green, Alexa-488), and activated caspase-3 (red, Alexa-647), with DAPI (blue). Lower panel, only activated caspase-3 channel. Scale (white bar): 40 µm. g Quantification of immunocytochemistry shown in ( f ). Proportion of DAPI and activated caspase-3-positive cells divided by total DAPI cells (mean ± SEM) (left to right, n = 11, 11, 7, 7, 9, 6 coverslips of cells) per treatment condition ( *p < 0.05 ,**p < 0.01, *** p < 0.001, one-way ANOVA with post-hoc Tukey’s test) (Tukey’s: G11 + vs G11 + MPX-004, p = 0.00282).
Article Snippet: Final concentrations of antagonists were 3 µM: TCN-201 (AdooQ Bioscience, A11947), MPX-004 (Alomone Labs, M280), and Ifenprodil (Sigma, I2892).
Techniques: MANN-WHITNEY, Immunocytochemistry, Transfection, Staining, Fluorescence, Incubation