Article Title: Lupus auto-antibodies act as positive allosteric modulators at NMDA receptors and induce spatial memory deficits
Figure Lengend Snippet: (A) Bar graphs (mean ± SEM) (from left to right, n = 6, 6, 8, 8, 6, 6) showing normalized whole-cell peak current amplitudes for diheteromeric NMDARs (N1/N2A-N2A or N1/N2B-N2B) or triheteromeric receptors (N1/N2A-N2B) at 10 μg/mL of B1 or G11 antibody (** p < 0.01, Mann-Whitney U test ). (B) A single charge reversal in the GluN2A DWEYS motif (DW D YS) (D285K) disrupts G11 binding. Representative images from immunocytochemistry of HEK293T cells not transfected (no DNA) or transfected with GluN1/GluN2A or with GluN1/GluN2A(D285K). Cells were stained with either B1 or G11 (10 μg/mL) (green, Alexa-488) under non-permeabilizing conditions with DAPI (blue) counterstain. Scale (white bar): 40 μm. (C) Quantification of mean fluorescence intensity in (B) (mean ± SEM) (n = 5, all conditions) (** p < 0.01, one-way ANOVA with post-hoc Tukey’s test ). (D) Single charge reversal prevents DNRAb (10 μg/mL) potentiation of whole cell currents (mean ± SEM, n = 5, all conditions). (E) Left , Current records of triheteromeric (N1/N2A-N2A(D285K)) or diheteromeric (N1/N2A(D285K)-N2A(D285K)) NMDARs. Currents displayed as in . Right , Bar graphs (mean ± SEM) (from left to right, n = 5, 6, 5, 7, 5, 6) showing normalized current amplitude for diheteromeric NMDARs (N1/N2A-N2A or N1/N2A(D285K)-N2A(D285K)) or triheteromeric receptors (N1/N2A-N2A(D285K)) at 10 μg/mL of B1 or G11 (* p < 0.05, Mann-Whitney U test ). (F) GluN2A specific antagonists are neuro-protective at moderate pathophysiological levels of DNRAbs. Immunocytochemistry of primary hippocampal neuronal cultures (DIV14) incubated either in control antibody (B1+vehicle) or in DNRAb (G11) at 10 μg/mL. G11 was incubated either alone (+vehicle), with GluN2A antagonists TCN-201 (not shown) or MPX-004, or with ifenprodil, a GluN2B negative allosteric modulator at 3 μM. Upper panels, representative images of hippocampal neurons stained with antibodies against neuronal-specific β-tubulin, Tubb3 (green, Alexa-488), and activated caspase-3 (red, Alexa-647), with DAPI (blue) counterstain. Lower panel, only activated caspase-3 channel is shown. Scale (white bar): 40 μm. (G) Quantification of immunocytochemistry results shown in (F). Proportion of DAPI and activated-caspase-3-positive cells divided by total DAPI cells (mean ± SEM) (from left to right, n = 11, 11, 7, 7, 9, 6) per treatment condition (* p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with post-hoc Tukey’s test ).
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, Binding Assay, Immunocytochemistry, Transfection, Staining, Fluorescence, Incubation