Figure 6.

RhuMab SSM5 reduces NMDAR‐mediated currents in vitro and induces severe memory deficits in mice. (A, B) RhuMab SSM5 reduces NMDAR‐mediated currents in transfected Xenopus oocytes. (A) Representative current traces measured in Xenopus oocytes expressing GluN1‐1a plus GluN2B in response to superfusion with a solution containing 100 μmol/L glutamate plus 10 μmol/L glycine in Ba²⁺ Ringer. All currents were measured at −70 mV. GluN1‐1a/GluN2B expressing oocytes were preincubated with 4 μg/mL patient‐derived rhuMab in bath medium for 1 h prior to measurement (“antibody”), whereas GluN1‐1a/GluN2B expressing control oocytes were not preincubated with antibody (“control”). Control oocytes were treated the same way, either preincubated with patient rhuMab (“uninjected, antibody,” n = 5) or not preincubated (“uninjected,” n = 7). Uninjected oocytes revealed no measurable currents. (B) GluN1‐1a/GluN2B amplitudes in oocytes either without (“control”) or with (“antibody”) preincubation with autoantibody were measured and are shown normalized to control currents. Data are given as mean ± SEM. Statistical testing was performed by two‐tailed t‐test with an α error of 0.05. Significant difference is indicated by *P < 0.05; n indicates number of oocytes measured. (C–E) Cerebroventricular infusion of anti‐NMDAR rhuMab SSM5 causes severe reversible memory deficits. (C) Infusion of rhuMab SSM5 into the cerebroventricular system of mice for a total of 14 days causes deficits of memory. Novel object recognition index in mice treated with the patient‐derived rhuMab (SSM5, red circles) and the control rhuMab (12D7, green circles) (each at 90 μg/mL in saline solution). A high index indicates better object recognition memory. Note that mice infused with patient‐derived rhuMab showed a progressive decrease of memory that was maximal on Day 18. The total time of exploration of both objects was similar in both groups (not shown). Data are presented as mean ± SEM. Number of animals: SSM5: n = 7, 12D7: n = 7. Significance of treatment effect was assessed by two‐way ANOVA with an α error of 0.05 and post hoc testing with Bonferroni adjustment (asterisks). ***P < 0.001. (D, E) Animals infused with rhuMab SSM5 have a progressive increase of human IgG bound to hippocampus. (D) Immunostaining of human IgG in sagittal brain sections showing the hippocampus of representative animals infused with anti‐NMDAR monoclonal antibody (SSM5) (right panels) and control antibody (12D7) (left panels), sacrificed at the indicated experimental days. In animals infused with the SSM5 antibody, a gradual increase of IgG immunostaining until Day 18, followed by decrease of immunostaining is observed. Scale bar represents 200 μm. (E) Quantification of intensity of human IgG immunolabeling in hippocampus of mice infused with SSM5 antibody (dark gray columns) and control CSF (pale gray columns) sacrificed at the indicated time points. Mean intensity of IgG immunostaining in the group with the highest value (animals treated with anti‐NMDAR monoclonal antibody (SSM5) and sacrificed at Day 18) was defined as 100%. Data are presented as mean ± SEM. Number of animals: 5 animals per condition and time point. Significance of treatment effect was assessed by two‐way ANOVA with an α error of 0.05 (o) and post hoc testing with Bonferroni adjustment (*). °°°P < 0.001, **P < 0.01, ****P < 0.0001.