Fig 2: Experimental Design(A) Studies were performed in 4 experimental groups of mice that received a continuous infusion (days 1–14) of controls' or patients' CSF via subcutaneously implanted osmotic pumps connected to the cerebroventricular system along with daily subcutaneous injection of vehicle (no drug) or SGE-301 (10 mg/kg diluted in vehicle) from days 11 to 19. Note that before starting treatment on day 11, there are 2 experimental groups (mice infused with controls' CSF or patients' CSF). (B) Timing of memory and locomotor tasks. Baseline novel object location (NOL) and locomotor activity (LA) were obtained before the infusion of controls' or patients' CSF (days -2, -1). The same tests were applied on days 10–11, 13–14, 18–19, and 25–26 after onset of infusion of CSF. The effects of patients' antibodies on the levels of NMDARs or LTP were examined in subsets of mice killed on days 10–11, 18–19, or 26. LTP = long-term potentiation; NMDAR = NMDA receptor.

Fig 3: Treatment With SGE-301 Reverses the Reduction of NMDARs Caused by Patients' CSF in the Hippocampus(A) Hippocampus of the mouse immunolabeled for NMDAR (green) and PSD95 (red). Images were merged to demonstrate colocalizing clusters (defined as synaptic NMDAR, white color). The 15 small white squares indicate the analyzed areas in CA1, CA3, and dentate gyrus (5 each). Each square is a 3D stack of 50 sections. Scale bar = 400 µm. (B) Four magnified squares (3D projection) of a CA1 region of hippocampus representing the 4 experimental conditions and showing the analysis of density of total cell surface NMDAR, PSD95, and synaptic NMDAR clusters on day 18. The images (NMDAR, green; PSD95, red) were postprocessed and used to calculate the density of the clusters (density = spots/µm3). Scale bar = 5 µm. Quantification of the density of total surface NMDAR and synaptic NMDAR clusters on day 10 (C), day 18 (D), and day 26 (E) in a pooled analysis of the 15 hippocampal areas (CA1, CA3, and dentate gyrus) for each experimental condition. Mean density of clusters in animals treated with controls' CSF + vehicle was defined as 100%. For each condition, 5 animals were examined. Box plots show the median and 25th and 75th percentiles; whiskers indicate the minimum and maximum values. Significance of the treatment effect was assessed using the Kruskal-Wallis test, ****p < 0.0001; ***p < 0.001; **p = 0.008. NMDAR = NMDA receptor.

Fig 4: Patient and commercial GluN1 antibodies were similarly distributed after immunostaining. (A) Sagittal mouse brain sections immunostained for GluN1 with 2 patient cerebrospinal fluid (CSF) samples (top) or 2 commercial anti-GluN1 antibodies against C-terminal epitopes (bottom). The pattern of N-methyl-D-aspartate receptor (NMDAR) localization is similar, with the greatest intensity of immunoreactivity in the hippocampus and less in cortex, striatum, and cerebellum. Scale bar = 1mm. (B) Cellular localization in hippocampal dentate gyrus neurons stained with patient CSF (left, green in overlay) or commercial anti-GluN1 (middle, red in overlay), demonstrating colabeling of NMDAR clusters throughout the neuropil. Scale bar = 20µm.

Fig 5: Patient antibodies do not acutely antagonize the N-methyl-D-aspartate receptor (NMDAR). (A) Representative traces of a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)-mediated and NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) from whole cell patch clamp recordings of hippocampal neurons following treatment with control or patient cerebrospinal fluid (CSF) for 30 minutes or F(ab) fragments for 24 hours. Recordings were made in the presence of tetrodotoxin, picrotoxin, and 0mM Mg2+ to isolate dual glutamatergic currents. In the lower traces, amino-phosphonovaleric acid (APV) was added to block the NMDAR-mediated portion of the mEPSC. (B) Representative averaged NMDAR-mediated mEPSCs recorded from neurons in different treatment conditions. The difference between the 0mM Mg2+ condition and the 0mM Mg2+ plus APV condition is plotted, showing the NMDAR current. (C) Quantification of NMDAR current amplitude following treatment (n = 5–10 cells per condition). Amplitude was not significantly different: 2.14 ± 0.62pA, control CSF; 1.26 ± 0.34pA, patient CSF; 1.79 ± 0.55pA, F(ab) fragments; 1-way analysis of variance, p > 0.05.