Article Figures & Data
Figures
- Figure 1 Patients' antibodies intravenously administered to pregnant mice reach fetal brain and bind to NMDAR
(A) Diagram of the experimental design. IV injection of pooled IgG was performed on gestational days E14, E15, and E16. Brain and blood samples from the fetus or offspring were collected on E16, E17, and E18 and PD 21, PD 70, and PD 155; blood samples of pregnant mice were collected on E16–E18. Brain tissue studies were performed on the same time points and electrophysiology on PD 21 and PD 70. Newborns were assessed daily for neurodevelopmental milestones from birth until PD 21. After breastfeeding withdrawal, mice underwent a battery of behavioral tests at age 1, 2, and 4 months. Gestational period is marked in pink and postnatal period in green. (B) Representative images of HEK293T cells expressing GluN1/2B immunolabeled with human IgG contained in serum of fetuses (E17) exposed to controls' or patients' IgG. Scale bar = 10 μm. (C and D) Representative human IgG immunolabeling in E17 fetal brain sections of animals exposed in utero to controls' (C) or patients' (D) IgG; the 2 insets (small squares in C and D, developing hippocampus) are shown enlarged on the right of each panel. Scale bar whole brain = 100 μm, insets = 10 μm. (E) Quantification of human IgG immunofluorescence intensity in the developing hippocampus of fetal brains obtained on gestational days E16-E18 exposed to controls' IgG or patients' IgG. Controls' IgG, E16, n = 5; E17, n = 13; E18, n = 8; patients' IgG, E16, n = 9; E17, n = 16; E18, n = 8. Median intensity of IgG immunofluorescence in brain of mice exposed to controls' IgG was defined as 100%. Data presented in box plots show the median, 25th, and 75th percentiles; whiskers indicate minimum and maximum. Significance of treatment effect was assessed by the Mann-Whitney U test. ****p < 0.0001. (F) Immunoprecipitated NMDAR-bound IgG from E17 and E18 brains of fetuses exposed in utero to controls' or patients' IgG. Lane brain tissue corresponds to homogenate of mouse brain used here as control. The band at ∼100 KDa corresponds to the GluN1 subunit of the NMDAR. Each lane corresponds to the immunoprecipitation of the brains from 4 fetuses, per each indicated condition. IgG = immunoglobulin G; NMDAR = NMDA receptor; PD = postnatal day.
- Figure 2 Intrauterine exposure to patients' IgG causes a transient reduction of cell-surface NMDARs and thinning of the cortical plate and cortical layers II–IV
(A–F) Quantification of total cell-surface (A–C) and synaptic (D–F) NMDAR cluster density in brains obtained on gestational days E16, E17, and E18 (A and D), PD 21 (B and E), and PD 70 (C and F) from animals exposed in utero to controls' IgG or patients' IgG. Median of NMDAR clusters in mice exposed to controls' IgG was defined as 100%. Controls' IgG, E16, n = 6; E17, n = 15; E18, n = 7; PD 21, n = 11; PD 70, n = 9; patients' IgG, E16, n = 13; E17, n = 21; E18, n = 5; PD 21, n = 14; PD 70, n = 10. (G) Representative cortical plate thickness (assessed with DAPI) in E17 and E18 brains of fetuses exposed in utero to controls' or patients' IgG. Scale bars = 100 μm. (H–I) Representative sections of the somatosensory cortex (H, PD 21; I, PD 70) of animals exposed in utero to controls' or patients' IgG, showing cortical layers II–IV (CUTL1 positive, green fluorescence) and cortical layer VI (FoxP2 positive, red fluorescence); arrows indicate the thickness of the indicated layers. Scale bars = 100 μm. (J) Quantification of cortical plate thickness in E16, E17, and E18 brains of fetuses exposed in utero to controls' IgG or patients' IgG. Controls' IgG, E16, n = 4; E17, n = 11; E18, n = 10; patients' IgG, E16, n = 8; E17, n = 14; E18, n = 8. (K and L) Quantification of the indicated cortical layers' thickness (K, PD 21; L, PD 70) in animals exposed to controls' IgG or patient's IgG. Controls' IgG, PD 21, n = 7; PD 70, n = 8; patients' IgG, PD 21, n = 15; PD 70, n = 10. For J–L, the mean thickness of the cortical plate or the indicated cortical layers in mice exposed in utero to controls' IgG was defined as 100%. Data presented in box plots show the median, 25th, and 75th percentiles; whiskers indicate minimum and maximum. Significance of treatment effect was assessed by the Mann-Whitney U test in A–F and independent sample t test in J–L. *p < 0.05, **p < 0.01, ****p < 0.0001. IgG = immunoglobulin G; NMDAR = NMDA receptor; PD = postnatal day.
- Figure 3 Intrauterine exposure to patients' IgG causes a reversible decrease in synaptic density and increase in activated microglia
(A) Representative z-stacks of brain sections obtained on PD 21 from mice exposed in utero to controls' or patients' IgG immunostained for Homer1 and Bassoon expression. Colocalization of both markers is defined as a glutamatergic synapse. Scale bar = 2 μm. (B) Quantification of synaptic density (Homer1/Bassoon colocalization) in brains obtained on PD 21 from animals exposed in utero to controls' IgG or patients' IgG. Median density of Homer1/Bassoon synapses in mice exposed in utero to controls' IgG was defined as 100%. Controls' IgG, n = 11; patients' IgG, n = 14. (C) Representative CD68 (macrophage specific) and Iba-1 (activated microglia specific) immunostainings in brains obtained on PD 21 from mice exposed in utero to controls' or patients' IgG. Scale bar = 20 μm. (D and E) Quantification of Iba-1 (D) and CD68 (E) immunostaining in brains obtained at PD 21 and PD 70 from mice exposed to controls' IgG or patients' IgG. Median surface density of immunostaining in mice exposed in utero to controls' IgG was defined as 100%. Controls' IgG, PD 21, n = 11; PD 70, n = 9; patients' IgG, PD 21, n = 14; PD 70, n = 10. Data presented in box plots show the median, 25th, and 75th percentiles; whiskers indicate the minimum and maximum. Significance of treatment effect was assessed by the Mann-Whitney U test. **p < 0.01, ****p < 0.0001. IgG = immunoglobulin G; PD = postnatal day.
- Figure 4 Intrauterine exposure to patients' IgG causes a transient increase in dendritic complexity and decrease in spine density
(A and B) Representative confocal image (A) and Sholl analysis (B) of a DiI-stained CA1 hippocampal pyramidal neuron showing in B the 10-μm spaced concentric rings used for quantification of dendritic complexity. Scale bar in A = 50 μm. (C and D) Number of dendrite intersections according to the indicated distance to the neuronal soma in cortical and hippocampal neurons examined at PD 21 and PD 70 of mice exposed in utero to controls' (Black) or patients' (blue) IgG. Number of mice exposed in utero to controls' IgG, PD 21, n = 6; PD 70, n = 4; number of mice exposed in utero to patients' IgG, PD 21, n = 6; PD 70, n = 4. Per each animal, condition, and brain region, 4 neurons were examined. Data presented as mean ± SEM. Significance of treatment effect was assessed by a generalized lineal model. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E–G) Representative images by confocal microscopy of a DiI-stained CA1 hippocampal pyramidal neuron (E) and a secondary apical dendrite (F), with morphologically classified dendritic spines (G, mushroom-shaped spines in green); Scale bar in E = 50 μm; scale bar in F = 5 μm. (H and I) Quantification of mushroom dendritic spine density in cortical and hippocampal neurons examined at PD 21 and PD 70 in mice exposed in utero to controls' or patients' IgG. The median density of mushroom spines (number of spines/μm) in mice exposed to controls' IgG was defined as 100%. Total number of dendrites examined/total number of mice exposed to controls' IgG: PD 21, cortex n = 122/6, hippocampus n = 120/6; PD 70, cortex n = 81/4, and hippocampus n = 81/4. Total dendrites/mice exposed to patients' IgG: PD 21, cortex n = 141/6, hippocampus n = 148/6; PD 70, cortex n = 70/4, and hippocampus n = 72/4. Data presented in box plots show the median, 25th, and 75th percentiles; whiskers indicate minimum and maximum. Significance of treatment effect was assessed by the Mann-Whitney U test in H and I. *p < 0.05. IgG = immunoglobulin G; PD = postnatal day.
- Figure 5 Intrauterine exposure to patients' IgG causes transient developmental delay and behavioral alterations in offspring
(A–D) Developmental milestone assessment (from birth to PD 21) in mice that were exposed in utero to controls' (Black) or patients' (blue) IgG including (A) body weight, (B) time needed for body righting using the surface righting reflex test (PD 1, 3, 5, 7, 9, and 11), (C) time needed for 180° turning to head up position using the negative geotaxis reflex (PD 2, 4, and 6), and (D) scored eye opening (PD 12–16). Number of mice exposed in utero to controls' IgG = 36; number of mice exposed to patients' IgG = 40. Data from A–C are represented as estimated mean ± SEM inferred by the GEE models described in Methods and in D are represented as median. Significance of treatment effect was assessed by a GEE adjusted model (A–C) and Mann-Whitney U test (D). *p < 0.05, ***p < 0.001. (E–L) Battery of behavioral tests performed at the indicated time points including novel object location index (E, 1 month; I, 2 months), nest building test (F, 1 month; J, 2 months), social interaction test (G, 1 month), rotarod test (H, 1 month), and tail suspension test (K, 2 months; L, 4 months). For E–I and K, number of mice exposed to controls' IgG = 18 and mice exposed to patients' IgG = 18. For J and L, number of mice exposed to controls' IgG = 8 and mice exposed to patients' IgG = 9. Data from panels E, F, and I–L are represented as box plots with median, 25th, and 75th percentiles; whiskers indicate minimum and maximum values; data from G and H are represented as mean ± SEM. For all behavioral tests, significance of treatment effect was assessed by GEE. *p < 0.05, **p < 0.01, ***p < 0.001. GEE = generalized estimated equation; IgG = immunoglobulin G; PD = postnatal day.
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