In utero exposure to endogenous maternal polyclonal anti-Caspr2 antibody leads to behavioral abnormalities resembling autism spectrum disorder in male mice

Abstract

The concept that exposure in utero to maternal anti-brain antibodies contributes to the development of autism spectrum disorders (ASD) has been entertained for over a decade. We determined that antibodies targeting Caspr2 are present at high frequency in mothers with brain-reactive serology and a child with ASD, and further demonstrated that exposure in utero to a monoclonal anti-Caspr2 antibody, derived from a mother of an ASD child, led to an-ASD like phenotype in male offspring. Now we propose a new model to study the effects of in utero exposure to anti-Caspr2 antibody. Dams immunized with the extracellular portion of Caspr2 express anti-Caspr2 antibodies throughout gestation to better mimic the human condition. Male but not female mice born to dams harboring polyclonal anti-Caspr2 antibodies showed abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as repetitive behaviors and impairments in novelty interest in the social preference test as adults. These data supporting the pathogenicity of anti-Caspr2 antibodies are consistent with the concept that anti-brain antibodies present in women during gestation can alter fetal brain development, and confirm that males are peculiarly susceptible.

Figure 1

Serum from Caspr2 immunized mice binds Caspr2. (A) IgG from serum of Caspr2 immunized mice, but not from Control mice, co-localized with human (upper panel) and mouse (second panel) Caspr2 on HEK 293T, and on HEK GnTI- (third panel) cells expressing turbo-green florescent protein (tGFP)-Caspr2. Serum of Control immunized mouse showed no binding (bottom panel). No staining was seen on cells expressing only tGFP or non-transfected cells (data not shown). (B) Serum from E18.5 fetuses of Caspr2-immunized mice bound both human (upper panel) and mouse (middle panel) Caspr2 measured by a cell based assay. Serum from Control immunized mice showed no binding (lower panel). (C) Serum from Caspr2-immunized mice show reduced staining in the hippocampal CA1 region of Caspr2^−/− compared to Caspr2^+/+mice.

Figure 2

Male mice born to dams immunized with Caspr2 show cortical abnormalities. (A-B) Male fetuses (E15.5) from dams immunized with Caspr2 (Anti-Caspr2) display (A) a reduced ratio of cortical plate (CP) to cortical width (CW) and (B) a decreased number of proliferating cells in the ventricular zone compared to male fetuses of Control dams. (A) Left, DAPI staining. White and yellow lines indicate CP, and CW, respectively. Right, quantification of the ratio of CP to CW. Mann Whitney, Male U = 1 *P < 0.05, Female U = 5, n.s. (B) Left, PH3 + staining of mitotic cells. Right, quantification of PH3 + . Mann Whitney, Male U = 1 *P < 0.05, Female U = 7, n.s (A,B) Male, Control n = 6, Anti-Caspr2 n = 4; Female, Control, Anti-Caspr2, n = 4; 3 litters each. (C) Anti-Caspr2 male fetuses (E15.5) display a reduced number of DAPI positive cells across the cortex. Control n = 10, Anti-Caspr2 n = 7, 3 litters each. t-test, t(15) = 2.433, * P < 0.05. (D) Analysis of the average of counts from 2 to 4 sections per male mouse of NeuN positive neurons in the deep layers of the entorhinal cortex . Dots represent number of NeuN positive cells counts in a section. Control n = 7, 5 litters, Anti-Caspr2 n = 7, 4 litters. t-test, t(12) = 2.929, *P < 0.05.

Figure 3

Male mice born to dams immunized with Caspr2 showed reduced dendritic arborization in pyramidal neurons and a reduced number of parvalbumin (PV) GABAergic interneurons. (A) Analysis of dendritic complexity in adult male mice exposed in utero to anti-Caspr2 (Anti-Caspr2, n = 5, 3 litters) or Control IgG (Control, n = 5, 4 litters). Left, tracings of CA1 neurons using Neurolucida360 (MBF, Williston VT), visualized with the Golgi method of silver staining. Right, Sholl analysis, male mice exposed in utero to Anti-Caspr2 IgG show decreased length of dendrites. Number of neurons = 44 per group. Mixed model linear analysis, P < 0.0001, ICC = 8%. (B) Analysis of the average number of synaptic dendritic spines per length in each mouse show reduced density of spines in CA1 neurons in mice exposed in utero to anti-Caspr2 IgG. Dots represent individual dendrites from which spines were counted, t-test, t(11) = 3.389, * P < 0.01. (Control, Anti-Caspr2, n = 4, 3 litters per group). (C) Analysis of the average counts of PV + GABAergic interneurons in the CA1 region. Dots represent number of individual PV + per section. t-test, t(9) = 6.639, * *P < 0.001, (Control n = 6, Anti-Caspr2 n = 5, 4 litters).

Figure 4

Behavioral phenotype of male mice born to dams harboring anti-Caspr2 IgG. (A) Grooming. Time spent grooming was recorded during two independent 15 min sessions and scored automatically using Ethovision software. Male mice born to Caspr2 immunized dams (Anti-Caspr2) spent overall more time grooming than male mice born to Control dams (Control). Control n = 19, Anti-Caspr2, n = 18, t-test, t(35) = 2.628, *P < 0.05. (B) Marble burying task. Anti-Caspr2 male mice buried more marbles than Control. Control, n = 22, Anti-Caspr2, n = 19, Mann Whitney, U = 66.5. ***P < 0.0001 (C) Sociability test. Control and Anti-Caspr2 offspring displayed a normal sociability defined as spending more time sniffing a mouse compared to an object. Control n = 20, Anti-Caspr2, n = 19. Two way repeated measures ANOVA, Immunization x Sociability Interaction: F (1, 37) = 7.828, P < 0.05, followed by Bonferroni post hoc test *P < 0.05, ***P < 0.0001. (D) Social Novelty test. Control offspring displayed a normal social novelty defined as spending more time sniffing a novel (Nov) mouse compared to a familiar (Fam) mouse, whereas offspring from Caspr2 immunized mice spent a similar time sniffing the two mice. Control n = 18, Anti-Caspr2, n = 18. Two way repeated measures ANOVA, Immunization × Novelty Interaction: F (1, 34) = 5.508, P < 0.05, followed by Bonferroni post hoc test *P < 0.05. (E) Distance traveled during the social Novelty test. Control and Anti-Caspr2 offspring did not show differences in total distance traveled during the social preference test Control n = 18, Anti-Caspr2, n = 18. t-test, t (34) = 0.07608, n.s. (A–E) Control, 9 litters, Anti-Caspr2, 7 litters, from two independent experiments. *P < 0.05, ***P < 0.001. Mean ± SEM.