Early developmental deletion of forebrain Ank2 causes seizure-related phenotypes by reshaping the synaptic proteome

Abstract

Rare genetic variants in ANK2, which encodes ankyrin-B, are associated with neurodevelopmental disorders (NDDs); however, their pathogenesis is poorly understood. We find that mice with prenatal deletion in cortical excitatory neurons and oligodendrocytes (Ank2^-/-:Emx1-Cre), but not with adolescent deletion in forebrain excitatory neurons (Ank2^-/-:CaMKIIα-Cre), display severe spontaneous seizures, increased mortality, hyperactivity, and social deficits. Calcium imaging of cortical slices from Ank2^-/-:Emx1-Cre mice shows increased neuronal calcium event amplitude and frequency, along with network hyperexcitability and hypersynchrony. Quantitative proteomic analysis of cortical synaptic membranes reveals upregulation of dendritic spine plasticity-regulatory proteins and downregulation of intermediate filaments. Characterization of the ankyrin-B interactome identifies interactors associated with autism and epilepsy risk factors and synaptic proteins. The AMPA receptor antagonist, perampanel, restores cortical neuronal activity and partially rescues survival in Ank2^-/-:Emx1-Cre mice. Our findings suggest that synaptic proteome alterations resulting from Ank2 deletion impair neuronal activity and synchrony, leading to NDDs-related behavioral impairments.

Keywords: CP: Developmental biology; CP: Neuroscience; LC-MS/MS; ankyrin; anti-epileptic drug; calcium imaging; postsynaptic interactome; proteomics; synchrony.

Figure 1.. Deletion of ankyrin-B in the forebrain during early development, but not in adolescent forebrain pyramidal neurons, leads to spontaneous seizures, juvenile mortality, hyperactivity, and impaired social behavior

(A) Representative Kaplan-Meier survival curves. ANK2^fl/fl, n = 66; ANK2^+/−:Emx1-Cre, n = 42; ANK2^−/−:Emx1-Cre, n = 73; ANK2^+/−:NEX-Cre, n = 7; ANK2^−/−:NEX-Cre, n = 16; ANK2^−/−:CaMKIIα-Cre, n = 36 (male and female). (B) Time point of seizure onset and death analyzed from long-term recordings. ANK2^fl/fl, n = 13; ANK2^−/−:Emx1-Cre, n = 5 (male and female). (C) Number of spontaneous seizures per hour. (D) Duration per seizure. Data were analyzed from the recorded movie files (B); ***p < 0.001; two-tailed unpaired t test. (E) Activity in open field test for 30 min (male and female). ***p < 0.001. (F) Quantification of time spent on social side (novel) or non-social side (empty) of social approach apparatus. (G) Time spent with familiar subject or novel subject.ANK2^fl/fl, n = 23–26; ANK2^+/−:Emx1-Cre (HT), n = 13–15; ANK2^−/−:Emx1-Cre (cKO), n = 15–20 (male and female); **p < 0.01; one-way ANOVA followed by a Bonferroni test. (H–J) Quantification of activity in open field (H), sociability (I), and social novelty (J) in ANK2^−/−:CaMKIIα-Cre (cKO) (male) (ANK2^fl/fl, n = 11; cKO, n = 10). All data are represented as mean ± SEM. See also Figures S2 and S3.

Figure 2.. Increased neuronal network activity and synchrony in cortical slices of ANK2^−/−:Emx1-Cre mice

(A) Schematic of the imaging of layer 2/3 in S1 somatosensory area and representative images using the Cal520/Sr101 dyes in ANK2^fl/fl and ANK2^−/−:Emx1-Cre brain slices. (B) Representative traces of 10 min recordings in ANK2^fl/fl and ANK2^−/−:Emx1-Cre mice slices in low-Mg aCSF. (C–E) Ca²⁺ event frequency (C), amplitude (D), and half-width (E) in ANK2^−/−:Emx1-Cre and ANK2^fl/fl slices. (F) Representative raster plots of the detected Ca²⁺ events during a 30-min recording session. (G) The number of neuronal co-activations normalized from 0 to 1 and color coded in a heatmap illustrates the higher degree of functional connectivity between neurons in ANK2^−/−:Emx1-Cre mice. (H and I) Maps of pairwise correlation coefficients R show an increased correlation between pairs of neurons in ANK2^−/−:Emx1-Cre vs. ANK2^fl/fl slices. (J) Network events are more frequent in ANK2^−/−:Emx1-Cre compared to ANK2^fl/fl slices. (K) ANK2^−/−:Emx1-Cre slices show higher percentage of co-active neurons during network events than ANK2^fl/fl. n = number of slices, six in ANK2^fl/fl, seven in ANK2^−/−:Emx1-Cre from three animals per genotype (male and female). Two-tailed unpaired t test was performed. *p < 0.05, **p < 0.01, ***p < 0.001. All data are represented as mean ± SEM. See also Figure S4.

Figure 3.. The proteome is remodeled in the cortical synapse of ANK2^−/−:Emx1-Cre mice

(A) Scheme of experimental workflow for 10 plex TMT LC-MS3 multinotch analysis from the cortex of ANK2^−/−:Emx1-Cre vs. ANK2^fl/fl male mice. (B) Volcano plot showing the protein level fold change relative to significance between ANK2^fl/fl and ANK2^−/−:Emx1-Cre mice. ANK2^fl/fl, n = 5; ANK2^−/−:Emx1-Cre, n = 5. Significantly upregulated proteins are in pink (p < 0.05), significantly downregulated proteins are in green (p < 0.05), and the other proteins are in gray. (C) Graph showing the significance of either upregulated or downregulated proteins present in psychiatric risk genes. Significance was tested by hypergeometric tests. Gene ontology (GO) analysis of biological processes by SynGO. (D) Scheme of the dysregulated proteomics in synapses. (E) Representative images of western blot from P2 fractionated samples. The graph shows relative abundance of proteins in cortex of 3-week-old ANK2^fl/fl and ANK2^−/−:Emx1-Cre mice (n = 4 per each group). *p < 0.05; **p < 0.01; two-tailed unpaired t test was performed. All data are represented as mean ± SEM. See also Figure S6. SFARI, Simons Foundation Autism Research Initiative; ASD, autism spectrum disorder, SZ, schizophrenia; BD, bipolar disorder.

Figure 4.. Mapping of the ankyrin-B interactome identifies synaptic protein partners

(A) Scheme of experimental workflow for the identification of proteins immunoprecipitated with an anti-ankyrin-B antibody from homogenized cortex using LC-MS/MS (n = 3 per each group). (B) Scatterplot showing the 78 identified proteins from IP-LC-MS/MS. Each sample set was analyzed by the t test (p < 0.05), and corresponding proteins (2-foldmore than IgG) are depicted in blue. (C) Enrichment analysis of ankyrin-B-interacting proteins in neurodevelopmental and psychiatric disorder risk factors. Significance was analyzed by hypergeometric tests. (D) Analysis of enrichment in synaptic cellular components in the interactome by SynGO. (E) Scheme of ankyrin-B in teractome at synapses. Epilepsy risk factors in blue letters and ASD risk factors in red borders are indicated. (F and G) Co-immunoprecipitation experiments with anti-ankyrin-B or anti-Shank3 (F) and anti-ankyrin-B or anti-GluA2 (G) from the P2 fraction. IgG, control IgG; IP, immunoprecipitation. See also Figure S8. SFARI, Simons Foundation Autism Research Initiative; ASD, autism spectrum disorder; SZ, schizophrenia; BD, bipolar disorder.

Figure 5.. Perampanel, an AMPA receptor antagonist, restores neuronal network activity and improves survival of ANK2^−/−:Emx1-Cre mice

(A) Representative heatmap of the effects of increasing doses of perampanel on cortical neuronal activity in brain slice. (B) Representative traces of three neurons in ANK2^fl/fl and ANK2^−/−:Emx1-Cre brain slices treated with DMSO (vehicle) or perampanel (10 μM). (C–E) number of events (C), amplitude (D), and percentage of co-active cells per network (E) of Ca²⁺ events in ANK2^−/−:Emx1-Cre brain slices compared to ANK2^fl/fl slices treated with 10 μM of perampanel. (F) Survival curve of ANK2^−/−:Emx1-Cre mice (male and female) chronically injected with perampanel (0.2 mg/kg). two-way ANOVA, Tukey post hoc test was performed. *p < 0.05, **p < 0.01, ***p < 0.001. All data are represented as mean ± SEM. See also Figure S9.