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FIGURE 1
Summary of reported AP3B2 mutations and inheritance patterns in patients. (a) Schematic representation of the human AP3B2 protein showing the locations and types of reported pathogenic variants. The conserved adaptin N terminal (Adaptin_N) and clathrin-adaptor complex-3 beta-1 subunit C-terminal (AP3B1_C) domains are indicated, with frameshift (green squares), missense (blue circle), nonsense (pink triangles), and splice-site (orange diamonds) mutations annotated along the protein. (b) Summary of reported inheritance patterns across 24 documented cases, 75% were homozygous for AP3B2 variants, while 25% are compound heterozygous. The DEE48 pathogenic variants in AP3B2 shown here were described in Assoum et al. (7), Anazi et al. (9), Dilber et al. (8), and Alizadeh et al. (10). |
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FIGURE 2
CRISPR/Cas9 disruption of ap3b2 triggers premature protein truncation resulting in hyperactivity and seizure-like behavior in CRISPants. (a) Schematic of the X. laevis Ap3b2. S protein showing the conserved Adaptin_N and AP3B1_C domains. sgRNA2 target site is indicated, along with representative Sanger sequencing traces show degradation at the sgRNA2 cut site. (b) Predicted protein outcomes for the three most observed indels (−7, −11, and −12 bp deletions) generated by sgRNA2. (c) Representative frames from behavioral recordings at 50 fps of an ap3b2 CRISPant tadpole. Frames highlighted in purple boxes show characteristic seizure-related C-shaped darting behavior observed in CRISPants. (d,e) Comparison of (d) mean swim velocity (mm/s) and (e) time spent in darting behavior (%) between ap3b2 CRISPants (N = 41) compared with sibling controls (N = 48), unpaired t-test with Mann–Whitney, *p < 0.05. Horizontal bars indicate group means and error bars denote SEM. (f) Summary of CRISPR/Cas9 editing outcomes in 7 arbitrarily selected ap3b2 CRISPant embryos from the same batch used in the behavior experiments, confirmed by Sanger sequencing and TIDE analysis. Raw count data and statistical analysis can be found in Supplementary File S1. |
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FIGURE 3
ap3b2 CRISPant brains have elevated spontaneous Ca2+ activity compared to unedited controls in CRISPants. (a) Representative still frames acquired every 10 s from ap3b2 CRISPant tadpole brain during in vivo widefield Ca2+ imaging. GCaMP6s fluorescence intensity (lighter shades indicate higher ΔF/F₀%) is shown. Forebrain (FB) and midbrain (MB) hemispheres are outlined; the hindbrain lies outside the field of view. Time (s) is indicated in each frame. (b) Representative raw and high-pass-filtered ΔF/F₀% traces from a control tadpole (left) and an ap3b2.S CRISPant (right). Significant Ca2+ events were detected using a global threshold defined as 3 × SD of all filtered control traces (black line); events exceeding this cutoff are marked by arrowheads. The red box indicates the time window in panel (a). (c,d) Comparison of Ca2+ event counts (c) and significant event amplitude (ΔF/F₀%) (d) in control (N = 12) and CRISPant tadpoles (N = 13). Individual data points represent single tadpoles; horizontal bars denote group means and error bars indicate SEM. Groups were compared using unpaired t-test with Mann Whitney, *p < 0.05, ***p < 0.001. (e) FFT-derived power spectral densities of whole-brain Ca2+ signals for control and ap3b2 CRISPant tadpoles. Solid lines represent group means and shaded envelopes indicate 95% confidence intervals. (f) Integrated low-frequency spectral power (0.01–1 Hz; (ΔF/F₀)2/Hz, calculated as the area under the power spectrum for each tadpole. Data are from control (N = 12) and ap3b2.S CRISPant tadpoles (N = 13). Individual data points represent single tadpoles; horizontal bars denote group means and error bars indicate SEM. Groups were compared using unpaired t-test with Mann–Whitney correction, ***p < 0.001, ****p < 0.0001. (g) Summary of CRISPR/Cas9 editing outcomes in the CRISPant group, determined by Sanger sequencing and TIDE analysis. Raw count data and full statistical details are provided in Supplementary File S1. |
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FIGURE 4
ap3b2 CRISPants exhibit increased interhemispheric synchrony in the midbrain and forebrain. (a) Schematic example illustrating the centroid-based regions of interest (ROIs) used for interregional correlation analysis. Fixed-area central ROIs (500 pixels each) were positioned at the geometric centroids of the left and right midbrain (MB), left and right forebrain (FB). (b,c) Group-averaged Pearson correlation coefficient matrices for unedited control tadpoles (b, N = 12) and ap3b2 CRISPants (c, N = 13). (d,e) Comparison of brain regional synchrony, quantified as Pearson correlation coefficients, in controls and ap3b2 CRISPants. Regions compared are (d) left–right MB and (e) left–right FB. Unpaired t-test with Mann Whitney, *p < 0.05, **p < 0.01. (f,g) Correlations between (f) right MB-FB and (g) left MB-FB, unpaired t-tests with Mann Whitney, ns, not significant. Raw correlation values and full statistical details are provided in Supplementary File S1. |
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FIGURE 5
ap3b2 disruption downregulates BBB transport, GABA signaling, and axon guidance pathways in CRISPant tadpole brains. (a) PCA plot of normalized read counts of the five control samples and four ap3b2−/− (mosaic) samples; each sample is derived from six pooled brains. (b) Volcano plot of EdgeR differentially expressed genes (DEG) with FDR threshold of <0.05 and Log2 fold change of >1. The 10 most significant up and down regulated genes are labelled. (c) Selected overrepresented ontologies calculated with EnrichR for down regulated DEG; the top 6 hits are shown for GO: biological process and KEGG_2021; for ClinVar, the only two significant hits are shown (PAdj <0.05). (d) Heatmap of z-scores for three down overrepresented ontologies, down regulated in CRISPants. PAdj ** < 0.01, *** < 0.001. (e) Summary of tadpole editing for ap3b2 CRISPant samples (pooled data corresponding to each of the 4 CRISPR brain transcriptomes), confirmed by Sanger sequencing and TIDE analysis. Supplementary Data for (c,e), as well as the custom brain background gene list, can be found in Supplementary File S1. |
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FIGURE 6
Rapid early sodium fluorescein dye leakage in ap3b2 CRISPants reveals a markedly compromised BBB. (a) Schematic dorsal view of an NF stage 47 X. laevis tadpole head showing forebrain (FB), midbrain (MB), hindbrain (HB), and spinal cord (SC), and the site of sodium fluorescein (NaF) microinjection into the 4th ventricle (red arrow). The dashed rectangle indicates the ROI outside the brain from which fluorescence intensity was quantified. (b) Representative images of NaF-injected controls (top row) and ap3b2.S CRISPants (bottom row) 2 min after microinjection (raw capture and green channel only). (c) Plot of mean fluorescence intensity (MFI) detected outside tadpole brain at 2, 5, 10, and 20 min post NaF injection in CRISPant tadpoles (N = 10) compared to controls (N = 7), repeated measures 2-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05. (d) Summary of CRISPR/Cas9 editing outcomes in the CRISPant group, determined by Sanger sequencing and TIDE analysis. Raw count data and full statistical details are provided in Supplementary File S1. |
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FIGURE 7
Losartan treatment reduces aberrant calcium activity and hyperactivity in ap3b2 CRISPants. (a) Conceptual schematic of ap3b2 CRISPant differentially expressed genes associated with loss of anti-inflammatory regulatory control, BBB dysfunction, and neuronal hyperexcitability relevant to TGF-β receptor–SMAD signaling, indicating a hypothetical mode of action for losartan (created in BioRender). (b) Paired comparison of mean swim velocity (mm/s, over 1 h) in ap3b2 CRISPant tadpoles before and after 10 mM losartan treatment. Lines connect individual tadpoles, and the effect of losartan was tested using a Wilcoxon paired t-test, *p < 0.05. (c) Summary of CRISPR/Cas9 editing outcomes for embryos used in the losartan phenotype experiments, determined by Sanger sequencing and TIDE analysis. (d) Representative whole-brain Ca2+ signals (raw and filtered ΔF/F₀ traces) showing activity from untreated ap3b2.S CRISPants (left) and losartan-treated CRISPants (1 h, 10 mM, right). Black lines indicate the global event detection threshold (3 × control SD), with arrowheads marking significant Ca2+ events. (e,f) Comparison of the numbers of Ca2+ events (e) and mean event amplitude (f) detected in untreated (N = 9) and losartan treated (10 mM; N = 10) ap3b2 CRISPant tadpoles. Groups were compared using unpaired Welch’s t-tests, ns = not significant (p > 0.05). (g) FFT-derived power spectral densities of spontaneous whole-brain Ca2+ activity in untreated and losartan-treated ap3b2 CRISPants, plotted on a logarithmic scale. Solid lines indicate group means and shaded envelopes represent 95% confidence intervals across animals. (h) Comparison of integrated low-frequency spectral power density (0.01–1 Hz; (ΔF/F₀)2/Hz) between untreated (N = 9) and losartan-treated (10 mM, N = 10) ap3b2 CRISPants. Individual data points on scatterplots represent single CRISPant tadpoles; horizontal bars denote group means and error bars indicate SEM. Statistical significance was assessed an unpaired Welch’s t-test, with the exact p value shown. (i,j) Distribution of CRISPR editing outcomes in untreated (i) and losartan-treated (j) CRISPant tadpoles, quantified by Sanger sequencing and TIDE analysis. Raw data and full statistical analyses are provided in Supplementary File S1. |
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Figure S 3. Workflow for cranial window preparation and calcium imaging in Xenopus tadpoles. a) Tadpole embedded dorsal-up in 2% LMP agarose with nares/mouth free. b) Specimen centered under a stereomicroscope. c) Initial skin tear at the hindbrain base using fine forceps (Dumont #5). d) Cranial window preparation: skin is peeled anteriorly to expose the dorsal brain (hindbrain → midbrain → forebrain) expressing GCaMP6s. e) Transfer to the fluorescence microscope for imaging. f) Calcium imaging of defined brain regions (ROIs); inset illustrates a representative field used for time-series extraction. Arrows indicate the order of operations. Created in BioRender. Banerjee, S. (2026) https://BioRender.com/1m35lxo. |
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Figure S 8. BBB [blood-brain barrier] figs for all timepoints, example of typical control unedited and ap3b2 CRISPant images. |
