A dual-reporter mouse for therapeutic discovery in Angelman syndrome
Hanna Vihma, Lucas M. James, Hannah C. Nourie, Audrey L. Smith, Siyuan Liang, Carlee A. Friar, Tasmai Vulli, Lei Xing, Dale O. Cowley, Alain C. Burette, Benjamin D. Philpot
Hanna Vihma, Lucas M. James, Hannah C. Nourie, Audrey L. Smith, Siyuan Liang, Carlee A. Friar, Tasmai Vulli, Lei Xing, Dale O. Cowley, Alain C. Burette, Benjamin D. Philpot
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Research Article Genetics Neuroscience

A dual-reporter mouse for therapeutic discovery in Angelman syndrome

Abstract

Angelman syndrome is a neurodevelopmental disorder caused by loss of the maternal UBE3A allele, the sole source of UBE3A in mature neurons owing to epigenetic silencing of the paternal allele. Although emerging therapies are being developed to restore UBE3A expression by activating the dormant paternal UBE3A allele, existing mouse models for such preclinical studies have limited throughput and utility, creating bottlenecks for both in vitro therapeutic screening and in vivo characterization. To address this, we developed the Ube3a-INSG dual-reporter knockin mouse, in which an IRES-Nanoluciferase-T2A-Sun1-sfGFP (INSG) cassette was inserted downstream of the endogenous Ube3a stop codon. The INSG model preserves UBE3A protein levels and function while enabling 2 complementary allele-specific readouts: Sun1-sfGFP and Nanoluciferase. We show that Sun1-sfGFP, a nuclear envelope–localized reporter, enables single-cell fluorescence analysis, whole-brain light-sheet imaging, and nuclear quantification by flow cytometry. Further, Nanoluciferase supports high-throughput luminescence assays for sensitive pharmacological profiling in cultured neurons and noninvasive in vivo bioluminescence imaging for pharmacodynamic assessment. By combining scalable screening, cellular analysis, and real-time in vivo monitoring in a single model, the Ube3a-INSG dual-reporter mouse provides a powerful platform to accelerate therapeutic development centered on UBE3A.

Authors

Hanna Vihma, Lucas M. James, Hannah C. Nourie, Audrey L. Smith, Siyuan Liang, Carlee A. Friar, Tasmai Vulli, Lei Xing, Dale O. Cowley, Alain C. Burette, Benjamin D. Philpot

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Figure 7

Relationship between UBE3A and Sun1-sfGFP labeling in individual cells in matINSG versus patINSG mice.

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Relationship between UBE3A and Sun1-sfGFP labeling in individual cells i...
(A) An illustration of the effectiveness of the deep learning algorithm used to quantify UBE3A and Sun1-sfGFP expression at single-cell resolution, even in densely packed areas like the hippocampal CA1 region. For easier visualization, each cell is outlined with a randomly assigned color to indicate both nuclear and cytoplasmic boundaries. Each of the 6 smaller panels overlays the cell segmentation results with 1 of the 5 input channels (DAPI, SOX9, GFP, NeuN, or UBE3A) used for segmentation, while the bottom right panel shows only the GFP channel without cell segmentations. The arrows point to a likely pyramidal neuron that expresses GFP, UBE3A, and the neuronal marker NeuN but not the astrocyte marker SOX9. The arrowheads indicate a small cell that is GFP, UBE3A, and SOX9 positive, but NeuN negative. The double arrows identify an outlier cell lacking all tested markers. (B and C) Scatterplots depicting the relationship between GFP and UBE3A fluorescence levels in individual cells within the hippocampal CA1 region at P30. (B) In SOX9-positive astrocytes, GFP and UBE3A intensities strongly correlate in matINSG and patINSG mice. (C) In NeuN-positive neurons, there is a strong correlation between GFP and UBE3A intensities in matINSG mice but not patINSG mice, apart from a small subset of cells, as most neurons in patINSG mice display GFP levels at background levels. The gray-shaded area marks the range of background GFP fluorescence observed in UBE3A-positive neurons from patINSG samples (C, right), which lack Sun1-sfGFP expression. This reference range is shown across all plots to facilitate comparison across genotypes and cell types. Scale bars: 10 μm.