Enhanced Nociception in Angelman Syndrome Model Mice

Abstract

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutation or deletion of the maternal UBE3A allele. The maternal UBE3A allele is expressed in nearly all neurons of the brain and spinal cord, whereas the paternal UBE3A allele is repressed by an extremely long antisense transcript (UBE3A-ATS). Little is known about expression of UBE3A in the peripheral nervous system, where loss of maternal UBE3A might contribute to AS phenotypes. Here we sought to examine maternal and paternal Ube3a expression in DRGs neurons and to evaluate whether nociceptive responses were affected in AS model mice (global deletion of maternal Ube3a allele; Ube3a^m-/p+). We found that most large-diameter proprioceptive and mechanosensitive DRG neurons expressed maternal Ube3a and paternal Ube3a-ATS In contrast, most small-diameter neurons expressed Ube3a biallelically and had low to undetectable levels of Ube3a-ATS Analysis of single-cell DRG transcriptomes further suggested that Ube3a is expressed monoallelically in myelinated large-diameter neurons and biallelically in unmyelinated small-diameter neurons. Behavioral responses to some noxious thermal and mechanical stimuli were enhanced in male and female AS model mice; however, nociceptive responses were not altered by the conditional deletion of maternal Ube3a in the DRG. These data suggest that the enhanced nociceptive responses in AS model mice are due to loss of maternal Ube3a in the central, but not peripheral, nervous system. Our study provides new insights into sensory processing deficits associated with AS.SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss or mutation of the maternal UBE3A allele. While sensory processing deficits are frequently associated with AS, it is currently unknown whether Ube3a is expressed in peripheral sensory neurons or whether maternal deletion of Ube3a affects somatosensory responses. Here, we found that Ube3a is primarily expressed from the maternally inherited allele in myelinated large-diameter sensory neurons and biallelically expressed in unmyelinated small-diameter neurons. Nociceptive responses to select noxious thermal and mechanical stimuli were enhanced following global, but not sensory neuron-specific, deletion of maternal Ube3a in mice. These data suggest that maternal loss of Ube3a affects nociception via a central, but not peripheral mechanism, with implications for AS.

Keywords: Angelman syndrome; Ube3a; nociception.

Figure 1.

Expression of Ube3a in the DRG. Immunostaining of UBE3A and NeuN in lumbar DRG from (A, B) WT (Ube3a^m+/p+) and (C, D) AS (Ube3a^m−/p+) mice. Scale bar, 50 μm.

Figure 2.

Expression of paternal UBE3A-YFP (Ube3a^m+/pYFP) relative to DRG neuron markers. A, D, G, J, UBE3A-YFP detected with GFP antibody in DRG sections and colabeled with one of the following: B, NeuN; E, IB4; H, CGRP; K, NF200. C, F, I, L, Merged images. Scale bar, 50 μm.

Figure 3.

Ube3a-ATS is highly expressed in large-diameter DRG neurons. A, In situ hybridization with an Ube3a-ATS probe in DRG. B, Merged with NeuN image. C, Table of each marker as a percentage of NeuN⁺ neurons >25 and <25 μm. D, In situ hybridization with Ube3a-ATS probe (green) and immunostained for IB4 (red) and UBE3A-YFP (blue). E, Costaining for UBE3A-YFP and DAPI, expanded from box in D. F, Merged image from box in D. Scale bar: D, 50 μm.

Figure 4.

Single-cell analysis of Ube3a-ATS expression in DRG. A, Expression of genes near Ube3a (chr7:59,223,946–60,142,803) in 11 DRG cell types, defined by single-cell transcriptomics (Usoskin et al., 2015). Expression values were log₁₀-transformed and averaged across all single cells for each cell type. Arrows indicate direction of transcription. Longer intergenic regions (dashed lines) are not shown. *Isoforms of Snord116 that are primarily expressed in neurofilament-heavy (NF⁺) cell types, which include PEP2. B, Expression of Ube3a-ATS across all 622 single cells of the DRG using the average expression of 18 genes that serve as a proxy for Ube3a-ATS expression. Horizontal lines indicate the average expression across each of the single cells of a given cell type.

Figure 5.

Ube3a^m−/p+ mice show enhanced responses to noxious mechanical and heat stimuli. Ube3a^m−/p+ mice were tested with mechanical stimuli. A, von Frey filaments of increasing force. B, Tail clip (p = 0.00054). C, Cotton swab. Mice were also tested with noxious heat stimuli. D, Tail immersion 46.5°C (p = 0.00053). E, Tail immersion 49°C (p = 6.30 × 10⁻⁶). F, Radiant heating of hindpaw with Hargreaves apparatus. t tests were used to compare responses between WT and Ube3a^m−/p+ mice. n = 10–12 males and 2–4 females/group, 12 weeks old. **p < 0.005; ***p < 0.0005.

Figure 6.

Ube3a^m−/p+ mice show heightened aversion to novel tactile environments. A, WT and Ube3a^m−/p+ mice were placed into the empty half of a rodent cage (inset). The other half contained a novel tactile environment (water, gravel, smooth stone, sand, or bedding). A, The amount of time spent exploring the novel environment (water, p = 0.014; sand, p = 0.0091; bedding, p = 0.00053). B, The mean number of entries into the novel environment was measured (water, p = 0.003; gravel, p = 0.029; sand, p = 0.0048; bedding, p = 0.022). A, Dashed line indicates half of the total trial time. B, Inset, Image of gravel, smooth stone, and sand used in texture assay. t tests were used to compare responses between WT and Ube3a^m−/p+ mice. *p < 0.05; **p < 0.005.

Figure 7.

Conditional deletion of maternal Ube3a in mouse DRG with Advillin-Cre. Immunostaining of UBE3A and NeuN in lumbar DRG from (A, B) WT (Ube3a^m+/p+ Advillin-Cre^+/−) and (C, D) Ube3a^FLOX/p+ Advillin-Cre^+/− mice. Scale bar: D, 50 μm. Confocal gain settings were equivalent to permit comparison of staining intensity between A, B and C, D.

Figure 8.

Conditional deletion of maternal Ube3a in somatosensory neurons does not alter nociceptive responses to mechanical or thermal stimuli. Ube3a^FLOX/p+ mice were tested with mechanical stimuli. A, von Frey filaments. B, Tail clip. C, Cotton swab. Mice were also tested with noxious heat stimuli. D, Tail immersion 46.5°C. E, Tail immersion 49°C. F, Radiant heating of the hindpaw. t tests were used to compare responses between WT and Ube3a^FLOX/p+ mice. n = 10 males and 4 females/group, 10–13 weeks old.

Figure 9.

Conditional deletion of maternal Ube3a in somatosensory neurons does not impair tactile discrimination or rotarod performance. A, The amount of time spent exploring the novel environment. B, Mean number of entries into the novel environment. A, Dashed line indicates half of the total trial time. C, Latency to fall from an accelerating rotarod. A retest was performed 48 h after the first test. n = 3 or 4 males and 3 or 4 females/group. t tests were used to compare responses between WT and Ube3a^FLOX/p+ mice. There were no significant differences between groups.