Altered ultrasonic vocalization and impaired learning and memory in Angelman syndrome mouse model with a large maternal deletion from Ube3a to Gabrb3

Abstract

Angelman syndrome (AS) is a neurobehavioral disorder associated with mental retardation, absence of language development, characteristic electroencephalography (EEG) abnormalities and epilepsy, happy disposition, movement or balance disorders, and autistic behaviors. The molecular defects underlying AS are heterogeneous, including large maternal deletions of chromosome 15q11-q13 (70%), paternal uniparental disomy (UPD) of chromosome 15 (5%), imprinting mutations (rare), and mutations in the E6-AP ubiquitin ligase gene UBE3A (15%). Although patients with UBE3A mutations have a wide spectrum of neurological phenotypes, their features are usually milder than AS patients with deletions of 15q11-q13. Using a chromosomal engineering strategy, we generated mutant mice with a 1.6-Mb chromosomal deletion from Ube3a to Gabrb3, which inactivated the Ube3a and Gabrb3 genes and deleted the Atp10a gene. Homozygous deletion mutant mice died in the perinatal period due to a cleft palate resulting from the null mutation in Gabrb3 gene. Mice with a maternal deletion (m-/p+) were viable and did not have any obvious developmental defects. Expression analysis of the maternal and paternal deletion mice confirmed that the Ube3a gene is maternally expressed in brain, and showed that the Atp10a and Gabrb3 genes are biallelically expressed in all brain sub-regions studied. Maternal (m-/p+), but not paternal (m+/p-), deletion mice had increased spontaneous seizure activity and abnormal EEG. Extensive behavioral analyses revealed significant impairment in motor function, learning and memory tasks, and anxiety-related measures assayed in the light-dark box in maternal deletion but not paternal deletion mice. Ultrasonic vocalization (USV) recording in newborns revealed that maternal deletion pups emitted significantly more USVs than wild-type littermates. The increased USV in maternal deletion mice suggests abnormal signaling behavior between mothers and pups that may reflect abnormal communication behaviors in human AS patients. Thus, mutant mice with a maternal deletion from Ube3a to Gabrb3 provide an AS mouse model that is molecularly more similar to the contiguous gene deletion form of AS in humans than mice with Ube3a mutation alone. These mice will be valuable for future comparative studies to mice with maternal deficiency of Ube3a alone.

Figure 1. Generation of mice with a deletion from Ube3a to Gabrb3 using chromosome engineering.

A. Genomic maps for Ube3a and Gabrb3 loci and design of targeting constructs (X, XbaI; H, HindIII; N, NotI; E, EcoRI; S, SacII; B, BamHI). The number in the rectangle indicates the exon number. B & C. Genomic DNA Southern analysis for Ube3a or Gabrb3 targeted ES cell clones using 5′-flanking probes and BamHI and HindIII restriction enzymes respectively. D. A genomic map from Ube3a to Gabrb3 and a scheme for chromosome engineering using half of Hprt and Cre/loxP strategy. The black bars indicate genomic probes used for DNA Southern analysis. E. Confirmation of a deletion from Ube3a to Gabrb3 by genomic DNA Southern analysis using probes shown in D. The probes P1 and P4 hybridize outside of the deleted interval and the probes P2, P3, and PD hybridize within the deleted interval. Four independent but indistinguishable ES cell clones (1–4, +/del) with a deletion were analyzed, and clone 5 (+ndel) was a Ube3a and Gabrb3 double targeted clone without a deletion. The DNA Southern analysis using the probe PD within the Atp10a gene showed no signal in the DNA from homozygous deletion (del/del or m−/p− in text) mice. “Co” was a control probe from the Snrpn gene that is outside of deleted region in chromosome 7.

Figure 2. Expression analysis of Ube3a, Atp10a, and Gabrb3.

A. Quantitative immunoblot analysis of Ube3a and Gabrb3 proteins in cerebellum. The m+/p+ is for wild-type, the m−/p+ is for maternal deletion, the m+/p− is for paternal deletion, and the m−/p− is for homozygous deletion fetus. The m+/p+ next to m−/p− was fetal brain tissue; others tissues were from adult mice. There was no detectable expression of Ube3a in m−/p+ and m−/p− mice indicating a null mutation in homozygotes and exclusively maternal expression of Ube3a. The expression of Ube3a in the m+/p− mice was similar to m+/p+ mice. The expression of Gabrb3 was reduced but readily detectable in m+/p− and m−/p− mice for both males and females compared to that of m+/p+ mice. The expression level of Gabrb3 normalized to β-actin is shown in 2B. The difference between m+/p+ and m−/p+, m+/p+ and m+/p− was significant (n = 5, p<0.01) but there was no significant difference between m−/p+ and m+/p− (n = 5, p>0.05). 2C–E. Expression analysis of Atp10a by regular and real-time RT-PCR analyses. The labels for genotype are the same as indicated in panel A. Data from regular RT-PCR analysis for Atp10a using RNA isolated from cerebral cortex are shown in 2C and from the cerebellum in 2D. The expression of Hprt was used as a control. The absence of RT-PCR product for Atp10a in the m−/p− fetus indicated a null mutation of Atp10a in mice with a deletion. The expression of Atp10a was detectable and not significantly different between m−/p+ and m+/p− mice. The expression of Atp10a normalized to β-actin by real-time RT-PCR analysis is shown in 2E. The level of expression of Atp10a in m−/p+ and m+/p− was significantly lower than that of m+/p+ both in males and females (n = 3, p<0.01) but there was no significant difference between m−/p+ and m+/p− (n = 3, p>0.05).

Figure 3. Perinatal lethality and cleft palate in newborn mice with a homozygous deletion from Ube3a to Gabrb3 (m−/p−).

A. Growth retardation of 2-day-old pup with a homozygous deletion from Ube3a to Gabrb3. B & C. Cleft palate in a pup with a homozygous deletion from Ube3a to Gabrb3. The black arrow points to the cleft palate in pup with a homozygous deletion (m−/p−).

Figure 4. Abnormal EEG in mice with a maternal deletion from Ube3a to Gabrb3 (m−/p+).

Multielectrode continuous EEG tracing shows representative generalized rapid cortical spike seizure discharge in freely moving m−/p+ deletion mice. Top-bottom: temporal (T), parietal (P), and occipital leads (O) alternating between left (L) and right hemisphere (R) indicated as LT, RT, LP, RP, LO, and RO. The six panels in the top tracing were continued in the lower section. Single arrow indicates the onset of sudden isolated giant spike-wave discharges with no behavioral accompaniment, and double arrow marks progression to higher spike discharge frequencies.

Figure 5. Abnormal light-dark exploration and impaired rotarod performance in mice with a maternal deletion from Ube3a to Gabrb3 (m−/p+) but not a paternal deletion (m+/p−).

A & B. Light-dark transition. The m−/p+ but not m+/p− mice showed a significantly lower number of light and dark transitions, shown in A (p = 0.003), and also spent significantly longer time in the dark chamber, shown in B (p = 0.008). There was no difference between m+/p+ of maternal and paternal group for both light-dark transition and percentage of time stayed in dark. C & D. Rotarod performance. The time that m−/p+ mice remained on the accelerating rotarod was significantly shorter than m+/p+ littermates for all trials, shown in C (p<0.0001). The overall performance of m+/p− mice was slightly worse than that of m+/p+ littermates, but the difference was not statistically significant, shown in D (p>0.05). * p<0.05.

Figure 6. Impaired learning and memory task in mice with a maternal deletion from Ube3a to Gabrb3 (m−/p+).

A & B. Fear conditioning. The m−/p+ but not m+/p− mice showed significantly less freezing behavior in contextual fear conditioning (A) but not for cued fear conditioning. C to G. Morris water maze task. The m−/p+ but not m+/p− mice showed significantly longer escape latency than m+/p+ littermates (p<0.001 for m−/p+ and p>0.05 for m+/p−). In the probe trial, analysis of platform crossing found that m+/p+ mice displayed a significantly biased searching strategy, shown in E (p<0.001), while m−/p+ mice displayed a random searching strategy, shown in F (p>0.05). Post hoc analysis of m+/p+ mice revealed that the difference was significant only between quadrant 4 and 2 (p<0.01), and quadrant 4 and 3 (p<0.02). The labeling of each quadrant and location of the hidden platform is illustrated in G. * p<0.05.

Figure 7. Increased ultrasonic vocalizations (USVs) of newborn pups with a maternal deletion (m−/p+) from Ube3a to Gabrb3.

A and B. Comparison of m−/p+ and m+/p− pups respectively to their m+/p+ littermates in different beddings. The m−/p+ pups emitted significantly more USVs than that of m+/p+ littermates at mother's bedding (p = 0.006) (2B) but not for m+/p− pups(2C, p>0.05). C. Comparison of USVs of m−/p+ to m+/p+ at different postnatal days. The m−/p+ pups emitted more USVs overall than m+/p+ mice but this was only statistically significantly on day 10 (p = 0.02) and day 12 (p = 0.05). The three-way (genotype X bedding X day) with mixed design ANOVA did not reveal significant interaction among them (p = 0.07). Similarly, two-way ANOVA did not reveal significant interactions between genotype and bedding (p = 0.15), and between genotype and day (p = 0.29). D. Comparison of USVs for m−/p+ and m+/p− pups recorded in clean bedding at low temperature (4–6°C) on day 10 and day 12. There was no significant difference between m−/p+ and their m+/p+ littermates (p = 0.82), or between m+/p− and their m+/p+ littermates (p = 0.3). * p<0.05.