Dissociating motor impairment from five-choice serial reaction time task performance in a mouse model of Angelman syndrome.

Angelman syndrome (AS) is a single-gene neurodevelopmental disorder associated with cognitive and motor impairment, seizures, lack of speech, and disrupted sleep. AS is caused by loss-of-function mutations in the UBE3A gene, and approaches to reinstate functional UBE3A are currently in clinical trials in children. Behavioral testing in a mouse model of AS (Ube3a m-/p+ ) represents an important tool to assess the effectiveness of current and future treatments preclinically. Existing behavioral tests effectively model motor impairments, but not cognitive impairments, in Ube3a m-/p+ mice. Here we tested the hypothesis that the 5-choice serial reaction time task (5CSRTT) can be used to assess cognitive behaviors in Ube3a m-/p+ mice. Ube3a m-/p+ mice had more omissions during 5CSRTT training than wild-type littermate controls, but also showed impaired motor function including open field hypoactivity and delays in eating pellet rewards. Motor impairments thus presented an important confound for interpreting this group difference in omissions. We report that despite hypoactivity during habituation, Ube3a m-/p+ mice had normal response latencies to retrieve rewards during 5CSRTT training. We also accounted for delays in eating pellet rewards by assessing omissions solely on trials where eating delays would not impact results. Thus, the increase in omissions in Ube3a m-/p+ mice is likely not caused by concurrent motor impairments. This work underscores the importance of considering how known motor impairments in Ube3a m-/p+ mice may affect behavioral performance in other domains. Our results also provide guidance on how to design a 5CSRTT protocol that is best suited for future studies in Ube3a mutants.

Multidimensional analysis of behavior predicts genotype with high accuracy in a mouse model of Angelman syndrome.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss of expression of the maternal copy of the UBE3A gene. Individuals with AS have a multifaceted behavioral phenotype consisting of deficits in motor function, epilepsy, cognitive impairment, sleep abnormalities, as well as other comorbidities. Effectively modeling this behavioral profile and measuring behavioral improvement will be crucial for the success of ongoing and future clinical trials. Foundational studies have defined an array of behavioral phenotypes in the AS mouse model. However, no single behavioral test is able to fully capture the complex nature of AS-in mice, or in children. We performed multidimensional analysis (principal component analysis + k-means clustering) to quantify the performance of AS model mice (n = 148) and wild-type littermates (n = 138) across eight behavioral domains. This approach correctly predicted the genotype of mice based on their behavioral profile with ~95% accuracy, and remained effective with reasonable sample sizes (n = ~12-15). Multidimensional analysis was effective using different combinations of behavioral inputs and was able to detect behavioral improvement as a function of treatment in AS model mice. Overall, multidimensional behavioral analysis provides a tool for evaluating the effectiveness of preclinical treatments for AS. Multidimensional analysis of behavior may also be applied to rodent models of related neurodevelopmental disorders, and may be particularly valuable for disorders where individual behavioral tests are less reliable than in AS.

Molecular and behavioral consequences of Ube3a gene overdosage in mice.

Chromosome 15q11.2-q13.1 duplication syndrome (Dup15q syndrome) is a severe neurodevelopmental disorder characterized by intellectual disability, impaired motor coordination, and autism spectrum disorder. Chromosomal multiplication of the UBE3A gene is presumed to be the primary driver of Dup15q pathophysiology, given that UBE3A exhibits maternal monoallelic expression in neurons and that maternal duplications typically yield far more severe neurodevelopmental outcomes than paternal duplications. However, studies into the pathogenic effects of UBE3A overexpression in mice have yielded conflicting results. Here, we investigated the neurodevelopmental impact of Ube3a gene overdosage using bacterial artificial chromosome-based transgenic mouse models (Ube3aOE) that recapitulate the increases in Ube3a copy number most often observed in Dup15q. In contrast to previously published Ube3a overexpression models, Ube3aOE mice were indistinguishable from wild-type controls on a number of molecular and behavioral measures, despite suffering increased mortality when challenged with seizures, a phenotype reminiscent of sudden unexpected death in epilepsy. Collectively, our data support a model wherein pathogenic synergy between UBE3A and other overexpressed 15q11.2-q13.1 genes is required for full penetrance of Dup15q syndrome phenotypes.

Evaluation of electroencephalography biomarkers for Angelman syndrome during overnight sleep.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss-of-function mutations in the maternal copy of the UBE3A gene. AS is characterized by intellectual disability, impaired speech and motor skills, epilepsy, and sleep disruptions. Multiple treatment strategies to re-express functional neuronal UBE3A from the dormant paternal allele were successful in rodent models of AS and have now moved to early phase clinical trials in children. Developing reliable and objective AS biomarkers is essential to guide the design and execution of current and future clinical trials. Our prior work quantified short daytime electroencephalograms (EEGs) to define promising biomarkers for AS. Here, we asked whether overnight sleep is better suited to detect AS EEG biomarkers. We retrospectively analyzed EEGs from 12 overnight sleep studies from individuals with AS with age and sex-matched Down syndrome and neurotypical controls, focusing on low frequency (2-4 Hz) delta rhythms and sleep spindles. Delta EEG rhythms were increased in individuals with AS during all stages of overnight sleep, but overnight sleep did not provide additional benefit over wake in the ability to detect increased delta. Abnormal sleep spindles were not reliably detected in EEGs from individuals with AS during overnight sleep, suggesting that delta rhythms represent a more reliable biomarker. Overall, we conclude that periods of wakefulness are sufficient, and perhaps ideal, to quantify delta EEG rhythms for use as AS biomarkers. LAY SUMMARY: Electroencephalography (EEG) is a safe and reliable way of measuring abnormal brain activity in Angelman syndrome. We found that low-frequency "delta" EEG rhythms are increased in individuals with Angelman syndrome during all stages of overnight sleep. Delta rhythms can be used as a tool to measure improvement in future clinical trials.

Visual Sequences Drive Experience-Dependent Plasticity in Mouse Anterior Cingulate Cortex.

Mechanisms of experience-dependent plasticity have been well characterized in mouse primary visual cortex (V1), including a form of potentiation driven by repeated presentations of a familiar visual sequence ("sequence plasticity"). The prefrontal anterior cingulate cortex (ACC) responds to visual stimuli, yet little is known about if and how visual experience modifies ACC circuits. We find that mouse ACC exhibits sequence plasticity, but in contrast to V1, the plasticity expresses as a change in response timing, rather than a change in response magnitude. Sequence plasticity is absent in ACC, but not V1, in a mouse model of a neurodevelopmental disorder associated with intellectual disability and autism-like features. Our results demonstrate that simple sensory stimuli can be used to reveal how experience functionally (or dysfunctionally) modifies higher-order prefrontal circuits and suggest a divergence in how ACC and V1 encode familiarity.

Electrophysiological Phenotype in Angelman Syndrome Differs Between Genotypes.

BACKGROUND: Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by either disruptions of the gene UBE3A or deletion of chromosome 15 at 15q11-q13, which encompasses UBE3A and several other genes, including GABRB3, GABRA5, GABRG3, encoding gamma-aminobutyric acid type A receptor subunits (ß3, α5, γ3). Individuals with deletions are generally more impaired than those with other genotypes, but the underlying pathophysiology remains largely unknown. Here, we used electroencephalography (EEG) to test the hypothesis that genes other than UBE3A located on 15q11-q13 cause differences in pathophysiology between AS genotypes. METHODS: We compared spectral power of clinical EEG recordings from children (1-18 years of age) with a deletion genotype (n = 37) or a nondeletion genotype (n = 21) and typically developing children without Angelman syndrome (n = 48). RESULTS: We found elevated theta power (peak frequency: 5.3 Hz) and diminished beta power (peak frequency: 23 Hz) in the deletion genotype compared with the nondeletion genotype as well as excess broadband EEG power (1-32 Hz) peaking in the delta frequency range (peak frequency: 2.8 Hz), shared by both genotypes but stronger for the deletion genotype at younger ages. CONCLUSIONS: Our results provide strong evidence for the contribution of non-UBE3A neuronal pathophysiology in deletion AS and suggest that hemizygosity of the GABRB3-GABRA5-GABRG3 gene cluster causes abnormal theta and beta EEG oscillations that may underlie the more severe clinical phenotype. Our work improves the understanding of AS pathophysiology and has direct implications for the development of AS treatments and biomarkers.

Abnormal coherence and sleep composition in children with Angelman syndrome: a retrospective EEG study.

Background: Angelman syndrome (AS) is a neurodevelopmental disorder characterized by intellectual disability, speech and motor impairments, epilepsy, abnormal sleep, and phenotypic overlap with autism. Individuals with AS display characteristic EEG patterns including high-amplitude rhythmic delta waves. Here, we sought to quantitatively explore EEG architecture in AS beyond known spectral power phenotypes. We were motivated by studies of functional connectivity and sleep spindles in autism to study these EEG readouts in children with AS. Methods: We analyzed retrospective wake and sleep EEGs from children with AS (age 4-11) and age-matched neurotypical controls. We assessed long-range and short-range functional connectivity by measuring coherence across multiple frequencies during wake and sleep. We quantified sleep spindles using automated and manual approaches. Results: During wakefulness, children with AS showed enhanced long-range EEG coherence across a wide range of frequencies. During sleep, children with AS showed increased long-range EEG coherence specifically in the gamma band. EEGs from children with AS contained fewer sleep spindles, and these spindles were shorter in duration than their neurotypical counterparts. Conclusions: We demonstrate two quantitative readouts of dysregulated sleep composition in children with AS-gamma coherence and spindles-and describe how functional connectivity patterns may be disrupted during wakefulness. Quantitative EEG phenotypes have potential as biomarkers and readouts of target engagement for future clinical trials and provide clues into how neural circuits are dysregulated in children with AS.

Enhanced Operant Extinction and Prefrontal Excitability in a Mouse Model of Angelman Syndrome.

Angelman syndrome (AS), a neurodevelopmental disorder associated with intellectual disability, is caused by loss of maternal allele expression of UBE3A in neurons. Mouse models of AS faithfully recapitulate disease phenotypes across multiple domains, including behavior. Yet in AS, there has been only limited study of behaviors encoded by the prefrontal cortex, a region broadly involved in executive function and cognition. Because cognitive impairment is a core feature of AS, it is critical to develop behavioral readouts of prefrontal circuit function in AS mouse models. One such readout is behavioral extinction, which has been well described mechanistically and relies upon prefrontal circuits in rodents. Here we report exaggerated operant extinction in male AS model mice, concomitant with enhanced excitability in medial prefrontal neurons from male and female AS model mice. Abnormal behavior was specific to operant extinction, as two other prefrontally dependent tasks (cued fear extinction and visuospatial discrimination) were largely normal in AS model mice. Inducible deletion of Ube3a during adulthood was not sufficient to drive abnormal extinction, supporting the hypothesis that there is an early critical period for development of cognitive phenotypes in AS. This work represents the first formal experimental analysis of prefrontal circuit function in AS, and identifies operant extinction as a useful experimental paradigm for modeling cognitive aspects of AS in mice.SIGNIFICANCE STATEMENT Prefrontal cortex encodes "high-level" cognitive processes. Thus, understanding prefrontal function is critical in neurodevelopmental disorders where cognitive impairment is highly penetrant. Angelman syndrome is a neurodevelopmental disorder associated with speech and motor impairments, an outwardly happy demeanor, and intellectual disability. We describe a behavioral phenotype in a mouse model of Angelman syndrome and related abnormalities in prefrontal cortex function. We hypothesize that robust and reliable prefrontally encoded behavior may be used to model cognitive impairments in Angelman syndrome.

Erratum to: Delta rhythmicity is a reliable EEG biomarker in Angelman syndrome: a parallel mouse and human analysis.

[This corrects the article DOI: 10.1186/s11689-017-9195-8.].

Delta rhythmicity is a reliable EEG biomarker in Angelman syndrome: a parallel mouse and human analysis.

BACKGROUND: Clinicians have qualitatively described rhythmic delta activity as a prominent EEG abnormality in individuals with Angelman syndrome, but this phenotype has yet to be rigorously quantified in the clinical population or validated in a preclinical model. Here, we sought to quantitatively measure delta rhythmicity and evaluate its fidelity as a biomarker. METHODS: We quantified delta oscillations in mouse and human using parallel spectral analysis methods and measured regional, state-specific, and developmental changes in delta rhythms in a patient population. RESULTS: Delta power was broadly increased and more dynamic in both the Angelman syndrome mouse model, relative to wild-type littermates, and in children with Angelman syndrome, relative to age-matched neurotypical controls. Enhanced delta oscillations in children with Angelman syndrome were present during wakefulness and sleep, were generalized across the neocortex, and were more pronounced at earlier ages. CONCLUSIONS: Delta rhythmicity phenotypes can serve as reliable biomarkers for Angelman syndrome in both preclinical and clinical settings.

GABAergic Neuron-Specific Loss of Ube3a Causes Angelman Syndrome-Like EEG Abnormalities and Enhances Seizure Susceptibility.

Loss of maternal UBE3A causes Angelman syndrome (AS), a neurodevelopmental disorder associated with severe epilepsy. We previously implicated GABAergic deficits onto layer (L) 2/3 pyramidal neurons in the pathogenesis of neocortical hyperexcitability, and perhaps epilepsy, in AS model mice. Here we investigate consequences of selective Ube3a loss from either GABAergic or glutamatergic neurons, focusing on the development of hyperexcitability within L2/3 neocortex and in broader circuit and behavioral contexts. We find that GABAergic Ube3a loss causes AS-like increases in neocortical EEG delta power, enhances seizure susceptibility, and leads to presynaptic accumulation of clathrin-coated vesicles (CCVs)-all without decreasing GABAergic inhibition onto L2/3 pyramidal neurons. Conversely, glutamatergic Ube3a loss fails to yield EEG abnormalities, seizures, or associated CCV phenotypes, despite impairing tonic inhibition onto L2/3 pyramidal neurons. These results substantiate GABAergic Ube3a loss as the principal cause of circuit hyperexcitability in AS mice, lending insight into ictogenic mechanisms in AS.

Metabotropic glutamate receptor signaling is required for NMDA receptor-dependent ocular dominance plasticity and LTD in visual cortex.

A feature of early postnatal neocortical development is a transient peak in signaling via metabotropic glutamate receptor 5 (mGluR5). In visual cortex, this change coincides with increased sensitivity of excitatory synapses to monocular deprivation (MD). However, loss of visual responsiveness after MD occurs via mechanisms revealed by the study of long-term depression (LTD) of synaptic transmission, which in layer 4 is induced by acute activation of NMDA receptors (NMDARs) rather than mGluR5. Here we report that chronic postnatal down-regulation of mGluR5 signaling produces coordinated impairments in both NMDAR-dependent LTD in vitro and ocular dominance plasticity in vivo. The data suggest that ongoing mGluR5 signaling during a critical period of postnatal development establishes the biochemical conditions that are permissive for activity-dependent sculpting of excitatory synapses via the mechanism of NMDAR-dependent LTD.

Fragile X mental retardation protein and synaptic plasticity.

Loss of the translational repressor FMRP causes Fragile X syndrome. In healthy neurons, FMRP modulates the local translation of numerous synaptic proteins. Synthesis of these proteins is required for the maintenance and regulation of long-lasting changes in synaptic strength. In this role as a translational inhibitor, FMRP exerts profound effects on synaptic plasticity.

Protein kinase a mediates activity-dependent Kv4.2 channel trafficking.

The A-type potassium channel subunit Kv4.2 influences hippocampal function through regulation of dendritic excitability, and changes in Kv4.2 surface expression alter synaptic plasticity. Recent data from our laboratory demonstrate that EGFP (enhanced green fluorescent protein)-tagged Kv4.2 channels located in dendritic spines are internalized in an activity-dependent manner after synaptic stimulation and during chemically induced long-term potentiation. However, the molecular trigger for Kv4.2 internalization remains unknown. Here we examined the role of protein kinase A (PKA) in Kv4.2 activity-dependent trafficking. In hippocampal neurons, PKA activation with forskolin or 8-Br-cAMP induced Kv4.2 internalization from dendritic spines, whereas PKA inhibition with H89 prevented AMPA-induced internalization. Furthermore, introduction of a point mutation at the C-terminal PKA phosphorylation site of Kv4.2 (S552A) prevented the AMPA-induced internalization of Kv4.2. Together, these data demonstrate that Kv4.2 activity-dependent internalization requires PKA phosphorylation of Kv4.2 at serine 522.