Dysfunctional sodium channel kinetics as a novel epilepsy mechanism in chromosome 15q11-q13 duplication syndrome.

OBJECTIVE: Duplication of the maternal chromosome 15q11.2-q13.1 region causes Dup15q syndrome, a highly penetrant neurodevelopmental disorder characterized by severe autism and refractory seizures. Although UBE3A, the gene encoding the ubiquitin ligase E3A, is thought to be the main driver of disease phenotypes, the cellular and molecular mechanisms that contribute to the development of the syndrome are yet to be determined. We previously established the necessity of UBE3A overexpression for the development of cellular phenotypes in human Dup15q neurons, including increased action potential firing and increased inward current density, which prompted us to further investigate sodium channel kinetics. METHODS: We used a Dup15q patient-derived induced pluripotent stem cell line that was CRISPR-edited to remove the supernumerary chromosome and create an isogenic control line. We performed whole cell patch clamp electrophysiology on Dup15q and corrected control neurons at two time points of in vitro development. RESULTS: Compared to corrected neurons, Dup15q neurons showed increased sodium current density and a depolarizing shift in steady-state inactivation. Moreover, onset of slow inactivation was delayed, and a faster recovery from both fast and slow inactivation processes was observed in Dup15q neurons. A fraction of sodium current in Dup15q neurons (~15%) appeared to be resistant to slow inactivation. Not unexpectedly, a higher fraction of persistent sodium current was also observed in Dup15q neurons. These phenotypes were modulated by the anticonvulsant drug rufinamide. SIGNIFICANCE: Sodium channels play a crucial role in the generation of action potentials, and sodium channelopathies have been uncovered in multiple forms of epilepsy. For the first time, our work identifies in Dup15q neurons dysfunctional inactivation kinetics, which have been previously linked to multiple forms of epilepsy. Our work can also guide therapeutic approaches to epileptic seizures in Dup15q patients and emphasize the role of drugs that modulate inactivation kinetics, such as rufinamide.

The role of UBE3A in the autism and epilepsy-related Dup15q syndrome using patient-derived, CRISPR-corrected neurons.

Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurodevelopmental disorder caused by maternal duplications of this region. Autism and epilepsy are key features of Dup15q. UBE3A, which encodes an E3 ubiquitin ligase, is likely a major driver of Dup15q because UBE3A is the only imprinted gene expressed solely from the maternal allele. Nevertheless, the exact role of UBE3A has not been determined. To establish whether UBE3A overexpression is required for Dup15q neuronal deficits, we generated an isogenic control line for a Dup15q patient-derived induced pluripotent stem cell line. Dup15q neurons exhibited hyperexcitability compared with control neurons, and this phenotype was generally prevented by normalizing UBE3A levels using antisense oligonucleotides. Overexpression of UBE3A resulted in a profile similar to that of Dup15q neurons except for synaptic phenotypes. These results indicate that UBE3A overexpression is necessary for most Dup15q cellular phenotypes but also suggest a role for other genes in the duplicated region.

Hyperexcitable Phenotypes in Induced Pluripotent Stem Cell-Derived Neurons From Patients With 15q11-q13 Duplication Syndrome, a Genetic Form of Autism.

BACKGROUND: Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurogenetic disorder caused by duplications of the maternal copy of this region. In addition to hypotonia, motor deficits, and language impairments, patients with Dup15q commonly meet the criteria for autism spectrum disorder and have a high prevalence of seizures. It is known from mouse models that synaptic impairments are a strong component of Dup15q pathophysiology; however, cellular phenotypes that relate to seizures are less clear. The development of patient-derived induced pluripotent stem cells provides a unique opportunity to study human neurons with the exact genetic disruptions that cause Dup15q. METHODS: Here, we explored electrophysiological phenotypes in induced pluripotent stem cell-derived neurons from 4 patients with Dup15q compared with 6 unaffected control subjects, 1 patient with a 15q11-q13 paternal duplication, and 3 patients with Angelman syndrome. RESULTS: We identified several properties of Dup15q neurons that could contribute to neuronal hyperexcitability and seizure susceptibility. Compared with control neurons, Dup15q neurons had increased excitatory synaptic event frequency and amplitude, increased density of dendritic protrusions, increased action potential firing, and decreased inhibitory synaptic transmission. Dup15q neurons also showed impairments in activity-dependent synaptic plasticity and homeostatic synaptic scaling. Finally, Dup15q neurons showed an increased frequency of spontaneous action potential firing compared with control neurons, in part due to disruption of KCNQ2 potassium channels. CONCLUSIONS: Together, these data point to multiple electrophysiological mechanisms of hyperexcitability that may provide new targets for the treatment of seizures and other phenotypes associated with Dup15q.

Disrupted neuronal maturation in Angelman syndrome-derived induced pluripotent stem cells.

Angelman syndrome (AS) is a neurogenetic disorder caused by deletion of the maternally inherited UBE3A allele and is characterized by developmental delay, intellectual disability, ataxia, seizures and a happy affect. Here, we explored the underlying pathophysiology using induced pluripotent stem cell-derived neurons from AS patients and unaffected controls. AS-derived neurons showed impaired maturation of resting membrane potential and action potential firing, decreased synaptic activity and reduced synaptic plasticity. These patient-specific differences were mimicked by knocking out UBE3A using CRISPR/Cas9 or by knocking down UBE3A using antisense oligonucleotides. Importantly, these phenotypes could be rescued by pharmacologically unsilencing paternal UBE3A expression. Moreover, selective effects of UBE3A disruption at late stages of in vitro development suggest that changes in action potential firing and synaptic activity may be secondary to altered resting membrane potential. Our findings provide a cellular phenotype for investigating pathogenic mechanisms underlying AS and identifying novel therapeutic strategies.

Gene expression analysis of human induced pluripotent stem cell-derived neurons carrying copy number variants of chromosome 15q11-q13.1.

BACKGROUND: Duplications of the chromosome 15q11-q13.1 region are associated with an estimated 1 to 3% of all autism cases, making this copy number variation (CNV) one of the most frequent chromosome abnormalities associated with autism spectrum disorder (ASD). Several genes located within the 15q11-q13.1 duplication region including ubiquitin protein ligase E3A (UBE3A), the gene disrupted in Angelman syndrome (AS), are involved in neural function and may play important roles in the neurobehavioral phenotypes associated with chromosome 15q11-q13.1 duplication (Dup15q) syndrome. METHODS: We have generated induced pluripotent stem cell (iPSC) lines from five different individuals containing CNVs of 15q11-q13.1. The iPSC lines were differentiated into mature, functional neurons. Gene expression across the 15q11-q13.1 locus was compared among the five iPSC lines and corresponding iPSC-derived neurons using quantitative reverse transcription PCR (qRT-PCR). Genome-wide gene expression was compared between neurons derived from three iPSC lines using mRNA-Seq. RESULTS: Analysis of 15q11-q13.1 gene expression in neurons derived from Dup15q iPSCs reveals that gene copy number does not consistently predict expression levels in cells with interstitial duplications of 15q11-q13.1. mRNA-Seq experiments show that there is substantial overlap in the genes differentially expressed between 15q11-q13.1 deletion and duplication neurons, Finally, we demonstrate that UBE3A transcripts can be pharmacologically rescued to normal levels in iPSC-derived neurons with a 15q11-q13.1 duplication. CONCLUSIONS: Chromatin structure may influence gene expression across the 15q11-q13.1 region in neurons. Genome-wide analyses suggest that common neuronal pathways may be disrupted in both the Angelman and Dup15q syndromes. These data demonstrate that our disease-specific stem cell models provide a new tool to decipher the underlying cellular and genetic disease mechanisms of ASD and may also offer a pathway to novel therapeutic intervention in Dup15q syndrome.