Hyperexcitability in Stem Cell-Derived Neurons from Dup15q Autism and Angelman Syndrome Patients

Thursday, May 12, 2016: 11:30 AM-1:30 PM
Hall A (Baltimore Convention Center)
J. Fink1, T. Robinson1, S. Chamberlain2 and E. Levine1, (1)Dept Neuroscience, University of Connecticut Health Center, Farmington, CT, (2)Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, CT
Background: Individuals with a duplication of the 15q11-q13 chromosomal region suffer from a neurodevelopmental disorder known as Dup15q syndrome, which represents the most common copy number variant associated with autism. In addition to the autistic-like symptoms such as intellectual disability and language delay, >50% of Dup15q patients suffer from some form of seizure disorder. Similarly, patients with maternal deletion of the same chromosomal region present with Angelman syndrome (AS), a related neurodevelopmental disorder in which a majority of the patients also develop seizures at some point in their life. Although the genetic cause of Angelman syndrome has been identified as the UBE3A gene, the specific gene or set of genes directly responsible for the Dup15q phenotype remains less clear, though UBE3A is thought to play an important role in Dup15q pathophysiology. Even less clear are the downstream UBE3A targets that might mediate these disease phenotypes, though several synaptic targets have been reported.

 Objectives: Given the seizure phenotype associated Dup15q and AS, we examined the excitability of neurons derived from these patients using induced pluripotent stem cell (iPSC) lines. Hyperexcitability can result from a variety of network and cell-intrinsic properties. For this reason we investigated resting membrane potential (RMP), spontaneous excitatory and inhibitory synaptic currents, and action potential (AP) firing in these cells.

 Methods: Cell Culture Preparation: Induced pluripotent stem cells (iPSCs) were derived from fibroblasts obtained from two AS subjects (1M/1F), 3 control subjects (2M/1F), and 2 Dup15q patients (2F) and differentiated into neurons and plated onto coverslips as previously described (Germain et al. 2014). Electrophysiology: Coverslips were individually transferred to a recording chamber and continuously perfused with carboxygenated artificial cerebrospinal fluid. Whole-cell patch recordings were obtained from morphologically-identified neurons. Cells were noted for RMP by injection with zero current, AP firing by holding at -40 mV or applying 10mV current injection steps from -70mV, and spontaneous synaptic activity via a holding potential of -70 mV. Calcium Imaging: Coverslips of neurons were incubated in Fluo-4 AM (10 µM) calcium dye for 45 minutes. Cells were then placed in a recording chamber and imaged for 40 minutes at a frequency of 10 Hz.

 Results: We have observed disruptions in the electrophysiological maturation of neurons derived from AS and Dup15q patients. Specifically, these neurons show depolarized RMPs, immature AP firing, and a reduction in spontaneous synaptic activity. Additionally, neurons derived from Dup15q patients displayed significant increases in spontaneous AP firing at -40 mV as well as increased network synchrony.

 Conclusions: Overall, data collected from both AS and Dup15q patients show significant differences compared to control subjects in a variety of electrophysiological properties throughout their development. These specific differences may contribute to hyperexcitability of these cells, a phenotype that could relate to the seizures associated with both AS and Dup15q syndrome. Therefore, these approaches may prove useful for identifying novel targets for drug discovery and for screening potential therapeutics aimed at reversing the seizures, movement disorders, and language and cognitive impairments in these patients.