Abstracts

Rationale: Untargeted genetic testing of patients with epilepsy has identified numerous gene and chromosomal copy number variants whose products are important for neurogenesis and synaptogenesis. These data support the hypothesis that genetic epilepsies involve global neurodevelopmental changes. The 15q11.2 locus is deleted in 1.5% of patients with genetic epilepsy and it confers risk for intellectual disability and schizophrenia, suggesting that this region is important for neuronal development. Of 4 genes at this locus, CYFIP1, a regulator of fragile X mental retardation protein and cytoskeleton remodeling, has been shown to regulate early neurogenesis and late synaptogenesis in mice. We examined whether there were differences in neurogenesis and neuronal activity in neurons derived from induced pluripotent stem cells (iPSCs) from patients with 15q11.2 deletion and whether deletion of the Cyfip1 gene was sufficient to increase seizure susceptibility in mice.

Methods: A forebrain specific neuronal differentiation protocol was used to generate primarily glutamatergic cortical neurons from iPSCs from control and 15q11.2 deletion patients. Neurons were cultured on 24 well multielectrode plates with rat astrocytes beginning at day in vitro (DIV) 37. Extracellular field potential recordings were obtained at weekly intervals until DIV 80. Sister cultures were immunostained to assess neuronal structure and network composition. Seizure susceptibility was determined by exposing 2-month-old mice with conditional forebrain neuron knockout of Cyfip1 to the GABAA receptor antagonist, flurothyl and quantification of time to seizure onset and progression to generalized tonic-clonic (GTC) seizure. Activity-regulated cytoskeletal gene (Arc) expression levels were assessed 2 hours post GTC by immunostaining of coronal sections of the hippocampus and forebrain.

Results: Over the first 80 days of differentiation there was a reduction in electrophsysiological bursting and a delay to the presence of synchronized bursting of culture networks in the 15q11.2 deleted human neurons compared to controls. This was associated with decreased single cell dendritic complexity and altered structural connectivity across culture networks as assessed with immunostaining for neurons specific beta tubulin III⁺ processes. Cyfip1 deficient mice demonstrated decreased latency to seizure in response to disinhibition by flurothyl. This was associated with increased expression of Arc compared to control mice.

Conclusions: These data suggest that deletion of the 15q11.2 region results in cell autonomous changes in neurons and synaptic maturation that contribute to pathologic changes in network excitability. As Arc expression is tightly regulated by neuronal activity and in turn, regulates structural plasticity at synapses, altered plasticity is either a cause or consequence of seizure susceptibility in Cyfip1 deficient mice. Together, these in vitro and in vivo studies indicate that 15q11.2 deletion results in impaired early neuronal development that may contribute to increased seizure susceptibility in humans with the deletion.

Funding: Please list any funding that was received in support of this abstract.: K08NS102526 NIH/NINDS, 2020 Doris Duke Charitable Foundation Clinical Scientist Award, CNCDP K12 (CWH).