Investigation Of Nav1.1 Mutation Effect And Epileptic Mechanism Using Patient IPSCs

Mutations in SCN1A, the gene encoding a subunit of Nav1.1 channel, can cause epilepsies with wide ranges of clinical phenotypes, which are associated with the contrasting effects of channel loss-of-function or gain-of-function. In this project, CRISPR/Cas9-and TALEN-mediated genome-editing techniques were applied to iPSC-based-disease model to explore mechanism of epilepsy caused by SCN1A loss-of-function mutation. By fluorescently labeling GABAergic subtype in iPSC-derived neurons using CRISPR/Cas9, we for the first time performed electrophysiological studies on SCN1A-expressing neural subtype and monitored the spontaneous postsynaptic activity of both inhibitory and excitatory types. We found that the mutation c.A5768G, which led to no current of Nav1.1 in exogenously transfected system, influenced the properties of not only Nav current amount, but also Nav activation in Nav 1.1-expressing GABAergic neurons. The two alterations in Nav further reduced the amplitudes and enhanced the thresholds of action potential in patient-derived GABAergic neurons, and led to weakened spontaneous inhibitory postsynaptic currents (sIPSCs) in patient-derived neuronal network. Although the spontaneous excitatory postsynaptic currents (sEPSCs) did not change significantly, when the frequencies of both sIPSCs and sEPSCs were further analyzed, we found the whole postsynaptic activity transferred from inhibition dominated state to excitation in patient-derived neuronal networks, suggesting changes in sIPSCs alone was sufficient to significantly reverse the excitatory level of spontaneous postsynaptic activity.The findings in this study were obtained on the selectively labeled mutant-expressing neural subtype and the neural network that was composed of distinct neural subtypes, which thus provides us the first accurate data about the mutation effect on its neural host and on the entire network. This finding fill the gap of our knowledge regarding the relationship between SCN1A mutation effects recorded on exogenously transfected cells and on Nav 1.1-expressing neurons. Additionally, our study revealed the physiological basis underlying epileptogenesis caused by SCN1A loss-of-function mutation, and showed us the origin of disease, which could provide practical instruction for clinical drug administration, as well as durg develpment and screening.