Jennifer Kearney, Ph.D.
Ted Cummins, Ph.D.
NaV and KV channels are critical for the initiation and propagation of action potentials in neurons, and serve as points of convergence in determining neuronal excitability. Functional characterization of a small fraction of epilepsy-associated NaV and KV channel variants has had great value in demonstrating the range of dysfunction. However, extrapolation of in vitro channel dysfunction to in vivo alterations of excitatory/inhibitory (E/I) balance is not necessarily straightforward. A central goal of our Center is to determine what information obtained from in vitro cellular models best predict neuronal dysfunction and drug responses in an intact and fully developed brain. Project 3 is developing representative animal models with prototypical NaV and KV channel variants in order to achieve this goal. We hypothesize that differences in the relative contribution of specific channels to excitability in various cell types within neuronal networks determines the net effect on E/I balance and influences drug responses. Mouse models of channelopathy-associated epilepsy will enable studies to evaluate the effect of channel variants in native cell neurons under control of endogenous regulatory elements at the whole animal, network and cellular level, as well as enable studies of anticonvulsant drug responses. The ultimate goal of this project is to provide mechanistic insight into the effects of channel dysfunction in a developed brain, which will facilitate translation of results from Project 1 (high-throughput in vitro functional evaluation of channel variants) and Project 2 (investigating induced pluripotent stem cell (iPSC)-derived neurons) into clinically actionable information for guiding precision treatment. In addition to the utility of these mouse models for addressing the specific research questions of our Center, the models are a readily-available resource for the wider research community in order to advance our understanding of the underlying pathophysiology of epilepsy.
Aim 1 implements a standardized experimental pipeline to elucidate epilepsy phenotypes and anticonvulsant drug responses in a series of mouse models of channelopathy-associated epilepsy. Two existing unpublished mouse models with novel variants in SCN2A and KCNB1 are enabling the initial studies. This will be followed by creation and characterization of additional models of variants selected based on studies performed by Project 1 and in collaboration with Core A. The work by Project 1 will establish that a channel variant produces the same or highly similar dysfunction in mouse and human orthologs. This is an important aspect of the rigor underlying our study that will serve to avoid generating mouse models in cases were channel dysfunction is divergent between species as has been observed in other genetic disorders such as cystic fibrosis.
In Aims 2 and 3, we are performing in-depth investigations of the cellular and neuronal network effects of channel variants in the developed brain. We hypothesize that NaV and KV channel variants contribute to neonatal and infantile seizure generation by impairing excitability of inhibitory interneurons, or by increasing excitability of principle cells or disinhibitory interneurons. Our approach can elucidate the cell-type specific dysfunction that manifests at the level of neuronal circuits, which we will compare with alternations in single-neuron excitability. Results from these endeavors will provide mechanistic insight into epilepsy pathogenesis for each studied ion channel variant. We also propose to determine the effectiveness of specific pharmacological agents at normalizing abnormalities in cellular excitability, excitation-inhibition ratio and neuronal network activity. Pharmacological agents will be prioritized based on work performed by Project 1 and/or clinical drug response information collated by Core A. In collaboration with Project 2, informative side-by-side comparisons of neurophysiological and pharmacological properties will be made between mouse and human neurons carrying the same variant.
Highlights of Project 3:
• Generation of several novel mouse models of channelopathy-associated epilepsy
• Combined expertise in mouse genetics, phenotyping, in vivo pharmacology, slice physiology
• Direct comparisons of channel dysfunction in different model systems