Thursday, April 18, 2024
4/18/2024
PROJECT SUMMARY Dravet Syndrome (DS) is a devastating form of epilepsy caused by loss of function of NaV1.1 (80-90% of cases), the predominant voltage-gated Na+ channel expressed in inhibitory (GABAergic) interneurons in the hippocampus and prefrontal cortex. This causes a decrease in the release of inhibitory neurotransmitter (GABA), resulting in hyperexcitability. The disease manifests itself within the first year of life and is usually triggered by hyperthermia causing frequent and prolonged seizures that result in a host of health problems including developmental delay, speech impairment, ataxia, hypotonia and sleep disturbances. Two small molecule monotherapies have been approved recently by the FDA: Epidiolex (Cannabidiol or CBD) in 2018 and Fintepla (fenfluramine or FA) in 2020 for patients two years of age and older. Even though they reduce the frequency of seizures, these drugs at their effective dosages cause multiple side effects. Their mechanism(s) of action to reduce epileptiform activity remain(s) unknown. G protein-gated inwardly rectifying K+ (GIRK) channels have been strongly implicated in epilepsy. They are activated by the Gß¿ dimer of G proteins and by [Na+]i in a synergistic manner. The basis of synergism lies in that they each work allosterically to control predominantly the two channel gates: Gß¿, the membrane gate and Na+, the cytosolic gate. In the case of the GABAergic interneurons, we hypothesize that NaV1.1 and GIRK1/2 are coupled, such that the Na+ entering through NaV1.1 promotes GIRK1/2 activity to hyperpolarize the cell and ensure removal of NaV1.1 inactivation for fast spiking. In DS this mechanism becomes compromised causing cell depolarization and inactivation of voltage-gated channels at large present in GABAergic neurons failing to compensate for the loss of NaV1.1. We use GAT1508, a specific activator of GIRK1/2 to compensate for the compromised Na+ entry. Since GAT1508 opens the cytosolic gate, we ask whether it synergizes with CBD (via CB1R) and FA (via 5-HT1DR) to open more fully the membrane gates. In Aim 1, experiments designed to test the hypothesis are aimed at the cellular level in both heterologous expression and in native GABAergic neurons. In Aims 2 and 3, we utilize a DS mouse model, heterozygous for the Scn1a gene that encodes NaV1.1, and in Aim 2, we test the hypothesis at the brain slice level, where synapses and transmitter release remain intact, and compare the DS model to a wild-type animal model. In Aim 3, we pursue experiments at the whole animal level (DS model), using simultaneous EEG and 2-photon microscopy to monitor the neuronal circuits involved.
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