| dc.description.abstract | Epilepsy is a chronic neurological disorder characterized by unpredicted recurrent seizures. Dysfunction of GABA-A receptors (GABA-ARs) in the brain plays a key role in the pathophysiology of epilepsy. Neurosteroids are powerful allosteric agonists of GABA-ARs and are more efficacious on extrasynaptic subunit-containing GABA-ARs that mediate tonic inhibition. Endogenous neurosteroids such as allopregnanolone (AP) have broad-spectrum anticonvulsant activity and possess therapeutic potential for epilepsy. There are many synthetic neurosteroids in trials. Ganaxolone (GX) is the 3-methylated synthetic analog of AP. Although GX was previously tested in recombinant GABA-ARs, its precise mechanisms of action on native neurons in the brain, including its ability to modulate extrasynaptic GABA-ARs, and its interactions with other molecules, remain unknown.
The main objective of this dissertation is to investigate the molecular, cell-specific, and phosphorylation-dependent mechanisms of action of synthetic neurosteroid GX and related neurosteroid agents at extrasynaptic GABA-ARs using pharmacological, electrophysiological, and behavioral approaches. In addition, structure-activity relationships of analogs are tested in native neurons. Several GABA-AR subunits contain phosphorylation sites regulated by protein kinases. Therefore, this study examines the influences of protein kinase activity on neurosteroid actions in native neurons. In addition, we explore the physiological interactions between neurosteroids and zinc and the potential combination strategy of neurosteroids with other clinical GABAergic agents.
We established concentration-response profiles of GX in two cell types: (i) -abundant dentate gyrus granule cells (DGGCs) and (ii) -rich CA1 pyramidal cells (CA1PCs). Our results show that GX and analogs are preferential allosteric modulators and direct activators of extrasynaptic GABA-ARs regulating network inhibition and seizures in
the dentate gyrus. The potentiation of tonic inhibition by GX is subunit-dependent and
protein kinase C activity-mediated. Zinc concentration-dependently blocks GX- potentiated GABA-gated currents in DGGCs and CA1PCs. Zinc reduces antiseizure activity of GX by the selective blockade of extrasynaptic GABA-AR-mediated tonic inhibition in the hippocampus. Combination therapies of neurosteroids with clinically used antiepileptic drugs tiagabine and midazolam exhibit strong synergistic effects on antiseizure activity and hippocampal tonic inhibition. Overall, these findings signify a unique role for extrasynaptic δGABA-ARs as key modulators of excitability in the brain and provide valuable mechanistic rationales for the clinical synergistic potential of neurosteroids in epilepsy and seizure disorders. | en |
