Florey Department of Neuroscience and Mental Health - Theses
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ItemEpileptic Encephalopathies: Identification and Characterization of Disease Mechanisms( 2022)Pathogenic mutations in the KCNQ2 gene, encoding the KV7.2 voltage - gated potassium channel, are known to cause neonatal seizure disorders, including severe Epileptic Encephalopathies. Epileptic Encephalopathies are characterised by pharmacoresistant seizures, developmental delay, and behavioural and cognitive deficits. Current therapies show limited efficacy in the treatment of seizures and fail to address the devastating comorbidities 1–5. KCNQ2 de novo variant K556E was identified in a patient with Epileptic Encephalopathies 6. In this research project we aimed to characterize the KCNQ2 K556E variant to better understand the underlying mechanisms of the disease and to set the stage for therapeutic screens that will help finding better treatments for this patient and for other patients like her. In order to assess the disease mechanisms of this variant, three disease models were used. Initially, the biophysical properties of the variant were investigated using in vitro expression in Xenopus oocytes followed by two - electrode Voltage - clamp Recordings. The data suggest a loss-of-function with no dominant negative effect caused by the variant. A loss of KV7 channel function indicate a significant reduction in the production of the potassium M - current. The M - current main biophysical role is setting the neuronal resting membrane potential and protecting against uncontrolled repetitive action potential firing. The loss-of-function might be the underlying cause of an excitable phenotype. We hypnotised that application of KV7 channels opener might rescue the significant reduction current seen in KCNQ2 K556E variant. However, retigabine, a KV7 opener, had no significant effect on the variant’s biophysical properties when the mutant channel was expressed as a homotetramer in Xenopus laevis oocytes. The second disease model we have generated and characterized is an in vivo disease model. A knock - in mouse model carrying the corresponding amino acid exchange in its KCNQ2 gene. Our observational studies revealed that both homozygous and heterozygous mice develop spontaneous seizures and present with increased mortality rates and premature death compared to wild-type littermate controls. The heterozygous mice are more susceptible to both heat induced seizures and chemically induced seizures. The heterozygous mice also expressed some behavioural changes when compared with wild-type littermate controls. The heterozygous mice bury less marbles in the marble burying test when compared with wild-type littermate controls. This might suggest a less anxious like behaviour and reduced cognitive abilities. Furthermore, the significant difference found between genotypes in this specific behavioural test could be used as an efficacy marker in a drug screening set of experiments in later stages. Lastly, we have generated and characterized patient specific in vitro iPSC-derived neuronal cultures. To this end we exploited a differentiation method based on the overexpression of Neurogenin 2 (NGN2) transcription factor to generate excitatory cortical neurons and investigated the electrophysiological characteristics of the neuronal cultures using whole cell patch clamping technique. Our findings suggest a more excitable phenotype of the patient-derived neurons in comparison with a control cell line from a healthy subject. In conclusion, the disease models indicate that a loss-of-function caused by the K556E variant likely leads to an increase in neuronal excitability which may be responsible for the increased susceptibility to epileptic seizures.