Pathophysiology of KCNA2-mediated epileptic encephalopathies and the effect of SCN1A variants on thalamocortical up-states

Abstract

Understanding the pathophysiological consequences of different ion-channel encoding gene mutations in the evolution of epileptic seizures remains a major goal in epilepsy research. Even after understanding the complex mechanisms that underlie epileptogenesis, the comorbidities associated with epilepsy still need to be studied. The development of new effective treatment options is totally dependent on the knowledge of these mechanisms. In this context, my thesis aimed to address the following two aspects of epilepsies:

(1) The pathophysiological mechanisms and development of treatment strategies for KCNA2-mediated developmental and epileptic encephalopathies: Developmental and epileptic encephalopathies (DEE) are disorders of early childhood, characterized by severe recurrent seizures and mental dysfunction. Often this progressive deterioration may be due to gene mutations induced effects or sometimes also to continuous seizures during development. KCNA2 has been identified as one of the candidate genes in DEE that encodes the voltage-gated potassium channel KV1.2, which is important in shaping neuronal repolarization. KV1.2 expresses in both excitatory and inhibitory neurons of the central nervous system (CNS). In the present work, I intended to unravel the pathophysiological mechanisms of both loss- and gain-of-function (LOF/GOF) KCNA2 variants for the morphological and electrophysiological properties of single neurons. Both forms of variants cause clinically distinguishable syndromes. Mouse models overexpressing (in utero) wildtype and mutated KV1.2 channel subunits in somatosensory cortical layer 2/3 pyramidal neurons were used to get insights into the related pathophysiology. Electrophysiological and histochemical approaches were used to decipher single neuronal level changes. My results showed a reduction in dendritic arborization and action potential firing in KV1.2 WT as well as GOF variant overexpressing neurons, probably due to a membrane hyperpolarization. Overexpression of LOF variant exhibited a considerable reduction in the number of APs probably due to the elongated repolarization phase, without significantly altering the morphological features as compared to control cells. The current study also analysed the application of therapeutic approaches using 4-Aminopyridine (4-AP) as a specific blocker for KV1 and KV4 potassium channels, which rescued the KV1.2 WT overexpressing neurons from their hypoexcitablity. These findings provided the leads in addressing the therapeutic manipulation of KCNA2 gene-related epileptic encephalopathy by using specific antagonist mechanisms.

(2) Effect of NaV1.1 mutations on thalamocortical up-states studied in genetic mouse models: Epilepsy and sleep are interrelated, and share the same thalamocortical (TC) loop as a regulatory mechanism. However, the interrelation between sleep and epilepsy at the molecular and cellular level is still unclear. The usage of antiepileptic drugs in patients has made it hard to understand this interconnection. Genetic mouse models carrying LOF and GOF mutations in SCN1A gene encoding NaV1.1 are possible windows to open this reciprocal interaction. Mutations in NaV1.1 channels have mainly altered inhibitory neuronal firing and thus led to uncontrolled firing in excitatory neurons and epilepsy. The characteristics of spontaneously generated TC up-states (<1Hz) within in vitro brain slices were studied through extracellular multi-unit (MU) neuronal recordings. The project had two objectives: (i) To understand the effect of SCN1A LOF on TC network dysfunction. (ii) To investigate the effect of SCN1A GOF (thus, an opposite molecular defect causing a rare sub-form of migraine) on TC up-states. I recorded TC up- and down-states from layer V neurons of somatosensory cortex (SSC) of acute and organotypic brain slices from SCN1A mouse. Recordings from both LOF and GOF conditions showed a reduction in the frequency of TC up states. This indicates the strong influence of inhibitory neurons in maintaining the balance of synchronized TC network firing, which is an important aspect of slow-wave sleep (SWS). However, this needs further validation experiments using in vivo models.

Overall, my thesis showed first pathophysiological neuronal mechanisms and 4-AP as an effective precision therapy (in vitro) in KCNA2-related DEE. The current work also showed an impact of SCN1A variants (LOF and GOF) on the TC network.