Understanding the role of Cnksr2 in Epilepsy-Aphasia Syndrome

Epilepsy-Aphasia Syndromes (EAS) are debilitating childhood focal epilepsies and epileptic encephalopathies with severe cognitive and language dysfunction. Patients with EAS experience loss of previously normal language, seizures, sleep-related EEG abnormalities, and cognitive deficits. Seizures are often refractory to treatments, and the prognosis of EAS is generally poor. Even when epilepsy is resolved, most patients have life-long language and cognitive disturbances. The link between EEG abnormalities, seizures, and cognito-behavioral comorbidities seen in this spectrum is poorly understood, making it more challenging to develop therapies. While the clinical manifestations of EAS have been long studied, the etiology of EAS has been unknown. Recently, loss of function mutations in X-linked gene CNKSR2(Connector Enhancer Of Kinase Suppressor Of Ras 2) have been implicated in EAS. A small number of in vitro studies suggest that CNKSR2 encodes for a putative scaffold protein that localizes to the synapse. Yet, there have been no functional studies of Cnksr2 to examine how its absence may lead to EAS. This dissertation focuses on uncovering the role of Cnksr2 in the brain and investigating the neuropathological effects of Cnksr2 loss in the context of EAS. Here, I first present that Cnksr2 is expressed in cortical, striatal, and cerebellar regions and is localized at excitatory and inhibitory postsynapses in the mouse brain. Next, we generate a novel transgenic Cnksr2 knockout mouse model and confirm the loss of Cnksr2. Then, using proteomics analysis, I demonstrate that Cnksr2 anchors key binding partners in synapses, and its loss results in significant alterations of synaptic proteins implicated in neurological disorders. I also find that loss of Cnksr2 leads to increased spontaneous activity of neurons, and Cnksr2 KO mice exhibit electrographic seizures and epileptiform discharges. Notably, Cnksr2 KO mice show significantly increased anxiety, impaired learning, and a progressive and dramatic loss of ultrasonic vocalizations. Next, I investigate the cell-specific contributions to the core Cnksr2-loss phenotypes in mice. Particularly, I seek to determine the effects of Cnksr2 deletion in excitatory vs. inhibitory cells using Cre/Lox system. Surprisingly, I discover that cell-specific deletion of Cnksr2 results in distinct phenotypic outcomes: glutamatergic neuron-specific deletion of Cnksr2 leads to behavioral deficits, and the GABAergic neuron-specific deletion leads to electrographic seizures. Taken together, my doctoral dissertation research validates that loss of CNKSR2 leads to EAS, as well as highlights the fundamental roles of Cnksr2 in synaptic protein organization and excitation of neuronal networks. Furthermore, I show that Cnksr2 KO mice exhibit electrophysiological and behavioral phenotypes similar to those of patients, providing an exciting new model for future therapeutics studies of EAS. Lastly, my research provides novel insights into the cellular mechanisms leading to distinct EAS-related pathology.