Dissecting the Molecular Mechanism Underlying Epilepsy Aphasia Syndrome

Abstract

Epilepsy Aphasia Syndrome (EAS) is a spectrum of childhood disorders that exhibit complex co-morbidities that include epilepsy and the emergence of cognitive and language disorders. Patients with EAS experience seizures, sleep related EEG abnormalities, cognitive deficits, and loss of language. Treatments for Epilepsy are often insufficient with poor outcomes. Interestingly as the disorder progresses and epilepsy resolves patients unfortunately suffer from life-long cognitive and language deficits. Finally, the connection between abnormalities in EEG, seizures, and the accompanying cognitive-behavioral issues within this spectrum remains unclear, complicating the development of effective treatments.Although the clinical features of EAS have been extensively researched for some time, the causes behind EAS remain unidentified. Studies have found CNKSR2 (Connector Enhancer of Kinase Suppressor of Ras 2) is an X-linked gene in which mutations are linked to EAS. A few in vitro investigations indicate that CNKSR2 is responsible for coding a scaffold protein that is found at the synapse. However, there are few practical studies conducted on Cnksr2 to explore the effects of its absence on the development of EAS. This thesis focuses on expanding upon the current role of Cnksr2 in the brain investigating cellular roles and how they contribute to different phenotypes found within EAS. We previously demonstrated Cnksr2 knockout (KO) mice model key phenotypes of EAS analogous to those present in clinical patients with mutations in the gene. Cnksr2 KO mice have increased seizures, impaired learning and memory, increased levels of anxiety, and loss of ultrasonic vocalizations (USV). The intricate interplay between these diverse phenotypes at the brain regional and cell type level remains unknown. Here we leverage conditional deletion of Cnksr2 in a neuronal cell type manner to demonstrate that anxiety and impaired USVs track with its loss from excitatory neurons. Conversely inhibitory knockout of Cnksr2 track to the seizure phenotype previously identified. I further investigate the links between excitatory neuronal Cnsksr2 and these phenotypes using temporal and brain-specific deletion of Cnksr2. Using glutamatergic cortical specific drivers, I find that anxiety and impaired USV calls can be further separated based brain region. I further narrow the essential role of Cnksr2 loss in USV deficits to excitatory neurons of the Anterior Cingulate Cortex (ACC), a region previously in mice implicated in USV production associated with specific emotional states or social contexts, such as mating calls, distress calls, or social bonding signals. I also utilized proximity labeling of endogenous Cnksr2 within glutamatergic or GABAergic neurons in vivo to reveal core Cnksr2 functions in excitatory synapse organization. Finally, utilizing in-vitro extracellular multielectrode array recordings we uncover that neurons with Cnksr2 deletion exhibit increase in neuronal firing. Taken together, my doctoral dissertation results reveal a Cnksr2-based mechanism that underlie USV impairments that suggest communication impairments can be dissociated from seizures or anxiety. Furthermore, we highlight the cortical circuitry important for initiating USVs, and the core functions of Cnksr2.