INVESTIGATING PCDH19 SYNAPTIC FUNCTION IN MOUSE EMBRYONIC STEM CELL-DERIVED NEURONS

Protocadherin-19 (PCDH19), an X-linked cadherin superfamily member, is associated with early-onset epilepsy characterized by infantile seizures, cognitive impairment, and neuropsychiatric comorbidities. The disorder predominantly affects heterozygous females, most likely due to cellular interference arising from mosaic expression of PCDH19 in the brain. We have uncovered a reduced calcium response in mouse embryonic stem cell (mESC)-derived neurons lacking PCDH19 (PCDH19 knockout, KO) compared with wild-type (WT) neurons, and a similar reduction in WT neurons when co-cultured with PCDH19 KO neurons; however, the molecular mechanisms underlying this effect remain unknown. We aim to investigate the molecular mechanisms by which PCDH19 regulates synaptic function using mESC-derived neurons cultured as WT, PCDH19 KO, and WT/KO mixed populations to model mosaicism in vitro. Experiments are performed on mature neurons at day in vitro 12. Plasma membrane protein–enriched samples and synaptosomal fractions are analyzed using data-independent acquisition mass spectrometry to assess changes in the levels of membrane and synaptic proteins, respectively. Preliminary differential expression analysis has revealed significant changes in protein abundance between mutant, wild-type, and mixed populations. We will now use these proteomic data to formulate hypotheses regarding possible mechanisms underlying the reduced intracellular calcium response, which we will test and validate using calcium imaging and western blotting. Together, this work aims to link PCDH19-dependent proteomic alterations to functional changes in calcium signalling and synaptic activity in a cellular model of PCDH19-related epilepsy.