Jasmeet Kaur Shergill

• EPS15 and its paralog EPS15R are endocytic adaptor proteins best known for initiating clathrin-mediated endocytosis and facilitating the uptake of EGFR, yet their neuronal roles have remained unclear. This thesis establishes that both proteins are critical components for synaptic receptor regulation, neurodevelopment, and ventricular homeostasis, suggesting that their functions are closely integrated with broader processes essential for brain development and maintenance. Comprehensive analyses showed that EPS15 and EPS15R are broadly expressed across the brain but display a distinct regional enrichment and a progressive postnatal upregulation. Both localize to pre- and postsynaptic compartments, supporting a dual function in synaptic vesicle recycling and neurotransmitter receptor trafficking. Their interaction with the AMPA receptor subunit GluA1, and the resulting GluA1 surface accumulation upon loss of EPS15/EPS15R, identify them as key regulators of AMPAR endocytosis with a potential influence on excitatory balance. In contrast, transferrin receptor uptake remained unaffected, indicating that global clathrin-mediated endocytosis is preserved. Unbiased proteomic analysis defined the molecular networks through which EPS15 and EPS15R operate. Both interactomes were strongly enriched for canonical endocytic components, confirming their roles in vesicle formation and cargo selection. However, in addition, the EPS15R interactome revealed unique partners such as CALCOCO1 and SEC16A, implicating it in autophagy and ER-associated membrane trafficking. These findings suggest a non-canonical role for EPS15R as a scaffold linking endocytic, secretory, and degradative pathways. At the organismal level, the forebrain-specific loss of EPS15 and EPS15R resulted in growth deficits, seizures, hind limb clasping, behavioral changes, alterations in brain anatomy, and reduced postnatal survival, highlighting their importance for overall brain function. Structural analyses revealed cortical and hippocampal lamination defects closely resembling those observed upon disruption of Reelin signaling. Supporting a mechanistic link, our biochemical analyses demonstrated that EPS15R physically associates with the Reelin receptor ApoER2, suggesting that it may participate in the trafficking or stabilization of Reelin receptor complexes, thereby influencing neuronal positioning and cortical layering during development. In addition, EPS15/15R knockout mice developed ventriculomegaly. Cell culture experiments revealed defective ependymal differentiation and multiciliogenesis as potential cause for this phenotype. Given that proper ependymal maturation requires controlled EGFR downregulation and Notch signaling modulation, the loss of EPS15/15R might disrupts these processes, thereby leading to impaired differentiation and compromised ventricular integrity. Together, these findings identify EPS15 and EPS15R as multifaceted adaptors that integrate endocytic trafficking with neuronal signaling and brain morphogenesis. By ensuring proper receptor internalization, neuronal lamination, and ependymal differentiation, they safeguard both the synaptic and structural integrity of the brain.