Mechano-Electrical Regulation of Neurogenesis - SUMMARY Dysregulation of neurogenesis during brain development can impact neuron numbers, subtypes, and connectivity, leading to a range of neurodevelopmental disorders. Our understanding of neuronal differentiation is still limited, largely centered on growth factors, morphogens, and lineage-specific transcription factors, while other fundamental aspects of cell biology such as plasma membrane properties and cytoskeleton mechanics are overlooked. Emerging evidence reveals a critical role of membrane-related electrical processes in regulating non-excitable cells such as neuroprogenitor cells (NPC) during corticogenesis. This R21 proposal explores the previously under-studied inner membrane surface charge as critical regulator of morphological transformation and neuronal lineage commitment. It is well known that anionic phospholipids are distributed asymmetrically between the inner and outer leaflets of the plasma membrane. This is maintained by an ATP-dependent process that leads to an overall negative surface charge of the inner membrane, known as zeta potential. This local potential is different from the Nernst potential, a more global electrophysiological transmembrane potential generated by diffusible ions. While the Nernst potential is well characterized in excitable cells such as neurons, the inner leaflet surface charge and the associated mechano-electrical coupling is under-studied. The asymmetric distribution of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2) in the inner leaflet can influence plasma membrane stability via cytoskeletal interactions; they also serve as signaling hubs for many downstream effectors. Our recent study revealed that NPC stiffness and cortical actin network can affect the timing of neuronal differentiation. Reducing cell stiffness and cortical actin can accelerate neuronal differentiation, likely through altered membrane surface charge, thus highlighting a novel link between mechano- electrical coupling at plasma membrane and neuronal differentiation. Here, we will test the central hypothesis that regulation of the inner membrane surface charge is critical during neurogenesis by affecting the organization of cortical actin and signaling cascades associated with membrane phospholipids. In Aim 1, we will use live-cell imaging, advanced fluorescent probes, and patch-clamp recordings to characterize the temporal and spatial distribution of inner leaflet surface charges, phospholipid composition, and cortical actin rearrangement in correspondence to morphological transformation during neurite outgrowth and neuronal differentiation. In Aim 2, we will manipulate membrane surface charge using our newly developed molecular actuators (ACTU- or ACTU+) to either increase or decrease the negative charge at the inner membrane surface and assess the impact on neuronal differentiation, maturation, survival, and functionality. In summary, this R21 proposal explores mechano-electrical regulation of neurogenesis, which will provide insights into neurodevelopmental disorders and improve disease modeling.
