CHARACTERIZING THE MOLECULAR COMPOSITION OF THE CISTERNAL ORGANELLE AT THE AXON INITIAL SEGMENT

The axon initial segment (AIS) is a specialized compartment at the proximal axon essential for action potential generation. Changes in neuronal activity induce structural and functional plasticity at the AIS and its subdomains. The cisternal organelle (CO), a specialized ER domain within the AIS, is suggested to regulate local Ca²⁺ events and to shape activity-dependent AIS remodeling. However, the molecular composition of the CO remains poorly characterized. Samples of human neocortex, obtained from access paths of neurosurgical procedures, were used to investigate the CO in human pyramidal neurons. Immunofluorescence and STED microscopy were employed to visualize synaptopodin, a CO-associated protein, together with an AIS marker and candidate proteins that are predicted to be part of the CO microenvironment. Candidate proteins were then quantified based on their spatial localization relative to synaptopodin in axial scans. The CO was found to be enriched in the Ca²⁺ ion pump SERCA, but not in RyR1. Clusters of the gap junction component connexin-43 were found to align with the CO. Furthermore, we observed a high frequency of membrane contact sites between the ER and axolema, formed by VAMP-associated protein A. Ongoing work will expand the molecular map of the human CO and investigate how the microdomain influences Ca²⁺ signaling and AIS remodeling.