Secretagogin, Driven by Neuronal Activity, Transiently Regulates Exocytosis During Development

Fluctuations of intracellular ionic concentrations are powerful signaling tools in living cells [1]. Among these ions, Ca²⁺ plays a well-documented and ubiquitous role in the regulation of cellular events, ranging from development and growth to intercellular signaling and information processing, and, ultimately, Ca²⁺ regulates cell survival or cell death. All these effects are mediated through the extended family of Ca²⁺-sensors.

A recent paper published by Alpar, Harkany, and their coworkers in Acta Physiologica [2] revealed a complex and developmentally regulated signaling network centered on dynamic changes of expression of the archetypal Ca²⁺ sensor, secretagogin. They show that changes in secretagogin expression are affected by neuronal activity, and therefore establish a novel link between excitation and intracellular signaling processing. The study of Hanics et al. critically challenges the notion of secretagogin, used widely as a neuronal marker, as a static marker by demonstrating its dynamic expression in response to neuronal activity during mammalian brain development. Using a combination of human foetal brain mapping, genetically modified mice (Scgn-iCre::Ai9), single-cell RNA sequencing, and both in vitro and in vivo activity manipulation models, the authors provide compelling evidence that secretagogin expression is both developmentally regulated and sensitive to neuronal activity.

One of the main functions of Ca²⁺ sensors in the information processing loops of the central nervous system is the regulation of vesicular neurotransmitter release. Fusion of the vesicle membrane with the plasma membrane, known as exocytosis, leads to the formation of a water-filled channel, the fusion pore, a conduit for molecules in the vesicle lumen to exit into the extracellular space. This evolutionary invention emerged in primordial eukaryotic cells [3-5] and mediates a myriad of processes, including the release of neurotransmitters, hormones, and other signaling molecules, all essential for maintaining cell-to-cell communication in multicellular organisms. The release of vesicle content may occur only if the fusion pore widens sufficiently. Over seven decades ago, Bernard Katz proposed that the fusion pore opens completely upon exocytosis. Therefore, since those days, the fusion mechanism has been intensively studied, mainly focusing on how the merger of the two membranes occurs. This led to the discovery of SNARE proteins, targets of proteolytic botulinum neurotoxins, and the concept that a rather complex array of events, including vesicle priming and docking at the active zone to fusion pore formation upon an increase in cytosolic Ca²⁺, mediates stimulus-secretion coupling to within milliseconds [6]. Secretagogin is linked to regulated exocytosis through an extended interactome which includes SNAP-23, DOC2α, ARFGAP2, rootletin, KIF5B, β-tubulin, DDAH-2, ATP-synthase, and many more. As a result, secretagogins can exert both stimulatory and inhibitory effects on vesicular release.

Molecular mechanisms of exocytosis inhibition (i.e., fusion pore narrowing) remain to be fully characterized. Experimental evidence revealed that the fusion pore may reversibly open and close, indicating that the fusion pore closure is regulated, also through the regulation of the SNARE complex [7, 8]. Moreover, the reversibly opening fusion pore may at rest attain a subnanometer diameter, too narrow to pass even glutamate or acetylcholine, termed unproductive exocytosis, and can swiftly become productive with fusion pore diameter widening. This indicates that the complex array of events leading to exocytosis may be lumped into the stage of unproductive exocytosis, where the fusion pore is pre-formed and only needs a stimulus to widen [4].

One of many inhibitory processes of exocytosis and fusion pore narrowing, still understudied, may relate to the content of cholesterol in the membrane. It was shown that elevating cholesterol reduces the fusion pore diameter, measured as fusion pore conductance, and reduces the probability of the fusion pore being open [9]. Moreover, in lysosomal storage diseases, cholesterol was observed to be accumulating in lysosomes. Under such conditions, the fusion pore geometry is also reduced, likely contributing to neurological symptoms [9]. The mechanism by which high vesicle cholesterol prevents the fusion pore diameter from enlarging is depicted in Figure 1.

In addition to cholesterol, some proteins may also inhibit the exocytotic fusion pore. One such candidate is amysin, which forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with synaptobrevin-2/VAMP2 for SNARE-complex assembly. Furthermore, amysin contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phosphatidylinositol 4,5-bisphosphate (generally known as PIP₂). Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function [10]. Secretagogin exerts multiple regulatory effects on secretion, for example, through binding to syntaxin-4 [11]. Secretagogin, however, also interacts with SNAP-25, preventing the formation of the SNARE complex, thus mediating an inhibition of exocytosis as determined in vitro [12].

As alluded to before, Hanics et al. [2] discovered dynamic regulation of neuronal secretagogin expression during development. Changes in secretagogin expression may transiently inhibit exocytotic fusion pore during forebrain development. A particularly striking finding is the delayed expression of secretagogin in fetuses with Down's syndrome, suggesting clinical relevance in neurodevelopmental disorders. Moreover, experimental manipulations like dark rearing and kainate administration revealed that sensory inputs and excitation can respectively suppress or enhance secretagogin levels, supporting an activity-dependent regulatory mechanism. These results refine our understanding of secretagogin not just as a static identity marker but as a dynamic player in neurodevelopment.

This work is significant for both basic neuroscience and clinical research. It cautions against the uncritical use of secretagogin as a sole marker of cell identity, emphasizing the need to account for environmental and activity-driven variability. Furthermore, it opens new avenues for exploring how calcium sensor proteins contribute to neuronal plasticity and disease states. Overall, the study is a thorough and impactful contribution that reshapes how we view secretagogin in the developing and mature brain. It also sheds new light on the role of development in controlling regulated exocytotic fusion pore.

The authors take full responsibility for this article.

The authors declare no conflicts of interest.