The Phosphoprotein Synapsin Ia Regulates the Kinetics of Dense-Core Vesicle Release.

Common fusion machinery mediates the Ca2+-dependent exocytosis of synaptic vesicles (SVs) and dense-core vesicles (DCVs). Previously, Synapsin Ia (Syn Ia) was found to localize to SVs, essential for mobilizing SVs to the plasma membrane through phosphorylation. However, whether (or how) the phosphoprotein Syn Ia plays a role in regulating DCV exocytosis remains unknown. To answer these questions, we measured the dynamics of DCV exocytosis by using single-vesicle amperometry in PC12 cells (derived from the pheochromocytoma of rats of unknown sex) overexpressing wild-type or phosphodeficient Syn Ia. We found that overexpression of phosphodeficient Syn Ia decreased the DCV secretion rate, specifically via residues previously shown to undergo calmodulin-dependent kinase (CaMK)-mediated phosphorylation (S9, S566, and S603). Moreover, the fusion pore kinetics during DCV exocytosis were found to be differentially regulated by Syn Ia and two phosphodeficient Syn Ia mutants (Syn Ia-S62A and Syn Ia-S9,566,603A). Kinetic analysis suggested that Syn Ia may regulate the closure and dilation of DCV fusion pores via these sites, implying the potential interactions of Syn Ia with certain DCV proteins involved in the regulation of fusion pore dynamics. Furthermore, we predicted the interaction of Syn Ia with several DCV proteins, including Synaptophysin (Syp) and soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. By immunoprecipitation, we found that Syn Ia interacted with Syp via phosphorylation. Moreover, a proximity ligation assay (PLA) confirmed their phosphorylation-dependent, in situ interaction on DCVs. Together, these findings reveal a phosphorylation-mediated regulation of DCV exocytosis by Syn Ia.SIGNIFICANCE STATEMENT Although they exhibit distinct exocytosis dynamics upon stimulation, synaptic vesicles (SVs) and dense-core vesicles (DCVs) may undergo co-release in neurons and neuroendocrine cells through an undefined molecular mechanism. Synapsin Ia (Syn Ia) is known to recruit SVs to the plasma membrane via phosphorylation. Here, we examined whether Syn Ia also affects the dynamics of DCV exocytosis. We showed that Syn Ia regulates the DCV secretion rate and fusion pore kinetics during DCV exocytosis. Moreover, Syn Ia-mediated regulation of DCV exocytosis depends on phosphorylation. We further found that Syn Ia interacts with Synaptophysin (Syp) on DCVs in a phosphorylation-dependent manner. Thus, these results suggest that Syn Ia may regulate the release of DCVs via phosphorylation.

Phosphorylation of cysteine string protein-α up-regulates the frequency of cholinergic waves via starburst amacrine cells.

During the first postnatal week in rodents, cholinergic retinal waves initiate in starburst amacrine cells (SACs), propagating to retinal ganglion cells (RGCs) and visual centers, essential for visual circuit refinement. By modulating exocytosis in SACs, dynamic changes in the protein kinase A (PKA) activity can regulate the spatiotemporal patterns of cholinergic waves. Previously, cysteine string protein-α (CSPα) is found to interact with the core exocytotic machinery by PKA-mediated phosphorylation at serine 10 (S10). However, whether PKA-mediated CSPα phosphorylation may regulate cholinergic waves via SACs remains unknown. Here, we examined how CSPα phosphorylation in SACs regulates cholinergic waves. First, we identified that CSPα1 is the major isoform in developing rat SACs and the inner plexiform layer during the first postnatal week. Using SAC-specific expression, we found that the CSPα1-PKA-phosphodeficient mutant (CSP-S10A) decreased wave frequency, but did not alter the wave spatial correlation compared to control, wild-type CSPα1 (CSP-WT), or two PKA-phosphomimetic mutants (CSP-S10D and CSP-S10E). These suggest that CSPα-S10 phosphodeficiency in SACs dampens the frequency of cholinergic waves. Moreover, the level of phospho-PKA substrates was significantly reduced in SACs overexpressing CSP-S10A compared to control or CSP-WT, suggesting that the dampened wave frequency is correlated with the decreased PKA activity. Further, compared to control or CSP-WT, CSP-S10A in SACs reduced the periodicity of wave-associated postsynaptic currents (PSCs) in neighboring RGCs, suggesting that these RGCs received the weakened synaptic inputs from SACs overexpressing CSP-S10A. Finally, CSP-S10A in SACs decreased the PSC amplitude and the slope to peak PSC compared to control or CSP-WT, suggesting that CSPα-S10 phosphodeficiency may dampen the speed of the SAC-RGC transmission. Thus, via PKA-mediated phosphorylation, CSPα in SACs may facilitate the SAC-RGC transmission, contributing to the robust frequency of cholinergic waves.

Presynaptic SNAP-25 regulates retinal waves and retinogeniculate projection via phosphorylation.

Patterned spontaneous activity periodically displays in developing retinas termed retinal waves, essential for visual circuit refinement. In neonatal rodents, retinal waves initiate in starburst amacrine cells (SACs), propagating across retinal ganglion cells (RGCs), further through visual centers. Although these waves are shown temporally synchronized with transiently high PKA activity, the downstream PKA target important for regulating the transmission from SACs remains unidentified. A t-SNARE, synaptosome-associated protein of 25 kDa (SNAP-25/SN25), serves as a PKA substrate, implying a potential role of SN25 in regulating retinal development. Here, we examined whether SN25 in SACs could regulate wave properties and retinogeniculate projection during development. In developing SACs, overexpression of wild-type SN25b, but not the PKA-phosphodeficient mutant (SN25b-T138A), decreased the frequency and spatial correlation of wave-associated calcium transients. Overexpressing SN25b, but not SN25b-T138A, in SACs dampened spontaneous, wave-associated, postsynaptic currents in RGCs and decreased the SAC release upon augmenting the cAMP-PKA signaling. These results suggest that SN25b overexpression may inhibit the strength of transmission from SACs via PKA-mediated phosphorylation at T138. Moreover, knockdown of endogenous SN25b increased the frequency of wave-associated calcium transients, supporting the role of SN25 in restraining wave periodicity. Finally, the eye-specific segregation of retinogeniculate projection was impaired by in vivo overexpression of SN25b, but not SN25b-T138A, in SACs. These results suggest that SN25 in developing SACs dampens the spatiotemporal properties of retinal waves and limits visual circuit refinement by phosphorylation at T138. Therefore, SN25 in SACs plays a profound role in regulating visual circuit refinement.