Liprin-α3 controls vesicle docking and exocytosis at the active zone of hippocampal synapses

The presynaptic active zone provides sites for vesicle docking and release at central nervous synapses and is essential for speed and accuracy of synaptic transmission. Liprin-α binds to several active zone proteins, and loss-of-function studies in invertebrates established important roles for Liprin-α in neurodevelopment and active zone assembly. However, Liprin-α localization and functions in vertebrates have remained unclear. We used stimulated emission depletion superresolution microscopy to systematically determine the localization of Liprin-α2 and Liprin-α3, the two predominant Liprin-α proteins in the vertebrate brain, relative to other active-zone proteins. Both proteins were widely distributed in hippocampal nerve terminals, and Liprin-α3, but not Liprin-α2, had a prominent component that colocalized with the active-zone proteins Bassoon, RIM, Munc13, RIM-BP, and ELKS. To assess Liprin-α3 functions, we generated Liprin-α3–KO mice by using CRISPR/Cas9 gene editing. We found reduced synaptic vesicle tethering and docking in hippocampal neurons of Liprin-α3–KO mice, and synaptic vesicle exocytosis was impaired. Liprin-α3 KO also led to mild alterations in active zone structure, accompanied by translocation of Liprin-α2 to active zones. These findings establish important roles for Liprin-α3 in active-zone assembly and function, and suggest that interplay between various Liprin-α proteins controls their active-zone localization.

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Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis

The Journal of Cell Biology ◽

10.1083/jcb.200711167 ◽

2008 ◽

Vol 181 (5) ◽

pp. 831-846 ◽

Cited By ~ 86

Author(s):

Sergio Leal-Ortiz ◽

Clarissa L. Waites ◽

Ryan Terry-Lorenzo ◽

Pedro Zamorano ◽

Eckart D. Gundelfinger ◽

...

Keyword(s):

Synaptic Vesicle ◽

Active Zone ◽

Hippocampal Neurons ◽

Synapse Formation ◽

Zone Formation ◽

Homologous Protein ◽

Synaptic Vesicle Exocytosis ◽

Vesicle Exocytosis ◽

Presynaptic Plasma Membrane ◽

And Function

Active zones are specialized regions of the presynaptic plasma membrane designed for the efficient and repetitive release of neurotransmitter via synaptic vesicle (SV) exocytosis. Piccolo is a high molecular weight component of the active zone that is hypothesized to participate both in active zone formation and the scaffolding of key molecules involved in SV recycling. In this study, we use interference RNAs to eliminate Piccolo expression from cultured hippocampal neurons to assess its involvement in synapse formation and function. Our data show that Piccolo is not required for glutamatergic synapse formation but does influence presynaptic function by negatively regulating SV exocytosis. Mechanistically, this regulation appears to be calmodulin kinase II–dependent and mediated through the modulation of Synapsin1a dynamics. This function is not shared by the highly homologous protein Bassoon, which indicates that Piccolo has a unique role in coupling the mobilization of SVs in the reserve pool to events within the active zone.

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PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure

Nature Communications ◽

10.1038/s41467-021-23116-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Javier Emperador-Melero ◽

Man Yan Wong ◽

Shan Shan H. Wang ◽

Giovanni de Nola ◽

Hajnalka Nyitrai ◽

...

Keyword(s):

Phase Separation ◽

Active Zone ◽

Neurotransmitter Release ◽

Hippocampal Neurons ◽

Phosphorylation Site ◽

Double Knockout ◽

Zone Structure ◽

Presynaptic Nerve Terminal ◽

Condensate Formation ◽

And Function

AbstractThe active zone of a presynaptic nerve terminal defines sites for neurotransmitter release. Its protein machinery may be organized through liquid–liquid phase separation, a mechanism for the formation of membrane-less subcellular compartments. Here, we show that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation in transfected HEK293T cells. Condensate formation is triggered by Liprin-α3 PKC-phosphorylation at serine-760, and RIM and Munc13 are co-recruited into membrane-attached condensates. Phospho-specific antibodies establish phosphorylation of Liprin-α3 serine-760 in transfected cells and mouse brain tissue. In primary hippocampal neurons of newly generated Liprin-α2/α3 double knockout mice, synaptic levels of RIM and Munc13 are reduced and the pool of releasable vesicles is decreased. Re-expression of Liprin-α3 restored these presynaptic defects, while mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented this rescue. Finally, PKC activation in these neurons acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. Our findings indicate that PKC-mediated phosphorylation of Liprin-α3 triggers its phase separation and modulates active zone structure and function.

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Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1521600113 ◽

2016 ◽

Vol 113 (29) ◽

pp. 8314-8319 ◽

Cited By ~ 22

Author(s):

Tae-Sun Lee ◽

Joo-Young Lee ◽

Jae Won Kyung ◽

Yoosoo Yang ◽

Seung Ju Park ◽

...

Keyword(s):

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Hippocampal Neurons ◽

Vesicle Fusion ◽

Synaptic Membrane ◽

Synaptic Vesicle Exocytosis ◽

Dependent Inhibition ◽

Vesicle Exocytosis ◽

Inositol Pyrophosphates

Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6. Synaptotagmin 1 (Syt1), a Ca2+ sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7. Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6. In addition, 5-IP7–dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca2+ levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca2+. These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.

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Liprin-α2 promotes the presynaptic recruitment and turnover of RIM1/CASK to facilitate synaptic transmission

The Journal of Cell Biology ◽

10.1083/jcb.201301011 ◽

2013 ◽

Vol 201 (6) ◽

pp. 915-928 ◽

Cited By ~ 62

Author(s):

Samantha A. Spangler ◽

Sabine K. Schmitz ◽

Josta T. Kevenaar ◽

Esther de Graaff ◽

Heidi de Wit ◽

...

Keyword(s):

Synaptic Vesicle ◽

Active Zone ◽

Molecular Complexes ◽

Ubiquitin Proteasome System ◽

Synaptic Activity ◽

Network Activity ◽

Synaptic Vesicle Exocytosis ◽

Vesicle Exocytosis ◽

Ubiquitin Proteasome ◽

Synaptic Vesicle Release

The presynaptic active zone mediates synaptic vesicle exocytosis, and modulation of its molecular composition is important for many types of synaptic plasticity. Here, we identify synaptic scaffold protein liprin-α2 as a key organizer in this process. We show that liprin-α2 levels were regulated by synaptic activity and the ubiquitin–proteasome system. Furthermore, liprin-α2 organized presynaptic ultrastructure and controlled synaptic output by regulating synaptic vesicle pool size. The presence of liprin-α2 at presynaptic sites did not depend on other active zone scaffolding proteins but was critical for recruitment of several components of the release machinery, including RIM1 and CASK. Fluorescence recovery after photobleaching showed that depletion of liprin-α2 resulted in reduced turnover of RIM1 and CASK at presynaptic terminals, suggesting that liprin-α2 promotes dynamic scaffolding for molecular complexes that facilitate synaptic vesicle release. Therefore, liprin-α2 plays an important role in maintaining active zone dynamics to modulate synaptic efficacy in response to changes in network activity.

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Caveolin-1 deficiency impairs synaptic transmission in hippocampal neurons

Molecular Brain ◽

10.1186/s13041-021-00764-z ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Soulmee Koh ◽

Wongyoung Lee ◽

Sang Myun Park ◽

Sung Hyun Kim

Keyword(s):

Synaptic Transmission ◽

Synaptic Vesicle ◽

Hippocampal Neurons ◽

Membrane Dynamics ◽

Synaptic Membrane ◽

Cellular Mechanisms ◽

Caveolin 1 ◽

Synaptic Vesicle Exocytosis ◽

Vesicle Dynamics ◽

Vesicle Exocytosis

AbstractIn addition to providing structural support, caveolin-1 (Cav1), a component of lipid rafts, including caveolae, in the plasma membrane, is involved in various cellular mechanisms, including signal transduction. Although pre-synaptic membrane dynamics and trafficking are essential cellular processes during synaptic vesicle exocytosis/synaptic transmission and synaptic vesicle endocytosis/synaptic retrieval, little is known about the involvement of Cav1 in synaptic vesicle dynamics. Here we demonstrate that synaptic vesicle exocytosis is significantly impaired in Cav1–knockdown (Cav1–KD) neurons. Specifically, the size of the synaptic recycled vesicle pool is modestly decreased in Cav1–KD synapses and the kinetics of synaptic vesicle endocytosis are somewhat slowed. Notably, neurons rescued by triple mutants of Cav1 lacking palmitoylation sites mutants show impairments in both synaptic transmission and retrieval. Collectively, our findings implicate Cav1 in activity-driven synaptic vesicle dynamics—both exocytosis and endocytosis—and demonstrate that palmitoylation of Cav1 is important for this activity.

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