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Huntingtin-associated protein-1 ( HAP1) is involved in intracellular trafficking, vesicle transport, and membrane receptor endocytosis. However, despite such diverse functions, the role of HAP1 in the synaptic vesicle ( SV) cycle in nerve terminals remains unclear. Here, we report that HAP1 functions in SV exocytosis, controls total SV turnover and the speed of vesicle fusion in nerve terminals and regulates glutamate release in cortical brain slices. We found that HAP1 interacts with synapsin I, an abundant neuronal phosphoprotein that associates with SVs during neurotransmitter release and regulates synaptic plasticity and neuronal development. The interaction between HAP1 with synapsin I was confirmed by reciprocal co-immunoprecipitation of the endogenous proteins. Furthermore, HAP1 co-localizes with synapsin I in cortical neurons as discrete puncta. Interestingly, we find that synapsin I localization is specifically altered in Hap1−/− cortical neurons without an effect on the localization of other SV proteins. This effect on synapsin I localization was not because of changes in the levels of synapsin I or its phosphorylation status in Hap1−/− brains. Furthermore, fluorescence recovery after photobleaching in transfected neurons expressing enhanced green fluorescent protein-synapsin Ia demonstrates that loss of HAP1 protein inhibits synapsin I transport. Thus, we demonstrate that HAP1 regulates SV exocytosis and may do so through binding to synapsin I. [ABSTRACT FROM AUTHOR]

Key points Huntingtin-associated protein 1 (HAP1) is expressed in neurons and endocrine cells, in which it is thought to regulate vesicle trafficking., HAP1 is a binding partner of the Huntington's disease (HD)-causing protein huntingtin, and binding is stronger in HD., Whether HAP1 regulates a significant end-point of vesicle transport, exocytosis, and what stage of exocytosis HAP1 may regulate, is unknown., We use mouse chromaffin cells to demonstrate that HAP1 regulates exocytosis via two potentially interlinked mechanisms: control of vesicle docking and the readily releasable vesicle pool, and regulation of fusion pore stabilization., These results establish HAP1 as a significant player in exocytosis control with potential relevance for HD and for a number of neuronal and homeostatic pathways., Abstract Huntingtin-associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study, we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon-fibre amperometry, we measured single vesicle exocytosis in chromaffin cells obtained from HAP1−/− and HAP1+/+ littermate mice. Numbers of Ca2+-dependent and Ca2+-independent full fusion events in HAP1−/− cells are significantly decreased compared with those in HAP1+/+ cells. We observed no change in the frequency of 'kiss-and-run' fusion events or in Ca2+ entry. Whereas release per full fusion event is unchanged in HAP1−/− cells, early fusion pore duration is prolonged, as indicated by the increased duration of pre-spike foot signals. Kiss-and-run events have a shorter duration, indicating opposing roles for HAP1 in the stabilization of the fusion pore during full fusion and transient fusion, respectively. We use electron microscopy to demonstrate a reduction in the number of vesicles docked at the plasma membrane of HAP1−/− cells, where membrane capacitance measurements reveal the readily releasable pool of vesicles to be reduced in size. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation, and the early stages of fusion pore formation. [ABSTRACT FROM AUTHOR]

We have previously shown that Regulator of Calcineurin 1 ( RCAN1) regulates multiple stages of vesicle exocytosis. However, the mechanisms by which RCAN1 affects secretory vesicle exocytosis and quantal release kinetics remain unknown. Here, we use carbon fibre amperometry to detect exocytosis from chromaffin cells and identify these underlying mechanisms. We observe reduced exocytosis with repeated stimulations in chromaffin cells over-expressing RCAN1 ( RCAN1ox), but not in wild-type ( WT) cells, indicating a negative effect of RCAN1 on vesicle recycling and endocytosis. Acute exposure to calcineurin inhibitors, cyclosporine A and FK-506, replicates this effect in WT cells but has no additional effect in RCAN1ox cells. When we chronically expose WT cells to cyclosporine A and FK-506 we find that catecholamine release per vesicle and pre-spike foot ( PSF) signal parameters are decreased, similar to that in RCAN1ox cells. Inhibiting calcineurin activity in RCAN1ox cells has no additional effect on the amount of catecholamine release per vesicle but further reduces PSF signal parameters. Although electron microscopy studies indicate these changes are not because of altered vesicle number or distribution in RCAN1ox cells, the smaller vesicle and dense core size we observe in RCAN1ox cells may underlie the reduced quantal release in these cells. Thus, our results indicate that RCAN1 most likely affects vesicle recycling and quantal release kinetics via the inhibition of calcineurin activity. [ABSTRACT FROM AUTHOR]