Phosphorylation of Synaptotagmin 4 captures transiting dense core vesicles at active synapses

by Vinita Bharat
Doctoral thesis
Date of Examination:2016-04-26
Date of issue:2017-03-03
Advisor:Dr. Camin Dean
Referee:Prof. Dr. Nils Brose
Referee:Prof. Dr. Reinhard Jahn
Persistent Address: http://dx.doi.org/10.53846/goediss-6173

 

 

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Abstract

English

Synaptic modulation requires fast recruitment of neuronal dense core vesicles (DCVs) containing various neuropeptides and neurotrophins at nerve terminals. DCVs undergo long-range trafficking in axons to deliver cargoes at release sites. However, the question of whether and how specific sites capture these transiting vesicles upon neuronal activity is open. In this study, we have used a Synaptotagmin (Syt) isoform, Syt4, as a DCV marker to investigate trafficking and activity-dependent capture of DCVs in hippocampal neurons. We found that Syt4-harboring vesicles are highly mobile on microtubules and switch directions only at the distal end of axons in hippocampal neurons. We examined the effects of phosphorylation of Syt4 at S135 on trafficking, capture and fusion of DCVs in mature neurons. We found that phosphomimetic Syt4 vesicles traffic less and are more concentrated at synapses. Conversely, phosphodeficient Syt4 vesicles had the most processivity and were least localized at synapses. We also found that disrupting actin, which is enriched at pre-synaptic sites, enhances the mobility of phosphomimetic vesicles. We found that the motor protein Kif1A is associated with Syt4 vesicles but phosphomimetic vesicles had less interaction with Kif1A. Over-expression of Kif1A rescued the trafficking of phosphomimetic Syt4 vesicles. In addition, we found that c-Jun N-terminal kinase (JNK) phosphorylates Syt4 at S135 specifically causing decreased motility of transiting DCVs. Furthermore, increased neuronal activity promoted capture of transiting vesicles at synapses via a JNK phosphorylation dependent mechanism. Phosphorylation of Syt4 did not affect the fusion of vesicles at synaptic and non-synaptic sites in hippocampal neurons. Together, this study reveals a JNK-dependent phosphorylation mechanism involved in trafficking and capture of Syt4 harboring DCVs in hippocampal neurons. We propose a mechanism whereby JNK at active synapses phosphorylates Syt4 at S135 on transiting DCVs, promoting destabilization of Syt4-Kif1A binding and allowing capture of DCVs at synapses by actin. This mechanism would potentially allow fast recruitment of dense core vesicles to active synapses, ensuring the efficient delivery of neuropeptides and neurotrophins to specific sites in hippocampal neurons whenever needed.
Keywords: Dense core vesicle; Vesicle trafficking; DCV fusion; Synaptotagmin 4
 
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