Growth factor-mediated regulation of neuronal exosome release depends on VAMP3/cellubrevin

Extracellular vesicles (EVs) are small membrane-derived vesicles that shuttle proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the CNS. Exosomes are small EVs (40-150 nm) that are derived from multi-vesicular bodies (MVBs) of the endo-lysosomal pathway, formed by inward budding of the limiting membrane into the MVB lumen and released into the extracellular space upon fusion of the MVB with the plasma membrane (PM). Previous research revealed certain effector molecules to be required for exosome release. For instance, RAB GTPases have been shown to control exosome release in a cell- and tissue-specific manner. In addition, exosome release appears to be evoked by membrane depolarization in conjunction with calcium influx and to depend on members of the SNARE family of proteins. However, the specific cellular and molecular factors that regulate neuronal exosome release and segregate it from the release of neurotransmitter vesicles are currently unknown. Here, we used a combination of molecular biology, patch-clamp electrophysiology and pH-sensitive dye imaging to examine the effect of the neuronal growth and differentiation factors basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on neuronal EV release. We found that bFGF-treatment of cultured hippocampal neurons increased the abundance of EVs in the culture medium as measured by Western blot and nanoparticle tracking analysis (NTA), without affecting the number or size of neuronal MVBs. The effect of bFGF depended on calcium and on receptor tyrosine kinase (RTK) activity as the calcium chelating agent BAPTA and the tyrosine kinase inhibitor genistein both abolished the bFGF-induced increase in EV release. In accord with these results, untreated neurons had a low rate of spontaneous and stimulus-evoked MVB-PM fusion events as measured by pH-sensitive dye imaging in conjunction with patch-clamp electrophysiology, but treatment with bFGF significantly increased the number of neurons exhibiting MVB-PM fusion events in response to a high-frequency stimulus. Proteomic analysis of neuronal EVs by Liquid Chromatography Mass Spectrometry (LC-MS) demonstrated bFGF to increase the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs, whereas VAMP2 was decreased. Conversely, knocking-down VAMP3 in cultured neurons abolished the effect of bFGF on EV release. Similar to bFGF, the classical neurotrophins BDNF and NGF increased neuronal EV release in a VAMP3-dependent manner. In summary, our results thus reveal a new function for neurotrophic factor signalling in controlling neuronal exosome release and support the investigation of growth factor-mediated signal transduction via EVs in the healthy and diseased CNS.

细胞外囊泡（Extracellular vesicles，EVs）是一类源自细胞膜的小型囊泡，可在胶质细胞与神经元之间转运蛋白质或核酸，从而促进中枢神经系统（Central Nervous System，CNS）内神经元的存活与可塑性。外泌体（Exosomes）是尺寸介于40至150 nm之间的小型细胞外囊泡，起源于内溶酶体通路（endo-lysosomal pathway）的多泡体（multi-vesicular bodies，MVBs）：其通过限制膜（limiting membrane）向内出芽进入多泡体腔（MVB lumen）形成，并随多泡体与质膜（plasma membrane，PM）融合而释放至细胞外间隙。既往研究表明，外泌体释放需要特定的效应分子。例如，RAB GTP酶（RAB GTPases）已被证实以细胞和组织特异性方式调控外泌体释放。此外，外泌体释放似乎可由膜去极化伴随钙内流所诱发，且依赖于SNARE蛋白家族（SNARE family of proteins）成员。然而，目前尚不清楚调控神经元外泌体释放并将其与神经递质囊泡（neurotransmitter vesicles）释放区分开来的特定细胞与分子因子。本研究结合分子生物学、膜片钳电生理学（patch-clamp electrophysiology）与pH敏感性染料成像（pH-sensitive dye imaging）技术，探究了神经元生长与分化因子——碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）、神经生长因子（nerve growth factor，NGF）以及脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）对神经元细胞外囊泡释放的影响。研究发现，对培养的海马神经元施以bFGF处理后，培养基中细胞外囊泡的丰度显著升高（通过蛋白质免疫印迹（Western blot）与纳米颗粒追踪分析（nanoparticle tracking analysis，NTA）检测），且该处理并未影响神经元多泡体的数量与尺寸。bFGF的该效应依赖于钙离子与受体酪氨酸激酶（receptor tyrosine kinase，RTK）的活性：钙螯合剂（calcium chelating agent）BAPTA与酪氨酸激酶抑制剂（tyrosine kinase inhibitor）金雀异黄素（genistein）均可抵消bFGF诱导的细胞外囊泡释放升高现象。与上述结果一致，未处理的神经元的自发及刺激诱发的多泡体-质膜融合事件发生率较低（通过pH敏感性染料成像结合膜片钳电生理学检测），但经bFGF处理后，响应高频刺激（high-frequency stimulus）而发生多泡体-质膜融合的神经元数量显著升高。通过液相色谱-质谱联用（Liquid Chromatography Mass Spectrometry，LC-MS）对神经元细胞外囊泡进行蛋白质组学分析发现，bFGF可提升细胞外囊泡上v-SNARE囊泡相关膜蛋白3（v-SNARE vesicle-associated membrane protein 3，VAMP3，又称cellubrevin）的丰度，而VAMP2的丰度则有所降低。反之，在培养的神经元中敲低VAMP3的表达可抵消bFGF对细胞外囊泡释放的调控效应。与bFGF类似，经典神经营养因子BDNF与NGF也以VAMP3依赖的方式提升神经元细胞外囊泡的释放。综上，本研究结果揭示了神经营养因子信号通路（neurotrophic factor signalling）在调控神经元外泌体释放中的全新功能，并支持针对健康与患病中枢神经系统中，通过细胞外囊泡介导生长因子信号转导（signal transduction）的相关研究。