Neurotrophin-3 Sorts to the Constitutive Secretory Pathway of Hippocampal Neurons and Is Diverted to the Regulated Secretory Pathway by Coexpression with Brain-Derived Neurotrophic Factor
Neurotrophin-3 Sorts to the Constitutive Secretory Pathway of Hippocampal Neurons and Is Diverted to the Regulated Secretory Pathway by Coexpression with Brain-Derived Neurotrophic Factor
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DOI：
10.1523/jneurosci.20-11-04059.2000
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发表时间：
2000-06
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影响因子：
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通讯作者：
Hooman Farhadi;S. Mowla;K. Petrecca;S. Morris;N. Seidah;R. A. Murphy
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作者：
Hooman Farhadi;S. Mowla;K. Petrecca;S. Morris;N. Seidah;R. A. Murphy
海马神经元通过组成性分泌途径释放神经生长因子（NGF），从而使蛋白质持续可用于促进神经细胞存活。相比之下，海马神经元使用调节分泌途径来处理脑源性神经营养因子（BDNF），其在从致密核心囊泡急性释放时改变突触活性。因此，了解神经元如何分类和传递神经营养因子可能会为它们在大脑中的功能提供线索。在这项研究中，我们监测了神经营养素-3（NT-3）的加工和传递。脉冲追踪研究，免疫细胞化学，和促分泌素诱导的释放实验进行培养的海马神经元和AtT-20细胞感染的牛痘病毒编码的NT-3前体（前NT-3）。结果表明，大多数新合成的NT-3是通过组成性分泌途径释放的结果，弗林蛋白酶介导的内切蛋白水解切割的前NT-3的trans-Golgi网络。当细胞用α1-PDX（一种弗林蛋白酶样酶的选择性抑制剂）处理时，或当通过瞬时转染方法增加pro-NT-3表达时，Pro-NT-3也可被转移到受调节的分泌途径中。在与编码前NT-3和前BDNF的病毒共感染的细胞中，NT-3被分选到调节途径中，储存在分泌颗粒中，并响应于细胞外信号与BDNF一起释放，显然是异源二聚化的结果，如免疫共沉淀数据所示。总之，这些数据表明，NT-3前体的分选可以发生在组成型和调节分泌途径，这是一致的NT-3既有生存促进和突触改变功能。
Hippocampal neurons release nerve growth factor (NGF) through the constitutive secretory pathway, thus allowing the protein to be continuously available for promoting nerve cell survival. In contrast, hippocampal neurons use the regulated secretory pathway to process brain-derived neurotrophic factor (BDNF), which alters synaptic activity when released acutely from dense-core vesicles. Thus, understanding how neurons sort and deliver neurotrophins may provide clues to their functions in brain. In this study, we monitored the processing and delivery of neurotrophin-3 (NT-3). Pulse–chase studies, immunocytochemistry, and secretagogue-induced release experiments were performed on cultured hippocampal neurons and AtT-20 cells infected with vaccinia viruses encoding the NT-3 precursor (pro-NT-3). Results show that most newly synthesized NT-3 is released through the constitutive secretory pathway as a result of furin-mediated endoproteolytic cleavage of pro-NT-3 in the trans-Golgi network. Pro-NT-3 can also be diverted into the regulated secretory pathway when cells are treated with α1-PDX, a selective inhibitor of furin-like enzymes, or when pro-NT-3 expression is increased by transient transfection methods. In cells coinfected with viruses coding for pro-NT-3 and pro-BDNF, NT-3 is sorted into the regulated pathway, stored in secretory granules, and released in response to extracellular cues together with BDNF, apparently as a result of heterodimerization, as suggested by coimmunoprecipitation data. Taken together, these data show that sorting of the NT-3 precursor can occur in both the constitutive and regulated secretory pathways, which is consistent with NT-3 having both survival-promoting and synapse-altering functions.