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

Hippocampal neurons release nerve growth factor (NGF) through the constitut ive secretory pathway, thus allowing the protein to be continuously availab le for promoting nerve cell survival. In contrast, hippocampal neurons use the regulated secretory pathway to process brain-derived neurotrophic facto r (BDNF), which alters synaptic activity when released acutely from dense-c ore vesicles. Thus, understanding how neurons sort and deliver neurotrophin s may provide clues to their functions in brain. In this study, we monitore d the processing and delivery of neurotrophin-3 (NT-3). Pulse-chase studies , immunocytochemistry, and secretagogue-induced release experiments were pe rformed on cultured hippocampal neurons and AtT-20 cells infected with vacc inia viruses encoding the NT-3 precursor (pro-NT-3). Results show that most newly synthesized NT-3 is released through the constitutive secretory path way as a result of furin-mediated endoproteolytic cleavage of pro-NT-3 in t he trans-Golgi network. Pro-NT-3 can also be diverted into the regulated se cretory pathway when cells are treated with alpha 1-PDX, a selective inhibi tor of furin-like enzymes, or when pro-NT-3 expression is increased by tran sient 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 se cretory granules, and released in response to extracellular cues together w ith BDNF, apparently as a result of heterodimerization, as suggested by coi mmunoprecipitation data. Taken together, these data show that sorting of th e 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.