Nerve growth factor, brain‐derived neurotrophic factor, and neurotrophin‐3 are sorted to dense‐core vesicles and released via the regulated pathway in primary rat cortical neurons

Abstract

Neurotrophins (NTs) play an important role in the modulation of synaptic transmission and in morphological changes in synaptic structures. Although there is agreement that brain-derived neurotrophic factor (BDNF) is sorted to large dense-core vesicles (LDCVs) and released via the regulated secretory pathway, there has been some dispute regarding the mode of secretion of nerve growth factor (NGF) and neurotrophin-3 (NT-3), two structurally related members of the NT family. In this study, we examined the subcellular localization and release characteristics of NGF, BDNF, and NT-3 in adenovirus-infected primary cortical neurons. We found that all members of the NT family colocalized with markers for the endoplasmic reticulum and Golgi within cell bodies and in a punctate manner with a marker for LDCVs within processes. Moreover, their release was triggered by depolarization, indicating that NGF, BDNF, and NT-3 are released via the regulated secretory pathway. When neurons were coinfected with two separate adenoviruses coding for NGF or BDNF, both NTs showed almost complete vesicular colocalization within single cells, suggesting that different NTs might be packaged into shared vesicles. We also examined whether the two splice variants of NGF, the short and long precursors, differ in their release characteristics. We found that neurons infected with viruses coding for either splice variant released NGF in a regulated way. Overall, our study supports the notion that all members of the NT family undergo activity-dependent regulated release from neurons, enabling them to act as "synaptotrophins" on electrically active neurons.

Differential Vesicular Targeting and Time Course of Synaptic Secretion of the Mammalian Neurotrophins

“…The intracellular fate and the sites of secretion of the other NTs, however, remained a highly controversial issue. Data from two laboratories suggested a similar intracellular targeting and release of all NTs (Heymach et al, 1996;Griesbeck et al, 1999;Wu et al, 2004). In contrast, NT-3, NT-4, and NGF were described by others to be released predominantly via the constitutive pathway (Mowla et al, 1999;Farhadi et al, 2000, Hibbert et al, 2003.…”

“…However, because of the low expression levels of endogenous BDNF, many details concerning BDNF localization were derived from BDNF-or BDNF-GFP-overexpressing CNS neurons. These studies indicated that BDNF is targeted to secretory granules in dendrites and axons of CNS neurons (Haubensak et al, 1998;Wu et al, 2004). Furthermore, BDNF vesicle clusters are found in the vicinity of active synapses and are released in response to synaptic stimulation Kohara et al, 2001;Kojima et al, 2001).…”

“…However, the routes of intracellular targeting of the other neurotrophins are discussed controversially. Overexpressed NGF and NT-3 have been described to be either targeted to secretory granules (Blochl and Thoenen, 1996;Wu et al, 2004) or were found in the constitutive pathway of secretion (Mowla et al, 1999;Farhadi et al, 2000). For NT-4, targeting to only the constitutive pathway was suggested by one previous study (Hibbert et al, 2003).…”

TrkB binds and tyrosine-phosphorylates Tiam1, leading to activation of Rac1 and induction of changes in cellular morphology

“…Cortical neurons were isolated from embryonic day-16 embryos of Sprague-Dawley rats according to the protocol of Wu et al (27). Briefly, cerebral cortices were dissected, dissociated with 0.25% trypsin (Invitrogen), triturated, and passed through 70-m-pore-size meshes.…”

Anterograde Transport and Secretion of Brain-Derived Neurotrophic Factor along Sensory Axons Promote Schwann Cell Myelination

“…cDNA for the myc-tagged BDNF was subcloned into pAdTrackCMV for the generation of recombinant adenovirus (pAdEasy system) (He et al, 1998;Wu et al, 2004). myc-BDNF was subcloned with SalI and XbaI, and large-scale virus preparations were performed in human embryonic kidney HEK293 cells using the cesium chloride banding method.…”