Activity-dependent BDNF release via endocytic pathways is regulated by synaptotagmin-6 and complexin

Brain-derived neurotrophic factor (BDNF) is known to modulate synapse development and plasticity, but the source of synaptic BDNF and molecular mechanisms regulating BDNF release remain unclear. Using exogenous BDNF tagged with quantum dots (BDNF-QDs), we found that endocytosed BDNF-QDs were preferentially localized to postsynaptic sites in the dendrite of cultured hippocampal neurons. Repetitive neuronal spiking induced the release of BDNF-QDs at these sites, and this process required activation of glutamate receptors. Down-regulating complexin 1/2 (Cpx1/2) expression eliminated activity-induced BDNF-QD secretion, although the overall activity-independent secretion was elevated. Among eight synaptotagmin (Syt) isoforms examined, down-regulation of only Syt6 impaired activity-induced BDNF-QD secretion. In contrast, activity-induced release of endogenously synthesized BDNF did not depend on Syt6. Thus, neuronal activity could trigger the release of endosomal BDNF from postsynaptic dendrites in a Cpx- and Syt6-dependent manner, and endosomes containing BDNF may serve as a source of BDNF for activity-dependent synaptic modulation.Brain-derived neurotrophic factor (BDNF), a member of neurotrophin family of secreted factors, is known to play important regulatory roles in neuronal survival and differentiation, synaptic development and plasticity, as well as many cognitive functions (1, 2). The findings that the synthesis and secretion of neurotrophins are regulated by neuronal activity prompted the suggestion that neurotrophins may regulate activity-dependent neural plasticity in the brain (3). Indeed, there is now substantial evidence indicating that activity-induced BDNF secretion at glutamatergic synapses is essential for long-term potentiation (LTP) (4), a cellular substrate for the learning and memory functions of neural circuits.The BDNF protein is first synthesized in the endoplasmic reticulum as a precursor protein, prepro-BDNF, which is then converted to pro-BDNF by removal of the signal peptide and further cleaved to generate the mature BDNF (5). Immunostaining and electron microscope studies using specific antibodies to the pro- and mature form of BDNF showed that pro-BDNF is colocalized with mature BDNF in secretory granules in presynaptic axon terminals (6), suggesting that the cleavage may occur in the secretory granule. However, under some experimental conditions, the processing of pro-BDNF into mature BDNF may occur extracellularly (7, 8). The secretory granule containing BDNF and pro-BDNF could undergo exocytosis upon neuronal excitation, as readily demonstrated in cell cultures using ELISA or fluorescent protein-tagged BDNF expressed in the neuron (9, 10). Besides secretory granules, neurotrophins within neuronal cytoplasm could also reside in endosomal compartments, resulting from endocytic uptake of extracellular neurotrophins secreted by the neuron itself or other nearby cells. Initially discovered as factors derived by target tissues, neurotrophins exert their actions via binding to neuronal surface receptors, including tropomyosin related kinase B (TrkB) and pan-neurotrophin receptor p75 (11). Neurotrophin binding to its receptor leads to cytoplasmic signaling as well as internalization of the neurotrophin-receptor complexes. These endocytosed neurotrophin-receptor complexes remain active in the form of “signaling endosomes” that could be transported over long distances within neuronal cytoplasm to exert its regulatory functions within the neuron (12–15). In this study, we have examined the possibility that these endosomes may undergo activity-dependent exocytosis at postsynaptic dendrites, thus providing an additional source of synaptic BDNF.To mark endosomes containing BDNF via the endocytic pathway, it is necessary to monitor BDNF trafficking in neurons. Although YFP-tagged BDNF has been used to study internalization of exogenous BDNF (16), such fluorescent protein-labeled BDNF was not suitable for real-time tracking of BDNF-containing endosomes at a high spatiotemporal resolution. In this study, we used BDNF linked to quantum dots (QDs), which are fluorescent nanoparticles with excellent photostability (17) and could be tracked in live cells with high signal-to-noise ratio and over unprecedented duration. This method has been used to examine endocytic recycling of synaptic vesicles (18) and axonal transport of endosomes containing neurotrophins (19, 20). In this study, we used time-lapse imaging of BDNF-QDs within cultured hippocampal neurons to monitor intracellular transport and localization of these endosomes. Furthermore, the sudden disappearance of cytoplasmic QD fluorescence in a solution containing fluorescence quencher was used to indicate the exocytosis of QD-containing endosomes. Previous studies have shown that extracellular false transmitters, soluble fluorescent markers, and membrane-bound fluorescent lipid dyes could be loaded into endosomes, which undergo exocytosis upon membrane depolarization (21–25). However, whether endosomes formed by receptor-mediated endocytosis is similarly regulated by activity remains unclear. Furthermore, the Ca²⁺-dependence and the kinetics of exocytosis of different endosomal vesicle populations may be differentially regulated by distinct vesicle-associated proteins.In the present study, we have explored the role of synaptotagmin (Syt) and complexin (Cpx) in regulating activity-induced exocytosis of BDNF-containing endosomes. As a universal cofactor in all Ca²⁺-triggered vesicular fusion reactions that have been examined (26), Cpx is known to serve both activating and clamping functions for vesicular exocytosis, by interacting with the Ca²⁺ sensor Syt and the assembled SNARE complexes at the plasma membrane (27). Various isoforms of Syt play distinct regulatory roles in various types of neurosecretion, presumably via their differential Ca²⁺ sensitivity. By manipulating the expression of various Syt and Cpx isoforms in cultured hippocampal neurons, we found that Syt6 and Cpx1/2 play essential regulatory roles in activity-dependent exocytosis of BDNF-containing endosomes. These results support the notion that BDNF-containing endosomes may serve as a source of extracellular BDNF for activity-dependent synaptic modulation and that Syt6 specifically regulates the exocytosis of BDNF-containing endosomes.