Neurotrophin receptors use endosomal pathways for signaling in neurons. However, how neurotrophins regulate the endosomal system for proper signaling is unknown. Rabs are monomeric GTPases that act as molecular switches to regulate membrane trafficking by binding a wide range of effectors. Among the Rab GTPases, Rab5 is the key GTPase regulating early endosomes and is the first sorting organelle of endocytosed receptors. The objective of our work was to study the regulation of Rab5-positive endosomes by BDNF at different levels, including dynamic, activity and protein levels in hippocampal neurons. Short-term treatment with BDNF increased the colocalization of TrkB in dendrites and cell bodies, increasing the vesiculation of Rab5-positive endosomes. Consistently, BDNF increased the number and mobility of Rab5 endosomes in dendrites. Cell body fluorescence recovery after photobleaching of Rab-EGFP-expressing neurons suggested increased movement of Rab5 endosomes from dendrites to cell bodies. These results correlated with the BDNF-induced activation of Rab5 in dendrites, followed by increased activation of Rab5 in cell bodies. Long-term treatment of hippocampal neurons with BDNF increased the protein levels of Rab5 and Rab11 in an mTOR-dependent manner. While BDNF regulation of Rab5a levels occurred at both the transcriptional and translational levels, Rab11a levels were regulated at the translational level at the time points analyzed. Finally, expression of a dominant-negative mutant of Rab5 reduced the basal arborization of nontreated neurons, and although BDNF was partially able to rescue the effect of Rab5DN at the level of primary dendrites, BDNF-induced dendritic branching was largely reduced. Our findings indicate that BDNF regulates the Rab5-Rab11 endosomal system at different levels and that these processes are likely required for BDNF-induced dendritic branching.

dynamics, in vivo tracking SUMMARY: Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a costeffective manner and is purified by affinity chromatography. BDNFavi is then directly monobiotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF. ABSTRACT: Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. We developed a reproducible protocol to directly mono-biotinylate BDNFavi with the enzyme BirA in a tube. In this reaction, monobiotinylated BDNFAvi retains its biological activity. Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow

Brain-derived neurotrophic factor (BDNF) induces activation of the TrkB receptor and several downstream pathways (MAPK, PI3K, PLC-γ), leading to neuronal survival, growth, and plasticity. It has been well established that TrkB signaling regulation is required for neurite formation and dendritic arborization, but the specific mechanism is not fully understood. The non-receptor tyrosine kinase c-Abl is a possible candidate regulator of this process, as it has been implicated in tyrosine kinase receptors’ signaling and trafficking, as well as regulation of neuronal morphogenesis. To assess the role of c-Abl in BDNF-induced dendritic arborization, wild-type and c-Abl-KO neurons were stimulated with BDNF, and diverse strategies were employed to probe the function of c-Abl, including the use of pharmacological inhibitors, an allosteric c-Abl activator, and shRNA to downregulates c-Abl expression. Surprisingly, BDNF promoted c-Abl activation and interaction with TrkB receptors. Furthermore, pharmacological c-Abl inhibition and genetic ablation abolished BDNF-induced dendritic arborization and increased the availability of TrkB in the cell membrane. Interestingly, inhibition or genetic ablation of c-Abl had no effect on the classic TrkB downstream pathways. Together, our results suggest that BDNF/TrkB-dependent c-Abl activation is a novel and essential mechanism in TrkB signaling.

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