mechanisms of long-distance control of dendritic growth by brain-derived neurotrophic factor (BDNF) in central neurons

Abstract

Brain Derived Neurotrophic Factor (BDNF) is broadly express in many circuits of the central nervous system (CNS) and binds its receptors TrkB and p75. BDNF triggers different signaling pathways, including the kinases ERK1/2, PI3K-mTOR and the PLCy-Ca+2 and inducing dendritic growth and synaptic plasticity. When binding to BDNF, TrkB and p75 are endocytosed to signaling endosomes allowing long-distance signaling by neurotrophin receptors. The long- distance signaling has been well-described in the peripheral nervous system for NGF/TrkA to mediate survival. However, whether there is a functional role for BDNF/TrkB signaling endosomes in central neurons is unknown. The general aim of this thesis was to study the role of signaling endosomes in the process of BDNF long-distance signaling in cortical neurons. The first objective of our work was to study the regulation of Rab5-positive endosomes by BDNF including activity and dynamics in neurons. 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. To study the role of Rab5 on the neuronal signaling of BDNF, we have done experiments with fluorescence microscopy in fixed cells and live imaging. In addition, we have evaluated whether the activity of the GTPase was required for the dendritic arborization induced by BDNF, by transducing the neurons with a dominant negative of Rab5. Here, we showed that short-term treatment with BDNF increased the colocalization of TrkB with Rab5 in dendrites, increasing the number and mobility of Rab5 endosomes. Additionally, we observed that BDNF upregulates the activation of Rab5. These results are complementary to parallel findings indicating that Rab5 activity was required for dendritic branching induced by BDNF. These findings suggest that BDNF regulates the dynamics of Rab5-positive early endosomes by increasing the activity of Rab5, processes required for BDNF-induced dendritic branching. Afterwards, we asked about the functional role of axonal BDNF signaling in cortical neurons and the possible role of signaling endosomes in this process. We found, that BDNF in axons increases dendritic branching in cell bodies. Using different models of transgenic animals and compartmentalized cultures, we found that this process is mostly mediated by TrkB receptors and not p75 NTR. In addition, we found that arborization depended on the activation of the CREB transcription factor in the nucleus and the PI3K-mTOR pathway in cell bodies increasing the synthesis of somatodendritic proteins. In contrast, the kinase activity of PI3K in axons was neither required for the transport of BDNF nor for the effect on arborization in the cell body. By using cortical neurons derived from TrkBF616A knock-in mice and using compartmentalized cultures, we showed that the activity of the TrkB receptor, activated in the axon by BDNF, is required for the dendritic effects observed in the cell body. On the other hand, the activities of Rab5 and dynein were required for these effects. All these results together, suggest the generation and transport of signaling endosomes for long-distance BDNF signaling in axons. Next, due to the little information available on how TrkB downstream routes regulate long-distance signaling, we studied the role of PLCg-Ca+2 in BDNF-mediated axonal signaling. Using compartmentalized cultures, we demonstrate that the axonal activity of PLCg is necessary for dendritic arborization and phosphorylation of CREB. These results were correlated with the increase in axonal levels of Ca+2 induced by BDNF in axons in a PLCg-dependent manner. Interestingly, we found that PLCy/Ca+2 pathway is necessary for the endocytosis of TrkB in axons. In summary, these results suggest that the axonal BDNF long-distance signaling has a functional role in cortical neurons, regulating the activation of key proteins to regulate the translation of protein and dendritic branching.