A common single-nucleotide polymorphism in the human brain-derived neurotrophic factor (BDNF) gene results in a Val66Met substitution in the BDNF prodomain region. This single-nucleotide polymorphism is associated with alterations in memory and with enhanced risk to develop depression and anxiety disorders in humans. Here we show that the isolated BDNF prodomain is detected in the hippocampus and that it can be secreted from neurons in an activity-dependent manner. Using nuclear magnetic resonance spectroscopy and circular dichroism we find that the prodomain is intrinsically disordered, and the Val66Met substitution induces structural changes. Surprisingly, application of Met66 (but not Val66) BDNF prodomain induces acute growth cone retraction and a decrease in Rac activity in hippocampal neurons. Expression of p75NTR and differential engagement of the Met66 prodomain to the SorCS2 receptor are required for this effect. These results identify the Met66 prodomain as a new active ligand which modulates neuronal morphology.

Brain-derived neurotrophic factor (BDNF) regulates neuronal differentiation, synaptic plasticity, and morphology, and modest changes in BDNF levels results in complex behavioral phenotypes. BDNF levels and intracellular localization in neurons are regulated by multiple mechanisms, including use of distinct promoters, mRNA and protein transport, and regulated cleavage of proBDNF to mature BDNF. Sortilin is an intracellular chaperone that binds to the prodomain of BDNF to traffic it to the regulated secretory pathway. However, sortilin binds to numerous ligands and plays a major role in mannose 6-phosphate receptor-independent transport of lysosomal hydrolases utilizing motifs in the intracellular domain that mediate trafficking from the Golgi and late endosomes. Sortilin is modified by ectodomain shedding, although the biological implications of this are not known. Here we demonstrate that ADAM10 is the preferred protease to cleave sortilin in the extracellular stalk region, to release the ligand binding sortilin ectodomain from the transmembrane and cytoplasmic domains. We identify sortilin shedding at the cell surface and in an intracellular compartment. Both sortilin and BDNF are trafficked to and degraded by the lysosome in neurons, and this is dependent upon the sortilin cytoplasmic tail. Indeed, expression of the sortilin ectodomain, which corresponds to the domain released after shedding, impairs lysosomal targeting and degradation of BDNF. These findings characterize the regulation of sortilin shedding and identify a novel mechanism by which sortilin ectodomain shedding acts as a regulatory switch for delivery of BDNF to the secretory pathway or to the lysosome, thus modulating the bioavailability of endogenous BDNF. Brain-derived neurotrophic factor (BDNF)2 is dynamically regulated in the central nervous system and is a critical factor in development and plasticity (1). Whereas mature BDNF promotes survival and long term potentiation, the proform induces long term depression and apoptosis (2, 3). This diversity of function suggests that BDNF is subject to sophisticated regulatory mechanisms, with studies showing dynamic regulation at the level of transcription, translation, enzymatic cleavage, and trafficking of mRNA and protein (4). In addition, numerous chaperone proteins regulate subcellular localization of BDNF mRNA and protein in neurons including sortilin, carboxypeptidase E, and translin (5-7).Sortilin, a member of the Vps10p domain containing family of proteins that includes sorLA and sorCS1-3, is highly expressed in the cortex and hippocampus (8 -10), where it can function as a cell surface receptor for proneurotrophins, neurotensin, and lipoprotein lipase (11-13). The majority of sortilin, however, is expressed within intracellular compartments, where it chaperones diverse ligands, including proBDNF and acid hydrolases (5,13,14). The sortilin cytoplasmic tail is highly homologous to mannose 6-phosphate receptor and is required for the intracellular trafficking of cargo proteins via interactions...

The neurotrophin receptor p75NTR has been implicated in mediating neuronal apoptosis after injury to the CNS. Despite its frequent induction in pathologic states, there is limited understanding of the mechanisms that regulate p75 NTR expression after injury. Here, we show that after focal cerebral ischemia in vivo or oxygen-glucose deprivation in organotypic hippocampal slices or neurons, p75 NTR is rapidly induced. A concomitant induction of proNGF, a ligand for p75 NTR , is also observed. Induction of this ligand/receptor system is pathologically relevant, as a decrease in apoptosis, after oxygen-glucose deprivation, is observed in hippocampal neurons or slices after delivery of function-blocking antibodies to p75 NTR or proNGF and in p75 NTR and ngf haploinsufficient slices. Furthermore, a significant decrease in infarct volume was noted in p75 NTR Ϫ/Ϫ mice compared with the wild type. We also investigated the regulatory mechanisms that lead to post-ischemic induction of p75 NTR . We demonstrate that induction of p75 NTR after ischemic injury is independent of transcription but requires active translation. Basal levels of p75 NTR in neurons are maintained in part by the expression of microRNA miR-592, and an inverse correlation is seen between miR-592 and p75 NTR levels in the adult brain. After cerebral ischemia, miR-592 levels fall, with a corresponding increase in p75 NTR levels. Importantly, overexpression of miR-592 in neurons decreases the level of ischemic injury-induced p75 NTR and attenuates activation of pro-apoptotic signaling and cell death. These results identify miR-592 as a key regulator of p75 NTR expression and point to a potential therapeutic candidate to limit neuronal apoptosis after ischemic injury.

Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb −/−) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb−/− embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.

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