Brain development requires the coordination of several complex and incompletely understood processes. These biological events include the generation of neural precursor cells, cell-type specification, induction of cell migration and differentiation, and integration into existing functional networks. In mammals, many studies have focused on how these actions are successfully orchestrated to form the stratified layers of the cerebral cortex.1,2 In rodent fetal brain, neurogenic precursor cells are generated in the ventricular zone and subventricular zone (SVZ), central regions of the brain in close proximity to the cerebrospinal fluid-filled ventricles. Starting at embryonic day nine (E9), newborn cortical neurons in the ventricular zone and SVZ migrate outwardly toward the pial surface, then turn and migrate inwardly to form the distinct cell layers of the cortical plate.3,4 What remain to be determined are the intrinsic and extrinsic factors that govern this migratory process.
Research suggests that TrkB, the receptor for Brain-derived Neurotrophic Factor (BDNF), has a central role during neuronal migration in the developing cortex. For example, inhibition of TrkB function via the expression of dominant-negative forms of the receptor has been shown to disrupt the migration of newborn mouse cortical neurons.5 Although the expression of TrkB in developing neurons of the ventricular zone and SVZ is well established, the role of BDNF during TrkB-dependent postnatal localization has not been clearly defined.6,7 Intriguingly, endogenous levels of BDNF do not reach maximal physiological levels until after birth, suggesting another ligand may stimulate TrkB-dependent migration.8
To test the hypothesis that BDNF is required for TrkB-dependent migration of newborn cortical neurons, Puehringer et al. studied the development of the neocortex in embryonic mice. Initial immunocytochemical and biochemical experiments verified that TrkB expression was detectable in the ventricular zone and SVZ from E11 onward.9 Phosphorylation of established downstream effectors of TrkB indicated that the receptor was activated. Specifically, cytoplasmic tyrosine residues that bind the Src Homology 2 Domain-containing Transforming Protein C (SHC)/Fibroblast Growth Factor Receptor Substrate 2 (FRS2) adaptor complex, and Phospholipase C-gamma (PLC-gamma), were shown to be phosphorylated.5 However, genetic depletion of BDNF (Bdnf-/-) had no effect on TrkB activation, supporting the theory that in E11 neural precursor cells TrkB is activated via another ligand.
To investigate the mechanism underlying TrkB activation, Puehringer and colleagues isolated neuronal cells from E11 forebrain. Characterization of the resulting cell population revealed a marker expression profile that was consistent with non-differentiated (MAP2-) neural precursors (Pax6+/Nestin+).10,11,12 In support of the BDNF knockout findings that showed BDNF-independent TrkB activation, TrkB stimulation following exposure to BDNF was low in E11 cells in vitro. In contrast, a much larger response was elicited when BDNF was pulsed onto cells isolated from E12-E15 brain, which were typically MAP2+ differentiated neurons. Thus, the authors tested the efficacy of other ligands that are known to transactivate TrkB.13,14
Studies with other ligands demonstrated rapid and prolonged TrkB activation following exposure to Epidermal Growth Factor (EGF), an effect could be blocked by the EGF receptor (EGF R) antagonist PD153035. Further pharmacological studies applying Src kinase inhibitors and lentiviral gene transfer experiments employing dominant-negative constructs supported the downstream involvement of c-Src and Fyn, known transactivators of TrkB.15 Transactivation of TrkB also appeared to be accompanied by TrkB translocation. Using stimulated emission depletion (STED) microscopy and Avidin pulldown experiments, EGF transactivation was shown to require a shift in TrkB localization from intracellular compartments to the plasma membrane.
To further investigate the significance of EGF R in TrkB transactivation, the authors examined the consequences of EGF R knockout (Egfr-/-). Neither activation of TrkB nor its localization at the plasma membrane were observed in cells isolated from Egfr-/- mice. Furthermore, in Egfr-/- mice at E16, the number of neurons in cortical layers V and VI was reduced and the number of TrkB+ cells remaining in the ventricular zone and SVZ was increased. These findings, coupled to the observation that there were no changes in total cell number in these brain regions, further supported the importance of EGF R-dependent transactivation for neuronal migration in the developing cerebral cortex.
Confocal analysis of wild-type E13 embryos revealed a brain region-dependent expression pattern for TrkB. Staining of TrkB in cells of the ventricular zone and SVZ indicated the receptor was retained in intracellular compartments. In contrast, TrkB was localized at the plasma membrane of cells that had successfully migrated to the cortical plate, suggesting subcellular translocation of TrkB might be functionally relevant during cortical migration. To test this possibility directly, Puehringer et al. used in vitro migration assays in which stripes of growth factor were applied on Laminin-coated coverslips. In these experiments, EGF stripes induced plasma membrane expression of phosphorylated TrkB, and levels of activated TrkB that were asymmetrically higher at the migrating front side of the cell. In initial experiments using unsupplemented media, little migration was observed toward BDNF stripes. However, when EGF was added to the growth medium there was a significant increase in the number of neurons that migrated towards areas of the coverslip that were coated with BDNF.
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EGF Receptor-dependent Transactivation of TrkB Stimulates Migration of Neural Precursor Cells. In embryonic rodent brain, EGF was shown to induce the migration of neural precursor cells from the ventricular zone and subventricular zone via a mechanism that required EGF Receptor-dependent signaling (1), activation of TrkB by c-Src and Fyn at intracellular membranes (2), deglycosylation of TrkB by PNGase F (3), and translocation of TrkB to the plasma membrane at the migratory front of the cell (4).9 This signaling cascade is believed to coordinate the migration of precursor cells so that BDNF-dependent differentiation and integration only occur at the correct location in the nascent neocortex (5).
Collectively, these novel findings suggest that TrkB functions as an integrative factor during development of the cerebral cortex. EGF-induced translocation of TrkB appears to be a critical step during neuronal differentiation that ensures the migratory precursor cells from the ventricular zone and SVZ are only fully BDNF-responsive when they arrive at their designated cortical layer. This mechanism facilitates BDNF-induced synaptogenesis and integration into extant neural circuits only when newborn neurons have completed their migratory journey. Interestingly, a recent study suggested that BDNF-induced synaptic protein synthesis during memory formation may involve degradation of Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) by Calpain-2.16 In rat hippocampal and cortical models, BDNF-dependent protein synthesis in dendrites was shown to require disinhibition of the Mammalian Target of Rapamycin (mTOR) pathway, following Calpain-2 cleavage of PTEN. It remains to be determined if the same mechanism underlies BDNF-induced synaptogenesis during development.
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