Tracy S. Tran

Distinct Roles for Secreted Semaphorin Signaling in Spinal Motor Axon Guidance

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.

Secreted Semaphorins Control Spine Distribution and Morphogenesis in the Postnatal CNS

The majority of excitatory synapses in the mammalian CNS (central nervous system) are formed on dendritic spines, and spine morphology and distribution are critical for synaptic transmission, synaptic integration and plasticity. Here, we show that a secreted semaphorin, Sema3F, is a negative regulator of spine development and synaptic structure. Mice with null mutations in genes encoding Sema3F, and its holoreceptor components neuropilin-2 (Npn-2, also known as Nrp2) and plexin A3 (PlexA3, also known as Plxna3), exhibit increased dentate gyrus (DG) granule cell (GC) and cortical layer V pyramidal neuron spine number and size, and also aberrant spine distribution. Moreover, Sema3F promotes loss of spines and excitatory synapses in dissociated neurons in vitro, and in Npn-2-/- brain slices cortical layer V and DG GCs exhibit increased mEPSC (miniature excitatory postsynaptic current) frequency. In contrast, a distinct Sema3A–Npn-1/PlexA4 signalling cascade controls basal dendritic arborization in layer V cortical neurons, but does not influence spine morphogenesis or distribution. These disparate effects of secreted semaphorins are reflected in the restricted dendritic localization of Npn-2 to apical dendrites and of Npn-1 (also known as Nrp1) to all dendrites of cortical pyramidal neurons. Therefore, Sema3F signalling controls spine distribution along select dendritic processes, and distinct secreted semaphorin signalling events orchestrate CNS connectivity through the differential control of spine morphogenesis, synapse formation, and the elaboration of dendritic morphology.

DAB1 and Reelin Effects on Amyloid Precursor Protein and ApoE Receptor 2 Trafficking and Processing

Numerous cytoplasmic adaptor proteins, including JIP1, FE65, and X11α, affect amyloid precursor protein (APP) processing and Aβ production. Dab1 is another adaptor protein that interacts with APP as well as with members of the apoE receptor family. We examined the effect of Dab1 on APP and apoEr2 processing in transfected cells and primary neurons. Dab1 interacted with APP and apoEr2 and increased levels of their secreted extracellular domains and their cytoplasmic C-terminal fragments. These effects depended on the NPXY domains of APP and apoEr2 and on the phosphotyrosine binding domain of Dab1 but did not depend on phosphorylation of Dab1. Dab1 decreased the levels of APP β-C-terminal fragment and secreted Aβ. Full-length Dab1 or its phosphotyrosine binding domain alone increased surface levels of APP, as determined by surface protein biotinylation and live cell staining. A ligand for apoEr2, the extracellular matrix protein Reelin, significantly increased the interaction of apoEr2 with Dab1. Surprisingly, we also found that Reelin treatment significantly increased the interaction of APP and Dab1. Moreover, Reelin treatment increased cleavage of APP and apoEr2 and decreased production of the β-C-terminal fragment of APP and Aβ. Together, these data suggest that Dab1 alters trafficking and processing of APP and apoEr2, and this effect is influenced by extracellular ligands.

Close Homolog of L1 and Neuropilin 1 Mediate Guidance of Thalamocortical Axons at the Ventral Telencephalon

We report a cooperation between the neural adhesion molecule close homolog of L1 (CHL1) and the semaphorin 3A (Sema3A) receptor, neuropilin 1 (Npn1), important for establishment of area-specific thalamocortical projections. CHL1 deletion in mice selectively disrupted the projection of somatosensory thalamic axons from the ventrobasal (VB) nuclei, causing them to shift caudally and target the visual cortex. At the ventral telencephalon, an intermediate target with graded Sema3A expression, VB axons were caudally shifted in CHL1 − embryos and in Npn1Sema−/− mutants, in which axons are nonresponsive to Sema3A. CHL1 colocalized with Npn1 on thalamic axons, and associated with Npn1 through a sequence in the CHL1 Ig1 domain that was required for Sema3A-induced growth cone collapse. These results identify a novel function for CHL1 in thalamic axon responsiveness to ventral telencephalic cues, and demonstrate a role for CHL1 and Npn1 in establishment of proper targeting of specific thalamocortical projections.

Interaction between Dab1 and CrkII is promoted by Reelin signaling.

Reelin-induced Dab1 tyrosine phosphorylation has been implicated in the regulation of neuronal positioning during brain development. The downstream consequences of Dab1 tyrosine phosphorylation are not fully understood, however. Here we identify CrkII, CrkL and Dock1 in complexes bound to tyrosine-phosphorylated Dab1, through mass spectrometry. The CrkII-Dab1 interaction requires tyrosine phosphorylation of Dab1 at residues 220 or 232 and is promoted by Reelin treatment of embryonic forebrain neurons. Unlike other CrkII binding proteins, such as paxillin and p130Cas, expression of Dab1 interfered with CrkII-dependent cell migration of Nara Bladder Tumor II (NBT-II) cells, in a tyrosine phosphorylation-site dependent manner. Overexpression of CrkIIGFP rescued the migration of these cells, suggesting that Dab1 makes Crk a limiting factor for migration. The Dock1-Dab1 association is indirect and requires CrkII. In organisms such as Drosophila melanogaster and Caenorhabditis elegans, signaling complexes, which contain Crk and Dock1 family members are conserved and act through Rac. We show that a rough-eye phenotype in Drosophila caused by exogenous expression of tyrosine-phosphorylated mouse Dab1RFP is partially rescued by a loss-of-function mutation in myoblast city, a Dock1-like gene in Drosophila. We propose a model that tyrosine-phosphorylated Dab1 engages the conserved Crk-Dock1-Rac signaling cassette, but when bound to Dab1 this signaling complex does not support migration.

Autism spectrum disorder susceptibility gene TAOK2 affects basal dendrite formation in the neocortex.

How neurons develop their morphology is an important question in neurobiology. Here we describe a new pathway that specifically affects the formation of basal dendrites and axonal projections in cortical pyramidal neurons. We report that thousand-and-one-amino acid 2 kinase (TAOK2), also known as TAO2, is essential for dendrite morphogenesis. TAOK2 downregulation impairs basal dendrite formation in vivo without affecting apical dendrites. Moreover, TAOK2 interacts with Neuropilin 1 (Nrp1), a receptor protein that binds the secreted guidance cue Semaphorin 3A (Sema3A). TAOK2 overexpression restores dendrite formation in cultured cortical neurons from Nrp1Sema− mice, which express Nrp1 receptors incapable of binding Sema3A. TAOK2 overexpression also ameliorates the basal dendrite impairment resulting from Nrp1 downregulation in vivo. Finally, Sema3A and TAOK2 modulate the formation of basal dendrites through the activation of the c-Jun N-terminal kinase (JNK). These results delineate a pathway whereby Sema3A and Nrp1 transduce signals through TAOK2 and JNK to regulate basal dendrite development in cortical neurons.

Semaphorin 3F Is a Bifunctional Guidance Cue for Dopaminergic Axons and Controls Their Fasciculation, Channeling, Rostral Growth, and Intracortical Targeting

Dopaminergic neurons in the mesodiencephalon (mdDA neurons) make precise synaptic connections with targets in the forebrain via the mesostriatal, mesolimbic, and mesoprefrontal pathways. Because of the functional importance of these remarkably complex ascending axon pathways and their implication in human disease, the mechanisms underlying the development of these connections are of considerable interest. Despite extensive in vitro studies, the molecular determinants that ensure the perfect formation of these pathways in vivo remain mostly unknown. Here, we determine the embryonic origin and ontogeny of the mouse mesoprefrontal pathway and use these data to reveal an unexpected requirement for semaphorin 3F (Sema3F) and its receptor neuropilin-2 (Npn-2) during mdDA pathway development using tissue culture approaches and analysis of sema3F−/−, npn-2−/−, and npn-2−/−;TH-Cre mice. We show that Sema3F is a bifunctional guidance cue for mdDA axons, some of which have the remarkable ability to regulate their responsiveness to Sema3F as they develop. During early developmental stages, Sema3F chemorepulsion controls previously uncharacterized aspects of mdDA pathway development through both Npn-2-dependent (axon fasciculation and channeling) and Npn-2-independent (rostral growth) mechanisms. Later on, chemoattraction mediated by Sema3F and Npn-2 is required to orient mdDA axon projections in the cortical plate of the medial prefrontal cortex. This latter finding demonstrates that regulation of axon orientation in the target field occurs by chemoattractive mechanisms, and this is likely to also apply to other neural systems. In all, this study provides a framework for additional dissection of the molecular basis of mdDA pathway development and disease.

Unique developmental patterns of GABAergic neurons in rat spinal cord

γ‐aminobutyric acid (GABA)ergic neurons have been postulated to compose an important component of local circuits in the adult spinal cord, yet their identity and axonal projections have not been well defined. We have found that, during early embryonic ages (E12–E16), both glutamic acid decarboxylase 65 (GAD65) and GABA were expressed in cell bodies and growing axons, whereas at older ages (E17–P28), they were localized primarily in terminal‐like structures. To determine whether these developmental changes in GAD65 and GABA were due to an intracellular shift in the distribution pattern of GAD proteins, we used a spinal cord slice model. Initial experiments demonstrated that the pattern of GABAergic neurons within organotypic cultures mimicked the expression pattern seen in embryos. Sixteen‐day‐old embryonic slices grown 1 day in vitro contained many GAD65‐ and GAD67‐labeled somata, whereas those grown 4 days in vitro contained primarily terminal‐like varicosities. When isolated E14–E16 slices were grown for 4 days in vitro, the width of the GAD65‐labeled ventral marginal zone decreased by 40–50%, a finding that suggests these GABAergic axons originated from sources both intrinsic and extrinsic to the slices. Finally, when axonal transport was blocked in vitro, the developmental subcellular localization of GAD65 and GAD67 was reversed, so that GABAergic cell bodies were detected at all ages examined. These data indicate that an intracellular redistribution of both forms of GAD underlie the developmental changes observed in GABAergic spinal cord neurons. Taken together, our findings suggest a rapid translocation of GAD proteins from cell bodies to synaptic terminals following axonal outgrowth and synaptogenesis. J. Comp. Neurol. 456:112–126, 2003.

NrCAM deletion causes topographic mistargeting of thalamocortical axons to the visual cortex and disrupts visual acuity.

NrCAM is a neural cell adhesion molecule of the L1 family that has been linked to autism spectrum disorders, a disease spectrum in which abnormal thalamocortical connectivity may contribute to visual processing defects. Here we show that NrCAM interaction with neuropilin-2 (Npn-2) is critical for semaphorin 3F (Sema3F)-induced guidance of thalamocortical axon subpopulations at the ventral telencephalon (VTe), an intermediate target for thalamic axon sorting. Genetic deletion of NrCAM or Npn-2 caused contingents of embryonic thalamic axons to misproject caudally in the VTe. The resultant thalamocortical map of NrCAM-null mutants showed striking mistargeting of motor and somatosensory thalamic axon contingents to the primary visual cortex, but retinogeniculate targeting and segregation were normal. NrCAM formed a molecular complex with Npn-2 in brain and neural cells, and was required for Sema3F-induced growth cone collapse in thalamic neuron cultures, consistent with a vital function for NrCAM in Sema3F-induced axon repulsion. NrCAM-null mice displayed reduced responses to visual evoked potentials recorded from layer IV in the binocular zone of primary visual cortex (V1), particularly when evoked from the ipsilateral eye, indicating abnormal visual acuity and ocularity. These results demonstrate that NrCAM is required for normal maturation of cortical visual acuity, and suggest that the aberrant projection of thalamic motor and somatosensory axons to the visual cortex in NrCAM-null mutant mice impairs cortical functions.

Ventrally Located Commissural Neurons Express the GABAergic Phenotype in Developing Rat Spinal Cord

Early‐forming commissural neurons are studied intensively as a model of axonal outgrowth and pathfinding, yet the neurotransmitter phenotype of the majority of these neurons is not known. The present study has determined that a substantial number of commissural neurons express the 65‐kDa isoform of glutamic acid decarboxylase (GAD65) as early as embryonic day 12 (E12). Patterns of GAD65 localization were compared with those of TAG‐1, the Transiently expressed Axonal Glycoprotein that is the best known marker of commissural axons. On E13, both GAD65‐ and TAG‐1‐labeled commissural axons emanate from similar lateral and ventromedial regions. However, dorsally located TAG‐1–positive commissural axons were GAD65‐negative. These results suggest that commissural neurons have both γ‐aminobutyric acid (GABA)ergic and non‐GABAergic phenotypes. The intensity of GAD65 staining within commissural somata and axons decreased between E14–15 and continued to decline during embryonic development, whereas terminal‐like structures in surrounding neuropil increased dramatically. This sudden loss of somatic and axonal GAD65 staining was unexpected and could be interpreted as commissural neurons only transiently expressing the GABAergic phenotype. Further experiments were undertaken to identify commissural neurons with other established GABAergic markers, GAD67 and GABA. When antibody labeling of the two GAD isoforms was compared, GAD67 was detected 1 day later than GAD65, and in a different subcellular distribution. In contrast to GAD65, GAD67 intensely stained somata but labeled few commissural axons. GABA immunoreactivity also was detected in commissural axons 1 day after GAD65, and the labeling pattern between E13 and E16 resembled that of GAD67 rather than GAD65. When GAD and GABA results were compared, it was clear that a number of ventrally located commissural neurons expressed and maintained the GABAergic phenotype during embryonic development. However, the early expression and subcellular redistribution of GAD65 suggests that the GAD isoforms are differentially regulated. The function of the transient GAD65 expression in commissural somata and axons is unknown, but its temporal expression pattern parallels the transient expression of TAG‐1, as both are expressed during the early stages of commissural axon outgrowth and pathfinding. J. Comp. Neurol. 409:285–298, 1999.