Axonal Identity Research Articles

Microtubule polarity is instructive for many aspects of neuronal polarity

Many neurons in bilaterian animals are polarized with functionally distinct axons and dendrites. Microtubule polarity, microtubule stability, and the axon initial segment (AIS) have all been shown to influence polarized transport in neurons. Each of these cytoskeletal cues could act independently to control axon and dendrite identity, or there could be a hierarchy in which one acts upstream of the others. Here we test the hypothesis that microtubule polarity acts as a master regulator of neuronal polarity by using a Drosophila genetic background in which some dendrites have normal minus-end-out microtubule polarity and others have the axonal plus-end-out polarity. In these mosaic dendrite arbors, we found that ribosomes, which are more abundant in dendrites than axons, were reduced in plus-end-out dendrites, while an axonal cargo was increased. In addition, we determined that microtubule stability was different in plus-end-out and minus-end-out dendrites, with plus-end-out ones having more stable microtubules like axons. Similarly, we found that ectopic diffusion barriers, like those at the AIS, formed at the base of dendrites with plus-end-out regions. Thus, changes in microtubule polarity were sufficient to rearrange other cytoskeletal features associated with neuronal polarization. However, overall neuron shape was maintained with only subtle changes in branching in mosaic arbors. We conclude that microtubule polarity can act upstream of many aspects of intracellular neuronal polarization, but shape is relatively resilient to changes in microtubule polarity in vivo.

Nucleating microtubules in neurons: Challenges and solutions.

The highly polarized morphology of neurons is crucial for their function and involves formation of two distinct types of cellular extensions, the axonal and dendritic compartments. An important effector required for the morphogenesis and maintenance and thus the identity of axons and dendrites is the microtubule cytoskeleton. Microtubules in axons and dendrites are arranged with distinct polarities, to allow motor-dependent, compartment-specific sorting of cargo. Despite the importance of the microtubule cytoskeleton in neurons, the molecular mechanisms that generate the intricate compartment-specific microtubule configurations remain largely obscure. Work in other cell types has identified microtubule nucleation, the de novo formation of microtubules, and its spatio-temporal regulation as essential for the proper organization of the microtubule cytoskeleton. Whereas regulation of microtubule nucleation usually involves microtubule organizing centers such as the centrosome, neurons seem to rely largely on decentralized nucleation mechanisms. In this review, I will discuss recent advances in deciphering nucleation mechanisms in neurons, how they contribute to the arrangement of microtubules with specific polarities, and how this affects neuron morphogenesis. While this work has shed some light on these important processes, we are far from a comprehensive understanding. Thus, to provide a coherent model, my discussion will include both well-established mechanisms and mechanisms with more limited supporting data. Finally, I will also highlight important outstanding questions for future investigation.

MTCL1 plays an essential role in maintaining Purkinje neuron axon initial segment.

The axon initial segment (AIS) is a specialized domain essential for neuronal function, the formation of which begins with localization of an ankyrin-G (AnkG) scaffold. However, the mechanism directing and maintaining AnkG localization is largely unknown. In this study, we demonstrate that invivo knockdown of microtubule cross-linking factor 1 (MTCL1) in cerebellar Purkinje cells causes loss of axonal polarity coupled with AnkG mislocalization. MTCL1 lacking MT-stabilizing activity failed to restore these defects, and stable MT bundles spanning the AIS were disorganized in knockdown cells. Interestingly, during early postnatal development, colocalization of MTCL1 with these stable MT bundles was observed prominently in the axon hillock and proximal axon. These results indicate that MTCL1-mediated formation of stable MT bundles is crucial for maintenance of AnkG localization. We also demonstrate that Mtcl1 gene disruption results in abnormal motor coordination with Purkinje cell degeneration, and provide evidence suggesting possible involvement of MTCL1 dysfunction in the pathogenesis of spinocerebellar ataxia.

Odorant receptors can mediate axonal identity and gene choice via cAMP-independent mechanisms.

Odorant receptors (ORs) control several aspects of cell fate in olfactory sensory neurons (OSNs), including singular gene choice and axonal identity. The mechanisms of OR-induced axon guidance have been suggested to principally rely on G-protein signalling. Here, we report that for a subset of OSNs, deleting G proteins or altering their levels of signalling does not affect axonal identity. Signalling-deficient ORs or surrogate receptors that are unable to couple to Gs/Golf still provide axons with distinct identities and the anterior–posterior targeting of axons does not correlate with the levels of cAMP produced by genetic modifications. In addition, we refine the models of negative feedback by showing that ectopic ORs can be robustly expressed without suppressing endogenous gene choice. In conclusion, our results uncover a new feature of ORs, showing that they can instruct axonal identity and regulate olfactory map formation independent of canonical G-protein signalling and cAMP production.

MouSensor: A Versatile Genetic Platform to Create Super Sniffer Mice for Studying Human Odor Coding

Typically, ∼0.1% of the total number of olfactory sensory neurons (OSNs) in the main olfactory epithelium express the same odorant receptor (OR) in a singular fashion and their axons coalesce into homotypic glomeruli in the olfactory bulb. Here, we have dramatically increased the total number of OSNs expressing specific cloned OR coding sequences by multimerizing a 21-bp sequence encompassing the predicted homeodomain binding site sequence, TAATGA, known to be essential in OR gene choice.Singular gene choice is maintained in these "MouSensors." Invivo synaptopHluorin imaging of odor-induced responses by known M71 ligands shows functional glomerular activation in an M71 MouSensor. Moreover, a behavioral avoidance task demonstrates that specific odor detection thresholds are significantly decreased in multiple transgenic lines, expressing mouse or human ORs. We have developed a versatile platform to study gene choice and axon identity, to create biosensors with great translational potential, and to finally decode human olfaction.

Nanoscale Architecture of the Axon Initial Segment Reveals an Organized and Robust Scaffold.

The axon initial segment (AIS), located within the first 30 μm of the axon, has two essential roles in generating action potentials and maintaining axonal identity. AIS assembly depends on a ßIV-spectrin/ankyrin G scaffold, but its macromolecular arrangement is not well understood. Here, we quantitatively determined the AIS nanoscale architecture by using stochastic optical reconstruction microscopy (STORM). First, we directly demonstrate that the 190-nm periodicity of the AIS submembrane lattice results from longitudinal, head-to-head ßIV-spectrin molecules connecting actin rings. Using multicolor 3D-STORM, we resolve the nanoscale organization of ankyrin G: its amino terminus associates with the submembrane lattice, whereas the C terminus radially extends (∼ 32 nm on average) toward the cytosol. This AIS nano-architecture is highly resistant to cytoskeletal perturbations, indicating its role in structural stabilization. Our findings provide a comprehensive view of AIS molecular architecture and will help reveal the crucial physiological functions of this compartment.

EphrinB2 sharpens lateral motor column division in the developing spinal cord.

BackgroundDuring sensori-motor circuit development, the somas of motoneurons (MN) are distributed in a topographic manner in the ventral horn of the neural tube. Indeed, their position within the lateral motor columns (LMC) correlates with axonal trajectories and identity of target limb muscles. The mechanisms by which this topographic distribution is established remains poorly understood. To address this issue, we assessed the role of ephrinB2 in MN topographic organization in the developing mouse spinal cord.ResultsFirst, we used a reporter mouse line to establish the spatio-temporal expression pattern of EfnB2 in the developing LMC. We show that early in LMC development, ephrinB2 is differentially expressed in MN of the lateral versus medial LMC, suggesting a possible role in MN sorting and/or migration. We demonstrate that while MN-specific excision of EfnB2 did not perturb specification or migration of MN, conditional loss of ephrinB2 led to the blurring of the LMC divisional boundary and to errors in the selection of LMC axon trajectory in the limb.ConclusionsAltogether, our study uncovered a novel cell autonomous role for ephrinB2 in LMC MN thus emphasizing the prevalent role of this ephrin member in maintaining cell population boundaries.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-015-0051-9) contains supplementary material, which is available to authorized users.

G protein-independent mechanisms are critical for odorant receptor-induced regulation of axonal wiring in the olfactory system.

Event Abstract Back to Event G protein-independent mechanisms are critical for odorant receptor-induced regulation of axonal wiring in the olfactory system. Kiavash Movahedi1, 2*, Xavier Grosmaitre3 and Paul Feinstein4 1 Vrije Universiteit Brussel - VIB, Myeloid Cell Immunology laboratory, Belgium 2 Max Planck Institute of Biophysics, Germany 3 Centre des Sciences du Goût et de l'Alimentation, France 4 Hunter College, United States Odorant receptors (ORs) regulate the axonal identity and glomerular formation of olfactory sensory neurons (OSNs). The mechanisms of OR-induced axon guidance remain enigmatic, but have been suggested to fully rely on G-protein signaling. Using G protein-coupling mutations in ORs via gene targeting, we now show that G-protein signaling is mainly required to promote OSN maturation and concomitant axon outgrowth. OSNs expressing signaling-deficient ORs remained immature and were unable to extend axons to the olfactory bulb. Neuronal maturation and axonal projections could be rescued via two complementary approaches: by the expression of a constitutively active G-protein mutant or through to the global co-expression of a second functional OR in all OSNs. In the latter approach, the forced expression of an OR did not suppress endogenous OR gene choice, even though the exogenous OR expression levels were high enough to support neuronal activity, alter the glomerular map and rescue the maturation and axonal projections of signaling-deficient ORs and a vomeronasal receptor expressed in OSNs. Our results show that while the OR was a critical determinant of axonal identity, the G-protein signaling capacity of OSNs did not correlate with the position of axonal convergence, indicating that the core determinants of axonal identity and anterior-posterior targeting relied on G protein-independent mechanisms. This was further corroborated by a new strategy to stochastically alter the levels of G-protein signaling in OSNs expressing functional ORs. Together, our results indicate that ORs rely on the interplay of distinct mechanistic pathways to regulate the development of the olfactory map. Keywords: Olfaction, odorant receptor, axon guidance, neuronal wiring, Olfactory receptor gene choice, G protein-coupled receptor, G-protein signaling, cAMP; Neurogenetics, Gene Choice, Singular Expression, Axon identity, Brainbow Conference: 11th National Congress of the Belgian Society for Neuroscience, Mons, Belgium, 22 May - 22 May, 2015. Presentation Type: Oral or Poster presentation Topic: Neuroscience Citation: Movahedi K, Grosmaitre X and Feinstein P (2015). G protein-independent mechanisms are critical for odorant receptor-induced regulation of axonal wiring in the olfactory system.. Front. Neurosci. Conference Abstract: 11th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2015.89.00013 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 May 2015; Published Online: 05 May 2015. * Correspondence: Dr. Kiavash Movahedi, Vrije Universiteit Brussel - VIB, Myeloid Cell Immunology laboratory, Brussel, 1050, Belgium, kiavash.movahedi@vub.ac.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kiavash Movahedi Xavier Grosmaitre Paul Feinstein Google Kiavash Movahedi Xavier Grosmaitre Paul Feinstein Google Scholar Kiavash Movahedi Xavier Grosmaitre Paul Feinstein PubMed Kiavash Movahedi Xavier Grosmaitre Paul Feinstein Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Truncation of the E3 ubiquitin ligase component FBXO31 causes non-syndromic autosomal recessive intellectual disability in a Pakistani family

In this study, we have performed autozygosity mapping on a large consanguineous Pakistani family segregating with intellectual disability. We identified two large regions of homozygosity-by-descent (HBD) on 16q12.2-q21 and 16q24.1-q24.3. Whole exome sequencing (WES) was performed on an affected individual from the family, but initially, no obvious mutation was detected. However, three genes within the HBD regions that were not fully captured during the WES were Sanger sequenced and we identified a five base pair deletion (actually six base pairs deleted plus one base pair inserted) in exon 7 of the gene FBXO31. The variant segregated completely in the family, in recessive fashion giving a LOD score of 3.95. This variant leads to a frameshift and a premature stop codon and truncation of the FBXO31 protein, p.(Cys283Asnfs*81). Quantification of mRNA and protein expression suggests that nonsense-mediated mRNA decay also contributes to the loss of FBXO31 protein in affected individuals. FBXO31 functions as a centrosomal E3 ubiquitin ligase, in association with SKP1 and Cullin-1, involved in ubiquitination of proteins targeted for degradation. The FBXO31/SKP1/Cullin1 complex is important for neuronal morphogenesis and axonal identity. FBXO31 also plays a role in dendrite growth and neuronal migration in developing cerebellar cortex. Our finding adds further evidence of the involvement of disruption of the protein ubiquitination pathway in intellectual disability.

The centrosomal E3 ubiquitin ligase FBXO31-SCF regulates neuronal morphogenesis and migration.

Neuronal development requires proper migration, polarization and establishment of axons and dendrites. Growing evidence identifies the ubiquitin proteasome system (UPS) with its numerous components as an important regulator of various aspects of neuronal development. F-box proteins are interchangeable subunits of the Cullin-1 based E3 ubiquitin ligase, but only a few family members have been studied. Here, we report that the centrosomal E3 ligase FBXO31-SCF (Skp1/Cullin-1/F-box protein) regulates neuronal morphogenesis and axonal identity. In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth. Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex. Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

Semaphorin 3A induces CaV2.3 channel-dependent conversion of axons to dendrites

Polarized neurites (axons and dendrites) form the functional circuitry of the nervous system. Secreted guidance cues often control the polarity of neuron migration and neurite outgrowth by regulating ion channels. Here, we show that secreted semaphorin 3A (Sema3A) induces the neurite identity of Xenopus spinal commissural interneurons (xSCINs) by activating Ca(V)2.3 channels (Ca(V)2.3). Sema3A treatment converted the identity of axons of cultured xSCINs to that of dendrites by recruiting functional Ca(V)2.3. Inhibition of Sema3A signalling prevented both the expression of Ca(V)2.3 and acquisition of the dendrite identity, and inhibition of Ca(V)2.3 function resulted in multiple axon-like neurites of xSCINs in the spinal cord. Furthermore, Sema3A-triggered cGMP production and PKG activity induced, respectively, the expression of functional Ca(V)2.3 and the dendrite identity. These results reveal a mechanism by which a guidance cue controls the identity of neurites during nervous system development.

Phosphorylation of E3 Ligase Smurf1 Switches Its Substrate Preference in Support of Axon Development

Ubiquitin E3 ligases serve for ubiquitination of specific substrates, and its ligase efficacy is regulated by interacting proteins or substrate modifications. Whether and how the ligases themselves are modified by cellular signaling is unclear. Here we report that protein kinase A (PKA)-dependent phosphorylation of Smad Ubiquitin Regulatory Factor 1 (Smurf1) can switch its substrate preference between two proteins of opposing actions on axon development. Extracellular factors that promote axon formation elevated Smurf1 phosphorylation at a PKA site Thr³⁰⁶, and preventing this phosphorylation reduced axon formation in cultured hippocampal neurons and impaired polarization of cortical neurons in vivo. Thr³⁰⁶-phosphorylation changed the relative affinities of Smurf1 for its substrates, leading to reduced degradation of polarity protein Par6 and increased degradation of growth-inhibiting RhoA. Thus, PKA-dependent phosphorylation of the E3 ligase could switch its substrate preference, contributing to selective protein degradation required for localized cellular function.

Posttranslational Modifications of Tubulin and the Polarized Transport of Kinesin-1 in Neurons

Polarized transport by microtubule-based motors is critical for neuronal development and function. Selective translocation of the Kinesin-1 motor domain is the earliest known marker of axonal identity, occurring before morphological differentiation. Thus, Kinesin-1-mediated transport may contribute to axonal specification. We tested whether posttranslational modifications of tubulin influence the ability of Kinesin-1 motors to distinguish microtubule tracks during neuronal development. We detected no difference in microtubule stability between axons and minor neurites in polarized stage 3 hippocampal neurons. In contrast, microtubule modifications were enriched in a subset of neurites in unpolarized stage 2 cells and the developing axon in polarized stage 3 cells. This enrichment correlated with the selective accumulation of constitutively active Kinesin-1 motors. Increasing tubulin acetylation, without altering the levels of other tubulin modifications, did not alter the selectivity of Kinesin-1 accumulation in polarized cells. However, globally enhancing tubulin acetylation, detyrosination, and polyglutamylation by Taxol treatment or inhibition of glycogen synthase kinase 3beta decreased the selectivity of Kinesin-1 translocation and led to the formation of multiple axons. Although microtubule acetylation enhances the motility of Kinesin-1, the preferential translocation of Kinesin-1 on axonal microtubules in polarized neuronal cells is not determined by acetylation alone but is probably specified by a combination of tubulin modifications.

Nuclear Receptor Unfulfilled Regulates Axonal Guidance and Cell Identity of Drosophila Mushroom Body Neurons

Nuclear receptors (NRs) comprise a family of ligand-regulated transcription factors that control diverse critical biological processes including various aspects of brain development. Eighteen NR genes exist in the Drosophila genome. To explore their roles in brain development, we knocked down individual NRs through the development of the mushroom bodies (MBs) by targeted RNAi. Besides recapitulating the known MB phenotypes for three NRs, we found that unfulfilled (unf), an ortholog of human photoreceptor specific nuclear receptor (PNR), regulates axonal morphogenesis and neuronal subtype identity. The adult MBs develop through remodeling of γ neurons plus de-novo elaboration of both α′/β′ and α/β neurons. Notably, unf is largely dispensable for the initial elaboration of γ neurons, but plays an essential role in their re-extension of axons after pruning during early metamorphosis. The subsequently derived MB neuron types also require unf for extension of axons beyond the terminus of the pruned bundle. Tracing single axons revealed misrouting rather than simple truncation. Further, silencing unf in single-cell clones elicited misguidance of axons in otherwise unperturbed MBs. Such axon guidance defects may occur as MB neurons partially lose their subtype identity, as evidenced by suppression of various MB subtype markers in unf knockdown MBs. In sum, unf governs axonal morphogenesis of multiple MB neuron types, possibly through regulating neuronal subtype identity.

Control of neuronal polarity and plasticity – a renaissance for microtubules?

Microtubules have been regarded as essential structures for stable neuronal morphology but new studies are highlighting their role in dynamic neuronal processes. Recent work demonstrates that the microtubule cytoskeleton has an active role during different phases of neuronal polarization - microtubules and their stability determine axon formation, they maintain the identity of axons and they regulate the dynamics of dendritic spines, the major sites of excitatory synaptic input. Although microtubules fulfill distinct cellular functions at different developmental stages, the underlying molecular mechanisms are remarkably similar. Reccurring themes are that microtubules direct specific membrane traffic and affect actin dynamics to locally organize axon growth and spine dynamics. We review the novel role of microtubules during neuronal development and discuss models for microtubule-dependent signaling in neuronal plasticity.

Neuronal polarity: consolidation for growth, growth for consolidation.

Neuronal polarity: consolidation for growth, growth for consolidation.

Mapping of Class I and Class II Odorant Receptors to Glomerular Domains by Two Distinct Types of Olfactory Sensory Neurons in the Mouse

The repertoire of approximately 1200 odorant receptors (ORs) is mapped onto the array of approximately 1800 glomeruli in the mouse olfactory bulb (OB). The spatial organization of this array is influenced by the ORs. Here we show that glomerular mapping to broad domains in the dorsal OB is determined by two types of olfactory sensory neurons (OSNs), which reside in the dorsal olfactory epithelium. The OSN types express either class I or class II OR genes. Axons from the two OSN types segregate already within the olfactory nerve and form distinct domains of glomeruli in the OB. These class-specific anatomical domains correlate with known functional odorant response domains. However, axonal segregation and domain formation are not determined by the class of the expressed OR protein. Thus, the two OSN types are determinants of axonal wiring, operate at a higher level than ORs, and contribute to the functional organization of the glomerular array.

Plasticity of Polarization: Changing Dendrites into Axons in Neurons Integrated in Neuronal Circuits

Developing neurons can change axonal and dendritic fate upon axonal lesion, but it is unclear whether neurons retain such plasticity when they are synaptically interconnected. To address whether polarity is reversible in mature neurons, we cut the axon of GFP-labeled hippocampal neurons in dissociated and organotypic cultures and found that a new axon arose from a mature dendrite. The regenerative response correlated with the length of the remaining stump: proximal axotomies (<35 microm) led to the transformation of a dendrite into an axon (identity change), whereas distal cuts (>35 microm) induced axon regrowth, similar to what is seen in young neurons. Searching for a putative landmark in the distal axon that could determine axon identity, we focused on the stability of microtubules, which regulate initial neuronal polarization during early development. We found that functionally polarized neurons contain a distinctively high proportion of stable microtubules in the distal axon. Moreover, pharmacological stabilization of microtubules was sufficient to induce the formation of multiple axons out of differentiated dendrites. Our data argue that mature neurons integrated in functional networks remain flexible in their polarity and that mechanisms acting during initial axon selection can be reactivated to induce axon growth out of functionally mature dendrites.

The JIP1 Scaffold Protein Regulates Axonal Development in Cortical Neurons

SummaryThe development of neuronal polarity is essential for the determination of neuron connectivity and for correct brain function. The c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1) is highly expressed in neurons and has previously been characterized as a regulator of JNK signaling. JIP1 has been shown to localize to neurites in various neuronal models, but the functional significance of this localization is not fully understood [1–4]. JIP1 is also a cargo of the motor protein kinesin-1, which is important for axonal transport [2, 4]. Here we demonstrate that before primary cortical neurons become polarized, JIP1 specifically localizes to a single neurite and that after axonal specification, it accumulates in the emerging axon. JIP1 is necessary for normal axonal development and promotes axonal growth dependent upon its binding to kinesin-1 and via a newly described interaction with the c-Abl tyrosine kinase. JIP1 associates with and is phosphorylated by c-Abl, and the mutation of the c-Abl phosphorylation site on JIP1 abrogates its ability to promote axonal growth. JIP1 is therefore an important regulator of axonal development and is a key target of c-Abl-dependent pathways that control axonal growth.

The Interaction of mPar3 with the Ubiquitin Ligase Smurf2 Is Required for the Establishment of Neuronal Polarity

The Par polarity complex consisting of the evolutionarily conserved proteins mPar3, mPar6, and aPKC regulates cell polarity in many cell types including neurons. Here we show that mPar3 is required for the establishment of neuronal polarity and links the Smurf2 to Kinesin-2. The HECT domain E3 ubiquitin ligase Smurf2 ensures that neurons extend only a single axon by initiating the degradation of inactive Rap1B through the ubiquitin/proteasome system. Its interaction with mPar3 is required to localize Smurf2 to growth cones and restrict Rap1B to the axon. Interfering with the binding of mPar3 to Kinesin-2 or Smurf2 to mPar3 and knockdown of mPar3 by RNAi disrupt the establishment of neuronal polarity through the failure to restrict Rap1B to a single neurite.