Growth Cone Extension Research Articles

Membrane tension propagation couples axon growth and collateral branching.

Neuronal axons must navigate a mechanically heterogeneous environment to reach their targets, but the biophysical mechanisms coupling mechanosensation, growth, and branching are not fully understood. Here, we show that local changes in membrane tension propagate along axons at approximately 20 μm/s, more than 1000-fold faster than in most other nonmotile cells where this property has been measured. Local perturbations to tension decay along the axon with a length constant of approximately 41 μm. This rapid and long-range mechanical signaling mediates bidirectional competition between axonal branch initiation and growth cone extension. Our data suggest a mechanism by which mechanical cues at one part of a growing axon can affect growth dynamics remotely.

Predicting neurite extension for varying extracellular matrix stiffness and topography

Neurite extension is a dynamic process and is dependent on the microenvironment. The mechanical properties of the extracellular matrix (ECM), such as stiffness and topography influence the microenvironment and affects neurite extension; however, the mechanistic basis for this dynamic response of neurite extension remains elusive. In this study, we develop a computational model that predicts neurite extension dynamics process as the stiffness and patterned topography of ECM changes. The model includes the contribution of receptors integrin and neural cellular adhesion molecule toward the growth of neurite tip. We use non-linear finite element analysis (FEA) to model the neuronal cell, neurite, and the ECM, which is then coupled to the force–deformation receptor properties obtained from molecular dynamics simulations. Using an empirical relation, we develop a neurite extension algorithm that simulates the dynamic process of growth cone induced by growth cone extension, receptor density, and rupture. We investigate the dependence of neurite extension on ECM stiffness using three distinct materials, the effect of width and spacing of continuous (cylindrical) and discontinuous (pillar) patterned topography, as well as the topography steepness and stiffness gradient. We find that an increasing stiffness and width of patterned topography results in increased neurite extension, but the magnitude of the increase differs depending on the growth cone extension and receptor density between them. These findings will aid in vitro studies in determining an ECM with appropriate mechanical properties, such as stiffness and topography that will improve neurite extension, thus resulting in the formation of functional neurons.

Analysis of growth cone extension in standardized coordinates highlights self-organization rules during wiring of the Drosophila visual system.

A fascinating question in neuroscience is how ensembles of neurons, originating from different locations, extend to the proper place and by the right time to create precise circuits. Here, we investigate this question in the Drosophila visual system, where photoreceptors re-sort in the lamina to form the crystalline-like neural superposition circuit. The repeated nature of this circuit allowed us to establish a data-driven, standardized coordinate system for quantitative comparison of sparsely perturbed growth cones within and across specimens. Using this common frame of reference, we investigated the extension of the R3 and R4 photoreceptors, which is the only pair of symmetrically arranged photoreceptors with asymmetric target choices. Specifically, we found that extension speeds of the R3 and R4 growth cones are inherent to their cell identities. The ability to parameterize local regularity in tissue organization facilitated the characterization of ensemble cellular behaviors and dissection of mechanisms governing neural circuit formation.

Coactosin Promotes F-Actin Protrusion in Growth Cones Under Cofilin-Related Signaling Pathway.

During brain development, axon outgrowth and its subsequent pathfinding are reliant on a highly motile growth cone located at the tip of the axon. Actin polymerization that is regulated by actin-depolymerizing factors homology (ADF-H) domain-containing family drives the formation of lamellipodia and filopodia at the leading edge of growth cones for axon guidance. However, the precise localization and function of ADF-H domain-containing proteins involved in axon extension and retraction remain unclear. We have previously shown that transcripts and proteins of coactosin-like protein 1 (COTL1), an ADF-H domain-containing protein, are observed in neurites and axons in chick embryos. Coactosin overexpression analysis revealed that this protein was localized to axonal growth cones and involved in axon extension in the midbrain. We further examined the specific distribution of coactosin and cofilin within the growth cone using superresolution microscopy, structured illumination microscopy, which overcomes the optical diffraction limitation and is suitable to the analysis of cellular dynamic movements. We found that coactosin was tightly associated with F-actin bundles at the growth cones and that coactosin overexpression promoted the expansion of lamellipodia and extension of growth cones. Coactosin knockdown in oculomotor neurons resulted in an increase in the levels of the inactive, phosphorylated form of cofilin and dysregulation of actin polymerization and axonal elongation, which suggests that coactosin promoted axonal growth in a cofilin-dependent manner. Indeed, the application of a dominant-negative form of LIMK1, a downstream effector of GTPases, reversed the effect of coactosin knockdown on axonal growth by enhancing cofilin activity. Combined, our results indicate that coactosin functions promote the assembly of protrusive actin filament arrays at the leading edge for growth cone motility.

Growth cone-localized microtubule organizing center establishes microtubule orientation in dendrites.

A polarized arrangement of neuronal microtubule arrays is the foundation of membrane trafficking and subcellular compartmentalization. Conserved among both invertebrates and vertebrates, axons contain exclusively 'plus-end-out' microtubules while dendrites contain a high percentage of 'minus-end-out' microtubules, the origins of which have been a mystery. Here we show that in Caenorhabditis elegans the dendritic growth cone contains a non-centrosomal microtubule organizing center (MTOC), which generates minus-end-out microtubules along outgrowing dendrites and plus-end-out microtubules in the growth cone. RAB-11-positive endosomes accumulate in this region and co-migrate with the microtubule nucleation complex γ-TuRC. The MTOC tracks the extending growth cone by kinesin-1/UNC-116-mediated endosome movements on distal plus-end-out microtubules and dynein clusters this advancing MTOC. Critically, perturbation of the function or localization of the MTOC causes reversed microtubule polarity in dendrites. These findings unveil the endosome-localized dendritic MTOC as a critical organelle for establishing axon-dendrite polarity.

Polydopamine/carboxylic graphene oxide-composited polypyrrole films for promoting adhesion and alignment of Schwann cells

Polydopamine (PDA)- and carboxylic graphene oxide (CGO)-composited polypyrrole (PPy) films were prepared via electrospinning of poly-l-lactic acid (PLLA), electrodeposition of PPy blended with CGO and polymerization of dopamine (PDA/CGO/PPy-PLLA), and its surface conductivity and elastic modulus reached to ∼17.3 S/cm and ∼260 MPa, respectively. PDA composition could maintain the higher conduction and elastic modulus of this film after the immersion of 3 w, due to the stronger force between PDA molecule and CGO sheets or PPy particles. The results of cell experiments indicated the better adhesion and higher expression of neural proteins in rat Schwann cells (RSCs) on PDA/CGO/PPy-PLLA films, because the composition of PDA provided more absorption and immobilization site for proteins and the regular coating of PDA particles on the CGO sheets reduced the potential damage of Graphene derivatives on the cell membrane integrity. Electrical stimulation (ES) could facilitate ∼31 % of RSCs to align along the current direction on PDA/CGO/PPy-PLLA films, significantly higher than those on PPy-PLLA and CGO/PPy-PLLA films. A mechanism about the cell alignment on films with ES was proposed: the movement of the cytomembrane proteins under ES and their linkage with serum proteins immobilized by PDA facilitated the extension of growth cone along the ES direction.

Growth Cone-Localized Microtubule Organizing Center Establishes Microtubule Orientation in Dendrites

A polarized arrangement of neuronal microtubule arrays is the foundation of membrane trafficking and subcellular compartmentalization. Conserved among both invertebrates and vertebrates, axons contain exclusively “plus-end-out” microtubules while dendrites contain a high percentage of “minus-end-out” microtubules, the origins of which have been a mystery. Here we show that the dendritic growth cone contains a non-centrosomal microtubule organizing center, which generates minus-end-out microtubules along outgrowing dendrites and plus-end-out microtubules in the growth cone. RAB-11-positive endosomes accumulate in this region and are responsible for localizing the microtubule nucleation complex γ-TuRC. The MTOC tracks the extending growth cone by kinesin-1/UNC-116-mediated endosome movements on distal plus-end-out microtubules and dynein clusters this advancing MTOC. Critically, perturbation of the function or localization of the MTOC causes reversed microtubule polarity in dendrites. These findings unveil the dendritic MTOC as a critical organelle for establishing axon-dendrite polarity.

Casein Kinase 1δ Stabilizes Mature Axons by Inhibiting Transcription Termination of Ankyrin.

After axon outgrowth and synapse formation, the nervous system transitions to a stable architecture. In C.elegans, this transition is marked by the appearance of casein kinase 1δ (CK1δ) in the nucleus. In CK1δ mutants, neurons continue to sprout growth cones into adulthood, leading to a highly ramified nervous system. Nervous system architecture in these mutants is completely restored by suppressor mutations in ten genes involved in transcription termination. CK1δ prevents termination by phosphorylating and inhibiting SSUP-72. SSUP-72 would normally remodel the C-terminal domain of RNA polymerase in anticipation of termination. The antitermination activity of CK1δ establishes the mature state of a neuron by promoting the expression of the long isoform of a single gene, the cytoskeleton protein Ankyrin.

Analysis of Epineurial Lidocaine Injection for Nerve Transfers in a Rat Sciatic Nerve Model

Nerve transfers for peripheral nerve injuries can result in variable outcomes. We investigated the neuroprotective effect of epineurial lidocaine injection in the donor nerve prior to transection, with the hypothesis that proximal axon loss would be decreased with consequent increased neuroregeneration and functional recovery. A rat sciatic nerve model was used with 4 intervention groups: (1) lidocaine; (2) lidocaine/calcium gluconate (CG); (3) CG; or (4) saline (control). Behavioral testing and qualitative and quantitative histological evaluation was performed at 8 and 12 weeks. Histological assays included transmission electron microscopy, retrograde fluorogold labeling, and whole mount immunostaining. Functional assessments through the sciatic functional index and Basso, Beattie, and Bresnahan scale showed a statistically significant increase in recovery at 8 and 12 weeks with lidocaine treatment. Significantly higher axonal counts were obtained in the lidocaine-treated groups. Fragmentation and increased myelin damage was present in the CG and saline groups. Retrograde fluorogold labeling showed a statistically significant increase in the number of L4-6 dorsal root ganglion neurons in the lidocaine-treated groups. Whole mount immunostaining identified extension of the axonal growth cone past the nerve coaptation site in lidocaine-treated groups, but not in CG and saline groups. Our results suggest that epineurial lidocaine injection prior to donor nerve transection for nerve transfer has a neuroprotective effect, resulting in increased proximal axon counts and improved functional recovery. These findings may have direct clinical application because epineurial lidocaine can be used in surgery as a simple and inexpensive intervention for promoting improved clinical outcomes after nerve transfer.

Syntaxin-1/TI-VAMP SNAREs interact with Trk receptors and are required for neurotrophin-dependent outgrowth.

SNARE proteins are essential components of the machinery that regulates vesicle trafficking and exocytosis. Their role is critical for the membrane-fusion processes that occur during neurotransmitter release. However, research in the last decade has also unraveled the relevance of these proteins in membrane expansion and cytoskeletal rearrangements during developmental processes such as neuronal migration and growth cone extension and attraction. Neurotrophins are neurotrophic factors that are required for many cellular functions throughout the brain, including neurite outgrowth and guidance, synaptic formation, and plasticity. Here we show that neurotrophin Trk receptors form a specific protein complex with the t-SNARE protein Syntaxin 1, both in vivo and in vitro. We also demonstrate that blockade of Syntaxin 1 abolishes neurotrophin-dependent growth of axons in neuronal cultures and decreases exocytotic events at the tip of axonal growth cones. 25-kDa soluble N-ethylmaleimide-sensitive factor attachment protein and Vesicle-associated membrane protein 2 do not participate in the formation of this SNARE complex, while tetanus neurotoxin-insensitive vesicle-associated membrane protein interacts with Trk receptors; knockdown of this (v) SNARE impairs Trk-dependent outgrowth. Taken together, our results support the notion that an atypical SNARE complex comprising Syntaxin 1 and tetanus neurotoxin-insensitive vesicle-associated membrane protein is required for axonal neurotrophin function.

Rapid Assembly of Presynaptic Materials behind the Growth Cone in Dopaminergic Neurons Is Mediated by Precise Regulation of Axonal Transport

SUMMARYThe proper assembly of neural circuits depends on the process of synaptogenesis, or the formation of synapses between partner neurons. Using the dopaminergic PDE neurons in C. elegans, we developed an in vivo system to study the earliest steps of the formation of en passant presynaptic specializations behind an extending growth cone. We find that presynaptic materials coalesce into puncta in as little as a few minutes and that both synaptic vesicle (SV) and active zone (AZ) proteins arrive nearly simultaneously at the nascent sites of synapse formation. We show that precise regulation of UNC-104/Kinesin-3 determines the distribution of SV proteins along the axon. The localization of AZ proteins to en passant puncta, however, is largely independent of the major axonal kinesins: UNC-104/Kinesin-3 and UNC-116/ Kinesin-1. Moreover, AZ proteins play a crucial role in recruiting and tethering SV precursors (SVPs).

Neuronal Growth Cone Size-Dependent and -Independent Parameters of Microtubule Polymerization.

Migration and pathfinding of neuronal growth cones during neurite extension is critically dependent on dynamic microtubules. In this study we sought to determine, which aspects of microtubule polymerization relate to growth cone morphology and migratory characteristics. We conducted a multiscale quantitative microscopy analysis using automated tracking of microtubule plus ends in migrating growth cones of cultured murine dorsal root ganglion (DRG) neurons. Notably, this comprehensive analysis failed to identify any changes in microtubule polymerization parameters that were specifically associated with spontaneous extension vs. retraction of growth cones. This suggests that microtubule dynamicity is a basic mechanism that does not determine the polarity of growth cone response but can be exploited to accommodate diverse growth cone behaviors. At the same time, we found a correlation between growth cone size and basic parameters of microtubule polymerization including the density of growing microtubule plus ends and rate and duration of microtubule growth. A similar correlation was observed in growth cones of neurons lacking the microtubule-associated protein MAP1B. However, MAP1B-null growth cones, which are deficient in growth cone migration and steering, displayed an overall reduction in microtubule dynamicity. Our results highlight the importance of taking growth cone size into account when evaluating the influence on growth cone microtubule dynamics of different substrata, guidance factors or genetic manipulations which all can change growth cone morphology and size. The type of large scale multiparametric analysis performed here can help to separate direct effects that these perturbations might have on microtubule dynamics from indirect effects resulting from perturbation-induced changes in growth cone size.

IRES ‐mediated translation of cofilin regulates axonal growth cone extension and turning

In neuronal development, dynamic rearrangement of actin promotes axonal growth cone extension, and spatiotemporal translation of local mRNAs in response to guidance cues directs axonal growth cone steering, where cofilin plays a critical role. While regulation of cofilin activity is well studied, regulatory mechanism for cofilin mRNA translation in neurons is unknown. In eukaryotic cells, proteins can be synthesized by cap-dependent or cap-independent mechanism via internal ribosome entry site (IRES)-mediated translation. IRES-mediated translation has been reported in various pathophysiological conditions, but its role in normal physiological environment is poorly understood. Here, we report that 5'UTR of cofilin mRNA contains an IRES element, and cofilin is predominantly translated by IRES-mediated mechanism in neurons. Furthermore, we show that IRES-mediated translation of cofilin is required for both axon extension and axonal growth cone steering. Our results provide new insights into the function of IRES-mediated translation in neuronal development.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in <em>Drosophila</em>

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila.

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Increased phosphorylation of collapsin response mediator protein-2 at Thr514 correlates with β-amyloid burden and synaptic deficits in Lewy body dementias.

Collapsin response mediator protein-2 (CRMP2) regulates axonal growth cone extension, and increased CRMP2 phosphorylation may lead to axonal degeneration. Axonal and synaptic pathology is an important feature of Lewy body dementias (LBD), but the state of CRMP2 phosphorylation (pCRMP2) as well as its correlations with markers of neurodegeneration have not been studied in these dementias. Hence, we measured CRMP2 phosphorylation at Thr509, Thr514 and Ser522, as well as markers of β-amyloid (Aβ), tau-phosphorylation, α-synuclein and synaptic function in the postmortem neocortex of a longitudinally assessed cohort of LBD patients characterized by low (Parkinson’s disease dementia, PDD) and high (dementia with Lewy bodies, DLB) burden of Alzheimer type pathology. We found specific increases of pCRMP2 at Thr514 in DLB, but not PDD. The increased CRMP2 phosphorylation correlated with fibrillogenic Aβ as well as with losses of markers for axon regeneration (β-III-tubulin) and synaptic integrity (synaptophysin) in LBD. In contrast, pCRMP2 alterations did not correlate with tau-phosphorylation or α-synuclein, and also appear unrelated to immunoreactivities of putative upstream kinases glycogen synthase kinase 3β and cyclin-dependent kinase 5, as well as to protein phosphatase 2A. In conclusion, increased pCRMP2 may underlie the axonal pathology of DLB, and may be a novel therapeutic target. However, antecedent signaling events as well as the nature of pCRMP2 association with Aβ and other neuropathologic markers require further study.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0264-9) contains supplementary material, which is available to authorized users.

DSCR1 is required for both axonal growth cone extension and steering.

Local information processing in the growth cone is essential for correct wiring of the nervous system. As an axon navigates through the developing nervous system, the growth cone responds to extrinsic guidance cues by coordinating axon outgrowth with growth cone steering. It has become increasingly clear that axon extension requires proper actin polymerization dynamics, whereas growth cone steering involves local protein synthesis. However, molecular components integrating these two processes have not been identified. Here, we show that Down syndrome critical region 1 protein (DSCR1) controls axon outgrowth by modulating growth cone actin dynamics through regulation of cofilin activity (phospho/dephospho-cofilin). Additionally, DSCR1 mediates brain-derived neurotrophic factor-induced local protein synthesis and growth cone turning. Our study identifies DSCR1 as a key protein that couples axon growth and pathfinding by dually regulating actin dynamics and local protein synthesis.

Hsc70 chaperone activity underlies Trio GEF function in axon growth and guidance induced by netrin-1.

During development, netrin-1 is both an attractive and repulsive axon guidance cue and mediates its attractive function through the receptor Deleted in Colorectal Cancer (DCC). The activation of Rho guanosine triphosphatases within the extending growth cone facilitates the dynamic reorganization of the cytoskeleton required to drive axon extension. The Rac1 guanine nucleotide exchange factor (GEF) Trio is essential for netrin-1-induced axon outgrowth and guidance. Here, we identify the molecular chaperone heat shock cognate protein 70 (Hsc70) as a novel Trio regulator. Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio. Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase-deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1-induced Rac1 activation. Hsc70 was required for netrin-1-mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex. These findings demonstrate that Hsc70 chaperone activity is required for Rac1 activation by Trio and this function underlies netrin-1/DCC-dependent axon outgrowth and guidance.

Signaling networks that regulate cell migration.

Stimuli that promote cell migration, such as chemokines, cytokines, and growth factors in metazoans and cyclic AMP in Dictyostelium, activate signaling pathways that control organization of the actin cytoskeleton and adhesion complexes. The Rho-family GTPases are a key convergence point of these pathways. Their effectors include actin regulators such as formins, members of the WASP/WAVE family and the Arp2/3 complex, and the myosin II motor protein. Pathways that link to the Rho GTPases include Ras GTPases, TorC2, and PI3K. Many of the molecules involved form gradients within cells, which define the front and rear of migrating cells, and are also established in related cellular behaviors such as neuronal growth cone extension and cytokinesis. The signaling molecules that regulate migration can be integrated to provide a model of network function. The network displays biochemical excitability seen as spontaneous waves of activation that propagate along the cell cortex. These events coordinate cell movement and can be biased by external cues to bring about directed migration.

Tanshinone IIA Rescued the Impairments of Primary Hippocampal Neurons Induced by BV2 Microglial Over-Activation.

Activated microglia plays an important role in monitoring the microenvironment and prune neural process in healthy neural tissue, in order to maintain synaptic homeostasis. However, hyperactive microglia may release various cytotoxic factors and induce neuroinflammation, which cause neuronal damages leading to neurodegenerative diseases. Tanshinone IIA (TSA), an extract from traditional Chinese medicine, features potent anti-apoptotic and anti-inflammatory effects both in vitro and in vivo. But little is known on the effects of TSA on microglial-over-activation-induced neural impairments. In this study, by employing murine BV2 cell lines as well as the combinations of ELISA assay, immunostaining, western blotting analysis and RT-PCR, we found that TSA has the potential to exhibit anti-inflammatory effects. We hereby demonstrated that TSA rescued neural growth and development in the primary cultured hippocampal neurons from impairments caused by BV2 microglial over-activation insult. The results show that TSA attenuated the BV2 cell activation by lipopolysaccharide (LPS) stimulation through suppressing the NF-кB signal pathway. Also, conditioned mediums (CM) from TSA treated and activated BV2 cells protected against LPS-CM-induced neuronal death. Furthermore, TSA treatment could recover the inhibitory effects of LPS-CM on growth cone extension, neurite sprouting and outgrowth, as well as spinogenesis. Our findings support that TSA is capable of inhibiting BV2 cell over-activation thus has potential protective effects in the cultured hippocampal neurons. This study may lay a foundation for using TSA to restore cerebral injuries after severe neuroinflammation.