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    Nerve conduction and microanatomy in the rabbit sciatic nerve after gradual limb lengthening-distraction neurogenesis.
    Novel ecological and climatic conditions drive rapid adaptation in invasive Florida Burmese pythons. Card Daren C,Perry Blair W,Adams Richard H,Schield Drew R,Young Acacia S,Andrew Audra L,Jezkova Tereza,Pasquesi Giulia I M,Hales Nicole R,Walsh Matthew R,Rochford Michael R,Mazzotti Frank J,Hart Kristen M,Hunter Margaret E,Castoe Todd A Molecular ecology Invasive species provide powerful in situ experimental systems for studying evolution in response to selective pressures in novel habitats. While research has shown that phenotypic evolution can occur rapidly in nature, few examples exist of genomewide adaptation on short "ecological" timescales. Burmese pythons (Python molurus bivittatus) have become a successful and impactful invasive species in Florida over the last 30 years despite major freeze events that caused high python mortality. We sampled Florida Burmese pythons before and after a major freeze event in 2010 and found evidence for directional selection in genomic regions enriched for genes associated with thermosensation, behaviour and physiology. Several of these genes are linked to regenerative organ growth, an adaptive response that modulates organ size and function with feeding and fasting in pythons. Independent histological and functional genomic data sets provide additional layers of support for a contemporary shift in invasive Burmese python physiology. In the Florida population, a shift towards maintaining an active digestive system may be driven by the fitness benefits of maintaining higher metabolic rates and body temperature during freeze events. Our results suggest that a synergistic interaction between ecological and climatic selection pressures has driven adaptation in Florida Burmese pythons, demonstrating the often-overlooked potential of rapid adaptation to influence the success of invasive species. 10.1111/mec.14885
    Correction: IGF-1 Induces GHRH Neuronal Axon Elongation during Early Postnatal Life in Mice. Decourtye Lyvianne,Mire Erik,Clemessy Maud,Heurtier Victor,Ledent Tatiana,Robinson Iain C,Mollard Patrice,Epelbaum Jacques,Meaney Michael J,Garel Sonia,Le Bouc Yves,Kappeler Laurent PloS one [This corrects the article DOI: 10.1371/journal.pone.0170083.]. 10.1371/journal.pone.0172915
    14-3-3ε and NAV2 interact to regulate neurite outgrowth and axon elongation. Marzinke Mark A,Mavencamp Terri,Duratinsky Joseph,Clagett-Dame Margaret Archives of biochemistry and biophysics Neuron navigator 2 (NAV2) is required for all-trans retinoic acid (atRA) to induce neurite outgrowth in human neuroblastoma cells. Further, ectopic overexpression of full-length human NAV2 rescues an axonal elongation defect in the Caenorhabditis elegans unc-53 (NAV2 ortholog) mutant. Using a region of NAV2 that independently associates with the cytoskeleton as bait in a yeast-two-hybrid screen, 14-3-3ε was identified as a novel NAV2 interacting partner. Amino acids 761-960 of NAV2 are sufficient to confer a positive interaction with 14-3-3ε as evidenced by a two-hybrid screen and co-immunoprecipitation assay. Knockdown of 14-3-3ε leads to a decrease in atRA-mediated neurite outgrowth, similar to the elongation defects observed when NAV2 is depleted or mutated. Likewise, posterior lateral microtubule (PLM) defects in C. elegans fed unc-53 RNAi are similar to those fed ftt-2 (14-3-3 homolog) RNAi. The discovery of an interaction between NAV2 and 14-3-3ε could provide insight into the mechanism by which NAV2 participates in promoting cell migration and neuronal elongation. 10.1016/j.abb.2013.10.012
    Synapsins regulate brain-derived neurotrophic factor-mediated synaptic potentiation and axon elongation by acting on membrane rafts. Kao Hung-Teh,Ryoo Kanghyun,Lin Albert,Janoschka Stephen R,Augustine George J,Porton Barbara The European journal of neuroscience In neurons, intracellular membrane rafts are essential for specific actions of brain-derived neurotrophic factor (BDNF), which include the regulation of axon outgrowth, growth cone turning and synaptic transmission. Virtually, all the actions of BDNF are mediated by binding to its receptor, TrkB. The association of TrkB with the tyrosine kinase, Fyn, is critical for its localization to intracellular membrane rafts. Here, we show that synapsins, a family of highly amphipathic neuronal phosphoproteins, regulate membrane raft lipid composition and consequently, the ability of BDNF to regulate axon/neurite development and potentiate synaptic transmission. In the brains of mice lacking all synapsins, the expression of both BDNF and TrkB were increased, suggesting that BDNF/TrkB-mediated signaling is impaired. Consistent with this finding, synapsin-depleted neurons exhibit altered raft lipid composition, deficient targeting of Fyn to rafts, attenuated TrkB activation, and abrogation of BDNF-stimulated axon outgrowth and synaptic potentiation. Conversely, overexpression of synapsins in neuroblastoma cells results in corresponding reciprocal changes in raft lipid composition, increased localization of Fyn to rafts and promotion of BDNF-stimulated neurite formation. In the presence of synapsins, the ratio of cholesterol to estimated total phospholipids converged to 1, suggesting that synapsins act by regulating the ratio of lipids in intracellular membranes, thereby promoting lipid raft formation. These studies reveal a mechanistic link between BDNF and synapsins, impacting early development and synaptic transmission. 10.1111/ejn.13552
    Scapinin-induced inhibition of axon elongation is attenuated by phosphorylation and translocation to the cytoplasm. Farghaian Hovik,Chen Yu,Fu Ada W Y,Fu Amy K Y,Ip Jacque P K,Ip Nancy Y,Turnley Ann M,Cole Adam R The Journal of biological chemistry Scapinin is an actin- and PP1-binding protein that is exclusively expressed in the brain; however, its function in neurons has not been investigated. Here we show that expression of scapinin in primary rat cortical neurons inhibits axon elongation without affecting axon branching, dendritic outgrowth, or polarity. This inhibitory effect was dependent on its ability to bind actin because a mutant form that does not bind actin had no effect on axon elongation. Immunofluorescence analysis showed that scapinin is predominantly located in the distal axon shaft, cell body, and nucleus of neurons and displays a reciprocal staining pattern to phalloidin, consistent with previous reports that it binds actin monomers to inhibit polymerization. We show that scapinin is phosphorylated at a highly conserved site in the central region of the protein (Ser-277) by Cdk5 in vitro. Expression of a scapinin phospho-mimetic mutant (S277D) restored normal axon elongation without affecting actin binding. Instead, phosphorylated scapinin was sequestered in the cytoplasm of neurons and away from the axon. Because its expression is highest in relatively plastic regions of the adult brain (cortex, hippocampus), scapinin is a new regulator of neurite outgrowth and neuroplasticity in the brain. 10.1074/jbc.M110.205781
    Distinct Functions for Mammalian CLASP1 and -2 During Neurite and Axon Elongation. Sayas Carmen Laura,Basu Sreya,van der Reijden Michael,Bustos-Morán Eugenio,Liz Marcia,Sousa Monica,van IJcken Wilfred F J,Avila Jesus,Galjart Niels Frontiers in cellular neuroscience Mammalian cytoplasmic linker associated protein 1 and -2 (CLASP1 and -2) are microtubule (MT) plus-end tracking proteins that selectively stabilize MTs at the edge of cells and that promote MT nucleation and growth at the Golgi, thereby sustaining cell polarity. analysis has shown that CLASPs are MT growth promoting factors. To date, a single CLASP1 isoform (called CLASP1α) has been described, whereas three CLASP2 isoforms are known (CLASP2α, -β, and -γ). Although CLASP2β/γ are enriched in neurons, suggesting isoform-specific functions, it has been proposed that during neurite outgrowth CLASP1 and -2 act in a redundant fashion by modulating MT dynamics downstream of glycogen synthase kinase 3 (GSK3). Here, we show that in differentiating N1E-115 neuroblastoma cells CLASP1 and CLASP2 differ in their accumulation at MT plus-ends and display different sensitivity to GSK3-mediated phosphorylation, and hence regulation. More specifically, western blot (WB) analysis suggests that pharmacological inhibition of GSK3 affects CLASP2 but not CLASP1 phosphorylation and fluorescence-based microscopy data show that GSK3 inhibition leads to an increase in the number of CLASP2-decorated MT ends, as well as to increased CLASP2 staining of individual MT ends, whereas a reduction in the number of CLASP1-decorated ends is observed. Thus, in N1E-115 cells CLASP2 appears to be a prominent target of GSK3 while CLASP1 is less sensitive. Surprisingly, knockdown of either CLASP causes phosphorylation of GSK3, pointing to the existence of feedback loops between CLASPs and GSK3. In addition, CLASP2 depletion also leads to the activation of protein kinase C (PKC). We found that these differences correlate with opposite functions of CLASP1 and CLASP2 during neuronal differentiation, i.e., CLASP1 stimulates neurite extension, whereas CLASP2 inhibits it. Consistent with knockdown results in N1E-115 cells, primary knockout (KO) neurons exhibit early accelerated neurite and axon outgrowth, showing longer axons than control neurons. We propose a model in which neurite outgrowth is fine-tuned by differentially posttranslationally modified isoforms of CLASPs acting at distinct intracellular locations, thereby targeting MT stabilizing activities of the CLASPs and controlling feedback signaling towards upstream kinases. In summary, our findings provide new insight into the roles of neuronal CLASPs, which emerge as regulators acting in different signaling pathways and locally modulating MT behavior during neurite/axon outgrowth. 10.3389/fncel.2019.00005
    Syntaphilin-Mediated Docking of Mitochondria at the Growth Cone Is Dispensable for Axon Elongation . Verreet Tine,Weaver Cory J,Hino Hiromu,Hibi Masahiko,Poulain Fabienne E eNeuro Mitochondria are abundantly detected at the growth cone, the dynamic distal tip of developing axons that directs growth and guidance. It is, however, poorly understood how mitochondrial dynamics relate to growth cone behavior , and which mechanisms are responsible for anchoring mitochondria at the growth cone during axon pathfinding. Here, we show that in retinal axons elongating along the optic tract in zebrafish, mitochondria accumulate in the central area of the growth cone and are occasionally observed in filopodia extending from the growth cone periphery. Mitochondrial behavior at the growth cone is dynamic, with mitochondrial positioning and anterograde transport strongly correlating with growth cone behavior and axon outgrowth. Using novel zebrafish mutant lines that lack the mitochondrial anchoring proteins Syntaphilin a and b, we further show that Syntaphilins contribute to mitochondrial immobilization at the growth cone. Syntaphilins are, however, not required for proper growth cone morphology and axon growth , indicating that Syntaphilin-mediated anchoring of mitochondria at the growth cone plays only a minor role in elongating axons. 10.1523/ENEURO.0026-19.2019
    Cofilin 1 activation prevents the defects in axon elongation and guidance induced by extracellular alpha-synuclein. Tilve Sharada,Difato Francesco,Chieregatti Evelina Scientific reports Impaired adult neurogenesis and axon traumatic injury participate in the severity of neurodegenerative diseases. Alpha-synuclein, a cytosolic protein involved in Parkinson's disease, may be released from neurons, suggesting a role for excess secreted alpha-synuclein in the onset and spread of the pathology. Here we provide evidence that long term exposure of young neurons to extracellular alpha-synuclein hampers axon elongation and growth cone turning. We show that actin turnover and the rate of movement of actin waves along the axon are altered, due to alpha-synuclein-induced inactivation of cofilin. Upon laser disruption of microfilaments, healing of axons is favored by the increased phosphorylation of cofilin, however, at later time points; the defect in neurite extension prevails, being lost the regulation of cofilin activity. Importantly, overexpression of the active form of cofilin in neurons exposed to alpha-synuclein is able to restore the movement of actin waves, physiological axon elongation and growth cone turning. Our study reveals the molecular basis of alpha-synuclein-driven deficits in growth and migration of newborn neurons, and in elongation and regeneration of adult neurons. 10.1038/srep16524
    Dendrosomatic Sonic Hedgehog Signaling in Hippocampal Neurons Regulates Axon Elongation. Yao Pamela J,Petralia Ronald S,Ott Carolyn,Wang Ya-Xian,Lippincott-Schwartz Jennifer,Mattson Mark P The Journal of neuroscience : the official journal of the Society for Neuroscience UNLABELLED:The presence of Sonic Hedgehog (Shh) and its signaling components in the neurons of the hippocampus raises a question about what role the Shh signaling pathway may play in these neurons. We show here that activation of the Shh signaling pathway stimulates axon elongation in rat hippocampal neurons. This Shh-induced effect depends on the pathway transducer Smoothened (Smo) and the transcription factor Gli1. The axon itself does not respond directly to Shh; instead, the Shh signal transduction originates from the somatodendritic region of the neurons and occurs in neurons with and without detectable primary cilia. Upon Shh stimulation, Smo localization to dendrites increases significantly. Shh pathway activation results in increased levels of profilin1 (Pfn1), an actin-binding protein. Mutations in Pfn1's actin-binding sites or reduction of Pfn1 eliminate the Shh-induced axon elongation. These findings indicate that Shh can regulate axon growth, which may be critical for development of hippocampal neurons. SIGNIFICANCE STATEMENT:Although numerous signaling mechanisms have been identified that act directly on axons to regulate their outgrowth, it is not known whether signals transduced in dendrites may also affect axon outgrowth. We describe here a transcellular signaling pathway in embryonic hippocampal neurons in which activation of Sonic Hedgehog (Shh) receptors in dendrites stimulates axon growth. The pathway involves the dendritic-membrane-associated Shh signal transducer Smoothened (Smo) and the transcription factor Gli, which induces the expression of the gene encoding the actin-binding protein profilin 1. Our findings suggest scenarios in which stimulation of Shh in dendrites results in accelerated outgrowth of the axon, which therefore reaches its presumptive postsynaptic target cell more quickly. By this mechanism, Shh may play critical roles in the development of hippocampal neuronal circuits. 10.1523/JNEUROSCI.1360-15.2015
    siRNA-Mediated Knockdown of the mTOR Inhibitor RTP801 Promotes Retinal Ganglion Cell Survival and Axon Elongation by Direct and Indirect Mechanisms. Morgan-Warren Peter J,O'Neill Jenna,de Cogan Felicity,Spivak Igor,Ashush Hagit,Kalinski Hagar,Ahmed Zubair,Berry Martin,Feinstein Elena,Scott Robert A H,Logan Ann Investigative ophthalmology & visual science PURPOSE:To investigate, using in vivo and in vitro models, retinal ganglion cell (RGC) neuroprotective and axon regenerative effects and underlying mechanisms of siRTP801, a translatable small-interfering RNA (siRNA) targeting the mTOR negative regulator RTP801. METHODS:Adult rats underwent optic nerve (ON) crush (ONC) followed by intravitreal siRTP801 or control siRNA (siEGFP) every 8 days, with Brn3a+ RGC survival, GFAP+ reactive gliosis, and GAP43+ regenerating axons analyzed immunohistochemically 24 days after injury. Retinal cultures, prepared from uninjured animals or 5 days after ONC to activate retinal glia, were treated with siRTP801/controls in the presence/absence of rapamycin and subsequently assessed for RGC survival and neurite outgrowth, RTP801 expression, glial responses, and mTOR activity. Conditioned medium was analyzed for neurotrophin titers by ELISA. RESULTS:Intravitreal siRTP801 enabled 82% RGC survival compared to 45% with siEGFP 24 days after ONC, correlated with greater GAP43+ axon regeneration at 400 to 1200 μm beyond the ONC site, and potentiated the reactive GFAP+ Müller glial response. In culture, siRTP801 had a direct RGC neuroprotective effect, but required GFAP+ activated glia to stimulate neurite elongation. The siRTP801-induced neuroprotection was significantly reduced, but not abolished, by rapamycin. The siRTP801 potentiated the production and release of neurotrophins NGF, NT-3, and BDNF, and prevented downregulation of RGC mTOR activity. CONCLUSIONS:The RTP801 knockdown promoted RGC survival and axon elongation after ONC, without increasing de novo regenerative sprouting. The neuroprotection was predominantly direct, with mTORC1-dependent and -independent components. Enhanced neurite/axon elongation by siRTP801 required the presence of activated retinal glia and was mediated by potentiated secretion of neurotrophic factors. 10.1167/iovs.15-17511
    Rab35 Functions in Axon Elongation Are Regulated by P53-Related Protein Kinase in a Mechanism That Involves Rab35 Protein Degradation and the Microtubule-Associated Protein 1B. Villarroel-Campos David,Henríquez Daniel R,Bodaleo Felipe J,Oguchi Mai E,Bronfman Francisca C,Fukuda Mitsunori,Gonzalez-Billault Christian The Journal of neuroscience : the official journal of the Society for Neuroscience UNLABELLED:Rab35 is a key protein for cargo loading in the recycling endosome. In neuronal immortalized cells, Rab35 promotes neurite differentiation. Here we describe that Rab35 favors axon elongation in rat primary neurons in an activity-dependent manner. In addition, we show that the p53-related protein kinase (PRPK) negatively regulates axonal elongation by reducing Rab35 protein levels through the ubiquitin-proteasome degradation pathway. PRPK-induced Rab35 degradation is regulated by its interaction with microtubule-associated protein 1B (MAP1B), a microtubule stabilizing binding protein essential for axon elongation. Consistently, axon defects found in MAP1B knock-out neurons were reversed by Rab35 overexpression or PRPK inactivation suggesting an epistatic relationship among these proteins. These results define a novel mechanism to support axonal elongation, by which MAP1B prevents PRPK-induced Rab35 degradation. Such a mechanism allows Rab35-mediated axonal elongation and connects the regulation of actin dynamics with membrane trafficking. In addition, our study reveals for the first time that the ubiquitin-proteasome degradation pathway regulates a Rab GTPase. SIGNIFICANCE STATEMENT:Rab35 is required for axonal outgrowth. We define that its protein levels are negatively regulated by p53-related protein kinase (PRPK). We show that microtubule-associated protein 1B (MAP1B) interacts with PRPK, preventing PRPK-dependent Rab35 proteasome degradation. We demonstrate that Rab35 regulates Cdc42 activity in neurons. This is the first evidence showing that a Rab protein is regulated by degradation dependent on the ubiquitin-proteasome system. 10.1523/JNEUROSCI.4064-15.2016
    IGF-1 Induces GHRH Neuronal Axon Elongation during Early Postnatal Life in Mice. Decourtye Lyvianne,Mire Erik,Clemessy Maud,Heurtier Victor,Ledent Tatiana,Robinson Iain C,Mollard Patrice,Epelbaum Jacques,Meaney Michael J,Garel Sonia,Le Bouc Yves,Kappeler Laurent PloS one Nutrition during the perinatal period programs body growth. Growth hormone (GH) secretion from the pituitary regulates body growth and is controlled by Growth Hormone Releasing Hormone (GHRH) neurons located in the arcuate nucleus of the hypothalamus. We observed that dietary restriction during the early postnatal period (i.e. lactation) in mice influences postnatal growth by permanently altering the development of the somatotropic axis in the pituitary gland. This alteration may be due to a lack of GHRH signaling during this critical developmental period. Indeed, underfed pups showed decreased insulin-like growth factor I (IGF-I) plasma levels, which are associated with lower innervation of the median eminence by GHRH axons at 10 days of age relative to normally fed pups. IGF-I preferentially stimulated axon elongation of GHRH neurons in in vitro arcuate explant cultures from 7 day-old normally fed pups. This IGF-I stimulating effect was selective since other arcuate neurons visualized concomitantly by neurofilament labeling, or AgRP immunochemistry, did not significantly respond to IGF-I stimulation. Moreover, GHRH neurons in explants from age-matched underfed pups lost the capacity to respond to IGF-I stimulation. Molecular analyses indicated that nutritional restriction was associated with impaired activation of AKT. These results highlight a role for IGF-I in axon elongation that appears to be cell selective and participates in the complex cellular mechanisms that link underfeeding during the early postnatal period with programming of the growth trajectory. 10.1371/journal.pone.0170083
    Presenilin/γ-secretase-dependent EphA3 processing mediates axon elongation through non-muscle myosin IIA. Javier-Torrent Míriam,Marco Sergi,Rocandio Daniel,Pons-Vizcarra Maria,Janes Peter W,Lackmann Martin,Egea Joaquim,Saura Carlos A eLife EphA/ephrin signaling regulates axon growth and guidance of neurons, but whether this process occurs also independently of ephrins is unclear. We show that presenilin-1 (PS1)/γ-secretase is required for axon growth in the developing mouse brain. PS1/γ-secretase mediates axon growth by inhibiting RhoA signaling and cleaving EphA3 independently of ligand to generate an intracellular domain (ICD) fragment that reverses axon defects in PS1/γ-secretase- and EphA3-deficient hippocampal neurons. Proteomic analysis revealed that EphA3 ICD binds to non-muscle myosin IIA (NMIIA) and increases its phosphorylation (Ser1943), which promotes NMIIA filament disassembly and cytoskeleton rearrangement. PS1/γ-secretase-deficient neurons show decreased phosphorylated NMIIA and NMIIA/actin colocalization. Moreover, pharmacological NMII inhibition reverses axon retraction in PS-deficient neurons suggesting that NMIIA mediates PS/EphA3-dependent axon elongation. In conclusion, PS/γ-secretase-dependent EphA3 cleavage mediates axon growth by regulating filament assembly through RhoA signaling and NMIIA, suggesting opposite roles of EphA3 on inhibiting (ligand-dependent) and promoting (receptor processing) axon growth in developing neurons. 10.7554/eLife.43646
    JIP3 Activates Kinesin-1 Motility to Promote Axon Elongation. Watt Dana,Dixit Ram,Cavalli Valeria The Journal of biological chemistry Kinesin-1 is a molecular motor responsible for cargo transport along microtubules and plays critical roles in polarized cells, such as neurons. Kinesin-1 can function as a dimer of two kinesin heavy chains (KHC), which harbor the motor domain, or as a tetramer in combination with two accessory light chains (KLC). To ensure proper cargo distribution, kinesin-1 activity is precisely regulated. Both KLC and KHC subunits bind cargoes or regulatory proteins to engage the motor for movement along microtubules. We previously showed that the scaffolding protein JIP3 interacts directly with KHC in addition to its interaction with KLC and positively regulates dimeric KHC motility. Here we determined the stoichiometry of JIP3-KHC complexes and observed approximately four JIP3 molecules binding per KHC dimer. We then determined whether JIP3 activates tetrameric kinesin-1 motility. Using an in vitro motility assay, we show that JIP3 binding to KLC engages kinesin-1 with microtubules and that JIP3 binding to KHC promotes kinesin-1 motility along microtubules. We tested the in vivo relevance of these findings using axon elongation as a model for kinesin-1-dependent cellular function. We demonstrate that JIP3 binding to KHC, but not KLC, is essential for axon elongation in hippocampal neurons as well as axon regeneration in sensory neurons. These findings reveal that JIP3 regulation of kinesin-1 motility is critical for axon elongation and regeneration. 10.1074/jbc.M115.651885
    FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b and Calm1. Wang Bin,Pan Lin,Wei Manyi,Wang Qiong,Liu Wen-Wen,Wang Nuoxin,Jiang Xing-Yu,Zhang Xu,Bao Lan Cell reports Subcellular targeting and local translation of mRNAs are critical for axon development. However, the precise local control of mRNA translation requires investigation. We report that the Fmr1-encoded protein, FMRP-mediated axonal delivery of miR-181d negatively regulates axon elongation by locally targeting the transcripts of MAP1B (Map1b) and calmodulin (Calm1) in primary sensory neurons. miR-181d affected the local synthesis of MAP1B and calmodulin in axons. FMRP was associated with miR-181d, Map1b, and Calm1. Both FMRP deficiency in Fmr1(I304N) mice and Fmr1 knockdown impeded the axonal delivery of miR-181d, Map1b, and Calm1 and reduced the protein levels of MAP1B and calmodulin in axons. Furthermore, nerve growth factor (NGF) induced Map1b and Calm1 release from FMRP and miR-181d-repressing granules, thereby promoting axon elongation. Both miR-181d overexpression and FMRP knockdown impaired NGF-induced axon elongation. Our study reveals a mechanism for the local regulation of translation by miR-181d and FMRP during axon development. 10.1016/j.celrep.2015.11.057
    Mechanisms of Axon Elongation Following CNS Injury: What Is Happening at the Axon Tip? Rodemer William,Gallo Gianluca,Selzer Michael E Frontiers in cellular neuroscience After an injury to the central nervous system (CNS), functional recovery is limited by the inability of severed axons to regenerate and form functional connections with appropriate target neurons beyond the injury. Despite tremendous advances in our understanding of the mechanisms of axon growth, and of the inhibitory factors in the injured CNS that prevent it, disappointingly little progress has been made in restoring function to human patients with CNS injuries, such as spinal cord injury (SCI), through regenerative therapies. Clearly, the large number of overlapping neuron-intrinsic and -extrinsic growth-inhibitory factors attenuates the benefit of neutralizing any one target. More daunting is the distances human axons would have to regenerate to reach some threshold number of target neurons, e.g., those that occupy one complete spinal segment, compared to the distances required in most experimental models, such as mice and rats. However, the difficulties inherent in studying mechanisms of axon regeneration in the mature CNS have caused researchers to rely heavily on extrapolation from studies of axon regeneration in peripheral nerve, or of growth cone-mediated axon development and . Unfortunately, evidence from several animal models, including the transected lamprey spinal cord, has suggested important differences between regeneration of mature CNS axons and growth of axons in peripheral nerve, or during embryonic development. Specifically, long-distance regeneration of severed axons may not involve the actin-myosin molecular motors that guide embryonic growth cones in developing axons. Rather, non-growth cone-mediated axon elongation may be required to propel injured axons in the mature CNS. If so, it may be necessary to use other experimental models to promote regeneration that is sufficient to contact a critical number of target neurons distal to a CNS lesion. This review examines the cytoskeletal underpinnings of axon growth, focusing on the elongating axon tip, to gain insights into how CNS axons respond to injury, and how this might affect the development of regenerative therapies for SCI and other CNS injuries. 10.3389/fncel.2020.00177
    Simultaneous Knockdown of Sprouty2 and PTEN Promotes Axon Elongation of Adult Sensory Neurons. Jamsuwan Sataporn,Klimaschewski Lars,Hausott Barbara Frontiers in cellular neuroscience Sprouty2 (Spry2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are both well-established regulators of receptor tyrosine kinase (RTK) signaling, and knockdown of Spry2 or PTEN enhances axon regeneration of dorsal root ganglia (DRG) neurons. The major role of Spry2 is the inhibition of the rat sarcoma RAS/extracellular signal-regulated kinase (ERK) pathway, whereas PTEN acts mainly as an inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In non-neuronal cells, Spry2 increases the expression and activity of PTEN, and PTEN enhances the amount of Spry2 by the inhibition of the microRNA-21 (miR-21) that downregulates Spry2. Applying dissociated DRG neuron cultures from wild-type (WT) or Spry2 deficient mice, we demonstrate that PTEN protein was reduced after 72 h during rapid axonal outgrowth on the laminin substrate. Furthermore, PTEN protein was decreased in DRG cultures obtained from homozygous Spry2-/- knockout mice. Vice versa, Spry2 protein was reduced by PTEN siRNA in WT and heterozygous Spry2+/- neurons. Knockdown of PTEN in DRG cultures obtained from homozygous Spry2-/- knockout mice promoted axon elongation without increasing axonal branching. Activation of Akt, but not ERK, was stronger in response to PTEN knockdown in homozygous Spry2-/- DRG neurons than in WT neurons. Together, our study confirms the important role of the signaling modulators Spry2 and PTEN in axon growth of adult DRG neurons. Both function as endogenous inhibitors of neuronal growth factor signaling and their simultaneous knockdown promotes axon elongation more efficiently than the single knockdown of each inhibitor. Furthermore, Spry2 and PTEN are reciprocally downregulated in adult DRG neuron cultures. Axon growth is influenced by multiple factors and our results demonstrate that the endogenous inhibitors of axon growth, Spry2 and PTEN, are co-regulated in adult DRG neuron cultures. Together, our data demonstrate that combined approaches may be more useful to improve nerve regeneration than targeting one single inhibitor of axon growth. 10.3389/fncel.2019.00583
    DCLK1 phosphorylates the microtubule-associated protein MAP7D1 to promote axon elongation in cortical neurons. Koizumi Hiroyuki,Fujioka Hiromi,Togashi Kazuya,Thompson James,Yates John R,Gleeson Joseph G,Emoto Kazuo Developmental neurobiology Doublecortin-like kinase 1 (DCLK1) is a member of the neuronal microtubule-associated doublecortin (DCX) family and functions in multiple stages of neural development including radial migration and axon growth of cortical neurons. DCLK1 is suggested to play the roles in part through its protein kinase activity, yet the kinase substrates of DCLK1 remain largely unknown. Here we have identified MAP7D1 (microtubule-associated protein 7 domain containing 1) as a novel substrate of DCLK1 by using proteomic analysis. MAP7D1 is expressed in developing cortical neurons, and knockdown of MAP7D1 in layer 2/3 cortical neurons results in a significant impairment of callosal axon elongation, but not of radial migration, in corticogenesis. We have further defined the serine 315 (Ser 315) of MAP7D1 as a DCLK1-induced phosphorylation site and shown that overexpression of a phosphomimetic MAP7D1 mutant in which Ser 315 is substituted with glutamic acid (MAP7D1 S315E), but not wild-type MAP7D1, fully rescues the axon elongation defects in Dclk1 knockdown neurons. These data demonstrate that DCLK1 phosphorylates MAP7D1 on Ser 315 to facilitate axon elongation of cortical neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017. 10.1002/dneu.22428
    c-Jun NH2-terminal kinase (JNK)-interacting protein-3 (JIP3) regulates neuronal axon elongation in a kinesin- and JNK-dependent manner. Sun Tao,Yu Nuo,Zhai Lu-Kai,Li Na,Zhang Chao,Zhou Liang,Huang Zhuo,Jiang Xing-Yu,Shen Ying,Chen Zhe-Yu The Journal of biological chemistry The development of neuronal polarity is essential for the establishment of the accurate patterning of neuronal circuits in the brain. However, little is known about the underlying molecular mechanisms that control rapid axon elongation during neuronal development. Here, we report that c-Jun NH2-terminal kinase (JNK)-interacting protein-3 (JIP3) is highly expressed at axon tips during the critical period for axon development. Using gain- and loss-of-function approaches, immunofluorescence analysis, and in utero electroporation, we find that JIP3 can enhance axon elongation in primary hippocampal neurons and cortical neurons in vivo. We further demonstrate that JIP3 promotes axon elongation in a kinesin- and JNK-dependent manner using several deletion mutants of JIP3. Next, we demonstrate that the successful transportation of JIP3 to axon tips by kinesin is a prerequisite for enhancing JNK phosphorylation in this area and therefore promotes axon elongation, constituting a novel mechanism for coupling JIP3 anterograde transport with JNK signaling at the distal axons and axon elongation. Finally, our immunofluorescence data suggest that the activation of JNK at axon tips facilitates axon elongation by modulating cofilin activity and actin filament dynamics. These findings may have important implications for our understanding of neuronal axon elongation during development. 10.1074/jbc.M113.464453
    Graphene Promotes Axon Elongation through Local Stall of Nerve Growth Factor Signaling Endosomes. Convertino Domenica,Fabbri Filippo,Mishra Neeraj,Mainardi Marco,Cappello Valentina,Testa Giovanna,Capsoni Simona,Albertazzi Lorenzo,Luin Stefano,Marchetti Laura,Coletti Camilla Nano letters Several works reported increased differentiation of neuronal cells grown on graphene; however, the molecular mechanism driving axon elongation on this material has remained elusive. Here, we study the axonal transport of nerve growth factor (NGF), the neurotrophin supporting development of peripheral neurons, as a key player in the time course of axonal elongation of dorsal root ganglion neurons on graphene. We find that graphene drastically reduces the number of retrogradely transported NGF vesicles in favor of a stalled population in the first 2 days of culture, in which the boost of axon elongation is observed. This correlates with a mutual charge redistribution, observed via Raman spectroscopy and electrophysiological recordings. Furthermore, ultrastructural analysis indicates a reduced microtubule distance and an elongated axonal topology. Thus, both electrophysiological and structural effects can account for graphene action on neuron development. Unraveling the molecular players underneath this interplay may open new avenues for axon regeneration applications. 10.1021/acs.nanolett.0c00571
    MicroRNA-30b promotes axon outgrowth of retinal ganglion cells by inhibiting Semaphorin3A expression. Han F,Huo Y,Huang C-J,Chen C-L,Ye J Brain research Semaphorin3A (Sema3A) is a major inhibitory factor of optic nerve (ON) regeneration post-injury. Many microRNAs (miRNAs) are expressed specifically in the mammalian brain and retina and are dynamically regulated during development, suggesting that this group of miRNAs may be associated with neural development. We found that microRNA-30b (miR-30b) bound to the three prime untranslated region (3' UTR) of Sema3A and inhibited the expression of Sema3A mRNA. The mRNA expression level of miR-30b and the protein expression levels of Sema3A, Neuropilin1 (NRP1), PlexinA1 (PlexA1), phosphorylated p38MAPK (p-p38MAPK), and active caspase-3 were all upregulated in retinas from rats with a damaged ON relative to those with an intact ON. Transfection of cultured retinal ganglion cells (RGCs) with an miR-30b mimic led to decreased levels of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 protein expression, as well as axon elongation and reduced levels of apoptosis. These findings provide evidence that miR-30b inhibits Sema3A expression. Decreased Sema3A expression promotes axon outgrowth in RGCs due to reduced levels of Sema3A binding to NRP1 and PlexA1 and simultaneously reduces apoptosis by inhibiting the p38MAPK and caspase-3 pathways. Our findings provide the first evidence that miR-30b-mediated Sema3A downregulation may serve as a new strategy for the clinical treatment of ON injury. 10.1016/j.brainres.2015.03.014
    Neural Stem Cells Direct Axon Guidance via Their Radial Fiber Scaffold. Kaur Navjot,Han Wenqi,Li Zhuo,Madrigal M Pilar,Shim Sungbo,Pochareddy Sirisha,Gulden Forrest O,Li Mingfeng,Xu Xuming,Xing Xiaojun,Takeo Yutaka,Li Zhen,Lu Kangrong,Imamura Kawasawa Yuka,Ballester-Lurbe Begoña,Moreno-Bravo Juan Antonio,Chédotal Alain,Terrado José,Pérez-Roger Ignacio,Koleske Anthony J,Sestan Nenad Neuron Neural stem cells directly or indirectly generate all neurons and macroglial cells and guide migrating neurons by using a palisade-like scaffold made of their radial fibers. Here, we describe an unexpected role for the radial fiber scaffold in directing corticospinal and other axons at the junction between the striatum and globus pallidus. The maintenance of this scaffold, and consequently axon pathfinding, is dependent on the expression of an atypical RHO-GTPase, RND3/RHOE, together with its binding partner ARHGAP35/P190A, a RHO GTPase-activating protein, in the radial glia-like neural stem cells within the ventricular zone of the medial ganglionic eminence. This role is independent of RND3 and ARHGAP35 expression in corticospinal neurons, where they regulate dendritic spine formation, axon elongation, and pontine midline crossing in a FEZF2-dependent manner. The prevalence of neural stem cell scaffolds and their expression of RND3 and ARHGAP35 suggests that these observations might be broadly relevant for axon guidance and neural circuit formation. 10.1016/j.neuron.2020.06.035
    Nerve conduction and microanatomy in the rabbit sciatic nerve after gradual limb lengthening-distraction neurogenesis. Yokota Atsushi,Doi Munekazu,Ohtsuka Hisashi,Abe Muneaki Journal of orthopaedic research : official publication of the Orthopaedic Research Society To clarify how the peripheral nerve adapts to elongation during gradual limb lengthening, electrophysiological and histomorphometric examinations were performed on the sciatic nerves in 18 rabbits. External fixators were used to lengthen the right femora by 30 mm (30%), at a daily rate of 0.5 mm (Group 1) or 2.0 mm (Group 2). Examinations were performed immediately after the limb lengthening procedure. Electrophysiologically, mild conduction slowing was observed in Group 1; a conduction block was evident in Group 2. Histologically, the mean diameter of myelinated fibers was unchanged in Group 1, but a significantly decreased diameter was observed in Group 2. Electron microscopy revealed that mild degenerative change of unmyelinated axons occurred sporadically in two cases in Group 2, but neither group showed evidence of thinning of myelin sheath of myelinated fibers. The mean internodal length (between nodes of Ranvier) of teased fibers was 1216+/-295 microm in the control contralateral side, 1484+/-347 microm in Group 1, and 1467+/-322 microm in Group 2. Thus the internodes were lengthened by 22.1% (Group 1) and 20.7% (Group 2) in comparison with those of the controls. Straightening of the geometry of paranodal myelin sheath was significantly correlated with the rate of distraction. These results indicate that myelinated nerve fibers adapt to gradual elongation by lengthening each Schwann cell body, not by proliferation of Schwann cells. 10.1016/S0736-0266(02)00100-6
    [New treatment for peripheral nerve defects: nerve elongation]. Kou Y H,Jiang B G Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences Peripheral nerve defects are still a major challenge in clinical practice, and the most commonly used method of treatment for peripheral nerve defects is nerve transplantation, which has certain limitations and shortcomings, so new repair methods and techniques are needed. The peripheral nerve is elongated in limb lengthening surgery without injury, from which we got inspirations and proposed a new method to repair peripheral nerve defects: peripheral nerve elongation. The peripheral nerve could beelongated by a certain percent, but the physiological change and the maximum elongation range were still unknown. This study discussed the endurance, the physiological and pathological change of peripheral nerve elongation in detail, and got a lot of useful data. First, we developed peripheral nerve extender which could match the slow and even extension of peripheral nerve. Then, our animal experiment result confirmed that the peripheral nerve had better endurance for chronic elongation than that of acute elongation and cleared the extensibility of peripheral nerve and the range of repair for peripheral nerve defects. Our result also revealed the histological basis and changed the rule for pathological physiology of peripheral nerve elongation: the most important structure foundation of peripheral nerve elongation was Fontana band, which was the coiling of nerve fibers under the epineurium, so peripheral nerve could be stretched for 8.5%-10.0% without injury because of the Fontana band. We confirmed that peripheral nerve extending technology could have the same repair effect as traditional nerve transplantation through animal experiments. Finally, we compared the clinical outcomes between nerve elongation and performance of the conventional method in the repair of short-distance transection injuries in human elbows, and the post-operative follow-up results demonstrated that early neurological function recovery was better in the nerve elongation group than in the conventional group. On the whole, all of these experimental results revealed the physiological phenomenon of peripheral nerve elongation, and described the physiological change and stretch range in detail. The systematic research results have filled the blank in this field, which is very helpful for clinical limb lengthening surgery, the design of elongation surgery and the evaluation of the peripheral nerve stretch injury. Peripheral nerve elongation will become an innovative treatment technology in repairing peripheral nerve defects.
    Role of sodium channels in recovery of sciatic nerve-stretch injury in rats. Hirofuji Shinji,Yokota Atsushi,Ohno Katsunori,Kinoshita Mitsuo,Neo Masashi Muscle & nerve INTRODUCTION:To elucidate the mechanism of functional recovery after gradual nerve-stretch injury, we used rats in which the femur length was increased by 15 mm at 1.5 mm/day. METHODS:We performed electrophysiology, mRNA analysis of tetrodotoxin-resistant voltage-gated sodium channels (TTX-R VGSCs) in dorsal root ganglia, and histology of unmyelinated sciatic nerve fibers and examined pain thresholds at 1, 10, 20, and 30 days after cessation of lengthening. RESULTS:Electrophysiology revealed conduction block after cessation that recovered after 30 days. TTX-R VGSC levels decreased immediately after cessation but were restored after 10 (Nav1.9) or 20 (Nav1.8) days. Histology revealed that injured unmyelinated nerve fibers regenerate 30 days after cessation. Pain threshold decreased gradually during lengthening but had not recovered to the control group level after 30 days. CONCLUSIONS:Early restoration of TTX-R VGSC mRNA in dorsal root ganglia preceded functional recovery of stretched nerves before regeneration of injured unmyelinated nerve fibers. 10.1002/mus.24172
    A novel method of lengthening the accessory nerve for direct coaptation during nerve repair and nerve transfer procedures. Tubbs R Shane,Maldonado Andrés A,Stoves Yolanda,Fries Fabian N,Li Rong,Loukas Marios,Oskouian Rod J,Spinner Robert J Journal of neurosurgery OBJECTIVE The accessory nerve is frequently repaired or used for nerve transfer. The length of accessory nerve available is often insufficient or marginal (under tension) for allowing direct coaptation during nerve repair or nerve transfer (neurotization), necessitating an interpositional graft. An attractive maneuver would facilitate lengthening of the accessory nerve for direct coaptation. The aim of the present study was to identify an anatomical method for such lengthening. METHODS In 20 adult cadavers, the C-2 or C-3 connections to the accessory nerve were identified medial to the sternocleidomastoid (SCM) muscle and the anatomy of the accessory nerve/cervical nerve fibers within the SCM was documented. The cervical nerve connections were cut. Lengths of the accessory nerve were measured. Samples of the cut C-2 and C-3 nerves were examined using immunohistochemistry. RESULTS The anatomy and adjacent neural connections within the SCM are complicated. However, after the accessory nerve was "detethered" from within the SCM and following transection, the additional length of the accessory nerve increased from a mean of 6 cm to a mean of 10.5 cm (increase of 4.5 cm) after cutting the C-2 connections, and from a mean of 6 cm to a mean length of 9 cm (increase of 3.5 cm) after cutting the C-3 connections. The additional length of accessory nerve even allowed direct repair of an infraclavicular target (i.e., the proximal musculocutaneous nerve). The cervical nerve connections were shown not to contain motor fibers. CONCLUSIONS An additional length of the accessory nerve made available in the posterior cervical triangle can facilitate direct repair or neurotization procedures, thus eliminating the need for an interpositional nerve graft, decreasing the time/distance for regeneration and potentially improving clinical outcomes. 10.3171/2016.10.JNS161106
    Simultaneous gradual lengthening of both proximal and distal nerve stumps for repair of peripheral nerve defect in rats. Saijilafu ,Nishiura Yasumasa,Hara Yuki,Yoshii Yuichi,Ochiai Naoyuki Muscle & nerve We investigated nerve regeneration following the repair of a segmental nerve defect induced by direct end-to-end neurorrhaphy after simultaneous gradual lengthening of both proximal and distal nerve stumps in rats. A 15-mm-long nerve segment was resected from the sciatic nerve of each rat. The proximal and distal nerve stumps, respectively, were directly lengthened at a rate of 1 mm/day using a custom-made external nerve-lengthening device. After being lengthened for 14 days, both nerve stumps were refreshed, and direct end-to-end neurorrhaphy was performed. For a control, 15-mm nerve grafting was performed immediately after nerve resection. Nerve regeneration was evaluated by motor nerve conduction velocity, muscle contraction force, and histological studies at 6, 8, and 14 weeks after initial nerve resection in both groups. As a result, at 8 and 14 weeks, the motor nerve conduction velocity was significantly higher in the nerve-lengthening group than in the autografting group. In addition, at 14 weeks, the tetanic force and wet weight of the gastrocnemius muscle were significantly higher in the nerve-lengthening group than in the autografting group. Histologically, the mean axonal diameter of myelinated nerve fibers and the total number of myelinated nerve fibers were also significantly higher in the nerve-lengthening group than in the autografting group for each evaluation period. It appears that the simultaneous gradual lengthening of both proximal and distal nerve stumps might have potential application in the repair of peripheral nerve defects. 10.1002/mus.21147