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Proceedings of the National Academy of... Feb 2017Although many aspects of optic pathway development are beginning to be understood, the mechanisms promoting the growth of retinal ganglion cell (RGC) axons toward visual...
Although many aspects of optic pathway development are beginning to be understood, the mechanisms promoting the growth of retinal ganglion cell (RGC) axons toward visual targets remain largely unknown. Down syndrome cell adhesion molecule () is expressed by mouse RGCs shortly after they differentiate at embryonic day 12 and is essential for multiple aspects of postnatal visual system development. Here we show that is also required during embryonic development for the fasciculation and growth of RGC axons. is expressed along the developing optic pathway in a pattern consistent with a role in regulating RGC axon outgrowth. In mice carrying spontaneous mutations in ( ; , RGC axons pathfind normally, but growth from the chiasm toward their targets is impaired, resulting in a delay in RGC axons reaching the dorsal thalamus compared with that seen in wild-type littermates. Conversely, gain of function results in exuberant growth into the dorsal thalamus. The growth of ipsilaterally projecting axons is particularly affected. Axon organization in the optic chiasm and tract and RGC growth cone morphologies are also altered in mutants. In vitro DSCAM promotes RGC axon growth and fasciculation, and can act independently of cell contact. In vitro and in situ DSCAM is required both in the RGC axons and in their environment for the promotion of axon outgrowth, consistent with a homotypic mode of action. These findings identify DSCAM as a permissive signal that promotes the growth and fasciculation of RGC axons, controlling the timing of when RGC axons reach their targets.
Topics: Animals; Axon Fasciculation; Axons; COS Cells; Cell Adhesion Molecules; Chlorocebus aethiops; Gene Expression Regulation, Developmental; Growth Cones; HEK293 Cells; Humans; In Situ Hybridization; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mutation; Optic Chiasm; Retina; Retinal Ganglion Cells; Visual Pathways
PubMed: 28137836
DOI: 10.1073/pnas.1618606114 -
Genetics Nov 2016The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made... (Review)
Review
The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment.
Topics: Actin Cytoskeleton; Animals; Axon Guidance; Caenorhabditis elegans; Nerve Regeneration
PubMed: 28114100
DOI: 10.1534/genetics.115.186262 -
Nagoya Journal of Medical Science Aug 2016Nogo receptor (NgR) is common in myelin-derived molecules, i.e., Nogo, MAG, and OMgp, and plays important roles in both axon fasciculation and the inhibition of axonal...
Nogo receptor (NgR) is common in myelin-derived molecules, i.e., Nogo, MAG, and OMgp, and plays important roles in both axon fasciculation and the inhibition of axonal regeneration. In contrast to NgR's roles in neurons, its roles in glial cells have been poorly explored. Here, we found a dynamic regulation of NgR1 expression during development and neuronal injury. NgR1 mRNA was consistently expressed in the brain from embryonic day 18 to postnatal day 25. In contrast, its expression significantly decreased in the spinal cord during development. Primary cultured neurons, microglia, and astrocytes expressed NgR1. Interestingly, a contusion injury in the spinal cord led to elevated NgR1 mRNA expression at the injury site, but not in the motor cortex, 14 days after injury. Consistent with this, astrocyte activation by TGFβ1 increased NgR1 expression, while microglia activation rather decreased NgR1 expression. These results collectively suggest that NgR1 expression is enhanced in a milieu of neural injury. Our findings may provide insight into the roles of NgR1 in glial cells.
Topics: Animals; Cells, Cultured; Neuroglia; Neurons; Nogo Receptor 1; Rats; Rats, Sprague-Dawley
PubMed: 27578914
DOI: No ID Found -
Journal of Cell Science Sep 2016Correct innervation of the main respiratory muscle in mammals, namely the thoracic diaphragm, is a crucial pre-requisite for the functionality of this muscle and the...
Correct innervation of the main respiratory muscle in mammals, namely the thoracic diaphragm, is a crucial pre-requisite for the functionality of this muscle and the viability of the entire organism. Systemic impairment of Sema3A-Npn-1 (Npn-1 is also known as NRP1) signaling causes excessive branching of phrenic nerves in the diaphragm and into the central tendon region, where the majority of misguided axons innervate ectopic musculature. To elucidate whether these ectopic muscles are a result of misguidance of myoblast precursors due to the loss of Sema3A-Npn-1 signaling, we conditionally ablated Npn-1 in somatic motor neurons, which led to a similar phenotype of phrenic nerve defasciculation and, intriguingly, also formation of innervated ectopic muscles. We therefore hypothesize that ectopic myocyte fusion is caused by additional factors released by misprojecting growth cones. Slit2 and its Robo receptors are expressed by phrenic motor axons and migrating myoblasts, respectively, during innervation of the diaphragm. In vitro analyses revealed a chemoattractant effect of Slit2 on primary diaphragm myoblasts. Thus, we postulate that factors released by motor neuron growth cones have an influence on the migration properties of myoblasts during establishment of the diaphragm.
Topics: Animals; Axon Fasciculation; Diaphragm; Embryo, Mammalian; Intercellular Signaling Peptides and Proteins; Mice; Motor Neurons; Muscle Development; Myoblasts; Nerve Tissue Proteins; Neuropilin-1; Phrenic Nerve; Receptors, Immunologic; Semaphorin-3A; Signal Transduction; Stem Cells; Tendons; Roundabout Proteins
PubMed: 27466379
DOI: 10.1242/jcs.186015 -
The Journal of Neuroscience : the... Jun 2016Voltage-gated sodium channel (VGSC) β subunits signal through multiple pathways on multiple time scales. In addition to modulating sodium and potassium currents, β...
UNLABELLED
Voltage-gated sodium channel (VGSC) β subunits signal through multiple pathways on multiple time scales. In addition to modulating sodium and potassium currents, β subunits play nonconducting roles as cell adhesion molecules, which allow them to function in cell-cell communication, neuronal migration, neurite outgrowth, neuronal pathfinding, and axonal fasciculation. Mutations in SCN1B, encoding VGSC β1 and β1B, are associated with epilepsy. Autosomal-dominant SCN1B-C121W, the first epilepsy-associated VGSC mutation identified, results in genetic epilepsy with febrile seizures plus (GEFS+). This mutation has been shown to disrupt both the sodium-current-modulatory and cell-adhesive functions of β1 subunits expressed in heterologous systems. The goal of this study was to compare mice heterozygous for Scn1b-C121W (Scn1b(+/W)) with mice heterozygous for the Scn1b-null allele (Scn1b(+/-)) to determine whether the C121W mutation results in loss-of-function in vivo We found that Scn1b(+/W) mice were more susceptible than Scn1b(+/-) and Scn1b(+/+) mice to hyperthermia-induced convulsions, a model of pediatric febrile seizures. β1-C121W subunits are expressed at the neuronal cell surface in vivo However, despite this, β1-C121W polypeptides are incompletely glycosylated and do not associate with VGSC α subunits in the brain. β1-C121W subcellular localization is restricted to neuronal cell bodies and is not detected at axon initial segments in the cortex or cerebellum or at optic nerve nodes of Ranvier of Scn1b(W/W) mice. These data, together with our previous results showing that β1-C121W cannot participate in trans-homophilic cell adhesion, lead to the hypothesis that SCN1B-C121W confers a deleterious gain-of-function in human GEFS+ patients.
SIGNIFICANCE STATEMENT
The mechanisms underlying genetic epilepsy syndromes are poorly understood. Closing this gap in knowledge is essential to the development of new medicines to treat epilepsy. We have used mouse models to understand the mechanism of a mutation in the sodium channel gene SCN1B linked to genetic epilepsy with febrile seizures plus. We report that sodium channel β1 subunit proteins encoded by this mutant gene are expressed at the surface of neuronal cell bodies; however, they do not associate with the ion channel complex nor are they transported to areas of the axon that are critical for proper neuronal firing. We conclude that this disease-causing mutation is not simply a loss-of-function, but instead results in a deleterious gain-of-function in the brain.
Topics: Animals; Animals, Newborn; Biotinylation; Cells, Cultured; Cerebral Cortex; Cysteine; Disease Models, Animal; Epilepsy; Fever; Gene Expression Regulation, Developmental; Immunoprecipitation; Mice; Mice, Transgenic; Neurons; Polymorphism, Single Nucleotide; Statistics, Nonparametric; Tryptophan; Voltage-Gated Sodium Channel beta-1 Subunit
PubMed: 27277800
DOI: 10.1523/JNEUROSCI.0405-16.2016 -
Journal of Neural Engineering Feb 2016Connectome disruption is a hallmark of many neurological diseases and trauma with no current strategies to restore lost long-distance axonal pathways in the brain. We...
OBJECTIVE
Connectome disruption is a hallmark of many neurological diseases and trauma with no current strategies to restore lost long-distance axonal pathways in the brain. We are creating transplantable micro-tissue engineered neural networks (micro-TENNs), which are preformed constructs consisting of embedded neurons and long axonal tracts to integrate with the nervous system to physically reconstitute lost axonal pathways.
APPROACH
We advanced micro-tissue engineering techniques to generate micro-TENNs consisting of discrete populations of mature primary cerebral cortical neurons spanned by long axonal fascicles encased in miniature hydrogel micro-columns. Further, we improved the biomaterial encasement scheme by adding a thin layer of low viscosity carboxymethylcellulose (CMC) to enable needle-less insertion and rapid softening for mechanical similarity with brain tissue.
MAIN RESULTS
The engineered architecture of cortical micro-TENNs facilitated robust neuronal viability and axonal cytoarchitecture to at least 22 days in vitro. Micro-TENNs displayed discrete neuronal populations spanned by long axonal fasciculation throughout the core, thus mimicking the general systems-level anatomy of gray matter-white matter in the brain. Additionally, micro-columns with thin CMC-coating upon mild dehydration were able to withstand a force of 893 ± 457 mN before buckling, whereas a solid agarose cylinder of similar dimensions was predicted to withstand less than 150 μN of force. This thin CMC coating increased the stiffness by three orders of magnitude, enabling needle-less insertion into brain while significantly reducing the footprint of previous needle-based delivery methods to minimize insertion trauma.
SIGNIFICANCE
Our novel micro-TENNs are the first strategy designed for minimally invasive implantation to facilitate nervous system repair by simultaneously providing neuronal replacement and physical reconstruction of long-distance axon pathways in the brain. The micro-TENN approach may offer the ability to treat several disorders that disrupt the connectome, including Parkinson's disease, traumatic brain injury, stroke, and brain tumor excision.
Topics: Animals; Biocompatible Materials; Brain; Cells, Cultured; Equipment Design; Equipment Failure Analysis; Guided Tissue Regeneration; Materials Testing; Miniaturization; Nerve Net; Neurons; Rats; Rats, Sprague-Dawley; Tissue Engineering; Tissue Scaffolds
PubMed: 26760138
DOI: 10.1088/1741-2560/13/1/016019 -
The Journal of Neuroscience : the... Mar 2015Retina ganglion cell (RGC) axons grow along a stereotyped pathway undergoing coordinated rounds of fasciculation and defasciculation, which are critical to establishing...
Retina ganglion cell (RGC) axons grow along a stereotyped pathway undergoing coordinated rounds of fasciculation and defasciculation, which are critical to establishing proper eye-brain connections. How this coordination is achieved is poorly understood, but shedding of guidance cues by metalloproteinases is emerging as a relevant mechanism. Secreted Frizzled Related Proteins (Sfrps) are multifunctional proteins, which, among others, reorient RGC growth cones by regulating intracellular second messengers, and interact with Tolloid and ADAM metalloproteinases, thereby repressing their activity. Here, we show that the combination of these two functions well explain the axon guidance phenotype observed in Sfrp1 and Sfrp2 single and compound mouse mutant embryos, in which RGC axons make subtle but significant mistakes during their intraretinal growth and inappropriately defasciculate along their pathway. The distribution of Sfrp1 and Sfrp2 in the eye is consistent with the idea that Sfrp1/2 normally constrain axon growth into the fiber layer and the optic disc. Disheveled axon growth instead seems linked to Sfrp-mediated modulation of metalloproteinase activity. Indeed, retinal explants from embryos with different Sfrp-null alleles or explants overexpressing ADAM10 extend axons with a disheveled appearance, which is reverted by the addition of Sfrp1 or an ADAM10-specific inhibitor. This mode of growth is associated with an abnormal proteolytic processing of L1 and N-cadherin, two ADAM10 substrates previously implicated in axon guidance. We thus propose that Sfrps contribute to coordinate visual axon growth with a dual mechanism: by directly signaling at the growth cone and by regulating the processing of other relevant cues.
Topics: Animals; Axons; Female; Frizzled Receptors; Intercellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Retinal Ganglion Cells; Visual Pathways
PubMed: 25788689
DOI: 10.1523/JNEUROSCI.3304-13.2015 -
Developmental Biology Feb 2015Sensory trigeminal growth cones innervate the cornea in a coordinated fashion during embryonic development. Polysialic acid (polySia) is known for its important roles...
Sensory trigeminal growth cones innervate the cornea in a coordinated fashion during embryonic development. Polysialic acid (polySia) is known for its important roles during nerve development and regeneration. The purpose of this work is to determine whether polySia, present in developing eyefronts and on the surface of sensory nerves, may provide guidance cues to nerves during corneal innervation. Expression and localization of polySia in embryonic day (E)5-14 chick eyefronts and E9 trigeminal ganglia were identified using Western blotting and immunostaining. Effects of polySia removal on trigeminal nerve growth behavior were determined in vivo, using exogenous endoneuraminidase (endoN) treatments to remove polySia substrates during chick cornea development, and in vitro, using neuronal explant cultures. PolySia substrates, made by the physical adsorption of colominic acid to a surface coated with poly-d-lysine (PDL), were used as a model to investigate functions of the polySia expressed in axonal environments. PolySia was localized within developing eyefronts and on trigeminal sensory nerves. Distributions of PolySia in corneas and pericorneal regions are developmentally regulated. PolySia removal caused defasciculation of the limbal nerve trunk in vivo from E7 to E10. Removal of polySia on trigeminal neurites inhibited neurite outgrowth and caused axon defasciculation, but did not affect Neural Cell Adhesion Molecule (NCAM) expression or Schwann cell migration in vitro. PolySia substrates in vitro inhibited outgrowth of trigeminal neurites and promoted their fasciculation. In conclusion, polySia is localized on corneal nerves and in their targeting environment during early developing stages of chick embryos. PolySias promote fasciculation of trigeminal axons in vivo and in vitro, whereas, in contrast, their removal promotes defasciculation.
Topics: Animals; Axons; Cell Movement; Cell Survival; Chick Embryo; Cornea; Embryonic Development; Fasciculation; Laminin; Neural Cell Adhesion Molecules; Neurites; Schwann Cells; Sensation; Sialic Acids; Trigeminal Nerve
PubMed: 25478909
DOI: 10.1016/j.ydbio.2014.11.020 -
Genes To Cells : Devoted To Molecular &... Jan 2015During central nervous system development, several guidance cues and receptors, as well as cell adhesion molecules, are required for guiding axons across the midline and...
During central nervous system development, several guidance cues and receptors, as well as cell adhesion molecules, are required for guiding axons across the midline and along the anterior-posterior axis. In Drosophila, commissural axons sense the midline attractants Netrin A and B (Net) through Frazzled (Fra) receptors. Despite their importance, lack of Net or fra affects only some commissures, suggesting that additional molecules can fulfill this function. Recently, planar cell polarity (PCP) proteins have been implicated in midline axon guidance in both vertebrate and invertebrate systems. Here, we report that the atypical cadherin and PCP molecule Flamingo/Starry night (Fmi/Stan) acts jointly with Net/Fra signaling during midline development. Additional removal of fmi strongly increases the guidance defects in Net/fra mutants. Rescue and domain deletion experiments suggest that Fmi signaling facilitates commissural pathfinding potentially by mediating axonal fasciculation in a partly homophilic manner. Altogether, our results indicate that contact-mediated cell adhesion via Fmi acts in addition to the Net/Fra guidance system during axon pathfinding across the midline, underlining the importance of PCP molecules during vertebrates and invertebrates midline development.
Topics: Animals; Axons; Cadherins; Cell Adhesion; Cell Communication; Central Nervous System; Drosophila; Drosophila Proteins; Mutation; Nerve Growth Factors; Nerve Tissue Proteins; Netrin Receptors; Netrin-1; Netrins; Neuroglia; Neurons; Phenotype; Protein-Tyrosine Kinases; Receptors, Cell Surface; Signal Transduction; Tumor Suppressor Proteins; rac GTP-Binding Proteins
PubMed: 25440577
DOI: 10.1111/gtc.12202 -
Scientific Reports Nov 2014The accuracy of axonal pathfinding and the formation of functional neural circuitry are crucial for an organism to process, store, and retrieve information from internal...
The accuracy of axonal pathfinding and the formation of functional neural circuitry are crucial for an organism to process, store, and retrieve information from internal networks as well as from the environment. Aberrations in axonal migration is believed to lead to loop formation and self-fasciculation, which can lead to highly dysfunctional neural circuitry and therefore self-avoidance of axons is proposed to be the regulatory mechanism for control of synaptogenesis. Here, we report the application of a newly developed non-contact optical method using a weakly-focused, near infrared laser beam for highly efficient axonal guidance, and demonstrate the formation of axonal loops in cortical neurons, which demonstrate that cortical neurons can self-fasciculate in contrast to self-avoidance. The ability of light for axonal nano-loop formation opens up new avenues for the construction of complex neural circuitry, and non-invasive guidance of neurons at long working distances for restoration of impaired neural connections and functions.
Topics: Animals; Axons; Cerebral Cortex; Embryo, Mammalian; Fasciculation; Infrared Rays; Kinetics; Lasers; Nerve Net; Neurogenesis; Photic Stimulation; Photons; Primary Cell Culture; Rats
PubMed: 25376602
DOI: 10.1038/srep06902