Local substitution of GDF-15 improves axonal and sensory recovery after peripheral nerve injury

The growth/differentiation factor-15, GDF-15, has been found to be secreted by Schwann cells in the lesioned peripheral nervous system. To investigate whether GDF-15 plays a role in peripheral nerve regeneration, we substituted exogenous GDF-15 into 10-mm sciatic nerve gaps in adult rats and compared functional and morphological regeneration to a vehicle control group. Over a period of 11?weeks, multiple functional assessments, including evaluation of pinch reflexes, the Static Sciatic Index and of electrophysiological parameters, were performed. Regenerated nerves were then morphometrically analyzed for the number and quality of regenerated myelinated axons. Substitution of GDF-15 significantly accelerated sensory recovery while the effects on motor recovery were less strong. Although the number of regenerated myelinated axons was significantly reduced after GDF-15 treatment, the regenerated axons displayed advanced maturation corroborating the results of the functional assessments. Our results suggest that GDF-15 is involved in the complex orchestration of peripheral nerve regeneration after lesion.     

The intrinsic axon regenerative properties of mature neurons after injury

Thousands of nerve injuries occur in the world each year.Axon regeneration is a very critical process for the restoration of the injured nervous system’s function.However,the precise molecular mechanism or signaling cascades that control axon regeneration are not clearly understood,especially in mammals.Therefore,there is almost no ideal treatment method to repair the nervous system’s injury until now.Mammalian axonal regeneration requires multiple signaling pathways to coordinately regulate gene expression in soma and assembly of the cytoskeleton protein in the growth cone.A better understanding of their molecular mechanisms,such as axon regeneration regulatory signaling cascades,will be helpful in developing new treatment strategies for promoting axon regeneration.In this review,we mainly focus on describing these regeneration-associated signaling cascades,which regulate axon regeneration.     

Robo2 determines subtype-specific axonal projections of trigeminal sensory neurons

How neurons connect to form functional circuits is central to the understanding of the development and function of the nervous system. In the somatosensory system, perception of sensory stimuli to the head requires specific connections between trigeminal sensory neurons and their many target areas in the central nervous system. Different trigeminal subtypes have specialized functions and downstream circuits, but it has remained unclear how subtype-specific axonal projection patterns are formed. Using zebrafish as a model system, we followed the development of two trigeminal sensory neuron subtypes: one that expresses trpa1b, a nociceptive channel important for sensing environmental chemicals; and a distinct subtype labeled by an islet1 reporter (Isl1SS). We found that Trpa1b and Isl1SS neurons have overall similar axon trajectories but different branching morphologies and distributions of presynaptic sites. Compared with Trpa1b neurons, Isl1SS neurons display reduced branch growth and synaptogenesis at the hindbrain-spinal cord junction. The subtype-specific morphogenesis of Isl1SS neurons depends on the guidance receptor Robo2. robo2 is preferentially expressed in the Isl1SS subset and inhibits branch growth and synaptogenesis. In the absence of Robo2, Isl1SS afferents acquire many of the characteristics of Trpa1b afferents. These results reveal that subtype-specific activity of Robo2 regulates subcircuit morphogenesis in the trigeminal sensory system.     

Acute exposure to high‐induction electromagnetic field affects activity of model peripheral sensory neurons

Exposure to repetitive low‐frequency electromagnetic field (LF‐EMF) shows promise as a non‐invasive approach to treat various sensory and neurological disorders. Despite considerable progress in the development of modern stimulation devices, there is a limited understanding of the mechanisms underlying their biological effects and potential targets at the cellular level. A significant impact of electromagnetic field on voltage‐gated calcium channels and downstream signalling pathways has been convincingly demonstrated in many distinct cell types. However, evidence for clear effects on primary sensory neurons that particularly may be responsible for the analgesic actions of LF‐EMF is still lacking. Here, we used F11 cells derived from dorsal root ganglia neurons as an in vitro model of peripheral sensory neurons and three different protocols of high‐induction magnetic stimulation to determine the effects on chemical responsiveness and spontaneous activity. We show that short‐term (<180 sec.) exposure of F11 cells to LF‐EMF reduces calcium transients in response to bradykinin, a potent pain‐producing inflammatory agent formed at sites of injury. Moreover, we characterize an immediate and reversible potentiating effect of LF‐EMF on neuronal spontaneous activity. Our results provide new evidence that electromagnetic field may directly modulate the activity of sensory neurons and highlight the potential of sensory neuron‐derived cell line as a tool for studying the underlying mechanisms at the cellular and molecular level.     

Impaired regenerative response of primary sensory neurons in ZPK/DLK gene-trap mice

Rapid and persistent activation of c-JUN is necessary for axonal regeneration after nerve injury, although upstream molecular events leading to c-JUN activation remain largely unknown. ZPK/DLK/MAP3K12 activates the c-Jun N-terminal kinase pathway at an apical level. We investigated axonal regeneration of the dorsal root ganglion (DRG) neurons of homozygous ZPK/DLK gene-trap mice. In vitro neurite extension assays using DRG explants from 14 day-old mice revealed that neurite growth rates of the ZPK/DLK gene-trap DRG explants were reduced compared to those of the wild-type DRG explants. Three ZPK/DLK gene-trap mice which survived into adulthood were subjected to sciatic nerve axotomy. At 24 h after axotomy, phosphorylated c-JUN-positive DRG neurons were significantly less frequent in ZPK/DLK gene-trap mice than in wild-type mice. These results indicate that ZPK/DLK is involved in regenerative responses of mammalian DRG neurons to axonal injury through activation of c-JUN.     

Nocistatin sensitizes TRPA1 channels in peripheral sensory neurons

The ability of sensory neurons to detect potentially harmful stimuli relies on specialized molecular signal detectors such as transient receptor potential (TRP) A1 ion channels. TRPA1 is critically implicated in vertebrate nociception and different pain states. Furthermore, TRPA1 channels are subject to extensive modulation and regulation - processes which consequently affect nociceptive signaling. Here we show that the neuropeptide Nocistatin sensitizes TRPA1-dependent calcium influx upon application of the TRPA1 agonist mustard oil (MO) in cultured sensory neurons of dorsal root ganglia (DRG). Interestingly, TRPV1-mediated cellular calcium responses are unaffected by Nocistatin. Furthermore, Nocistatin-induced TRPA1-sensitization is likely independent of the Nocistatin binding partner 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) as assessed by siRNA-mediated knockdown in DRG cultures. In conclusion, we uncovered the sensitization of TRPA1 by Nocistatin, which may represent a novel mechanism how Nocistatin can modulate pain.     

Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system

The vomeronasal system mediates pheromonal effects in mammals. We have employed gene targeting technology to introduce mutations in a putative pheromone receptor gene, VR2, in the germline of mice. By generating alleles differentially tagged with the histological markers taulacZ and tauGFP, we show that VR2 is monoallelically expressed in a given neuron. Axons of VR2-expressing neurons converge onto numerous glomeruli in the accessory olfactory bulb. The pattern of axonal projections is complex and variable. This wiring diagram is substantially different from that of the main olfactory system. The projection pattern is disrupted by deleting the coding region of VR2, but an unrelated seven-transmembrane protein, the odorant receptor M71, can partially substitute for VR2.     

Early peripheral sensory neurons in the development of trochozoan animals

Neuronal development of the majority of trochozoan animals with biphasic pelago-bentic life cycle starts from transient peripheral neurons, which do not belong to the central nervous system and are mainly located in the apical sensory organ and in the hyposphere. Some of these neurons are pioneer and send neurites that form a scaffold upon which the adult central nervous system later develops. In representative species of molluscs and polychaetes, immunolabelling with the antibodies against neurotransmitters serotonin and FMRFamide, and acetylated α-tubulin revealed that the structure of almost all early peripheral neurons is typical for sensory, most probably chemosensory cells: flask shape, and cilia at the end of the apical dendrite or inside the distal ampoule. Morphology, transmitter specificity, location and projections of the early sensory cells differ in trochophores of different species thus suggesting different origin of these cells. In polychaete larvae, pharmacological inhibition of serotonin synthesis in early peripheral neurons did not affect the development, whereas its increase resulted in developmental arrest and neural malformations, suggesting that early peripheral sensory neurons are involved in developmental regulation.     

BPAG1n4 is essential for retrograde axonal transport in sensory neurons

Disruption of the BPAG1 (bullous pemphigoid antigen 1) gene results in progressive deterioration in motor function and devastating sensory neurodegeneration in the null mice. We have previously demonstrated that BPAG1n1 and BPAG1n3 play important roles in organizing cytoskeletal networks in vivo. Here, we characterize functions of a novel BPAG1 neuronal isoform, BPAG1n4. Results obtained from yeast two-hybrid screening, blot overlay binding assays, and coimmunoprecipitations demonstrate that BPAG1n4 interacts directly with dynactin p150Glued through its unique ezrin/radixin/moesin domain. Studies using double immunofluorescent microscopy and ultrastructural analysis reveal physiological colocalization of BPAG1n4 with dynactin/dynein. Disruption of the interaction between BPAG1n4 and dynactin results in severe defects in retrograde axonal transport. We conclude that BPAG1n4 plays an essential role in retrograde axonal transport in sensory neurons. These findings might advance our understanding of pathogenesis of axonal degeneration and neuronal death.     

Characterization of retrograde axonal transport of antibodies in central and peripheral neurons

Retrograde axonal transport of antibodies against synaptic membrane glycoproteins was studied in the hypoglossal nerve and several CNS pathways of the rat. Injection into the tongue of polyclonal antibodies against synaptic membrane glycoproteins produced immunocytochemically labeled cells in the hypoglossal nucleus 4-5 hr later. Immunoreactive staining increased through 48 hr after injection and then declined. Injections of Fab preparations of the antibody gave labeling patterns indistinguishable from those of the whole antibody. The specificity of this method is shown by control studies in which antibodies against antigens that are not known to be present on the surface of presynaptic membranes were injected and gave no retrograde labeling. Retrograde labeling was also demonstrated in CNS pathways. However, labeling was never as intense as that seen in the hypoglossal nucleus, and some CNS pathways failed to show any retrograde labeling. Furthermore, retrograde labeling after control injections could be demonstrated in some cases. To determine if antibodies were also transported anterogradely, injections were made into the vitreous body of the eye, and the superior colliculus was processed for immunocytochemistry. Unlike wheat-germ agglutinin and several other tracers, antibodies were not found to be anterogradely transported in the optic nerve.     

Glial and axonal regeneration following spinal cord injury

Spinal cord injury (SCI) has been regarded clinically as an irreversible damage caused by tissue contusion due to a blunt external force. Past research had focused on the analysis of the pathogenesis of secondary injury that extends from the injury epicenter to the periphery, as well as tissue damage and neural cell death associated with secondary injury. Recent studies, however, have proven that neural stem (progenitor) cells are also present in the brain and spinal cord of adult mammals including humans. Analyses using spinal cord injury models have also demonstrated active dynamics of cells expressing several stem cell markers, and methods aiming at functional reconstruction by promoting the potential self-regeneration capacity of the spinal cord are being explored. Furthermore, reconstruction of the neural circuit requires not only replenishment or regeneration of neural cells but also regeneration of axons. Analysis of the tissue microenvironment after spinal cord injury and research aiming to remove axonal regeneration inhibitors have also made progress. SCI is one of the simplest central nervous injuries, but its pathogenesis is associated with diverse factors, and further studies are required to elucidate these complex interactions in order to achieve spinal cord regeneration and functional reconstruction.     

Glial and axonal regeneration following spinal cord injury

Spinal cord injury (SCI) has been regarded clinically as an irreversible damage caused by tissue contusion due to a blunt external force. Past research had focused on the analysis of the pathogenesis of secondary injury that extends from the injury epicenter to the periphery, as well as tissue damage and neural cell death associated with secondary injury. Recent studies, however, have proven that neural stem (progenitor) cells are also present in the brain and spinal cord of adult mammals including humans. Analyses using spinal cord injury models have also demonstrated active dynamics of cells expressing several stem cell markers, and methods aiming at functional reconstruction by promoting the potential self-regeneration capacity of the spinal cord are being explored. Furthermore, reconstruction of the neural circuit requires not only replenishment or regeneration of neural cells but also regeneration of axons. Analysis of the tissue microenvironment after spinal cord injury and research aiming to remove axonal regeneration inhibitors have also made progress. SCI is one of the simplest central nervous injuries, but its pathogenesis is associated with diverse factors, and further studies are required to elucidate these complex interactions in order to achieve spinal cord regeneration and functional reconstruction.Key words: glia, regeneration, spinal cord, injury, axon     

Neurofilaments and microtubules in sensory neurons after peripheral nerve section

Summary Sensory neurons were examined in spinal ganglia of the rat 1 to 55 days after section of the plexus brachialis nerves. Only light neurons of the type A were investigated. Maximal reaction to axotomy was found 7 to 14 days after the operation. The majority of the axotomized perikarya developed central chromatolysis. In such neurons, Nissl bodies virtually disappeared from the central area of the neuron and formed a more or less continuous zone at the cell circumference. The cytocentrum became filled with large numbers of mitochondria, dense bodies and other organelles. Neurofilaments and microtubules were disarranged and ran at random among the accumulated particles. Microtubules were often more prominent in chromatolytic areas than neurofilaments. Both these organelles were rare in the peripheral areas filled with massed Nissl substance.Part of the neurons that did not show typical chromatolysis contained increased numbers of neurofilaments among Nissl bodies dispersed throughout the cytoplasm. Neurofilaments were roughly arrayed in bundles up to several microns wide; they were linked by cross-bridges and separated by distances of about 500 Å. Microtubules were rarely found in the filamentous areas. However, they were numerous in the axon hillock and in the initial segment where they formed fascicles similar to those described in normal neurons of other types.During the period from 14 to 55 days after axotomy, many perikarya recovering from chromatolysis contained enlarged bundles of neurofilaments with occasional microtubules among the restored Nissl bodies.Mean diameters of sensory neurons, measured 7 to 55 days after axotomy, in no instance exceeded those of contralateral control neurons. It thus appears that sensory perikarya do not increase in size either during the chromatolytic process or during the period of recovery.This project was supported by a grant from the Muscular Dystrophy Association of America, Inc. The main part of this study was done while the author was a Research Fellow in Anatomy at the Harvard Medical School, Boston. The author wishes to thank prof. S. L. Palay for his valuable advice and help received during her stay at the Department of Anatomy at the Harvard Medical School, under NIH training grant NBO5591.     

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.     

Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.     

Influence of human skin injury on regeneration of sensory neurons

The regeneration of sensory nerve fibres is regulated by trophic factors released from their target tissue, particularly the basal epidermis, and matrix molecules. Means to modulate this response may be useful for the treatment of neuromas and painful hypertrophic scars and of sensory deficits in skin grafts and flaps. We have developed an in vitro model of sensory neuron regeneration on human skin in order to study the mechanisms of sensory dysfunction in pathological conditions. Adult rat sensory neurons were co-cultured with unfixed cryosections of normal or injured (crushed) human skin for 72 h. Neurons were immunostained for growth-associated protein-43 and the neurite lengths of neuronal cell bodies situated in various skin regions were measured. Two-way analysis of variance was performed. Neurites of sensory cell bodies on epidermis of normal skin were the shortest, with a mean +/- SEM of 75+/-10 micrometer, whereas those of cells on the dermo-epidermal junction were the longest, with a mean +/- SEM of 231+/-18 micrometer. Neurons on the dermo-epidermal junction of injured skin had significantly longer neurites than those on the same region of normal skin (mean +/- SEM = 289+/-21 micrometer). Regeneration of sensory neurons may be influenced by extracellular matrix molecules, matrix-binding growth factors and trophic factors. Altered substrate or trophic factors in injured skin may explain the increase of neurite lengths. This in vitro model may be useful for studying the molecular mechanisms of sensory recovery and the development of neuropathic pain following peripheral nerve injury.     

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Injured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.