Activin and bone morphogenetic proteins are present in perinatal sensory neuron target tissues that induce neuropeptides

Previous studies have shown that sensory target tissues induce neuropeptides in na?ve sensory neurons, and that activin and bone morphogenetic proteins (BMPs) are capable of inducing neuropeptides associated with nociception in embryonic sensory neurons in vitro. The goal of the present study was to learn if these ligands were available in native sensory neuron target tissues at correct developmental periods to play this inductive role in vivo. Sensory neurons initially contact their peripheral target tissues and begin to express neuropeptides during late embryogenesis, and we demonstrate that activin and BMPs are present in the embryo and neonate to regulate sensory neuron differentiation. Native embryonic and neonatal target tissues were analyzed by immunoblot and immunohistochemical studies using ligand-specific antibodies. Although activin was easily solubilized, BMPs were detected only after high salt extraction, suggesting that BMPs were bound to extracellular moieties and were capable of acting only locally in native tissues. One inhibitor, noggin, was present in both embryonic skin and muscle. In combination, these data suggest that neuronal differentiation is unlikely to be regulated by simple expression of ligand, but that the functional availability of ligand is a critical component confering biological activity.     

Wounds increase activin in skin and a vasoactive neuropeptide in sensory ganglia

Successful healing of skin wounds requires sensory innervation and the release of vasoactive neuropeptides that dilate blood vessels and deliver serum proteins to the wound, and that cause pain that protects from further injury. Activin has been proposed as a target-derived regulator of sensory neuropeptides during development, but its role in the mature nervous system is unknown. While adult skin contains a low level of activin, protein levels in skin adjacent to a wound increase rapidly after an excision. Neurons containing the neuropeptide calcitonin gene-related peptide (CGRP) increased in sensory ganglia that projected to the wounded skin, but not in ganglia that projected to unwounded skin, suggesting that neurons respond to a local skin signal. Indeed, many adult sensory neurons respond with increased CGRP expression to the application of activin in vitro and utilize a smad-mediated signal transduction pathway in this response. A second skin-derived factor nerve growth factor (NGF) also increased in wounded skin and increased CGRP in cultured adult dorsal root ganglia (DRG) neurons but with lower efficacy. Together, these data support the hypothesis that activin made by skin cells regulates changes in sensory neuropeptides following skin injury, thereby promoting vasodilation and wound healing.     

Skin cell induction of calcitonin gene-related peptide in embryonic sensory neurons in vitro involves activin

Target skin cells induce the neuropeptide calcitonin gene-related peptide (CGRP) in na?ve embryonic dorsal root ganglion (DRG) neurons in vitro, but the molecular basis of that induction is not known. Recombinant activin or bone morphogenetic proteins (BMPs) dramatically increase the number of sensory neurons with CGRP and substance P in vitro (X. Ai et al., 1999, Mol. Cell. Neurosci. 14, 506-518). These experiments were designed to test if activin or BMPs accounted for the CGRP-inductive activity by skin cells. To identify factors from skin that induce CGRP, we developed a bioassay in which embryonic DRG neurons isolated before peripheral target contact in vivo are challenged in vitro with specific factors. Conditioned medium from an embryonic rat skin cell line induced neuronal CGRP expression, and induction was blocked by follistatin, implicating transforming growth factor family members. Immunoblot analysis revealed that the skin cell line medium contained several activin and bone morphogenetic protein moieties. Antibody specific to activin neutralized most of the CGRP-inductive activity in skin conditioned medium. These data indicate that the CGRP-inductive action of skin cells involves activin and establish activin as a candidate regulator of this sensory neuropeptide phenotype during development.     

CaMKK-CaMK1a,a New Post-Traumatic Signalling Pathway Induced in Mouse Somatosensory Neurons

Neurons innervating peripheral tissues display complex responses to peripheral nerve injury. These include the activation and suppression of a variety of signalling pathways that together influence regenerative growth and result in more or less successful functional recovery. However, these responses can be offset by pathological consequences including neuropathic pain. Calcium signalling plays a major role in the different steps occurring after nerve damage. As part of our studies to unravel the roles of injury-induced molecular changes in dorsal root ganglia (DRG) neurons during their regeneration, we show that the calcium calmodulin kinase CaMK1a is markedly induced in mouse DRG neurons in several models of mechanical peripheral nerve injury, but not by inflammation. Intrathecal injection of NRTN or GDNF significantly prevents the post-traumatic induction of CaMK1a suggesting that interruption of target derived factors might be a starter signal in this de novo induction. Inhibition of CaMK signalling in injured DRG neurons by pharmacological means or treatment with CaMK1a siRNA resulted in decreased velocity of neurite growth in vitro. Altogether, the results suggest that CaMK1a induction is part of the intrinsic regenerative response of DRG neurons to peripheral nerve injury, and is thus a potential target for therapeutic intervention to improve peripheral nerve regeneration.     

Glial influences on neural stem cell development: cellular niches for adult neurogenesis

Neural stem cells continually generate new neurons in very limited regions of the adult mammalian central nervous system. In the neurogenic regions there are unique and highly specialized microenvironments (niches) that tightly regulate the neuronal development of adult neural stem cells. Emerging evidence suggests that glia, particularly astrocytes, have key roles in controlling multiple steps of adult neurogenesis within the niches, from proliferation and fate specification of neural progenitors to migration and integration of the neuronal progeny into pre-existing neuronal circuits in the adult brain. Identification of specific niche signals that regulate these sequential steps during adult neurogenesis might lead to strategies to induce functional neurogenesis in other brain regions after injury or degenerative neurological diseases.     

μ-Opioid Receptor Antibody Reveals Tissue-Dependent Specific Staining and Increased Neuronal μ-Receptor Immunoreactivity at the Injured Nerve Trunk in Mice

Neuropathic pain is a debilitating chronic disease often resulting from damage to peripheral nerves. Activation of opioid receptors on peripheral sensory neurons can attenuate pain without central nervous system side effects. Here we aimed to analyze the distribution of neuronal μ-opioid receptors, the most relevant opioid receptors in the control of clinical pain, along the peripheral neuronal pathways in neuropathy. Hence, following a chronic constriction injury of the sciatic nerve in mice, we used immunohistochemistry to quantify the μ-receptor protein expression in the dorsal root ganglia (DRG), directly at the injured nerve trunk, and at its peripheral endings in the hind paw skin. We also thoroughly examined the μ-receptor antibody staining specificity. We found that the antibody specifically labeled μ-receptors in human embryonic kidney 293 cells as well as in neuronal processes of the sciatic nerve and hind paw skin dermis, but surprisingly not in the DRG, as judged by the use of μ/δ/κ-opioid receptor knockout mice. Therefore, a reliable quantitative analysis of μ-receptor expression in the DRG was not possible. However, we demonstrate that the μ-receptor immunoreactivity was strongly enhanced proximally to the injury at the nerve trunk, but was unaltered in paws, on days 2 and 14 following injury. Thus, μ-opioid receptors at the site of axonal damage might be a promising target for the control of painful neuropathies. Furthermore, our findings suggest a rigorous tissue-dependent characterization of antibodies'' specificity, preferably using knockout animals.     

IKAP/Elp1 is required in vivo for neurogenesis and neuronal survival, but not for neural crest migration

Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III) manifests from a failure in development of the peripheral sensory and autonomic nervous systems. The disease results from a point mutation in the IKBKAP gene, which encodes the IKAP protein, whose function is still unresolved in the developing nervous system. Since the neurons most severely depleted in the disease derive from the neural crest, and in light of data identifying a role for IKAP in cell motility and migration, it has been suggested that FD results from a disruption in neural crest migration. To determine the function of IKAP during development of the nervous system, we (1) first determined the spatial-temporal pattern of IKAP expression in the developing peripheral nervous system, from the onset of neural crest migration through the period of programmed cell death in the dorsal root ganglia, and (2) using RNAi, reduced expression of IKBKAP mRNA in the neural crest lineage throughout the process of dorsal root ganglia (DRG) development in chick embryos in ovo. Here we demonstrate that IKAP is not expressed by neural crest cells and instead is expressed as neurons differentiate both in the CNS and PNS, thus the devastation of the PNS in FD could not be due to disruptions in neural crest motility or migration. In addition, we show that alterations in the levels of IKAP, through both gain and loss of function studies, perturbs neuronal polarity, neuronal differentiation and survival. Thus IKAP plays pleiotropic roles in both the peripheral and central nervous systems.     

Monocyte chemoattractant protein-1 functions as a neuromodulator in dorsal root ganglia neurons

It has previously been observed that expression of chemokine monocyte chemoattractant protein-1 (MCP-1/CC chemokine ligand 2 (CCL2)) and its receptor CC chemokine receptor 2 (CCR2) is up-regulated by dorsal root ganglion (DRG) neurons in association with rodent models of neuropathic pain. MCP-1 increases the excitability of nociceptive neurons after a peripheral nerve injury, while disruption of MCP-1/CCR2 signaling blocks the development of neuropathic pain, suggesting MCP-1 signaling is responsible for heightened pain sensitivity. To define the mechanisms of MCP-1 signaling in DRG, we studied intracellular processing, release, and receptor-mediated signaling of MCP-1 in DRG neurons. We found that in a focal demyelination model of neuropathic pain both MCP-1 and CCR2 were up-regulated by the same neurons including transient receptor potential vanilloid receptor subtype 1 (TRPV1) expressing nociceptors. MCP-1 expressed by DRG neurons was packaged into large dense-core vesicles whose release could be induced from the soma by depolarization in a Ca²⁺-dependent manner. Activation of CCR2 by MCP-1 could sensitize nociceptors via transactivation of transient receptor potential channels. Our results suggest that MCP-1 and CCR2, up-regulated by sensory neurons following peripheral nerve injury, might participate in neural signal processing which contributes to sustained excitability of primary afferent neurons.     

RSK1 promotes mammalian axon regeneration by inducing the synthesis of regeneration-related proteins

In contrast to the adult mammalian central nervous system (CNS), the neurons in the peripheral nervous system (PNS) can regenerate their axons. However, the underlying mechanism dictating the regeneration program after PNS injuries remains poorly understood. Combining chemical inhibitor screening with gain- and loss-of-function analyses, we identified p90 ribosomal S6 kinase 1 (RSK1) as a crucial regulator of axon regeneration in dorsal root ganglion (DRG) neurons after sciatic nerve injury (SNI). Mechanistically, RSK1 was found to preferentially regulate the synthesis of regeneration-related proteins using ribosomal profiling. Interestingly, RSK1 expression was up-regulated in injured DRG neurons, but not retinal ganglion cells (RGCs). Additionally, RSK1 overexpression enhanced phosphatase and tensin homolog (PTEN) deletion-induced axon regeneration in RGCs in the adult CNS. Our findings reveal a critical mechanism in inducing protein synthesis that promotes axon regeneration and further suggest RSK1 as a possible therapeutic target for neuronal injury repair.

This study shows that p90 ribosomal S6 kinase 1 (RSK1) responds differentially to nerve injury in the peripheral and central nervous systems, and identifies it as a crucial regulator of axonal regeneration; mechanistically, RSK1 preferentially induces the synthesis of regeneration-related proteins via the RSK1-eEF2K-eEF2 axis.     

Expression and histochemical localization of ciliary neurotrophic factor in cultured adult rat dorsal root ganglion neurons

Ciliary neurotrophic factor (CNTF) is abundantly expressed in Schwann cells in adult mammalian peripheral nerves, but not in neurons. After peripheral nerve injury, CNTF released from disrupted Schwann cells is likely to promote neuronal survival and axonal regeneration. In the present study, we examined the expression and histochemical localization of CNTF in adult rat DRG in vivo and in vitro. In contrast to the restricted expression in Schwann cells in vivo, we observed abundant CNTF mRNA and protein expression in DRG neurons after 3 h, 2, 7, and 15 days in dissociated cell culture. At later stages (7 and 15 days) of culture, CNTF immunoreactivity was detected in both neuronal cell bodies and regenerating neurites. These results suggest that CNTF is synthesized and transported to neurites in cultured DRG neurons. Since we failed to observe CNTF immunoreactivity in DRG neurons in explant culture, disruption of cell–cell interactions, rather than the culture itself, may be an inducible factor for localization of CNTF in the neurons.     

Galectin-1 plays essential roles in adult mammalian nervous tissues. Roles of oxidized galectin-1

Previous data have suggested that galectin-1 is expressed widely in nervous tissues at embryonic stages but becomes restricted mainly to peripheral nervous tissues with maturation. Though the expression is intense in adult mammalian peripheral neurons, there had been no report about functions of galectin-1 there. Recently we discovered a factor that enhanced peripheral axonal regeneration. The factor was identified as oxidized galectin-1 with three intramolecular disulfide bonds and showed no lectin activity. Oxidized recombinant human galectin-1 (rhGAL-1/Ox) showed the same nerve growth promoting activity at very low concentrations (pg/ml). rhGAL-1/Ox at similarly low concentration was also effective in in vivo experiments of axonal regeneration. Moreover, the application of functional anti-rhGAL-1 antibody strongly inhibited the axonal regeneration in vivo as well as in vitro. Since galectin-1 is expressed in the regenerating sciatic nerves as well as in both sensory neurons and motor neurons, these results suggest that galectin-1 is secreted into the extracellular space to be oxidized, and then, in its oxidized form, to regulate initial repair after axotomy. The administration of oxidized galectin-1 effectively promoted functional recovery after sciatic nerve injury in vivo. Oxidized galectin-1, hence, appears to play an important role in promoting axonal regeneration, working as a kind of cytokine, not as a lectin. Recent reports indicated additional roles of cytosolic galectin-1 in neural diseases, such as ALS. Furthermore galectin-1 has been proved to be a downstream target of DeltaFosB. In hippocampus of rat brain, expression of DeltaFosB is induced immediately after ischemia-reperfusion, suggesting that galectin-1 may also play important roles in central nervous system after injury.     

Effects of the neurotrophic factor artemin on sensory afferent development and sensitivity

Artemin is a neuronal survival and differentiation factor in the glial cell line-derived neurotrophic factor family.Its receptor GFRα3 is expressed by a subpopulation of nociceptor type sensory neurons in the dorsal root and trigeminal ganglia(DRG and TG).These neurons co-express the heat,capsaicin and proton-sensitive channel TRPV1 and the cold and chemical-sensitive channel TRPA1.To further investigate the effects of artemin on sensory neurons,we isolated transgenic mice(ARTN-OE mice) that overexpress art...     

Roles of extracellular signal-regulated protein kinases 5 in spinal microglia and primary sensory neurons for neuropathic pain

Neuropathic pain that occurs after peripheral nerve injury is poorly controlled by current therapies. Increasing evidence shows that mitogen-activated protein kinase (MAPK) play an important role in the induction and maintenance of neuropathic pain. Here we show that activation of extracellular signal-regulated protein kinases 5 (ERK5), also known as big MAPK1, participates in pain hypersensitivity caused by nerve injury. Nerve injury increased ERK5 phosphorylation in spinal microglia and in both damaged and undamaged dorsal root ganglion (DRG) neurons. Antisense knockdown of ERK5 suppressed nerve injury-induced neuropathic pain and decreased microglial activation. Furthermore, inhibition of ERK5 blocked the induction of transient receptor potential channels and brain-derived neurotrophic factor expression in DRG neurons. Our results show that ERK5 activated in spinal microglia and DRG neurons contributes to the development of neuropathic pain. Thus, blocking ERK5 signaling in the spinal cord and primary afferents has potential for preventing pain after nerve damage.     

Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons

Peripheral nerve injury results in the increased synthesis and axonal trasnport of the growth-associated protein GAP-43 in dorsal root ganglion (DRG) neurons, coincident with regenerative growth of the injured peripheral axon branches. To determine wheter the injury-associated signalling mechanism which leads to GAP-43 induction also operates through the central branches of DRG axons, we used immunocytochemistry to compare the expression of GAP-43 in adult rat DRG neurons 2 weeks after dorsal root crush lesions (central axotomy) or peripheral nerve crush lesions (peripheral axotomy). In uninjured ganglia, a subpopulation of smaller DRG neurons expresses moderate levels of GAP-43, whereas larger neurons generally do not. At 2 weeks following peripheral axotomy, virtually all axotomized neurons, large and small, express high levels of GAP-43. At 2 weeks following dorsal root lesions, no increase in GAP-43 expression is detected. Thus, the injury-associated up-regulation of GAP-43 expression in DRG neurons is triggered by a mechanism that is responsive to injury of only the peripheral, and not the central, axon branches. These findings support the hypothesis that GAP-43 induction in DRG neurons is caused by disconnection from peripheral target tissue, not by axon injury per se. © 1993 John Wiley & Sons, Inc.     

Co-administration of Monosialoganglioside and Skeletal Muscle Cells on Dorsal Root Ganglion Neuronal Phenotypes In Vitro

The neuropeptide-immunoreactive (IR) and neurofilament-IR neurons are two major phenotypical classes in dorsal root ganglion (DRG). Targets of neuronal innervation play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. Monosialoganglioside (GM1) has been considered to have a neurotrophic factor-like activity. Both GM1 and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons. However, whether target SKM cells and GM1, alone or associated, generate neuropeptide or neurofilament expression remains unclear. The aim of the present study is to investigate the effects of GM1 or/and SKM on DRG neuronal phenotypes. DRG neurons containing the neuropeptide substance P (SP) and neurofilament 200 (NF-200) were quantified using immunofluorescent labeling in cultures of DRG, which was dissected out at times before (at embryonic days 12.5, E12.5) and after (at E19.5) sensory neurons contact peripheral targets in vivo. DRG neurons were cultured in absence or presence of GM1 or/and SKM cells. In this experiment, we found that: (1) GM1 promoted expression of SP and NF-200 in E12.5 DRG cultures; (2) SKM cells promoted expression of NF-200 but not SP in E12.5 DRG cultures; (3) GM1 and target SKM cells had additive effects on expression of SP and NF-200 in E12.5 DRG cultures; and (4) SKM or/and GM1 did not have effects on expression of SP and NF-200 in E19.5 DRG cultures. These results suggested that GM1 could influence DRG, two major neuronal phenotypes, before sensory neurons contact peripheral targets in vivo. Target SKM cells could only influence neurofilament-expressed neuronal phenotype before sensory neurons contact peripheral targets in vivo. GM1 and SKM cells had the additive effects on two major DRG neuronal classes, which express neuropeptide or neurofilament when DRG cells were harvested before sensory neurons contact peripheral targets in vivo. These results offered new clues for a better understanding of the association of GM1 or/and SKM with neuronal phenotypes.