ABSTRACT
Slit-Robo signaling has been characterized as a repulsive signal for precise axon pathfinding and cell migration during embryonic development. Here, we describe a role for Sox2 in the regulation of Robo1 in Schwann cells and for Slit3-Robo1 signaling in controlling axon guidance within the newly formed nerve bridge following peripheral nerve transection injury. In particular, we show that macrophages form the outermost layer of the nerve bridge and secrete high levels of Slit3, while migratory Schwann cells and fibroblasts inside the nerve bridge express the Robo1 receptor. In line with this pattern of Slit3 and Robo1 expression, we observed multiple axon regeneration and cell migration defects in the nerve bridge of Sox2-, Slit3-, and Robo1-mutant mice. Our findings have revealed important functions for macrophages in the peripheral nervous system, utilizing Slit3-Robo1 signaling to control correct peripheral nerve bridge formation and precise axon targeting to the distal nerve stump following injury.
Subject(s)
Axon Guidance , Macrophages/metabolism , Membrane Proteins/metabolism , Nerve Regeneration , Peripheral Nerves/metabolism , Animals , Cell Movement , Cells, Cultured , Female , Fibroblasts/metabolism , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Peripheral Nerves/physiology , Rats , Rats, Wistar , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Schwann Cells/metabolism , Signal Transduction , Roundabout ProteinsABSTRACT
BACKGROUND: Neurofibromatosis type 2 (NF2) is a genetic tumor-predisposition disorder caused by NF2/merlin tumor suppressor gene inactivation. The hallmark of NF2 is formation of bilateral vestibular schwannomas (VS). Because merlin modulates activity of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, we investigated repurposing drugs targeting MEK1 and/or MEK2 as a treatment for NF2-associated schwannomas. METHODS: Mouse and human merlin-deficient Schwann cell lines (MD-MSC/HSC) were screened against 6 MEK1/2 inhibitors. Efficacious drugs were tested in orthotopic allograft and NF2 transgenic mouse models. Pathway and proteome analyses were conducted. Drug efficacy was examined in primary human VS cells with NF2 mutations and correlated with DNA methylation patterns. RESULTS: Trametinib, PD0325901, and cobimetinib were most effective in reducing MD-MSC/HSC viability. Each decreased phosphorylated pERK1/2 and cyclin D1, increased p27, and induced caspase-3 cleavage in MD-MSCs. Proteomic analysis confirmed cell cycle arrest and activation of pro-apoptotic pathways in trametinib-treated MD-MSCs. The 3 inhibitors slowed allograft growth; however, decreased pERK1/2, cyclin D1, and Ki-67 levels were observed only in PD0325901 and cobimetinib-treated grafts. Tumor burden and average tumor size were reduced in trametinib-treated NF2 transgenic mice; however, tumors did not exhibit reduced pERK1/2 levels. Trametinib and PD0325901 modestly reduced viability of several primary human VS cell cultures with NF2 mutations. DNA methylation analysis of PD0325901-resistant versus -susceptible VS identified genes that could contribute to drug resistance. CONCLUSION: MEK inhibitors exhibited differences in antitumor efficacy resistance in schwannoma models with possible emergence of trametinib resistance. The results support further investigation of MEK inhibitors in combination with other targeted drugs for NF2 schwannomas.
Subject(s)
Azetidines/pharmacology , Drug Resistance, Neoplasm/drug effects , Neuroma, Acoustic , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridones/pharmacology , Pyrimidinones/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Humans , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 2/antagonists & inhibitors , Mice , Neurofibromatosis 2/complications , Neuroma, Acoustic/etiologyABSTRACT
The regenerative capacity of peripheral nerves declines during aging, contributing to the development of neuropathies, limiting organism function. Changes in Schwann cells prompt failures in instructing maintenance and regeneration of aging nerves; molecular mechanisms of which have yet to be delineated. Here, we identified an altered inflammatory environment leading to a defective Schwann cell response, as an underlying mechanism of impaired nerve regeneration during aging. Chronic inflammation was detected in intact uninjured old nerves, characterized by increased macrophage infiltration and raised levels of monocyte chemoattractant protein 1 (MCP1) and CC chemokine ligand 11 (CCL11). Schwann cells in the old nerves appeared partially dedifferentiated, accompanied by an activated repair program independent of injury. Upon sciatic nerve injury, an initial delayed immune response was followed by a persistent hyperinflammatory state accompanied by a diminished repair process. As a contributing factor to nerve aging, we showed that CCL11 interfered with Schwann cell differentiation in vitro and in vivo. Our results indicate that increased infiltration of macrophages and inflammatory signals diminish regenerative capacity of aging nerves by altering Schwann cell behavior. The study identifies CCL11 as a promising target for anti-inflammatory therapies aiming to improve nerve regeneration in old age.
Subject(s)
Aging/pathology , Inflammation/pathology , Nerve Regeneration , Peripheral Nerves/pathology , Peripheral Nerves/physiopathology , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Aspirin/pharmacology , Aspirin/therapeutic use , Chemokine CCL11/pharmacology , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Mice, Inbred C57BL , Myelin Sheath/metabolism , Nerve Crush , Nerve Regeneration/drug effects , Peripheral Nerve Injuries/pathology , Peripheral Nerve Injuries/physiopathology , Peripheral Nerves/drug effects , Schwann Cells/drug effects , Schwann Cells/metabolism , Schwann Cells/pathology , Sciatic Nerve/drug effects , Sciatic Nerve/metabolism , Sciatic Nerve/pathology , Sciatic Nerve/physiopathologyABSTRACT
Correct myelination is crucial for the function of the peripheral nervous system. Both positive and negative regulators within the axon and Schwann cell function to ensure the correct onset and progression of myelination during both development and following peripheral nerve injury and repair. The Sox2 transcription factor is well known for its roles in the development and maintenance of progenitor and stem cell populations, but has also been proposed in vitro as a negative regulator of myelination in Schwann cells. We wished to test fully whether Sox2 regulates myelination in vivo and show here that, in mice, sustained Sox2 expression in vivo blocks myelination in the peripheral nerves and maintains Schwann cells in a proliferative non-differentiated state, which is also associated with increased inflammation within the nerve. The plasticity of Schwann cells allows them to re-myelinate regenerated axons following injury and we show that re-myelination is also blocked by Sox2 expression in Schwann cells. These findings identify Sox2 as a physiological regulator of Schwann cell myelination in vivo and its potential to play a role in disorders of myelination in the peripheral nervous system.
Subject(s)
Macrophages/metabolism , Myelin Sheath/metabolism , Peripheral Nerves/metabolism , SOXB1 Transcription Factors/metabolism , Schwann Cells/metabolism , Animals , Biomarkers/metabolism , Cadherins/metabolism , Cell Proliferation , Early Growth Response Protein 2/metabolism , Green Fluorescent Proteins/metabolism , Mice, Transgenic , Motor Activity , Neural Conduction , Peripheral Nerve Injuries/metabolism , Peripheral Nerve Injuries/pathology , Peripheral Nerves/pathology , Peripheral Nerves/ultrastructure , Proto-Oncogene Proteins c-jun/metabolism , Rats , Recovery of Function , Schwann Cells/pathology , Transgenes , beta Catenin/metabolismABSTRACT
Loss of the Merlin tumor suppressor and activation of the Hippo signaling pathway play major roles in the control of cell proliferation and tumorigenesis. We have identified completely novel roles for Merlin and the Hippo pathway effector Yes-associated protein (YAP) in the control of Schwann cell (SC) plasticity and peripheral nerve repair after injury. Injury to the peripheral nervous system (PNS) causes a dramatic shift in SC molecular phenotype and the generation of repair-competent SCs, which direct functional repair. We find that loss of Merlin in these cells causes a catastrophic failure of axonal regeneration and remyelination in the PNS. This effect is mediated by activation of YAP expression in Merlin-null SCs, and loss of YAP restores axonal regrowth and functional repair. This work identifies new mechanisms that control the regenerative potential of SCs and gives new insight into understanding the correct control of functional nerve repair in the PNS.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Proliferation , Crush Injuries/metabolism , Nerve Regeneration , Neurofibromin 2/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Schwann Cells/metabolism , Sciatic Nerve/metabolism , Sciatic Neuropathy/metabolism , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , Axons/metabolism , Axons/pathology , Cell Cycle Proteins , Crush Injuries/genetics , Crush Injuries/pathology , Crush Injuries/physiopathology , Disease Models, Animal , Female , Genotype , Hippo Signaling Pathway , Male , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , Motor Activity , Myelin Sheath/metabolism , Nerve Growth Factors/metabolism , Neurofibromin 2/deficiency , Neurofibromin 2/genetics , Neuronal Plasticity , Phenotype , Phosphoproteins/deficiency , Phosphoproteins/genetics , Proto-Oncogene Proteins c-jun/metabolism , Recovery of Function , Schwann Cells/pathology , Sciatic Nerve/injuries , Sciatic Nerve/pathology , Sciatic Nerve/physiopathology , Sciatic Neuropathy/genetics , Sciatic Neuropathy/pathology , Sciatic Neuropathy/physiopathology , Signal Transduction , Time Factors , YAP-Signaling ProteinsABSTRACT
Myelination in the peripheral nervous system (PNS) is controlled by both positive and negative regulators within Schwann cells to ensure timely onset and correct myelin thickness for saltatory conduction by neurons. Transcription factors such as Sox10, octamer-binding transcription factor 6 (Oct6) and Krox20 form a positive regulatory network, whereas negative regulators such as cJun and Sox2 oppose myelination in Schwann cells. The role of the p38 MAPK pathway has been studied in PNS myelination, but its precise function remains unclear, with both positive and negative effects of p38 activity reported upon both myelination and processes of nerve repair. To clarify the role of p38 MAPK in the PNS, we have analysed mice with a Schwann cell-specific ablation of the major p38 isoform, p38alpha. In line with previous findings of an inhibitory role for p38 MAPK, we observe acceleration of post-natal myelination in p38alpha null nerves, a delay in myelin down-regulation following injury, together with a small increase in levels of re-myelination following injury. Finally we explored roles for p38alpha in controlling axonal regeneration and functional repair following PNS injury and observe that loss of p38alpha function in Schwann cells does not appear to affect these processes as previously reported. These studies therefore provide further proof for a role of p38 MAPK signalling in the control of myelination by Schwann cells in the PNS, but do not show an apparent role for signalling by this MAP kinase in Schwann cells controlling other elements of Wallerian degeneration and functional repair following injury. Cover Image for this issue: doi: 10.1111/jnc.13793.
Subject(s)
Mitogen-Activated Protein Kinase 14/physiology , Nerve Fibers, Myelinated/enzymology , Peripheral Nerve Injuries/enzymology , Peripheral Nerves/enzymology , Recovery of Function/physiology , Schwann Cells/enzymology , Animals , Animals, Newborn , Cells, Cultured , Female , Male , Mice , Nerve Fibers, Myelinated/pathology , Peripheral Nerve Injuries/pathology , Peripheral Nerves/pathology , Rats , Schwann Cells/pathologyABSTRACT
Schwannomas are predominantly benign nerve sheath neoplasms caused by Nf2 gene inactivation. Presently, treatment options are mainly limited to surgical tumor resection due to the lack of effective pharmacological drugs. Although the mechanistic understanding of Nf2 gene function has advanced, it has so far been primarily restricted to Schwann cell-intrinsic events. Extracellular cues determining Schwann cell behavior with regard to schwannoma development remain unknown. Here we show pro-tumourigenic microenvironmental effects on Schwann cells where an altered axonal microenvironment in cooperation with injury signals contribute to a persistent regenerative Schwann cell response promoting schwannoma development. Specifically in genetically engineered mice following crush injuries on sciatic nerves, we found macroscopic nerve swellings in mice with homozygous nf2 gene deletion in Schwann cells and in animals with heterozygous nf2 knockout in both Schwann cells and axons. However, patient-mimicking schwannomas could only be provoked in animals with combined heterozygous nf2 knockout in Schwann cells and axons. We identified a severe re-myelination defect and sustained macrophage presence in the tumor tissue as major abnormalities. Strikingly, treatment of tumor-developing mice after nerve crush injury with medium-dose aspirin significantly decreased schwannoma progression in this disease model. Our results suggest a multifactorial concept for schwannoma formation-emphasizing axonal factors and mechanical nerve irritation as predilection site for schwannoma development. Furthermore, we provide evidence supporting the potential efficacy of anti-inflammatory drugs in the treatment of schwannomas.
Subject(s)
Axons/pathology , Neurilemmoma/pathology , Schwann Cells/pathology , Sciatic Nerve/pathology , Tumor Microenvironment/physiology , Animals , Mice, Transgenic , Myelin Sheath/pathology , Neurilemmoma/genetics , Neurofibromatosis 2/genetics , Tumor Microenvironment/geneticsABSTRACT
Repair in the peripheral nervous system (PNS) depends upon the plasticity of the myelinating cells, Schwann cells, and their ability to dedifferentiate, direct axonal regrowth, remyelinate, and allow functional recovery. The ability of such an exquisitely specialized myelinating cell to revert to an immature dedifferentiated cell that can direct repair is remarkable, making Schwann cells one of the very few regenerative cell types in our bodies. However, the idea that the PNS always repairs after injury, in contrast to the central nervous system, is not true. Repair in patients after nerve trauma can be incredibly variable, depending on the site and type of injury, and only a relatively small number of axons may fully regrow and reinnervate their targets. Recent research has shown that it is an active process that drives Schwann cells back to an immature state after injury and that this requires activity of the p38 and extracellular-regulated kinase 1/2 mitogen-activated protein kinases, as well as the transcription factor cJun. Analysis of the events after peripheral nerve transection has shown how signaling from nerve fibroblasts forms Schwann cells into cords in the newly generated nerve bridge, via Sox2 induction, to allow the regenerating axons to cross the gap. Understanding these pathways and identifying additional mechanisms involved in these processes raises the possibility of both boosting repair after PNS trauma and even, possibly, blocking the inappropriate demyelination seen in some disorders of the peripheral nervous system.
Subject(s)
Neuronal Plasticity/physiology , Peripheral Nervous System/pathology , Peripheral Nervous System/physiopathology , Schwann Cells/pathology , Wound Healing , Animals , Humans , MAP Kinase Signaling System , Myelin Sheath/metabolism , Peripheral Nervous System/enzymology , Schwann Cells/enzymologyABSTRACT
Physical damage to the peripheral nerves triggers Schwann cell injury response in the distal nerves in an event termed Wallerian degeneration: the Schwann cells degrade their myelin sheaths and dedifferentiate, reverting to a phenotype that supports axon regeneration and nerve repair. The molecular mechanisms regulating Schwann cell plasticity in the PNS remain to be elucidated. Using both in vivo and in vitro models for peripheral nerve injury, here we show that inhibition of p38 mitogen-activated protein kinase (MAPK) activity in mice blocks Schwann cell demyelination and dedifferentiation following nerve injury, suggesting that the kinase mediates the injury signal that triggers distal Schwann cell injury response. In myelinating cocultures, p38 MAPK also mediates myelin breakdown induced by Schwann cell growth factors, such as neuregulin and FGF-2. Furthermore, ectopic activation of p38 MAPK is sufficient to induce myelin breakdown and drives differentiated Schwann cells to acquire phenotypic features of immature Schwann cells. We also show that p38 MAPK concomitantly functions as a negative regulator of Schwann cell differentiation: enforced p38 MAPK activation blocks cAMP-induced expression of Krox 20 and myelin proteins, but induces expression of c-Jun. As expected of its role as a negative signal for myelination, inhibition of p38 MAPK in cocultures promotes myelin formation by increasing the number as well as the length of individual myelin segments. Altogether, our data identify p38 MAPK as an important regulator of Schwann cell plasticity and differentiation.