Immunodominant myelin basic protein fragments induce mechanical hypersensitivity.
ABSTRACT: An insulating myelin sheath ensures saltatory conduction of mechanosensory A afferents. Myelin damage results in the electrical instability of A fibers and the ability to generate pain in response to light touch/pressure (mechanical allodynia). We have hypothesized and then established that the release of T cell epitopes of myelin basic protein (MBP) enables nociceptive circuitry in myelinated fibers. Thus, mass spectrometry analysis of the rat sciatic nerve proteome followed by bioinformatics examination of the datasets revealed a loss of MBP and activation of T-helper cell signaling in the nerves undergoing chronic constriction injury (CCI). Matrix metalloproteinase-9 (MMP-9) proteolysis resulted in the MBP digest peptides, including the MBP84-104 and MBP68-86 regions, which exhibit prominent immunogenic epitopes. Myelin-forming Schwann cells and paranodal areas accumulated MHCII, MMP-9 and the degraded MBP at the sciatic nerve injury site. Administration of the immunodominant MBP84-104 and MBP68-86 peptides but not of the control peptides in a naïve rat sciatic nerve produced robust mechanical allodynia. Allodynia was accompanied by the T cell infiltration and an increase in MHCII, IL-17A and TNF- levels at the nerve injection site and the segmental ganglia. The pro-nociceptive activity of the synthetic MBP84-104 diminished in athymic nude rats lacking T cells. SB-3CT, an antagonist of MMP-9, inhibited mechanical allodynia, neuroinflammation and spinal sensitization after CCI. Collectively, our novel data implicate, for the first time, MMP-mediated cleavage of MBP and the resulting MBP digest fragments as a major cause of neuropathic pain. Gene extression profiling of total RNAs extracted from rat sciatic nerves, dorsal root ganglion and spinal cords after MBP84-104 peptide injection
Project description:An insulating myelin sheath ensures saltatory conduction of mechanosensory A afferents. Myelin damage results in the electrical instability of A fibers and the ability to generate pain in response to light touch/pressure (mechanical allodynia). We have hypothesized and then established that the release of T cell epitopes of myelin basic protein (MBP) enables nociceptive circuitry in myelinated fibers. Thus, mass spectrometry analysis of the rat sciatic nerve proteome followed by bioinformatics examination of the datasets revealed a loss of MBP and activation of T-helper cell signaling in the nerves undergoing chronic constriction injury (CCI). Matrix metalloproteinase-9 (MMP-9) proteolysis resulted in the MBP digest peptides, including the MBP84-104 and MBP68-86 regions, which exhibit prominent immunogenic epitopes. Myelin-forming Schwann cells and paranodal areas accumulated MHCII, MMP-9 and the degraded MBP at the sciatic nerve injury site. Administration of the immunodominant MBP84-104 and MBP68-86 peptides but not of the control peptides in a naïve rat sciatic nerve produced robust mechanical allodynia. Allodynia was accompanied by the T cell infiltration and an increase in MHCII, IL-17A and TNF- levels at the nerve injection site and the segmental ganglia. The pro-nociceptive activity of the synthetic MBP84-104 diminished in athymic nude rats lacking T cells. SB-3CT, an antagonist of MMP-9, inhibited mechanical allodynia, neuroinflammation and spinal sensitization after CCI. Collectively, our novel data implicate, for the first time, MMP-mediated cleavage of MBP and the resulting MBP digest fragments as a major cause of neuropathic pain. Overall design: Gene extression profiling of total RNAs extracted from rat sciatic nerves, dorsal root ganglion and spinal cords after MBP84-104 peptide injection
Project description:BACKGROUND:The myelin sheath provides electrical insulation of mechanosensory Aβ-afferent fibers. Myelin-degrading matrix metalloproteinases (MMPs) damage the myelin sheath. The resulting electrical instability of Aβ-fibers is believed to activate the nociceptive circuitry in Aβ-fibers and initiate pain from innocuous tactile stimulation (mechanical allodynia). The precise molecular mechanisms, responsible for the development of this neuropathic pain state after nerve injury (for example, chronic constriction injury, CCI), are not well understood. METHODS AND RESULTS:Using mass spectrometry of the whole sciatic nerve proteome followed by bioinformatics analyses, we determined that the pathways, which are classified as the Infectious Disease and T-helper cell signaling, are readily activated in the nerves post-CCI. Inhibition of MMP-9/MMP-2 suppressed CCI-induced mechanical allodynia and concomitant TNF-α and IL-17A expression in nerves. MMP-9 proteolysis of myelin basic protein (MBP) generated the MBP84-104 and MBP68-86 digest peptides, which are prominent immunogenic epitopes. In agreement, the endogenous MBP69-86 epitope co-localized with MHCII and MMP-9 in Schwann cells and along the nodes of Ranvier. Administration of either the MBP84-104 or MBP68-86 peptides into the naïve nerve rapidly produced robust mechanical allodynia with a concomitant increase in T cells and MHCII-reactive cell populations at the injection site. As shown by the genome-wide expression profiling, a single intraneural MBP84-104 injection stimulated the inflammatory, immune cell trafficking, and antigen presentation pathways in the injected naïve nerves and the associated spinal cords. Both MBP84-104-induced mechanical allodynia and characteristic pathway activation were remarkably less prominent in the T cell-deficient athymic nude rats. CONCLUSIONS:These data implicate MBP as a novel mediator of pain. Furthermore, the action of MMPs expressed within 1 day post-injury is critical to the generation of tactile allodynia, neuroinflammation, and the immunodominant MBP digest peptides in nerve. These MBP peptides initiate mechanical allodynia in both a T cell-dependent and -independent manner. In the course of Wallerian degeneration, the repeated exposure of the cryptic MBP epitopes, which are normally sheltered from immunosurveillance, may induce the MBP-specific T cell clones and a self-sustaining immune reaction, which may together contribute to the transition of acute pain into a chronic neuropathic pain state.
Project description:Remyelination is a key step in functional nerve regeneration performed by Schwann cells (SC). We have demonstrated that matrix metalloproteinase (MMP)-9 is a major regulator of signal transduction and phenotypic switching in SCs. Herein, genome-wide transcriptional profiling, followed by Ingenuity Pathway Analysis revealed the MMP-9 signaling network and its endogenous inhibitor, TIMP-1, among the top induced genes of the injured sciatic nerve, that co-distributed with MMP-9 in myelinating SCs and the paranodal/nodal areas of myelinated fibers. Homo- and heterodimers of the active and proMMP-9 were purified from injured nerves using gelatin-sepharose. MMP-9 gene deletion increased the number of immature, GFAP+ mSC and post-mitotic cell counts that correlate with shorter myelin internodes in remyelinated fibers lacking MMP-9. MMP-9 is essential to nodal clustering of voltage-gated Na+ (Nav) channels. MMP inhibitor therapy diminished the expression of Nav 1.7 and 1.8. These data established the essential role of MMP-9 in guiding SC differentiation toward myelin production and in molecular assembly of the myelin domains. Modification of Nav channels in myelinated fibers may thus provide an important therapeutic approach for a number of facilitates regeneration and attenuated neuropathic pain. Gene expression profiling of total RNAs extracted from murine sciatic nerves, dorsal root ganglion and spinal cords at day 1 and day 5 post injury.
Project description:Myelin basic protein (MBP) is an auto-antigen able to induce intractable pain from innocuous mechanical stimulation (mechanical allodynia). The mechanisms provoking this algesic MBP activity remain obscure. Our present study demonstrates that membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) releases the algesic MBP peptides from the damaged myelin, which then reciprocally enhance the expression of MT1-MMP in nerve to sustain a state of allodynia. Specifically, MT1-MMP expression and activity in rat sciatic nerve gradually increased starting at day 3 after chronic constriction injury (CCI). Inhibition of the MT1-MMP activity by intraneural injection of the function-blocking human DX2400 monoclonal antibody at day 3 post-CCI reduced mechanical allodynia and neuropathological signs of Wallerian degeneration, including axon demyelination, degeneration, edema and formation of myelin ovoids. Consistent with its role in allodynia, the MT1-MMP proteolysis of MBP generated the MBP69-86-containing epitope sequences in vitro. In agreement, the DX2400 therapy reduced the release of the MBP69-86 epitope in CCI nerve. Finally, intraneural injection of the algesic MBP69-86 and control MBP2-18 peptides differentially induced MT1-MMP and MMP-2 expression in the nerve. With these data we offer a novel, self-sustaining mechanism of persistent allodynia via the positive feedback loop between MT1-MMP and the algesic MBP peptides. Accordingly, short-term inhibition of MT1-MMP activity presents a feasible pharmacological approach to intervene in this molecular circuit and the development of neuropathic pain.
Project description:Mechanosensory fibers are enveloped by myelin, a unique multilamellar membrane permitting saltatory neuronal conduction. Damage to myelin is thought to contribute to severe pain evoked by innocuous tactile stimulation (i.e., mechanical allodynia). Our earlier (Liu et al., 2012) and present data demonstrate that a single injection of a myelin basic protein-derived peptide (MBP84-104) into an intact sciatic nerve produces a robust and long-lasting (>30days) mechanical allodynia in female rats. The MBP84-104 peptide represents the immunodominant epitope and requires T cells to maintain allodynia. Surprisingly, only systemic gabapentin (a ligand of voltage-gated calcium channel ?2?1), but not ketorolac (COX inhibitor), lidocaine (sodium channel blocker) or MK801 (NMDA antagonist) reverse allodynia induced by the intrasciatic MBP84-104. The genome-wide transcriptional profiling of the sciatic nerve followed by the bioinformatics analyses of the expression changes identified interleukin (IL)-6 as the major cytokine induced by MBP84-104 in both the control and athymic T cell-deficient nude rats. The intrasciatic MBP84-104 injection resulted in both unilateral allodynia and unilateral IL-6 increase the segmental spinal cord (neurons and astrocytes). An intrathecal delivery of a function-blocking IL-6 antibody reduced the allodynia in part by the transcriptional effects in large-diameter primary afferents in DRG. Our data suggest that MBP regulates IL-6 expression in the nervous system and that the spinal IL-6 activity mediates nociceptive processing stimulated by the MBP epitopes released after damage or disease of the somatosensory nervous system.
Project description:We applied Solexa sequencing technology to identify rat microRNA genes in proximal sciatic nerve following sciatic nerve resection. Using Solexa sequencing, computational analysis and Q-PCR verification, 93 novel miRNAs in rats were discovered and identified, of which 42 novel miRNAs were first reported in proximal sciatic nerve of rat and 51 novel miRNAs were produced at days 1, 4, 7 and 14 after sciatic nerve resection. These data provide an important resource relating to the role and regulation of miRNAs for future studies relating to peripheral nerve injury and regeneration. Keywords: Small RNA sequencing 18-30 nt small RNAs from proximal sciatic nerve of 30 Thirty Sprague-Dawley (SD) rats were sequenced at one Solexa lane
Project description:Axon regeneration in the central nervous system (CNS) requires reactivating injured neurons’ intrinsic growth state and enabling growth in an inhibitory environment. Using an inbred mouse neuronal phenotypic screen, we find that CAST/Ei mouse adult dorsal root ganglion neurons extend axons more on CNS myelin than the other eight strains tested, especially when pre-injured. Injury-primed CAST/Ei neurons also regenerate markedly in the spinal cord and optic nerve more than those from C57BL/6 mice and show greater spouting following ischemic stroke. Heritability estimates indicate that extended growth in CAST/Ei neurons on myelin is genetically determined, and two whole-genome expression screens yield the Activin transcript Inhba as most correlated with this ability. These screens are presented here. Biological quadruplicate - Mouse tissue - Naïve Dorsal Root Ganglia (DRG) and 5 day post sciatic nerve crush DRG - x9 strains.
Project description:The NuRD complex is required for efficient and timely myelination in the peripheral nervous system. ChIP-chip assays were performed on rat sciatic nerve at P15, a peak timepoint of myelination, for binding of Chd4 to genes involved in regulating myelin formation. This experiment includes two custom ChIP-chip design incorporating many genes that are dynamically regulated during myelination. The antibodies used in this platform were Chd3/4 (Santa Cruz sc-11378) Chd4 (gift from Paul Wade), Mta2 (Santa Cruz sc-9447), and Nab2 (Santa Cruz sc-22815). Chd4 ChIP samples from experimental and input samples were hybridized.
Project description:To shed light on the early processes of immune response to peripheral nerve injury, we first used genome-wide transcriptional profiling and bioinformatics (Ingenuity, NextBio) pathway analyses of the proximal (P; regenerating) and distal (D; degenerating) nerve stumps at day 1 in the sciatic nerve axotomy model in rats. We identified a number of specific immunomodulatory genes and pathways that were regulated shortly post-injury in both the P and D segments, including all members of the interleukin (IL), chemokine, tumor necrosis factor (TNF), matrix metalloproteinase (MMP), toll-like receptor (TLR), tissue inhibitor of metalloproteinase (TIMP), ion channel and myosin families. Immunomodulatory calcium-binding S100A8 and S100A9 were the top up-regulated genes in both the D and P segments. In cultured Schwann cells stimulated with the purified S100A8/A9 heterodimer we recorded a high level of similarity of the activated genes and pathways with that of the injured nerve, especially in the activation of the chemokine and cytokine gene networks that support agranulocyte and granulocyte chemotaxis, adhesion, transmigration and rolling signaling pathways. We also confirmed activation of multiple cell death related gene networks supporting TNFR1, natural killer cell receptor and death receptor apoptosis signaling in the D stump, and the gluconeogenesis/glycolysis and cytoskeletal motility signaling in the P stump, corroborated by activation of ERK, PI3K and JNK kinase pathways. As compared to the D segment, multiple additional pathways were more efficiently upregulated in the P stump, including the IL-6 and -17, MMP-9, calcium, activated agranulocyte, leukocyte rolling and glutathione-mediated detoxification signaling pathways. These data suggest that shortly after nerve injury, upregulation of S100A8/A9 is responsible for the expression and release of chemokines and cytokines by Schwann cells, necessary to generate the initial chemotactic gradient and guide the hematogenous immune cells into the injury site. Gene expression profiling of total RNAs extracted from injured and non-injured rat sciatic nerves, and primary rat Schwann cells stimulated with S100A8/A9 proteins