18 research outputs found
Matrix Metalloproteinase Inhibition Enhances the Rate of Nerve Regeneration In Vivo by Promoting Dedifferentiation and Mitosis of Supporting Schwann Cells
Spinal Glia Division Contributes to Conditioning LesionβInduced Axon Regeneration Into the Injured Spinal Cord
Immunodominant fragments of myelin basic protein initiate T cell-dependent pain
AbstractBackgroundThe myelin sheath provides electrical insulation of mechanosensory AΞ²-afferent fibers. Myelin-degrading matrix metalloproteinases (MMPs) damage the myelin sheath. The resulting electrical instability of AΞ²-fibers is believed to activate the nociceptive circuitry in AΞ²-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 resultsUsing 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-Ξ± 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 naΓ―ve 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 naΓ―ve 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.ConclusionsThese data implicate MBP as a novel mediator of pain. Furthermore, the action of MMPs expressed within 1βday 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
The MMP-9/TIMP-1 Axis Controls the Status of Differentiation and Function of Myelin-Forming Schwann Cells in Nerve Regeneration
Background: Myelinating Schwann cells (mSCs) form myelin in the peripheral nervous system. Because of the works by us and others, matrix metalloproteinase-9 (MMP-9) has recently emerged as an essential component of the Schwann cell signaling network during sciatic nerve regeneration. Methodology/Principal Findings: In the present study, using the genome-wide transcriptional profiling of normal and injured sciatic nerves in mice followed by extensive bioinformatics analyses of the data, we determined that an endogenous, specific MMP-9 inhibitor [tissue inhibitor of metalloproteinases (TIMP)-1] was a top up-regulated gene in the injured nerve. MMP-9 capture followed by gelatin zymography and Western blotting of the isolated samples revealed the presence of the MMP-9/TIMP-1 heterodimers and the activated MMP-9 enzyme in the injured nerve within the first 24 h post-injury. MMP-9 and TIMP-1 co-localized in mSCs. Knockout of the MMP-9 gene in mice resulted in elevated numbers of de-differentiated/immature mSCs in the damaged nerve. Our comparative studies using MMP-9 knockout and wild-type mice documented an aberrantly enhanced proliferative activity and, accordingly, an increased number of post-mitotic Schwann cells, short internodes and additional nodal abnormalities in remyelinated nerves of MMP-9 knockout mice. These data imply that during the first days post-injury MMP-9 exhibits a functionally important anti-mitogenic activity in the wildtype mice. Pharmacological inhibition of MMP activity suppressed the expression of Nav1.7/1.8 channels in the crushe
MMP-9/TIMP-1 relationship in the sciatic nerve.
<p><b>A,</b> Left, Multiple murine MMP-9 species are detected by gelatin zymography. The total extracts (10 Β΅g total protein each) of the naΓ―ve (N), distal (D) and proximal (P) segments of the transected nerves were analyzed by gelatin zymography. Right lane, the purified MMP-9 sample isolated from an aliquot (25 Β΅g total protein) of the proximal nerve extract (P) is provided as a reference. Middle, the purified MMP-9 samples isolated from the naΓ―ve (N) and crushed (I) nerve (50 Β΅g and 25 Β΅g total protein, respectively) 1, 3, 6 and 24 h post-injury were analyzed by gelatin zymography. Note a 2Γ protein loading of the naΓ―ve samples relative to the injured samples. Right, 2% agarose gel-electrophoresis of the RT-PCR products. The MMP-9 and TIMP-1 genes were amplified using the naive and crushed samples in the RT-PCR reactions. The unique primers were used in a 35-cycle PCR reaction to generate a 153 bp MMP-9 fragment, and 129 bp and 141 bp TIMP-1 fragments. GAPDH was used for sample normalization. Right lane (cDNA), the first strand cDNA control synthesized from the total mouse spleen RNA in the RT-PCR reaction. <b>B,</b> High molecular weight forms of MMP-9 in the injured sciatic nerve. The reduced (+DTT) and unreduced (βDTT) purified MMP-9 samples (15 Β΅g total protein each) isolated from the injured sciatic nerve 0, 1, 3, 6 and 24 h post-injury were analyzed by Western blotting with the MMP-9 antibody. <b>C,</b> Immunofluorescence for MMP-9 and TIMP-1 co-localize in mSCs of crushed nerve 24 h post-injury. Upper panel, MMP-9 (green) and TIMP-1 (red) co-localize in crescent structures of mSCs (arrows). Circular structures are MMP-9 but not TIMP-1 reactive (arrowheads). Lower panel, MMP-9 (red) localizes in the cytoplasm of mSCs, marked with MBP (green). MMP-9 is also detected in axoplasm of mSCs (arrows). Scale bar, 10 Β΅m.</p
TIMP-1: MMP-9 ratio in the nerve samples.
<p>The normalized signal intensity of the MMP-9 and TIMP-1 genes was derived from the genome-wide transcriptional profiling data. Note the dramatic increase of the TIMP-1βΆ MMP-9 signal intensity ratio on days 1 and 5 post-injury (D1 and D5, respectively) in the peripheral nerve sample. DRG, dorsal root ganglion, day 5 post-injury.</p
MNCV in MMP-9<sup>β/β</sup> and MMP-9<sup>+/+</sup> mice.
<p>Motor nerve conduction velocity (MNCV) expressed as mean Β± SEM (nβ=β7/group) analyzed with a two-tailed, unpaired t-test or an unpaired t-test with Welch's correction when variances were unequal. NS, not significant.</p
MMPi therapy blocks Na<sub>v</sub>1.7 and 1.8 channel induction in nerve.
<p><i>Taqman</i> qPCR for Na<sub>v</sub>1.8 and 1.7 in normal or crushed (day 5) rat sciatic nerve normalized to GAPDH. GM6001 or vehicle was administered i.p. immediately and then daily at days 1β5 after crush. The mean fold increase Β± SEM to normal nerves (*, p<0.05; **, p<0.01, ANOVA, Tukey's post-hoc test of nβ=β6/group). NS, not significant.</p
Heatmap of the genome-wide transcriptional profiling data of the murine sciatic nerve and the corresponding L4/5 DRG from the same animals.
<p>Red and blue correspond to the high and the low expression levels, respectively. Color map inset shows the signal intensity scale. Only the genes with the BβΆA ratio >4 are shown in a descending order. A, sham nerve; B, nerve day 1 post-transection; C, nerve day 5 post-transection; D, DRG corresponding to the sham-operated nerve; E, DRG corresponding to day 5 post-transection. The green dots point to TIMP-1, TNC, NGAL, CD44, and MMP-9 induced in the injured nerve, and MMP-13 in the DRG. DRG, dorsal root ganglia.</p
Top 50 upregulated genes in transected nerve.
<p>The fold-increase was calculated in transected (distal and proximal, pooled) relative to the sham-operated nerve samples. TIMP-1, tenascin (Tnc), Cd44 and MMP-16 are in bold. DRG, dorsal root ganglia.</p