Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.     

Effects of facial nerve injury on mouse motoneurons lacking the p75 low-affinity neurotrophin receptor

When motoneuron axons in peripheral nerves are injured, the expression of the p75 low-affinity neurotrophin receptor (p75) increases in their cell bodies and axons, as well as in the Schwann cells undergoing Wallerian degeneration in the distal excised nerve segment. To understand the role of p75 in the events following nerve injury, we have examined the survival and regeneration of motoneurons in mice lacking the p75 receptor. In adult p75 (−/−) mice, functional recovery of whiskers movement following a facial nerve crush occurred slightly earlier than in p75 (+/+) mice, and some recovery of function over a 25-day interval following a nerve cut occurred more frequently in p75 (−/−) mice. Motoneuron profile numbers were slightly reduced in p75 (−/−) mice, and there were correspondingly fewer axons in the facial nerve. At 25 days following axotomy, profile survival in the adult p75 (−/−) mice was significantly improved compared to p75 (+/+) mice (mean 85% ± standard error of the mean 3%, n = 11 vs. 67 ± 5%, n = 11 in CD-1 mice and 68.0 ± 4%, n = 6 in balb/c mice), and significantly more regenerating axons were present in the distal facial nerve. After axotomy on postnatal day 1, there was almost total loss of motoneuron profiles in the lateral facial nucleus in p75 (+/+) mice (1.7 ± 0.3% remained, n = 5), while significantly more survived in p75 (−/−) mice (17 ± 2.5%, n = 6) . We conclude that expression of p75 in motoneurons or Schwann cells following facial nerve injury is not necessary for motoneuron survival or prompt regeneration of their axons; rather, p75 may increase their risk of dying. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 1–9, 1998     

Axons regulate Schwann cell expression of the major myelin and NGF receptor genes

The elaboration of myelin by Schwann cells is triggered by contact with appropriate peripheral axons. Among the most prominent features of this interaction is the activation and high-level expression of the genes encoding the major myelin proteins P0 and Myelin Basic Protein (MBP). Although the initial induction of these genes is thought to be dependent upon contact with axons, neither the inductive signal of the axon nor the receptor and associated second messenger system of the Schwann cell that transduces this signal has been identified. In this report, we demonstrate that expression of the P0 and MBP genes in rapidly myelinating Schwann cells is sharply reduced upon withdrawal of axons, but that this expression can be substantially restored by agents that raise the intracellular concentration of cyclic AMP. We further show that Schwann cell expression of a third gene, i.e. that encoding the Nerve Growth Factor receptor, is strongly activated by the withdrawal of axons, and that this activation is largely independent of cAMP.     

The Expression of Nerve Growth Factor Receptor on Schwann Cells and the Effect of These Cells on Regeneration of Axons in Traumatically Injured Human Spinal Cord

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Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and myelin-associated glycoprotein) in regenerating adult mouse sciatic nerve

The localization of the neural cell adhesion molecules L1, N-CAM, and the myelin-associated glycoprotein was studied by pre- and postembedding staining procedures at the light and electron microscopic levels in transected and crushed adult mouse sciatic nerve. During the first 2-6 d after transection, myelinated and nonmyelinated axons degenerated in the distal part of the proximal stump close to the transection site and over the entire length of the distal part of the transected nerve. During this time, regrowing axons were seen only in the proximal, but not in the distal nerve stump. In most cases L1 and N-CAM remained detectable at cell contacts between nonmyelinating Schwann cells and degenerating axons as long as these were still morphologically intact. Similarly, myelin-associated glycoprotein remained detectable in the periaxonal area of the degenerating myelinated axons. During and after degeneration of axons, nonmyelinating Schwann cells formed slender processes which were L1 and N-CAM positive. They resembled small-diameter axons but could be unequivocally identified as Schwann cells by chronical denervation. Unlike the nonmyelinating Schwann cells, only few myelinating ones expressed L1 and N-CAM. At the cut ends of the nerve stumps a cap developed (more at the proximal than at the distal stump) that contained S-100-negative and fibronectin-positive fibroblast-like cells. Most of these cells were N-CAM positive but always L1 negative. Growth cones and regrowing axons expressed N-CAM and L1 at contact sites with these cells. Regrowing axons of small diameter were L1 and N-CAM positive where they made contact with each other or with Schwann cells, while large-diameter axons were only poorly antigen positive or completely negative. 14 d after transection, when regrowing axons were seen in the distal part of the transected nerve, regrowing axons made L1- and N-CAM-positive contacts with Schwann cells. When contacting basement membrane, axons were rarely found to express L1 and N-CAM. Most, if not all, Schwann cells associated with degenerating myelin expressed L1 and N-CAM. In crushed nerves, the immunostaining pattern was essentially the same as in the cut nerve. During formation of myelin, the sequence of adhesion molecule expression was the same as during development: L1 disappeared and N-CAM was reduced on myelinating Schwann cells and axons after the Schwann cell process had turned approximately 1.5 loops around the axon. Myelin-associated glycoprotein then appeared both periaxonally and on the turning loops of Schwann cells in the uncompacted myelin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of reciprocally regulated gene modules in regenerating dorsal root ganglion neurons and activated peripheral or central nervous system glia

Differential gene expression in the rat after injury of dorsal root ganglion neurons in vivo, and simulation injury of Schwann cells and oligodendrocytes in vitro was analyzed using high-density cDNA microarrays. The analyses were carried out to study the genetic basis of peripheral nerve regeneration, and to compare gene regulation in glia of the central (oligodendrocyte) and peripheral (Schwann cell) nervous systems. The genes showing significant differential regulation in the three study groups represented all aspects of cellular metabolism. However, two unexpected observations were made. Firstly, a number of identical genes were differentially regulated in activated Schwann cells, activated oligodendrocytes and regenerating DRG neurons. Specifically, a group of 113 out of 210 genes that were down-regulated in Schwann cells upon lipopolysaccharide (LPS) treatment, were identical to genes up-regulated in the injured, regenerating DRG. Furthermore, a group of 53 out of 71 genes that were down-regulated in interferon gamma (IFN-gamma)/LPS-activated oligodendrocytes, were identical to genes up-regulated in the DRG neurons. Finally, 22 genes were common to these three groups, i.e., down-regulated in activated oligodendrocytes, down-regulated in activated Schwann cells, and up-regulated in regenerating DRG neurons. Secondly, a group of 16 cell-cycle and proliferation-related genes were up-regulated in the DRG following sciatic nerve crush, despite the absence of cells undergoing mitosis in the DRG, or any significant presence of apoptosis-related gene expression. Therefore, it appears that in these three cell types, large sets of genes are reciprocally regulated upon injury and/or activation. This suggests that the activation of the injury-related gene expression program in cell derivatives of the neuroectoderm involves, in part, highly conserved genetic elements.     

Deletion of p75NTR impairs regeneration of peripheral nerves in mice

AimsAfter peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves.Main methodsIn p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry.Key findingsThe results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice.SignificanceOur data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.     

Visualizing Peripheral Nerve Regeneration by Whole Mount Staining

Peripheral nerve trauma triggers a well characterised sequence of events both proximal and distal to the site of injury. Axons distal to the injury degenerate, Schwann cells convert to a repair supportive phenotype and macrophages enter the nerve to clear myelin and axonal debris. Following these events, axons must regrow through the distal part of the nerve, re-innervate and finally are re-myelinated by Schwann cells. For nerve crush injuries (axonotmesis), in which the integrity of the nerve is maintained, repair may be relatively effective whereas for nerve transection (neurotmesis) repair will likely be very poor as few axons may be able to cross between the two parts of the severed nerve, across the newly generated nerve bridge, to enter the distal stump and regenerate. Analysing axon growth and the cell-cell interactions that occur following both nerve crush and cut injuries has largely been carried out by staining sections of nerve tissue, but this has the obvious disadvantage that it is not possible to follow the paths of regenerating axons in three dimensions within the nerve trunk or nerve bridge. To try and solve this problem, we describe the development and use of a novel whole mount staining protocol that allows the analysis of axonal regeneration, Schwann cell-axon interaction and re-vascularisation of the repairing nerve following nerve cut and crush injuries.     

Schwann cells in the regenerating fish optic nerve: Evidence that CNS axons,not the glia,determine when myelin formation begins

Fish optic nerve fibres quickly regenerate after injury, but the onset of remyelination is delayed until they reach the brain. This recapitulates the timetable of CNS myelinogenesis during development in vertebrate animals generally, and we have used the regenerating fish optic nerve to obtain evidence that it is the axons, not the myelinating glial cells, that determine when myelin formation begins. In fish, the site of an optic nerve injury becomes remyelinated by ectopic Schwann cells of unknown origin. We allowed these cells to become established and then used them as reporters to indicate the time course of pro-myelin signalling during a further round of axonal outgrowth following a second upstream lesion. Unlike in the mammalian PNS, the ectopic Schwann cells failed to respond to axotomy and to the initial outgrowth of new optic axons. They only began to divide after the axons had reached the brain. Shortly afterwards, small numbers of Schwann cells began to leave the dividing pool and form myelin sheaths. More followed gradually, so that by 3 months remyelination was almost completed and few dividing cells were left. Moreover, remyelination occurred synchronously throughout the optic nerve, with the same time course in the pre-existing Schwann cells, the new ones that colonised the second injury, and the CNS oligodendrocytes elsewhere. The optic axons are the only common structures that could synchronise myelin formation in these disparate glial populations. The responses of the ectopic Schwann cells suggest that they are controlled by the regenerating optic axons in two consecutive steps. First, they begin to proliferate when the growing axons reach the brain. Second, they leave the cell cycle to differentiate individually at widely different times during the ensuing 2 months, during the critical period when the initial rough pattern of axon terminals in the optic tectum becomes refined into an accurate map. We suggest that each axon signals individually for myelin ensheathment once it completes this process.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

Transcriptional profile of the human peripheral nervous system by serial analysis of gene expression

The peripheral nerve contains both nonmyelinating and myelinating Schwann cells. The interactions between axons, surrounding myelin, and Schwann cells are thought to be important for the correct functioning of the nervous system. To get insight into the genes involved in human myelination and maintenance of the myelin sheath and nerve, we performed a serial analysis of gene expression of human sciatic nerve and cultured Schwann cells. In the sciatic nerve library, we found high expression of genes encoding proteins related to lipid metabolism, the complement system, and the cell cycle, while cultured Schwann cells showed mainly high expression of genes encoding extracellular matrix proteins. The results of our study will assist in the identification of genes involved in maintenance of myelin and peripheral nerve and of genes involved in inherited peripheral neuropathies.     

Laminin A, B1, and B2 Chain Gene Expression in Transected and Regenerating Nerves: Regulation by Axonal Signals

Abstract: Laminin A, B1, and B2 chain mRNA levels in degenerating and regenerating mouse sciatic nerves were examined using northern blot analysis. In normal intact nerves, B1 and B2 mRNA steady-state levels were high, but when the nerves were crushed, the steady-state levels of B1 and B2 mRNA per milligram wet tissue weight of the distal segments of the nerves increased five- to eightfold over that of control levels as the total RNA and β-actin mRNA levels increased, suggesting that these increases were the consequence of Schwann cell proliferation after axotomy. When the steady-state levels of B1 and B2 mRNA were normalized as the ratio to total RNA or β-actin mRNA levels, however, they drastically decreased to about 20% of the normal nerve levels in the nerve segments distal to both the crush and transaction sites 1 day after injury. In the crushed nerves, B1 and B2 mRNA levels gradually increased as the regenerating nerves arrived at the distal segments and reestablished normal axon–Schwann cell contact, and then returned to normal levels on the 21 st day. In the transected nerves, where Schwann cells continued to be disconnected from axons, both B1 and B2 mRNA levels remained low. Cultured Schwann cells expressed detectable levels of B1 and B2 chain mRNA which significantly increased when the cells were cocultured with sensory neurons. However, mRNA for A chain was not detectable in the normal, axotomized nerves or in cultured Schwann cells. These data indicate that Schwann cells express laminin B1 and B2 chain mRNA that are up-regulated by axonal or neuronal contact, but they do not express A chain mRNA.     

A role for Nogo receptor in macrophage clearance from injured peripheral nerve

We report a role for Nogo receptors (NgRs) in macrophage efflux from sites of inflammation in peripheral nerve. Increasing numbers of macrophages in crushed rat sciatic nerves express NgR1 and NgR2 on the cell surface in the first week after injury. These macrophages show reduced binding to myelin and MAG in vitro, which is reversed by NgR siRNA knockdown and by inhibiting Rho-associated kinase. Fourteen days after sciatic nerve crush, regenerating nerves with newly synthesized myelin have fewer macrophages than cut/ligated nerves that lack axons and myelin. Almost all macrophages in the cut/ligated nerves lie within the Schwann cell basal lamina, while in the crushed regenerating nerves the majority migrate out. Furthermore, crush-injured nerves of NgR1- and MAG-deficient mice and Y-27632-treated rats show impaired macrophage efflux from Schwann cell basal lamina containing myelinated axons. These data have implications for the resolution of inflammation in peripheral nerve and CNS pathologies.     

Expression of P30, a protein with adhesive properties, in Schwann cells and neurons of the developing and regenerating peripheral nerve

P30 is a heparin-binding protein with adhesive and neurite outgrowth-promoting properties present at high levels in the developing rat central nervous system (Rauvala, H., and R. Pihlaskari. 1987 J. Biol. Chem. 262:16625-16635). Partial sequencing of p30 has revealed homology or identity with HMG-1 (Rauvala, H., J. Merenmies, R. Pihlaskari, M. Korkolainen, M.-L. Huhtala, and P. Panula. 1988. J. Cell Biol. 107:2292-2305), a 28-kD protein that was originally purified from the thymus (Goodwin, G.H., C. Sanders, and E. W. Johns. 1973. Eur. J. Biochem. 38:14-19) which binds DNA in vitro. We have analyzed the distribution of p30 in the developing rat peripheral nervous system (PNS). P30 was detected by immunohistochemistry and Western blot analysis using antibodies raised against intact p30 and against a synthetic peptide corresponding to the amino terminus of the p30 molecule. P30 was localized to nonnuclear compartments of neurons and peripheral glial cells (Schwann cells). P30 immunoreactivity of PNS neurons persisted into adulthood. In contrast, Schwann cell staining decreased after the second postnatal week and was not detectable in adult animals. Neuron-Schwann cell contact was correlated with diminished p30 levels in Schwann cells. Schwann cells of the normal adult sciatic nerve did not express p30; however, when deprived of axonal contact by nerve transection, the Schwann cells of the distal nerve stained intensely for p30. In addition, when Schwann cells and dorsal root ganglion neurons were grown in coculture, Schwann cells that were associated with neurites were not as intensely stained by anti-p30 as Schwann cells that were not in contact with neurons. The pattern of p30 expression during development and regeneration, and its apparent regulation by cell-cell contact suggests that p30 plays a role in the interaction between neurons and Schwann cells during morphogenesis of peripheral nerves.     

NGF receptor-mediated reduction in axonal NGF uptake and retrograde transport following sciatic nerve injury and during regeneration.

Injury to the rat sciatic nerve leads to the induction of nerve growth factor (NGF) receptors on the denervated Schwann cells and their disappearance on the regenerating axons of the axotomized, normally NGF-sensitive sensory and sympathetic neurons. This disappearance in the axonal expression and retrograde transport of NGF receptors is associated with a similarly dramatic reduction in the axonal uptake and retrograde transport of NGF following axotomy and during regeneration. In view of the massive NGF synthesis occurring in the injured nerve, these results suggest that, while sensory and sympathetic neurons are the primary targets of NGF in the normal peripheral nervous system, the denervated Schwann cells may become its primary target in the aftermath of nerve injury.     

Axonal Transport of Choline Lipids in Normal and Regenerating Rat Sciatic Nerve

Abstract: Biochemical methods were used to study the time course of transport of choline phospholipids (labeled by the injection of [³H]choline into the ventral horn of the lumbar spinal cord) in rat sciatic nerve. Autoradiographic methods were used to localize the transported lipid within motor axons. Transported phospholipid, primarily phosphatidylcholine, present in the nerve at 6 h, continued to accumulate over the following 12 days. No discrete waves of transported lipid were observed (a small wave of radioactive phospholipid moving at the high rate would have been missed); the amounts of radioactive lipid increased uniformly along the entire sciatic nerve. In light-microscope autoradiographs, a class of large-caliber axons, presumably motor axons, retained the labeled lipid. Some lipid, even at 6 h, was seen within the myelin sheaths. Later, the labeling of the myelin relative to axon increased. The continued accumulation of choline phospholipids in the axons probably signifies their prolonged release from cell bodies and their retention in various axonal membranes, including the axolemma. The build-up of these phospholipids in myelin probably represents their transfer from the axons to the myelin sheaths surrounding them. When nerves are crushed and allowed to regenerate for 6 or 12 days, choline phospholipids transported during these times enter the regenerating nerve. In light and electron microscope autoradiographs, transported lipid was seen to be localized primarily in the regenerating axons. However, grains overlay the adjacent Schwann cell cytoplasm, indicating transported lipids were transferred from the regenerating axons to the associated Schwann cells. In addition, some cells not associated with growing axons were labeled, suggesting that phosphatidylcholine and possibly acetylcholine, carried to the regenerating axons by axonal transport, were actively metabolized in the terminal, with released choline label being used by other cells. These results demonstrate that axonal transport supplies mature and growing axons and their glial cells with choline phospholipids.