LITAF (SIMPLE) regulates Wallerian degeneration after injury but is not essential for peripheral nerve development and maintenance: Implications for Charcot-Marie-Tooth disease

Missense mutations affecting the LITAF gene (also known as SIMPLE) lead to the dominantly inherited peripheral neuropathy Charcot‐Marie‐Tooth disease type 1C (CMT1C). In this study, we sought to determine the requirement of Litaf function in peripheral nerves, the only known affected tissue in CMT1C. We reasoned that this knowledge is a prerequisite for a thorough understanding of the underlying disease mechanism with regard to potential contributions by Litaf loss of function. In addition, we anticipated to obtain valuable information about the basic function of the Litaf protein in peripheral nerves. To address these issues, we generated mice without Litaf expression using gene disruption in embryonic stem cells and analyzed Litaf‐deficient peripheral nerves during development, in maintenance, and after injury. Our results show that Litaf function is not absolutely required for peripheral nerve development and maintenance. In injured nerves, however, we found that lack of Litaf led to increased numbers of macrophages during Wallerian degeneration, accelerated myelin destruction, and the emergence of more axonal sprouts. Consistent with these data, the migration of Litaf‐deficient macrophages was increased upon chemokine stimulation. We conclude that loss of Litaf function is unlikely to be a major contributor to CMT1C, but modulating effects of macrophages need to be considered in the etiology of the disease. © 2012Wiley Periodicals, Inc.     

Myelin phagocytosis and remyelination of macrophage-enriched central nervous system aggregate cultures.

An increased level of myelin basic protein (MBP) degradation peptide 80-89, representative of myelin breakdown, is detected in myelinating foetal rat brain aggregate cultures supplemented with peritoneal macrophages at a time coinciding with the onset of myelination. During the period of myelination, the proportion of activated macrophages/microglia in the aggregates decreases, accompanied by a reduction in the content of MBP degradation products. During the recovery period following a demyelinating episode, the rate of MBP synthesis in antibody-treated standard aggregates was greater than in their medium controls. However, the rate of MBP accumulation was not as efficient in macrophage-enriched aggregates and was associated with persistently raised MBP peptide levels. Thus, as occurs in multiple sclerosis lesions, attempts at remyelination appear to be counterbalanced by macrophage-mediated demyelination, with the continued presence of degraded myelin rendering a local environment that is not fully conducive to remyelination.     

Glial cell transplanatation and remyelination of the central nervous system

Glial cell transplantation has proved to be a powerful tool in the study of glial cell biology. The extent of myelination achieved by transplanting myelin-producing cells into the CNS of myelin mutants, or into focal demyelinating lesions has raised hope that such a strategy may have therapeutic applications. Oligodendrocytes or Schwann cells could be used for repair. It is likely that the immature stages of the oligodendrocyte lineage have the best phenotypic characteristics for remyelination when transplanted, either as primary cells or as immortalized cells or cell lines. Prior culturing and growth factor treatment provides opportunities to expand cell populations before transplantation as dissociated cell preparations. Cell lines are attractive candidates for transplantation, but the risk of transformation must be monitored. The application of this technique to human myelin disorders may requier proof that migration, division and stable remyelination of axons by the tranplanted cells can occur in the presence of gliosis and inflammation.     

The fatty acid binding protein FABP7 is required for optimal oligodendrocyte differentiation during myelination but not during remyelination

The major constituents of the myelin sheath are lipids, which are made up of fatty acids (FAs). The hydrophilic environment inside the cells requires FAs to be bound to proteins, preventing their aggregation. Fatty acid binding proteins (FABPs) are one class of proteins known to bind FAs in a cell. Given the crucial role of FAs for myelin sheath formation we investigated the role of FABP7, the major isoform expressed in oligodendrocyte progenitor cells (OPCs), in developmental myelination and remyelination. Here, we show that the knockdown of Fabp7 resulted in a reduction of OPC differentiation in vitro. Consistent with this result, a delay in developmental myelination was observed in Fabp7 knockout animals. This delay was transient with full myelination being established before adulthood. FABP7 was dispensable for remyelination, as the knockout of Fapb7 did not alter remyelination efficiency in a focal demyelination model. In summary, while FABP7 is important in OPC differentiation in vitro, its function is not crucial for myelination and remyelination in vivo.     

Differential expression of interleukin-10 mRNA in Wallerian degeneration and immune-mediated inflammation of the rat peripheral nervous system

Interleukin-10 (IL-10) is a potent immunosuppressant cytokine which downregulates MHC class II antigen expression and inflammatory cytokine production. In this study we localized mRNA for IL-10 in the rat peripheral nervous system (PNS) by nonradioactive in situ hybridization using a digoxygenin-labeled riboprobe specific for rat IL-10. IL-10 mRNA was expressed by some Schwann cells (SCs) in the normal sciatic nerve. During Wallerian degeneration, SCs strongly expressed IL-10 mRNA between days 2 and 4 after transection. By day 14 only occasional cells were positive for IL-10 mRNA. The vast majority of ED1-positive macrophages were IL-10 negative after axotomy. Contrastingly, infiltrating macrophages expressed IL-10 mRNA coincident with beginning clinical recovery in experimental autoimmune neuritis (EAN), the rat model of human Guillain-Barré syndrome. Our data suggest that SCs provide a constitutive immunosuppressant system in the PNS. In EAN additional macrophage-derived IL-10 may be important for the resolution of the T cell-mediated immune response. © 1996 Wiley-Liss, Inc.     

Induction of myelin genes during peripheral nerve remyelination requires a continuous signal from the ingrowing axon

The effect of a permanent transection on myelin gene expression in a regenerating sciatic nerve and in an adult sciatic nerve was compared to establish the degree of axonal control exerted upon Schwann cells in each population. First, the adult sciatic nerve was crushed, and the distal segment allowed to regenerate. At 12 days post-crush, the sciatic nerve was transected distal to the site of crush to disrupt the Schwann cell-axonal contacts that had reformed. Messenger RNA (mRNA) levels coding for five myelin proteins were assayed in the distal segment of the crush-transected nerve after 9 days and were compared to corresponding levels in the distal segments of sciatic nerves at 21 days post-crush and 21 days post-transection using Northern blot and slot-blot analysis. Levels of mRNAs found in the distal segment of the transected and crush-transected nerve suggested that Schwann cells in the regenerating nerve and in the mature adult nerve are equally responsive to axonal influences. The crush-transected model allowed the genes that were studied to be classified according to their response to Schwann cell-axonal contact. The levels of mRNAs were (1) down-regulated to basal levels (PO and MBP mRNAs), (2) down-regulated to undetectable levels (myelin-associated glycoprotein mRNAs), (3) upregulated (mRNAs encoding 2′3′-cyclic nucleotide phosphodiesterase and β-actin), or (4) not stringently controlled by the removal of Schwann cell-axonal contact (proteolipid protein mRNAs). This novel experimental model has thus provided evidence that the expression of some of the important myelin genes during peripheral nerve regeneration is dependent on continuous signals from the ingrowing axons. © 1993 Wiley-Liss, Inc.     

Prostaglandin D2 synthase modulates macrophage activity and accumulation in injured peripheral nerves

We have previously reported that prostaglandin D2 Synthase (L-PGDS) participates in peripheral nervous system (PNS) myelination during development. We now describe the role of L-PGDS in the resolution of PNS injury, similarly to other members of the prostaglandin synthase family, which are important for Wallerian degeneration (WD) and axonal regeneration. Our analyses show that L-PGDS expression is modulated after injury in both sciatic nerves and dorsal root ganglia neurons, indicating that it might play a role in the WD process. Accordingly, our data reveals that L-PGDS regulates macrophages phagocytic activity through a non-cell autonomous mechanism, allowing myelin debris clearance and favoring axonal regeneration and remyelination. In addition, L-PGDS also appear to control macrophages accumulation in injured nerves, possibly by regulating the blood–nerve barrier permeability and SOX2 expression levels in Schwann cells. Collectively, our results suggest that L-PGDS has multiple functions during nerve regeneration and remyelination. Based on the results of this study, we posit that L-PGDS acts as an anti-inflammatory agent in the late phases of WD, and cooperates in the resolution of the inflammatory response. Thus, pharmacological activation of the L-PGDS pathway might prove beneficial in resolving peripheral nerve injury.     

Short‐term low‐frequency electrical stimulation enhanced remyelination of injured peripheral nerves by inducing the promyelination effect of brain‐derived neurotrophic factor on Schwann cell polarization

Electrical stimulation (ES) has been found to aid repair of nerve injuries and have been shown to increase and direct neurite outgrowth during stimulation. However, the effect of ES on peripheral remyelination after nerve damage has been investigated less well, and the mechanism underlying its action remains unclear. In the present study, the crush‐injured sciatic nerves in rats were subjected to 1 hr of continuous ES (20 Hz, 100 μsec, 3 V). Electron microscopy and nerve morphometry were performed to investigate the extent of regenerated nerve myelination. The expression profiles of P0, Par‐3, and brain‐derived neurotrophic factor (BDNF) in the injuried sciatic nerves and in the dorsal root ganglion neuron/Schwann cell cocultures were examined by Western blotting. Par‐3 localization in the sciatic nerves was determined by immunohistochemistry to demonstrate Schwann cell polarization during myelination. We reported that 20‐Hz ES increased the number of myelinated fibers and the thickness myelin sheath at 4 and 8 weeks postinjury. P0 level in the ES‐treated groups, both in vitro and in vivo, was enhanced compared with the controls. The earlier peak of Par‐3 in the ES‐treated groups indicated an earlier initiation of Schwann cell myelination. Additionally, ES significantly elevated BDNF expression in nerve tissues and in cocultures. ES on the site of nerve injury potentiates axonal regrowth and myelin maturation during peripheral nerve regeneration. Furthermore, the therapeutic actions of ES on myelination are mediated via enhanced BDNF signals, which drive the promyelination effect on Schwann cells at the onset of myelination. © 2010 Wiley‐Liss, Inc.     

The chemokine receptor CCR2 is involved in macrophage recruitment to the injured peripheral nervous system

Wallerian degeneration is one of the most elementary reactions of the nervous system after transection of axons, leading to the recruitment of mononuclear cells from the systemic circulation. However, the exact mechanisms regulating this cell invasion have not yet been clarified in detail. Chemokines and their receptors play a central role in leukocyte trafficking, in particular the chemokine MCP-1 has been strongly implicated in macrophage recruitment to the injured nervous system. The present study investigates the course of Wallerian degeneration after transection of the sciatic nerve in mice deficient in two chemokine receptors: CCR2, the main receptor for MCP-1, and CCR5, a marker for Th1 T lymphocytes but also present on macrophages. The number of invading macrophages was determined by immunocytochemistry for three typical macrophage antigens (F4/80, Mac-1, LFA-1). The chemokine receptor CCR2 was expressed by infiltrating cells in the transected nerve stumps. Macrophage invasion was significantly impaired in CCR2-knockout mice when compared with wildtype controls and CCR5-deficient mice. Subsequently, there was a corresponding decrease in myelin phagocytosis due to the reduced invasion of phagocytic macrophages. These data demonstrate the involvement of the chemokine receptor CCR2 in macrophage recruitment to the injured nervous system.     

Comparison of Schwann cell and sciatic nerve transcriptomes indicates that mouse is a valid model for the human peripheral nervous system

High-throughput gene expression analyses of murine models of the peripheral nervous system (PNS), and its cellular components, have yielded enormous amounts of expression data of the PNS in various conditions. These data provided clues for future research directions to further decipher this complex organ in relation to acquired and inherited PNS diseases. Various studies addressing the validity of mouse models for human conditions in other tissues and cell types have indicated that in many cases the mouse model only poorly represents the human situation. To determine how well the mouse can serve as model to study the biological processes occurring in the PNS, we compared the gene expression profiles that we generated for mouse and human sciatic nerve and cultured Schwann cells derived thereof. A two-way analysis based on the differentially expressed genes between the sciatic nerve and the cultured Schwann cell, and which takes into account the differential expression between mouse and man, indicates that the human PNS is well represented by that of the mouse in terms of the "biological processes" ontology.     

Local production of serum amyloid a is implicated in the induction of macrophage chemoattractants in Schwann cells during wallerian degeneration of peripheral nerves

The elevation of serum levels of serum amyloid A (SAA) has been regarded as an acute reactive response following inflammation and various types of injuries. SAA from the liver and extrahepatic tissues plays an immunomodulatory role in a variety of pathophysiological conditions. Inflammatory cytokines in the peripheral nerves have been implicated in the Wallerian degeneration of peripheral nerves after injury and in certain types of inflammatory neuropathies. In the present study, we found that a sciatic nerve axotomy could induce an increase of SAA1 and SAA3 mRNA expression in sciatic nerves. Immunohistochemical staining showed that Schwann cells are the primary sources of SAA production after nerve injury. In addition, interleukin‐6‐null mice, but not tumor necrosis factor‐α‐null mice showed a defect in the production of SAA1 in sciatic nerve following injury. Dexamethasone treatment enhanced the expression and secretion of SAA1 and SAA3 in sciatic nerve explants cultures, suggesting that interleukin‐6 and corticosteroids might be major regulators for SAA production in Schwann cells following injury. Moreover, the stimulation of Schwann cells with SAA1 elicited the production of the macrophage chemoattractants, Ccl2 and Ccl3, in part through a G‐protein coupled receptor. Our findings suggest that locally produced SAA might play an important role in Wallerian degeneration after peripheral nerve injury. © 2012 Wiley Periodicals, Inc.     

The neurotrophin receptor p75NTR in Schwann cells is implicated in remyelination and motor recovery after peripheral nerve injury

The function of the p75(NTR) neurotrophin receptor (p75(NTR)) in nervous system regeneration is still controversial. Part of that controversy may be due to the fact that p75(NTR) is expressed by both neuronal and glial cell types and may have very distinct and even contradictory roles in each population. In this study, to elucidate the in vivo function of p75(NTR) in Schwann cells during remyelination after peripheral nerve injury, we established a new animal model for p75(NTR)-deficient Schwann cell transplantation in nude mice. We performed quantitative assessments of the functional, histological, and electrophysiological recovery after sciatic nerve injury, and compared them with those of the p75(NTR)(+/+) Schwann cell transplanted animals. At 7-10 weeks after injury, the motor recovery in the p75(NTR)(-/-) Schwann cell transplanted animals was significantly impaired compared with that in the p75(NTR)(+/+) Schwann cell transplanted animals. The lower number of the retrogradely labeled motoneurons and the hypomyelination in the p75(NTR)(-/-) Schwann cell transplanted animals were evident at 6 and 10 weeks after injury. At 10 weeks after injury, the radial growth in the axon caliber was also impaired in the p75(NTR)(-/-) Schwann cell transplanted animals. Measurement of the amount of myelin proteins and the nerve conduction velocity at 10 weeks after injury reflected these results. In summary, the p75(NTR) expression in Schwann cells is important for remyelination process, and the motor recovery after injury is impaired due to impaired axonal growth, remyelination, and radial growth in the axon calibers.     

Tenascin-R is expressed by Schwann cells in the peripheral nervous system

The extracellular matrix glycoprotein tenascin-R (TN-R) has been implicated in a variety of cell-matrix interactions involved in the molecular control of axon guidance and neural cell migration during development and regeneration of the central nervous system (CNS). Whereas TN-R is amply expressed in the early postnatal and adult mammalian CNS, the protein has so far not been detected in different compartments of the peripheral nervous system (PNS). Here we provide first evidence that TN-R (predominantly TN-R 160 isoform) is transiently expressed in the sciatic nerve of late embryonic (E14-18) and neonatal mice, while at later developmental stages, both protein and mRNA are downregulated. In vitro, TN-R protein was found to be expressed by both undifferentiated and neuronally differentiated PC12 cells and by L1-positive Schwann cells (SC), but not by other neural and non-neural cell types in cell cultures derived from embryonic (E17/18) hindlimbs and neonatal sciatic nerves. In the developing PNS, TN-R expression correlated with axon growth and SC migration during the period of skeletal muscle innervation. Based on different in vitro approaches, we found that the substrate-bound glycoprotein selectively inhibits the fibronectin-dependent: (1) neurite outgrowth from dorsal root ganglion neurons (strongly expressing alpha5beta1 integrin and the disialoganglioside GD3) by a ganglioside-sensitive signaling mechanism; and (2) migration of primary myoblasts and other non-neuronal cells in a ganglioside-independent manner. Our findings suggest the functional role of TN-R in PNS pattern formation during distinct stages of axon pathfinding and skeletal muscle innervation.     

Schwann cell‐derived exosomes enhance axonal regeneration in the peripheral nervous system

Axonal regeneration in the peripheral nervous system is greatly supported by Schwann cells (SCs). After nerve injury, SCs dedifferentiate to a progenitor‐like state and efficiently guide axons to their original target tissues. Contact and soluble factors participate in the crosstalk between SCs and axons during axonal regeneration. Here we show that dedifferentiated SCs secrete nano‐vesicles known as exosomes which are specifically internalized by axons. Surprisingly, SC‐derived exosomes markedly increase axonal regeneration in vitro and enhance regeneration after sciatic nerve injury in vivo. Exosomes shift the growth cone morphology to a pro‐regenerating phenotype and decrease the activity of the GTPase RhoA, involved in growth cone collapse and axon retraction. Altogether, our work identifies a novel mechanism by which SCs communicate with neighboring axons during regenerative processes. We propose that SC exosomes represent an important mechanism by which these cells locally support axonal maintenance and regeneration after nerve damage. GLIA 2013;61:1795–1806     

Expression of protein 4.1G in Schwann cells of the peripheral nervous system

The membrane-associated cytoskeletal proteins, including protein 4.1 family, play important roles in membrane integrity, protein targeting, and signal transduction. Although protein 4.1G (4.1G) is expressed ubiquitously in mammalian tissues, it can have very discrete distributions within cells. The present study investigated the expression and distributions of 4.1G in rodent sciatic nerve. Northern and Western blot analysis detected abundant 4.1G mRNA and protein in rat sciatic nerve extracts. Immunohistochemical staining with a 4.1G-specific antibody and double immunolabeling with E-cadherin, betaIV spectrin, and connexin 32 detected 4.1G in paranodal loops, Schmidt-Lanterman incisures, and periaxonal, mesaxonal, and abaxonal membranes of rodent sciatic nerve. Immunoelectron microscopy confirmed the immunodistribution of 4.1G in Schwann cells. In developing mouse sciatic nerves, 4.1G was diffusely distributed in immature Schwann cells and gradually localized at paranodes, incisures, and periaxonal and mesaxonal membranes during their maturation. These data support the concept that 4.1G plays an important role in the membrane expansion and specialization that occurs during formation and maintenance of myelin internodes in the peripheral nervous system.     

Sodium‐dependent Vitamin C transporter 2 deficiency impairs myelination and remyelination after injury: Roles of collagen and demethylation

Peripheral nerve myelination involves rapid production of tightly bound lipid layers requiring cholesterol biosynthesis and myelin protein expression, but also a collagen‐containing extracellular matrix providing mechanical stability. In previous studies, we showed a function of ascorbic acid in peripheral nerve myelination and extracellular matrix formation in adult mice. Here, we sought the mechanism of action of ascorbic acid in peripheral nerve myelination using different paradigms of myelination in vivo and in vitro. We found impaired myelination and reduced collagen expression in Sodium‐dependent Vitamin C Transporter 2 heterozygous mice (SVCT2^+/‐) during peripheral nerve development and after peripheral nerve injury. In dorsal root ganglion (DRG) explant cultures, hypo‐myelination could be rescued by precoating with different collagen types. The activity of the ascorbic acid‐dependent demethylating Ten‐eleven‐translocation (Tet) enzymes was reduced in ascorbic acid deprived and SVCT2^+/‐ DRG cultures. Further, in ascorbic acid‐deprived DRG cultures, methylation of a CpG island in the collagen alpha1 (IV) and alpha2 (IV) bidirectional promoter region was increased compared to wild‐type and ascorbic acid treated controls. Taken together, these results provide further evidence for the function of ascorbic acid in myelination and extracellular matrix formation in peripheral nerves and suggest a putative molecular mechanism of ascorbic acid function in Tet‐dependent demethylation of collagen promoters.     

Overexpression of P2X4 receptor in Schwann cells promotes motor and sensory functional recovery and remyelination via BDNF secretion after nerve injury

Of the seven P2X receptor subtypes, P2X4 receptor (P2X4R) is widely distributed in the central nervous system, including in neurons, astrocytes, and microglia. Accumulating evidence supports roles for P2X4R in the central nervous system, including regulating cell excitability, synaptic transmission, and neuropathic pain. However, little information is available about the distribution and function of P2X4R in the peripheral nervous system. In this study, we find that P2X4R is mainly localized in the lysosomes of Schwann cells in the peripheral nervous system. In cultured Schwann cells, TNF-a not only enhances the synthesis of P2X4R protein but also promotes P2X4R trafficking to the surface of Schwann cells. TNF-a-induced BDNF secretion in Schwann cells is P2X4R dependent. in vivo experiments reveal that expression of P2X4R in Schwann cells of injured nerves is strikingly upregulated following nerve crush injury. Moreover, overexpression of P2X4R in Schwann cells by genetic manipulation promotes motor and sensory functional recovery and accelerates nerve remyelination via BDNF release following nerve injury. Our results suggest that enhancement of P2X4R expression in Schwann cells after nerve injury may be an effective approach to facilitate the regrowth and remyelination of injured nerves.     

Dynamic expression of the Slit‐Robo GTPase activating protein genes during development of the murine nervous system

We investigated the expression of the three known Slit‐Robo GTPase activating protein (srGAP) genes in the developing murine nervous system using in situ hybridization. The three genes are expressed during embryonic and early postnatal development in the murine nervous system, showing a distinct pattern of expression in the olfactory system, the eye, forebrain and midbrain structures, the cerebellum, the spinal cord, and dorsal root ganglia, which we discuss in relation to Slit‐Robo expression patterns and signaling pathways. We also report srGAP2 expression in zones of neuronal differentiation and srGAP3 in ventricular zones of neurogenesis in many different tissues of the central nervous system (CNS). Compared to srGAP2 and srGAP3, the onset of srGAP1 expression is later in most CNS tissues. We propose that these differences in expression point to functional differences between these three genes in the development of neural tissues. J. Comp. Neurol. 513:224–236, 2009. © 2009 Wiley‐Liss, Inc.     

Localization of annexin II in the paranodal regions and Schmidt-Lanterman incisures in the peripheral nervous system

Annexin II (AX II) is a member of the family of calcium-dependent actin- and phospholipid-binding proteins implicated in numerous intracellular functions such as signal transduction, membrane trafficking, and mRNA transport, as well as in the regulation of membrane/cytoskeleton contacts and extracellular functions. AX II is expressed in the central nervous system (CNS) and is upregulated in some pathological conditions. However, expression and localization of this protein in the peripheral nervous system (PNS) is still uncertain. In the present study, we examined the expression and distribution of AX II in the PNS. By western blot analysis, we found that a higher level of AX II was present in sciatic nerve homogenates than in brain homogenates. RT-PCR of total RNA from rat sciatic nerves revealed that AX II was synthesized within the nerves. Immunohistological analysis showed the characteristic distribution of AX II in Schmidt-Lanterman incisures (SLI) as well as in the paranodal regions. Localization of AX II in the PNS was examined in two mutant mouse models, shiverer and cerebroside sulfotransferase knockout mice, both of which show increased numbers of SLI. The paranodal axo-glial junction is also disrupted in the latter. Interestingly, the staining intensities of AX II in these regions were increased markedly in both mutants, suggesting that not only the numbers but also AX II content in each incisure and paranodal loop were affected. From its characteristic distribution and molecular features, AX II may be important for myelin function in the PNS.