Temporal expression pattern of peripheral myelin protein 22 during in vivo and in vitro myelination

Peripheral myelin protein 22 (PMP22) was initially described as a minor component of peripheral myelin. Mutations affecting the PMP22 gene cause demyelinating neuropathies, supporting a role for the protein in PNS myelination. Furthermore, PMP22 carries the L2/HNK-1 carbohydrate epitope suggesting an adhesion/recognition function. Despite advances in characterizing the PMP22 gene, the specific role(s) of the protein in myelin remains unknown. In this study we determined the temporal expression pattern of PMP22 in comparison to galactocerebroside (GalC) and myelin associated glycoprotein (MAG), early constituents of PNS myelin, and to protein zero (P0) and myelin basic protein (MBP), late components of myelin. In sciatic nerve lysates, PMP22 was detected at postnatal day 3, after MAG, but before MBP expression. The same results were obtained in cocultures of dorsal root ganglion neurons and Schwann cells (SCs). Low levels of PMP22 were found in early, anti-MAG and anti-GalC immunoreactive, myelinating cocultures. However, PMP22 could only be detected in the SC plasma membrane after basal lamina formation. In long-term myelinating cocultures PMP22 levels continued to increase and the protein was found in anti-P0 and anti-MBP immunoreactive myelin segments. Furthermore, PMP22, MBP, and P0 protein levels were greatly enhanced by progesterone treatment of the cocultures. The highest levels of PMP22 expression were associated with late stages of myelination; however the presence of the protein in nonmyelinating SCs and in SCs commencing myelination supports multiple roles for PMP22 in peripheral nerve biology.     

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.     

Expression of myelin proteins in the adult heterozygous Trembler mouse

The Trembler mouse (Tr) suffers from a dominantly inherited autosomal mutation (glycine to aspartic acid, G150D) affecting the PMP22 gene, which results in an abnormal myelination of the peripheral nervous system. The Tr mouse represents an animal model for the human hereditary neuropathy, Charcot-Marie-Tooth disease. We compared the expression of PMP22, P0, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) in nerve biopsies from a 1-year-old heterozygous Tr mouse (Tr/+) with that of a control mouse of the same age. Quantitative and qualitative ultrastructural immunocytochemical analysis showed a significant decrease in PMP22, P0 and MBP and an abnormal location of the MAG in the sciatic nerve of the Tr/+ mouse. We also noted a marked reduction in number of myelinated fibers associated with the presence of two types of myelinated axons: a population of abnormally thin myelin sheaths with lower PMP22 labeling than that observed in myelinated fibers from the control mouse, and some others fibers with normal sheaths for axons of comparable diameter to those of normal mouse with no difference in the PMP22 immunolabeling. This pointed to an involvement of PMP22 in the structure of myelin sheaths. Received: 27 October 1998 / Revised, accepted: 20 January 1999     

Presence of alpha-globin mRNA and migration of bone marrow cells after sciatic nerve injury suggests their participation in the degeneration/regeneration process

We have previously reported that in the distal stump of ligated sciatic nerves, there is a change in the distribution of myelin basic protein (MBP) and P0 protein immunoreactivities. These results agreed with the studies of myelin isolated from the distal stump of animals submitted to ligation of the sciatic nerve, showing a gradual increase in a 14 kDa band with an electrophoretic mobility similar to that of an MBP isoform, among other changes. This band, which was resolved into two bands of 14 and 15 kDa using a 16% gel, was found to contain a mixture of MBP fragments and peptides with great homology with alpha- and beta-globins. In agreement with these results, we have demonstrated that the mRNA of alpha-globin is present in the proximal and distal stumps of the ligated nerve. It is also detected at very low levels in Schwann cells isolated from normal nerves. These results could be due to the presence of alpha- and/or beta-globin arising from immature cells of the erythroid series. Also, they could be present in macrophages, which spontaneously migrate to the injured nerve to promote the degradation of myelin proteins. Cells isolated from normal adult rat bone marrow which were injected intraortically were found to migrate to the injured area. These cells could contribute to the remyelination of the damaged area participating in the removal of myelin debris, through their transdifferentiation into Schwann cells or through their fusion with preexisting Schwann cells in the distal stump of the injured sciatic nerve.     

Synthesis of progesterone in Schwann cells: regulation by sensory neurons

In peripheral nerves, progesterone synthesized by Schwann cells has been implicated in myelination. In spite of such an important function, little is known of the regulation of progesterone biosynthesis in the nervous system. We show here that in rat Schwann cells, expression of the 3 beta-hydroxysteroid dehydrogenase and formation of progesterone are dependent on neuronal signal. Levels of 3 beta-hydroxysteroid dehydrogenase mRNA and synthesis of [3H]progesterone from [3H]pregnenolone were low in purified Schwann cells prepared from neonatal rat sciatic nerves. However, when Schwann cells were cultured in contact with sensory neurons, both expression and activity of the 3 beta-hydroxysteroid dehydrogenase were induced. Regulation of 3 beta-hydroxysteroid dehydrogenase expression by neurons was also demonstrated in vivo in the rat sciatic nerve. 3 beta-hydroxysteroid dehydrogenase mRNA was present in the intact nerve, but could no longer be detected 3 or 6 days after cryolesion, when axons had degenerated. After 15 days, when Schwann cells made new contact with the regenerating axons, the enzyme was re-expressed. After nerve transection, which does not allow axonal regeneration, 3 beta-hydroxysteroid dehydrogenase mRNA remained undetectable. The regulation of 3 beta-hydroxysteroid dehydrogenase mRNA after lesion was similar to the regulation of myelin protein zero (P0) and peripheral myelin protein 22 (PMP22) mRNAs, supporting an important role of locally formed progesterone in myelination.     

Influence of aging on peripheral nerve function and regeneration

Aging deeply influences several morphologic and functional features of the peripheral nervous system (PNS). Morphologic studies have reported a loss of myelinated and unmyelinated nerve fibers in elderly subjects, and several abnormalities involving myelinated fibers, such as demyelination, remyelination and myelin balloon figures. The deterioration of myelin sheaths during aging may be due to a decrease in the expression of the major myelin proteins (P0, PMP22, MBP). Axonal atrophy, frequently seen in aged nerves, may be explained by a reduction in the expression and axonal transport of cytoskeletal proteins in the peripheral nerve. Aging also affects functional and electrophysiologic properties of the PNS, including a decline in nerve conduction velocity, muscle strength, sensory discrimination, autonomic responses, and endoneurial blood flow. The age-related decline in nerve regeneration after injury may be attributed to changes in neuronal, axonal, Schwann cell and macrophage responses. After injury, Wallerian degeneration is delayed in aged animals, with myelin remnants accumulated in the macrophages being larger than in young animals. The interaction between Schwann cells and regenerative axons takes longer, and the amount of trophic and tropic factors secreted by reactive Schwann cells and target organs are lower in older subjects than they are in younger subjects. The rate of axonal regeneration becomes slower and the density of regenerating axons decrease in aged animals. Aging also determines a reduction in terminal and collateral sprouting of regenerated fibers, further limiting the capabilities for target reinnervation and functional restitution. These age-related changes are not linearly progressive with age; the capabilities for axonal regeneration and reinnervation are maintained throughout life, but tend to be delayed and less effective with aging.     

The α-chemokine CXCL14 is up-regulated in the sciatic nerve of a mouse model of Charcot–Marie–Tooth disease type 1A and alters myelin gene expression in cultured Schwann cells

At present the pathogenesis of CMT1A neuropathy, caused by the overexpression of PMP22, has not yet been entirely understood. The PMP22-overexpressing C61 mutant mouse is a suitable animal model, which mimics the human CMT1A disorder. We observed that myelin gene expression in the sciatic nerve of the C61 mouse was up-regulated at postnatal day 4 to 7 (P4–P7). When investigating the morphology of peripheral nerves in C61 and wildtype mice at early stages of postnatal development, hypermyelination could be detected in the femoral quadriceps and sciatic nerve of transgenic animals at postnatal day 7 (P7). In order to identify genes, other than Pmp22, that are modulated in sciatic nerve of P7 transgenic mice, we applied microarray technology. Amongst the regulated genes, the gene encoding the α-chemokine CXCL14 was most prominently up-regulated. We report that Cxcl14 was expressed exclusively by Schwann cells of the sciatic nerve, as well as by cultured Schwann cells triggered to differentiate. Furthermore, in cultured Schwann cells CXCL14 modulated the expression of myelin genes and altered cell proliferation. Our findings demonstrate that early overexpression of PMP22, in a mouse model of CMT1A, results in a strong up-regulation of CXCL14, which seems to play a novel regulatory role in Schwann cell differentiation.     

The temporospatial expression of peripheral myelin protein 22 at the developing blood-nerve and blood-brain barriers

Peripheral myelin protein 22 (PMP22), also known as growth arrest-specific gene 3 (gas3), is a tetraspan membrane protein whose misexpression is associated with demyelinating peripheral neuropathies. Although the function of PMP22 in Schwann cells is unknown, the protein is found at intercellular junctions of various epithelia and endothelia. To begin to elucidate the role of PMP22 at cell junctions, we examined the temporal expression and protein localization during development and maturation of the rat blood-nerve barrier (BNB) and blood-brain barrier (BBB). Developing and adult rat sciatic nerves and brains were coimmunostained for PMP22 and known junctional proteins including zonula occludens-1 (ZO-1), occludin, and claudin-5. Prior to the maturation of the BNB and BBB and detection of the tight junction protein occludin, PMP22 is present at ZO-1 positive endothelial junctions of the sciatic nerve and brain cortex. The subcellular localization of PMP22 in cultured brain endothelia was confirmed by internalization with ZO-1 after EGTA-induced disruption of cell junctions. In choroid epithelia, PMP22 is detected along with occludin and ZO-1 as early as embryonic day 15 (E15). In agreement, PMP22 message is elevated in P1 rat brain microvasculature and choroid epithelia, compared with total cortex. Additionally, neuroepithelial cell junctions in the embryonic rat brain are immunoreactive for PMP22, ZO-1, and beta-catenin but not occludin. Together, these studies identify PMP22 as an early constituent of intercellular junctions in the developing and mature rat BNB and BBB.     

Neuroactive steroids and peripheral myelin proteins

The present review summarizes observations obtained in our laboratories which underline the importance of neuroactive steroids (i.e., progesterone (PROG), dihydroprogesterone (5α-DH PROG), tetrahydroprogesterone (3α, 5α-TH PROG), testosterone (T), dihydrotestosterone (DHT) and 5α-androstan-3α,17β-diol (3α-diol)) in the control of the gene expression of myelin proteins (i.e. glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22)) in the peripheral nervous system. Utilizing different in vivo (aged and adult male rats) and in vitro (Schwann cell cultures) experimental models, we have observed that neuroactive steroids are able to stimulate the mRNA levels of Po and PMP22. The effects of these neuroactive steroids, which are able to interact with classical (progesterone receptor, PR, and androgen receptor, AR) and non-classical (GABA_A receptor) steroid receptors is further supported by our demonstration in sciatic nerve and/or Schwann cells of the presence of these receptors. On the basis of the observations obtained in the Schwann cells cultures, we suggest that the stimulatory effect of neuroactive steroids on Po is acting through PR, while that on PMP22 needs the GABA_A receptor. The present findings might be of importance for the utilization of specific receptor ligands as new therapeutical approaches for the rebuilding of the peripheral myelin, particularly in those situations in which the synthesis of Po and PMP22 is altered (i.e. demyelinating diseases like Charcot–Marie–Tooth type 1A and type 1B, hereditary neuropathy with liability to pressure palsies and the Déjérine–Sottas syndrome, aging, and after peripheral injury).     

Stimulation of myelin gene expression in vitro and of sciatic nerve remyelination by interleukin-6 receptor-interleukin-6 chimera

Induction of myelin gene expression denotes the last stage of differentiation of myelinating glial cells. Following peripheral nerve transection, Schwann cells (SC) lose myelin gene expression and proliferate, resembling premyelinating embryonic SC (eSC). We show that a fusion protein of the soluble interleukin-6 receptor to interleukin-6 (IL6RIL6), a potent activator of the gp130 signaling receptor, is an inducer of MBP and Po gene products in rat E18 embryonic dorsal root ganglia (DRG) 3 day cultures. Cells whose growth is dependent on the IL6RIL6 chimera were isolated from DRG. These cells (designated CH cells) express Krox-20, as do promyelinating and myelinating SC (mSC). IL6RIL6 induces Po and MBP in CH cells and their cocultures with neurons. In addition, IL6RIL6 leads to a disappearance of Pax-3, a marker of eSC and nonmyelinating Schwann cells (nmSC). Glial fibrillary acidic protein, present in nmSC, is not significantly induced by IL6RIL6. The CH cells acquire glial morphology when exposed to IL6RIL6 and cover axons in cocultures. In a sciatic nerve-derived SC line, IL6RIL6 also induces Po and triggers a rapid attachment along axons. In vivo administration of IL6RIL6 intraperitoneally to rats after sciatic nerve transection and resuture increases 4-fold the number of myelinated nerve fibers (MF) measured on day 12, 2.5-5 mm distal to the suture. The stimulation by IL6RIL6 treatment is highest (7.1-fold) at the more distant 5 mm site, and the thickness of myelin sheaths is increased. Compared to known SC growth factors, the gp130 activator IL6RIL6 appears to combine both in vitro mitogenic effects and promotion of myelin gene expression.     

Cytokine regulation of MMP-9 in peripheral glia: implications for pathological processes and pain in injured nerve

Matrix metalloproteinase-9 (MMP-9) is an extracellular protease that is induced in Schwann cells hours after peripheral nerve injury and controls axonal degeneration and macrophage recruitment to the lesion. Here, we report a robust (90-fold) increase in MMP-9 mRNA within 24 h after rat sciatic nerve crush (1 to 60 days time-course). Using direct injection into a normal sciatic nerve, we identify the proinflammatory cytokines TNF-alpha and IL-1beta as potent regulators of MMP-9 expression (Taqman qPCR, zymography). Myelinating Schwann cells produced MMP-9 in response to cytokine injection and crush nerve injury. MMP-9 gene deletion reduced unstimulated neuropathic nociceptive behavior after one week post-crush and preserved myelin thickness by protecting myelin basic protein (MBP) from degradation, tested by Western blot and immunofluorescence. These data suggest that MMP-9 expression in peripheral nerve is controlled by key proinflammatory cytokine pathways, and that its removal protects nerve fibers from demyelination and reduces neuropathic pain after injury.     

Glial cells in degenerating and regenerating optic nerve of the adult rat

The glial cell reaction both in degenerating and regenerating adult rat optic nerve was studied by immunohistochemistry and electron microscopy. Degeneration in the optic nerve was achieved by complete transection, and the retinal stump was then analyzed. The regeneration was observed by autotransplantation of a sciatic nerve segment to the transected retinal stump. In both cases, optic nerve axons were labeled anterogradely with rhodamine, followed by immunohistochemical staining. Glial fibrillary acidic protein-positive astrocytes covered the transected end of degenerating optic nerve, whereas in the regenerating optic nerve they enwrapped axonal bundles emerging from the optic nerve stump and migrated together into the transitional zone intervening between the retinal stump and graft. In electron microscopy, direct attachment of astrocyte and Schwann cell was found within the transitional zone, whereby these cells were holding axons between them. Decrease of 04 immunoreactivity, which labels oligodendrocytes, was apparent in the transected end of retinal stump during the regeneration. The ED1 -positivity, which labels microglia/macrophages, was found in cells accumulated in the transitional zone of degenerating optic nerve, whereas during regeneration, ED1-immunoreactive cells were also distributed in the retinal stump. These results suggest that astrocytes, usually considered to interfere with optic nerve regeneration, change their characteristics in the presence of peripheral nerve graft and guide the regenerating axons in cooperation with Schwann cells. The response of oligodendrocytes and microglia/macrophages may also be modulated by peripheral nerve.     

Axons modulate the expression of transforming growth factor-betas in Schwann cells

We have investigated the expression of transforming growth factor (TGF)-β1,-β2, and -β3 in developing, degenerating, and regenerating rat peripheral nerve by immunohistochemistry and Northern blot analysis. In normal adult sciatic nerve, TGF-β1, -β2, and -β3 are detected in the cytoplasm of Schwann cells, and the levels of TGF-β1 and -β3 mRNAs are constant during post-natal development. When sciatic nerves are transected to cause axonal degeneration and prevent axonal regeneration, the level of TGF-β1 mRNA in the distal nerve-stump increases markedly and remains elevated, whereas the level of TGF-β3 mRNA falls modestly and remains depressed. When sciatic nerves are crushed to cause axonal degeneration and allow axonal regeneration, the level of TGF-β1 mRNA initially increases as axons degenerate, and then falls as axons regenerate. TGF-β2 mRNA was not detected in developing or lesioned sciatic nerves at any time. Cultured Schwann cells have high levels of TGF-β1 mRNA, the amount of which is reduced by forskolin, which mimicks the effect of axonal contact. These data demonstrate that Schwann cells express TGF-β1, -β2, and -β3, and that TGF-β1 and -β3 mRNA predominate over TGF-β2 mRNA in peripheral nerve. Axonal contact and forskolin decrease the expression of TGF-β1 in Schwann cells. © 1993 Wiley-Liss, Inc.     

β4 integrin and other Schwann cell markers in axonal neuropathy

Schwann cell gene expression is dynamically regulated after peripheral nerve injury and during regeneration. We hypothesized that the changes in protein expression described after rat peripheral nerve injury could be used to identify single Schwann cell-axon units in human axonal neuropathy. Therefore, we performed immunofluorescence staining on sections of injured rat sciatic nerves compared with sections of neuropathic human sural nerves. We chose the markers β4 integrin, P0 glycoprotein, and glial fibrillary acidic protein (GFAP) to characterize Schwann cells, and neurofilament-heavy (NF-H) to recognize axons. Normal rat or human myelin-forming units demonstrated a sharp ring of β4 staining at their outer surface, P0 staining in the myelin sheath, and NF-H staining in the axon. Acutely denervated rat units transited from broken rings of β4 and P0 staining, to diffuse β4 and absent P0 and NF-H staining. Chronically denervated rat Schwann cells re-expressed β4 more highly, but in a diffuse, non-polarized pattern. In contrast, regenerating units re-expressed β4, P0, and NF-H; β4 staining was polarized to the outer surface of Schwann cells. Finally, GFAP staining increased progressively after injury and decreased during regeneration in the distal nerve stump. In neuropathic human sural nerves, we identified units exhibiting each of these β4, P0, and NF-H staining patterns; the proportion of each pattern correlated best with the extent and chronicity of axonal injury. Thus, synchronous injury of rat sciatic nerve predicts patterns of Schwann cell marker expression in human axonal neuropathy. In addition, the unique changes in the polarity of β4 integrin expression, in combination with changes in P0 and NF-H expression, may distinguish normal from denervated or reinnervated myelin-forming Schwann cells in human sural nerve biopsies. © 1996 Wiley-Liss, Inc.     

Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents

Following a peripheral nerve injury, brain-derived neurotrophic factor (BDNF) and the p75 neurotrophin receptor are upregulated in Schwann cells of the Wallerian degenerating nerves. However, it is not known whether the endogenous BDNF is critical for the functions of Schwann cells and regeneration of injured nerve. Treatment with BDNF antibody was shown to retard the length of the regenerated nerve from injury site by 24%. Histological and ultrastructural examination showed that the number and density of myelinated axons in the distal side of the lesion in the antibody-treated mice was reduced by 83%. In the BDNF antibody-treated animals, there were only distorted and disorganized myelinated fibres in the injured nerve where abnormal Schwann cells and phagocytes were present. As a result of nerve degeneration in BDNF antibody-treated animals, subcellular organelles, such as mitochondria, disappeared or were disorganized and the laminal layers of the myelin sheath were loosened, separated or collapsed. Our in situ hybridization revealed that BDNF mRNA was expressed in Schwann cells in the distal segment of lesioned nerve and in the denervated muscle fibres. These results indicate that Schwann cells and muscle fibres may contribute to the sources of BDNF during regeneration and that the deprivation of endogenous BDNF results in an impairment in regeneration and myelination of regenerating axons. It is concluded that endogenous BDNF is required for peripheral nerve regeneration and remyelination after injury.