Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve

The cellular and subcellular localization of the neural cell adhesion molecules L1, N-CAM, and myelin-associated glycoprotein (MAG), their shared carbohydrate epitope L2/HNK-1, and the myelin basic protein (MBP) were studied by pre- and post-embedding immunoelectron microscopic labeling procedures in developing mouse sciatic nerve. L1 and N-CAM showed a similar staining pattern. Both were localized on small, non-myelinated, fasciculating axons and axons ensheathed by non- myelinating Schwann cells. Schwann cells were also positive for L1 and N-CAM in their non-myelinating state and at the onset of myelination, when the Schwann cell processes had turned approximately 1.5 loops. Thereafter, neither axon nor Schwann cell could be detected to express the L1 antigen, whereas N-CAM was found in the periaxonal area and, more weakly, in compact myelin of myelinated fibers. Compact myelin, Schmidt-Lanterman incisures, paranodal loops, and finger-like processes of Schwann cells at nodes of Ranvier were L1-negative. At the nodes of Ranvier, the axolemma was also always L1- and N-CAM-negative. The L2/HNK-1 carbohydrate epitope coincided in its cellular and subcellular localization most closely to that observed for L1. MAG appeared on Schwann cells at the time L1 expression ceased. MAG was then expressed at sites of axon-myelinating Schwann cell apposition and non-compacted loops of developing myelin. When compaction of myelin occurred, MAG remained present only at the axon-Schwann cell interface; Schmidt- Lanterman incisures, inner and outer mesaxons, and paranodal loops, but not at finger-like processes of Schwann cells at nodes of Ranvier or compacted myelin. All three adhesion molecules and the L2/HNK-1 epitope could be detected in a non-uniform staining pattern in basement membrane of Schwann cells and collagen fibrils of the endoneurium. MBP was detectable in compacted myelin, but not in Schmidt-Lanterman incisures, inner and outer mesaxon, paranodal loops, and finger-like processes at nodes of Ranvier, nor in the periaxonal regions of myelinated fibers, thus showing a complementary distribution to MAG. These studies show that axon-Schwann cell interactions are characterized by the sequential appearance of cell adhesion molecules and MBP apparently coordinated in time and space. From this sequence it may be deduced that L1 and N-CAM are involved in fasciculation, initial axon-Schwann cell interaction, and onset of myelination, with MAG to follow and MBP to appear only in compacted myelin. In contrast to L1, N- CAM may be further involved in the maintenance of compact myelin and axon-myelin apposition of larger diameter axons.     

Expression of the neural cell adhesion molecules L1 and N-CAM and their common carbohydrate epitope L2/HNK-1 during development and after transection of the mouse sciatic nerve

The expression of the neural cell adhesion molecules L1 and N-CAM and of their shared carbohydrate epitope L2/HNK-1 was studied during the development and after the transection of mouse sciatic nerves. During development, L1 and N-CAM were detectable on most, if not all, Schwann cells at embryonic day 17, the earliest stage tested. With increasing age, the immunoreactivity was reduced being confined to non-myelinating Schwann cells by post-natal day 10, at which stage the staining pattern resembled that seen in adult sciatic nerves. Double-immunolabelling experiments revealed a complete overlap between L1 and N-CAM antibodies. The L2/HNK-1 epitope was not detectable in developing sciatic nerves until the end of the 2nd post-natal week, when it appeared to be associated with the outer profiles of thick myelin sheets, as also seen in adult sciatic nerves. Three days after the transection of adult sciatic nerves, L1 antigen and N-CAM was detectable in more Schwann cells in the distal nerve end than in untreated control nerves. The peak level of the reappearance of L1 antigen and N-CAM in Schwann cells occurred between 2 and 4 weeks after transection. The reduction of L1-antigen expression to its normal adult level took more than a year, thus recapitulating normal development, but on a more protracted time scale. Similarly, the L2/HNK-1 epitope remained undetectable until the transected nerve had returned to its normal state of myelination, i.e. approximately 1 year after transection.     

Gpr126 is essential for peripheral nerve development and myelination in mammals

In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.     

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)     

Schwann cell myelination requires timely and precise targeting of P(0) protein

This report investigated mechanisms responsible for failed Schwann cell myelination in mice that overexpress P(0) (P(0)(tg)), the major structural protein of PNS myelin. Quantitative ultrastructural immunocytochemistry established that P(0) protein was mistargeted to abaxonal, periaxonal, and mesaxon membranes in P(0)(tg) Schwann cells with arrested myelination. The extracellular leaflets of P(0)-containing mesaxon membranes were closely apposed with periodicities of compact myelin. The myelin-associated glycoprotein was appropriately sorted in the Golgi apparatus and targeted to periaxonal membranes. In adult mice, occasional Schwann cells myelinated axons possibly with the aid of endocytic removal of mistargeted P(0). These results indicate that P(0) gene multiplication causes P(0) mistargeting to mesaxon membranes, and through obligate P(0) homophilic adhesion, renders these dynamic membranes inert and halts myelination.     

Endogenous Lectin Cerebellar Soluble Lectin Involved in Myelination Is Absent from Nonmyelinating Schwann Cells

In the sciatic nerve, two major classes of Schwann cells are present which differ in their capability to produce myelin. Myelinating Schwann cells surround most of the axons with the formation of a typical myelin sheath. Nonmyelinating Schwann cells serve to insulate individual axons without formation of myelin. These dissimilarities between the two types of Schwann cells provided an interesting model for studying mechanisms underlying myelination and the formation of contacts between axons and myelinating cells. It is demonstrated here that the endogenous lectin cerebellar soluble lectin (CSL), implicated in myelin stabilization and in formation of contact between axon and myelinating cells in the CNS and in the sciatic nerve, is undetectable in non-myelinating Schwann cells. In contrast, most axons surrounded by these cells contained the major axonal glycoprotein ligand of CSL, a 31-kDa glycoprotein which is present in large amounts. The possible relationship between the presence of CSL in Schwann cells and their capacity to interact with axons and to produce myelin are discussed.     

Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons.

We have used homologous recombination in embryonic stem cells to generate mice carrying a mutation in the gene encoding P0, an immunoglobulin-related recognition molecule and the major protein of peripheral nervous system myelin. These mice are deficient in normal motor coordination and exhibit tremors and occasional convulsions. Axons in their peripheral nerves are severely hypomyelinated and a subset of myelin-like figures and axons degenerate. The mutation leads to an abnormal regulation of some, but not all, molecules involved in myelination. These results demonstrate that P0 is essential for the normal spiraling, compaction, and maintenance of the peripheral myelin sheath and the continued integrity of associated axons. They further suggest that this protein conveys a signal that regulates Schwann cell gene expression.     

Distribution of the myelin-associated glycoprotein and P0 protein during myelin compaction in quaking mouse peripheral nerve

Ultrastructural studies have shown that during early stages of Schwann cell myelination mesaxon membranes are converted to compact myelin lamellae. The distinct changes that occur in the spacing of these Schwann cell membranes are likely to be mediated by the redistribution of (a) the myelin-associated glycoprotein, a major structural protein of mesaxon membranes; and (b) P0 protein, the major structural protein of compact myelin. To test this hypothesis, the immunocytochemical distribution of these two proteins was determined in serial 1-micron-thick Epon sections of ventral roots from quaking mice and compared to the ultrastructure of identical areas in an adjacent thin section. Ventral roots of this hypomyelinating mouse mutant were studied because many fibers have a deficit in converting mesaxon membranes to compact myelin. The results indicated that conversion of mesaxon membranes to compact myelin involves the insertion of P0 protein into and the removal of the myelin-associated glycoprotein from mesaxon membranes. The failure of some quaking mouse Schwann cells to form compact myelin appears to result from an inability to remove the myelin-associated glycoprotein from their mesaxon membranes.     

In vivo knockdown of ErbB3 in mice inhibits Schwann cell precursor migration

The myelin sheath insulates neuronal axons and markedly increases the nerve conduction velocity. In the peripheral nervous system (PNS), Schwann cell precursors migrate along embryonic neuronal axons to their final destinations, where they eventually wrap around individual axons to form the myelin sheath after birth. ErbB2 and ErbB3 tyrosine kinase receptors form a heterodimer and are extensively expressed in Schwann lineage cells. ErbB2/3 is thought to be one of the primary regulators controlling the entire Schwann cell development. ErbB3 is the bona fide Schwann cell receptor for the neuronal ligand neuregulin-1. Although ErbB2/3 is well known to regulate both Schwann cell precursor migration and myelination by Schwann cells in fishes, it still remains unclear whether in mammals, ErbB2/3 actually regulates Schwann cell precursor migration. Here, we show that knockdown of ErbB3 using a Schwann cell-specific promoter in mice causes delayed migration of Schwann cell precursors. In contrast, littermate control mice display normal migration. Similar results are seen in an in vitro migration assay using reaggregated Schwann cell precursors. Also, ErbB3 knockdown in mice reduces myelin thickness in sciatic nerves, consistent with the established role of ErbB3 in myelination. Thus, ErbB3 plays a key role in migration, as well as in myelination, in mouse Schwann lineage cells, presenting a genetically conservative role of ErbB3 in Schwann cell precursor migration.     

神经退变和再生的构筑变化

将夹伤的大鼠坐骨神经分离成单根纤维,观察98d内轴突和许旺细胞的构筑变化过程发现,损伤既使轴浆转运阻断、积累的细胞器退变,也使髓鞘板层,特别是斯兰氏切迹撕裂、变形或侵入轴突。轴突或髓鞘虽可各呈单一的退变,但以两者并存多见。伤后1d即出现富含微管的再生芽,它被增殖的许旺细胞突起及其基底膜包绕,并逐步发育成熟。根据再生的特征性构筑变化,提出了再生芽、无髓和有髓纤维、斯兰氏切迹、朗氏结与神经小束的初见、发育和成熟高峰期的时间顺序。无髓纤维的发育成熟早于有髓纤维。     

Immunocytochemical localization of P0 protein in Golgi complex membranes and myelin of developing rat Schwann cells

P0 protein, the dominant protein in peripheral nervous system myelin, was studied immunocytochemically in both developing and mature Schwann cells. Trigeminal and sciatic nerves from newborn, 7-d, and adult rats were processed for transmission electron microscopy. Alternating 1- micrometer-thick Epon sections were stained with paraphenylenediamine (PD) or with P0 antiserum according to the peroxidase-antiperoxidase method. To localize P0 in Schwann cell cytoplasm and myelin membranes, the distribution of immunostaining observed in 1-micrometer sections was mapped on electron micrographs of identical areas found in adjacent thin sections. The first P0 staining was observed around axons and/or in cytoplasm of Schwann cells that had established a 1:1 relationship with axons. In newborn nerves, staining of newly formed myelin sheaths was detected more readily with P0 antiserum than with PD. Myelin sheaths with as few as three lamellae could be identified with the light microscope. Very thin sheaths often stained less intensely and part of their circumference frequently was unstained. Schmidt-Lanterman clefts found in more mature sheaths also were unstained. As myelination progressed, intensely stained myelin rings became much more numerous and, in adult nerves, all sheaths were intensely and uniformly stained. Particulate P0 staining also was observed in juxtanuclear areas of Schwann cell cytoplasm. It was most prominent during development, then decreased, but still was detected in adult nerves. The cytoplasmic areas stained by P0 antiserum were rich in Golgi complex membranes.     

Schwann cells infected with a recombinant retrovirus expressing myelin-associated glycoprotein antisense RNA do not form myelin.

To elucidate the role of myelin-associated glycoprotein (MAG) in the axon-Schwann cell interaction leading to myelination, neonatal rodent Schwann cells were infected in vitro with a recombinant retrovirus expressing MAG antisense RNA or MAG sense RNA. Stably infected Schwann cells and uninfected cells were then cocultured with purified sensory neurons under conditions permitting extensive myelination in vitro. A proportion of the Schwann cells infected with the MAG antisense virus did not myelinate axons and expressed lower levels of MAG than control myelinating Schwann cells, as measured by immunofluorescence. Electron microscopy revealed that the affected cells failed to segregate large axons and initiate a myelin spiral despite having formed a basal lamina, which normally triggers Schwann cell differentiation. Cells infected with the MAG sense virus formed normal compact myelin. These observations strongly suggest that MAG is the critical Schwann cell component induced by neuronal interaction that initiates peripheral myelination.     

RETARDATION OF PERIPHERAL NERVE MYELINATION IN MICE TREATED WITH INHIBITORS OF CHOLESTEROL BIOSYNTHESIS : A Quantitative Electron Microscopic Study

The effect of two inhibitors of cholesterol biosynthesis, triparanol and AY 9944, on peripheral nerve myelination, was studied. Suckling mice were intraperitoneally injected with both drugs on 3 consecutive days and were sacrificed 6 hr after the last injection; others were suckled by an injected mother and sacrificed at 2½ days of age. A single mouse which had been injected with both drugs at 1, 2, and 3 days of age was sacrificed 2 wk after the last injection. Membranous and crystalline intracytoplasmic inclusions were observed in the Schwann cells of the sciatic nerves of all the experimental animals. Both the number of unmyelinated single axons and the number of myelin lamellae around each myelinating axon in the sciatic nerves were recorded for treated mice and of mice suckled by treated mothers. The sciatic nerve of the experimental mice contained a larger proportion of unmyelinated single axons and smaller numbers of myelin lamellae around the myelinating axons, when compared with age-matched controls. The results suggest that a decrease of endogenous cholesterol in suckling mice may affect peripheral nerve myelination in two ways: by retarding the "triggering" of myelination in unmyelinated axons and by decreasing the rate of myelination already in progress.     

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.     

Alteration of ethanolamine glycerophospholipid turnover in trembler dysmyelinating mutant: An analysis of the sciatic nerve by biochemistry and radioautography

The turnover of phospholipids was compared in peripheral nerves of Trembler dysmelinating mutant and control mice, after intraperitoneal and local injection of labeled ethanolamine. In the mutant sciatic nerve, neurochemical analysis showed that [¹⁴C]ethanolamine is incorporated into EGP (ethanolamine glycerophospholipids) of the sciatic nerve at a much higher rate in Trembler mutant than in control mice. Furthermore the decay rate of ¹⁴C-labeled EGP is faster in Trembler than in normal animals. The accelerated turnover of EGP in Trembler sciatic nerve affects the diacyl-EGP while the renewal of the alkenylacyl-EGP (plasmalogens) is slower than in controls. Quantitative radioautographic study at the ultrastructural level corroborate that the initial increase of the label in Trembler nerve fibers was different in axons, Schwann cells and myelin sheaths. EM radioautographs reveal indeed that the high label content observed in Trembler axons takes place preferentially in the myelinated portions of axons and drops within 1 week. In both myelinated and unmyelinated segments of the axons, the majority of the radioactivity was contained in axolemma and smooth axoplasmic reticulum. The 10-fold increase of label found in the myelin sheath of Trembler nerve fibers at 1 day raises the question of the origin of the labeled EGP, either by a stimulated synthesis in Schwann cells or by transfer from axonally transported phospholipids. In contrast, the label of axons, Schwann cells and myelin sheaths of control nerve remains stable during the same period.     

The effects of cAMP on differentiation of cultured Schwann cells: progression from an early phenotype (04+) to a myelin phenotype (P0+, GFAP-, N-CAM-, NGF-receptor-) depends on growth inhibition

The present experiments were designed to clarify the relationship between cAMP elevation, proliferation and differentiation in Schwann cells. They were carried out on short-term cultures of cells obtained from neonatal rat sciatic nerves. It was found that the myelin-related phenotype was expressed in response to agents that elevate or mimic intracellular cAMP (forskolin, cholera toxin, cAMP analogues), provided cell division was absent. This phenotype included upregulation of the major myelin protein P0 and downregulation of GFAP, N-CAM, A5E3, and NGF receptor. In contrast, when cells were cultured in conditions where cell division occurred, elevation of intracellular cAMP produced an alternative response, characterized by DNA synthesis and absence of myelin-related differentiation. The cAMP mediated induction of an early Schwann cell antigen, 04, followed a different pattern since it was induced equally in dividing and nondividing cells. These observations are consistent with the proposal that during development of the rat sciatic nerve: (a) cAMP elevation, possibly induced by axon-associated factors, is a primary signal responsible for the induction of 04 expression in proliferating Schwann cells during the premyelination period; (b) subsequent withdrawal of cells associated with the larger axons from the cell cycle acts as a permissive secondary signal for induction of myelination, since in quiescent cells the ongoing cAMP elevation will trigger myelination.     

N-WASP is required for membrane wrapping and myelination by Schwann cells

During peripheral nerve myelination, Schwann cells sort larger axons, ensheath them, and eventually wrap their membrane to form the myelin sheath. These processes involve extensive changes in cell shape, but the exact mechanisms involved are still unknown. Neural Wiskott-Aldrich syndrome protein (N-WASP) integrates various extracellular signals to control actin dynamics and cytoskeletal reorganization through activation of the Arp2/3 complex. By generating mice lacking N-WASP in myelinating Schwann cells, we show that N-WASP is crucial for myelination. In N-WASP-deficient nerves, Schwann cells sort and ensheath axons, but most of them fail to myelinate and arrest at the promyelinating stage. Yet, a limited number of Schwann cells form unusually short internodes, containing thin myelin sheaths, with the occasional appearance of myelin misfoldings. These data suggest that regulation of actin filament nucleation in Schwann cells by N-WASP is crucial for membrane wrapping, longitudinal extension, and myelination.     

Death of oligodendrocytes and microglial phagocytosis of myelin precede immigration of Schwann cells into the spinal cord

Small, circumscribed electrolytic lesions were made in the upper cervical corticospinal tract in adult rats. In the centre of the lesion, the axons and all other tissue elements were totally destroyed. Surrounding this region of destruction is an area of tissue which is only partially damaged. In this area TUNEL positive staining of contiguous rows of tract glial cells indicates massive oligodendrocytic apoptosis at 1–3 days after operation, but axons, astrocytes and blood vessels survive. From around 4 days, the corticospinal axons in this area are demyelinated, and the microglia contain ingested myelin, identified in electron micrographs as characteristic MBP immunoreactive laminar cytoplasmic bodies. After around 3 weeks, large numbers of Schwann cells, continuous with those on the pial surface of the spinal cord, accumulate along the lesion track and selectively infiltrate the perilesional reactive area, where they mingle intimately with the phagocytic microglia. Electron micrographs show that at this time basal lamina-enclosed Schwann cell processes establish non-myelinated ensheathment of axons. From around 4 weeks after operation, prominent Schwann cell myelination is indicated by P0 immunoreactivity, and peripheral type, one-to-one myelination in electron micrographs. Thus the effect of the selective loss of oligodendrocytes is to first activate microglia, and then to induce a replacement of myelin by Schwann cells.