Schwann cell caveolin-1 expression increases during myelination and decreases after axotomy

The caveolins are a family of related proteins that form the structural framework of caveolae. They have been implicated in the regulation of signal transduction, cell cycle control, and cellular transport processes, particularly cholesterol trafficking. Caveolin-1 is expressed by a variety of cell types, including Schwann cells, although its expression is greatest in differentiated cell types, such as endothelial cells and adipocytes. In the present work, we characterize caveolin-1 expression both during rat sciatic nerve development and after axotomy. Schwann cells express little caveolin-1 on postnatal days 1 and 6. By P30, myelinating Schwann cells express caveolin-1, which is localized in the outer/abaxonal myelin membranes as well as intracellularly. After axotomy, Schwann cell caveolin-1 expression in the distal nerve stump decreases as Schwann cells revert to a premyelinating (p75-positive) phenotype; residual caveolin-1 within the nerve largely localizes to myelin debris and infiltrating macrophages. We speculate that caveolin-1 plays a role in the biology of myelinating Schwann cells.     

Myelin-associated glycoprotein in development and disease

The myelin-associated glycoprotein (MAG) is a 100 K-dalton, integral membrane glycoprotein containing 30% carbohydrate that is in central and peripheral myelin sheaths. Recent research on its function during normal myelinogenesis and possible roles in the pathology of demyelinating diseases is reviewed. Emphasis is placed on studies supporting a role for MAG in glia-axon interactions. Also, a highly immunogenic carbohydrate antigen on MAG, that is shared with natural killer cells and binds IgM paraproteins associated with neuropathy, is considered in detail.     

IGF-1 stimulates de novo fatty acid biosynthesis by Schwann cells during myelination

Schwann cell (SC) differentiation to the myelinating phenotype is characterized by the elaboration of a lipid-rich membrane and the expression of myelin-specific proteins. Insulin-like growth factor-1 (IGF-1) has been identified as a growth factor that stimulates the early events of myelination in SCs that signals via the PI3K/Akt pathway. Given the role of IGF-1 in promoting myelination, we performed studies to determine if the fatty acid biosynthetic pathway was a target of IGF-1 signaling in the formation of myelin membrane in dorsal root ganglion neuron/Schwann cell (DRG/SC) cocultures. We report that the fatty acid profile of lipid extracts of cocultures treated with IGF-1 match that reported for native myelin membrane by electrospray mass spectroscopy analysis. We also demonstrate de novo fatty acid biosynthesis in response to IGF-1 treatment in DRG/SC cocultures metabolically labeled with (13)C-acetate as a carbon source for fatty acid synthesis. Consistent with this finding, Western blot analysis of lysates from both cocultures and purified SCs reveal that IGF-1 stimulates two key fatty acid synthesizing enzymes. Additionally, we show that stimulation of fatty acid synthesizing enzymes is mediated by the PI3K/Akt signaling pathway. We also show that the fatty acid synthesizing enzymes and associated signaling pathways are elevated during the period of myelin membrane formation in sciatic nerve. Collectively, these findings demonstrate that IGF-1 plays an important regulatory function during myelin membrane formation.     

Monocarboxylate transporter 1 in Schwann cells contributes to maintenance of sensory nerve myelination during aging

Schwann cell (SC)-specific monocarboxylate transporter 1 (MCT1) knockout mice were generated by mating MCT1 ^f/f mice with myelin protein zero (P0)-Cre mice. P0-Cre^+/−, MCT1 ^f/f mice have no detectable early developmental defects, but develop hypomyelination and reduced conduction velocity in sensory, but not motor, peripheral nerves during maturation and aging. Furthermore, reduced mechanical sensitivity is evident in aged P0-Cre^+/−, MCT1 ^f/f mice. MCT1 deletion in SCs impairs both their glycolytic and mitochondrial functions, leading to altered lipid metabolism of triacylglycerides, diacylglycerides, and sphingomyelin, decreased expression of myelin-associated glycoprotein, and increased expression of c-Jun and p75-neurotrophin receptor, suggesting a regression of SCs to a less mature developmental state. Taken together, our results define the contribution of SC MCT1 to both SC metabolism and peripheral nerve maturation and aging.     

Schwann cells depleted of galactocerebroside express myelin-associated glycoprotein and initiate but do not continue the process of myelination.

Two peripheral myelin components, galactocerebroside (GalC) and myelin-associated glycoprotein (MAG), are known to be expressed early in Schwann cell differentiation, prior to the formation of definitive myelin segments containing compacted membrane. To discern the relative roles of these myelin components, cultures of Schwann cells and dorsal root ganglion neurons were treated with antigalactocerebroside mAbs in order to remove GalC from the Schwann cell surface (Ranscht et al., 1987). In the continuous presence of anti-GalC antibodies and in a medium containing serum plus ascorbic acid, Schwann cells assemble a basal lamina and progress to the one:one stage of Schwann cell:axon interaction but do not differentiate further. Immunostaining with anti-MAG antibodies revealed that GalC-depleted Schwann cells expressed high levels of MAG. Double staining with anti-MAG and anti-P0 antibodies showed that there was essentially no P0 immunoreactivity in the same cells. In those Schwann cells that had attained a one:one association with large-diameter axons, the inner-axon-related cytoplasmic process had passed under the outer mesaxon but had not completed a full turn around the axon. The expression of MAG on the single cytoplasmic process apposed to the axon in Schwann cells depleted of GalC further implicates MAG in the initial envelopment of the axon during myelination.     

Cthrc1 is a negative regulator of myelination in Schwann cells

The analysis of the molecular mechanisms involved in the initial interaction between neurons and Schwann cells is a key issue in understanding the myelination process. We recently identified Cthrc1 (Collagen triple helix repeat containing 1) as a gene upregulated in Schwann cells upon interaction with the axon. Cthrc1 encodes a secreted protein previously shown to be involved in migration and proliferation in different cell types. We performed a functional analysis of Cthrc1 in Schwann cells by loss-of- and gain-of-function approaches using RNA interference knockdown in cell culture and a transgenic mouse line that overexpresses the gene. This work establishes that Cthrc1 enhances Schwann cell proliferation but prevents myelination. In particular, time-course analysis of myelin formation intransgenic animals reveals that overexpression of Cthrc1 in Schwann cells leads to a delay in myelin formation with cells maintaining a proliferative state. Our data, therefore, demonstrate that Cthrc1 plays a negative regulatory role, fine-tuning the onset of peripheral myelination.     

A novel P0 glycoprotein transgene activates expression of lacZ in myelin-forming Schwann cells

P0 glycoprotein, the most abundant protein in peripheral nerve, is expressed specifically in the Schwann cell lineage. Upstream of the rat P0 gene 1.1 kb of DNA can activate expression of cDNAs specifically in Schwann cells in transgenic mice. However, the expression of P0 promoter-based transgenes has been inconsistent. As much as 9 kb of 5' flanking sequence fused to lacZ never yielded detectable levels of beta-galactosidase in multiple lines of mice. We describe transgenic mice that express lacZ in peripheral nerve, using the complete mouse P0 gene, including 6 kb of 5' flanking sequence, all exons and introns, and the natural polyadenylation signal. This vector activated lacZ expression specifically in cultured Schwann cells, and myelin-forming Schwann cells in four out of six transgenic lines. Transgene expression paralleled that of the endogenous P0 gene, both during development and after Wallerian degeneration. lacZ expression was lower than endogenous P0 expression, and was not detected in neural crest or Schwann cell precursors, where low levels of P0 mRNA are present. However, when the same vector contained a small myc tag instead of the 3.2-kb lacZ insert, the resulting transgenic mRNA was expressed at levels comparable to endogenous P0 mRNA. These data suggest that intragenic or 3' flanking sequences are necessary to generate the remarkable levels of endogenous P0 gene expression.     

Disrupting insulin signaling in Schwann cells impairs myelination and induces a sensory neuropathy

Although diabetic mice have been studied for decades, little is known about the cell type specific contributions to diabetic neuropathy (DN). Schwann cells (SCs) myelinate and provide trophic support to peripheral nervous system axons. Altered SC metabolism leads to myelin defects, which can be seen both in inherited and DNs. How SC metabolism is altered in DN is not fully understood, but it is clear that insulin resistance underlies impaired lipid metabolism in many cell types throughout the body via the phosphoinositide 3-kinase/protein kinase b (PKB)/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Here, we created an insulin resistant SC by deleting both insulin receptor (INSR) and insulin-like growth factor receptor 1 (IGF1R), to determine the role of this signaling pathway in development and response to injury in order to understand SC defects in DN. We found that myelin is thinner throughout development and adulthood in INSR/IGF1R Schwann cell specific knock out mice. The nerves of these mutant mice had reduced expression of key genes that mediate fatty acid and cholesterol synthesis due to reduced mTOR–sterol regulatory element-binding protein signaling. In adulthood, these mice show sensory neuropathy phenotypes reminiscent of diabetic mice. Altogether, these data suggest that SCs may play an important role in DN and targeting their metabolism could lead to new therapies for DN.     

Myelin-associated glycoprotein immunoreactive material: an early neuronal marker of dorsal root ganglion cells during chick development

Immunostaining of myelin-associated glycoprotein (MAG) was performed in chick dorsal root ganglia (DRG) during development. The MAG-immunoreactive material appeared first around 7 days of incubation in immature neurons of DRG. Immunoprecipitates first confined to one pole of nucleus were gradually redistributed in the perinuclear Golgi apparatus of small DRG cells. Thus MAG may be used in the chick embryo as an early marker of primary sensory neurons of class B.     

Arrest of myelination and reduced axon growth when Schwann cells lack mTOR

In developing peripheral nerves, differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years, there has been an increased understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination, together with a growing appreciation of some of the signaling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal postnatal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signaling in both the longitudinal and radial growth of the myelinating Schwann cell.     

Transduced Schwann cells promote axon growth and myelination after spinal cord injury

We sought to directly compare growth and myelination of local and supraspinal axons by implanting into the injured spinal cord Schwann cells (SCs) transduced ex vivo with adenoviral (AdV) or lentiviral (LV) vectors encoding a bifunctional neurotrophin molecule (D15A). D15A mimics actions of both neurotrophin-3 and brain-derived neurotrophic factor. Transduced SCs were injected into the injury center 1 week after a moderate thoracic (T8) adult rat spinal cord contusion. D15A expression and bioactivity in vitro; D15A levels in vivo; and graft volume, SC number, implant axon number and cortico-, reticulo-, raphe-, coerulo-spinal and sensory axon growth were determined for both types of vectors employed to transduce SCs. ELISAs revealed that D15A-secreting SC implants contained significantly higher levels of neurotrophin than non-transduced SC and AdV/GFP and LV/GFP SC controls early after implantation. At 6 weeks post-implantation, D15A-secreting SC grafts exhibited 5-fold increases in graft volume, SC number and myelinated axon counts and a 3-fold increase in myelinated to unmyelinated (ensheathed) axon ratios. The total number of axons within grafts of LV/GFP/D15A SCs was estimated to be over 70,000. Also 5-HT, DbetaH, and CGRP axon length was increased up to 5-fold within D15A grafts. In sum, despite qualitative differences using the two vectors, increased neurotrophin secretion by the implanted D15A SCs led to the presence of a significantly increased number of axons in the contusion site. These results demonstrate the therapeutic potential for utilizing neurotrophin-transduced SCs to repair the injured spinal cord.     

Myelin-associated glycoprotein is phosphorylated by protein kinase C

The myelin-associated glycoprotein (MAG) is a neural recognition molecule involved in heterophilic interactions between myelin-forming cells and neurons. To characterize the molecular mechanisms underlying post-translational modifications which may be instrumental in signal transduction following the recognition event, we have studied the stimuli leading to modification of ³²P-orthophosphate incorporation into MAG in cultures of oligodendrocytes or transformed differentiated Schwann cells. Here we show that in oligodendrocytes both the 67 and 72 kD isoforms of MAG were phosphorylated exclusively on serine, while in the transformed Schwann cells only the 67 kD isoform was found to be present and phosphorylated. The phorbol-12-myristoyl-13-acetate (PMA) did not affect biosynthesis of the protein backbone, but enhanced incorporation of phosphate by a factor of 2–3, indicating the involvement of protein kinase C. Exclusive phosphorylation of serine residues was also observed, when purified MAG was incubated with protein kinase C in the presence of [γ-³²P] ATP. In searching for the physiological stimuli which may trigger phosphorylation of MAG, cultures of oligodendrocytes were exposed to extracellular signals, such as coculture with dorsal root ganglion and spinal cord neurons carrying the MAG receptor, to membrane fractions of these neurons, monoclonal MAG antibody 513 binding to the recognition site of MAG, or platelet-derived growth factor. None of these additives modified the phosphorylation of MAG. These observations point to the possibility that phosphorylation of MAG is controlled by yet unknown intracellular cues rather than by extracellular signals interacting with cell surface receptors of oligodendrocytes. © 1993 Wiley-Liss, Inc.     

Myelin-associated glycoprotein is produced before myelin basic protein in cultured oligodendrocytes

Mixed glial cell cultures from neonatal dog cerebellum were harvested daily between 3 and 21 days after seeding and studied with immunocytochemical techniques for the demonstration of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP). Both MAG and MBP were detected in the cultures and by means of double labelling techniques shown to be produced by the same cells. MAG+ cells occurred earlier and were always more numerous than MBP+ cells. These results suggest that the oligodendrocyte in vitro expresses MAG before MBP. The findings are discussed in respect to oligodendroglial differentiation and myelination in vivo.     

Caveolin-1 expression in Schwann cells.

Caveolae are non-clathrin-coated invaginations of the plasma membrane, which are present in most cell types. An integral component of caveolae is the caveolin family of related proteins, which not only forms the structural framework of caveolae, but also likely subserves its functional roles, including regulation of signal transduction and cellular transport, in particular, cholesterol trafficking. Although caveolae have been identified ultrastructurally in the peripheral nervous system (PNS), caveolin expression has not previously been studied. To date, three caveolin genes have been reported. Here, we show for the first time that caveolin-1 is expressed by Schwann cells (SC) as well as several SC-derived cell lines. Caveolin-1 is enriched in the buoyant, detergent-insoluble membranes of rat sciatic nerve (SN) and SC, a hallmark of the caveolar compartment. Caveolin-1 exists as both soluble and insoluble forms in rat SN and SC, and localizes to SC cytoplasm and abaxonal myelin. SC caveolin-1 decreases after axotomy, when SC revert to a premyelinating phenotype. We speculate that caveolin-1 may regulate signal transduction and/or cholesterol transport in myelinating SC.     

Myelin-associated glycoprotein in multiple sclerosis lesions: a quantitative and qualitative analysis

Myelin-associated glycoprotein (MAG), myelin basic protein (MBP), and proteolipid protein (PLP) were quantitated by immunoassays in nine plaque, inner periplaque, outer periplaque, and normal-appearing white matter regions from brains of five multiple sclerosis patients and compared with the levels found in white matter samples of control subjects matched for age, postmortem time, and brain region. In plaque and inner periplaque regions, all three proteins were substantially reduced due to extensive myelin loss. In outer periplaque regions, MBP and PLP were close to control levels, but MAG was significantly reduced to a mean of 57% of control. All three proteins were close to control levels in the normal-appearing white matter samples. MAG in the various regions was qualitatively examined on Western blots by binding of lectins and by immunostaining with polyclonal and monoclonal antibodies against carbohydrate and protein epitopes of MAG. Densitometric scanning of these blots did not reveal any qualitative differences in the oligosaccharide or polypeptide moieties of MAG between samples from control subjects and those from multiple sclerosis patients. However, a high proportion of the MAG in the multiple sclerosis samples was often in the form of dMAG, a proteolytic derivative of MAG that is formed by a myelin-associated, Ca2+-activated, neutral protease. The preferential loss of MAG at the periphery of multiple sclerosis plaques may be initiated by its proteolytic conversion to dMAG.     

Induction of microtubule-associated protein 1B expression in Schwann cells during nerve regeneration

Microtubule-associated protein 1B (MAP1B) is expressed at high levels during development of the nervous system and is localized primarily in neurons while specific phosphorylated isoforms of MAP1B are localized exclusively in growing axons. The levels of MAP1B are down regulated in most regions of the adult CNS, but remain high in neurons and axons of the PNS. This study demonstrates that the expression of MAP1B is induced in adult Schwann cells following sciatic nerve lesion and regeneration. High levels of both mRNA and the MAP1B protein were detected in Schwann cells associated with the axotomized distal stump. Expression of MAP1B was also observed in cultured primary Schwann cells from neonatal rats. The properties of the MAP1B protein in cultured Schwann cells were further characterized by Western blot analysis using specific antibodies that recognize the N-terminal, middle and C-terminal domains of MAP1B. All of these antibodies detected a protein of 320-340 kDa demonstrating that MAP1B expressed by Schwann cells is very similar, or identical, to MAP1B expressed by neurons. The phosphorylation of MAP1B in Schwann cells was also studied using monoclonal antibodies (mAb) that recognize specific phosphorylation epitopes. The results indicated that the expression of MAP1B in Schwann cells exhibited a differential phosphorylation state that was recognized by mAb 1B6 but not by other mAbs, including 1B-P, 150 and RT97, that recognize phosphorylated MAP1B in growing axons. We therefore conclude that MAP1B is expressed in Schwann cells during both development and axonal regeneration, suggesting that the developmental pattern of MAP1B in these cells is recapitulated in adult Schwann cells during the early stages of regeneration and remyelination of injured peripheral axons. The presence of MAP1B in Schwann cells may support morphological changes of these cells, particularly the formation of processes prior to their differentiation into myelin forming Schwann cells.