Impairment of PMP22 transgenic Schwann cells differentiation in culture: implications for Charcot-Marie-Tooth type 1A disease

Charcot-Marie-Tooth type 1A (CMT1A) is a hereditary demyelinating neuropathy due to an increased genetic dosage of the peripheral myelin protein 22 (PMP22). The mechanisms leading from PMP22 overexpression to impairment of myelination are still unclear. We evaluated expression and processing of PMP22, viability, proliferation, migration, motility and shaping properties, and ability of forming myelin of PMP22 transgenic (PMP22(tg)) Schwann cells in culture. In basal conditions, PMP22(tg) Schwann cells, although expressing higher PMP22 levels than control ones, show normal motility, migration and shaping properties. Addition of forskolin to the media induces an additional stimulation of PMP22 expression and results in an impairment of cells migration and motility, and a reduction of cell area and perimeter. Similarly, co-culturing transgenic Schwann cells with neurons causes an altered cells differentiation and an impairment of myelin formation. In conclusion, exposure of PMP22(tg) Schwann to the axon or to axonal-mimicking stimuli significantly affects the transition of transgenic Schwann cells to the myelinating phenotype.     

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.     

Sex-dimorphic effects of progesterone and its reduced metabolites on gene expression of myelin proteins by rat Schwann cells

Data obtained in our and other laboratories have indicated that progesterone (P) and its derivatives, dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), stimulate the expression of two myelin proteins of the peripheral nervous system (PNS) [i.e., glycoprotein zero (P0) and peripheral myelin protein 22 (PMP22)]. We have now considered the effects of P and its derivatives on these and other myelin proteins [i.e., myelin-associated glycoprotein (MAG) and myelin and lymphocyte protein (MAL)] in sex-specific cultures of rat Schwann cells. Gene expression of myelin proteins was assessed by RNase protection assay. Treatment with P or DHP induced a stimulatory effect on P0 mRNA levels in male but not in female Schwann cells. In contrast, treatment with THP increased gene expression of P0 exclusively in female Schwann cells. A similar sex-difference was also evident for other myelin proteins. Indeed, PMP22 expression was stimulated by treatment with P in male cultures, whereas THP induced an increase of mRNA levels in female cultures. Moreover, MAG was stimulated by THP treatment in male cultures only, whereas MAL expression was unaffected by neuroactive steroid treatment in both male and female cultures. In conclusion, the present observations indicate that the effects of neuroactive steroids on myelin proteins are sexually dimorphic. This finding might represent an important background for sex-specific therapies of acquired and inherited peripheral neuropathies.     

Localization and functional roles of PMP22 in peripheral nerves of P0-deficient mice

Peripheral nerves of P0-deficient (P0(-)) mice show a severe dysmyelination and altered expression of several cell surface molecules. In the present study we investigated the subcellular localization of the peripheral myelin protein (PMP)22 in the abnormal axon-Schwann cell units of the mutants. We show by postembedding immunoelectron microscopy that PMP22 is expressed in both noncompacted and abnormally compacted myelin-like regions of P0(-) mice. By the generation of mice deficient for both P0 and PMP22 (P0(-)/PMP22(-) double mutants) we investigated the functional role(s) of PMP22 in P0(-) mice. In 4-week-old double mutants, some abnormally compacted myelin-like sheaths showed slight alterations in compaction with collapsed intraperiod lines, whereas the totally uncompacted axon-Schwann cell units displayed a more irregular cytoarchitecture owing to the presence of more cytoplasm within the loose Schwann cell loops. These findings show an only subtle impact of PMP22 on the structure of P0-deficient myelin-like sheaths. During early stages of myelin formation, peripheral nerves of P0(-)/PMP22(-) mice resembled those of PMP22(-) mutants in that they were characterized by a strongly retarded spiralling of Schwann cell processes. Thus, P0 appears to be the major determinant of myelin structure, whereas PMP22 is the predominant regulator of the timely correct initiation of myelination.     

TRANSFECTION OF PRIMARY SCHWANN CELL CULTURES AND OF ORGANOTYPIC DORSAL ROOT GANGLIA CULTURES: PRELIMINARY RESULTS AND TECHNICAL CONSIDERATIONS

The most common forms of Charcot-Marie-Tooth (CMT) disease are due to duplication/deletion of the myelin protein 22 gene (PMP22); however, several patients harbor point mutations in PMP22 or in other myelin related proteins. To study the mechanism by which dominant point mutations affect myelin formation and maintenance, we are attempting to optimize gene transfer protocols into Schwann cells (SC) that will introduce expression constructs carrying mutated cDNA.
Initial experiments were carried out on pure primary cultures of SC from sciatic nerves of newborn rats, co-cultured with primary sensory neurons under conditions inducing myelination in vitro. Using the EGFP expression vector, which induces endogenous green fluorescence in the transfected cells, we have tested a number of gene transfer approaches, including electroporation and polycationic reagents (FuGene). Best results are obtained with the FuGene reagent (Boheringer) (up to 25%), while the electroporation is less efficient (5%–12%). However, the difficulties in selecting and expanding SC after transfection need to be overcome. Thus, we are optimizing gene transfer procedures on organotypic cultures of dorsal root ganglia from 15 day-old-rat embryos. SC and neurons are identified by their morphology; immunostaining with S100 and antibodies to phosphorlated neurophylaments are used to recognize, respectively, SC and axons. Using either Lipofectamine 2000 (GIBCO) and FuGene, polycationic reagents, SC are trasfected to levels of efficiency comparable or better than pure culture protocols. The same cultures maintained for 15 days after transfection showed extensive axonal growth and initial myelin formation. Scattered SC were still expressing the transgene at a visible level.
These advances will likely help in understanding the role of specific genes in myelination and learning how their mutated forms lead to hereditary demyelinating neuropathies.     

Developmental abnormalities in the nerves of peripheral myelin protein 22-deficient mice

Peripheral myelin protein 22 (PMP22) is a tetraspan glycoprotein whose misexpression is associated with a family of hereditary peripheral neuropathies. In a recent report, we have characterized a novel PMP22-deficient mouse model in which the first two coding exons were replaced by the lacZ reporter. To investigate further the myelin abnormalities in the absence of PMP22, sciatic nerves and dorsal root ganglion (DRG) neuron explant cultures from PMP22-deficient mice were studied at various stages of myelination. Throughout the first 3 months of postnatal development, myelin protein and beta4 integrin levels are dramatically reduced, whereas p75 and beta1 integrin remain elevated. By immunostaining, the distributions of several glial proteins, including beta4 integrin, the voltage-gated potassium channel Kv1.1, and E-cadherin, are altered. Schwann cells from PMP22-deficient mice are able to produce limited amounts of myelin in DRG explant cultures, yet the internodal segments are dramatically fewer and shorter. The comparison of PMP22-deficient mice with other PMP22 mutant models reveals that the decrease in beta4 integrin is specific to an absence of PMP22. Furthermore, whereas lysosome-associated membrane protein 1 and ubiquitin are notably up-regulated in nerves of PMP22-deficient mice, heat shock protein 70 levels remain constant or decrease compared with wild-type or PMP22 mutant samples. Together these results support a role for PMP22 in the early events of peripheral nerve myelination. Additionally, although myelin abnormalities are a commonality among PMP22 neuropathic models, the underlying subcellular mechanisms are distinct and depend on the specific genetic abnormality.     

Glucocorticosteroids stimulate the activity of the promoters of peripheral myelin protein-22 and protein zero genes in Schwann cells

To better understand the mechanism by which glucocorticosteroids (GLUC) could enhance myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P(0)) genes. GLUC stimulated the activity of the P(0) promoter and the PMP22 promoters 1 and 2. The effect of GLUC was specific as estradiol and testosterone did not activate the promoters. The antagonist RU486 did not abolish the effect of GLUC, but instead stimulated promoter activities by itself. In the mammary carcinoma cell line 34i, which expresses GLUC receptors, GLUC did not stimulate the P(0) and PMP22 promoters while the promoter of the mouse mammary tumor virus was strongly activated. Thus, the activation by GLUC of the promoter activities of two peripheral myelin protein genes is Schwann cell-specific.     

Distinct elements of the peripheral myelin protein 22 (PMP22) promoter regulate expression in Schwann cells and sensory neurons

Genetic disease mechanisms in the demyelinating peripheral neuropathies Charcot-Marie-Tooth disease type 1A (CMTA) and hereditary neuropathy with liability to pressure palsies (HNPP) as well as transgenic animals with altered PMP22 gene dosage revealed that alterations in PMP22 gene expression have profound effects on the development and maintenance of peripheral nerves. Consequently, the regulation of PMP22 is a crucial aspect in understanding the function of this protein in health and disease. In this study, we dissected and analyzed different cis-acting elements in the 5'-flanking region of the Pmp22 gene in vivo. We found two separate elements that contribute to different aspects of Pmp22 expression. The first is located 5' distally to promoter 1 and is involved in gene regulation during late phases of myelination in development ["late myelination Schwann cell-specific element" (LMSE)] and in remyelination after injury. The second element was identified upstream of promoter 2 and guides Pmp22 expression in sensory neurons. These results suggest that multiple distinct signaling pathways regulating Pmp22 expression in myelination as well as in neurons converge on distinct segments of the PMP22 promoter region. The underlying molecular mechanisms are likely to be crucially involved in the maintenance of the integrity of myelinated peripheral nerves.     

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.     

MAG-deficient schwann cells myelinate dorsal root ganglion neurons in culture

The myelin-associated glycoprotein (MAG) has been postulated to play a crucial role during myelin formation. Evidence supporting this hypothesis was provided by infecting rat Schwann cells with a retrovirus expressing MAG antisense RNA; these Schwann cells showed reduced levels of MAG expression and failed to myelinate DRG neurons in vitro. However, when MAG expression was disrupted by generating MAG-deficient mice, normal myelin sheaths were formed in peripheral nerves in vivo. In the present study we investigated whether myelination is compromised in MAG-deficient Schwann cells in vitro, i.e., under similar conditions where Schwann cells expressing MAG antisense RNA failed to myelinate. We show that MAG-deficient Schwann cells do myelinate DRG neurons in vitro and express the myelin-specific glycolipid galactocerebroside (Gal-C) and the myelin proteins P0 and MBP. Furthermore, myelin sheaths appear morphologically normal with both compacted and uncompacted aspects when investigated by electron microscopy. Quantitative analysis revealed that the number of myelin sheaths was similar in cultures from MAG-deficient and wild-type mice. These findings support the view that MAG is not essential for myelin formation in the PNS. GLIA 22:213–220, 1998. © 1998 Wiley-Liss, Inc.     

Identification of the regulatory region of the peripheral myelin protein 22 (PMP22) gene that directs temporal and spatial expression in development and regeneration of peripheral nerves

Minor changes in PMP22 gene dosage have profound effects on the development and maintenance of peripheral nerves. This is evident from the genetic disease mechanisms in Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) as well as transgenic animals with altered PMP22 gene dosage. Thus, regulation of PMP22 is a crucial aspect in understanding the function of this protein in health and disease. In this study, we have generated transgenic mice containing 10 kb of the 5'-flanking region of the PMP22 gene, including the two previously identified alternative promoters, fused to a lacZ reporter gene. We show that this part of the PMP22 gene contains the necessary information to mirror the endogenous expression pattern in peripheral nerves during development and regeneration and in mouse models of demyelination due to genetic lesions. Transgene expression is strongly regulated during myelination, demyelination, and remyelination in Schwann cells, demonstrating the crucial influence of neuron-Schwann cell interactions in the regulation of PMP22. In addition, the region of the PMP22 gene present on this transgene confers also neuronal expression in sensory and motor neurons. These results provide the crucial basis for further dissection of the elements that direct the temporal and spatial regulation of the PMP22 gene and to elucidate the molecular basis of the master program regulating peripheral nerve myelination.     

Aggresome formation in neuropathy models based on peripheral myelin protein 22 mutations

Alterations in peripheral myelin protein 22 (PMP22) gene expression are associated with demyelinating peripheral neuropathies. Overexpression of wild type (wt) PMP22 or inhibition of proteasomal degradation lead to the formation of aggresomes, intracellular ubiquitinated PMP22 aggregates. Aggresome formation has now been observed with two mutant PMP22s, the Tr- and TrJ-PMP22 when the proteasome is inhibited. The formation of these aggresomes required intact microtubules and involved the recruitment of chaperones, including Hsp40, Hsp70, and alphaB-crystallin. Spontaneously formed ubiquitinated PMP22 aggregates were also observed in Schwann cells of homozygous TrJ mice. Significant upregulation of both the ubiquitin-proteasomal and lysosomal pathways occurred in affected nerves suggesting that two pathways of PMP22 degradation are present. Thus, the presence of aggresomes appears to be a common finding in neuropathy models of PMP22 overexpression and of some point mutations known to cause neuropathy in mice and humans.     

Characterization of peripheral myelin protein 22 in zebrafish (zPMP22) suggests an early role in the development of the peripheral nervous system.

Peripheral myelin protein 22 (PMP22) is a component of compact myelin of the peripheral nervous system (PNS). Mutations affecting PMP22 are associated with hereditary neuropathies in humans and rodents. Although mammalian PMP22 is expressed in several tissues, the disease pathology is restricted to the PNS. We describe the characterization of a PMP22-related cDNA from zebrafish and the distribution of its cognate mRNA. Phylogenetic considerations and mRNA expression in cranial nerves are consistent with the interpretation that the encoded protein is the orthologue of mammalian PMP22. In situ hybridization analysis during development showed zebrafish PMP22 expression in embryonic sclerotome cells, in neural crest cells, and in migratory derivatives of both populations. Based on this specific expression pattern prior to the onset of myelination, we hypothesize that zebrafish PMP22 may play a role in early PNS development and that disturbance of such functions may contribute to the PNS-restricted defects caused by mutations in the mammalian PMP22 gene.     

Improved culture methods to expand schwann cells with altered growth behaviour from CMT1A patients

A duplication of the gene for myelin protein PMP22 is by far the most common cause of the hereditary demyelinating neuropathy CMT1A. A role for PMP22 in cell growth in addition to its function as a myelin protein has been suggested because PMP22 is homologous to a gene specifically upregulated during growth arrest. Furthermore, transfected rat Schwann cells overexpressing PMP22 show reduced growth. In addition, abnormal Schwann cell differentiation has been described in nerve biopsies from CMT1A patients. To analyse whether the duplication of the PMP22 gene in CMT1A neuropathy primarily alters Schwann cell differentiation and to exclude nonspecific secondary responses, we improved human Schwann cell culturing. This allowed us long-term passaging of human Schwann cells with unchanged phenotype, assessed by expression of different Schwann cell markers. Subsequently we established Schwann cell cultures from CMT1A nerve biopsies. We find decreased proliferation of Schwann cells from different CMT1A patients in all passages. We also demonstrate PMP22 mRNA overexpression in cultured CMT1A Schwann cells. We conclude that decreased proliferation in cultured Schwann cells that carry the CMT1A duplication indicates abnormal differentiation of CMT1A Schwann cells. The identification of an abnormal phenotype of CMT1A Schwann cells in culture could possibly lead to an in vitro disease model. GLIA 23:89–98, 1998. © 1998 Wiley-Liss, Inc.