Myelinating and demyelinating phenotype of Trembler-J mouse (a model of Charcot-Marie-Tooth human disease) analyzed by atomic force microscopy and confocal microscopy

The accumulation of misfolded proteins is associated with various neurodegenerative conditions. Mutations in PMP-22 are associated with the human peripheral neuropathy, Charcot-Marie-Tooth Type 1A (CMT1A). PMP-22 is a short-lived 22 kDa glycoprotein, which plays a key role in the maintenance of myelin structure and compaction, highly expressed by Schwann cells. It forms aggregates when the proteasome is inhibited or the protein is mutated. This study reports the application of atomic force microscopy (AFM) as a detector of profound topographical and mechanical changes in Trembler-J mouse (CMT1A animal model). AFM images showed topographical differences in the extracellular matrix and basal lamina organization of Tr-J/+ nerve fibers. The immunocytochemical analysis indicated that PMP-22 protein is associated with type IV collagen (a basal lamina ubiquitous component) in the Tr-J/+ Schwann cell perinuclear region. Changes in mechanical properties of single myelinating Tr-J/+ nerve fibers were investigated, and alterations in cellular stiffness were found. These results might be associated with F-actin cytoskeleton organization in Tr-J/+ nerve fibers. AFM nanoscale imaging focused on topography and mechanical properties of peripheral nerve fibers might provide new insights into the study of peripheral nervous system diseases.     

Rapid screening of myelin genes in CMT1 patients by SSCP analysis: identification of new mutations and polymorphisms in the P0 gene

Charcot-Marie-Tooth type (CMT1) disease or hereditary motor and sensory neuropathy type I (HMSNI) is an autosomal dominant peripheral neuropathy. In most CMT1 families, the disease cosegregates with a 1.5-Mb duplication on chromosome 17p11.2 (CMT1A). A few patients have been found with mutations in the peripheral myelin protein 22 (PMP-22) gene located in the CMT1A region. In other families mutations have been identified in the major peripheral myelin protein p_o gene localized on chromosome Iq21-q23 (CMT1B). We performed a rapid mutation screening of the PMP-22 and P₀ genes in non-duplicated CMT1 patients by single-strand conformation polymorphism analysis followed by direct polymerase chain reaction sequencing of genomic DNA. Six new single base changes in the P₀ gene were observed: two missense mutations in, respectively, exons 2 and 3, two nonsense mutations in exon 4, and two silent mutations or polymorphisms in, respectively, exons 3 and 6.     

Characterization of a novel peripheral nervous system myelin protein (PMP-22/SR13)

We have recently described a novel cDNA, SR13 (Welcher, A. A., U. Suter, M. De Leon, G. J. Snipes, and E. M. Shooter. 1991. Proc. Natl. Acad. Sci. USA. 88:7195-7199), that is repressed after sciatic nerve crush injury and shows homology to both the growth arrest-specific mRNA, gas3 (Manfioletti, G., M. E. Ruaro, G. Del Sal, L. Philipson, and C. Schneider, 1990. Mol. Cell Biol. 10:2924-2930), and to the myelin protein, PASII (Kitamura, K., M. Suzuki, and K. Uyemura. 1976. Biochim. Biophys. Acta. 455:806-816). In this report, we show that the 22-kD SR13 protein is expressed in the compact portion of essentially all myelinated fibers in the peripheral nervous system. Although SR13 mRNA was found in the central nervous system, no corresponding SR13 protein could be detected by either immunoblot analysis or by immunohistochemistry. Northern and immunoblot analysis of SR13 mRNA and protein expression during development of the peripheral nervous system reveal a pattern similar to other myelin proteins. Furthermore, we demonstrate by in situ mRNA hybridization on tissue sections and on individual nerve fibers that SR13 mRNA is produced predominantly by Schwann cells. We conclude that the SR13 protein is apparently exclusively expressed in the peripheral nervous system where it is a major component of myelin. Thus, we propose the name Peripheral Myelin Protein-22 (PMP-22) for the proteins and cDNA previously designated PASII, SR13, and gas3.     

Evidence for genetic heterogeneity underlying hereditary neuropathy with liability to pressure palsies

Hereditary neuropathy with liability to pressure palsies (HNPP) is a disorder of the peripheral nervous system, the cause of which has recently been identified as a deletion on chromosome 17p. The deletion corresponds to the duplication that is commonly observed in patients with hereditary motor and sensory neuropathy type Ia (HMSNIa, 17p11.2–p12). Therefore, the gene for peripheral myelin protein 22 (PMP-22) is a candidate gene for both HMSNIa and HNPP. Here, we show that a similar deletion is present in one family with HNPP but is clearly absent in another family. Affected members of this family carry the expected two copies of the PMP-22 gene and the surrounding region. Furthermore, linkage analyses of this family exclude a large part of 17p, spanning the area deleted in other families with HNPP, as the location for the disease gene. These data strongly argue for the existence of genetic heterogeneity underlying HNPP. Results from two-point linkage analysis with markers on chromosome 1q are inconsistent with a possible involvement of the locus for HMSNIb in the present family.     

Isolation and sequence determination of cDNA encoding PMP-22 (PAS-II/SR13/Gas-3) of human peripheral myelin.

A full length cDNA of PMP-22 (PAS-II/SR13/Gas-3) of peripheral myelin has been isolated from a cDNA library of human fetus spinal cord. The clone is 1823 base pairs (bp) in length and contains a 480 bp open reading frame encoding a polypeptide of 160 residues. The deduced amino acid sequence is highly homologous to PMP-22 from bovine (PAS-II), rat (SR13) and mouse (Gas-3).     

Myelin Gene Expression in Immortalized Schwann Cells: Relationship to Cell Density and Proliferation

Abstract: Myelin gene expression was investigated in the immortalized S16 Schwann cell line grown in the presence and absence of serum and at different densities. Protein expression was monitored by western blotting, and message levels were determined by RNase protection assays. To study cell proliferation rates at different cell densities and serum conditions. [³H]thymidine uptake assays and cell counts were performed. Although serum deprivation decreased cell proliferation as expected, the proliferation of S16 cells was unchanged or slightly increased at high density under the conditions of our experiments in either serum-containing or serum-free medium. This increased cell division at high density appeared to be due to greater release of an autocrine growth factor to the medium by dense cell populations. For both sparse and dense cells, substantially more P₀ glycoprotein (P₀) and myelin-associated glycoprotein (MAG) per milligram of total cellular protein were expressed when the cells were proliferating slowly in defined medium in comparison with more rapidly proliferating cells in serum-containing medium. Furthermore, in both serum-containing and defined media, dense cell populations expressed more MAG and P₀ than sparse ones. P₀ mRNA and MAG mRNA levels generally paralleled protein levels. The level of mRNA for peripheral myelin protein-22 (PMP-22) was also increased at high cell density but did not change much when proliferation was decreased by serum deprivation. PMP-22 protein was not detected under any of the growth conditions. The changes in expression of these genes with growth conditions may be specific for myelin proteins, because the expression of a nonmyelin glycoprotein, L1, remained constant. The level of cyclic AMP in the cells did not change with the different growth conditions tested. The results indicate that the S16 Schwann cell line mimics primary or secondary Schwann cells by down-regulating myelin gene expression when it proliferates more rapidly in the presence of serum. Furthermore, in both the presence and absence of serum, there was greater expression of myelin genes at high cell density that was not associated with a decreased proliferative rate. Because evidence for a role of secretory factors in affecting myelin gene expression was not obtained by treating sparse S16 cells with medium conditioned by dense S16 cells, the results suggest that the higher expression of myelin genes at high density may be mediated by cell-to-cell contact.     

Elevated Protein Carbonylation,and Misfolding in Sciatic Nerve from db/db and Sod1?/? Mice: Plausible Link between Oxidative Stress and Demyelination

Diabetic peripheral polyneuropathy is associated with decrements in motor/sensory neuron myelination, nerve conduction and muscle function; however, the mechanisms of reduced myelination in diabetes are poorly understood. Chronic elevation of oxidative stress may be one of the potential determinants for demyelination as lipids and proteins are important structural constituents of myelin and highly susceptible to oxidation. The goal of the current study was to determine whether there is a link between protein oxidation/misfolding and demyelination. We chose two distinct models to test our hypothesis: 1) the leptin receptor deficient mouse (dbdb) model of diabetic polyneuropathy and 2) superoxide dismutase 1 knockout (Sod1^−/−) mouse model of in vivo oxidative stress. Both experimental models displayed a significant decrement in nerve conduction, increase in tail distal motor latency as well as reduced myelin thickness and fiber/axon diameter. Further biochemical studies demonstrated that oxidative stress is likely to be a potential key player in the demyelination process as both models exhibited significant elevation in protein carbonylation and alterations in protein conformation. Since peripheral myelin protein 22 (PMP22) is a key component of myelin sheath and has been found mutated and aggregated in several peripheral neuropathies, we predicted that an increase in carbonylation and aggregation of PMP22 may be associated with demyelination in dbdb mice. Indeed, PMP22 was found to be carbonylated and aggregated in sciatic nerves of dbdb mice. Sequence-driven hydropathy plot analysis and in vitro oxidation-induced aggregation of purified PMP22 protein supported the premise for oxidation-dependent aggregation of PMP22 in dbdb mice. Collectively, these data strongly suggest for the first time that oxidation-mediated protein misfolding and aggregation of key myelin proteins may be linked to demyelination and reduced nerve conduction in peripheral neuropathies.     

COMPARATIVE STUDIES ON THE MYELIN PROTEINS OF BOVINE PERIPHERAL NERVE AND SPINAL CORD

Abstract— (1) Two myelin fractions of bovine peripheral nerve and spinal cord have been studied comparatively. Cholesterol as well as cerebroside content per mg of protein in the peripheral nerve myelin was less than that in the spinal cord myelin, while no significant difference in the total phospholipid content was noted.
(2) The basic proteins in myelin fractions were quantitatively estimated by disc gel electrophoresis. Around one-fourth of the total myelin protein in the bovine peripheral nerve was a basic protein with a mobility of 1.07 relative to lysozyme by Reisfeld's disc gel electrophoresis.
(3) The myelin proteins in the peripheral nerve were less completely solubilized than those of the spinal cord by treatment with deoxycholate as well as by Triton-salt solution. The protein fractions obtained from the peripheral nerve myelin by techniques similar to that for obtaining the proteolipids from the spinal cord myelin, contained different types of protein.
(4) 2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the peripheral nerve myelin was only one tenth of that in the spinal cord myelin. The Triton-salt insoluble fraction showed remarkable high activity among subfractions of the spinal cord myelin.
(5) By immunological studies, it may be concluded that an antigenic substance for experimental allergic neuritis was localized in the peripheral nerve myelin, but not in its basic protein.     

Rafts in adult peripheral nerve myelin contain major structural myelin proteins and myelin and lymphocyte protein (MAL) and CD59 as specific markers

The myelin and lymphocyte protein (MAL) proteolipid is localized in central and peripheral compact myelin membranes, as well as in apical membranes of particular polarized cells. In this study, we addressed the question whether MAL and other peripheral myelin proteins are sorted and targeted to myelin membranes using mechanisms similar to those observed in polarized epithelial cells. To investigate the presence of raft-mediated sorting pathways in Schwann cells, we have isolated and analysed their composition in myelin membranes. Here, we show that rafts are present in adult human and rat peripheral compact myelin membranes and contain MAL, the GPI-anchored protein CD59, and substantial amounts of the PMP22 and P0. Colocalization studies show that CD59, and MAL have an almost identical expression pattern within compact myelin. Moreover, immuno-electron microscopy revealed that MAL, besides its localization in compact myelin, is also localized to Schmidt-Lanterman incisures. Taken together, our results demonstrate the presence of detergent-insoluble glycolipid-enriched complexes (DIGs) in different compartments of myelin membranes and indicate an important role for DIG-mediated transport mechanisms in the maintenance of the adult myelin sheath.     

Myelin tetraspan family proteins but no non-tetraspan family proteins are present in the ascidian (Ciona intestinalis) genome

Several of the proteins used to form and maintain myelin sheaths in the central nervous system (CNS) and the peripheral nervous system (PNS) are shared among different vertebrate classes. These proteins include one-to-several alternatively spliced myelin basic protein (MBP) isoforms in all sheaths, proteolipid protein (PLP) and DM20 (except in amphibians) in tetrapod CNS sheaths, and one or two protein zero (P0) isoforms in fish CNS and in all vertebrate PNS sheaths. Several other proteins, including 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP), myelin and lymphocyte protein (MAL), plasmolipin, and peripheral myelin protein 22 (PMP22; prominent in PNS myelin), are localized to myelin and myelin-associated membranes, though class distributions are less well studied. Databases with known and identified sequences of these proteins from cartilaginous and teleost fishes, amphibians, reptiles, birds, and mammals were prepared and used to search for potential homologs in the basal vertebrate, Ciona intestinalis. Homologs of lipophilin proteins, MAL/plasmolipin, and PMP22 were identified in the Ciona genome. In contrast, no MBP, P0, or CNP homologs were found. These studies provide a framework for understanding how myelin proteins were recruited during evolution and how structural adaptations enabled them to play key roles in myelination.     

Effects of neuroactive steroids on myelin of peripheral nervous system

Peripheral nervous system (PNS) possess both classical (e.g. progesterone receptor, PR, androgen receptor, AR) and non-classical (e.g. GABA_A receptor) steroid receptors and consequently may represent a target for the action of neuroactive steroids. Our data have indicated that neuroactive steroids, like for instance, progesterone, dihydroprogesterone, tetrahydroprogesterone, dihydrotestosterone and 3-diol, stimulate both in vivo and in vitro (Schwann cell cultures), the expression of two important proteins of the myelin of peripheral nerves, the glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22). It is important to highlight that the mechanisms by which neuroactive steroids exert their effects on the expression of Po and PMP22 involve different kind of receptors depending on the steroid and on the myelin protein considered. In particular, at least in culture of Schwann cells, the expression of Po seems to be under the control of PR, while that of PMP22 needs the GABA_A receptor.

Because Po and PMP22 play an important physiological role for the maintenance of the multilamellar structure of the myelin of the PNS, the present observations might suggest the utilization of neuroactive steroids as new therapeutically approaches for the rebuilding of the peripheral myelin.     

Polymorphisms in the PMP-22 gene region (17p11.2–12) are crucial for simplified diagnosis of duplications/deletions

DNA duplications and deletions of a 1.5-Mb region in chromosome 17p11.2–12 comprising the gene encoding peripheral myelin protein 22 (PMP-22) are the common mutations in Charcot-Marie-Tooth disease type 1 (CMT1) and hereditary neuropathy with liability to pressure palsies (HNPP). A 1.7-kb recombination hotspot region has been identified within misaligned flanking repeats (CMT1-REP elements) by detection of CMT- and HNPP-specific junction fragments in Southern blot analyses. In order to simplify routine diagnosis we introduce a polymerase chain reaction-based method to identify directly specific REP junction fragments. Using this test, specific fragments were detected in ∼ 67% of both CMT duplication and HNPP deletion cases. Polymorphism within a specific restriction enzyme recognition site is crucial for both Southern blot and PCR analyses of junction fragments. Received: 25 October 1996 / Revised: 16 December 1996     

HISTOCHEMISTRY OF MYELIN XIV PERIPHERAL NERVE MYELIN PROTEINS: ELECTROPHORETIC AND HISTOCHEMICAL CORRELATIONS

Abstract— Myelin from the peripheral nervous system has been shown to contain two basic protein components and an electrophoretically slower-moving major protein, the 'J' band. The 'J' band protein cannot be selectively removed by aqueous or organic solvents and does not correspond to proteolipid or acidic protein. Histochemical stains applied to peripheral nervous systems myelin proteins separated by polyacrylamide electrophoresis indicate that 'J' band protein is analogous with the neurokeratin of the nerve sheath. Trypanophilia observed histochemically in unfixed myelin is principally due to basic proteins. With prolonged tryptic digestion 'J' band protein is degraded. Thus, previous classifications of myelin proteins based on trypsin sensitivity have been modified. All peripheral nervous system myelin proteins should be regarded as trypsin-sensitive, the basic protein being relatively more and the 'J' band protein relatively less susceptible.     

Steroid effects on the gene expression of peripheral myelin proteins

The present article summarizes recent observations obtained in our laboratory which clearly indicate that sex steroids exert relevant effects on the peripheral nervous system. In particular, the following important points have emerged: (1) Steroids exert stimulatory actions on the synthesis of the proteins proper of the peripheral myelin (e.g., glycoprotein Po and peripheral myelin protein 22) in vivo and on the Schwann cells in culture; (2) in many cases the actions of hormonal steroids are not due to their native molecular forms but rather to their metabolites (e.g., dihydroprogesterone and tetrahydroprogesterone in the case of progesterone; dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol in the case of testosterone); (3) the mechanism of action of the various steroidal molecules may involve both classical (progesterone and androgen receptors) and nonclassical steroid receptors (GABA(A) receptor); and finally, (4) the stimulatory action of steroid hormones on the proteins of the peripheral myelin might have clinical significance in cases in which the rebuilding of myelin is needed (e.g., aging, peripheral injury, demyelinating diseases, and diabetic neuropathy).     

Functional Recovery Occurs Even After Partial Remyelination of Axon-Meshed Median and Ulnar Nerves in Mice

Upper limb nerve injuries are common, and their treatment poses a challenge for physicians and surgeons. Experimental models help in minimum exploration of the functional characteristics of peripheral nerve injuries of forelimbs. This study was conducted to characterize the functional recovery (1, 3, 7, 10, 14, and 21 days) after median and ulnar nerve crush in mice and analyze the histological and biochemical markers of nerve regeneration (after 21 days). Sensory–functional impairments appeared after 1 day. The peripheral nerve morphology, the nerve structure, and the density of myelin proteins [myelin protein zero (P0) and peripheral myelin protein 22 (PMP22)] were analyzed after 21 days. Cold allodynia and fine motor coordination recovery occurred on the 10th day, and grip strength recovery was observed on the 14th day after injury. After 21 days, there was partial myelin sheath recovery. PMP22 recovery was complete, whereas P0 recovery was not. Results suggest that there is complete functional recovery even with partial remyelination of median and ulnar nerves in mice.

     

Myelin and lymphocyte protein (MAL/MVP17/VIP17) and plasmolipin are members of an extended gene family

An increasing number of four-transmembrane proteins has been found to be associated with CNS and PNS myelin. Some of these proteins play crucial roles in the development and maintenance of the nervous system. In the CNS, proteolipid protein (PLP) is mutated in the myelin disorder Pelizaeus-Merzbacher disease and in spastic paraplegia, while in the PNS, peripheral myelin protein 22 (PMP22) and connexin32 (C×32) are culprit genes in the most frequent forms of hereditary peripheral neuropathies. Myelin and lymphocyte protein (MAL; also called MVP17 or VIP17) and plasmolipin are additional tetraspan proteins that are highly expressed by myelinating glial cells. However, little is known about the role of these proteins in the nervous system. As a prerequisite for functional genetic approaches in the mouse, we have isolated and characterized a mouse MAL cDNA and the corresponding structural MAL gene. Computer-aided analysis and database searches revealed that MAL belongs to a larger gene family which also includes plasmolipin, BENE and the expressed sequence tag (EST) H09290. While the overall amino acid sequence identities between mouse MAL and the related proteins are relatively low (29–37%), the conserved motif -[Q/Y-G-W-V-M-F/Y-V]- which is found at the junction of the first extracellular loop and the second membrane-associated domain serves as a fingerprint for the MAL protein family. Expression analysis of the members of the MAL gene family indicates widespread expression in various tissues, suggesting a common role of these proteins in cell biology.     

Common themes in peripheral neuropathy disease genes

After a century of study, mutations in connexin32, peripheral myelin protein22, and protein zero are now known to culminate in the prototypical phenotype of Charcot-Marie-Tooth disease. Many of these mutations have been modeled in rodents and in tissue culture. Consequently, structure-function predictions for these mutations are now possible and detailed analyses of many of them are ongoing. Despite the marked differences in the functions of these three proteins, it is profitable to consider the many similarities between them, including the types of mutational mechanisms and their effects on myelin structure and function. Accordingly, the biology and genetics of Charcot-Marie-Tooth disease and other inherited peripheral neuropathies due to mutations in these proteins are reviewed.     

Oral curcumin mitigates the clinical and neuropathologic phenotype of the Trembler-J mouse: a potential therapy for inherited neuropathy

Mutations in myelin genes cause inherited peripheral neuropathies that range in severity from adult-onset Charcot-Marie-Tooth disease type 1 to childhood-onset Dejerine-Sottas neuropathy and congenital hypomyelinating neuropathy. Many myelin gene mutants that cause severe disease, such as those in the myelin protein zero gene (MPZ) and the peripheral myelin protein 22 gene (PMP22), appear to make aberrant proteins that accumulate primarily within the endoplasmic reticulum (ER), resulting in Schwann cell death by apoptosis and, subsequently, peripheral neuropathy. We previously showed that curcumin supplementation could abrogate ER retention and aggregation-induced apoptosis associated with neuropathy-causing MPZ mutants. We now show reduced apoptosis after curcumin treatment of cells in tissue culture that express PMP22 mutants. Furthermore, we demonstrate that oral administration of curcumin partially mitigates the severe neuropathy phenotype of the Trembler-J mouse model in a dose-dependent manner. Administration of curcumin significantly decreases the percentage of apoptotic Schwann cells and results in increased number and size of myelinated axons in sciatic nerves, leading to improved motor performance. Our findings indicate that curcumin treatment is sufficient to relieve the toxic effect of mutant aggregation-induced apoptosis and improves the neuropathologic phenotype in an animal model of human neuropathy, suggesting a potential therapeutic role in selected forms of inherited peripheral neuropathies.     

Dichloroacetate causes reversible demyelination in vitro: potential mechanism for its neuropathic effect

Dichloroacetate (DCA) is an investigational drug for genetic mitochondrial diseases whose use has been mitigated by reversible peripheral neuropathy. We investigated the mechanism of DCA neurotoxicity using cultured rat Schwann cells (SCs) and dorsal root ganglia (DRG) neurons. Myelinating SC-DRG neuron co-cultures, isolated SCs and DRG neurons were exposed to 1-20 mm DCA for up to 12 days. In myelinating co-cultures, DCA caused a dose- and exposure-dependent decrease of myelination, as determined by immunolabeling and immunoblotting for myelin basic protein (MBP), protein zero (P0), myelin-associated glycoprotein (MAG) and peripheral myelin protein 22 (PMP22). Partial recovery of myelination occurred following a 10-day washout of DCA. DCA did not affect the steady-state levels of intermediate filament proteins, but promoted the formation of anti-neurofilament antibody reactive whirls. In isolated SC cultures, DCA decreased the expression of P0 and PMP22, while it increased the levels of p75(NTR) (neurotrophin receptor), as compared with non-DCA-treated samples. DCA had modest adverse effects on neuronal and glial cell vitality, as determined by the release of lactate dehydrogenase. These results demonstrate that DCA induces a reversible inhibition of myelin-related proteins that may account, at least in part, for its clinical peripheral neuropathic effects.     

Competitive binding of triplex-forming oligonucleotides in the two alternate promoters of the PMP22 gene

Overexpression of the 22-kDa peripheral myelin protein (PMP22) causes the inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A). In an attempt to alter PMP22 gene expression as a possible therapeutic strategy for CMT1A, antiparallel triplex-forming oligonucleotides (TFO) were designed to bind to purine-rich target sequences in the two PMP22 gene promoters, P1 and P2. Target region I in P1 and region V in P2 were also shown to specifically bind proteins in mammalian nuclear extracts. Competition for binding of these targets by TFO vs. protein(s) was compared by exposing proteins to their target sequences after triplex formation (passive competition) or by allowing TFO and proteins to simultaneously compete for the same targets (active competition). In both formats, TFO were shown to competitively interfere with the binding of protein to region I. Oligonucleotides directed to region V competed for protein binding by a nontriplex-mediated mechanism, most likely via the formation of higher-order, manganese-destabilizable structures. Given that the activity of the P1 promoter is closely linked to peripheral nerve myelination, TFO identified here could serve as useful reagents in the investigation of promoter function, the role of PMP22 in myelination, and possibly as rationally designed drugs for the therapy of CMT1A. The nontriplex-mediated action of TFO directed at the P2 promoter may have wider implications for the use of such oligonucleotides in vivo.