PNS Myelin Research Articles

Gadofluorine M-enhanced magnetic resonance nerve imaging: Comparison between acute inflammatory and chronic degenerative demyelination in rats

Nerve imaging by magnetic resonance imaging (MRI) is an emerging tool for the diagnostic work-up of patients with PNS disorders. We have recently shown that the experimental MR contrast agent gadofluorine M (Gf, Bayer Schering Pharma AG, Berlin) accumulates in nerves undergoing Wallerian degeneration and in areas of acute focal demyelination allowing in-vivo assessment of nerve pathology. The exact pathomechanism underlying Gf accumulation in peripheral nerve disorders is unknown so far. In the present study we compared nerve signal alterations on T2-w and Gf-enhanced T1-w MRI in two different models of acute inflammatory and chronic degenerative demyelination: experimental autoimmune neuritis (EAN) induced by immunization with PNS myelin and experimental Charcot–Marie-Tooth (CMT) disease in rats overexpressing the myelin protein PMP22. During the acute stage of inflammation and demyelination, strong Gf enhancement on T1-w MRI was seen in nerve roots and peripheral nerves in EAN, which resolved with completed remyelination. Similarly, Gf accumulation was seen in CMT rats during early stages with active demyelination at 6 weeks while at chronic stages (9 months) Gf enhancement decreased despite numerous demyelinated axons and onion bulb formation. At all disease stages no signal alterations were seen on T2-w MRI. In conclusion, our data show that the novel MR contrast agent Gf, but not Gadolinium (Gd)–DTPA, facilitates detection of ongoing demyelination by MR neurography independent from the underlying pathology. It appears that the extent of Gf enhancement depends on the acuity of demyelination and is probably related to a transient disturbance of the blood–nerve barrier. Clinical development of Gf may help to further improve the sensitivity of nerve lesion assessment by MRI in patients with peripheral neuropathies.

Peripheral myelin of Xenopus laevis: Role of electrostatic and hydrophobic interactions in membrane compaction

P0 glycoprotein is the major structural protein of peripheral nerve myelin where it is thought to modulate inter-membrane adhesion at both the extracellular apposition, which is labile upon changes in pH and ionic strength, and the cytoplasmic apposition, which is resistant to such changes. Most studies on P0 have focused on structure-function correlates in higher vertebrates. Here, we focused on its role in the structure and interactions of frog (Xenopus laevis) myelin, where it exists primarily in a dimeric form. As part of our study, we deduced the full sequence of X. laevis P0 (xP0) from its cDNA. The xP0 sequence was found to be similar to P0 sequences of higher vertebrates, suggesting that a common mechanism of PNS myelin compaction via P0 interaction might have emerged through evolution. As previously reported for mouse PNS myelin, a similar change of extracellular apposition in frog PNS myelin as a function of pH and ionic strength was observed, which can be explained by a conformational change of P0 due to protonation-deprotonation of His52 at P0's putative adhesive interface. On the other hand, the cytoplasmic apposition in frog PNS myelin, like that in the mouse, remained unchanged at different pH and ionic strength. The contribution of hydrophobic interactions to stabilizing the cytoplasmic apposition was tested by incubating sciatic nerves with detergents. Dramatic expansion at the cytoplasmic apposition was observed for both frog and mouse, indicating a common hydrophobic nature at this apposition. Urea also expanded the cytoplasmic apposition of frog myelin likely owing to denaturation of P0. Removal of the fatty acids that attached to the single Cys residue in the cytoplasmic domain of P0 did not change PNS myelin structure of either frog or mouse, suggesting that the P0-attached fatty acyl chain does not play a significant role in PNS myelin compaction and stability. These results help clarify the present understanding of P0's adhesion role and the role of its acylation in compact PNS myelin.

Myelin Structure and Composition in Zebrafish

To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability.

Evolution of a neuroprotective function of central nervous system myelin

The central nervous system (CNS) of terrestrial vertebrates underwent a prominent molecular change when a tetraspan membrane protein, myelin proteolipid protein (PLP), replaced the type I integral membrane protein, P0, as the major protein of myelin. To investigate possible reasons for this molecular switch, we genetically engineered mice to express P0 instead of PLP in CNS myelin. In the absence of PLP, the ancestral P0 provided a periodicity to mouse compact CNS myelin that was identical to mouse PNS myelin, where P0 is the major structural protein today. The PLP–P0 shift resulted in reduced myelin internode length, degeneration of myelinated axons, severe neurological disability, and a 50% reduction in lifespan. Mice with equal amounts of P0 and PLP in CNS myelin had a normal lifespan and no axonal degeneration. These data support the hypothesis that the P0–PLP shift during vertebrate evolution provided a vital neuroprotective function to myelin-forming CNS glia.

Peripheral neuropathies caused by mutations in the myelin protein zero

Charcot-Marie-Tooth disease type 1B (CMT1B) is caused by mutations in the major PNS myelin protein myelin protein zero (MPZ). MPZ is a member of the immunoglobulin supergene family and functions as an adhesion molecule helping to mediate compaction of PNS myelin. Mutations in MPZ appear to either disrupt myelination during development, leading to severe early onset neuropathies, or to disrupt axo–glial interactions leading to late onset neuropathies in adulthood. Identifying molecular pathways involved in early and late onset CMT1B will be crucial to understand how MPZ mutations cause CMT1B so that rational therapies for both early and late onset neuropathies can be developed.

Identification and functional characterization of mouse TPO1 as a myelin membrane protein

TPO1 is a member of the AIGP family, a unique group of proteins that contains 11 putative transmembrane domains. Expression of the rat TPO1 gene is upregulated in cultured oligodendrocytes (OLs) during development from pro-oligodendroblasts to postmitotic OLs. However, the distribution of native TPO1 protein in cultured OLs and in the brain has not been elucidated. We investigated the distribution and cellular function of TPO1 in myelinating cells of the nervous system. In mice, TPO1 gene expression was detected in the central (CNS) and peripheral (PNS) nervous systems and was markedly upregulated at postnatal days 10–20, an early phase of myelination in the mouse brain. To investigate TPO1 localization, we generated affinity-purified antibodies to synthetic peptides derived from mouse TPO1. Immunohistochemical analysis showed that TPO1 was expressed in OLs and Schwann cells but not in neurons and astrocytes. Schwann cells from trembler mice, which lack PNS myelin, had significantly decreased TPO1 expression and an altered localization pattern, suggesting that TPO1 is a functional myelin membrane protein. In OL lineage cell cultures, TPO1 was detected in A2B5(+) bipolar early progenitors, A2B5(+) multipolar Pro-OLs, GalC(+) immature OLs and MBP(+) mature OLs. The subcellular localization of TPO1 in OL lineage cells was mapped to the GM130(+) Golgi in cell bodies and Fyn(+) cell processes and myelin-like sheets. Furthermore, TPO1 selectively colocalized with non-phosphorylated Fyn and promoted Fyn autophosphorylation in COS7 cells, suggesting that TPO1 may play a role in myelin formation via Fyn kinase activation in the PNS and CNS.

Expression of P0 glycoprotein in CNS glia: Effects of overexpression in N20.1 cells

To examine effects of expression of the PNS myelin P0 glycoprotein in glial cells of CNS lineage, we transfected murine N20.1 glial cells with a rat P0 cDNA. A stably transfected cell line expressing high levels of P0 message showed P0 immunostaining, along with changes in morphology. Polymerase chain reaction (PCR) identified the predicted rat P0 sequence in the transfected N20.1 cells and further revealed low levels of mouse P0 message in the nontransfected cells and in primary mouse astrocytes. This is the first evidence of endogenous expression of message for P0 glycoprotein in CNS glia. Quantitative RT-PCR confirmed the expression of rat P0 mRNA in the transfected N20.1 cells, at levels about 400 times greater than murine P0 in nontransfected cells. A 27-kD band was detected in the transfected cells by Western blot with P0 antibody, but not in mock-transfected or nontransfected N20.1 cells. Immunocytochemistry following permeabilization showed intracellular vesicular localization of P0 in the cytoplasm and perinuclear rings in transfected cells, with a similar pattern but much lower levels in nontransfected cells. Faint surface staining for P0 protein without permeabilization was seen only on the transfected cells. A few transfected cells with membrane sheets stained more intensely for surface P0. Quantitative RT-PCR was used to determine if P0 overexpression altered expression of other myelin-related genes compared with glial fibrillary acidic protein (GFAP); the ratios of myelin basic protein (MBP)/GFAP and proteolipid protein (PLP)/GFAP were increased 2- to 3-fold in the P0-transfected cells. We conclude that P0 overexpression alters N20.1 gene expression and cell morphology, and shifts the cells from astroglial to oligodendroglial phenotype.

Junctions tighten myelin sheaths

![][1] Claudin-19 (green) helps form tight junction structures that seal PNS myelin sheaths. Tight junction-like structures run along myelin sheaths in both the central and peripheral nervous systems (CNS and PNS), but have only been functionally characterized in the CNS. Miyamoto et

Proteolipid plasmolipin: localization in polarized cells, regulated expression and lipid raft association in CNS and PNS myelin.

The proteolipid plasmolipin is member of the expanding group of tetraspan (4TM) myelin proteins. Initially, plasmolipin was isolated from kidney plasma membranes, but subsequent northern blot analysis revealed highest expression in the nervous system. To gain more insight into the functional roles of plasmolipin, we have generated a plasmolipin-specific polyclonal antibody. Immunohistochemical staining confirms our previous observation of glial plasmolipin expression and proves plasmolipin localization in the compact myelin of rat peripheral nerve and myelinated tracts of the CNS. Western blot analysis indicates a strong temporal correlation of plasmolipin expression and (re-) myelination in the PNS and CNS. However, following axotomy plasmolipin expression is also recovered in non-regenerating distal nerve stumps. In addition, we detected plasmolipin expression in distinct neuronal subpopulations of the CNS. The observed asymmetric distribution of plasmolipin in compact myelin, as well as in epithelial cells of kidney and stomach, indicates a polarized cellular localization. Therefore, we purified myelin from the CNS and PNS and demonstrated an enrichement of phosphorylated plasmolipin protein in detergent-insoluble lipid raft fractions, suggesting selective targeting of plasmolipin to the myelin membranes. The present data indicate that the proteolipid plasmolipin is a structural component of apical membranes of polarized cells and provides the basis for further functional analysis.

The SOX10 transcription factor: evaluation as a candidate gene for central and peripheral hereditary myelin disorders.

The SOX10 transcription factor is involved in development of neural crest derivatives and fate determination in glial cells. SOX10 mutations have been found in patients with intestinal aganglionosis and depigmentation with deafness (Waardenburg-Hirschsprung). Associated neurological signs have been reported in some cases, including a patient exhibiting a central and peripheral myelin deficiency. Therefore, we screened for SOX10 mutations in a large cohort of patients with peripheral and central myelin disorders. 56 were affected by classical demyelinating Charcot-Marie-Tooth disease without identified mutations in the genes encoding PNS myelin proteins (PMP22, P0), connexin 32 and the zinc-finger transcription factor, EGR2. 88 patients with undetermined leukodystrophy were selected from a large European prospective study. Associated clinical, magnetic resonance imaging and electrophysiological signs were consistent with a defect in CNS myelination in 83 and with an active degeneration of the CNS myelin in 5. No abnormalities in the proteolipid protein gene (PLP) were found. The absence of SOX100 mutation in this large cohort of patients suggests that this gene is not frequently involved in peripheral or central inherited myelin disorders.

Functional analysis for peripheral myelin protein PASII/PMP22: is it a member of claudin superfamily?

Two major glycoproteins, P0 and PASII/PMP22, are specifically expressed in peripheral myelin. Point mutations of these proteins and over or under expression of PASII/PMP22 cause various hereditary peripheral neuropathies. P0 is well characterized as a major adhesion molecule in PNS myelin, but the function of PASII/PMP22 is still unknown. Recently, an oligodendrocyte-specific protein (OSP) was identified as a member of the claudin family and as a component of tight junctions of central myelins. Since PASII/PMP22 shows similarity in structure to OSP, which is a tetraspan membrane protein, we speculated if PASII/PMP22 could be a member of claudin superfamily. The primary structure of PASII/PMP22 showed a significant homology of 48% and a 21% identity with the OSP sequence. Exogenous expression of PASII/PMP22 in C6 cells significantly inhibited BrdU incorporation to the cells. The C6 cells stably transfected with PASII/PMP22 cDNA showed no homophilic cell adhesive activity. When dorsal root ganglion (DRG) neurons were cocultured on PASII/PMP22 expressing cells, both neurite extension and branching of DRG neurons were significantly inhibited. These results indicate that PASII/PMP22 may play a role in a turning point of Schwann cell development from proliferation to differentiation. On the other hand, the cells expressing claudin family proteins are reported to show strong cell adhesive activity and an ability to form tight junctions with neighboring cells. For this reason, we currently do not have any functional data supporting that PASII/PMP22 is the member of claudin superfamily.

Structure of a major oligosaccharide of PASII/PMP22 glycoprotein in bovine peripheral nerve myelin.

The amino acid sequence of the glycopeptide obtained from bovine PASII/PMP22 protein in the PNS myelin was determined to be Gln-Asn-Cys-Ser-Thr, where the asparagine was glycosylated. To eliminate all the contaminated P(o) glycopeptides from the PASII/PMP22 glycopeptide preparation, we used a fluorescent probe, N-[2-(2-pyridylamino)ethyl]maleimide, which reacts with the cysteine of the PASII/PMP22 glycopeptides. The labeled PASII/PMP22 glycopeptides were isolated by HPLC and were digested further with glycopeptidase A. The resultant oligosaccharides were conjugated with 2-aminopyridine (PA) as a fluorescent tag. One major PA-oligosaccharide, OPPE1, was purified by HPLC. The structure of OPPE1 was elucidated by fast atom bombardment mass spectrometry and (1)H-NMR studies and comparing the derivatives of PA-OPPE1 and PA-oligosaccharides of gamma-globulin on HPLC. The structure, SO(4)-3GlcAbeta1-3Galbeta1-4GlcNAcbeta1-2Manalpha1+ ++-6(GlcNAcbeta1-4) (GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-6)GlcNAc- PA, was identical to the pyridylaminated form of the major oligosaccharide D8 of bovine P(o) previously reported.

Solubilization of PNS myelin membrane proteins by detergents.

We have applied 25 different detergents to the PNS myelin membrane. The extracts produced were analyzed regarding the total amount of protein solubilized, and which myelin glycoproteins were solubilized. The degree of protein extraction was correlated with the critical micelle concentration. For some detergents the protein extraction ability depends clearly on critical micelle concentration. These detergents are more potent if critical micelle concentration is small. The other type of extraction is independent on the critical micelle concentration, but extractability of proteins is low. The best detergents for solubilization of myelin proteins are neutral of the alkyl chain length n =9-11, as follows: dodecyl-beta-D-maltopyranoside, decyl-beta-D-maltopyranoside, n-dodecylsulfobetaine. We expect that these detergents will also be suitable for crystallization of P0 and PASII/PMP22 glycoproteins.

Tyrosine phosphorylation of PNS myelin P(0) occurs in the cytoplasmic domain and is maximal during early development.

P(0), the major protein of PNS myelin, is considered to play a critical role in the compaction and stabilization of myelin lamellae. The protein undergoes extensive posttranslational modifications, including phosphorylation at multiple serine moieties in the cytoplasmic region. Recently, we demonstrated that P(0) is phosphorylated on one or more tyrosine residues in rat nerve homogenates after incubation. In this study, we show that P(0) phosphorylated on tyrosine is also present in the intact animal. The proportion of P(0) molecules phosphorylated on tyrosine is highest during the first postnatal week, a period that coincides with the most rapid period of myelin deposition in the PNS. A peptide that constitutes the cytoplasmic domain was isolated from purified P(0) and shown by immunochemical and chemical means to be phosphorylated on the tyrosine corresponding to Y(191) in the intact protein. No evidence was obtained supporting the possibility that P(0) is phosphorylated on other tyrosine residues. The sequence of amino acids surrounding Y(191) resemble known substrate phosphorylation sites for some nonreceptor cytoplasmic tyrosine kinases, as well as tyrosine-based recognition signals associated with clathrin vesicle-mediated cndocytosis.

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.

On the molecular architecture of myelinated fibers.

Schwann cells and oligodendrocytes make the myelin sheaths of the PNS and CNS, respectively. Their myelin sheaths are structurally similar, consisting of multiple layers of specialized cell membrane that spiral around axons, but there are several differences. (1) CNS myelin has a "radial component" composed of a tight junction protein, claudin-11/oligodendrocyte-specific protein. (2) Schwann cells have a basal lamina and microvilli. (3) Although both CNS and PNS myelin sheaths have incisures, those in the CNS lack the structural as well as the molecular components of "reflexive" adherens junctions and gap junctions. In spite of their structural differences, the axonal membranes of the PNS and CNS are similarly organized. The nodal axolemma contains high concentrations of voltage-dependent sodium channels that are linked to the axonal cytoskeleton by ankyrin(G). The paranodal membrane contains Caspr/paranodin, which may participate in the formation of axoglial junctions. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, their associated beta2 subunit, as well as Caspr2, which is closely related to Caspr. The myelin sheath probably organizes these axonal membrane-related proteins via trans interactions.

Contact with Central Nervous System Myelin Inhibits Oligodendrocyte Progenitor Maturation

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated during development through the proliferation and differentiation of a distinct progenitor population. Not all oligodendrocyte progenitors generated during development differentiate, however, and large numbers of oligodendrocyte progenitors are present in the adult CNS, particularly in white matter. These “adult progenitors” can be identified through expression of the NG2 proteoglycan. Adult oligodendrocyte progenitors are thought to develop from the original pool of progenitors and in vitro are capable of differentiating into oligodendrocytes. Why these cells fail to differentiate in the intact CNS is currently unclear. Here we show that contact with CNS myelin inhibits the maturation of immature oligodendrocyte progenitors. The inhibition of oligodendrocyte progenitor maturation is a characteristic of CNS myelin that is not shared by several other membrane preparations including adult and neonatal neural membrane fractions, PNS myelin, or liver. This inhibition is concentration dependent, is reversible, and appears not to be mediated by either myelin basic protein or basic fibroblast growth factor. Myelin-induced inhibition of oligodendrocyte progenitor maturation provides a mechanism to explain the generation of a residual pool of immature oligodendrocyte progenitors in the mature CNS.

New Vistas on the Pathomechanism of Charcot-Marie-Tooth and Related Peripheral Neuropathies.

A gene-dosage mechanism in CMT1A and HNPP has been postulated previously. Here, recent findings are discussed concerning (i) the functional consequences of altered PMP22 expression on Schwann cell growth regulation and on the capacity of genetically modified Schwann cells to myelinate peripheral axons, (ii) the cell physiological effects caused by the expression of certain disease-related missense mutations of PMP22 that are known to alter the Schwann cell phenotype and impair myelination in vivo, and (iii) the pathomechanism of CMT1 in light of findings on a novel association between PMP22 and P0 in PNS myelin.

Peripheral Myelin Protein 22 and Protein Zero: a Novel Association in Peripheral Nervous System Myelin

Mutations found in the two major glycosylated transmembrane proteins of the PNS myelin, the peripheral myelin protein zero (P0) and peripheral myelin protein 22 (PMP22), have been independently associated with the most common hereditary demyelinating peripheral neuropathies. Genotype-phenotype correlations in humans and transgenic animals have provided functional evidence that P0 and PMP22 are involved in formation and maintenance of compact myelin. Here, we demonstrate for the first time that P0 and PMP22 proteins form complexes in the myelin membrane, as shown by coimmunoprecipitation experiments, and that glycosylation is not involved in mediating these interactions. Complex formation was also detected when the two proteins were coexpressed in heterologous cells. In transfected cells, P0 and PMP22 are recruited and colocalize at the apposed plasma membranes of expressors as shown by confocal microscopy. These findings provide a new basis for a better understanding of myelin assembly and of the pathomechanisms involved in demyelinating peripheral neuropathies. Furthermore, these results propose a possible explanation why alterations in either of these molecules are sufficient to destabilize the myelin structure and cause a similar disease 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. GLIA 25:358–369, 1999. © 1999 Wiley-Liss, Inc.