Anthony M. Heape

Myelin synthesis in the peripheral nervous system

By imposing saltatory conduction on the nervous impulse, the principal role of the myelin sheath is to allow the faster propagation of action potentials along the axons which it surrounds. Peripheral nervous system (PNS) myelin is formed by the differentiation of the plasma membrane of Schwann cells. One of the biochemical characteristics that distinguishes myelin from other biological membranes is its high lipid-to-protein ratio. All the major lipid classes are represented in the myelin membrane, while several myelin-specific proteins have been identified. During development, the presence of axons is required for the initiation of myelination, but the nature of the axonal signal is still unknown. The only certainties are that this signal is synthesized by axons whose diameter is greater than 0.7 microm, and that the signal(s) include(s) a diffusible molecule. Morphological studies have provided us with information concerning the timing of myelination, the mechanism by which immature Schwann cells differentiate into a myelinating phenotype and lay down the myelin sheath around the axon, and the accumulation and the structure of the myelin membrane. The last 20 years have seen the identification and the cDNA and gene cloning of the major PNS myelin proteins, which signalled the beginning of the knock-out decade: transgenic null-mutant mice have been created for almost every protein gene. The study of these animals shows that the formation of myelin is considerably less sensitive to molecular alterations than the maintenance of myelin. During the same period, important data has been gathered concerning the synthesis and function of lipids in PNS myelin, although this field has received relatively little attention compared with that of their protein counterparts.

Myelination in mouse dorsal root ganglion/Schwann cell cocultures.

The established protocols for in vitro studies of peripheral nerve myelination with rat embryonic dorsal root ganglia (DRG) and postnatal Schwann cell cocultures do not work with mouse cells. Consequently, the full potential of this model, which would allow to perform cell type-specific, mixed genotype cocultures without cross-breeding the animals, cannot be exploited. We determined the conditions required to promote full myelination in cocultures of pre-purified mouse embryonic DRG and neonatal Schwann cells, and present a method which consistently yields 50-200 mature myelin sheaths/culture. Causes for the failure of the existing protocols to yield satisfactory results with mouse cells fell into three categories: the lack of adherent support provided by the substratum, growth factor and hormone deficiencies, and the high serum content of the media. For optimal results, mouse cocultures require a 3-dimensional substratum, a myelination-promoting culture medium containing pituitary extract, N2 supplement and forskolin, and a low serum concentration.

Lack of Collagen XV Impairs Peripheral Nerve Maturation and, When Combined with Laminin-411 Deficiency, Leads to Basement Membrane Abnormalities and Sensorimotor Dysfunction

Although the Schwann cell basement membrane (BM) is required for normal Schwann cell terminal differentiation, the role of BM-associated collagens in peripheral nerve maturation is poorly understood. Collagen XV is a BM zone component strongly expressed in peripheral nerves, and we show that its absence in mice leads to loosely packed axons in C-fibers and polyaxonal myelination. The simultaneous lack of collagen XV and another peripheral nerve component affecting myelination, laminin α4, leads to severely impaired radial sorting and myelination, and the maturation of the nerve is permanently compromised, contrasting with the slow repair observed in Lama4−/− single knock-out mice. Moreover, the Col15a1−/−;Lama4−/− double knock-out (DKO) mice initially lack C-fibers and, even over 1 year of age have only a few, abnormal C-fibers. The Lama4−/− knock-out results in motor and tactile sensory impairment, which is exacerbated by a simultaneous Col15a1−/− knock-out, whereas sensitivity to heat-induced pain is increased in the DKO mice. Lack of collagen XV results in slower sensory nerve conduction, whereas the Lama4−/− and DKO mice exhibit increased sensory nerve action potentials and decreased compound muscle action potentials; x-ray diffraction revealed less mature myelin in the sciatic nerves of the latter than in controls. Ultrastructural analyses revealed changes in the Schwann cell BM in all three mutants, ranging from severe (DKO) to nearly normal (Col15a1−/−). Collagen XV thus contributes to peripheral nerve maturation and C-fiber formation, and its simultaneous deletion from neural BM zones with laminin α4 leads to a DKO phenotype distinct from those of both single knock-outs.

Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP–CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein–protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP–CaM interaction, including a 3D model of the complex between full-length proteins.

A quantitative developmental study of the peripheral nerve lipid composition during myelinogenesis in normal and trembler mice

The quantitative evolution of 10 polar lipids was examined in the sciatic nerves of normal and trembler mice between the ages of 3 days and 60 days. In normal nerves, the polar lipids accumulated slowly until the age of 9 days. A period of rapid accumulation then took place until 18 days of age, after which the phospholipids plateaued, while the glycolipid content continued to increase at a slower rate. The results obtained for the sciatic nerves of trembler mice show that the accumulation of all the polar lipids studied, except phosphatidylcholine and hydroxysulfatides, is abnormal from the earliest stages of postnatal development, and strongly support the view that the primary disorder in the trembler peripheral nervous system is one of dysmyelination. With the exception of cardiolipin, all the lipids in the trembler nerves stopped accumulating at the age of 18 days. The cerebrosides were the lipids the most affected severely at all ages.

A quantitative developmental study of neutral lipids during myelinogenesis in the peripheral nervous system of normal and trembler mice

The quantitative accumulation of neutral lipids during the period of myelination in the peripheral nervous system was studied in normal and trembler mouse sciatic nerves, between the ages of 5 and 27 days. Neutral lipids were resolved by high-performance thin-layer chromatography, using the solvent mixture hexane/diethyl ether/acetic acid (90:15:2, v/v/v). The lipids were quantitated, after copper acetate/phosphoric acid charring, by densitometric scanning, using an external standard technique. Cholesterol and triacylglycerols accumulated in normal nerves throughout the period studied, while cholesteryl esters were not observed at any age. In trembler nerves, the accumulation of cholesterol took place at a much lower rate than in normal nerves and this lipid was deficient from the earliest stages of development. Triacylglycerols were not significantly deficient in trembler nerves during the first 2-3 weeks, but, after the age of 18 days, their quantity diminished significantly. Cholesteryl esters were first detected in the mutant nerves at the age of 18 days. These results, in agreement with those of a previous developmental study of the polar lipids, are strongly in favour of the view that the trembler mutation directly induces a process of dysmyelination and that demyelination is a secondary event.

Isolation, purification and expansion of myelination-competent, neonatal mouse Schwann cells.

Most studies of peripheral nerve myelination using culture models are performed with dorsal root ganglion neurons and Schwann cells pre‐purified from the rat. The potential of this model is severely compromised by the lack of rat myelin mutants and the published protocols work poorly with mouse cells, for which numerous myelin mutants are available. This is partly due to difficulties in obtaining sufficient quantities of myelination‐competent mouse Schwann cells. Here, we describe the isolation, purification and expansion of wild‐type, myelination‐competent Schwann cells from the sciatic nerves of 4‐day‐old mouse pups. The method consistently yields 1.9–3.3 × 106 of ∼95% pure Schwann cells from the sciatic nerves of 12–15 4‐day‐old mouse pups, within 14–20 days. The Schwann cell proliferation rate ranges from 2.7‐ to 4.30‐fold growth/week. Proliferation ceases within 4 weeks, when the cells become quiescent. Growth is reinduced by the presence of neurons; neuregulin is not sufficient for this effect. The Schwann cells isolated by this protocol are able to form compact myelin in culture, as judged by the segregated expression patterns of early (myelin‐associated glycoprotein) and late (myelin basic protein) myelination markers in a three‐dimensional neuron/Schwann cell coculture model. The Schwann cell batch yields are sufficient to perform 100–150 individual myelinating coculture assays. Employing mixed phenotype/genotype mouse neuron/Schwann cell cocultures, it will be possible to analyse the cell specificity of a mutation, and the cumulative effects of different mutations, without having to cross‐breed the animals.

The small myelin-associated glycoprotein binds to tubulin and microtubules

The myelin-associated glycoprotein (MAG) exists as two isoforms, differing only by their respective cytoplasmic domains, that have been suggested to function in the formation and maintenance of myelin. In the present study, a 50 kDa protein binding directly to the small MAG (S-MAG) cytoplasmic domain was detected and identified as tubulin, the core component of the microtubular cytoskeleton. In vitro, the S-MAG cytoplasmic domain slowed the polymerization rate of tubulin and co-purified with assembled microtubules. A significant sequence homology was found between the tau family tubulin-binding repeats and the carboxy-terminus of S-MAG. Our results indicate that S-MAG is the first member of the Ig superfamily that can be classified as a microtubule-associated protein, and place S-MAG in a dynamic structural complex that could participate in linking the axonal surface and the myelinating Schwann cell cytoskeleton.

S100β inhibits the phosphorylation of the L-MAG cytoplasmic domain by PKA

The myelin-associated glycoprotein (MAG) is a cell adhesion molecule expressed by myelinating glia, existing as two isoforms that differ only by their cytoplasmic domains. We have studied the in vitro phosphorylation of recombinant rat MAG cytoplasmic domains by three kinases for which consensus sequences exist within this domain, revealing phosphorylation of the L-MAG-specific domain by protein kinase A (PKA). Phosphorylation of the L-MAG cytoplasmic domain by PKA was decreased in the presence of S100beta, providing a functional significance to the interaction between L-MAG and S100beta, and further indicating that L-MAG may play a role in myelinating glial cell signalling processes.

Correlation between the morphology and the lipid and protein compositions in the peripheral nervous system of individual 8-day-old normal and trembler mice

The hereditary, hypertrophic interstitial neuropathy which afflicts the trembler mouse manifests itself about two weeks after birth. Consequently, the identification of these mutant mice was not possible before this age, except when double mutants were available. We show that the trembler mice can be easily distinguished from their normal littermates before the clinical symptoms appear by using an HPTLC/densitometry technique that allows the simple and rapid analysis of the polar lipids extracted from one sciatic nerve. The results presented in this paper demonstrate important differences between the polar lipid compositions of sciatic nerves from 8-day-old normal and trembler littermates, whose phenotypes were confirmed by the morphological analysis of the contralateral sciatic nerves. The small amount of material that is needed for this identification makes it possible to use the remaining nerve material for other studies. Furthermore, important differences between the sciatic nerve protein compositions of normal and trembler mice, identified according to their polar lipid composition, were also observed and these differences can, therefore, also be employed for the identification of the mutants before the manifestation of the clinical symptoms of the trembler neuropathy.