Proteolipid/DM-20 proteins bearing the paralytic tremor mutation in peripheral nerves and transfected Cos-7 cells

Paralytic tremor (Plp-pt) is a missense mutation of the myelin proteolipid gene (Plp) in rabbits. The myelin yield in the Plp-pt brain is reduced and the protein and lipid composition of central nervous system (CNS) myelin is abnormal. We studied the intracellular transport of the normal and Plp-pt mutant PLP and DM-20 in transiently transfected Cos-7 cells. While the mutant PLP accumulates in the rough endoplasmic reticulum and does not reach the plasma membrane, the spliced isoform of PLP, mutant DM-20, is normally transported to the cell surface and integrated into the membrane. Analysis of rabbit sciatic nerves revealed that concentration of peripheral nervous system (PNS) myelin proteins is normal in Plp-pt myelin. In the PNS like in the CNS, the level of Plp gene products is subnormal. But this does not affect myelination, in the PNS where PLP, present in low concentration, is not a structural component of compact myelin. The normal level of Plp gene expression in Schwann cells is low and these results suggest that, in the Plp-pt PNS, Schwann cell function is not affected by the deficiency in PLP and/or the impairment of intracellular PLP transport. Special issue dedicated to Dr Marion E. Smith.     

Protein Composition of PNS Myelin: Developmental Comparison of Control and Quaking Mice

Protein compositions were determined for sciatic nerve myelin isolated from young and adult control and quaking (Qk) mice. Age-related changes in the relative amounts of large (Pl) and small (Pr) basic proteins were found. In control animals, the ratio Pr/Pl increased with age, a change similar to that observed for the large (Bl) and small (Bs) CNS myelin basic proteins of adult mice. Pr/Pl also increased with age in the Qk mouse sciatic nerve, but only to the point that the value in the adult Qk mouse was similar to that observed for young control animals, a situation reminiscent of the effect of the Qk mutation on CNS basic proteins. Thus, our data suggest that the Qk mutation has a similar effect on peripheral nervous system (PNS) and CNS basic proteins. Our findings are consistent with recent electrophoretic and immunochemical data showing that PNS and CNS myelin basic proteins in rodents are analogous, and they suggest that the genetic program controlling basic protein expression is common to oligodendroglia and Schwann cells.     

The Glycoprotein Composition of Peripheral Nervous System Myelin Subfractions

Two fractions were isolated by continuous density gradient centrifugation from total particulate matter of rabbit sciatic nerves: a minor fraction, B, consisting of small-sized membrane fragments and a major fraction, C, of characteristic multilayered myelin figures, with maxima at 0.33 and 0.58 M-sucrose, respectively. In comparison with C, fraction B was enriched in CNPase and alkaline phosphatase activities and the P0, 23K and Z proteins, but was virtually devoid of basic protein. The glycoprotein composition of all fractions was examined with four fluorescein isothiocyanate-labelled lectins (WGA, Con A, RCA-60, U.E.). These revealed the presence of six glycoproteins in all fractions with similar lectin binding capacities and molecular weights ranging from 35,500 to 16,000, of which P0 was the predominant component. Material found on the heavy side of fraction C was characterized by the presence of a multitude of glycoproteins which bound variable proportions of the four different lectins, suggesting substantial variations in their carbohydrate moieties. Their absence from the central portion of fraction C points to a location other than that of compact PNS myelin.     

Tumor promoters accentuate phosphorylation of PO: Evidence for the presence of protein kinase C in purified PNS myelin

The effects of carbon tetrachloride, methylene chloride and chloroform on phosphorylation of PO was examined. The results of the dose response curve revealed that carbon tetrachloride (0.67%), methylene chloride (2%) and chloroform (1%) induced phosphorylation of PO by approximately 4, 6, and 12-fold, respectively. PO was found to be phosphorylated on the serine residue, and the phosphorylation of the serine residue was markedly increased when PO was phosphorylated in the presence of these compounds. Since tumor promoters, carbon tetrachloride and chloroform, have been shown to activate protein kinase C in platelets it is postulated that the increased phosphorylation of PO may result from the activation of myelin associated protein kinase C. The presence of phospholipid sensitive Ca²⁺-dependent protein kinase (protein kinase C) in purified nerve myelin was demonstrated by increased phosphorylation of PO in the presence of Ca²⁺ and phosphatidylserine.     

Panaxydol treatment enhances the biological properties of Schwann cells in vitro

Schwann cells (SCs), the glial cells of the peripheral nerve system, play a key role in the regeneration of injured peripheral nerves. However, problems with the use of SCs to repair peripheral nerves include attenuated biologic properties and impaired function with ageing. Panaxydol (PND) effectively protects neurons against injury in degenerative diseases. We investigated the protective role of PND in SCs through immunocytochemistry and ELISA assay. PND promoted the expression and secretion of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) by SCs in a dose-dependent manner at doses of 2.5-20 and 5.0-20 μM, respectively. The effects on both factors were maximal at 10 μM. PND also enhanced the synthesis of actin, a key component of the cytoskeleton. When we examined mitochondria in SCs with probes marked with rhodamine-123, fluorescence intensity was stronger in the PND group than in a control group, indicating a stabilized mitochondrial transmembrane potential. PND modified cytoskeleton dynamics and induced SCs to secrete and express neurotrophic factors (NTFs), and to resist high energy consumption in a dose-dependent manner. It exerted its maximum effect at 10 μM. PND treatment of SCs might be promising strategies for the application of these cells in repairing PNS injury by enhancing the biological properties.     

Internodal myelination during development quantitated using X-ray diffraction

Characterizing the formation, accretion, and stability of myelin during development, maturation, and senescence is important for better understanding critical periods in the function of the nervous system in normal growth and following environmental insult or genetic mutation. Although there are numerous studies on the ultrastructural, biochemical, and genetic aspects of myelin development and maturation, few have used X-ray diffraction (XRD), which can rapidly provide unique metrics about internodal myelin based on measurements from whole, unfixed tissue. Besides periodicity (the classic attribute of internodal myelin measured by XRD), other parameters include: relative amount of myelin, membrane dimensions, and packing disorder. To provide a baseline for future experiments on myelin structural integrity, we used XRD to characterize internodal myelin as a function of age (from 5 to 495 days) in the mouse, a species increasingly used for developing transgenic models of human neurological diseases. As expected, the relative amount of myelin increased with age in both PNS and CNS, with the most rapid accumulation occurring in the youngest age group. Changes in rate of myelin accretion yielded three distinct age brackets during which small but significant changes in structural parameters were detected: in PNS, myelin period increased, packing distortion decreased, width of extracellular apposition (EXT) decreased, and widths of cytoplasmic apposition (CYT) and lipid bilayer (LPG) increased; in CNS, myelin period decreased, packing distortion decreased, EXT and CYT decreased, and LPG increased. We propose that the data obtained here can serve as a basis for rapidly detecting abnormal pathologies during myelination.     

In Situ Fluorescence Imaging of Myelination

We describe a novel fluorescent dye, 3-(4-aminophenyl)-2H-chromen-2-one (termed case myelin compound or CMC), that can be used for in situ fluorescent imaging of myelin in the vertebrate nervous system. When administered via intravenous injection into the tail vein, CMC selectively stained large bundles of myelinated fibers in both the central nervous system (CNS) and the peripheral nervous system (PNS). In the CNS, CMC readily entered the brain and selectively localized in myelinated regions such as the corpus callosum and cerebellum. CMC also selectively stained myelinated nerves in the PNS. The staining patterns of CMC in a hypermyelinated mouse model were consistent with immunohistochemical staining. Similar to immunohistochemical staining, CMC selectively bound to myelin sheaths present in the white matter tracts. Unlike CMC, conventional antibody staining for myelin basic protein also stained oligodendrocyte cytoplasm in the striatum as well as granule layers in the cerebellum. In vivo application of CMC was also demonstrated by fluorescence imaging of myelinated nerves in the PNS. (J Histochem Cytochem 58:611–621, 2010)     

GFAP transgenic zebrafish

We have generated transgenic zebrafish that express green fluorescent protein (GFP) in glial cells driven by the zebrafish glial fibrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was first visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage ( approximately 12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (approximately 15 hpf). At 24 hpf GFP-expressing cells were in the retina and lens. By 72 hpf GFP expression levels were strong and localized to the glia of the brain, neural retina, spinal cord, and ventral spinal nerves, with moderate expression in the enteric nervous system and weaker levels in the olfactory sensory placode and otic capsule. GFP expression in glia co-localized with anti-GFAP antibodies, but did not co-localize with the neuronal antibodies HuC/D or calretinin in mature neurons.