Rolf Schiff

Subtle myelin defects in PLP-null mice †

This study explores subtle defects in the myelin of proteolipid protein (PLP)‐null mice that could potentially underlie the functional losses and axon damage known to occur in this mutant and in myelin diseases including multiple sclerosis. We have compared PLP‐null central nervous system (CNS) myelin with normal myelin using ultrastructural methods designed to emphasize fine differences. In the PLP‐null CNS, axons large enough to be myelinated often lack myelin entirely or are surrounded by abnormally thin sheaths. Short stretches of cytoplasm persist in many myelin lamellae. Most strikingly, compaction is incomplete in this mutant as shown by the widespread presence of patent interlamellar spaces of variable width that can be labeled with ferricyanide, acting as an aqueous extracellular tracer. In thinly myelinated fibers, interlamellar spaces are filled across the full width of the sheaths. In thick myelin sheaths, they appear filled irregularly but diffusely. These patent spaces constitute a spiral pathway through which ions and other extracellular agents may penetrate gradually, possibly contributing to the axon damage known to occur in this mutant, especially in thinly myelinated fibers, where the spiral path length is shortest and most consistently labeled. We show also that the “radial component” of myelin is distorted in the mutant (“diagonal component”), extending across the sheaths at 45° instead of 90°. These observations indicate a direct or indirect role for PLP in maintaining myelin compaction along the external surfaces of the lamellae and to a limited extent, along the cytoplasmic surfaces as well and also in maintaining the normal alignment of the radial component.

Myelin structure in proteolipid protein (PLP)-null mouse spinal cord.

Fixed preparations of proteolipid protein (PLP)‐null mouse spinal cord show myelin sheaths which in some regions consist of typical alternating major dense lines (MDLs) and intermediate lines (ILs) with a repeat period of 10.3 nm. More commonly, the lamellar structure consists of what appears to be a single population of dense lines, having a repeat period of 5.2 nm. These apparently equivalent lines are, however, sometimes distinguishable as MDLs or ILs based on continuity with cytoplasmic or extracellular regions. Focal separations of lamellae at the intermediate line are common. MDLs too may be replaced focally by cytoplasmic pockets, sometimes in the same quadrant over several lamellae, resembling Schmidt‐Lanterman clefts. Occasional densities reminiscent of the “radial component” can be seen. Otherwise, this structure, which is prominent in wild‐type myelin, is conspicuously absent. Redundant folding of some lamellae but not others may occur in the same sheath. These observations conform to those made previously on the isolated myelin segments that occur in the myelin‐deficient rat central nervous system (CNS), which also lacks PLP. Thus, a compact lamellar structure can be seen in fixed PLP‐null myelin, but defects in the apposition of both the extracellular and the cytoplasmic surfaces of the myelin membranes are common. The abnormalities seen suggest a lack of firm intermembrane bonding, resulting in structural instability. PLP‐null myelin may therefore be more susceptible than normal myelin to disruption by mechanical or osmotic stresses. Although PLP is not essential for the formation of either major dense lines or intermediate lines, it may play a role in stabilizing the compact structure.

Inhibition of CNS myelin developmentin vivo by implantation of anti-GalC hybridoma cells

SummaryImplantation of hybridoma cells that secrete a monoclonal antigalactocerebroside into the dorsal columns of ⩽ 9-day-old rat spinal cord results in failure of development of dorsal column myelin in the vicinity of the implant. Clusters of apparently undamaged amyelinated axons remain among the hybridoma cells. Ventral myelin is unaffected. These in vivo results support antibody-mediated inhibition of myelin formation as a potential mechanism underlying failure of remyelination in multiple sclerosis.

Myelin formation by mouse glia in myelin-deficient rats treated with cyclosporine

SummaryPrevious attempts to generate myelin in the myelin-deficient rat spinal cord by transplanting mouse glia were not successful. In order to determine whether this result was due to graft rejection or to interspecies mismatch of cellular or molecular components at the axoglial junction, we have repeated the experiment in cyclosporine-treated rats. Our results show that in the immunosuppressed hosts, foetal glial xenografts form an abundance of myelin within the dorsal columns at or near the injection site about two weeks after the operation. In some cases, myelination extends virtually across the entire width of the dorsal columns. Ultrastructurally, the myelin sheaths are normal in all respects, including the presence of the ‘radial component’. The lateral edges of the myelin lamellae form typical paranodal axoglial junctions, some displaying periodic ‘transverse bands’. We infer that previous mouse to rat xenograft failures reflect host immune response rather than mismatch of heterologous junctional components. We also compared foetal, early post-natal and adult xenografts. Foetal donor cells, containing an abundance of precursors but virtually no mature oligodendrocytes, are more effective than neonatal donor cells in forming myelin, and after adult grafts, we found no myelin formation. Thus, in xenografts, as in allografts, foetal precursor cells are far more suitable than glia from mature donors in generating significant amounts of myelin.

Role of transverse bands in maintaining paranodal structure and axolemmal domain organization in myelinated nerve fibers: Effect on longevity in dysmyelinated mutant mice

The consequences of dysmyelination are poorly understood and vary widely in severity. The shaking mouse, a quaking allele, is characterized by severe central nervous system (CNS) dysmyelination and demyelination, a conspicuous action tremor, and seizures in ∼25% of animals, but with normal muscle strength and a normal lifespan. In this study we compare this mutant with other dysmyelinated mutants including the ceramide sulfotransferase deficient (CST−/−) mouse, which are more severely affected behaviorally, to determine what might underlie the differences between them with respect to behavior and longevity. Examination of the paranodal junctional region of CNS myelinated fibers shows that “transverse bands,” a component of the junction, are present in nearly all shaking paranodes but in only a minority of CST−/− paranodes. The number of terminal loops that have transverse bands within a paranode and the number of transverse bands per unit length are only moderately reduced in the shaking mutant, compared with controls, but markedly reduced in CST−/− mice. Immunofluorescence studies also show that although the nodes of the shaking mutant are somewhat longer than normal, Na+ and K+ channels remain separated, distinguishing this mutant from CST−/− mice and others that lack transverse bands. We conclude that the essential difference between the shaking mutant and others more severely affected is the presence of transverse bands, which serve to stabilize paranodal structure over time as well as the organization of the axolemmal domains, and that differences in the prevalence of transverse bands underlie the marked differences in progressive neurological impairment and longevity among dysmyelinated mouse mutants. J. Comp. Neurol. 518:2841–2853, 2010.

Effects of osmolality on PLP-null myelin structure: implications re axon damage.

In order to test the adhesiveness of PLP-null compact myelin lamellae we soaked aldehyde-fixed CNS specimens from PLP-null and control mice overnight in distilled water, in Ringers solution or in Ringers solution with added 1 M sucrose. Subsequent examination of the tissue by EM showed that both PLP-null and control white matter soaked in Ringer remained largely compact. After the distilled water soak, control myelin was virtually unchanged, but PLP-null myelin showed some decompaction, i.e., separation of myelin lamellae from one another. After the sucrose/Ringer soak, normal myelin developed foci of decompaction, but the great majority of lamellae remained compact. In the PLP-null specimens, in contrast, many of the myelin sheaths became almost completely decompacted. Such sheaths became thicker overall and were comprised of lamellae widely separated from one another by irregular spaces. Thus, in normal animals, fixed CNS myelin lamellae are firmly adherent and resist separation; PLP-null myelin lamellae, in contrast, are poorly adherent and more readily separated. Mechanisms by which impaired adhesiveness of PLP-null myelin lamellae and fluctuations in osmolality in vivo might underlie slowing of conduction and axon damage are discussed.

Antibody-mediated CNS demyelination: focal spinal cord lesions induced by implantation of an IgM anti-galactocerebroside-secreting hybridoma

O1 hybridoma cells, which secrete an IgM antigalactocerebroside, were implanted into the spinal cord of cyclosporine-treated juvenile or adult rats, and the animals were sacrificed ∼2–3 wk later. About half the recipient animals developed myelin lesions. In some, sharply circumscribed foci of demyelination formed within the dorsal columns. Cellular reaction consisted of macrophages containing refractile globules in the parenchyma and within enlarged perivascular spaces as well as thickened endothelial cells. “Shadow plaques” also developed, i.e. regions in which axons were surrounded by thin myelin sheaths, compatible with remyelination. In addition, we found damaged axons, some of which were swollen with organelles, comparable to the enlarged axon profiles seen at sites of constriction or interruption. Compromise of the blood-brain barrier at sites of hybridoma growth was demonstrated by extravasation of Evans blue dye. Discontinuation of cyclosporine was followed by an anti-hybridoma, complement-fixing antibody response within 2–3 d. This model of focal CNS demyelination and remyelination, with evidence of some axon damage, is mediated by a defined IgM antiglycolipid monoclonal antibody secreted within the spinal cord parenchyma. The lesions, which are similar to those of multiple sclerosis, probably result from the interaction between the intrathecally secreted IgM antibody and complement entering from the circulation at foci of compromised blood-brain barrier plus activation of endogenous or hematogenous macrophages via their complement receptors.

Distribution of myelin lipid antigens in adult and developing rat spinal cord

We examined the distribution of myelin antigens recognized by monoclonal antibodies (mAbs) 01 and 04 in the developing ventral white matter of the cervical spinal cord of the rat using immunogold-labeled ultrathin cryosections. From the beginning of myelination after birth to multilamellar myelin in adult animals, we observed colocalization of 04 and 01 label in myelin. In the oligodendrocyte soma, immunolabel was found primarily over Golgi cisternae. In the oligodendrocyte processes, immunolabeling was also found in the cytoplasm and along the plasmalemma. More cytoplasmic 04 and 01 label was found in the external loop of myelin than in the internal loop. The amount of 01 and 04 label increased over compact myelin in proportion to the number of lamellae, but the label density per unit length of membrane remained approximately the same in compact myelin as in oligodendrocyte plasmalemma. We did not see a concentration gradient for either 04 or 01 label across, or along multilamellar myelin sheaths.

Spinal cord dysmyelination induced in vivo by IgM antibodies to three different myelin glycolipids.

It was shown previously (Rosenbluth et al.: J. Neurosci. 16:2635–2641, 1996) that implantation of hybridoma cells that produce an IgM antigalactocerebroside into the spinal cord of young rats results in the development of myelin sheaths with a repeat period ∼2–3× normal, similar to the abnormal peripheral myelin sheaths seen in human IgM gammopathies. We now present evidence that this effect can be reproduced in the spinal cord by implanting either of two other hybridomas, O4 and A2B5, that secrete, respectively, antisulfatide and antiganglioside IgM antibodies. The formation of expanded CNS myelin thus does not depend on antibodies to galactocerebroside specifically but can be mediated by IgM antibodies that react with other myelin glycolipids as well. GLIA 19:58–66, 1997.

Distribution and morphology of transgenic mouse oligodendroglial-lineage cells following transplantation into normal and myelin-deficient rat CNS

Glial cells from neonatal MβP5 transgenic mice, which express bacterial β‐galactosidase (lacZ) under control of the myelin basic protein (MBP) promoter (Gow et al, 1992 ), were transplanted into the spinal cord or cerebral hemisphere of immunosuppressed normal and myelin‐deficient (md) rats in order to assess the ability of the donor cells to survive, migrate, and differentiate within normal compared with myelin‐deficient central nervous system (CNS). LacZ+ cells were detected as early as 6–7 days after transplantation into the low thoracic cord and by 10 days had spread rostrally to the brainstem and caudally to the sacral spinal cord. Initially, compact lacZ+ cells, lacking processes, were found associated with small blood vessels and with the glia limitans. Cells of this type persisted throughout the experiment. Later, lacZ+ cells with processes were seen along fiber tracts in the dorsal columns and, after intracerebral injection, subjacent to ventricular ependyma, as well as scattered in cerebral white and gray parenchyma. The extent of spread was comparable in md and normal rats, but in the md group, the success rate was higher, and more cells differentiated into process‐bearing oligodendrocytes. Acceptance of xenografts in immunosuppressed recipients equaled that of allografts. The overall spread of grafted cells exceeded that of injected charcoal, indicating active migration. In contrast to earlier studies that identified oligodendrocytes based on morphology alone, this study has allowed us to identify and track oligodendrocytes based on myelin gene expression. We show some oligodendrocytes whose morphology is consistent with classical morphological descriptions, some that resemble astrocytes, and a class of compact perivascular oligodendrocyte‐lineage cells that we suggest are migratory. J. Comp. Neurol. 446:46–57, 2002.