Rudolf Martini

Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons

We have used homologous recombination in embryonic stem cells to generate mice carrying a mutation in the gene encoding P0, an immunoglobulin-related recognition molecule and the major protein of peripheral nervous system myelin. These mice are deficient in normal motor coordination and exhibit tremors and occasional convulsions. Axons in their peripheral nerves are severely hypomyelinated and a subset of myelin-like figures and axons degenerate. The mutation leads to an abnormal regulation of some, but not all, molecules involved in myelination. These results demonstrate that P0 is essential for the normal spiraling, compaction, and maintenance of the peripheral myelin sheath and the continued integrity of associated axons. They further suggest that this protein conveys a signal that regulates Schwann cell gene expression.

Mice deficient for the myelin-associated glycoprotein show subtle abnormalities in myelin

Using homologous recombination in embryonic stem cells, we have generated mice with a null mutation in the gene encoding the myelin-associated glycoprotein (MAG), a recognition molecule implicated in myelin formation. MAG-deficient mice appeared normal in motor coordination and spatial learning tasks. Normal myelin structure and nerve conduction in the PNS, with N-CAM overexpression at sites normally expressing MAG, suggested compensatory mechanisms. In the CNS, the onset of myelination was delayed, and subtle morphological abnormalities were detected in that the content of oligodendrocyte cytoplasm at the inner aspect of most myelin sheaths was reduced and that some axons were surrounded by two or more myelin sheaths. These observations suggest that MAG participates in the formation of the periaxonal cytoplasmic collar of oligodendrocytes and in the recognition between oligodendrocyte processes and axons.

Crucial Role for the Myelin‐associated Glycoprotein in the Maintenance of Axon‐Myelin Integrity

It has recently been shown that mice deficient in the gene for myelin‐associated glycoprotein develop normal myelin sheaths in the peripheral nervous system. Here we report that in mutant mice older than 8 months the maintenance of axon‐myelin units is disturbed, resulting in both axon and myelin degeneration. Morphological features include those typically seen in human peripheral neuropathies, where demyelination‐induced Schwann cell proliferation and remyelination lead to the formation of so‐called onion bulbs. Expression of tenascin‐C, a molecule indicative of peripheral nerve degeneration, was up‐regulated by axon‐deprived Schwann cells and regenerating axons were occasionally seen. Myelin‐associated glycoprotein thus appears to play a crucial role in the long‐term maintenance of the integrity of both myelin and axons.

Disruption of the Gene for the Myelin-Associated Glycoprotein Improves Axonal Regrowth along Myelin in C57BL/Wlds Mice

The myelin-associated glycoprotein (MAG) has been shown to be inhibitory for certain neurons in vitro (Mukhopadhyay et al., 1994; McKerracher et al., 1994). To investigate whether MAG is an inhibitory component in peripheral myelin in vivo, MAG-deficient mutant mice were cross-bred with C57BL/Wlds mice that have delayed lesion-induced myelin degeneration and axon regrowth. While in crushed nerves of C57BL/Wlds mice expressing MAG, only 16% of myelin sheaths were associated with regrowing axons, this number was doubled in MAG-deficient C57BL/Wlds mice. These observations suggest that the absence of MAG may contribute to the improved axonal regrowth in the double mutants. Therefore, degeneration of MAG-containing myelin might be an important prerequisite to optimize axonal regrowth after peripheral nerve injury.

Immune Cells Contribute to Myelin Degeneration and Axonopathic Changes in Mice Overexpressing Proteolipid Protein in Oligodendrocytes

Overexpression of the major myelin protein of the CNS, proteolipid protein (PLP), leads to late-onset degeneration of myelin and pathological changes in axons. Based on the observation that in white matter tracts of these mutants both CD8+ T-lymphocytes and CD11b+ macrophage-like cells are numerically elevated, we tested the hypothesis that these cells are pathologically involved in the primarily genetically caused neuropathy. Using flow cytometry of mutant brains, CD8+ cells could be identified as activated effector cells, and confocal microscopy revealed a close association of the T-cells with MHC-I+ (major histocompatibility complex class I positive) oligodendrocytes. Crossbreeding the myelin mutants with mice deficient in the recombination activating gene-1 (RAG-1) lacking mature T- and B-lymphocytes led to a reduction of the number of CD11b+ cells and to a substantial alleviation of pathological changes. In accordance with these findings, magnetic resonance imaging revealed less ventricular enlargement in the double mutants, partially because of more preserved corpora callosa. To investigate the role of CD8+ versus CD4+ T-lymphocytes, we reconstituted the myelin-RAG-1 double mutants with bone marrow from either CD8-negative (CD4+) or CD4-negative (CD8+) mice. The severe ventricular enlargement was only found when the double mutants were reconstituted with bone marrow from CD8+ mice, suggesting that the CD8+ lymphocytes play a critical role in the immune-related component of myelin degeneration in the mutants. These findings provide strong evidence that a primary glial damage can cause secondary immune reactions of pathological significance as it has been suggested for some forms of multiple sclerosis and other leukodystrophies.

Absence of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM) interferes with the maintenance, but not with the formation of peripheral myelin

Abstract.We have previously shown that mice deficient in the gene for the myelin-associated glycoprotein (MAG) develop normal myelin in the peripheral nerves, but show axon and myelin degeneration at eight months of age, suggesting that MAG is involved in the maintenance of axon-Schwann cell integrity. The search for molecules that might replace MAG during myelination revealed an overexpression of the neural cell adhesion molecule (N-CAM) at those aspects where MAG is detectable in wild type mice. To test whether N-CAM might compensate for MAG during myelination in MAG-deficient mice, double mutants deficient in both MAG and N-CAM (MAG−/N-CAM−mice) were generated by cross-breeding the single mutants. Whereas alterations of myelin development were not detectable in either of the single or double mutants, degeneration of myelin and axons occurred approximately 4 weeks earlier in MAG−/N-CAM−than in MAG−mutants. Furthermore, at 8 weeks of age, single fiber preparation and electron microscopy revealed that the number of profiles indicative of degeneration was substantially increased in MAG−/N-CAM−mutants when compared to MAG−mice. These data suggest that in MAG-deficient mice N-CAM does not compensate for MAG in myelin formation but partially substitutes for it in the maintenance of axon-myelin integrity.

UP-REGULATION OF A CHONDROITIN SULPHATE EPITOPE DURING REGENERATION OF MOUSE SCIATIC NERVE : EVIDENCE THAT THE IMMUNOREACTIVE MOLECULES ARE RELATED TO THE CHONDROITIN SULPHATE PROTEOGLYCANS DECORIN AND VERSICAN

After transection of adult mouse sciatic nerve, the expression of a chondroitin sulphate epitope recognized by the monoclonal antibody 473‐HD (mAb 473‐HD) was found to be up‐regulated. The epitope was localized immunocytochemically mainly in Schwann cell basal laminae and, more weakly, also in the endoneurium. In cultures of mouse dorsal root ganglion cells, Schwann cells expressed high levels but fibroblasts only low levels of the epitope. To identify the molecule(s) carrying this chondroitin sulphate epitope, human sciatic nerves were extracted with phosphate‐buffered saline and shown to contain two chondroitin sulphate proteoglycans of apparent molecular weights of 130 and 900 kDa. The 900 kDa and, more weakly, the 130 kDa proteoglycan were reactive with mAb 473‐HD, which was found to recognize chondroitin‐6‐sulphate as epitope. Following chondroitinase ABC treatment of the 130 kDa proteoglycan, a core protein of ∼45 kDa was seen and shown to react with polyclonal antibodies against the chondroitin‐dermatan sulphate proteoglycan decorin from human fibroblasts. Chondroitinase ABC treatment of the 900 kDa proteoglycan yielded a core protein with a molecular weight of ∼400 kDa that was recognized by polyclonal antibodies against recombinantly expressed fusion proteins from human versican. After transection of adult mouse sciatic nerves, the distal nerve stumps showed up‐regulation of the chondroitin‐6‐sulphate epitope of the 900 kDa proteoglycan, whereas the core protein of this proteoglycan did not show any detectable change in the level of expression. In contrast, the core protein of the 130 kDa proteoglycan was up‐regulated in expression. These observations suggest that versican‐ and decorinlike molecules may contribute to successful regeneration in the peripheral nervous system of mammals.

Chapter 16 Functions of the L2/HNK-1 carbohydrate in the nervous system

Publisher Summary This chapter discusses the functions of L2/HNK-1 (L2) carbohydrate in the nervous system. The L2 carbohydrate is carried by several neural recognition molecules. Its receptors are present on cell surface and extracellular matrix recognition molecules. Thus, the L2 carbohydrate is an important ligand in cell interactions in the nervous system and in the immune system, possibly triggering interactions between the cells of the neural and lymphoid lineages. The finely tuned regulation of L2 expression by the subpopulations of myelinating Schwann cells in the adult peripheral nervous system of mice raises the possibility that it may favor target preferential reinnervation by motor axons. The fact that recognition molecules can be both receptors and donors of functionally important carbohydrates points to an intricate network of multiple combinations in cell-to-cell and cell-to-matrix interactions in the nervous system, particularly because carbohydrate structures are subject to finely tuned-developmentally and cell type-specifically regulated synthetic mechanisms. The L2 carbohydrate is an eminent example of the ingenuity of nature to use carbohydrate structures in functional contexts.

Tenascin-C expression during Wallerian degeneration in C57BL/Wlds mice: possible implications for axonal regeneration

SummarySchwann cells in the distal stumps of lesioned peripheral nerves strongly express the extracellular matrix glycoprotein tenascin-C. To gain insights into the relationship between Wallerian degeneration, lesion induced tenascin-C upregulation and regrowth of axons we have investigated C57BL/Wlds (C57BL/Ola) mice, a mutant in which Wallerian degeneration is considerably delayed. Since we found a distinct difference in the speed of Wallerian degeneration between muscle nerves and cutaneous nerves in 16-week-old C57BL/Wlds mice, as opposed to 6-week-old animals in which Wallerian degeneration is delayed in both, we chose the older animals for closer investigation. Five days post lesion tenascin-C was upregulated in the muscle branch (quadriceps) but not in the cutaneous branch (saphenous) of the femoral nerve in 16-week-old animals. In addition myelomonocytic cells displaying endogenous peroxidase activity invaded the muscle branch readily whereas they were absent from the cutaneous branch at this time. We could further show that it is only a subpopulation of axon-Schwann cell units (mainly muscle efferents) in the muscle branch which undergo Wallerian degeneration and upregulate tenascin-C at normal speed and that the remaining axon-Schwann cell units (mainly afferents) displayed delayed Wallerian degeneration and no tenascin-C expression. Regrowing axons could only be found in the tenascin-C-positive muscle branch where they always grew in association with axon-Schwann cell units undergoing Wallerian degeneration. These observations indicate a tight relationship between Wallerian degeneration, upregulation of tenascin-C expression and regrowth of axons, suggesting an involvement of tenascin-C in peripheral nerve regeneration.

The L2/HNK‐1 Carbohydrate is Carried by the Myelin Associated Glycoprotein and Sulphated Glucuronyl Glycolipids in Muscle but not Cutaneous Nerves of Adult Mice

We have previously shown that myelinating Schwann cells associated with motor, but not sensory, axons in peripheral nerves of adult mice express the L2/HNK‐1 carbohydrate epitope. This carbohydrate structure carried by glycolipids and neural cell adhesion molecules has been suggested to specifically foster regrowth of motor as opposed to sensory axons after infliction of a lesion. To determine which molecular components may be the carriers of the L2 carbohydrate in motor axon‐associated myelinating Schwann cells, we have isolated the purely sensory, cutaneous branch and the mixed sensory and motor muscle branch of the femoral nerve of adult mice, isolated the myelin fraction thereof and analysed the molecules expressing the L2 carbohydrate by several immunochemical methods. L2 immunoreactivity in myelin of the muscle branch was four to five times higher than that of the cutaneous branch. The 110 kDa L2‐immunoreactive glycoprotein in myelin of the muscle branch, which is not L2‐immunoreactive in the cutaneous branch, was identified as the myelin‐associated glycoprotein by a combination of immunoprecipitation and Western blot analysis. Myelin extraction with organic solvents additionally revealed the two L2‐carrying glycolipids, which amounted to‐40 ng glycolipid/mg dry weight in myelin of the muscle branch, whereas no significant amounts of the L2 glycolipids were found in myelin of the cutaneous branch. These observations suggest an astonishing degree of differential regulation of carbohydrate‐synthesizing activities in myelinating Schwann cells.