Hyperplasia of oligodendrocytes in quaking mice.

The number of neuroglial cells in selected fiber tracts of 90-day-old quaking and normal mice was determined by a combination of light and electron microscopy. Oligodendrocytes of quaking mice are normal in number in the anterior commissure and corticospinal tract (in the cervical spinal cord) but are increased two- to fourfold in the optic nerve and the fasciculi cuneatus and gracilis (in the cervical spinal cord). The nuclei and perikarya are normal in size or smaller than normal. Those tracts with the greatest hyperplasia of oligodendrocytes also have the greatest content of myelin, suggesting that cell number influences content of myelin. However, the volume of myelin per oligodendrocyte also varies, between 2 and 11% of normal, in the different tracts of the mutant. The hyperplasia of oligodendrocytes in quaking mice may arise as compensation for their decreased production of myelin and reflect a normal plasticity in the processes of myelination. If so, the mutant may be a useful system for study of the regulation of myelogenesis.     

Auditory-evoked far-field potentials in myelin deficient mutant quaking mice

Auditory evoked far-field potentials were recorded from mature quaking mice and their littermate controls. There was a significant prolongation in the latencies of all FFP peaks in the quaking mutants. Examination of the interaction between group (normal, quaking) and for selected pair-wise peak comparisons showed that the degree of retardation was not uniform for each FFP peak. In view of the reported histological and biochemical findings that the CNS of the quaking mutant is specifically deficient in myelin, we conclude that slowed conduction in the quaking mouse brain is due to myelin deficiency and that non-uniform retardation of each FFP peak reflects regional variation in the extent of myelin deficiency.These data suggest that the changes in interpeak latencies of far-field potentials may be useful in identifying the demyelinated region(s) of the diseased central nervous system.     

Withanoside IV improves hindlimb function by facilitating axonal growth and increase in peripheral nervous system myelin level after spinal cord injury

Although methylprednisolone is the clinically standard medication and almost the only therapy for spinal cord injury (SCI), its effect on functional recovery remains questionable. Transplantation strategies using sources such as neural stem cells and embryonic spinal cord still have some hurdles to overcome before practical applications become available. We therefore aimed to develop a practical medication for SCI. Per oral treatment with withanoside IV, which was previously shown to regenerate neuronal networks in the brain, improved locomotor functions in mice with SCI. In the spinal cord after SCI, axons were crushed in the white matter and gray matter, and central nervous system (CNS) myelin level decreased. In mice treated with withanoside IV (10micromol/kg body weight/day, for 21 days), axonal density and peripheral nervous system (PNS) myelin level increased. The loss of CNS myelin and increase in reactive gliosis were not affected by withanoside IV. These results suggest that oral administration of withanoside IV may ameliorate locomotor functions by facilitating both axonal regrowth and increase in PNS myelin level.     

Morphological study of disordered myelination and the degeneration of nerve fibers in the spinal cord of mice lacking complex gangliosides

Gangliosides, a family of glycosphingolipids that contain sialic acid, are abundant on the neuronal cell membranes, but their precise functions in the central nervous system remain largely undefined. In a previous study of GalNAc-T(-/-) mice engineered to lack beta1,4-N-acetylgalactos-aminyltransferase (GM2/GD2 synthase) to abolish any, complex gangliosides, we observed the reduction of nerve conduction velocity but did not find any obvious morphological change in the brain. In the present study, we observed morphological changes in the nerve fiber tracts of the spinal cord in these mice. In GalNAc-T(-/-) mice, the number of degenerated axons was markedly increased in the dorsal funiculus, tract of Lissauer, and dorsolateral funiculus of the cervical segment of the spinal cord as well as the dorsal funiculus and tract of Lissauer of the lumbar segment of the spinal cord. There were also increased numbers of unmyelinated fibers in GalNAc-T(-/-) mice. Loosened myelin sheaths and myelin sheaths separated from axons by wide spaces were also observed in GalNAc-T(-/-) mice. These results provide a morphological basis for the previously observed reduction in the nerve conduction velocity and suggest that complex gangliosides are essential for the maintenance of myelin and the integrity of nerve fibers of the spinal cord.     

The twitcher mouse. Central nervous system pathology after bone marrow transplantation

Effects of bone marrow transplantation (BMT) on the pathology of the central nervous system were evaluated, at light and electron microscope levels, in the homozygous twitcher mouse (twi/twi), an authentic murine model of globoid cell leukodystrophy (GLD, Krabbe disease) in humans. In the twitcher mice with BMT examined at ages 44 and 71 days (transplanted at the day 9 to 12), degeneration of oligodendrocytes and myelin was still present. In these mice, oligodendrocytes and macrophages containing typical tubular and crystalloid inclusions of GLD were observed in the white matter, in particular that of the cerebellum, brainstem, and spinal cord. However, significant morphologic improvement was observed in the central nervous system of the twitcher with BMT which survived more than 100 days. In the cerebellum, brainstem, and spinal cord of these mice, myelin degeneration and infiltration of macrophages containing typical GLD inclusions were either absent or only rarely observed. Instead, many foamy macrophages, either scattered or clustered around blood vessels, were conspicuous. Many nerve fibers with thin myelin sheaths indicative of remyelination were present in the white matter. GLD inclusions were, however, still found in the cytoplasm of apparently remyelinating oligodendrocytes. These results suggest that the foamy macrophages are of donor origin, since they are able to digest degraded myelin components completely, because of a sufficient galactosylceramidase activity, and are contributing to the improvement of the central nervous system pathology in the twitcher mouse following BMT, by supplying the deficient enzyme, galactosylceramidase. However, the presence of inclusions in oligodendrocytes in the twitcher with BMT, even after 100 days of age indicates that the basic enzymatic defect involving oligodendrocytes could not be completely corrected with BMT.     

Native myelin oligodendrocyte glycoprotein promotes severe chronic neurological disease and demyelination in Biozzi ABH mice

Myelin oligodendrocyte glycoprotein (MOG) is a powerful encephalitogen for experimental autoimmune demyelination. However, the use of MOG peptides or recombinant proteins representing part of the protein fails to fully address the possible pathogenic role of the full-length myelin-derived protein expressing post-translational modifications. Immunization of mice with central nervous system tissues from wild-type (WT) and MOG-deficient (MOG(-/-)) mice demonstrates that MOG in myelin is necessary for the development of chronic demyelinating experimental autoimmune encephalomyelitis (EAE) in mice. While immunization with WT spinal cord homogenate (SCH) resulted in a progressive EAE phenotype, MOG(-/-) SCH induced a mild self-limiting acute disease. Following acute EAE with MOG(-/-) SCH, mice developed T cell responses to recombinant mouse MOG (rmMOG), indicating that MOG released from myelin is antigenic; however, the lack of chronic disease indicates that such responses were not pathogenic. Chronic demyelinating EAE was observed when MOG(-/-) SCH was reconstituted with a dose of rmMOG comparable to MOG in myelin (2.5% of total white matter-derived protein). These data reveal that while immunization with the full-length post-translational modified form of MOG in myelin promotes the development of a more chronic autoimmune demyelinating neurological disease, MOG (and/or other myelin proteins) released from myelin during ongoing disease do not induce destructive autoimmunity.     

Pathogenesis of axonal dystrophy and demyelination in alphaA-crystallin-expressing transgenic mice

We recently described a transgenic mouse strain overexpressing hamster alphaA-crystallin, a small heat shock protein, under direction of the hamster vimentin promoter. As a result myelin was degraded and axonal dystrophy in both central nervous system (especially spinal cord) and peripheral nervous system occurred. Homozygous transgenic mice developed hind limb paralysis after 8 weeks of age and displayed progressive loss of myelin and axonal dystrophy in both the central and peripheral nervous system with ongoing age. Pathologically the phenotype resembled, to a certain extent, neuroaxonal dystrophy. The biochemical findings presented in this paper (activity of the enzymes superoxide dismutase, catalase and transglutamase, myelin protein zero expression levels and blood sugar levels) confirm this pathology and exclude other putative pathologies like Amyothrophic Lateral Sclerosis and Hereditary Motor and Sensory Neuropathy. Consequently, an excessive cytoplasmic accumulation of the transgenic protein or a disturbance of the normal metabolism are considered to cause the observed neuropathology. Therefore, extra-ocular alphaA-crystallin-expressing transgenic mice may serve as a useful animal model to study neuroaxonal dystrophy.     

Leukodystrophy caused by plasmalogen deficiency rescued by glyceryl 1‐myristyl ether treatment

Plasmalogens are the most abundant form of ether phospholipids in myelin and their deficiency causes Rhizomelic Chondrodysplasia Punctata (RCDP), a severe developmental disorder. Using the Gnpat‐knockout (KO) mouse as a model of RCDP, we determined the consequences of a plasmalogen deficiency during myelination and myelin homeostasis in the central nervous system (CNS). We unraveled that the lack of plasmalogens causes a generalized hypomyelination in several CNS regions including the optic nerve, corpus callosum and spinal cord. The defect in myelin content evolved to a progressive demyelination concomitant with generalized astrocytosis and white matter‐selective microgliosis. Oligodendrocyte precursor cells (OPC) and mature oligodendrocytes were abundant in the CNS of Gnpat KO mice during the active period of demyelination. Axonal loss was minimal in plasmalogen‐deficient mice, although axonal damage was observed in spinal cords from aged Gnpat KO mice. Characterization of the plasmalogen‐deficient myelin identified myelin basic protein and septin 7 as early markers of dysmyelination, whereas myelin‐associated glycoprotein was associated with the active demyelination phase. Using in vitro myelination assays, we unraveled that the intrinsic capacity of oligodendrocytes to ensheath and initiate membrane wrapping requires plasmalogens. The defect in plasmalogens was rescued with glyceryl 1‐myristyl ether [1‐O‐tetradecyl glycerol (1‐O‐TDG)], a novel alternative precursor in the plasmalogen biosynthesis pathway. 1‐O‐TDG treatment rescued myelination in plasmalogen‐deficient oligodendrocytes and in mutant mice. Our results demonstrate the importance of plasmalogens for oligodendrocyte function and myelin assembly, and identified a novel strategy to promote myelination in nervous tissue.     

牛MBP诱导BALB/c和C57BL/6小鼠实验性变态性脑脊髓炎样反应的研究

为了进一步研究实验性变态反应性脑脊髓炎(EAE)动物模型,采用从新鲜牛脊髓中提取的MBP免疫诱导非敏感品系BALB/c小鼠和C57BL/6小鼠,通过对实验动物的症状、中枢神经系统的病理改变及细胞因子水平变化的检测,较成功地建立了C57BL/6、BALB/c小鼠的EAE模型,实验方法稳定可靠。     

Programmed Cell Death in the Dysmyelinating Mutants

A large number of genetic mutants that are missing a particular myelin protein or that have an aberrant myelin protein composition have been described. These mutations usually cause dysmyelination in the PNS or CNS. Similarly, the nervous system of animals experimentally altered to block synthesis of myelin proteins have recently been generated that show aberrations in the myelin sheath. For both groups of animals, the numbers of myelinating cells remain relatively stable and glial cell death is minimal. The exception is animals with mutations in the proteolipid protein (PLP) gene which exhibit extensive death of oligodendrocytes (OLs). The degree of OL death in the PLP mutants generally correlates with the amount of dysmyelination. Dying OLs in the PLP mutants exhibit the classical features of apoptotic cells. Programmed cell death (PCD) is often, but not necessarily, manifested by cleavage of DNA into abundant oligonucleosomal fragments. Detection of these abundant DNA fragments was examined in normal and jimpy (jp) mice using the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) method. In normal spinal cord and brain, at least twice as many cells exhibited DNA fragmentation when compared to numbers of pyknotic glia observed microscopically. In jp spinal cord and brain, roughly onehalf of cells exhibited DNA fragmentation when compared to numbers of pyknotic glia observed microscopically. PCD of cells in normal development involving DNA fragmentation has been previously described and our results support that conclusion. The data obtained from the jp mice suggests that the mechanism of apoptosis due to PLP mutations is somewhat different from that which occurs in normal development.     

Visualization of myelination in GFP‐transgenic zebrafish

The insulation of axons in the vertebrate nervous system by myelin is essential for efficient axonal conduction. Myelination disruption and remyelination failure can cause human diseases, such as multiple sclerosis and hereditary myelin diseases. However, despite progress in understanding myelination regulation, many important questions remain unanswered. To investigate the mechanisms underlying myelination in vivo, we generated transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under the control of the mbp promoter. This transgenic fish displayed faithful EGFP expression in oligodendrocytes and Schwann cells in embryonic and adult zebrafish. Interestingly, although myelination progressed continuously in the postembryonic central nervous system, some of the spinal cord regions were filled with unmyelinated axons even in the adult spinal cord, suggesting functional differences between myelinated and unmyelinated axons. Our results suggest that this transgenic zebrafish could be a valuable animal model to study oligodendrocyte differentiation and myelination in vivo. Developmental Dynamics 239:592–597, 2010. © 2009 Wiley‐Liss, Inc.     

Fractionation of encephalitogenic polypeptides from bovine spinal cord by gel filtration in phenol—acetic acid—water

Basic peptides capable of causing experimental allergic encephalomyelitis have been isolated from bovine spinal cord. From gel filtration experiments on Sephadex with phenol—acetic acid—water they appeared to have a molecular weight of about 3000. It is not known yet exactly how many of these basic polypeptides are encephalitogenic. Their predominant basic amino acid was lysine and there was a high content of glycine and serine.

Reasons are given for the assumption that they are naturally occurring substances of central nervous tissue and that they are not artificial degradation products of extracted cord proteins. A possible physiological role for these compounds in myelin is discussed.

     

Downregulation of membrane type-matrix metalloproteinases in the inflamed or injured central nervous system

Background  

Matrix metalloproteinases (MMPs) are thought to mediate cellular infiltration in central nervous system (CNS) inflammation by cleaving extracellular matrix proteins associated with the blood-brain barrier. The family of MMPs includes 23 proteinases, including six membrane type-MMPs (MT-MMPs). Leukocyte infiltration is an integral part of the pathogenesis of autoimmune inflammation in the CNS, as occurs in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE), as well as in the response to brain trauma and injury. We have previously shown that gene expression of the majority of MMPs was upregulated in the spinal cord of SJL mice with severe EAE induced by adoptive transfer of myelin basic protein-reactive T cells, whereas four of the six MT-MMPs (MMP-15, 16, 17 and 24) were downregulated. The two remaining MT-MMPs (MMP-14 and 25) were upregulated in whole tissue.     

Chronic relapsing experimental autoimmune encephalomyelitis with a delayed onset and an atypical clinical course,induced in PL/J mice by myelin oligodendrocyte glycoprotein (MOG)-derived peptide: Preliminary analysis of MOG T cell epitopes

Myelin basic protein (MBP) and proteolipid protein (PLP), the most abundant proteins of central nervous system (CNS) myelin, have been extensively studied as possible primary target antigens in multiple sclerosis (MS), a primary demyelinating autoimmune disease of the CNS. However, there is increasing evidence to suggest that autoimmune reactivity against the quantitatively minor myelin component, myelin oligodendrocyte glycoprotein (MOG), can also play a role in the pathogenicity of MS. We recently demonstrated a predominant response to MOG by peripheral blood lymphocytes from patients with MS tested for their reactivity against various myelin antigens, including MBP and PLP. To ascertain whether or not T cell reactivity to MOG in MS is a potentially pathogenic response, we have tested the ability of synthetic MOG peptides (pMOG) representing potential T cell epitopes, to induce neurological disease in mice. Both strains of mice tested (SJL/J and PL/J mice) were able to mount a primary T cell response to some of the five MOG peptides synthesized, pMOG 1–21, 35–55, 67–87, 104–117 and 202–218. T cell lines could be raised in both strains to pMOG 35–55 and 67–87, but epitope definition revealed that each strain recognized a different minimal epitope within these two peptides. T cell lines to pMOG 1–21 and 202–218 could also be raised in SJL/J and PL/J mice, respectively. T cell reactivity to pMOG 104–117 was not observed in either mouse strain. None of the peptides tested induced detectable clinical signs in SJL/J mice. In contrast, an MS-like chronic relasping-remitting disease could be induced in PL/J mice with pMOG 35–55. The disease presented with a delayed onset and with clinical signs which differed significantly in their progression and expression from the typical ascending paralysis of experimental autoimmune encephalomyelitis induced with other myelin components, such as MBP and PLP. Histological examination of CNS tissue from mice injected with pMOG 35–55 revealed only mild neuropathological signs with few inflammatory foci in brain and spinal cord. Some myelin splitting and edema were detected upon electron microscopic examination in the spinal cord and cerebellum. Transfer of pMOG 35–55 reactive T cells into naive PL/J mice resulted in pathological changes characterized by inflammatory foci in the brain and spinal cord. This passively induced disease was clinically silent, as was also reported for Lewis rats injected with T cells specific for the same MOG peptide. These data, which demonstrate unequivocally the encephalitogenic activity of MOG, support our contention that MOG may be as important as MBP or PLP in disease pathogenesis and could be a primary target antigen in autoimmune diseases of the CNS.     

Anti-TWEAK monoclonal antibodies reduce immune cell infiltration in the central nervous system and severity of experimental autoimmune encephalomyelitis

TWEAK is a member of the TNF family, constitutively expressed in the central nervous system (CNS), with pro-inflammatory, proliferative or apoptotic effects depending upon cell types. Its receptor, Fn14, is expressed in CNS by endothelial cells, reactive astrocytes and neurons. We showed that TWEAK and Fn14 mRNA expression increased in spinal cord during experimental autoimmune encephalomyelitis (EAE). We investigated the role of TWEAK during EAE using neutralizing anti-TWEAK antibody in myelin oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice. We observed a reduction of disease severity and leukocyte infiltration when mice were treated after the priming phase.     

Active immunization with proteolipid protein (190-209) induces ascending paralysing experimental autoimmune encephalomyelitis in C3H/HeJ mice

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system (CNS) that shares clinical and pathophysiological feature with multiple sclerosis (MS) and is commonly used as an animal model for the human disease. Upon active immunization, different myelin proteins and other neuronal antigens are known to induce EAE in susceptible mouse strains. However, there are rodent strains reputed to be resistant to actively-induced EAE and the correct combination of animal strains and their respective autoantigen is absolutely critical as some antigens are encephalitogenic in one animal strain, but not in another. Here we describe actively-induced EAE in C3H/HeJ mice with different myelin peptides. Whereas no clinical signs could be found by immunization with myelin oligodendrocyte glycoprotein 35-55, significant weight loss as well as rapidly occurring severe ascending paralysis was found in animals immunized with proteolipid protein 190-209 (PLP(190-209)). Histologically, this form of EAE was characterized by predominant involvement of the spinal cord. As PLP is one of the major lipid antigens putatively involved in the pathogenesis of MS, this model may be useful for further studies of the disease.     

Protection of p27(Kip1) mRNA by quaking RNA binding proteins promotes oligodendrocyte differentiation

The quaking (Qk) locus expresses a family of RNA binding proteins, and the expression of several alternatively spliced isoforms coincides with the development of oligodendrocytes and the onset of myelination. Quaking viable (Qk(v)) mice harboring an autosomal recessive mutation in this locus have uncompacted myelin in the central nervous system owing to the inability of oligodendrocytes to properly mature. Here we show that the expression of two QKI isoforms, absent from oligodendrocytes of Qk(v) mice, induces cell cycle arrest of primary rat oligodendrocyte progenitor cells and differentiation into oligodendrocytes. Injection of retroviruses expressing QKI into the telencephalon of mouse embryos induced differentiation and migration of multipotential neural progenitor cells into mature oligodendrocytes localized in the corpus callosum. The mRNA encoding the cyclin-dependent kinase (CDK)-inhibitor p27(Kip1) was bound and stabilized by QKI, leading to an increased accumulation of p27(Kip1) protein in oligodendrocytes. Our findings demonstrate that QKI is upstream of p27(Kip1) during oligodendrocyte differentiation.     

Nerve fibre regeneration across the peripheral–central transitional zone

Neurons cannot negotiate an elongation across the peripheral (PNS)–central nervous system (CNS) transitional zone and grow into or out of the spinal cord in the mature mammal. The astrocytic rich CNS part of the spinal nerve root is most effective in preventing regeneration even of nerve fibres from transplanted embryonic ganglion cells. Regeneration of severed nerve fibres into the spinal cord occurs when the transition zone is absent as in the immature animal. Before the establishment of a transition zone there is also new growth of neuronal processes from dorsal horn neurons distally to the injured dorsal root. Thus the experimental strategy to reestablish spinal cord to peripheral nerve connectivity has been to delete the transitional region and implant severed ventral or dorsal roots into the spinal cord. Dorsal root implantation resulted in reestablished afferent connectivity by new neuronal processes from secondary sensory neurons in the dorsal horn of the spinal cord extending into the PNS. The ability for plasticity in these cells allowed for a concurrent retention of their original rostral projection. Ventral root implantation into the spinal cord corrected deficit motor function. In a long series of experiments performed in different species, the functional restitution was demonstrated to depend on an initial regrowth of motor neuron axons through spinal cord tissue (CNS). These findings have led to the design of a new surgical strategy in cases of traumatic spinal nerve root injuries.     

Kallikrein 6 regulates early CNS demyelination in a viral model of multiple sclerosis

Kallikrein 6 (Klk6) is a secreted serine protease that is elevated in active multiple sclerosis lesions and patient sera. To further evaluate the involvement of Klk6 in chronic progressive demyelinating disease, we determined its expression in the brain and spinal cord of SJL mice infected with Theiler's murine encephalomyelitis virus (TMEV) and assessed the effects of Klk6-neutralizing antibodies on disease progression. Klk6 RNA expression was elevated in the brain and spinal cord by 7 days postinfection (dpi). Thereafter, Klk6 expression persisted primarily in the spinal cord reaching a peak of fivefold over controls at mid-chronic stages (60?dpi-120?dpi). Significant elevations in Klk6 RNA were also induced in splenocytes stimulated with viral capsid proteins in vitro and in activated human acute monocytic leukemia cells. Klk6-neutralizing antibodies reduced TMEV-driven brain and spinal cord pathology and delayed-type hypersensitivity (DTH) responses when examined at early chronic time points (40?dpi). Reductions in spinal cord pathology included a decrease in activated monocytes/microglia and reductions in the loss of myelin basic protein (MBP). By 180?dpi, pathology scores no longer differed between groups. These findings point to regulatory activities for Klk6 in the development and progression of central nervous system (CNS) inflammation and demyelination that can be effectively targeted through the early chronic stages with neutralizing antibody.