Neuropsychiatric assessment of patients with hyperkinetic and hypokinetic movement disorders

Exposure of weanling rats to a diet containing 1% elemental tellurium causes segmental demyelination of peripheral nerve, and an inhibition of squalene epoxidase. This inhibition is thought to be the mechanism of action leading to demyelination. Tellurite appears to be the active inhibitory species in a cell-free system but the active species in vivo is unknown. We examined potassium tellurite (K2TeO3) and three organotellurium compounds for their ability to inhibit squalene epoxidase in Schwann cell cultures and to induce demyelination in weanling rats. K2TeO3 had no effect on squalene epoxidase activity in cultured Schwann cells and caused no demyelination in vivo. All three organotellurium compounds caused inhibition of squalene epoxidase in vitro and caused demyelination in vivo. (CH3)2TeCl2 was the most potent of these compounds and its neuropathy most resembled that caused by elemental tellurium. These data are consistent with the hypothesis that tellurium-induced demyelination is a result of squalene epoxidase inhibition and suggest that a dimethyltelluronium compound may be the neurotoxic species presented to Schwann cells in vivo.     

Inflammatory cytokines inhibit upregulation of glycolipid expression by Schwann cells in vitro

OBJECTIVE: To determine whether products of inflammatory cells can inhibit differentiation and synthesis of myelin glycolipids by Schwann cells. BACKGROUND: Infiltration of the peripheral nervous system by inflammatory cells is a feature of acquired demyelinating neuropathies. It is not clear what role these cells have in causing demyelination or inhibiting myelin synthesis. METHODS: Nonmyelinating rat Schwann cells were incubated with 1) different concentrations of activated supernatants (AS) from mitogen-activated inflammatory cells; 2) 8-bromo cyclic adenosine monophosphate (8Br cAMP), known to induce Schwann cell differentiation and synthesis of glycolipids; 3) 8Br cAMP and varying concentrations of AS; 4) 8Br cAMP and cytosine arabinoside (Ara C), which inhibits Schwann cell proliferation; 5) 8Br cAMP, AS, and Ara C; or 6) additional medium. RESULTS: AS inhibits the capacity of cAMP to induce Schwann cell expression of myelin-associated glycolipids. Inhibition of glycolipid expression was independent of the capacity of these AS to induce Schwann cell proliferation. CONCLUSIONS: These data suggest that inflammatory mediators are capable of inhibition of Schwann cell differentiation and synthesis of myelin.     

Changes in peripheral nerves of rats four months after induction of streptozotocin diabetes. A qualitative and quantitative study

Eight streptozotocin-injected Wistar rats and eight controls were fixed by whole-body perfusion 4 months after beginning of the experiment, the nervus radialis was dissected and processed for light and electron microscopy. After light-microscopic study standard photographs of nerve cross sections were measured by means of a semiautomatic image analyzer. The following measurements were obtained: (1) surface of fibers, axons, and myelin sheaths, (2) ratio of myelin to axon surface, and (3) percent of endoneural space. Group means and standard deviations were calculated, and cumulated size class distributions were made. Representative nerve specimens from all animals were also studied by electron microscopy. The quantitative study revealed in the diabetics a severe reduction of the average myelin surface, a mild increase of axonal cross section and of endoneural space, a reduction of myelin/axon ratio and a mild reduction in cross section of the nerve. Ultrastructural lesions of minor degree were found in the cytoplasm of Schwann and mesenchymal cells, no lesion was observed in axons. These findings demonstrate the presence of neuropathy 4 months after induction of diabetes and support the pathogenetic role of the Schwann cell in our experimental model.     

The influence of acupuncture stimulation on plasma levels of LH, FSH, progesterone and estradiol in normally ovulating women

Myelinated cultures of mouse spinal cord were exposed to sera obtained from rabbits affected by experimental allergic encephalomyelitis following challenge with whole white matter in complete Freund's adjuvant. In the presence of complement, the tissue response begins with an increased birefringence of its myelin sheaths. This is rapidly followed by a gamut of changes leading to demyelination. This study reports that, in the absence of complement, the response is arrested at the stage of increased birefringence. In this way, this early stage of the demyelinating process was available for detailed examination by light and electron microscopy. The brightened myelin sheaths appeared with a few hours of exposure and were seen around all axons and sometimes around cell bodies. This was often accompanied by abrupt breaks in the sheaths and angularly shaped myelin figures. Examination by electron microscope revealed a uniform increase in the myelin period from 11 nm. to 22 nm. The normally double intraperiod line was increased to four electron-dense leaflets, the additional two appearing to be derived from the close apposition of an additional electron-dense layer on the outer surface of the myelin sheath or oligodendrocytic membrane. Oligondendrocytes responsed with a prolific growth of processes whose membranes compacted to form swollen myelin. Neurons, astrocytes, and neuropil showed no changes. In its early stages, at least, the swelling was reversible. It would appear, therefore, that we have isolated the first stage of antiserum-induced demyelination in vitro, a stage which is now available for further study.     

An in-frame deletion in peripheral myelin protein-22 gene causes hypomyelination and cell death of the Schwann cells in the new Trembler mutant mice

Cloning and sequencing of the peripheral myelin protein-22 cDNA and genomic DNA from newly found Trembler mice revealed an in-frame deletion including exon IV which codes for the second (TM2) and a part of third (TM3) transmembrane domain of peripheral myelin protein-22. This mutation was distinct from those in both other allelic Trembler and Trembler-J mice, which carry point mutations within the putative transmembrane spanning regions of peripheral myelin protein-22. Inheritance was autosomal dominant. The affected mice revealed an abnormal gait, which appeared at 15-20 days of age, followed by motor and sensory ataxia, which remained throughout life. Most of the affected mice could survive more than one year. One of the most notable pathological phenotypes was a giant vacuolar formation in the sciatic nerve of homozygotes. They vary in size within the cytoplasm of Schwann cells, which failed to assemble myelin at any ages studied. Heterozygotes showed normal myelination during the early postnatal stages, followed by a segmental demyelination at an advanced stage. Vacuolar formation was not so frequent as in the homozygotes. These results suggest that the missing of transmembrane spanning region (TM2 and TM3) of peripheral myelin protein-22 may disturb a dual biological function of peripheral myelin protein-22, leading to a dysmyelination of axons and to a vacuolar formation within the cytoplasm of the Schwann cells. The latter phenotype is discussed in conjunction with the disruption of an intracellular transport system and subsequent cell death.     

BDNF attenuates functional and structural disorders in nerves of galactose-fed rats

Galactose intoxication of rats was used to disrupt metabolism of Schwann cells and skeletal muscle, two sites that contain the polyol-forming enzyme aldose reductase (AR). Galactose-fed rats develop a neuropathy characterized by nerve conduction deficits and axonal atrophy. To investigate the possibility that galactose metabolism by AR influences axonal function and structure by altering production of neurotrophic factors, the impact of galactose intoxication on nerve and muscle BDNF levels and the effects of exogenous BDNF treatment on galactose neuropathy were examined using biochemical, electrophysiologic and morphometric techniques. Galactose feeding increased BDNF protein in peripheral nerve and muscle. Exogenous BDNF treatment attenuated motor nerve conduction velocity deficits in the sciatic nerve of galactose-fed animals and myelin splitting of motor axons in the ventral root. In contrast, sensory nerve conduction velocity (SNCV) deficits in the sciatic nerve and myelin splitting in the central projections of sensory neurons were not prevented by BDNF treatment. BDNF treatment did not attenuate reduced axonal caliber in the sciatic nerve, but did ameliorate the diminution of the caliber of central sensory projections in the dorsal root. These findings point to the potential use of BDNF in the treatment of peripheral neuropathies.     

Pathways of migration of transplanted Schwann cells in the demyelinated mouse spinal cord

We have studied the behavior of Schwann cells transplanted at a distance from an induced myelin lesion of the adult mouse spinal cord. These transplanted cells were mouse Schwann cells arising from an immortalized cell line (MSC80) which expresses several Schwann cell phenotypes including the ability to produce myelin. The behavior of MSC80 cells was compared to that of purified rat Schwann cells transplanted in the same conditions. Schwann cells were labeled in vitro with the nuclear fluorochrome Hoechst 33342 and were transplanted at distances of 2-8 mm from a lysolecithin-induced myelin lesion in the spinal cord of shiverer and normal mice. Our results show that transplanted MSC80 cells migrated toward the lesion, in both shiverer and normal mouse spinal cord, preferentially along the ependyma, meninges, and blood vessels. They also migrated along white matter tracts but traveled a longer distance in shiverer (8 mm) than in normal (2-3 mm) white matter. Using these different pathways, MSC80 cells arrived within the lesion of shiverer and normal mouse spinal cord at the average speed of 166 microns/hr (8 mm/48 hr). Migration was most efficient along the ependyma and the meninges where it attained up to 250 microns/hr. Migration was much slower in white matter tracts (95 microns/hr +/- 54 in the shiverer and only 38 microns/hr +/- 3 in the normal mouse). We also provide evidence for the specific attraction of MSC80 cells by the lysolecithin-induced lesion since 1) their number increased progressively with time in the lesion, and 2) MSC80 cells left their preferential pathways of migration specifically at the level of the lesion. Finally, combining the Hoechst Schwann cell labeling method with the immunohistochemical detection of the peripheral myelin protein, P0, we show that some of the MSC80 cells which have reached the lesion participate in myelin repair in both shiverer and normal lesioned mouse spinal cord. A series of control experiments performed with rat Schwann cells indicate that the migrating behavior of transplanted MSC80 cells was identical to that of purified but non-immortalized rat Schwann cells.     

Spontaneous central nervous system remyelination in strain A mice after infection with the Daniel's (DA) strain of Theiler's virus

Intracerebral infection of susceptible SJL/J (H-2s) mice with the Daniel's strain of Theiler's murine encephalomyelitis virus produces chronic, progressive, inflammatory central nervous system demyelination, with minimal spontaneous remyelination. To assess the role of host genetic factors in spontaneous myelin repair following chronic infection with the Daniel's strain of Theiler's virus, we examined demyelination and spontaneous remyelination in strain A mice after infection with Theiler's virus. We found that A.BY/SnJ (H-2a), and A.SW/SnJ (H-2s) mice all developed chronic demyelination with substantial spontaneous remyelination 90 days after infection. In the spinal cords of both A/J and A/WySnJ mice, one quarter of the total lesion area showed spontaneous remyelination, whereas in A.SW/SnJ mice, the extent of remyelination increased to two thirds of the total lesion area. The spontaneous remyelination seen in strain A mice was consistent with myelin repair by oligodendrocytes and Schwann cells, and occurred despite the presence of persistent virus antigen. These results indicate that host-pathogen interactions play an important role in myelin regeneration after virus-induced demyelination, and suggest that host genetic factors influence spontaneous remyelination.     

Connexin43 is another gap junction protein in the peripheral nervous system

That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 microM forskolin, whereas that of Po increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although Po expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with Po, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

Purification of the insecticidal toxin in crystals of Bacillus thuringiensis

Newly transected or denervated segments of isogeneic rat tibial nerve were implanted into the rat midbrain and sampled at weekly intervals up to 6 weeks post-operation. By 3 weeks, the peripheral nervous system (PNS) grafts were well-vascularized and contained Schwann cells, axons associated with Schwann cell processes, and macrophages. From 3 to 6 weeks, many axons within both the fresh and predegenerated grafts were myelinated by Schwann cells. The nerve fiber arrangement within the implant was similar to that of regenerating peripheral nerve in situ. The central nervous system (CNS) border of the implant was clearly demarcated by a rim of astrocytes behind which was a layer of regenerating oligodendrocytes and axons. Extending from the CNS margin were radial bridges of astroglial tissue which apprarently guided regenerating axons into the implant. Between the CNS and the PNS implant, abundant collagen deposition was present. The findings suggest that regenerating CNS axons grow via astroglial bridges into transplanted PNS tissue and are capable of stimulating the implanted Schwann cells to form myelin. Even Schwann cells deprived of axonal contact for prolonged periods were still capable of PNS myelin formation.     

A journey from blame to empathy in a family assessment of a mother and her sons

Ultrastructural analysis of myelin from 8-month-old mice deficient in the myelin-associated glycoprotein revealed pronounced and characteristic alterations of the periaxonal oligodendrocyte processes, consisting of intracytoplasmic deposition of vesicular material, multivesicular bodies, mitochondria, and lipofuscin granules, as well as granular or paracrystalline inclusions. These alterations are similar to those described before as "dying-back oligodendrogliopathy" in diseases of toxic or immune-mediated demyelination including multiple sclerosis.     

Ultrastructural localization of relaxin in the corpus luteum of the pregnant and early lactating tammar wallaby, Macropus eugenii

Electron-microscope immunocytochemistry was used to determine the subcellular distribution and presence of immunoreactive relaxin throughout pregnancy and early lactation in the corpus luteum of a marsupial, the tammar wallaby. Membrane-bound, electron-dense granules were a prominent feature of the luteal cell cytoplasm. The highest numbers of granules were observed between days 20 and 24 of the 26-day gestation, with a rapid clearance immediately after birth. Relaxin immunogold particles were present only in small, electron-dense granules (200-350 nm in diameter), with no particles observed in larger granules (>400 nm diameter), nuclei or mitochondria. Relaxin immunoreactivity was low throughout early and mid pregnancy but increased markedly between days 21 and 22 and remained high over the last 4 days of pregnancy. The number of granules containing relaxin immunogold particles and the density of immunostaining were both reduced on the day of expected births (day 26). Our data demonstrate that electron-dense granules in the luteal cell cytoplasm of a pregnant marsupial contain relaxin. The peptide is produced in greatest amounts at the end of pregnancy, consistent with a role in parturition.     

A previously undescribed side effect of icodextrin: overestimation of glycemia by glucose analyzer

In a study designed to identify the neuropathological features typical of chronic inflammatory demyelinating polyneuropathy (CIDP), we reviewed the sural nerve biopsy findings in 105 patients with this disorder. The patients' mean age at biopsy was 49 years. In 65% of patients the disease had a progressive and in 35% a relapsing-remitting course. In 47% of cases the disorder was idiopathic; the remainder had various concurrent conditions. All sural nerve biopsy specimens showed varying amounts of active demyelination associated with onion bulbs (48% of cases), endoneurial edema (55%) and inflammatory infiltrates (25%). The immunopathological hallmarks were T cell infiltration with macrophagic activation and up-regulation of major histocompatibility complex (MHC) class II expression, without B cell infiltration or immunoglobulin deposition on myelin sheaths. In 30% of cases some myelin sheaths showed C3d deposition. Analysis of proinflammatory cytokine expression invariably showed interleukin-1 in perivascular and endoneurial ramified cells and tumor necrosis factor-alpha prevalently in epineurial macrophages, whereas it detected interferon-gamma only in samples with perivascular inflammatory cells. This immunological pattern suggests that the cellular components of immunity play the major role in CIDP. In 19% of cases the neuropathological changes had a focal distribution. This distinctive feature corresponded to more active demyelination, more frequent detection of inflammatory infiltrates and more prominent immunological activation, suggesting that focal involvement is a possible step in the course of the disease.     

Basis for phospholipid incorporation into peripheral nerve myelin

To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline-, glycerol-, and ethanolamine-labeled phospholipids into four subfractions: microsomes, mitochondria, myelin-like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23-24-day-old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin-containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline-labeled) accumulated in myelin-rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol-labeled) and plasmalogen (ethanolamine-labeled) accumulated in the myelin-rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin-like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P0 (Golgi) and myelin basic proteins (more direct).     

The use of transplanted glial cells to reconstruct glial environments in the CNS

Transplantation studies have demonstrated that glia-depleted areas of the CNS can be reconstituted by the introduction of cultured cells. Thus, the influx of Schwann cells into glia-free areas of demyelination in the spinal cord can be prevented by the combined introduction of astrocytes and cells of the O-2A lineage. Although Schwann cell invasion of areas of demyelination is associated with destruction of astrocytes, the transplantation of rat tissue culture astrocytes ("type-1") alone cannot suppress this invasion, indicating a role for cells of the O-2A lineage in reconstruction of glial environments. By transplanting different glial cell preparations and manipulating lesions so as to prevent meningeal cell and Schwann cell proliferation it is possible to demonstrate that the behaviour of tissue culture astrocytes ("type-1") and astrocytes derived from O-2A progenitor cells ("type-2") is different. In the presence of meningeal cells, tissue culture astrocytes clump together to form cords of cells. In contrast, type-2 astrocytes spread throughout glia-free areas in a manner unaffected by the presence of meningeal cells or Schwann cells. Thus, progenitor-derived astrocytes show a greater ability to fill glia-free areas than tissue culture astrocytes. Similarly, when introduced into infarcted white matter in the spinal cord, progenitor-derived astrocytes fill the malacic area more effectively than tissue culture astrocytes, although axons do not regenerate into the reconstituted area.     

In vitro and in vivo characterisation of glial cells immortalised with a temperature sensitive SV40 T antigen-containing retrovirus

An oncogene-carrying replication-defective retrovirus was used to establish immortalised lines of murine glial cells. Primary cultures of early postnatal cerebellar cells were infected with a retrovirus based on the Murine Moloney Leukemia Virus containing a temperature-sensitive mutant of the Simian Virus 40 large T antigen (SV40 T) oncogene and a gene coding for resistance to the antibiotic G418. Infected cells were selected in G418 and after several in vitro passages cells expressing the O4 antigen were established as a cell line. At a later time point O4-positive single-cell clones were established. Two different types of clones were obtained: 1) "plastic" clones consisting of cells which initially had a morphological and antigenic phenotype of young glial precursor cells but which gradually lost these features, and 2) "stable" cell clones including a clone with the immunological and electrophysiological characteristics of Schwann cells. Culture of the latter cells in the presence of 1 mM dibutyryl cyclic adenosine monophosphate for a period of at least 10 days induced a change in shape and a shift in antigen expression towards a more "differentiated" maturation stage. When the SV40 T O4-positive immortalised cell line isolated on the cell sorter was transplanted into demyelinated lesions in adult rats, cells were observed ensheathing axons and forming limited amounts of PNS-type myelin. Glial cells immortalised with a temperature-sensitive mutant of the SV40 T oncogene thus retain many physiological properties of their primary culture counterparts and can be induced to undergo limited differentiation in vitro and in vivo. These cell lines, which represent immature CNS glia or Schwann cells, are providing useful tools for investigating the role of cell surface antigens involved in neuron-glial interactions.     

Heterozygous P0 knockout mice develop a peripheral neuropathy that resembles chronic inflammatory demyelinating polyneuropathy (CIDP)

Demyelinating peripheral neuropathies are clinically divided into inherited and acquired types. Inherited demyelinating neuropathies are caused by mutations in genes expressed by myelinating Schwann cells, whereas acquired ones, including chronic inflammatory demyelinating polyneuropathy (CIDP), are probably caused by autoimmune mechanisms. We find that heterozygous P0 knockout (P0+/-) mice develop a neuropathy that resembles CIDP. By one year of age, P0+/- mice develop severe, asymmetric slowing of motor nerves, with temporal dispersion or conduction block, which are features of acquired demyelinating neuropathies including CIDP. Moreover, morphological analysis of affected nerves reveals severe and selective demyelination of motor fibers, focal regions of demyelination, and inflammatory cells. These data suggest that immune-mediated mechanisms may contribute to the pathogenesis of the neuropathy in P0+/- mice.     

Mouse models of myelin diseases

Dys- and demyelination are the common endpoints of several inherited diseases of glial cells, which elaborate myelin and which maintain the myelin sheath very much like an "external" cellular organelle. Whereas some of the genes that are affected by mutations appear to be glial-specific, other genes are expressed in many cell types but their defect is restricted to oligodendrocytes or Schwann cells. Many of the disease genes and their encoded proteins have been studied with the help of mouse models, and a number of different molecular pathomechanisms have emerged which have been summarized in Figure 8. Some of the new concepts in the field, which have been addressed in this review, have only emerged because similar pathomechanisms were discovered for different myelin proteins. Mouse models have clearly helped to address both, the molecular pathology of myelin diseases and the normal function of myelin genes, but as discussed in this review, these questions turned out to be very different. Despite the progress in understanding the role of the abundant myelin proteins, there also remain a number of open questions that concern, among other things, the initial axon-glia recognition, the assembly process of the myelin sheath, and the long-term interaction of axons with their myelinating glia. Finally, animal models of human neurological diseases should not be restricted to the study of pathology, but they should also contribute to the development of experimental treatments. It is encouraging that a few attempts have been made.     

Glucocorticoids and progestins signal the initiation and enhance the rate of myelin formation

Dexamethasone and progesterone have been found to accelerate the time of initiation and enhance the rate of myelin synthesis in Schwann cell/neuronal cocultures. The expression of mRNA for cytochrome P450scc (converts cholesterol to pregnenolone), 3beta-hydroxysteroid dehydrogenase (converts pregnenolone to progesterone), and the progesterone receptor were detected and markedly induced during peak myelin formation in the cocultures. The mRNA for the glucocorticoid receptor was detected, but was found to be constituitively expressed. In addition, the specific activity of 3beta-hydroxysteroid dehydrogenase was measured and found to increase by 10-fold. The mRNA for cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase also were found to be induced during the differentiation of O-2A precursor cells to oligodendrocytes. Fibroblast growth factor and platelet-derived growth factor were found to have proliferative effects on Schwann cells, but they had no effect on the initiation or the rate of myelin formation. These results demonstrate that myelin-forming cells have inducible enzymes responsible for steroid biosynthesis and suggest a critical role for endogenous steroid hormones in signaling the initiation and enhancing the rate of myelin formation.