Implantable data logging system for heart rate and body temperature: its application to the estimation of field metabolic rates in Antarctic predators

Mutations affecting the peripheral myelin protein 22 (PMP22) gene are associated with inherited motor and sensory neuropathies in mouse (Trembler and Trembler-J) and human (Charcot-Marie-Tooth disease type 1A and Dejerine-Sottas syndrome). Although genetic studies have established a critical role of PMP22 in the formation and/or maintenance of myelin in the peripheral nervous system, the biological function of PMP22 in myelin and in non-myelin forming cells remains largely enigmatic. In this Mini-Review, we will summarize the current knowledge about PMP22 and discuss its hypothetical function(s) in a broad context.     

A novel PMP22 point mutation causing HNPP phenotype: studies on nerve xenografts

BACKGROUND: Hereditary neuropathy with liability to pressure palsies (HNPP) in most cases is caused by a deletion in chromosome 17p11.2-12 or, rarely, mutations resulting in a functional loss of one copy of the peripheral myelin protein 22 (PMP22) gene. Point mutations that lie deep within transmembrane (TM) domains causing major structural changes in PMP22 are associated with severe neuropathy. METHODS: A 25-year-old asymptomatic woman with a normal neurologic examination volunteered as a control subject. Electrophysiologic studies showed multiple entrapment neuropathies, prompting a search for a genetic defect. In addition, sural nerve fascicles from the subject were grafted into the cut ends of the sciatic nerve of nude mice and studied at 2, 6, and 8 weeks and compared with controls. RESULTS: Direct sequencing of the PMP22 gene revealed a G-->A transition at position 202 in axon 3 of the PMP22 gene. To determine if this was a causative mutation rather than a polymorphism, 102 DNA samples from controls were studied; none showed a similar base pair change. In the nerve xenografts, there was a marked delay at the onset of myelination and an impairment in the regenerative capacity of the nude mice axons engulfed by the mutant human Schwann cells. The axon tips were enlarged and demonstrated neurofilament density increase. Neurofilament density distribution histograms were bimodal in xenografts as well as in the subject's sural nerve. CONCLUSION: This study provides unequivocal evidence that a base pair change causing a Val30Met substitution at the junction of the first TM domain and the extracellular loop of PMP22 results in the HNPP phenotype.     

Progesterone stimulates the activity of the promoters of peripheral myelin protein-22 and protein zero genes in Schwann cells

To understand better the mechanisms by which progesterone (PROG) promotes myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P0) genes. PROG stimulated the P0 promoter and promoter 1, but not promoter 2, of PMP22. The effect of PROG was specific, as estradiol and testosterone only weakly activated promoters. Dose-response curves for stimulation of both promoter constructs by PROG were biphasic. RU486, a PROG antagonist, did not abolish the effect of PROG, but stimulated promoter activities by itself. In the human carcinoma cell line T47D expressing high levels of PROG receptor, PROG did not stimulate the P0 and PMP22 promoters, whereas the promoter region of the mouse mammary tumor virus was fully activated. Thus, the activation by PROG of promoter activity of two peripheral myelin protein genes is Schwann-cell specific.     

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.     

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.     

Formation and maintenance of the myelin sheath in the peripheral nerve: roles of cell adhesion molecules and the gap junction protein connexin 32

Based on previous in vitro studies, the cell adhesion molecules L1, N-CAM, MAG, and P0, which all belong to the immunoglobulin (Ig)-superfamily, have been suggested to mediate myelin formation in the peripheral nervous system. Unexpectedly, studies in mice deficient for the corresponding molecules revealed that only P0 plays pivotal roles during the formation of peripheral nerve myelin in vivo, while L1-, N-CAM-, and MAG-deficient mice develop myelin of normal ultrastructure. However, MAG turned out to be important for the maintenance of myelin, as reflected by degeneration of myelin and axons in MAG-deficient mice older than 6 months. The MAG-mediated maintenance of myelin is backed up by N-CAM, since mice deficient in both MAG and N-CAM show an earlier and more prominent myelin degeneration than MAG single mutants. Another peripheral nerve component involved in the maintenance of myelinated fibers is connexin 32 (Cx32), a gap junction channel protein that does not belong to the Ig-superfamily. Mice deficient in Cx32 initially form normal myelin, which then develops blown-up periaxonal collars and abnormally shaped non-compacted regions followed by myelin and axonal degeneration. Our findings strongly support the view that very few myelin components are necessary for myelin formation whereas the maintenance of myelin is much more sensitive to molecular alterations. In addition, it became evident that myelin molecules can fulfill functionally overlapping roles that ensure that myelination takes place even under conditions in which there is a deficiency in the normal molecular components of myelin.     

Effect of static compression on proteoglycan biosynthesis by chondrocytes transplanted to articular cartilage in vitro

Mutant mice that lack either protein zero (P0) or connexin 32 (Cx32) were generated previously to investigate the function of these myelin proteins in peripheral nerves and to assess the value of these mice as animal models for hereditary human peripheral neuropathies. Mice that are completely devoid of P0 expression (P0(+/0)) show a complex phenotype that is characterized by hypomyelination, compromised myelin compaction, and degeneration of myelin and axons early in life. In contrast, young mouse mutants that have retained one wild-type allele of the P0 gene (P0(+/0)) reveal morphologically normal myelin but start to develop signs of demyelination and remyelination at 4 months of age. A similar late-onset myelin deficiency was observed in Cx32-deficient mice (Cx32(0/0)). We have now generated mice deficient for Cx32 and P0. In animals that lack both proteins (Cx32(0/0)/P0(0/0), the phenotype is morphologically identical to mice that solely lack P0. Animals that lack Cx32 and carry one functional P0 allele (Cx32(0/0/P0(+/0)) revealed demyelination and remyelination as evidenced by thin myelin and Schwann cell onion bulb formation already at the age of 4 weeks, a time point when no pathology was observed in the single mutants. These morphological deficits were also more prominent in 4-month-old Cx32(0/0)/P0(+/0)animals compared to the single mutants. Our data support the view that Cx32 and P0 are crucial molecules for the maintenance of myelin. Furthermore, the function of Cx32 in the peripheral nervous system appears to be largely dispensable when myelin compaction is impaired.     

Peripheral nerve glycoproteins and myelin fine structure during development of rat sciatic nerve

Developmental changes in relative amounts of peripheral nerve proteins and glycoproteins have been correlated with the degree of morphological myelination at various ages during the first 25 postnatal days in rat sciatic nerve. At birth there is virtually no major myelin glycoprotein (P0), but there is a protein which migrates to the same point on sodium dodecyl sulphate (SDS) polyacrylamide gels as the small myelin basic protein (P2). During the time myelin is being formed in the nerve, the P0 protein increases and the P2 protein appears to decrease in relative amount in the nerve. The accumulation of P0 protein in the nerve correlates extremely well with the degree of myelination in sciatic nerve. At 4-6 days postnatal, smooth membrane profiles are observed which are located within axons and in the inner Schwann cell cytoplasm. Such profiles are also observed to fuse with the axolemma-Schwann cell interface. The profiles may represent membrane material being added to or deleted from the axolemma or myelin during myelination.     

An autoradiographic study of the renewal of mouse bronchiolar epithelium following bromobenzene exposure

We report an experimental model of germanium dioxide (GeO2)-induced neuropathy in rats. More than 6 months administration of GeO2 to young rats produced neuropathy characterized by segmental demyelination/remyelination and nerve edema. Electron microscopic studies demonstrated that changes in Schwann cells, such as an increased cytoplasmic volume or disintegration of the cytoplasm, were the earliest pathological findings. Schwann cell mitochondria contained high electron-dense materials. Subsequent removal of necrotic Schwann cell debris and myelin by invading macrophages was evident. These findings suggested that the Schwann cells themselves are the primary target of the toxin. The deposition of electron-dense granules in the intra-axonal vesicles, which was suggestive of glycogen granules in mitochondria, was observed in the advanced stage of the neuropathy. The findings of endoneurial edema with splitting of myelin lamellae were noted at the early stage of demyelination. Nerve edema may be the result of GeO2-induced endothelial cell injury.     

The cytokine network of wallerian degeneration: IL-10 and GM-CSF

Wallerian degeneration (WD) is the inflammatory response of peripheral nerves to injury. Evidence is provided that granulocyte macrophage colony stimulating factor (GM-CSF) contributes to the initiation and progression of WD by activating macrophages and Schwann, whereas IL-10 down-regulates WD by inhibiting GM-CSF production. A significant role of activated macrophages and Schwann for future regeneration is myelin removal by phagocytosis and degradation. We studied the timing and magnitude of GM-CSF and IL-10 production, macrophage and Schwann activation, and myelin degradation in C57BL/6NHSD and C57BL/6-WLD/OLA/NHSD mice that display normal rapid-WD and abnormal slow-WD, respectively. We observed the following events in rapid-WD. The onset of GM-CSF production is within 5 h after injury. Production is steadily augmented during the first 3 days, but is attenuated thereafter. The onset of production of the macrophage and Schwann activation marker Galectin-3/MAC-2 succeeds that of GM-CSF. Galectin-3/MAC-2 production is up-regulated during the first 6 days, but is down-regulated thereafter. The onset of myelin degradation succeeds that of Galectin-3/MAC-2, and is almost complete within 1 week. IL-10 production displays two phases. An immediate low followed by a high that begins on the fourth day, reaching highest levels on the seventh. The timing and magnitude of GM-CSF production thus enable the rapid activation of macrophages and Schwann that consequently phagocytose and degrade myelin. The timing and magnitude of IL-10 production suggest a role in down-regulating WD after myelin is removed. In contrast, slow-WD nerves produce low inefficient levels of GM-CSF and IL-10 throughout. Therefore, deficient IL-10 levels cannot account for inefficient GM-CSF production, whereas deficient GM-CSF levels may account, in part, for slow-WD.     

Age-induced decrease of glycoprotein Po and myelin basic protein gene expression in the rat sciatic nerve. Repair by steroid derivatives

The data here reported show that the gene expression of the glycoprotein Po and of the myelin basic protein, the major components of myelin in the peripheral nervous system, dramatically decreases with ageing in the sciatic nerve of normal male rats. A one-month treatment with dihydroprogesterone, the 5alpha-reduced derivative of progesterone, is able to partially restore the fall in Po gene expression occurring in the sciatic nerve of aged male rats, without significantly modifying the gene expression of the myelin basic protein. In cultures of neonatal Schwann cells (the peripheral nervous system elements involved in the synthesis of myelin), the addition of progesterone and of dihydroprogesterone significantly increases Po gene expression; the 3alpha-reduced metabolite of dihydroprogesterone, tetrahydroprogesterone proved to be even more effective. These data suggest that the effect of progesterone is linked to its conversion into dihydroprogesterone and especially into tetrahydroprogesterone, since Schwann cells possess the 5alpha-reductase-3alpha-hydroxysteroid dehydrogenase system. The data provide the first demonstration that ageing decreases the gene expression of two major components of the peripheral myelin in the sciatic nerve; they also show that this phenomenon may be partially reversed by progesterone derivatives, which might act by stimulating Po gene expression in the Schwann cells.     

Autonomic neuropathy in the trembler mouse

There is little detailed histological information concerning the autonomic nervous system in hereditary demyelinating neuropathies in man. An opportunity was therefore taken to study the autonomic nervous system of the trembler mouse which suffers from a dominantly inherited peripheral neuropathy. Schwann cell myelination in trembler vagus and splanchnic nerves was abnormal. Morphometric analysis of myelinated and unmyelinated fibres in these nerves showed a marked reduction in myelinated fibre density distribution, whilst unmyelinated fibre densities were within the control range. The trembler vagus contained increased numbers of large diameter unmyelinated fibres probably as a result of trembler Schwann cell failure to form myelin around axons of the appropriate diameter for myelination. The trembler splanchnic nerve, however, contained increased numbers of small diameter unmyelinated fibres, possibly postganglionic fibres which fail to achieve their expected diameters.     

Dystonin is an essential component of the Schwann cell cytoskeleton at the time of myelination

A central role for the Schwann cell cytoskeleton in the process of peripheral nerve myelination has long been suggested. However, there is no genetic or biological evidence as yet to support this assumption. Here we show that dystonia musculorum (dt) mice, which carry mutations in dystonin, a cytoskeletal crosslinker protein, have hypo/amyelinated peripheral nerves. In neonatal dt mice, Schwann cells were arrested at the promyelinating stage and had multiple myelinating lips. Nerve graft experiments and primary cultures of Schwann cells demonstrated that the myelination abnormality in dt mice was autonomous to Schwann cells. In culture, dt Schwann cells showed abnormal polarization and matrix attachment, and had a disorganized cytoskeleton. Finally, we show that the dt mutation was semi-dominant, heterozygous animals presenting hypo- and hyper-myelinated peripheral nerves. Altogether, our results suggest that dt Schwann cells are deficient for basement membrane interaction and demonstrate that dystonin is an essential component of the Schwann cell cytoskeleton at the time of myelination.     

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.     

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.     

Molecular basis of Charcot-Marie-Tooth neuropathy

Charcot-Marie-Tooth neuropathy (CMT) is the most common inherited peripheral neuropathy. CMT is classified into type types on the basis of pathological and electrophysiological findings: type 1(CMT1), characterized by decreased nerve conduction velocities and by "onion bulb" formation: type 2(CMT2), in which nerve conduction velocities are normal and "onion bulb" formations are rarely seen. CMT1 loci map to chromosome 17 (CMT1A), chromosome 1(CMT1B), another unknown autosome (CMT1C) and the X chromosome (CMTX). Recent work has identified the gene products corresponding to CMT1A, CMT1B and CMTX as peripheral myelin protein-22(PMP22), Po and connexin 32, respectively. Dejerine-Sottas disease has been identified as being caused by the mutation of PMP-22 or Po gene.     

Characterization of dp6troglycan-laminin interaction in peripheral nerve

Dystoroglycan is encoded by a single gene and cleaved into two proteins, alpha and beta-dystroglycan, by posttranslational processing. The 120kDa peripheral nerve isoform of alpha-dystroglycan binds laminin-2 comprised of the alpha 2, beta 1, and gamma 1 chains. In congenital muscular dystrophy and dy mice deficient in laminin alpha 2 chain, peripheral myelination is disturbed, suggesting a role for the dystroglycan- laminin interaction in peripheral myelinogenesis. To begin to test this hypothesis, we have characterized the dystroglycan-laminin interaction in peripheral nerve. We demonstrate that (1) alpha-dystroglycan is an extracellular peripheral membrane glycoprotein that links beta-dystroglycan in the Schwann cell outer membrane with laminin-2 in the endoneurial basal lamina, and (2) dystrophin homologues Dp116 and utrophin are cytoskeletal proteins of the Schwann cell cytoplasm. We also present data that suggest a role for glycosylation of alpha-dystroglycan in the interaction with laminin.