Biochemical Demonstration of the Myelin-Associated Glycoprotein in the Peripheral Nervous System

Abstract: Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [³H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, WI, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [³H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [¹⁴C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and Wl fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole US-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS.     

Biochemical demonstration of the myelin-associated glycoprotein in the peripheral nervous system

Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [3H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, W1, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [3H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [14C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and W1 fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole LIS-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS.     

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm Microstomum lineare with antiserum to FMRF-amide

Summary The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.     

Bony Fish Myelin: Evidence for Common Major Structural Glycoproteins in Central and Peripheral Myelin of Trout

Peripheral nervous system (PNS) myelin from the rainbow trout (Salmo gairdneri) banded at a density of 0.38 M sucrose. The main myelin proteins consisted of (1) two basic proteins, BPa and BPb (11,500 and 13,000 MW, similar to those of trout central nervous system (CNS) myelin proteins BP1 and BP2), and (2) two glycosylated components, IPb (24,400 MW) and IPc (26,200 MW). IPc comigrated with trout CNS myelin protein IP2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas trout CNS myelin protein IP1 had a lower molecular weight (23,000). Following two-dimensional separation, however, both IPb and IPc from PNS showed two components; the more acidic component of IPc comigrated with IP2 from CNS. PNS tissue autolysis led to the formation of IPa (20,000 MW), consisting of two components in isoelectric focusing of which again the more acidic one comigrated with the CNS autolysis product IP0. Limited enzymatic digestion of isolated IP proteins from PNS and CNS led to closely similar degradation patterns, being most pronounced in the case of IP2 and IPc. Immunoblotting revealed that all IP components from trout PNS and CNS myelins reacted with antibodies to trout IP1 (CNS) and bovine P0 protein (PNS) whereas antibodies to rat PLP (CNS) were entirely unreactive. All BP components from trout PNS and CNS myelins bound to antibodies against human myelin basic protein. On the basis of these studies trout PNS and CNS myelins contain at least one common IP glycoprotein, whereas other members of the IP myelin protein family appear closely related. In the CNS myelin of trout the IP components appear to replace PLP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of Gene Expression Following Sciatic Nerve Crush and Spinal Cord Hemisection in the Mouse by Microarray Expression Profiling

1. The responses of periphery (PNS) and central nervous systems (CNS) towards nerve injury are different: while injured mammalian periphery nerons can successfully undergo regeneration, axons in the central nervous system are usually not able to regenerate.2. In the present study, the genes which were differentially expressed in the PNS and CNS following nerve injury were identified and compared by microarray profiling techniques.3. Sciatic nerve crush and hemisection of the spinal cord of adult mice were used as the models for nerve injury in PNS and CNS respectively.4. It was found that of all the genes examined, 14% (80/588) showed changes in expression following either PNS or CNS injury, and only 3% (18/588) showed changes in both types of injuries.5. Among all the differentially expressed genes, only 8% (6/80) exhibited similar changes in gene expression (either up- or down-regulation) following injury in both PNS and CNS nerve injuries.6. Our results indicated that microarray expression profiling is an efficient and useful method to identify genes that are involved in the regeneration process following nerve injuries, and several genes which are differentially expressed in the PNS and/or CNS following nerve injuries were identified in the present study.     

Proton Nuclear Magnetic Resonance Spectroscopy of Primary Cells Derived from Nervous Tissue

Abstract: Cell culture techniques, high-resolution in vitro ¹H nuclear magnetic resonance (NMR) spectroscopy, and chromatographic analyses were used to compare the properties of purified cell populations derived from the PNS and cortical neurones. Cell cultures were immunocytochemically characterised with specific antibodies to ensure purity of the individual cultures. Spectra of perchloric acid extracts of cultured Schwann cells, perineural fibroblasts, dorsal root ganglion neurones, and cortical neurones displayed several common features. However, statistically significant differences were found by ¹H NMR spectroscopy in most metabolites among the cell types studied. In addition, cells could be distinguished by the presence or absence of certain amino acids. For example, N -acetylaspartate was present in dorsal root ganglion neurones and cortical neurones, γ-aminobutyric acid was present in large amounts in cortical neurones, and Schwann cell spectra displayed a large signal from glycine. These results extend our earlier findings that different cell types of the CNS exhibit highly characteristic metabolite profiles to now include the major cell types of the PNS. These latter cell types also exhibit characteristic metabolite compositions, such that even Schwann cells and oligodendrocyte type 2 astrocyte (O-2A) progenitor cells—precursors of the myelinating cells of the CNS and PNS, respectively—can be readily distinguished from each other.     

Comparison of the Major Proteins of Shark Myelin with the Proteins of Higher Vertebrates

Abstract: On gel electrophoresis in dodecyl sulphate solutions shark CNS myelin showed four bands close in mobility to the proteolipid protein of bovine CNS myelin. They had apparent molecular weights of 21,000, 26,000, 27,000, and 31,500. Unlike bovine proteolipid protein, all of these shark proteins were shown to be glycosylated by staining gels with the periodate-Schiff reagent. Amino acid analyses of the polypeptides eluted from polyacrylamide gels indicated a high content of apolar amino acids and a composition approximating that of the P_o protein of bovine peripheral nervous system (PNS) myelin, rather than that of the CNS proteolipid protein. The shark poly-peptide of apparent molecular weight 31,500 was obtained by elution from dodecyl sulphate gels and antibodies raised against it in rabbits. By probing of electroblots with this antiserum the four shark CNS bands were shown to share common determinants with each other, with a major shark PNS protein and with sheep and chicken major PNS glycoproteins (P_o). The binding of antibody was unaffected by deglycosylation of the shark CNS polypeptides with anhydrous hydrogen fluoride. Together, these results appeared to establish that shark CNS myelin contains four proteins that are closely related to a major shark PNS protein and to the P_o protein of higher species.     

Myelin-Associated Glycoprotein Is the Antigen for a Monoclonal IgM in Polyneuropathy

Recent studies show that IgM monoclonal antibody from patients with IgM paraproteinemia and peripheral neuropathy reacts with a protein component of human PNS myelin and an analogous component or components of human CNS myelin. We have now demonstrated that the antigen for this antibody is a specific glycoprotein component of myelin, referred to as myelin-associated glycoprotein (MAG). Human PNS and CNS myelin proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on pore-gradient slabs, and MAG was identified by the immuno-electroblot procedure with rabbit anti-MAG (rat). The identical band(s) were stained by an analogous procedure with patient serum as the first antibody. Human PNS MAG had an apparent molecular weight of 107,000. Human CNS MAG appeared as three bands: 113,000, 107,000, and 92,000. Passage of myelin proteins through a concanavalin A-Sepharose column removed the staining component. Purified patient IgM, added to a lithium diiodosalicylate extract of myelin, immunoprecipitated MAG. This antibody also cross-reacted with MAG from bovine CNS, but not from rabbit, rat, or mouse.     

Satellite cells of dorsal root ganglia are multipotential glial precursors

The evolutionary origin of myelinating cells in the vertebrate nervous system remains a mystery. A clear delineation of the developmental potentialities of neuronal support cells in the CNS and PNS might aid in formulating a hypothesis about the origins of myelinating cells. Although a glial-precursor cell in the CNS can differentiate into oligodendrocytes (OLs), Schwann cells (SCs) and astrocytes, a homologous multipotential cell has not yet been found in the PNS. Here, we identify a cell type of embryonic dorsal root ganglia (DRG) of the PNS - the satellite cell - that develops into OLs, SCs and astrocytes. Interestingly,satellite-cell-derived OL precursors were found in cultures prepared from embryonic day 17 (E17) to postnatal day 8 (P8) ganglia,but not from adult DRGs, revealing a narrow developmental window for multipotentiality. We suggest that compromising the organization of the ganglia triggers a differentiation pathway in a subpopulation of satellite cells, inducing them to become myelinating cells with either a CNS or PNS phenotype. Our data provide an additional, novel piece in the myelinating cell-precursor puzzle, and lead to the concept that cells in the CNS and PNS that function to ensheath neuronal cell bodies and axons can differentiate into OLs, SCs and astrocytes. In sum, it appears that glial fate might be determined over and above the CNS/PNS dichotomy. Last, we suggest that primordial ensheathing cells form the original cell population in which the myelination program first evolved.     

Endogenous Peptides That Inhibit Brain Cyclic AMP Phosphodiesterase

Peptide extracts of rat brain powerfully inhibited the cyclic AMP phosphodiesterase activity of rat brain homogenate. Similar extracts of ox brain showed comparable although less potent activity. Preliminary investigation of the physicochemical properties of brain extracts indicated that the rat brain extract contained an active peptide of low molecular weight (about 1400), whereas ox brain contained two such peptides (about 1400 and 900). These studies indicate that endogenous oligopeptides that inhibit cyclic AMP phosphodiesterase are present in brain. Experiments on several pure peptides known to be present in brain. Experiments on several pure peptides known to be present in the CNS showed that the majority were inactive against brain phosphodiesterase, but ACTH(1-24), somatostatin, substance P and Lys8-vasopressin, in descending order of potency, were active. To help distinguish the peptides found in rat and ox brain extracts from known peptides, preliminary analyses of amino acid composition were performed. These suggested that the peptides found in brain extracts were distinct from known peptides having the ability to inhibit cyclic AMP phosphodiesterase.     

In Vivo imaging reveals distinct inflammatory activity of CNS microglia versus PNS macrophages in a mouse model for ALS

Mutations in the enzyme superoxide dismutase-1 (SOD1) cause hereditary variants of the fatal motor neuronal disease Amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous: neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring non-neuronal cells. In vivo imaging by two-photon laser-scanning microscopy was used to compare the role of microglia/macrophage-related neuroinflammation in the CNS and PNS using ALS-linked transgenic SOD1(G93A) mice. These mice contained labeled projection neurons and labeled microglia/macrophages. In the affected lateral spinal cord (in contrast to non-affected dorsal columns), different phases of microglia-mediated inflammation were observed: highly reactive microglial cells in preclinical stages (in 60-day-old mice the reaction to axonal transection was ～180% of control) and morphologically transformed microglia that have lost their function of tissue surveillance and injury-directed response in clinical stages (reaction to axonal transection was lower than 50% of control). Furthermore, unlike CNS microglia, macrophages of the PNS lack any substantial morphological reaction while preclinical degeneration of peripheral motor axons and neuromuscular junctions was observed. We present in vivo evidence for a different inflammatory activity of microglia and macrophages: an aberrant neuroinflammatory response of microglia in the CNS and an apparently mainly neurodegenerative process in the PNS.     

Peptidome Analysis of Cerebrospinal Fluid by LC-MALDI MS

We report on the analysis of endogenous peptides in cerebrospinal fluid (CSF) by mass spectrometry. A method was developed for preparation of peptide extracts from CSF. Analysis of the extracts by offline LC-MALDI MS resulted in the detection of 3,000-4,000 peptide-like features. Out of these, 730 peptides were identified by MS/MS. The majority of these peptides have not been previously reported in CSF. The identified peptides were found to originate from 104 proteins, of which several have been reported to be involved in different disorders of the central nervous system. These results support the notion that CSF peptidomics may be viable complement to proteomics in the search of biomarkers of CNS disorders.     

Selective interaction of peripheral and central nervous system cells with two distinct cell-binding domains of fibronectin

Mechanisms of cell interaction with fibronectin have been studied with proteolytic fibronectin fragments that have well-defined ligand binding properties. Results of a previous study (Rogers, S. L., J. B. McCarthy, S. L. Palm, L. T. Furcht, and P. C. Letourneau, 1985, J. Neurosci., 5:369-378) demonstrated that (a) central (CNS) and peripheral (PNS) nervous system neurons adhere to, and extend neurites on a 33-kD carboxyl terminal fibronectin fragment that also binds heparin, and (b) neurons from the PNS, but not the CNS, have stable interactions with a 75-kD cell-binding fragment and with intact fibronectin. In the present study domain-specific reagents were used in inhibition assays to further differentiate cell surface interactions with the two fibronectin domains, and to define the significance of these domains to cell interactions with the intact fibronectin molecule. These reagents are (a) a soluble synthetic tetrapeptide Arg-Gly-Asp-Ser (RGDS; Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) representing a cell-binding determinant in the 75-kD fragment, and (b) an antibody raised against the 33-kD fragment that binds specifically to that fragment. Initial cell attachment to, and neurite extension upon, fibronectin and the two different fragments was evaluated in the presence and absence of the two reagents. Attachment of both PNS and CNS cells to intact fibronectin was reduced in the presence of RGDS, the former more so than the latter. In contrast, the antibody to the 33-kD fragment did not affect attachment of PNS cells to fibronectin, but significantly decreased attachment of CNS cells to the molecule. RGDS inhibited attachment of CNS cells to the molecule. RGDS inhibited attachment of both cell types to the 75-kD fragment to a greater degree than it did attachment to the intact molecule. Cell interaction with the 33-kD fragment was not affected by RGDS. Reduction of neurite lengths (determined after 24 h of culture) by the domain-specific reagents paralleled the reduction in initial adhesion to each substratum. Therefore, it appears that (a) both PNS and CNS cells have receptors for each cell-binding domain of fibronectin, (b) the receptor(s) for the two domains are distinct, with attachment to the 33-kD fragment being independent of RGDS, and (c) the relative importance of each domain to cell interaction with intact fibronectin is different for CNS and PNS cells.     

In Vitro Reconstruction of Neuronal Networks Derived from Human iPS Cells Using Microfabricated Devices

Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions.     

Peptides and neurotransmission in the central nervous system

Radioimmunoassays of brain extracts have shown that several peptides occur in high concentrations in the CNS. The releasing-factor peptides TRF, LRF, somatostatin, CRF and GRF have the highest concentration in the hypothalamic extracts. High levels of somatostatin, CCK octapeptide, neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) are found in cortical extracts. Substance P, CCK, NPY, and enkephalins are present in high concentrations in basal ganglia and mesolimbic areas. Pharmacological doses of these peptides result in several behavioural and vegetative effects. Immunocytochemical studies show that the CNS peptides are localised in neurones and in synaptic vesicles. In vitro studies with brain tissues show that peptides are capable of modifying the ongoing classical neurotransmission. In depressive patients several neuropeptides (CCK, CRF and NPY) have been shown to have low CSF levels. Patients dying of senile dementia have low cortical levels of somatostatin, CRF and substance P. In schizophrenic patients CCK peptides have shown to improve some symptoms. At present the therapeutic potentials of peptides are poorly known. More studies are required to understand their role in neurotransmission and related pathological states.     

Conditional mutation of Rb causes cell cycle defects without apoptosis in the central nervous system

Targeted disruption of the retinoblastoma gene in mice leads to embryonic lethality in midgestation accompanied by defective erythropoiesis. Rb(-/-) embryos also exhibit inappropriate cell cycle activity and apoptosis in the central nervous system (CNS), peripheral nervous system (PNS), and ocular lens. Loss of p53 can prevent the apoptosis in the CNS and lens; however, the specific signals leading to p53 activation have not been determined. Here we test the hypothesis that hypoxia caused by defective erythropoiesis in Rb-null embryos contributes to p53-dependent apoptosis. We show evidence of hypoxia in CNS tissue from Rb(-/-) embryos. The Cre-loxP system was then used to generate embryos in which Rb was deleted in the CNS, PNS and lens, in the presence of normal erythropoiesis. In contrast to the massive CNS apoptosis in Rb-null embryos at embryonic day 13.5 (E13.5), conditional mutants did not have elevated apoptosis in this tissue. There was still significant apoptosis in the PNS and lens, however. Rb(-/-) cells in the CNS, PNS, and lens underwent inappropriate S-phase entry in the conditional mutants at E13.5. By E18.5, conditional mutants had increased brain size and weight as well as defects in skeletal muscle development. These data support a model in which hypoxia is a necessary cofactor in the death of CNS neurons in the developing Rb mutant embryo.     

Restricted neural epidermal growth factor-like like 2 (NELL2) expression during muscle and neuronal differentiation

We have identified a secreted glycoprotein, neural epidermal growth factor-like like 2 (NELL2), in a screen designed to isolate molecules regulating sensory neuron genesis and differentiation in the dorsal root ganglia (DRG). In investigating NELL2 expression during embryogenesis, we demonstrate here that NELL2 is highly regulated spatially and temporally, being only transiently expressed in discrete regions of the central (CNS) and peripheral nervous systems (PNS) and in a subset of mesoderm derived structures during their peak periods of development. In the CNS and PNS, NELL2 is maximally expressed as motor and sensory neurons differentiate. Interestingly, its expression is restricted to sublineages of the neural crest, being strongly expressed throughout the immature DRG, but excluded from sympathetic ganglia. Similarly during muscle development, NELL2 is specifically expressed by hypaxial muscle precursor cells in the differentiating somite and derivatives in the forelimbs and body wall, but not by epaxial muscle precursors. Furthermore, NELL2 is differentially regulated in the CNS and PNS; in the CNS, NELL2 is only expressed by nascent, post-mitotic neurons as they commence their differentiation, yet in the PNS, NELL2 is expressed by subsets of progenitor cells in addition to nascent neurons. Based on this restricted spatial and temporal expression pattern, functional studies are in progress to determine NELL2's role during neuronal differentiation in both the PNS and CNS.