Martin G. Rumsby

N-WASP regulates extension of filopodia and processes by oligodendrocyte progenitors, oligodendrocytes, and Schwann cells—implications for axon ensheathment at myelination

The molecular mechanisms used by oligodendrocyte precursor cells (OPCs), oligodendrocytes (OLs), and Schwann cells (SCs) to advance processes for motility in the developing nervous system and to ensheath axons at myelination are currently not well defined. Here we demonstrate that OPCs, OLs, and SCs express the major proteins involved in actin polymerization‐driven protrusion; these key proteins including F‐actin, the Arp2/3 complex, neural‐Wiskott Aldrich Syndrome protein (N‐WASP) and WAVE proteins, and the RhoGTPases Rac and Cdc42 are present at the leading edges of processes being extended by OPCs, OLs, and SCs. We reveal by real‐time PCR that OLs and SCs have different dominant WAVE isoforms. Inhibition of the WASP/WAVE protein, N‐WASP, with wiskostatin that prevents activation of the Arp2/3 complex, blocks process extension by OPCs and SCs. Inhibition of N‐WASP also causes OPC and SC process retraction, which is preceded by retraction of filopodia. This implicates filopodia in OPC and SC process stability and also of N‐WASP in OPC and SC process dynamics. We also demonstrate that p34 (a component of the Arp2/3 complex), WASP/WAVE proteins, actin, α‐tubulin, Rac, Cdc42, vinculin, and focal adhesion kinase are detected in water‐shocked myelin purified from brain. Inhibition of N‐WASP with wiskostatin decreases the number of axons undergoing initial ensheathment in intact optic nerve samples and reduces the Po content of dorsal root ganglia:SC co‐cultures. Our findings indicate that OPCs, OLs, and SCs extend processes using actin polymerization‐driven protrusion dependent on N‐WASP. We hypothesize that inner mesaxons of OLs and SCs use the same mechanism to ensheath axons at myelination.

The effect of Freund's adjuvants on blood-cerebrospinal fluid barrier permeability☆

In guinea pigs with experimental allergic encephalomyelitis induced by spinal cord homogenate--complete Freunds adjuvant (CFA) emulsions an increase in the albumin permeability of the blood--cerebrospinal fluid barrier occurred from day 10 post-inoculation (p.i.) onward. In animals inoculated with CFA alone an increased albumin permeability was also demonstrated but only between days 5 and 10 after inoculation; by day 14-16 p.i. the barrier permeability had returned to control values. A similar change was seen in animals inoculated with incomplete Freunds adjuvant (IFA) only. However, both CFA and CFA-cord induced a strong humoral immune response which was not seen in animals inoculated with IFA alone. These results may have important consequences for the understanding of the development of inflammatory diseases of the central nervous system.

Accessibility of Galactosyl Ceramides to Probe Reagents in Central Nervous System Myelin

The suitability of isolated central nerve myelin preparations for probe labelling studies was assessed and the accessibility of galactosyl ceramides in myelin to galactose oxidase and sodium periodate was determined. Isolated myelin preparations present a uniform external membrane surface to added probes because lamellae in the myelin sheath separate at their external apposition surfaces exclusively during isolation. The cytoplasmic apposition remains intact in isolated myelin. Cationised ferritin can gain access along external apposition regions of inner lamellae in multilamellar fragments of isolated myelin, indicating that proteins and lipids on the external membrane surface will be accessible to probes. Over 50% of the total galactosyl ceramides of myelin are accessible to galactose oxidase attack; hydroxy fatty acid‐ and nonhydroxy fatty acid‐containing cerebrosides are equally attacked. Sodium periodate attacks over 90% of the galactosyl ceramides in isolated myelin at 20°C and electron micrographs of the periodate‐treated myelin reveal changes at the external apposition only. Galactosyl ceramides in vesicles of myelin lipid vesicles are not so readily attacked by periodate. The disposition of galactosyl ceramides in the myelin lamellae is discussed.

Chronic relapsing experimental allergic encephalomyelitis: Immunological and blood-cerebrospinal fluid barrier-dependent changes in the cerebrospinal fluid

Cerebrospinal fluid (CSF) and plasma were taken from strain 13 guinea pigs in various stages of chronic relapsing experimental allergic encephalomyelitis using techniques which allowed repeated sampling from the same animal. Samples were assayed for albumin and IgG and the corresponding CSF/plasma quotients evaluated graphically using a method which could discriminate between blood-CSF barrier dysfunction and local IgG synthesis in the central nervous system (CNS). During the disease a 2-3-fold increase in plasma IgG concentration developed and an increase in blood-CSF permeability was noted. Isoelectric focusing revealed an oligoclonal IgG pattern identical in both plasma and CSF. The results provided no evidence for a local production of IgG in the CNS like that which is known to occur in multiple sclerosis.

Immunocytochemical characterisation of cell cultures grown from dissociated 1–2-day post-natal rat cerebral tissue *: A developmental study

A range of cell-specific markers have been employed with immunocytochemical methods to characterise and quantitate the cell types present in mixed brain cell cultures derived from dissociated 1-2-day post-natal rat cerebral hemispheres and grown in the presence of FCS. Protoplasmic astrocytes (GFAP+, A2B5-) were the major cell type to develop in culture, a confluent monolayer forming in 5-8 days. A population of smaller round cells of oligodendrocyte-like morphology appeared on this astrocyte layer. Greater than 70% of these smaller cells were GC- and thus were not oligodendrocytes. The GC- cells were A2B5+ and, in early cultures, may therefore be progenitor glial cells. Examination of GFAP and A2B5 co-expression by these smaller cells was difficult due to the dense underlying GFAP+ astrocyte layer. In less dense areas of older cultures these smaller cells with processes were GFAP+ and A2B5+: these are Type 2, fibrous astrocytes. GC+ oligodendrocytes, comprising 5-10% of the total identified cell population, were initially distributed over the astrocyte monolayer; in older cultures (after about 8 days) GC+ cells were observed in clumps over places where NF+ cells were identifiable. Such GC+ cells mostly became MBP+. Neurones accounted for about 6% of the identifiable cells in early cultures but a lower percentage in older cultures. Minor populations of ependymal cells and macrophages were present; cells displaying fibronectin, fibroblasts, were rarely identified. Use of horse serum in place of FCS gave lower yields of GC+ cells in cultures, slowed down astrocyte development, and resulted in the formation of trunks of GFAP+ cells throughout cultures. Other sera gave lower numbers of GC+ cells.

THE INTRINSIC FLUORESCENCE CHARACTERISTICS OF THE MYELIN BASIC PROTEIN

—The encephalitogenic basic protein has been isolated from the myelin sheath of ox brain white matter and the purity and amino acid composition have been verified. The intrinsic fluorescence characteristics of the purified basic protein have been determined and the results interpreted in terms of current ideas on the structure of the protein. Fluorescence data obtained from the basic protein in aqueous solution indicate that the tyrosine and tryptophan residues are largely exposed to the solvent and that resonance energy transfer from tyrosine to tryptophan is very inefficient. Denaturing conditions in 8 m‐urea have little effect on the fluorescence properties of the protein. The ionic detergent, sodium dodecyl sulphate, interacts with the basic protein and alters the fluorescence properties in a manner which indicates that the tryptophan residue is in the hydrocarbon chain region of the detergent while the local positive charge around the tyrosine residues is neutralized by the negatively charged sulphate head‐groups. The fluorescence results suggest that the basic protein can be used as a natural, non‐perturbing probe which will report on its environment after it has reacted with other membrane components.

Protein expression of the α, γ, δ and ε subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum

Total protein kinase C (PKC) activity and protein expression of the α and δ subspecies of PKC increases markedly as C6 glioma cells grow from low cell density to the contact‐inhibited quiescent state (also known as G0) in the presence of serum. At the same time protein expression of PKC subspecies γ and ϵ decreases while the β I, β II, ι and ζ subspecies did not change. Serum deprivation of growing C6 glioma cells does not induce the same changes in PKC subspecies protein expression. The findings support the growing view that there are significant differences between the G0 states brought about by contact inhibition or serum deprivation.

Evidence of free radical damage in the central nervous system of guinea-pigs at the prolonged acute and early relapse stages of chronic relapsing experimental allergic encephalomyelitis

Central nervous system tissue from guinea-pigs in various stages of chronic relapsing experimental allergic encephalomyelitis (CR-EAE) has been analysed for evidence of free radical damage using the thiobarbituric acid test as an indicator of oxidative changes. Levels of thiobarbituric acid-reactive materials in brain and spinal cord regions from prolonged acute and early relapse phases of CR-EAE were significantly higher than in central nervous system tissue from Freunds adjuvant control and normal uninoculated animals. In the late relapse phase of CR-EAE, levels of thiobarbituric acid-reactive materials in CNS samples were at control values. Fluorescence analysis of central nervous system tissue confirmed evidence of increased free radical damage in prolonged acute and early relapse stages of CR-EAE. The increased oxidative damage observed in the central nervous system in CR-EAE is discussed in relation to the role of macrophages and their oxidative burst.

Rat brain glial cells in primary culture and subculture contain the δ, ϵ and ζ subspecies of protein kinase C as well as the conventional subspecies

Abstract We raised polyclonal antibodies against the C-terminal peptides of protein kinase C (PkC) subspecies α, β 1 , β 2 , γ, δ, ϵ, and ζ and checked their specificity against brain extracts using Western immunoblot analysis. With equal amounts of protein applied to gels PkC subspecies β 1 , δ, ϵ and ζ were detected in primary cultures of mixed glial cells: bands for the α and β 2 subspecies were less prominent. PkC γ was not detected in primary glial cultures. The ϵ and ζ subspecies of PkC were detected in subcultures of type 1 astrocytes with weaker bands for the α, β 1 and β 2 subspecies. Blots of O-2A-lineage glia contained PkCs δ and ζ as prominent bands: the α, β 1 and ϵ subspecies were also present. All PkC subspecies including PkC γ were detected in C6 glioma cells.

Phorbol ester-induced translocation of PKC epsilon to the nucleus in fibroblasts: identification of nuclear PKC epsilon-associating proteins

We show that phorbol ester treatment of NIH 3T3 fibroblasts induces rapid translocation of PKCε from a perinuclear site to the nucleus, extending findings in PC12 and NG108‐15 cells and in myocytes. We have immunoprecipitated the PKCε from nuclei isolated from phorbol ester‐treated fibroblasts and identified six proteins which associate with nuclear PKCε. These have been characterised as matrin 3, transferrin, Rac GTPase activating protein 1, vimentin, β‐actin and annexin II by MALDI–TOF–MS. We have confirmed that these proteins associate with PKCε by gel overlay and/or dot blotting assays. The role of these PKCε‐associating proteins in the nucleus and their interaction with PKCε are considered.