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S Szuchet

Publications and source records attributed to S Szuchet.

At least 37 records · Page 2Linked to original sources
PubMed1988

Plasma membrane of cultured oligodendrocytes: I. Isolation, purification, and initial characterization.

Oligodendrocytes generate myelin as extensions of the plasma membrane. Myelin has been well characterized, yet little is known concerning oligodendrocyte plasma membrane. We have developed a reproducible method for the isolation of an oligodendrocyte plasma membrane-rich fraction (F2.2). F2.2 has a 25-fold enrichment in K+-dependent p-nitrophenyl phosphatase, a plasma membrane marker. Impurities are composed of Golgi elements (8-12%), microsomes (4-6%), and lysosomal membranes (1-5%). Our starting material was oligodendrocytes kept in culture in a nonattached state for 3 to 5 d. After disrupting the cells and removing nuclei (P1), the supernatant (SP1) was fractionated on a self-generating 20% Percoll gradient into three bands: F1, F2, and F3. F1 had only 3% of the applied protein and was not characterized. F2, with 11% of the protein, was fivefold enriched in plasma membrane. F3 had 27% of initial protein; it consisted of a crude mitochondrial and lysosomal fraction. F2 was further purified by first washing it hypotonically, treating it with Mg2+, and then fractionating it on a Ficoll step gradient that yielded F2.2 at the interphase. Morphologically F2.2 comprises 1) membranous sheets, often with more than one membrane in close apposition; 2) membrane vesicles of various sizes and shapes frequently filled with amorphous material; 3) Golgi elements; and 4) unrecognizable profiles. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile of F2.2 reveals CNPase as a major component in agreement with the high CNPase specific activity (3,860 mumol/mgP/h) found in F2.2. Other significant polypeptides have Mr = 170,000, 135,000, 108,000, 80,000, 53,000, 38,500, 32,000, and 22,200.

Animals↗
PubMed1988

Plasma membrane of cultured oligodendrocytes: II. Possible structural and functional domains.

An oligodendrocyte plasma membrane-rich fraction, F2.2, was resolved by equilibrium density centrifugation on a linear sucrose gradient from 0.5 M to 1.3 M into three fractions, F2.2a, F.2.2b, F2.2c, and a pellet F2.2p. F2.2a and F.2.2b were enriched 1.5-fold relative to F2.2 in plasma membrane markers at the expense of F2.2c and F2.2p, which became correspondingly impoverished. This gave F2.2a and F2.2b a 42-fold and 37-fold enrichment, respectively, in plasma membrane markers relative to the initial cell homogenate. F2.2c had a sevenfold enrichment in a Golgi marker; together with F2.2p, they contained all the Golgi marker initially present in F2.2. Preliminary data indicated that the F2.2-subfractions differed from one another in their molar ratios of cholesterol to phospholipids and protein to lipids but had similar protein profiles when examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Their content of fucosylated glycoproteins appeared also to be different. Morphologically, F2.2a and F2.2b were very similar: they contained large membrane vesicles, membrane sheets, and vesicles entrapped within other vesicles. Membrane-membrane interaction was apparent in these fractions. F2.2c had many of the same elements, but most of the membrane structures contained amorphous material. F2.2p differed morphologically from the other fractions in that it had principally electron-dense structures. It is postulated that F2.2a, F2.2b, and perhaps F2.2c represent different domains of oligodendrocyte plasma membrane. Alternatively, these fractions might correspond to the plasma membrane of oligodendrocyte subtypes.

Animals↗
PubMed1988

Plasma membrane of cultured oligodendrocytes: III. Relatedness to myelin.

We have compared highly purified fractions of oligodendrocyte plasma membrane to myelin by one- and two dimensional gel electrophoresis and found them to be distinct. The major myelin proteins--proteolipid protein (PLP), DM-20, and myelin basic protein (MBP), which dominate the sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern of myelin--were minor components of the plasmalemma. However, 2',3', cyclic nucleotide phosphodiesterase (CNPase) and myelin-associated glycoprotein (MAG) were represented equally in both membranes. Labeling the cells with various precursors followed by isolation of plasmalemma revealed that newly synthesized PLP, DM-20, CNPase, and MAG were incorporated into the plasma membrane of "floating" oligodendrocytes (i.e., nonattached to substratum). This was not so with MBP. Nevertheless, scattered patches of MBP were localized on the plasma membrane of intact cells using the immunogold method at the electron microscopic level. The data are consistent with the notion that MBP is not a constituent of the plasma membrane of mature oligodendrocytes (the MBP patches on intact cells are likely remnants from past association with myelin) but is rapidly associated with the plasmalemma of myelinating oligodendrocytes (i.e., attached cells). It is suggested that phosphorylation of MBP provides the triggering signal for plasma membrane association. In order to analyze the minor proteins in myelin and compare them to the plasma membrane by two-dimensional gel electrophoresis, myelin was extracted with chloroform:methanol to remove PLP, DM-20, and MBP. Even in the absence of PLP, DM-20, and MBP the pattern of extracted myelin still differed from that of plasmalemma indicating that their minor protein compositions were not the same. Myelin was characterized by a group of proteins that clustered at pI 5.5-6.5 and Mr 40,000-60,000 of which alpha-tubulins, beta-tubulins, and actin are part: the plasmalemma had tubulins and actin but in different proportions. Our findings indicate that in addition to PLP, DM-20, and MPB, myelin is also enriched relative to the plasmalemma in another group of proteins.

Animals↗
PubMed1988

Forskolin and phorbol esters decrease the same K+ conductance in cultured oligodendrocytes.

Cultured ovine oligodendrocytes (OLGs) express a number of voltage-dependent potassium currents after they attach to a substratum and as they begin to develop processes. At 24-48 hours following plating, an outward potassium current can be identified that represents a composite response of a rapidly inactivating component and a steady-state or noninactivating component. After 4-7 days in culture, OLGs also develop an inward rectifier current. We studied the effects of forskolin and phorbol 12-myristate 13-acetate (PMA) on OLG outward currents. These compounds are known to alter the myelinogenic metabolism of OLGs. PMA, an activator of protein kinase C (PK-C), has been shown to enhance myelin basic protein phosphorylation while forskolin acting on adenylate cyclase, and thereby increasing cAMP, inhibits it. Both forskolin and PMA increase the phosphorylation of 2'3'-cyclic nucleotide phosphodiesterase, an OLG/myelin protein. We found that forskolin decreased the steady-state outward current at 120 mV by 10% at 100 nM, and by 72% at 25 microM from a holding potential of -80 mV. The time course of inactivation of the peak currents was decreased, affecting both the fast and slow time constants. There was no significant change in the steady-state parameters of current activation and inactivation. The effect of forskolin was attenuated when the adenylate cyclase inhibitor adenosine (2 mM) was present in the intracellular/pipette filling solution. The results of PMA experiments were similar to those obtained with forskolin. Whereas the amplitude of the currents in the presence of PMA was reduced by 28% at 1.5 nM and 60% and 600 nM, the decay phase of the peak currents was less affected. The PMA effect could still be seen when the intracellular Ca2+ was reduced to less than or equal to 10 nM with 5 mM BAPTA, but was inhibited when the cells were pre-exposed to 50 microM psychosine, a PK-C inhibitor. It is postulated that the potassium currents in OLG can be physiologically modulated by two distinct second-messenger systems, perhaps converging at the level of a common phosphorylated enzyme or regulatory protein.

Animals↗
PubMed1988

Oligodendrocyte substratum adhesion modulates expression of adenylate cyclase-linked receptors.

The molecular mechanisms of myelin formation/reformation in the central nervous system are unknown. In previous work we have demonstrated that mature oligodendrocytes (OLG) respond to a signal(s), elicited by their adhesion to a substratum, by turning on a myelinogenic metabolism. Events occurring within 24 hr of adhesion include generation of diacylglycerol, activation of protein kinase C, phosphorylation of myelin basic protein, and enhanced synthesis of myelin lipids and proteins. To elucidate the mechanism(s) of signal transduction, we have investigated whether OLG-substratum interaction influences the level of basal cAMP and the expression of receptors coupled to adenylate cyclase. By using ovine brain OLG we have found that adhesion to a polylysine-coated surface for 24 hr increased the basal level of cAMP 2-fold and altered the expression (assessed by cAMP production) of receptors coupled to adenylate cyclase. Isoproterenol (beta-adrenergic agonist) augmented cAMP from 4 to 26 pmol/mg of protein in adhering OLG but had no such effect in nonattached OLG. Adhesion of OLG was accompanied by rapid synthesis of ethanolamine plasmalogen, a class of lipids believed to be associated with beta-adrenergic receptors. Nonattached OLG responded to prostaglandin E1 with only a 3-fold stimulation in their cAMP content; in attached OLG, 6-fold stimulation was observed. In contrast, vasoactive intestinal polypeptide elicited a 3-fold increase in cAMP in nonattached OLG but, following 24 hr of attachment, OLG did not respond to vasoactive intestinal polypeptide. The increase of cellular cAMP levels was accompanied by a 2.5-fold gain in protein kinase A. OLG-substratum adhesion resulted also in phosphorylation of the OLG/myelin protein, 2',3'-cyclic nucleotide 2'-phosphodiesterase, which proved to be a substrate for cAMP and phospholipid-, Ca2+-dependent protein kinases. These findings, in conjunction with our earlier work, implicate cAMP and diacylglycerol in signaling myelinogenesis; they suggest that phosphorylation/dephosphorylation of myelin basic protein and 2',3'-cyclic nucleotide 2'-phosphodiesterase may be key processes in the cascade of events that are initiated by adhesion of OLG to a polylysine surface (possibly acting as a surrogate for axons) and culminate in the reformation of myelin.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1988

A peanut agglutinin binding glycoprotein in CNS myelin and oligodendrocytes.

We isolated and characterized a 120-kd PNA-binding polypeptide from the human CNS. This polypeptide is linked to membranes through a PI linkage. After release from membranes by PLC it measures 105 kd, 30 kd of which appear to be contributed by N-linked carbohydrates. We isolated the polypeptide by the use of PLC and PNA affinity chromatography and used it to raise polyclonal antibodies and to determine the N-terminal sequence. Immunohistochemical and immunochemical studies using these antibodies showed that 120 kdpp is present in both myelin and oligodendrocytes.

Amino Acid Sequence↗
PubMed1988

Voltage-gated potassium currents in cultured ovine oligodendrocytes.

Cultured oligodendrocytes (OLGs) develop processes and form myelin following attachment to a substratum. We applied the whole-cell voltage-clamp technique to identify and characterize the ionic currents of OLGs in culture. Within 2 d after attachment, OLGs extended processes and began to express an outward current that represents a composite response of an inactivating/transient component and a non-inactivating component. The current had a reversal potential of -66 mV and was sensitive to potassium channel blockers. After 4-5 d in culture, the transient component was less prominent, often accompanied by an increase in noninactivating or steady-state outward current. In addition, there was an increase in inward rectifier current. Four of 7 cells that failed to develop processes exhibited only linear high-resistance membranes. We conclude that cultured OLGs express 3 voltage-gated potassium conductances: (1) a transient outward current, (2) a noninactivating outward current, and (3) an inward rectifier current. The sequential appearance of the several currents may relate, at least in part, to process formation.

4-Aminopyridine↗
PubMed1986

Oligodendrocyte adhesion activates protein kinase C-mediated phosphorylation of myelin basic protein.

When isolated adult oligodendrocytes adhere to a substratum myelinogenesis occurs. Investigation of the mechanism by which this happens indicated that the oligodendrocyte-substratum interaction activated protein kinase C-dependent phosphorylation of myelin basic protein and promoted the synthesis of myelin basic protein. In addition, when agents that activate protein kinase C (second messenger diacylglycerol or a tumor-promoting phorbol ester) were added to nonattached oligodendrocytes, they mimicked the influence of the substratum by inducing phosphorylation of myelin basic protein; and reagents that increase cellular adenosine 3', 5'-monophosphate (cyclic AMP) inhibited phosphorylation of myelin basic protein. Thus, at least in vitro, the interaction between oligodendrocytes and the substratum may mediate myelinogenic events, and phosphorylation of myelin basic protein may be an early requirement in the sequence of steps that ultimately results in myelin formation.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Cultured oligodendrocytes. A role for cell-substratum interaction in phenotypic expression.

Oligodendrocytes can be maintained in two states: nonattached; we call these cells B3.f; morphologically they resemble freshly isolated cells; attached; we refer to the latter as B3.fA. Profound morphological, ultrastructural, and biochemical changes take place upon adhesion to a competent surface (Szuchet, S., Yim, S. H., and Monsma, S. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7019-7023). Here we present evidence that the transition from B3.f to B3.fA has important consequences for the expression of myelinogenic properties by these cells. We have examined the incorporation of [3H]leucine, [35S]methionine, and [35S]cysteine into polypeptide chains by B3.f and B3.fA cells from 3 days after isolation up to 8 weeks in culture. Specific antisera against myelin and cytoskeletal proteins were used to identify the newly synthesized proteins. Our results indicate that: overall incorporation expressed as cpm/mg of protein remains essentially constant and independent of the state of adhesion or time in culture; B3.f cells keep a low profile in the synthesis of the major myelin proteins but have a high uptake of precursors into 2',3'-cyclic nucleotide phosphodiesterase, actin, and tubulin; adhesion of oligodendrocytes to a polylysine substratum activates the synthesis and phosphorylation of myelin basic protein, and the synthesis and acylation of proteolipid protein and DM-20; over time in culture there is an increased synthesis and accumulation of these proteins and of myelin-associated glycoprotein. We conclude that B3.f cells exhibit a behavior that is distinct from that of B3.fA cells. Our results are consistent with the notion that upon adhesion to a substratum, oligodendrocytes undergo a transition from myelin-maintaining cells (B3.f) to that of myelin-forming cells (B3.fA). This conclusion is substantiated by the finding of myelin membranes in these cultures.

Animals↗
PubMed1986

Lipid and glycolipid metabolism of cultured astrocytes: a time course study.

Primary cultures of astrocytes free of neurons and containing less than 1% of oligodendrocytes were examined for their ability to incorporate labeled precursors into lipids and glycolipids. At selected developmental stages cultures were double-labeled with either [3H]glycerol and [14C]acetate or with [3H]galactose and Na2[35SO4] for a total of 72 hr. Lipids were extracted with CHCl3/CH3OH, fractionated on a silicic acid column, and further resolved by two-dimensional thin-layer chromatography. It was found that cultured astrocytes actively incorporate acetate and glycerol into various phospholipids; they have very limited ability to utilize galactose and virtually lack the synthetic machinery to use Na2SO4 for the synthesis of sulfated sphingogalactolipids; and their overall lipid metabolism is very distinct from that of oligodendrocytes. It was also found that cultured astrocytes have low levels of 2',3' cyclic phosphodiesterase and glycerol phosphate dehydrogenase activities; the latter is less than one fifth of that in oligodendrocytes.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1986

Mature oligodendrocytes cultured in the absence of neurons recapitulate the ontogenic development of myelin membranes.

We have maintained isolated oligodendrocytes in culture for up to 5 months. Over time cultured cells acquire conspicuous membranous profiles that either emanate from cell processes and perikarya or are apposed to them. These membranous profiles have biochemical and ultrastructural features characteristic of myelin. Because the cells used in this study are post-myelination (cells are isolated from white matter of mature brains), we argue that this is a case of resynthesis of myelin membranes. Because this process takes place in the absence of neurons, we have termed it myelin palingenesis in order to distinguish it from remyelination, a term commonly used to designate reensheathment of axons. Myelin palingenesis may occur seemingly without wrapping and spiraling, but membranes wrapped around oligodendrocyte processes and soma are also observed. Irrespective of the modes of formation, those membranes that are compact have the fine structure of normal myelin. Thus, long-term cultures of mature oligodendrocytes should be a good model system in helping to decipher the molecular and cellular mechanisms involved in myelination and in remyelination.

Animals↗
PubMed1985

Detection of visna virus antigens and RNA in glial cells in foci of demyelination.

Visna is a slow virus infection of sheep in which the characteristic pathological change is demyelination in foci of inflammation. The latter is thought to be the result of an immunopathological process directed against cellular and antigenic targets that have been difficult to define because of restricted viral gene expression. A new simultaneous detection assay is used to demonstrate viral RNA in cells identified unambiguously as oligodendrocytes and astrocytes. These cells were found in inflammatory foci. With a new strain of virus that causes a rapid form of visna in Icelandic sheep, viral antigens were demonstrated in cells in the inflammatory lesions. These findings are consistent with the postulated immunopathological mechanism of demyelination: cells that maintain intact myelin sheaths in the central nervous system are destroyed by the inflammatory response to viral antigens expressed in these cells.

Animals↗
PubMed1985

A monoclonal antibody that binds to both astrocytes and myelin sheaths.

A monoclonal antibody designated III 5H8 was shown to bind both to astrocytes and to myelin sheaths as studied with immunocytochemical techniques on brain sections and cell cultures. Binding to astrocytes was confirmed by double immunofluorescent labelling of frozen sections and cell cultures with anti-GFAP, and appeared to be sensitive to formalin treatment. Binding to myelin sheaths was confirmed by comparing sections labelled with III 5H8 with sections labelled with antibodies against axons and myelin basic protein as well as by staining of sections of hypomyelinated spinal cord with III 5H8. On immunoblots of separated white matter III 5H8 revealed two bands, while on immunoblots of purified myelin only one band was seen. The findings are discussed with respect to the function of astrocytes in white matter and shared antigenic determinants between astrocytes and oligodendrocytes.

Animals↗
PubMed1984

Characterization of a subset of oligodendrocytes separated on the basis of selective adherence properties.

A subset of oligodendrocytes (B3,f) was isolated by taking advantage of selective cell-substratum interaction. B3,f cells were characterized morphologically, biochemically, and immunocytochemically. Oligodendrocytes were isolated from 4-to-6-month-old lamb brains by a modified version of our published procedure [Szuchet et al, J Neurosci Methods 3:7-19, 1980]. Freshly isolated cells from band III were plated on plastic culture plates at a concentration of 2 X 10(6) cells/ml. Approximately 40% of the cells attached to the plate under these conditions. The remaining cells formed small floating clusters. We refer to the latter as B3,f oligodendrocytes. After 4 to 5 days, the supernatant containing B3,f cells was removed and centrifuged, and the pellet was resuspended in culture medium and replated on polylysine-coated petri dishes. B3,f oligodendrocytes attached to this surface and extended an intricate network of processes. The purity of the cultures, judged by the number of cells staining with a monoclonal antibody against galactocerebroside was 98-99%. This high degree of cell homogeneity was maintained throughout the life of the cultures. B3,f cells appeared to be highly differentiated and remained so in vitro. This is surmised by the expression of oligodendrocytic characteristic functions such as high levels of CNPase activity typically, 5 microM/min/mgP; high incorporation of H2 35SO4 into sulfatides, an overall lipid metabolism that mimics events associated with myelinogenesis [Szuchet et al, PNAS 80:7019-7023, 1983]; the presence, detected immunocytochemically, of myelin-associated glycoprotein and myelin basic proteins. It is concluded that this culture system offers an opportunity for studying the biology of interfascicular oligodendrocytes and their interaction with neurons and/or astrocytes. It also should open up a way of examining the relevance of oligodendrocyte polymorphism.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1984

Cell-cell interactions of isolated and cultured oligodendrocytes: formation of linear occluding junctions and expression of peculiar intramembrane particles.

Oligodendrocytes were isolated from lamb brain. Freshly isolated cells and cultured cells, either 1- to 4-day-old unattached or 1- to 5-week-old attached, were examined by thin section and freeze-fracture electron microscopy. Freeze-fracture of freshly isolated oligodendrocytes showed globular and elongated intramembrane particles similar to those previously described in oligodendrocytes in situ. Enrichment of these particles was seen at sites of inter-oligodendrocyte contact. Numerous gap junctions and scattered linear tight junctional arrays were apparent. Gap junctions were connected to blebs of astrocytic plasma membrane sheared off during isolation, whereas tight junctions were facing extracellular space or blebs of oligodendrocytic plasma membrane. Thin sections of cultured, unattached oligodendrocytes showed rounded cell bodies touching one another at points without forming specialized cell junctions. Cells plated on polylysine-coated aclar dishes attached, emanated numerous, pleomorphic processes, and expressed galactocerebroside and myelin basic protein, characteristic markers for oligodendrocytes. Thin sections showed typical oligodendrocyte ultrastructure but also intermediate filaments not present in unattached cultures. Freeze-fracture showed intramembrane particles similar to but more numerous, and with a different fracture face repartition, than those seen in oligodendrocytes, freshly isolated or in situ. Gap junctions were small and rare. Apposed oligodendrocyte plasma membrane formed linear tight junctions which became more numerous with time in culture. Thus, cultured oligodendrocytes isolated from ovine brains develop and maintain features characteristic of mature oligodendrocytes in situ and can be used to explore formation and maintenance of tight junctions and possibly other classes of cell-cell interactions important in the process of myelination.

Animals↗
PubMed1983

A simple cell disrupter designed for small cells with relatively large nuclei.

A simple cell disrupter that is particularly suitable for breaking small cells with relatively large nuclei is described. Cells are disrupted by the shearing forces set up as they are pushed by a positive N2 pressure through a 0.8- to 1.5-micrometer stainless-steel filter. The total procedure takes only a few minutes, it is highly reproducible, and the yield is good. The cell homogenate obtained is a good starting source for the isolation of plasma membranes, intracellular organelles, and nuclei.

Animals↗
PubMed1983

Lipid metabolism of isolated oligodendrocytes maintained in long-term culture mimics events associated with myelinogenesis.

Oligodendrocytes isolated from ovine white matter according to a published procedure [Szuchet, S., Stefansson, K., Wollmann, R. L., Dawson, G. & Arnason, B. G. W. (1980) Brain Res. 200, 151-164] were cultured for up to 35 days and their capacity to incorporate precursors into lipids was investigated. At various times, cultures were double labeled with [3H]glycerol/[14C]acetate or [3H]galactose/35SO2-4. The cells were harvested 72 hr later and lipids were fractionated using standard procedures. The time course of incorporation for each precursor was distinct. In the days after attachment to substratum, oligodendrocytes preferentially incorporated [3H]glycerol into phospholipids and [14C]acetate into cholesterol while uptake of 35SO2-4 and [3H]galactose into glycolipids was modest. A switch in phospholipid metabolism from preferential incorporation into phosphatidylcholine to incorporation into phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol occurred at about the 10th day in vitro. After 20 days, uptake of [3H]glycerol into phospholipids and [14C]acetate into cholesterol had stabilized but incorporation of 35SO2-4 into glycolipids had increased. 35SO2-4 incorporation into glycolipids was even greater at 35 than at 20 days. Uptake of [3H]galactose did not change over time. An attempt was made to correlate changes in lipid metabolism with morphologic developments. High incorporation into phospholipids and cholesterol coincided in time with the extensive membrane synthesis required for cell attachment and process extension. Differentiation of these newly formed membranes, as assessed by the incorporation of myelin-characteristic glycolipids, galactocerebrosides, and sulfatides, occurred at a time when an intricate network of processes had already been established. The sequence of metabolic events observed in vitro parallels that observed at the onset of myelinogenesis in vivo. We postulate that mature oligodendrocytes can reenact those early events associated with myelinogenesis.

Acetates↗