摘要:

AbstractPrevious studies have demonstrated that inhibitors of glycoprotein processing glucosidases interfere with the development of oligodendrocyte properties in primary cultures of embryonic rat brain cells (Bhat, J Neurosci Res 20:158–164, 1988). The present study examines the effect of castanospermine, an inhibitor of the processing glucosidases, on the development and differentiation of isolated oligodendrocyte progenitor cells. Treatment of oligodendrocyte progenitors with castanospermine did not affect the developmental progression of the precursors to become committed oligodendrocytes as revealed by comparable increases in the percentages of cells positive for galactocerebroside (a surface marker for terminally differentiated oligodendrocytes) in control and drugtreated cultures. On the other hand, there was an impairment of the expression of differentiated properties of oligodendrocytes [i.e., sulfolipid synthesis, myelin basic protein (MBP)] and 2′3′‐cyclic nucleotide 3′‐phosphohydrolase in the drug‐treated cultures. Immunocytochemical analysis with anti‐MBP antibodies revealed a reduced number of MBP‐positive cells in inhibitor‐treated cultures. Furthermore, a majority of MBP‐positive cells in such cultures displayed immunoreactive MBP in their cell body and not the processes, unlike in control cultures where both cell body and the processes of oligodendrocytes stained intensely for MBP. The strong inhibitory effect of castanospermine on the expression of oligodendrocyte‐specific activities was contrasted with a relatively smaller effect of swainsonine, a mannosidase inhibitor on oligodendrocyte differentiation. Both castanospermine and swainsonine, however, effectively blocked the formation of complex‐type oligosaccharides, suggesting thereby a lack of correlation between the inhibition of the formation of complex‐type oligosaccharides and oligodendrocyte differentiation. It is suggested, therefore, that early trimming reactions involving the removal of glucose residues from the high mannose oligosaccharides in the endoplasmic reticulum may be essential for the cell surface localization and function of glycoproteins critically involved in surface interactions of oligodendrocytes with each other and/or with the substratum. Copyr

ISSN:0360-4012    

DOI:10.1002/jnr.490390102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractUsing a rapid (0.5 ml/min) flow rate superfusion system, the dopamine (DA) transporter mediated release of DA is further explored, and compared to the depolarization evoked release of DA in rat striatal synaptosomes preloaded with radioactive DA (3H‐DA). In this system external DA in the low μM range efficaciously releases the preloaded transmitter, the maximal response being reached at 3 μM DA. The external DA stimulated release is Ca2+‐independent, Cl−‐dependent, and blocked by both bupropion and nomifensine. The atypical antidepressant bupropion inhibits3H‐DA accumulation to rat striatal synaptosomes with a calculated IC50of 1.3 × 10−6M. Among DA uptake blockers some are known to act as DA releasing agents. Here we found that the DA uptake blocker nomifensine (30 μM) is unable to modify the baseline release of3H‐DA, whereas bupropion (10 μM) clearly elevates the baseline release of3H‐DA in a Ca2+‐independent and Cl−‐dependent manner. The non releasing agent nomifensine blocks the release of3H‐DA induced by bupropion. The Ca2+‐dependent, high K+depolarization evoked release of3H‐DA is not modified by nomifensine and does not depend on the external Cl−concentration. When the depolarizing medium contains DA the carrier mediated release of3H‐DA induced by the external DA is additive to the high K+induced response. A drastic drop in the external Cl−concentration induces3H‐DA release. This release of3H‐DA induced by low external Cl−levels is completely blocked by nomifensine, which only slightly diminished the release of3H‐DA induced by the absence of external Na+. On the basis of these results, it is concluded that: (1) Rapid perfusion flow rates eliminate DA reuptake. (2) DA uptake inhibitors either with or without DA releasing capabilities block the release of DA induced by μM levels of external DA. (3) By preventing translocation of the DA transporter mobile moiety, nomifensine may inhibit the release of DA induced by external DA or bupropion and by drastic drops in the external Cl−w concentration. (4) In the absence of nomifensine, the DA transporter works under both resting and depolarized conditions, but in contrast to the GABA transporter (Sitges et al.: Neurochem Res 18:1081–1087, 1993), the DA transporter does not contribute to the amount of the DA released by depolarization. (5) Reversal of the DA uptake carrier is favored by conditions increasing the internal DA levels. (6) Cl−rather than Na+is a major determinant in3H‐DA movements throu

ISSN:0360-4012    

DOI:10.1002/jnr.490390103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe distal stump of an injured peripheral nerve supports regenerating axons by offering a favourable growth substratum and several cell‐produced growth factors. Deprivation of cellular factors alone has been shown not to prevent fairly rapid axonal elongation after nerve injury if the growth substratum was preserved. The present study examined possible long‐term untoward effects of cell support deprivation during an early phase of nerve regeneration. Rat sciatic nerve was crushed and a 25 mm long distal nerve segment was made acellular by freezing‐thawing, while the integrity of the growth substratum for the regenerating axons was preserved. Toe‐spreading reflex and skin sensitivity to pinch in the foot were monitored to follow recovery of motor and sensory function, respectively. The number of myelinated axons was determined in the sciatic nerve proximally to the lesion site, and distally in the predominantly sensory sural nerve as well as in the mixed motor nerve to the soleus muscle. Except for a short delay in the onset of recovery, explainable by the reduced elongation rate of axons growing through the acellular nerve segment, we found no deleterious effect of cell support deprivation on sensory or motor function recovery after nerve crush. Most of regenerating sensory neurons did not critically depend on the distal stump cell support. However, a 15% and 25% loss of myelinated axons both proximally to the lesion and distally in the sensory sural nerve, respectively, indicated that a corresponding minor loss of injured sensory neurons occurred when they were deprived of such cell support even if provided with a favorable growth substratum for successful regeneration. In these animals, Dexamethasone application in a dose shown to reduce nerve growth factor (NGF) production in the proximal nerve segment did not detectably affect nerve regeneration. Under the same conditions of cell support deprivation, however, no axon loss could be demonstrated in the nerve to the soleus muscle, suggesting that the regenerating proprioceptive sensory neurons are less dependent on the distal stump cell support than the cutaneous ones. Copyright © 1994 Wiley

ISSN:0360-4012    

DOI:10.1002/jnr.490390104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe chemokines, macrophage inflammatory protein‐1 (MIP‐1) and its subunit MIP‐1β, induce an intense fever in the rat when they are injected directly into the anterior hypothalamic, pre‐optic area (AH/POA), a region containing thermosensitive neurons. The purpose of this study was to compare the central action on body temperature (Tb) of MIP‐1β with that of interleukin‐6 (IL‐6), which also has been implicated in the cerebral mechanism underlying the pathogenesis of fever. Following the stereotaxic implantation in the AH/POA of guide cannulae for repeated micro‐injections, radio transmitters which monitor Tbcontinuously were inserted intraperitoneally in each of 15 male Sprague‐Dawley rats. Each micro‐injection was made in a site in the AH/POA in a volume of 1.0 μl of pyrogen‐free artificial CSF, recombinant murine MIP‐1β, or recombinant human IL‐6. MIP‐1β in a dose of 25 pg evoked an intense fever characterized by a short latency, a mean maximum rise in Tbof 2.4 ± 0.21°C reached by 3.7 ± 0.42 hr, and a duration exceeding 6.5 hr. Injected into homologous sites in the AH/POA, IL‐6 induced a dose dependent fever of similar latency and a mean maximal increase in Tbof 1.2 ± 0.25°C, 1.8 ± 0.15°C, and 2.1 ± 0.22°C and duration of 6.2 ± 1.28 hr, 6.7 ± 0.49 hr, and 6.8 ± 0.65 hr when given in doses of 25, 50, and 100 ng, respectively. These results show that MIP‐1β and the highest dose of IL‐6 induce a fever of comparable intensity, but MIP‐1β exerts its action in a much lower concentration. Thus, the de novo synthesis and subsequent action of the MIP‐1 family of cytokines on neurons of the AH/POA in response to a pyrogen challenge apparently play a functional role in the pathogenesis of fever. Further, the endogenous activity of IL‐6 in the hypothalamus which is enhanced in response to a lipopolysaccharide also may reflect its essential part in the acute phase response

ISSN:0360-4012    

DOI:10.1002/jnr.490390105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractGlutamate neurotoxicity is correlated with an increase of cytosolic free Ca2+. In some cell systems, activation of Ca2+dependent endonucleases or formation of free radicals can damage DNA and activate the chromatin bound enzyme poly(ADP‐ribose) polymerase (pADPRP). We have investigated whether pADPRP may be involved in glutamate neurotoxicity in vitro. Cerebellar granule cells at 12 days in culture when treated with a toxic dose of glutamate (100 μM) showed a rapid and transient increase of poly ADP‐ribose immunoreactivity. Cellular immunostaining was heterogeneous and returned to control levels after washout of glutamate. In the same cell preparations glutamate elicited a marked increase in enzyme protein immunoreactivity which persisted at later times. Non‐toxic doses of glutamate did not affect immunostaining. In another set of experiments, pADPRP mRNA was increased 30 min after glutamate. In order to investigate the role of pADPRP in glutamate‐mediated neurotoxicity, structurally different inhibitors of pADPRP (3‐aminobenzamide, benzamide, 3‐aminophthalhydrazide) and their inactive analogues (benzoic acid and phthalimide) were tested in this model. Addition of the inhibitors to cultures 60 min before and during the 30 min of glutamate treatment prevented neuronal death by 60–100%, assessed 24 hr later. Glutamate‐induced Ca2+influx was not affected. Inactive analogues failed to afford neuroprotection. These data indicate that not only is pADPRP activated by the early, possibly Ca2+‐mediated mechanisms initiated by glutamate, but that it might also actively contribute to the subsequent neuronal death. © 1

ISSN:0360-4012    

DOI:10.1002/jnr.490390106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe ability of neonatal astrocytes to promote neurite outgrowth in vitro and in vivo diminishes as astrocytes mature. This property correlates with the developmental loss of the central nervous system's ability to regenerate after injury. Cell lines representative of immature and mature astrocytes would be useful for studies to determine differences between these two populations. Previous work on immortalization of bipotential neural/glial precursors and fully differentiated glial cells suggests that immortalization of astrocytes at timed intervals of culture may yield cell lines trapped in different maturation states. To test this, neonatal mouse cortical astrocytes were immortalized by retrovirus‐mediated transfer of the SV40 T antigen (Tag) gene at 2, 6 and 17 days of culture. The clonal cell lines express Tag and are contact‐inhibited. Three phenotypes that change as a function of astrocyte maturation were examined to determine the fidelity with which the cell lines represent immature and mature astrocytes. These were: (1) cell morphology, growth pattern and size, (2) level of glial fibrillary acidic protein (GFAP) expression, and (3) neurite outgrowth promotion. First, immature and mature lines resemble mortal type 1 astrocytes of corresponding ages with respect to morphology and growth pattern, and retain a quantitative difference in cell size (mature cells are larger). Second, the pattern of GFAP expression is preserved, with immature lines expressing lower levels than mature cell lines, but the overall GFAP levels are significantly lower in immortalized cell lines compared to mortal cells. Finally, promotion of neurite outgrowth from embryonic chick retinal ganglion cells on monolayers of the cell lines was examined. While all neurite outgrowth measures are significantly greater for the immortalized lines than for control 3T3 cells, they are attenuated relative to mortal astrocytes. The age‐related pattern of stronger outgrowth support on immature astrocytes is retained for neurite initiation, but not retained for mean neurite length. Thus, SV40 Tag‐immortalized astrocytes have a complex phenotype characterized by retention of age‐related differences in morphology, growth pattern and cell size, and by a marked attenuation of some astrocyte‐specific characteristics but retention of age‐related differences in the expression level of these same characteristics, and finally, loss of the ability to support neurite extension at levels characteristic of immature astrocytes. © 1994 W

ISSN:0360-4012    

DOI:10.1002/jnr.490390107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractChanges in synaptic density in various brain regions were assessed among different age groups of rats maintained in ordinary small cages, as determined by synaptophysin assay. The synaptophysin content in hippocampus decreases as early as in the adult stage. The most remarkable decrement occurs in occipital cortex. In other regions, synaptophysin contents decrease in senescence to 60–77% of the respective peak values during young and adult stages. The other rat group reared under enriched environment in a large cage until 30 months of age was examined for synaptic density, and was revealed to maintain the similar levels as in young, or even higher levels in frontal, temporal, entorhinal cortices and hippocampus. These results indicate that the synaptic density in cerebrum decreases in senescence and this decrease can be prevented by rearing under enriched environment. © 1994 Wiley‐Liss,

ISSN:0360-4012    

DOI:10.1002/jnr.490390108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractHomophilic interactions of the major integral membrane protein of peripheral nerve myelin, P0‐glycoprotein, are thought to mediate membrane adhesion and compaction. Molecular modeling of its extracellular domain (P0‐ED), based on its resemblance to an immunoglobulin variable domain and on X‐ray diffraction measuements of inter‐membrane spacings of myelin, has suggested which amino acid sidechains may be involved in the homophilic adhesion. Recently identified point‐mutations in the human P0 gene result in amino acid substitutions in P0 protein and correlate with demyelinating motor and sensory neuropathies. The molecular model explains how these changes result in disrupted P0‐P0 interactions; indicates how compensatory changes in amino acids, as occur in P0‐ED of other species, preserve normal homophilic interactions; and predicts what other residue substitutions might underlie additional cases of demyelinating neuropathies. Copyright © 1994 W

ISSN:0360-4012    

DOI:10.1002/jnr.490390109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe brain creatine kinase (CKB) gene is expressed in a variety of tissues with highest expression seen in the brain. We have previously shown in primary rat brain cell cultures that CKB mRNA levels are high in oligodendrocytes and astrocytes and low in neurons (Molloy et al.: J Neurochem 59:1925–1932, 1992). In this report we show that treatment of human U87 glioblastoma cells with forskolin and IBMX, to elevate intracellular cAMP, induces expression of CKB mRNA from the transiently transfected rat CKB gene by 14‐fold and also increases expression from the endogenous human CKB gene. This induction of CKB mRNA (i) is due to increased transcription; (ii) occurs rapidly (with maximal induction after 6 hr; iii) requires the activity of protein kinase A (PKA), but (iv) does not require de novo protein synthesis and, in fact, is superinduced in the presence of cycloheximide. Given the role of oligodendrocytes in the energy‐demanding process of myelination and of astrocytes in ion transport, these results have physiological significance, since they suggest that changes in cellular energy requirements in the brain during events, such as glial cell differentiation and increased neuronal activity, may in part be met by a cAMP‐mediated modulation of CKB gene expression. Of particular importance is the possible modulation of CKB gene expression during myelinogenesis, since oligodendrocyte differentiation has been shown previously to be stimulated by increases in cAMP. Copyright © 1994 Wiley

ISSN:0360-4012    

DOI:10.1002/jnr.490390110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe extension of cellular processes from the oligodendrocyte soma is an early and critical event in myelin formation. Previous reports from this laboratory have implicated a role for protein kinase C (PKC) as an important intracellular mediator of this critical step in myelinogenesis. In the current study, the regrowth of fibers by adult human oligodendrocytes was examined and was found to be significantly enhanced by the PKC stimulator, 4β‐phorbol‐12,13‐didecanoate (PDB); this was accompanied by a 400–500% increase in oligodendroglial PKC activity. In contrast to other cell types, the increased PKC activity in oligodendrocytes was not followed by subsequent down‐regulation of the enzyme. The role of PKC in oligodendroglial process formation was further demonstrated by the ability of inhibitors of PKC to block the basal‐ or PDB‐enhanced fiber outgrowth. As well, studies employing isoform‐specific agonists implicated PKCα as the major determinant of fiber outgrowth by oligodendrocytes. The potential significance of PKC in myelin formation was further underscored by the observation that the synthesis of myelin basic protein, a prerequisite component for myelinogenesis, was increased by 2‐fold in PDB‐treated oligodendrocytes. Collectively, these observations suggest that PKC, in particular the α isoform, constitutes an important mediator in the initiation of myelin formation.

ISSN:0360-4012    

DOI:10.1002/jnr.490390111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY