bluuebunny/crossref_metadata_2025 huggingface dataset:
Alkylcatechols regulate NGF gene expression in astroglial cells via both protein kinase C‐and cAMP‐independent mechanisms
Authors: Furukawa Y. , Furukawa S. , Omae F. , Awatsuji H. , Hayashi K.
Published: January 2005

It has been previously demonstrated that, in mouse astroglial cells and fibroblast cells in culture, alkylcatechols cause a rapid increase in the nerve growth factor (NGF) mRNA level followed by a marked increase in the amount of NGF released into the medium. To understand the mechanism of this alkylcatechol effect on NGF gene expression in astroglial cells, we examined the effects of protein kinases that influence intracellular signal transduction and of their inhibitors. The reagents to increase the intracellular content of cyclic AMP (cAMP) such as dibutyryl cyclic AMP (Bt2cAMP), forskolin, or cholera toxin did not mimic alkylcatechol induction of NGF gene expression. Phorbol ester, a direct activator of protein kinase C (PKC), caused an increase in the NGF synthesis/secretion. The stimulatory effect of homocatechol (4‐methylcatechol) on NGF synthesis was not completely inhibited by staurosporine, an inhibitor of PKC. The treatment of cells with homocatechol resulted in the translocation of PKC from cytosol to membrane‐associated fractions, although the levels of the subcellular location of PKC were not correlated with the level of the induction of NGF gene expression. The concomitant administration of homocatechol (10−4M) and PMA (10−8−10−6 M) evoked a drastic and prolonged increase in the NGF mRNA level, and also markedly increased the amounts of NGF secreted by the cells (approximately 150‐fold). This synergism was inhibited in part by staurosporine, but the level of increase in NGF mRNA and in NGF protein was rather greater than that of activation of PKC.

These results suggest that neither PKC‐dependent pathway nor cAMP‐dependent pathway is dominant in the stimulatory effect of alkylcatechol on NGF synthesis. © 1993 Wiley‐Liss, Inc.
Transient requirement for vimentin in neuritogenesis: Intracellular delivery of anti‐vimentin antibodies and antisense oligonucleotides inhibit neurite initiation but not elongation of existing neurites in neuroblastoma
Authors: Shea T. B. , Beermann M. L. , Fischer I.
Published: January 2005

Vimentin is initially expressed by nearly all neuronal precursors in vivo, and is gradually replaced by neurofilaments shortly after the immature neurons become postmitotic (Cochard and Paulin, 1984, J Neurosci 4:2080; Tapscott et al., 1981, Dev Biol 86:40). A transient increase in neuritic vimentin filaments occurs within the first day of dbcAMP‐mediated neurite induction in NB2a/d1 neuroblastoma, after which vimentin levels rapidly decline and neurofilaments increase (Shea, 1990, Brain Res 521:343). In the present study, we tested the possibility that vimentin filaments may function in neurite elaboration by inducing neuritogenesis under conditions where vimentin expression and assembly was inhibited. Intracellular delivery of anti‐vimentin antiserum into transiently permeabilized NB2a/d1 cells prevented the initial elaboration of neurites, but did not retract existing neurites. By contrast, intracellular delivery of antiserum directed against the low molecular weight neurofilament subunit or normal rabbit antiserum did not affect neurite outgrowth. Treatment with vimentin antisense oligonucleotides reversibly depleted vimentin synthesis and steady‐state levels, and prevented neurite initiation, but did not induce retraction of existing neurites. These findings point toward an hitherto undetected role for vimentin in the initiation of neurite outgrowth. © 1993 Wiley‐Liss, Inc.
Proliferation and differentiation properties of bipotent glial progenitor cell lines immortalized with the adenovirus E1A gene
Authors: Galiana E. , Bernard R. , Borde I. , Rouget P. , Evrard C.
Published: January 2005

Bipotent glial progenitors have been immortalized by the transfer of the adenovirus E1A gene into primary cultured cells from embryonic rat brain. The lines obtained are phenotypically untransformed, retain growth contact‐inhibition, and are able to differentiate, unless they are surtransfected with transforming oncogenes. Depending on the growth conditions, these immortalized cells express differentially either oligodendrocyte or astrocyte‐specific markers and genes. After being seeded in serum‐free medium, they display gangliosides recognized by A2B5 monoclonal antibody, and then they express sequentially O4 epitopes, galactocerebroside, and the myelin protein DM20. When grown in serum‐supplemented medium, the cells express at first A2B5 epitopes, and then transiently O4 and galactocerebroside; after reaching confluence, O4 and galactocerebroside become undetectable, whereas the cells begin to coexpress glial fibrillary acidic protein and glutamine synthetase. These results indicate that the cell lines can undergo a differentiation reminiscent both of O‐2A progenitors and of plastic process‐bearing glial subpopulations. The cells were also genetically marked by the stable introduction of the nlslacZreporter gene. Thus, the lines could be useful for studying direct interactions in vitro, or for post‐grafting investigations. They should also provide a model for studying the mechanisms involved in the commitment and in the control of proliferation and differentiation of this cell lineage. This suggestion is consistent with the data indicating a growth arrest‐dependent differential expression of a novel gene encoding a protein with a helix‐loop‐helix domain. © 1993 Wiley‐Liss, Inc.
Phosphorylation and disruption of intermediate filament proteins in oligodendrocyte precursor cultures treated with calyculin A
Authors: Almazan G. , Afar D. E. H. , Bell J. C.
Published: January 2005

Treatment of primary cultures of oligodendrocyte precursors with calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, caused the phosphorylation of two intermediate filament components, nestin and vimentin. Phosphoamino acid analysis demonstrated that phosphorylation took place mainly on serine and to a lesser extent on threonine residues. In addition, calyculin A treatment caused a shift in the distribution of the two proteins from the Triton‐X‐100 insolube fraction to the detergent soluble fraction as demonstrated by immunoblotting. This redistribution, which was evident within 15 min after treatment and was nearly completed by 90 min, was accompanied by a disruption of the intermediate filament network. Thus, both nestin and vimentin retracted from the cytoplasmic processes to form a large perikaryal ring as shown by immunocytochemical analysis. Both morphological and biochemical changes were reversed 2–5 hr after removal of calyculin A from the culture medium. © 1993 Wiley‐Liss, Inc.
Nitric oxide synthase in cultured endothelial cells of cerebrovascular origin: Cytochemistry
Authors: Morin A. M. , Stanboli A.
Published: January 2005

Cultured cells derived from the cerebromicrovasculature can be shown to contain the enzyme nitric oxide synthase (NOS) by NADPH diaphorase staining. Nos is located largely as a distinct crescent adjacent to the nuclear membrane. NOS in these cells appears to be associated with the microtubule and microfilament structures and the Golgi apparatus of the cell as opposed to being freely soluble in the cytoplasm or bound to the plasma membrane. Disruption of the integrity of these structures with colchicine or cytochalasin B causes a change in the subcellular localization of NOS as well as a change in the intensity of staining. © 1993 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Expression of mRNA for neurotrophic factors and their receptors in the rat dorsal root ganglion and sciatic nerve following nerve injury
Authors: Sebert M. E. , Shooter E. M.
Published: January 2005

The adult rat dorsal root ganglion (DRG) produces mRNA for the neurotrophic factors nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) and contains large populations of neurons responsive to these factors. We report that following a focal crush injury of the sciatic nerve, NGF mRNA expression increases threefold and BDNF mRNA twofold, in the ipsilateral L4 and L5 DRGs. The mRNAs encoding the cognate neurotrophin receptors, p75NGFR, trkA, and trkB were expressed in the DRG throughout the post‐injury time course, suggesting that DRG neurons remain responsive to both NGF and BDNF. p75NGFR mRNA levels became transiently depressed in the DRG during the first several days after the lesion but returned to normal within 1 week. trkB mRNA was expressed in the normal sciatic nerve and levels were not altered by nerve crush. RNase protection assays detected both full‐length and truncated trkB transcripts in the DRG, but only truncated trkB mRNA, lacking the tyrosine kinase domain, was detected in the sciatic nerve. Likewise, trkA transcripts were not detected by RNase protection in normal sciatic nerve or in a segment of nerve distal to the crush site. These results are consistent with a model in which regenerating sensory neurons are supported by neurotrophic factors synthesized within the DRG. © 1993 Wiley‐Liss, Inc.
Glutamate neurotoxicity in mesencephalic dopaminergic neurons in culture
Authors: Kikuchi S. , Kim S. U.
Published: January 2005

The neurotoxic effect of glutamate in cultured mouse mesencephalic dopaminergic neurons was investigated. Neuron‐rich cell cultures were prepared from 13–14‐day‐old fetal mouse ventral mesencephalic tissue. Cultures were exposed to glutamate for 10 min and evaluated for glutamate neurotoxicity (GNT) 18–24 hr later by tyrosine hydroxylase (TH) immunostaining, microtubule associated protein‐2 (MAP2)immunostaining, and radiolabeled dopamine uptake assay.

In glutamate‐exposed cultures, the number of TH‐positive neurons and the level of dopamine uptake were reduced to 40% (35–45%) and 50% (47–52%), respectively, of control cultures. The number of MAP2‐positive neurons was also reduced to 47%, indicating that the GNT was not restricted or selective to dopaminergic neurons.

It is concluded that GNT was mediated by the N‐methyl‐D‐aspartic acid (NMDA) receptor from the following observations: (1) GNT was completely blocked by MK‐801, an NMDA receptor antagonist; (2) NMDA itself was as toxic as glutamate; (3) 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (CNQX), an antagonist of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid/kainate (AMPA/KA) receptor, did not block GNT; (4) kainate did not show neurotoxicity at a low concentration; and 5) two modulators of the NMDA receptor, 7‐chlorokynurenic acid and magnesium, were effective in blocking GNT.

Protective effects of phorbol myristate acetate, a tumor promoter, and gangliosides (GM1 and GT1b on GNT were also demonstrated. Possible interactions between GNT and several protein kinase cascades were also investigated. Forskolin, an activator of adenyl cyclase and protein kinase A, showed some protective effect on GNT. But okadaic acid, an inhibitor of phosphatases, and genistein, a tyrosine kinase inhibitor, did not show any protective effect.

These results suggest that (1) glutamate is capable of causing neuronal death in the substantia nigra; (2) GNT on dopaminergic neurons is mainly mediated by the NMDA receptor under the conditions of our study; (3) protein kinase C translocation is a key mechanism of GNT; and (4) there is an interplay of a signal transduction system in the pathomechanism of GNT. © 1993 Wiley‐Liss, Inc.
NADPH‐Diaphorase histochemistry in the postnatal mouse cerebellum suggests specific developmental functions for nitric oxide
Authors: Brüning G.
Published: January 2005

NADPH‐diaphorase histochemistry has been applied for the localization of nitric oxide synthase during the postnatal development of the mouse cerebellum. Staining for NADPH‐diaphorase during the first week after birth was confined to some but not all laminae of the immature cerebellum: NADPH‐diaphorase activity was located in the molecular/Purkinje cell layer and in inner parts of the internal granular layer. The external granular layer and the developing white matter were essentially unstained. Expression was earliest and very strong in parallel fibers and in the internal granule layer of the ventral part of the pyramis and the dorsal part of the uvula. Staining in the Purkinje cell layer was observed throughout the cerebellum. The presence of formazan deposits within Purkinje cells was verified by colocalization with calbindin D‐28k immunoreactivity. The distribution of NADPH‐diaphorase activity changed into the adult pattern between 8 and 12 days of age: Within the molecular layer, basket cells and their processes became strongly stained. Reaction product within Purkinje cells gradually disappeared. Likewise, strongly stained parallel fibers were no longer detectable. These results suggest that nitric oxide is involved in different processes in cerebellar development. © 1993 Wiley‐Liss, Inc.
Characterization of a Schwann cell neurite‐promoting activity that directs motoneuron axon outgrowth
Authors: Bolin L. M. , Shooter E. M.
Published: January 2005

Schwann cells support and facilitate axonal growth during development and successful regeneration in the peripheral nerve. In the regenerating rat sciaticnerve, Schwann cells provide a trophic milieu for primary sensory, sympathetic, and motoneurons. We have characterized a neurotrophic activity produced by adult rat sciatic nerve Schwann cells and a spontaneously immortal Schwann cell clone (iSC). This activity elicits neurite outgrowth from chick embryo explants of both CNS and PNS. The iSC activity has been concentrated by cation‐exchange chromatography and compared to known neurotrophins in bioassay. Pooled bound fractions elicit neurite outgrowth from sympathetic, ciliary and motoneurons. In collagen matrix cocultures of iSC and E4 ventral horn(before motor axon extension to muscle targets), the iSC activity can direct the initial axonal extension from motoneurons. The data presented suggest that Schwann cell‐produced activity may mediate motoneuron axonal extension before contact with their peripheral source of neurotrophin. © 1994 Wiley‐Liss, Inc.
Guanosine‐5′‐(3‐O‐thio)triphosphate‐mediated stimulation of phosphoinositidase C in solubilized rat peripheral nerve myelin and its alteration in streptozotocin induced diabetes
Authors: Mathew J. , Eichberg J.
Published: January 2005

The regulation of phosphoinositidase C (PIC) activity by guanosine‐5′‐(3‐O‐thio)triphosphate (GTPγgMS) was characterized in a cholate‐solubilized peripheral myelin‐enriched fraction from rat sciatic nerve. The GTP analog maximally enhanced PIC‐catalyzed hydrolysis of exogenous phosphatidylinositol‐4,‐5‐bisphosphate (PIP2) in a dose‐dependent manner only within a narrow range of cholate concentrations. Maximal stimulation was attained at 0.6 μM GTPγS and could be completely prevented by 1 μM guanosine‐5′‐(2‐O‐thio)diphosphate. Neither adenylyl‐imidodiphosphate nor adenosine triphosphate (ATP) enhanced PIC activity. Carbamoylcholine (1 mM)added together with GTPγS increased the extent of PIP2 hydrolysis over that elicited by GTPγ increased the extent of PIP2 hydrolysis over that elicited by GTPγS alone and this stimulation was blocked by the muscarinic receptor antagonist, atropine (50 μM). In detergent solubilized myelin preparations from streptozotocin induced diabetic rats, a higher concentration of the guanine nucleotide analogn was required to achieve stimulation comparable to that obtained with corresponding preparations from normal animals. These results suggest that sciatic nerve myelin possesses muscarinic receptors coupled via a GTP‐binding protein to PIC and that this system can be reconstituted in detergent‐solubilized extracts. It is possible that the function of G Proteins in cell signaling is impaired in experimental diabetic neuropathy. © 1994 Wiley‐Liss, Inc.
Platelet‐derived growth factor receptor is expressed by cells in the early oligodendrocyte lineage
Authors: Ellison J. A. , de Vellis J.
Published: January 2005

We report the loaclization of PDGFRα mRNA (PDGFRα) in phenotypically defined cells during the first postanatal week of rat forebrain development. Using a method of combined immunocytochemistry and in situ hybridization we have demonstrated the cellular colocalization of PDGFRα mRNA with GD3 ganglioside or 04 sulfatide, phenotypic markers of oligodendrocytes, in the gray and white matter of the dorsal cerebral cortex at all ages studies. Population analysis of the PDGFRα+/G3+ and PDGFRα+/analysis of the PDGFRα+/GD3+and PDGFRα+/04+ cells revealed that three populations express PDGFRα: GD3+, GD3+/04+, and 04+, corresponding to two lineage stages, progenitor and preoligodendrocyte, in oligodendrocyte development. Immature oligodendrocytes, identifice by galactocerebroside immunoreactivity, did not express detectable levels of PDGFR α mRNA. Post‐mitotic neurons, identifice by immunoperodxidase localization of the 68 kD neurofilament, and astrocytes identified by S‐100 or GFAP immunoreactivity were also negative for PDGFRα mRNA occured in oligodendrocyte cell populations which are post‐migratory and proliferative, but which do not express myelin proteins characteristic of post‐mitotic oligodendrocytes. © 1994 Wiley‐Liss, Inc.
Satellite cells in slow and fast rat muscles differ in respect to acetylcholinesterase regulation mechanisms they convey to their descendant myofibers during regeneration
Authors: Dolenc I. , Črne‐Finderle N. , Eržen I. , Sketelj J.
Published: January 2005

The hypothesis of satellite cell diversity in slow and fast mammalian muscles was tested by examining acetylcholinesterase (AChE) regulation in muscles regenerating (1) under conditions of muscle disuse (tenotomy, leg immobilization) in which the pattern of neural stimulation is changed, and (2) after cross‐transplantation when the regenerating muscle develops under a foreign neural stimulation pattern. Soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were allowed to regenerate after ischemic‐toxic injury either in their own sites or had been cross‐transplanted to the site of the other muscle. Molecular forms of AChE in regenerating muscles were analyzed by velocity sedimentation in linear sucrose gradients. Neither tenotomy nor limb immobilization significantly affected the characteristic pattern of AChE molecular forms in regenerating SOL muscles, suggesting that the neural stimulation pattern is probably not decisive for its induction. During an early phase of regeneration, the general pattern of AChE molecular forms in the cross‐transplanted regenerating muscle was predominantly determined by the type of its muscle of origin, and much less by the innervating nerve which exerted only a modest modifying effect. However, alkali‐resistant myofibrillar ATPase activity on which the separation of muscle fibers into type I and type II is based, was determined predominantly by the motor nerve innervating the regenerating muscle. Mature regenerated EDL muscles (13 weeks after injury) which had been innervated by the SOL nerve became virtually indistinguishable from the SOL muscles in regard to their pattern of AChE molecular forms. However, AChE patterns of mature regenerated SOL muscles that had been innervated by the EDL nerve still displayed some features of the SOL pattern. In regard to AChE regulation, muscle satellite cells from slow or fast rat muscles convey to their descendant myotubes the information shifting their initial development in the direction of either slow or fast muscle, respectively. The satellite cells in fast or slow muscles are, therefore, intrinsically different. Intrinsic information is expressed mostly during an early phase of regeneration whereas later on the regulatory influence of the motor nerve more or less predominates. © 1994 Wiley‐Liss, Inc.
Differential expression patterns of mRNAs for members of the fibroblast growth factor receptor family, FGFR‐1–FGFR‐4, in rat brain
Authors: Yazaki N. , Hosoi Y. , Kawabata K. , Miyake A. , Minami M. , Satoh M. , Ohta M. , Kawasaki T. , Itoh N.
Published: January 2005

We have examined the region‐specific expression of mRNAs for four members of rat FGF receptor family, FGFR‐1, FGFR‐2, FGFR‐3, and FGFR‐4, in rat brain by in situ hybridization. The FGFR‐1, FGFR‐2, and FGFR‐3 mRNAs were expressed widely but differentially in the brain. However, the FGFR‐4 mRNA was not expressed in the brain. The FGFR‐1 mRNA was strongly expressed in several regions including the hippocampus, cerebellum, and pedunculoptine tegmental nucleus. The FGFR‐2 mRNA expression was high in the choroid plexus, and moderate in the fiber‐rich regions (the corpus callosum, external capsule, and internal capsule) and the olfactory bulb. The FGFR‐3 mRNA was expressed diffusely in the brain. We have also examined the cellular localization of these mRNAs in the brain. Although the FGFR‐1 mRNA was expressed preferentially in neurons, the FGFR‐2 and FGFR‐3 mRNAs were expressed preferentially in glial cells. The present findings that the FGFR‐1, FGFR‐2, and FGFR‐3 mRNAs were expressed widely but with region‐and cell‐specificity in the brain indicate that these receptors have different roles in the brain. © 1994 Wiley‐Liss, Inc.
Differential expression of two mRNA species indicates a dual function of peripheral myelin protein PMP22 in cell growth and myelination
Authors: Bosse F. , Zoidl G. , Wilms S. , Gillen C. P. , Kuhn H. G. , Müller H. W.
Published: January 2005

Two peripheral myelin protein PMP22 transcripts, CD25 and Sr13, have been identified by Northern blot and RNA‐polymerase chain reaction (PCR) methods in rat. The CD25 and SR13 mRNA species (each approximately 1.8 kb in size) differ significantly in their 5′‐untranslated region (5′‐UTR) sequences but encode the same protein. While CD25 mRNA is largely confined to the peripheral nervous system, the Sr13 transcript is more ubiquitously expressed in rat tissues. Both transcripts are differentially expressed during postnatal sciatic nerve development, while CD25 expression steadily increases from low levels in neonates up to a maximum at postnatal day 14, SR13 mRNA levels are elevated at birth but decrease throughout adulthood. CD25 and Sr13 transcripts are expressed at very low constant levels in developing and adult brain. In degenerating and regenerating segments of injured peripheral nerve changes in CD25 mRNA levels clarly resemble the expression pattern of other myelin genes, whereas expression of SR13 is inversely correlated with the time course of Schwann cell proliferation. In cultured rat meningeal fibroblast SR13 mRNA expression is strictly growth arrest‐specific and independent of forskolin. On the other hand, regulation of CD25 mRNA levels in these cells is more complex with respect to interfering effects of serum and forskolin. In cultured Schwann cells neither CD25 nor SR13 expression is growth arrest‐specific. However, both transcript levels are consistently enhanced by forskolin under all conditions of cell growth tested. Expression of CD25 (but not SR13) depends on high Schwann cell density. Our results substantiate the hypothesis that PMP22 serves two biological functions, one related to cell growth (SR13) and another to myelination (CD25). © 1994 Wiley‐Liss, Inc.
Changes of β‐amyloid precursor protein splice patterns in brain cell aggregate cultures
Authors: Gebicke‐Haerter P. J. , Appel K. , Honegger P. , Berger M.
Published: January 2005

The splice pattern of β‐amyloid precursor protein (β‐APP) has been studied in a variety of neuronal and glial cells and in brain cell aggregate cultures by the polymerase chain reaction (PCR). The brain‐typical pattern, in which β‐APP695 is the dominant form, has been found only in aggregate cultures but not in any of the other cell types including neuronal cell lines. Selective elimination of glial cells from aggregates resulted in increased quantities of β‐APP695, whereas removal of neurons led to a reduction of β‐APP695 and to an elevation of β‐APP751 and β‐APP770. This shift of splice pattern was not observed in cocultures of the neuronal cell line PC 12 with primary astrocytes combined in a variety of cellular ratios. Blood serum, which is an essential component of these cultures, tested on aggregates, did not reduce the amount of β‐APP695 or have any marked effects on splice patterns generally. From these results it is concluded that investigations on brain‐typical splicing of β‐APP require primary neurons. Neuronal cell lines may be no suitable model systems. Splicing events favoring production of β‐APP695 may mark an important, very early step of amyloid formation in the brain. © 1994 Wiley‐Liss, Inc.
Identification of Gal(β1–3)GalNAc bearing glycoproteins at the nodes of ranvier in peripheral nerve
Authors: Apostolski S. , Sadiq S. A. , Hays A. , Corbo M. , Suturkova‐Milosevic L. , Chaliff P. , Stefansson K. , Lebaron R. G. , Ruoslahti E. , Hays A. P. , Latov N.
Published: January 2005

A subset of human anti‐GM1 ganglioside antibodies cross‐reacts with Gal(β1–3)GalNAc bearing glycoproteins in peripheral nerve and spinal cord. The same oligosaccharide determinant is recognized by the lectin peanut agglutinin (PNA) which binds at the nodes of Ranvier in intact peripheral nerve. The Gal(β1–3)GalNAc bearing glycoproteins were isolated using PNA lectin affinity chromatography followed by separation on Western blot, and the proteins were subjected to partial amino acid sequence analysis. Two major PNA binding glycoproteins were identified in peripheral nerve and spinal cord; one had an approximate molecular weight of 120 kD and had sequence homology to the oligodendrocyte‐myelin glycoprotein (OMgp). The other migrated between 70 and 80 kD and had sequence homology to the hyaluronate binding domain of versican, which has been reported to share sequence homology with the 70 kD proteins hyaluronectin and the glial hyaluronic acid binding protein (GHAP). By immunocytochemistry, OMgp was localized to the paranodal region of myelin, and the protein homologous to the hyaluronate binding domain of versican was localized to the nodal gap in peripheral nerve. These PNA binding glycoproteins might be target antigens for autoantibodies in peripheral nerve. © 1994 Wiley‐Liss, Inc.
Type‐2 astrocytes have much greater susceptibility to heat stress than type‐1 astrocytes
Authors: Juurlink B. H. J.
Published: January 2005

The present investigations were undertaken to examine the susceptibility of type‐2 astrocytes to elevated temperature. Type‐2 astrocytes are much more easily injured by temperature elevation than type‐1 astrocytes. This may be related to cellular redox potential. Type‐1 astrocytes have a greater cytosolic NAD redox potential (i.e., higher NADH:NAD levels) than type‐2 astrocytes as evidenced by a 9‐fold higher ratio of lactate to pyruvate released into the medium by type‐1 astrocytes than type‐2 astrocytes. Heat stress causes the induction of hsp‐72 in both type‐2 and type‐1 astrocytes; however, hsp‐72 protein expression is retained for a longer period of time by the type‐2 astrocyte. A possible basis for the greater sensitivity of type‐2 astrocytes to stress may be a poorer ability to scavenge free radicals. This differential sensitivity of one neural cell type relative to another to elevated temperature may be of significance in understanding the effects of hyperthermia on the developing brain. © 1994 Wiley‐Liss, Inc.
Differential expression of immediate early genes in distinct layers of rat cerebral cortex after selective immunolesion of the forebrain cholinergic system
Authors: Roßner S. , Perez‐Polo J. R. , Wiley R. G. , Schliebs R. , Bigl V.
Published: January 2005

The aim of this study was to show whether reduction or loss of cortical cholinergic activity results in any particular change in the expression of the proto‐on‐cogenes c‐fos and/or c‐jun. To produce cortical cholinergic hypofunction, the monoclonal antibody, 1921gG, to the low‐affinity nerve growth factor receptor p75NGFR coupled to a cytotoxin, saporin, was used as an efficient and selective immunotoxin for cholinergic neurons in rat basal forebrain. Brain sections of adult rats that had received an intracerebro‐ventricular injection of 4 μg of the 1921gG‐saporin were subjected to in situ hybridization using oligonucleotides to detect c‐fos and c‐jun mRNA. Autoradiographs obtained were evaluated by quantitative image analysis. Seven days following injection of the immunotoxin there was a dramatic loss in acetylcholinesterase staining in frontal, parietal, piriform, temporal, and occipital cortices, hippocampus, and olfactory bulb, but not in the striatum and cerebellum. In situ hybridization revealed a considerable increase in the level of c‐fos mRNA in the lateral septum following the cholinergic lesion, whereas in the medial septum both c‐fos and c‐jun mRNA were elevated. Immunolesioning led to a distinct and specific increase in the level of c‐jun but not c‐fos mRNA in the parietal and occipital cortex that was restricted to cortical layer IV.

These data suggest that reduced cortical cholinergic activity differentially regulates expression of c‐fos/c‐jun genes in distinct cortical regions of the rat brain. © 1994 Wiley‐Liss, Inc.
Gonadal steroid control of preprocholecystokinin mRNA expression in the limbic‐hypothalamic circuit: Comparison of adult with neonatal steroid treatments
Authors: Micevych P. E. , Abelson L. , Fok H. , Ulibarri C. , Priest C. A.
Published: January 2005

The neuropeptide cholecystokinin (CCK) is involved in the regulation of female, but not male, reproductive behavior. In both sexes, estrogen regulates the expression of CCK in adulthood within the bed nucleus of the stria terminalis and medial amygdaloid nucleus. These areas are parts of an interconnected limbic system‐hypothalamic circuit, the development of which is influenced by estrogen during the early postnatal period. This is the same period during which central nervous system (CNS) expression of CCK is dramatically increased, suggesting that the male and female patterns of CCK expression may be the result of early postnatal exposure to estrogen. In the present experiment, the expression of preprocholecystokinin (pCCK) mRNA was determined by in situ hybridization with an isotopically labeled pCCK complementary RNA and emulsion autoradiography in animals whose neonatal and adult gonadal steroid levels had been manipulated. The number of pCCK‐expressing cells in animals that were gonadectomized as adults was determined by neonatal estrogen, but stimulation with steroids in adulthood induced a similar number of pCCK‐expressing cells in both sexes in the medial amygdala and bed nucleus of the stria terminalis. Neonatal treatment of females with estrogen or testosterone, followed by ovariectomy in adulthood, eliminated the sex difference in pCCK mRNA expression. Males treated neonatally with the aromatase inhibitor androstenedione (to block metabolism of testosterone to estrogen) and orchidectomized in adulthood had a level of pCCK mRNA expression that was similar to that of ovariectomized females. These data suggest that, during neonatal development, estrogen determines the constitutive expression of pCCK mRNA in the medial amygdala and bed nucleus of the stria terminalis, resulting in higher levels of pCCK mRNA expression in males than in females. However, exogenous gonadal steroids induce the same levels of pCCK mRNA expression in adult females, indicating that the levels of gonadal steroids and the patterns of their secretion are the predominant influences on the sexually dimorphic adult levels of pCCK mRNA expression. © 1994 Wiley‐Liss, Inc.
Activation of adult human derived microglia by myelin phagocytosis in vitro
Authors: Williams K. , Ulvestad E. , Waage A. , Antel J. P. , McLaurin J.
Published: January 2005

The present study was designed to determine the extent to which cultured glial cells phagocytose normal central nervous system (CNS) myelin and CNS myelin opsonized with serum or purified antibody against myelin basic protein (MBP). Glial cells studied were mixed cultures (consisting of astrocytes, microglia, and oligodendrocytes) and enriched microglia established from adult human brain specimens and enriched astrocytes from fetal human brain. A human monocytic cell line, THP‐1, was included as a control. Uptake of 125I‐labelled myelin was followed over a 24 hr time period. An assay of oxidative burst (30 min) and cytokine bioassays measuring IL‐1, IL‐6, and tumor necrosis factor (TNF) production (6‐‐48 hr) were used to investigate short‐ and longterm activation of phagocytosing cells. Maximum myelin uptake by phagocytosing glial cells occurred within 12‐‐24 hr following myelin incubation. Opsonization of myelin prior to the phagocytosis assay resulted in greater myelin uptake by mixed glial cell cultures, microglia, and THP‐1 cells over that of nontreated myelin. The magnitude of myelin phagocytosis by astrocytes was considerably lower than microglia and THP‐, and was not affected by myelin opsonization. Within 30 min of myelin phagocytosis, microglia and THP‐1 cells underwent oxidative burst; opsonization of myelin by purified anti‐MBP IgG and heat‐inactivated serum enhanced the microglial oxidative burst activity. Production of IL‐1, TNF, and most markedly IL‐6 by microglia was increased following 12‐‐24 hr of myelin ingestion. Our data demonstrate that myelin phagocytosis by adult human‐derived microglia occurs in vitro, is augmented when myelin is opsonized, and results in the activation of microglia as assessed by oxidative burst and cytokine production. © 1994 Wiley‐Liss, Inc.
A new oligodendrocyte specific plasma membrane surface protein identified by a monoclonal antibody produced in vitro
Authors: Birling M.‐C. , Nussbaum F. , Nussbaum J.‐L.
Published: January 2005

This paper describes a novel monoclonal antibody (C1G5F2) derived from mice splenocytes immunized in vitro with a wheat germ agglutinin glycoprotein fraction isolated from bovine central nervous system (CNS) myelin. Immunohistochemical reactions with C1G5F2 were investigated on rat brain sections during the active period of myelination. From day 10 to 13 postnatally, no stained structures were observed throughout the whole brain. The first immunolabeled myelin fibers were detected within the pons at day 14, and the white matter areas in the cerebrum started to be stained some days later. White matter areas of the cerebellum were clearly immunopositive after the third week. There was a strong positive signal on myelin fibers in the cerebrum at day 30. By contrast, no immunolabeled cell bodies of oligodendrocytes were observed throughout the brain. The other neural cell types were also not labeled. This C1G5F2 monoclonal antibody bound mainly to the extracytosolic membrane surface of the processes of live cultured oligodendrocytes derived from newborn rat brain but was unreactive with live or fixed astrocytes and neurons maintained in culture. No immunostaining was detected in the peripheral nervous system or in the spleen, liver, or pancreas. The C1G5F2 epitope containing antigen may therefore be considered as a CNS myelin/oligodendrocyte specific molecule. Sodium deoxycholate‐Tween 20 extracts of secondary oligodendrocyte cultures, biotinylated with biotin hydrazide, were used to attempt the purification of the antigen with C1G5F2 IgMs linked to antimouse IgM agarose. A main broad biotinylated protein band of 54–58 kDa molecular mass was noted. In a second approach, the antigen was immunopurified from cultured oligodendrocytes as an immune complex using biotinylated C1G5F2 IgMs. A distinct protein doublet of 53–56 kDa was also observed. It is postulated that this antigen may play an essential role in myelin formation and could be a possible target in diseases restricted to CNS myelin. © 1994 Wiley‐Liss, Inc.
Toxic axonal degeneration occurs independent of neurofilament accumulation
Authors: Sickles D. W. , Pearson J. K. , Beall A. , Testino A.
Published: January 2005

Alteration of neurofilament (NF) proteins is considered a critical component and a causative factor for a number of neuropathologies, especially certain neurotoxicities. Correlative observations have supported this hypothesis; the current study tests this relationship by exposure of neurotoxicants to crayfish, a species lacking NFs. Morphological and immunological tests verified the absence of NFs in crayfish peripheral nerve axons. Tail injections of acrylamide (ACR), 2,5‐hexanedione (2,5‐HD), or 3,4‐dimethyl‐2,5‐HD (3,4‐DMHD) produced ataxia and paralysis. Morphological expression of axonal degeneration in a spatial and temporal pattern of progression comparable to mammalian species possessing NFs was observed. With gamma‐diketones, time to onset was slower than observed in mammals but relative potency between neurotoxic analogues was maintained. Non‐neurotoxic analogues failed to produce any functional signs of neurotoxicity. These data are consistent with the conclusion that NF accumulations are not cause‐effect related to axonal degeneration in these models of neurotoxicity and raise questions as to the relationship between accumulation of NF proteins and axonal degeneration in other neuropathological conditions. Copyright © 1994 Wiley‐Liss, Inc.
Human IMR‐32 neuroblastoma cells as a model cell line in Alzheimer's disease research
Authors: Neill D. , Hughes D. , Edwardson J. A. , Rima B. K. , Allsop D.
Published: January 2005

The present study investigated expression and processing of amyloid precursor protein by neuronally differentiated IMR‐32 neuroblastoma cells. APP mRNA in these cells was found to consist of approximately 58% APP695, 38% APP751, and <4% APP770. APP‐immunoreactive bands detected in western blots of cellular protein extracts were only detected by anti‐APP antibodies to peptides with strong homology to APLP2, suggesting that these bands represent APP‐like proteins and not APP itself. This result suggests that previous studies claiming immunodetection of cellular forms of APP may have to be re‐evaluated. Four main species of C‐terminal truncated, secreted APP were detected in blots of protein extracts from medium conditioned by these cells. The immunoreactive profile of these bands suggested a cleavage site N‐terminal to the Lys16‐Leu17 bond of α‐secretase. This, together with differences in number and molecular mass of APP‐immunoreactive bands between secreted APP from IMR‐32 cells and that from the commonly used PC‐12 cells, suggests differences in APP processing between these two neuronally differentiated cell lines. In theory, IMR‐32 cells being of human neuronal origin may be a more appropriate cell line to study APP‐processing in relation to Alzheimer's disease than the rat phaeo‐chromocytoma PC‐12 cell line. Therefore, these detected differences warrant further investigation. Additionally IMR‐32 cells under certain tissue culture conditions can form intracellular fibrillary material that reacts with anti‐PHF specific antibodies. Neuronally differentiated IMR‐32 cells could therefore be used as a model system to investigate possible interactions between APP‐processing and PHF formation. © 1994 Wiley‐Liss, Inc.
Monoclonal antibody Alz‐50 reacts with bovine and human serum albumin
Authors: Davis P. K. , Johnson G. V. W.
Published: January 2005

Alz‐50, a monoclonal antibody originally prepared using Alzheimer brain homogenates, reacts with PHF‐tau and normal tau on immunoblots, and stains specific neuronal populations in sections from Alzheimer's disease brain. Although the Alz‐50 epitope has been mapped to amino acids 2–10 present in all human tau isoforms, minimal Alz‐50 immunoreactivity is present in tissue from control brain, suggesting Alz‐50 binding may be dependent on tau conformational differences. The absence of conclusive results concerning Alz‐50 binding presents the possibility of Alz‐50 immunoreactivity with proteins other than tau. The present study demonstrates Alz‐50 crossreactivity with denatured bovine serum albumin (BSA) and human serum albumin (HSA). Using LA‐N‐5 neuroblastoma cells, BSA from serum‐containing media was present in cell homogenates and was found to be Alz‐50‐reactive on immunoblots. In fact, Alz‐50 (0.1 μg/ml) recognized as little as 78 ng of BSA and 312 ng of HSA. Since Alz‐50 does not recognize native BSA, blocking of immunoblots with 3% BSA did not alter Alz‐50 reactivity with tau from LA‐N‐5 cells. On SDS‐polyacrylamide gels, HSA (∼ 69 kDa) migrates very closely to the pattern of A68 (PHF‐tau) from Alzheimer brain homogenates. Hence, the presence of BSA or other albumins in cell or brain homogenates may be an important concern when using the Alz‐50 antibody. © 1994 Wiley‐Liss, Inc.
Neurite outgrowth and GAP‐43 mRNA expression in cultured adult rat dorsal root ganglion neurons: Effects of NGF or prior peripheral axotomy
Authors: Hu‐Tsai M. , Winter J. , Emson P. C. , Woolf C. J.
Published: January 2005

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth‐associated protein GAP‐43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP‐43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP‐43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increase in GAP‐43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF‐independent, peripheral nerve injury‐provoked growth associated with substantial GAP‐43 upregulation, and an NGF‐dependent growth that may underlie branching or sprouting of NGF‐sensitive neurons, but which is not associated with increased levels of GAP‐43 mRNA. © 1994 Wiley‐Liss, Inc.
Monoclonal antibody PHF‐1 recognizes tau protein phosphorylated at serine residues 396 and 404
Authors: Otvos L. , Feiner L. , Lang E. , Szendrei G. I. , Goedert M. , Lee V. M‐Y.
Published: January 2005

The microtubule‐associated protein τ is hyperphosphorylated in the paired helical filaments (PHFs) of Alzheimer's disease. Immunological and direct chemical studies have identified Ser396 and Ser404 as two of the phosphorylated sites. Previously, we have demonstrated, using synthetic τ peptides containing phosphorylated Ser396, that this site is recognized by the monoclonal antibody PHF‐1. The present sudy extends this observation by showing that PHF‐1 recognizes τ peptides containing either individually phosphorylated Ser396 or Ser404, but that there is a >10‐fold increase in the sensitivity of detection of τ peptides by PHF‐1 when both serines are phosphorylated. The recognition of singly or doubly phosphorylated Ser396 and Ser404 in τ by PHF‐1 can also be demonstrated in Chinese hamster ovary cells transfected with full‐length wild‐type τ constructs or mutant constructs with Ala substituted for Ser396 or Ser404. We conclude that the PHF‐1 epitope contains both phosphorylated Ser396 and Ser404. © 1994 Wiley‐Liss, Inc.
Reassembly of the 66 kD neurofilament protein in vitro following isolation and purification from bovine spinal cord
Authors: Balin Brain J. , Miller M. E.
Published: January 2005

NF‐66, also known as α‐internexin, has been characterized as a 66 kD mammalian neurofilament (NF) protein whose expression in developing rat brain precedes that of the low molecular weight NF protein (NF‐L). NF‐66 is thought to assemble into 10 nm diameter intermediate filaments in vitro, although the precise nature of the assembly process remains obscure. Likewise, the ability of NF‐66 to polymerize with the low (NF‐L), middle (NF‐M), and high (NF‐H) MrNF proteins has not been defined. This investigation describes the reassembly of bovine NF‐66 regarding its formation into 10 nm diameter filaments as well as its potential for polymerization with other type IV intermediate filaments. NF‐66 and the NF triplet proteins were isolated from bovine spinal cord using established biochemical extraction and isolation procedures (Balin et al., Brain Res 556:181–195, 1991), and purified by a combination of high performance liquid chromatography (HPLC) (DEAE anion exchange and hydroxylapatite column chromatography) and gel elution strategies. In vitro reassembly experiments revealed that NF‐66 formed ˜ 10 nm diameter filaments of varying length; immunoelectron microscopy demonstrated labeling of these filaments by a monoclonal antibody to intermediate filament antigen (IFA), a polyclonal antibody against rat NF‐66 and by a monoclonal antibody generated against the core region of NF‐M but cross‐reactive with NF‐66. This report is the first investigation to look at the in vitro interaction between NF‐66 and other type IV intermediate filament proteins (NF‐H, ‐M, and ‐L) and establishes that NF‐66 forms heteropolymeric filaments with these other neurofilament proteins, as confirmed by double immunolabeling. These studies suggest that NF‐66 could provide a nucleation site for the polymerization of later‐expressed proteins during neuronal development. © 1995 Wiley‐Liss, Inc.
Macrophage recruitment in different models of nerve injury: Lysozyme as a marker for active phagocytosis
Authors: Venezie R. D. , Toews A. D. , Morell Pierre
Published: January 2005

Macrophages play critical roles in both degenerative and regenerative processes following peripheral nerve injury. These include phagocytosis of debris, stimulation of Schwann cell dedifferentiation and proliferation, and salvage of myelin lipids for reutilization during regeneration. To better define the role of macrophages, we studied models of primary demyelination (tellurium intoxication) and secondary demyelination (nerve crush and cut). Sections of paraformaldehyde‐fixed rat sciatic nerves at various stages of demyelination were stained with monoclonal antibody ED1, a standard macrophage marker, and a polyclonal antiserum specific for lysozyme (LYS). Near the peak of demyelination in all three models, LYS immunoreactivity colocalized with ED1 staining. Macrophages present in nerve after the period of maximal phagocytosis of myelin were much less immunoreactive for LYS. These results suggest LYS is a good marker for macrophages which are active in phagocytosis. Tellurium intoxication, which causes synchronous demyelination and subsequent remyelination of only about 25% of myelin internodes, recruited more macrophages (and induced more lysozyme expression) than either nerve crush or cut, which cause demyelination of all internodes distal to the injury site. This suggests that Schwann cells may recruit macrophages soon after metabolic insult and prior to actual demyelination. The final signal for macrophage recruitment is not directly related to the amount of damaged myelin. In the models listed above, steady state mRNA levels for apolipoprotein E (ApoE; possible mediator of cholesterol salvage), LYS, and Po (major structural protein of PNS myelin), were analyzed by Northern blot analysis. LYS mRNA levels peaked sharply in all models, with a temporal pattern consistent with the expected presence of activated, phagocytic macrophages. The temporal pattern for ApoE mRNA levels differed in the 3 models, but ApoE expression was consistent with its proposed role in salvage of cholesterol during remyelination. © 1995 Wiley‐Liss, Inc.