bluuebunny/crossref_metadata_2025 huggingface dataset:
Tau protein induces bundling of microtubules in vitro: Comparison of different tau isoforms and a tau protein fragment
Authors: Scott C. W. , Klika A. B. , Lo M. M. S. , Norris T. E. , Caputo C. B.
Published: January 2005

Expression of tau protein in non‐neuronal cells can result in a redistribution of the microtubule cytoskeleton into thick bundles of tau‐containing microtubules (Lewis et al.: Nature 342:498–505, 1989; Kanai et al.: J Cell Biol 109:1173–1184, 1989). We reconstituted microtubule bundles using purified tubulin and tau in order to study the assembly of these structures. Taxol‐stabilized tubulin polymers were incubated with various concentrations of recombinant human tau and examined by electron microscopy. With increasing concentrations of τ3 (tau isoform containing three microtubule binding domains) or τ4 (isoform containing four microtubule binding domains) the microtubules changed orientation from a random distribution to loosely and tightly packed parallel arrays and then to thick cables. In contrast, τ4L, the tau isoform containing four microtubule binding domains plus a 58‐amino acid insert near the N‐terminus, showed minimal bundling activity. τ4‐induced bundling could be inhibited by the addition of 0.5M NaCl or 0.4 mM estramustine phosphate, conditions which are known to inhibit tau binding to microtubules. A tau construct that contained only the microtubule binding domains plus 19 amino acids to the C‐terminus was fully capable of bundling microtubules. Phosphorylation of τ3 with cAMP‐dependent protein kinase had no effect on its ability to induce microtubule bundling. These results indicate that tau protein is directly capable of bundling microtubules in vitro, and suggests that different tau isoforms differ in their ability to bundle microtubule filaments. © 1992 Wiley‐Liss, Inc.
Antibody to beta‐amyloid precursor protein recognizes an intermediate filament‐associated protein in alzheimer's and control fibroblasts
Authors: Dooley N. P. , Gauthier S. , Durham H. D.
Published: January 2005

The expression of beta‐amyloid precursor protein (BAPP) and its mRNAs was studied in fibroblasts obtained from patients afflicted with Alzheimer's disease (AD) and age‐matched controls. Using reverse transcriptase‐polymerase chain reaction (RT‐PCR), transcripts corresponding to 770, 751, 714, and 695 amino acids were detected in both AD and control fibroblasts. Antibody 22C11 against BAPP (Boehringer Mannheim) labeled an intracellular protein, specifically localized to the intermediate filament network. In addition to bands of the predicted molecular weights for BAPP (120–135 kDa), Western blotting revealed a 57 kDa band which was not evident in samples of human brain. As cytoskeletal elements are vital in maintaining cellular architecture and various cell interactions, localization of BAPP or a related molecule to the cytoskeleton suggests a possible structural role for this protein within the cell. © 1992 Wiley‐Liss, Inc.
Nerve growth factor (NGF) receptors in a central nervous system glial cell line: Upregulation by NGF and brain‐derived neurotrophic factor
Authors: Spoerri P. E. , Romanello S. , Petrelli L. , Negro A. , Dal Toso R. , Leon A. , Skaper S. D.
Published: January 2005

The neurotrophic proteins nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) are related in their primary amino acid structures. In this study we investigated the extent to which the low‐affinity NGF receptor (LNGFR) in C6 glioma cells can discriminate between the neurotrophins NGF and BDNF. LNGFR‐immunoreactivity (IR) was studied in C6 cells treated for 16 hr with NGF (50 ng/ml) or BDNF (10 ng/ml), using immunogold labeling and electron microscopic morphometric analysis. The cells were exposed to the anti‐NGFR antibody 192‐IgG, followed by immunoglobulin conjugated with colloidal gold. Untreated C6 cells exhibited some surface gold label (positive LNGFR‐IR). Cells treated with NGF or BDNF displayed significantly increased LNGFR‐IR on all surfaces in terms of gold labeling, which was more pronounced in NGF‐treated cells. LNGFR‐IR was also localized in coated endocytotic vesicles, in smooth endoplasmic reticulum, and in secondary multivesicular lysosomes in neurotrophin‐treated and untreated cells. The increase in LNGFR protein was further substantiated by a correspondingly higher content of LNGFR mRNA detected after 15 hr of either NGF or BDNF treatment. These results suggest that the LNGFR in glial cells can be upregulated by the structurally related neurotrophins NGF and BDNF. © 1992 Wiley‐Liss, Inc.
Changes in chromatin proteins during optic nerve regeneration in the goldfish
Authors: Gossels J. M. , Lewis S. E. , Perrone‐Bizzozero N. I. , Benowitz L. I.
Published: January 2005

Regeneration of the goldfish optic nerve involves massive changes in the structure and pattern of macromolecular synthesis in the retinal ganglion cells. To explore the mechanisms that underlie these events, we investigated the changes in chromatin proteins during the course of regeneration. Three major retinal chromatin proteins, two with apparent molecular weights of 58 kDa (C1 and C2) and one at 51 kDa (C3), all having isoelectric points around 5.5, showed a fourfold increase in their synthesis and/or accumulation by 14 days of regeneration. Synthesis of C1 and C3 decreased by day 32, the time at which the axons have grown back to the optic tectum and have formed many of their synapses; synthesis of C2 remained high through day 32. All three proteins bound to DNA‐cellulose and required high salt concentrations (0.2–0.5 M KCl) to be eluted. C1 and C2 had similar proteolytic digestion patterns and reacted with monoclonal antibodies that recognize the goldfish intermediate filament proteins of the ON complex. The proteins identified here could be involved in structural alterations in the chromatin, or might serve as transcription factors to regulate gene expression during nerve regeneration. © 1992 Wiley‐Liss, Inc.
Neuronal migration in cerebellar microcultures is inhibited by antibodies against a neurite outgrowth domain of laminin
Authors: Liesi P. , Seppälä I. , Trenkner E.
Published: January 2005

The functional role of laminin in neuronal migration was investigated by using polyclonal antibodies or their divalent (Fab′)2 fragments to a neurite outgrowth promoting domain of the B2 chain of laminin in a cerebellar microculture system widely recognized as a model for neuronal migration. We show here that these antibodies or their (Fab′)2 fragments totally inhibit migration of the mouse cerebellar granule cells along the glial and other neuronal cell processes. Antibodies to native laminin or other control antibodies have no inhibitory effect. Immunocytochemical analysis of the cerebellar microcultures indicates that the functional role of these antibodies may relate to the fact that the punctate deposits of laminin and its neurite outgrowth promoting domain accumulate in between the migrating neurons and the glial cells. These data provide the first direct evidence for the functional role of laminin and its neurite outgrowth domain in neuronal migration in the mammals. They further suggest that a neuronal cell surface contact with the extracellular deposits of a neurite outgrowth domain of the B2 chain of laminin may mediate neuronal‐glial interactions. © 1992 Wiley‐Liss, Inc.
Developmental expression of major myelin protein genes in the CNS of X‐Linked hypomyelinating mutant rumpshaker
Authors: Mitchell L. S. , Gillespie S. C. , McAllister F. , Fanarraga M. L. , Kirkham D. , Kelly B. , Brophy P. J. , Griffiths I. R. , Montague P. , Kennedy P. G. E.
Published: January 2005

Rumpshaker (rsh) is an X‐linked mutation causing hypomyelination of the CNS of mice and has recently been identified as an allele of jimpy (jp). The mutation (known as jprsh) differs in several respects from other X‐linked myelin mutants, including jp, in that mice have normal longevity, oligodendrocyte numbers are not decreased, and cell death is not a feature. Myelin sheaths are deficient in immunostainable PLP protein. The present study examines the developmental expression of the major myelin protein genes and translatability of PLP and MBP mRNA. Differences between the spinal cord and brain of mutants are evident in that mRNA levels are more markedly decreased in the brain. Protein levels are severely reduced in both locations and to a proportionately greater extent than the mRNA, particularly in the spinal cord where PLP RNA and protein are approximately 80% and 10–20%, respectively, of age‐matched wild type mice. DM‐20 protein, the other major product of the PLP gene, is disproportionately expressed in rumpshaker as is a 10 kDa proteolipid. In vitro translation studies indicate a marked decrease in PLP translation products from mutant RNA. There is no deficiency in the numbers of PLP mRNA‐expressing oligodendrocytes although the abundance per cell is reduced. The data suggest that the phenotypic effects of the mutation may be associated with reduced translation of major myelin proteins, in particular PLP and its incorporation into compact myelin. However, the mutation is compatible with survival of oligodendrocytes and their differentiation to the stage of expressing PLP/DM‐20 mRNA. © Wiley‐Liss, Inc.
Recombinant human ciliary neurotrophic factor alters the threshold of hippocampal pyramidal neuron sensitivity to excitotoxin damage: Synergistic effects of monosialogangliosides
Authors: Skaper S. D. , Negro A. , Dal Toso R. , Facci L.
Published: January 2005

Ciliary neurotrophic factor (CNTF) is a multifunctional protein which not only promotes neuronal survival in vitro and in vivo but also controls cell division of neuronal precursors, transmitter differentiation, and glial cell differentiation. Recent studies have indicated that neurotrophic factors can alter hippocampal neuronal threshold to excitotoxin sensitivity. To examine such a role for CNTF, cultures of rat embryonic hippocampal neurons were maintained with recombinant human CNTF for different times, prior to exposure to a toxic dose of glutamate at 5 days in vitro for a further 24 hr. The cytotoxic action of 200 μM glutamate (approximately 40% of pyramidal neurons remaining after 24 hr) was reduced in a concentration‐dependent manner in cultures receiving a prior exposure to CNTF within the first 3 days of cell plating: 30 ng/ml CNTF permitted about 75% of the initial number of pyramidal neurons to survive. Presentation of CNTF less than 48 hr before glutamate challenge was ineffective at up to 100 ng/ml. When pyramidal neurons were cultured with a subthreshold concentration (2 ng/ml) of CNTF together with 10 μM of the monosialoganglioside GM1 (or its inner ester form) in the same paradigm, the resulting neuronal survival was similar to that seen with 30 ng/ml CNTF in the face of a glutamate challenge. Such low doses of either CNTF or ganglioside alone were ineffective. The ability of trophic factors to influence the threshold of neuronal sensitivity to excitatory amino acid injury suggests that these proteins could play an important role in the reparative capacity of acutely traumatized central neurons and in neurodegenerative diseases linked to an excitotoxic mechanism. Furthermore, monosialogangliosides may represent useful pharmacological agents for facilitating neurotrophic factor‐initiated nerve cell recovery processes. © Wiley‐Liss, Inc.
Stereoselective effects of AMOA on non‐NMDA receptors expressed in Xenopus Oocytes
Authors: Wahl P. , Nielsen B. , Krogsgaard‐Larsen P. , Hansen J. J. , Schousboe A. , Miledi R.
Published: January 2005

Pharmacological characterization of the action of the novel non‐N‐methyl‐D‐aspartate non‐NMDA) antagonist AMOA (2‐amino‐3‐[3‐ (carboxymethoxy)‐5‐methylisoxazol‐4‐yl]propionate on glutamate receptors was investigated in Xenopus ooccytes injected with mouse brain mRNA. AMOA (150 μM) produced a nearly parallel shift to the right of the dose‐response curve for kainate‐induced currents. AMOA was found to have two different effects on AMPA receptors: (1) currents elicited by low concentrations of AMPA (6 μM) were inhibited by AMOA with an IC50 value of 160 ± 19 μM and (2) currents elicited by high concentrations of AMPA (100 μM) were potentiated with an IC50 value of 88 ± 22 μM. The maximal potentiating effect of AMOA on AMPA currents was around 170%. Furthermore, the two opposing effects of AMOA on AMPA responses are specific for the L‐configuration of AMOA. This unusual antagonistic/agonistic property of AMOA may explain its unusual properties with regard to antagonism of non‐NMDA receptor‐mediated events previously described.
Opposing influences of protein kinase activities on neurite outgrowth in human neuroblastoma cells: Initiation by kinase A and restriction by kinase C
Authors: Shea T. B. , Beermann M. L. , Leli U. , Nixon R. A.
Published: January 2005

The respective roles of cAMP‐dependent protein kinase (protein kinase A [PKA]) and protein kinase C (PKC) in the early stages of neurite outgrowth were examined in SH‐SY‐5Y human neuroblastoma cells. Forskolin or dbcAMP, agents that increase intracellular cAMP levels, and intracellular delivery of PKA catalytic subunit induced neurite outgrowth. The PKA inhibitor, N‐(2‐guanidinoethyl)‐5‐isoquinolinesulfonamide (HA 1004), prevented the increases, and decreased further the percentage of cells possessing short, filopodia‐like neurites in the absence of inducers. In contrast to effects on PKA activation, PKC activation by 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) reduced the percentage of filopodia‐like neurites elaborated by otherwise untreated cells, and prevented neurite outgrowth induced by PKA activators. PKC inhibitors 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine dihydrochloride (H7), staurosporine, and sphingosine induced neurite outgrowth.

Neurites induced by PKA activation contained higher levels of tubulin immunoreactivity than those induced by PKC inhibition. Furthermore, PKA‐induced neurites rapidly retracted in the presence of colchicine, while those elaborated following PKC inhibition were more resistant. These data suggest that neurites elaborated in response to PKA activation are dependent upon microtubule polymerization, and that neurite induction following PKC inhibition is mediated by a different mechanism.

PKA activators and PKC inhibitors exerted additive effects on neurite outgrowth, suggesting that the distinct pathways regulated by these two kinases function cooperatively during neuritogenesis. © Wiley‐Liss, Inc.
Anomalous phosphorylated neurofilament aggregations in central and peripheral axons of hens treated with tri‐Ortho‐cresyl phosphate (TOCP)
Authors: Jensen K. F. , Lapadula D. M. , Knoth Anderson J. , Haykal‐Coates N. , Abou‐Donia M. B.
Published: January 2005

Previous biochemical studies demonstrated a dramatic increase in phosphorylation of cytoskeletal proteins that occurs early in organophosphorus ester‐induced delayed neurotoxicity (OPIDN). In this report we present immunohistochemical evidence that there is anomalous aggregation of phosphorylated neurofilaments within central and peripheral axons following organophosphate exposure. The morphology, location, and time of appearance of these aggregations are consistent with the hypothesis that the aberrant phosphorylation of cytoskeletal elements is an antecedent to the focal axonal swelling and degeneration characteristic of OPIDN. © Wiley‐Liss, Inc.
Autoradiographic analysis of second messenger and neurotransmitter receptor bindings in the strionigral system of the postischemic rat brain
Authors: Nagasawa H. , Araki T. , Kogure K.
Published: January 2005

We studied the postischemic alterations of second messenger and receptor systems focusing on the striobigral pathway in order to clarify the mechanism of the delayed neuronal changes in remote areas of the rat brain after transient focal ischemia. Chronological changes of [3H]forskolin and [3H]SCH 23390 binding sites and 45Ca accumulation were determined by using autoradiographic methods after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by differnt periods of recirculation. After the ischemic insult, 45Ca accumulation extended to the lateral segment of the caudate putamen (CPu‐L) and to the cerebral cortex, both supplied by the occluded MCA. After the ischemia, [3H]forskolin binding sites were found to be markedly decreased in the early stage in the CPu‐L, the ischemic focus in this model, but reduction of the dopamine D‐1 receptor sites was first detected there 1 day after the ischemia. On the contrary, in the exo‐focal remote areas, there was no alteration of either [3H]forskolin of D‐1 receptor binding sites on day 1. However, 3 days after the ischemia, marked reduction of both these binding sites was first observed in the ipsilateral substantia nigra, which had not been directly affected by the original ischemic insult. These postischemic delayed phenomena observed in the substantia nigra developed concurrently with abnormal 45Ca accumulation. These results suggest that strionigral terminal degeneration in the substantia nigra is caused by precedent ischemic damage of the ipsilateral caudate putamen and that intracellular signal transduction including both second messenger and receptor systems may be involved prior to the neuronal damage in the exo‐focal postischemic brain areas. © 1992 Wiley‐Liss, Inc.
Ethanol administration during early embryogenesis affects neuronal phenotypes at a time when neuroblasts are pluripotential
Authors: Kentroti S. , Vernadakis A.
Published: January 2005

Our previous studies have reported that ethanol administration during a critical period of development profoundly affects the expression of neuronal phenotypes in whole brains of chick embryos. The present study examines (a) the long‐lasting effects on anatomically discrete regions of the developing chick CNS. Ethanol (10 mg/50 μl) was administered to embryos via the air sac from E1 to E3. Embryos were sacrificed on days 4, 8, 10, or 15 of embryonic development (E4, E8, E10, E15) and assayed in specific regions of the CNS for glutamate decarboxylase (GAD) or choline acetyltransferase (ChAT) as markers for GABAergic or cholinergic neurons, respectively. The magnitude of the developmental profile for ChAT was highest in spinal cord (SC), with similar profiles observed for cerebral hemispheres (CH) and optic lobes (OL). In contrast, the developmental profile for GAD was highest in OL and lowest in SC. Thus, neuronal phenotypes inhabit specific CNS areas from early primordial stages of development. Furthermore, cholinergic neuronal populations in discrete CNS areas reached mature levels by E10, whereas GABAergic populations continued to increase throughout the experimental period. We suggest that GABAergic precursor neuoblasts may differentiate at a later embryonic age and that specific regional factors may play a role in neuronal distribution and the rate of maturation. As reported previously, primordial CNS areas exposed to ethanol (E1‐E3) exhibited a differential sensitivity. Cholinergic neuronal expression in CH remained retarded throughout the experimental period examined, whereas the early decline observed at E4 in SC cholinergic expression was reversed by E15. In contrast, the enhanced GABAergic expression exhibited at E4 continued up to E15. This differential neuronal sensitivity to ethanol is interpreted to indicate that (a) ethanol may shift neuronal fates during early neuronal commitment and (b) regional factors may play an important role for neuronal recovery following ethanol insult. © 1992 Wiley‐Liss, Inc.
Molecular analysis of the nerve growth factor inducible ornithine decarboxylase gene in PC12 cells
Authors: Muller S. R. , Huff S. Y. , Goode B. L. , Marschall L. , Chang J. , Feinstein Stuart C.
Published: January 2005

In an effort to understand molecular mechanisms by which nerve growth factor (NGF) regulates gene expression, we have isolated a full‐length rat cDNA clone encoding ornithine decarboxylase (ODC) and utilized this probe to identify and examine the transcriptionally active, NGF inducible ODC gene in rat PC12 cells. This same gene is also responsive to epidermal growth factor, basic fibroblast growth factor, and dibutyryl cAMP. Primer extension analysis demonstrates that both basal and NGF induced transcription of the ODC gene utilize the same major transcriptional start site, demonstrating that NGF acts to increase transcriptional activity at the basal start site as opposed to unmasking an alternative, stronger start site. Functional promoter analysis reveals the presence of a constitutive core promoter residing between positions −201 and +390, relative to the start site of transcription. Additional analyses reveal that sequences in the region −7800 to +2257 are insufficient to mediate NGF induced transcriptional activation, demonstrating that at least some of the regulatory sequences necessary for NGF mediated transcriptional induction of the ODC gene must reside at relatively enormous distances from the transcriptional start site. Such a long distance transcriptional regulatory mechanism is unique when compared with other NGF responsive genes that have been similarly analyzed. © 1993 Wiley‐Liss, Inc.
Establishment of an astrocyte progenitor cell line: Induction of glial fibrillary acidic protein and fibronectin by transforming growth factor‐β1
Authors: Yoshida T. , Takeuchi M.
Published: January 2005

An immortalized clonal cell line (AP‐16) has been established from glial cultures obtained from neonatal mouse cerebra by multipassages under serum‐free conditions. Immunofluorescent experiments showed that AP‐16 cells expressed a marker for glial progenitors (A2B5) and did not express markers for oligodendrocytes (galactocerebroside) or mature astrocytes (glial fibrillary acidic protein: GFAP). Treatment with transforming growth factor‐β1 (TGF‐β1) or fetal calf serum (FCS) for 2 days induced AP‐16 cells to differentiate into A2B5‐negative, GFAP‐positive, phenotypically mature astrocytes. AP‐16 cells depended on epidermal growth factor for survival, and their growth was inhibited by FCS. These results indicate that AP‐16 cells retained the properties of astrocyte progenitors. An enzyme‐linked immunosorbent assay showed that AP‐16 cells synthesized fibronectin and laminin, and that the expression of fibronectin was increased by TGF‐β1. AP‐16 cells should be useful for studying the roles of TGF‐β1 in the differentiation of astrocyte progenitors. © 1993 Wiley‐Liss, Inc.
The differentiation and survival of murine neurons in vitro is promoted by soluble factors produced by an astrocytic cell line
Authors: Kilpatrick T. J. , Talman P. S. , Bartlett P. F.
Published: January 2005

The influence of accessory cells on the generation of neurons and neuronal survival has been studied in vitro using an immortalised, cloned cell line, Ast‐1, which has many of the functional and phenotypic characteristics of cells of the astrocytic lineage. It was found that monolayers of Ast‐1 cells were equivalent to monolayers of primary astrocytes in their ability to promote the generation of neurofilament positive neurons from neuroepithelial cells obtained from embryonic day 10 (E10) mice; and both were superior to NIH 3T3 cells. Ast‐1 cell monolayers were also found to provide a suitable substrate for the prolonged survival (at least 3 days in vitro) of neurofilament positive neurons obtained from E17 mice, whereas neurons plated onto NIH 3T3 cells were all dead after 2 days. Medium conditioned by Ast‐1 cells displayed similar biological activities to that of the monolayers: It increased the number of neurons generated from the E10 neuroepithelial cells, whether they were plated directly onto glass coverslips or onto monolayers of NIH 3T3 cells; and it increased the survival of E17 neurons plated directly onto glass coverslips. In addition, the Ast‐1 conditioned medium was shown to promote the survival of the neuroepithelial cells. These results confirm that one of the mechanisms by which astrocytes or their precursors may regulate neuronal development is by secreting soluble growth factors, as has been previously documented in the case of fibroblast growth factor (FGF) (Hatten et al., 1988; Drago et al., 1991a). However, it appears in this system that FGF is not responsible for the demonstrated biological activities, and the Ast‐1 action appears to be mediated by putatively novel factor(s). © 1993 Wiley‐Liss, Inc.
Nitric oxide induces calcium‐dependent release of [3H]dopamine release from striatal slices
Authors: Lonart G. , Cassels K. L. , Johnson K. M.
Published: January 2005

Hydroxylamine (0.01–30 mM), a nitric oxide (NO) generator, produced a concentration‐dependent release of [3H]dopamine ([3H]DA) from rat striatal slices. Hemoglobin (10 μM), a NO scavenger, reduced basal [3H]DA release and blocked hydroxylamine (100 μM)‐stimulated [3H]DA efflux. Tetrodotoxin (0.5 μM) had no significant effect. Sodium cyanide was used as a model compound to test the possibility that NO acted through blockade of mitochondrial electron transport. Calcium‐free experimental buffer (1 mM EGTA) reduced basal release and the hydroxylamine response, while sodium cyanide‐induced release did not change under these experimental conditions. Cadmium (200 μM), a non‐selective inhibitor of voltage‐dependent calcium channels, reduced the hydroxylamine response by 69%. Methylene blue (10 μM), an inhibitor of guanylate cyclase, produced a 3‐fold increase in the basal release but had no significant effect on the hydroxylamine response. These data suggest that NO induces calcium‐dependent [3H]DA release from the striatum via a mechanism which is independent of blockade of electron transport or activation of guanylate cyclase. © 1993 Wiley‐Liss, Inc.
Axon growth is enhanced by NCAM lacking the VASE exon when expressed in either the growth substrate or the growing axon
Authors: Liu L. , Haines S. , Shew R. , Akeson R. A.
Published: January 2005

The neural cell adhesion molecule NCAM exists as several related peptides formed by alternative splicing of the single NCAM gene. Here the ability of NCAM containing and lacking the alternatively spliced VASE exon to act as a permissive growth substrate was tested by examining retinal axon outgrowth on normal L cell fibroblasts and L cells expressing stably transfected 140 kD NCAM ± VASE. L cells expressing either NCAM form were a more permissive substrate than control L cells. At higher substrate cell densities, greater axon outgrowth occurred on substrate cells expressing NCAM − VASE than on those expressing NCAM + VASE. Similar experiments tested retinal axon growth on neuronal substrates by utilizing clonal B35 cells, C3 cells that are NCAM lacking variants of B35, and C3 cells into which 140 kD NCAM ± VASE has been restored by transfection. Axon growth on C3 cells transfected with NCAM − VASE was greater than that on all other substrates including cells transfected with NCAM + VASE. In these experiments C3 cells and transfected C3 expressing NCAM + VASE cell promoted similar outgrowth. The influence on neurite growth of the NCAM isoform of the neurite itself was tested by examining neurite formation using combinations of C3 cells and C3 NCAM transfectants both in the growth monolayer and as responding cells. C3 cells were able to extend neurites, indicating NCAM is not required for neurite growth. However, C3 derivatives transfected with NCAM ± VASE had greater neurite outgrowth. The most extensive neurite growth was found when NCAM − VASE was expressed by both substrate cells and the responding neurite growing cells. Thus NCAM enhances axon or neurite outgrowth when present either in the growth substrate or on the growing axon. NCAM − VASE has a significantly greater growth promoting capability than NCAM + VASE. The expression of NCAM + VASE by more mature neural cells could thus be a © Wiley‐Liss, Inc. significant factor in the reduced axonation capabilities of mature neurons. © 1993 Wiley‐Liss, Inc.
Retinoic acid induces cholinergic differentiation of cultured newborn rat sympathetic neurons
Authors: Berrard S. , Biguet N. Faucon , Houhou L. , Lamouroux A. , Mallet J.
Published: January 2005

Many studies provide evidence that retinoic acid (RA), an endogenous derivative of vitamin A, plays a role in the development of the nervous system. We now report that RA controls the neurotransmitter phenotype of post‐mitotic rat sympathetic neurons in cell culture. RA added to the culture medium increased the specific activity of choline acetyltransferase (ChAT) and the level of acetylcholine (ACh). Concomitantly, RA reduced the specific activities of two catecholamine synthetic enzymes, tyrosine hydroxylase (TH) and dopamine β‐hydroxylase (DBH) and the level of norepinephrine (NE). After a 2 week treatment with 5 μM RA, ChAT was increased by 5–10 fold, whereas TH and DBH were decreased by 10–15 fold and 2–3 fold, respectively, as compared to sympathetic neurons grown in the absence of RA. The modulation of the activity of the three enzymes was dose‐dependent and followed a similar time course. The decrease of TH expression was demonstrated to be due to a decreased number of TH molecules. © 1993 Wiley‐Liss, Inc.
Vesicular transport of myelin proteolipid and cerebroside sulfates to the myelin membrane
Authors: Brown M. C. , Moreno M. Besio , Bongarzone E. R. , Cohen P. D. , Soto E. F. , Pasquini J. M.
Published: January 2005

The possibility that cerebroside sulfates and myelin proteolipid (PLP) could be simultaneously located in transport vesicles destined to be assembled in myelin was investigated in the brain of 20 day old rats. The brain was homogenized and fractionated according to Burkart et al. (J Biol Chem 257:3151–3156, 1982) to obtain a microsomal fraction that was further subfractionated in a linear sucrose density gradient following the procedure of Siegrist et al. (J Neurochem 33:497–504, 1979) to obtain a vesicular fraction which has been shown to transport cerebroside sulfates (Burkart et al., as above). This fraction was associated with acid hydrolase activity and had a lipid composition different from that of myelin and microsomal fractions. Studied by slab gel electrophoresis, dot blot, and Western blot analysis, using a highly specific anti‐PLP antibody, it was found to contain myelin PLP. In view of previous findings of several laboratories including our own, the presence of myelin proteolipid in a vesicular fraction which is related to the transport of cerebroside sulfates gives further support to the hypothesis that the delivery of both constituents to the myelin membrane could be associated. © 1993 Wiley‐Liss, Inc.
Changes of amino acid and monoamine levels after neonatal 6‐hydroxydopamine denervation in rat basal ganglia, substantia nigra, and raphe nuclei
Authors: Molina‐Holgado E. , Dewar K. M. , Grondin L , van Gelder N. M. , Reader T. A.
Published: January 2005

The effects of a neonatal dopaminergic deafferentation with the neurotoxin 6‐hydroxydopamine (6‐OHDA) on endogenous tissue levels of catecholamines, indoleamines, and amino acids were investigated in discrete rat brain regions. After producing the lesion at postnatal day 3 by intraventricular injections of 6‐OHDA, with a desipramine pretreatment to protect noradrenaline neurons, the animals were kept for 3 months. Their brains were dissected to obtain samples of neostriatum, Globus pallidus, Substantia nigra, and Raphe nuclei, which were then analyzed by high‐performance liquid chromatography, coupled either to electrochemical detection for aromatic monoamines, or to post‐column ninhydrin derivatization with spectrophotometry for amino acids. The neonatal 6‐OHDA treatment depleted dopamine (DA) levels in neostriatum, Globus pallidus, and Substantia nigra, but in Raphe nuclei DA was increased. The main metabolites of DA were also decreased in neostriatum, Globus pallidus, and Substantia nigra but remained unchanged in Raphe nuclei. Serotonin (5‐HT) and its metabolite 5‐hydroxyindole‐3‐acetic acid increased in neostriatum and Raphe nuclei; in Substantia nigra there was a slight increase in 5‐HT only. The 6‐OHDA lesion caused heterogeneous alterations in amino acid contents, which varied according to the region. In the neostriatum there were increases of γ‐aminobutyric acid (GABA), aspartic acid, and glycine. In the Globus pallidus taurine, GABA, glutamic acid, glutamine, aspartic acid, serine, and alanine were elevated. In the Substantia nigra only increases in taurine, GABA, glutamic acid, and glutamine could be documented. This study shows important changes in amino acid levels and in some of their ratios, occurring in different anatomical subdivisions of the basal ganglia and related brainstem nuclei following a neonatal treatment with 6‐OHDA. The results thus demonstrate major biochemical modifications in amino acids in the aftermath of a DA denervation and/or a 5‐HT hyperinnervation during an early developmental period. © 1993 Wiley‐Liss, Inc.
mRNAs Coding for neurotransmitter receptors in rabbit and rat visual areas
Authors: Matute C. , Nguyen Q.‐T. , Miledi R.
Published: January 2005

Levels of mRNAs encoding neurotransmitter receptors in the visual cortex, lateral geniculate nucleus, and superior colliculus of the rabbit and rat, and properties of the receptors expressed, were studied using Xenopus laevis oocytes. mRNA extracted from these areas was injected into the oocytes, which then acquired functional receptors. Electrical recordings of neurotransmitter‐induced membrane currents reflect the relative amounts of mRNAs encoding the corresponding receptors. Receptors to gamma aminobutyric acid (GABA), kainate, glutamate, and serotonin exhibited uniformly high levels of expression, whereas expression of receptors to glycine and N‐methyl‐D‐aspartate was uniformly low. In contrast, the expression of receptors to acetylcholine and substance P was highly non‐uniform. Expression of acetylcholine receptors was high in oocytes injected with mRNA from the visual cortex, low for the lateral geniculate nucleus, and very low or absent for the superior colliculus. Conversely, the currents elicited by substance P were large in oocytes injected with superior colliculus mRNA, but were small or absent in oocytes injected with mRNAs from the other regions. Immunohistochemical analysis, at the light and electron microscopic levels, was used to localize choline acetyltransferase, the acetylcholine‐synthesizing enzyme, and substance P‐containing synaptic boutons in the three visual areas. Their presence closely paralleled the potency of mRNAs coding for acetylcholine and substance P receptors. The ability of rat mRNA, from each visual area, to induce neurotransmitter receptors was similar to that observed in the corresponding rabbit mRNAs. In addition to the marked differential distribution of mRNA encoding neurotransmitter receptors in the visual system, our findings reveal the probable existence of as yet uncharacterized receptors, whose new molecular forms may be revealed by further study. Our results also provide the basic information required for subsequent studies on the effect of monocular deprivation on the expression of neurotransmitter receptors in the visual system. © 1993 Wiley‐Liss, Inc.
Nerve growth factor and p75NGFR factor receptor mRNA change in rodent CNS following stress activation of the hypothalamo‐pituitary‐adrenocortical axis
Authors: Foreman P. J. , Taglialatela G. , Angelucci L. , Turner C. P. , Perez‐Polo J. R.
Published: January 2005

The synthesis of nerve growth factor (NGF) by the hippocampus raises the possibility that NGF may play a role in the regulation of the hypothalamic‐pituitary‐adrenal axis (HPAA). Subchronic cold stress has been shown to activate the HPAA in a mild noninvasive manner, to stimulate serum glucocorticoid levels, and to perturb NGF binding in hippocampus and basal forebrain. One or repeated episodes of cold stress increased NGF mRNA levels in the hippocampus and p75NGFR mRNA levels in the basal forebrain. These changes were not due to elevated serum glucocorticoid levels since treatment with exogenous corticosterone had no effect on NGF and p75NGFR mRNA levels. Adrenalectomy did not prevent the stress induced increases in NGF and p75NGFR mRNA. © 1993 Wiley‐Liss, Inc.
Possible role of myelin‐associated neuraminidase in membrane adhesion
Authors: Saito M. , Yu R. K.
Published: January 2005

Previous studies from our laboratory have demonstrated the presence of a specific interaction between myelin‐associated neuraminidase and GM1 (Saito and Yu, J. Neurochem 47:632–641, 1986). In the present study, we further characterized this neuraminidase‐GM1 interaction and examined its role in the adhesion of rat oligodendroglial cells to GM1. Hydrolysis of N‐acetylneuramin‐lactitol by the enzyme was inhibited by GM1 in a competitive manner; GM1 itself was not hydrolyzed, suggesting that GM1 may serve as a competitive inhibitor of the enzyme. Asialo‐GM1 had no inhibitory effect. When a soluble enzyme preparation was applied to a GM1‐linked affinity column, the enzyme activity was retained on the column and was recovered from the column only by elution with a buffer containing 5 mM 2,3‐dehydro‐2‐deoxy‐N‐acetylneuraminic acid (Neu2en5Ac), a competitive inhibitor of neuraminidase. A binding study with 51 Cr‐labeled rat oligodendroglial cells showed that oligodendroglial cells bound preferentially to GM1 developed on a thin‐layer plate, but not to other gangliosides such as GM3, GD1a, GD1b, and GT1b. The binding reaction to GM1 was inhibited by Neu2en5Ac (5 mM). These results suggest that myelin‐associated neuraminidase specifically interacts with GM1 and may be involved in adhesion of oligodendroglial cells to GM1. This neuraminidase‐GM1 interaction may play an important role in the formation and stabilization of the multilamellar structure of the myelin sheath. © 1993 Wiley‐Liss, Inc.
Effect of hyaluronidase on brain extracellular matrix in vivo and optic nerve regeneration
Authors: Tona A. , Bignami A.
Published: January 2005

In the rat, intracerebral injection of bacterial hyaluronidase resulted in the almost complete disappearance of hyaluronic acid (HA) and glial hyaluronate‐binding protein (GHAP) from cerebral hemispheres, brain stem, and cerebellum (but not from optic nerves and chiasm) starting 2–3 hr after the injection. HA and GHAP reappeared throughout the brain in characteristic patches 2–3 days after the injection. The patches gradually became confluent and after 12 days the brain appeared virtually normal. In normal rat optic nerve, staining for HA and GHAP ceased abruptly in the region of the lamina cribrosa. The retina was completely negative. HA and GHAP disappeared from hyaluronidase‐injected optic nerve, chiasm, and contralateral optic nerve. In hyaluronidase‐injected crushed optic nerves, regenerated axons were able to grow for short distances (about 500μm) into the distal stump undergoing Wallerian degeneration. No such growth was observed in saline‐injected controls. © 1993 Wiley‐Liss, Inc.
Calcitonin induces a decreased Na+ conductance in identified neurons of Aplysia
Authors: Sawada M. , Ichinose M. , Ishikawa S. , Sasayama Y.
Published: January 2005

The ionic mechanism of the effect of extracellularly ejected calcitonin (CT) on the membrane of identified neurons R9 and R10 of Aplysia was investigated with voltage‐clamp, micropressure ejection, and ion substitution techniques. Micropressure‐ejected CT caused a marked hyperpolarization in the unclamped neuron. Heat‐inactivated CT was without effect. Clamping the same neuron at its resting potential level (−60mV) and re‐ejecting CT with the same dose produced a slow outward current (Io(CT), 30–40 sec in duration, 4–6 nA in amplitude) associated with a decrease in input membrane conductance. Io (CT) was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential of Io was approximately +10mV. Io (CT) was sensitive to changes in the external Na+ concentration but not to changes in K+, Ca2+, and CI− concentrations. Micropressure‐ejected forskolin produced a slow outward current, which, like the current to CT, was associated with a decrease in input membrane conductance, and was sensitive to changes in the external Na+ concentration. Io (CT) was prolonged by bath‐applied isobutylmethylxanthine (IBMX) but was not affected by I‐oleoyl‐2‐acetylglycerol (OAG) and calphostin C. Neither superfusion of the neuron with nordihydroguaiaretic acid (NDGA) nor superfusion with indomethacin caused any changes in Io(CT). These results suggest that extracellular CT can induce a slow outward current associated with a decrease in Na+ conductance, mediated by a receptor‐controlled increase in intracellular cyclic adenosine 3′, 5′‐monophosphate. © 1993 Wiley‐Liss, Inc.
Cell type‐ and differentiation‐dependent expression from the mouse acetylcholine receptor ϵ‐subunit promoter
Authors: Sunyer T. , Merlie J. P.
Published: January 2005

The nicotinic acetylcholine receptor (AChR) in adult skeletal muscle is composed of α‐, β‐, ϵ‐, and δ‐sub‐units and is localized at the neuromuscular junction; in contrast, the more diffusely distributed fetal form is composed of α‐, β‐, γ‐, and δ‐subunits. To define sequences necessary for the transcriptional regulation of the mouse ϵ‐subunit gene, we sequenced and analyzed 1036 bp upstream of the transcription start site. Using deletion analysis of the 5′‐flanking region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfection of the resulting constructs into established cell lines, we demonstrate that a 151 bp fragment exhibits cell type‐ and differentiation‐specific promoter activity. This activity was independent of a myogenic factor putative binding site (E‐box). However, transactivation experiments with recombinant myoD, myogenin, or MRF4 showed that the E‐box was functional and that MRF4 preferentially transactivates the ϵ‐promoter. Thus, like other AChR promoters, the proximal region of the ϵ‐promoter contains information for cell type‐specific and developmental regulation of CAT and can be transactivated by myogenci factors in cultured cell lines. Unlike the other AChR promoters characterized to date, ϵ‐promoter function can be partially independent of myogenic factors of the helix‐loop‐helix class. © 1993 Wiley‐Liss, Inc.
Induction of c‐fos by excitatory amino acids in developing chick retina is affected by changes in cellular interactions
Authors: Pearlman E. M. , Seigel G. M. , Notter M. F. D.
Published: January 2005

Cell contact is important for normal maturation of chicken retinal Muller cells. In order to gain a better understanding as to how this occurs, we examined the ability of retinal cells with altered cell contacts to respond to an environmental stimulus. The response of Muller cells cultured under conditions which alter cell contacts was measured by activating intracellular signaling systems leading to induction of the earlyinducible gene c‐fos. Chicken retinal cells were cultured as explants, reaggregates, and monolayers and exposed to extracellular stimuli in the form of the excitatory amino acids D, L‐alpha aminoadipic acid (AAA) and N‐methyl‐D, L‐aspartic acid (NMDA). Each culture was exposed to 1.25 mM AAA, 2.5 mM AAA,50 μM NMDA, or 100 μM NMDA. Toxicity was assessed histologically and by immunocytochemical labeling of Müller cells after 2 days of exposure. Activation of c‐fos was determined by Western blot analysis for Fos protein after 30,60, and 120 minutes of exposure. Exposure to AAA led to a loss of Muller cells in explant and reaggregate cultures; however, Muller cells in monolayer culture; however, Muller cells in monolayer culture were not susceptible to AAA at either dose. NMDA was toxic to a specific population of neurons under all three culture conditions. Fos protein expression paralleled the histologic findings. Fos protein was significantly elevated after exposure to either dose of AAA in explant and reaggregate cultures but not in monolayer cultures. Monolayer and reaggregate cultures did not exhibit a significant increase in Fos levels in response to either dose of NMDA. Explant cultures exhibited an equivical response to only the 50 μM NMDA. These results suggest that the ability of Müller cells to respond to AAA by increasing levels of Fos protein requires that specific cell contacts occur during development. © 1993 Wiley‐Liss, Inc.
Myelin‐associated glycoprotein is phosphorylated by protein kinase C
Authors: Kirchhoff F. , Hofer H.‐W. , Schachner M.
Published: January 2005

The myelin‐associated glycoprotein (MAG) is a neural recognition molecule involved in heterophilic interactions between myelin‐forming cells and neurons. To characterize the molecular mechanisms underlying post‐translational modifications which may be instrumental in signal transduction following the recognition event, we have studied the stimuli leading to modification of 32P‐orthophosphate incorporation into MAG in cultures of oligodendrocytes or transformed differentiated Schwann cells. Here we show that in oligodendrocytes both the 67 and 72 kD isoforms of MAG were phosphorylated exclusively on serine, while in the transformed Schwann cells only the 67 kD isoform was found to be present and phosphorylated. The phorbol‐12‐myristoyl‐13‐acetate (PMA) did not affect biosynthesis of the protein backbone, but enhanced incorporation of phosphate by a factor of 2–3, indicating the involvement of protein kinase C. Exclusive phosphorylation of serine residues was also observed, when purified MAG was incubated with protein kinase C in the presence of [γ‐32P] ATP. In searching for the physiological stimuli which may trigger phosphorylation of MAG, cultures of oligodendrocytes were exposed to extracellular signals, such as coculture with dorsal root ganglion and spinal cord neurons carrying the MAG receptor, to membrane fractions of these neurons, monoclonal MAG antibody 513 binding to the recognition site of MAG, or platelet‐derived growth factor. None of these additives modified the phosphorylation of MAG. These observations point to the possibility that phosphorylation of MAG is controlled by yet unknown intracellular cues rather than by extracellular signals interacting with cell surface receptors of oligodendrocytes. © 1993 Wiley‐Liss, Inc.
Transforming growth factor beta expression in reactive spinal cord microglia and meningeal inflammatory cells during experimental allergic neuritis
Authors: Kiefer R. , Gold R. , Gehrmann J. , Lindholm D. , Wekerle H. , Kreutzberg G. W.
Published: January 2005

Experimental allergic neurities (EAN), an inflammatory demyelinating disorder of the peripheral nervous system, is preceded and accompanied by a massive microglial reaction in the spinal cord which occurs in the absence of inflammatory cells infiltrating the cord parenchyma. Since transforming growth factor beta (TGF‐β) has been shown to play a beneficial role in experimental autoimmune disease and might be involved in the regulation of glial activity, we have investigated the expression of TGF‐β in EAN spinal cord and nerve root tissue. Adoptive transfer EAN was induced by the injection of neurotogenic T‐cells specific for the P2 myelin protein. In normal spinal cord tissue, both TGF‐β1 and TGF‐β3 mRNA were constitutively expressed at low levels. Already 3 days following injection of P2‐specific T‐cells, TGF‐β1 mRNA levels began to increase, peaked at day 6 at levels about tenfold above normal, and thereafter declined. TGF‐β3 was induced even earlier with a sharp rise at day 3 and a peak fourfold above normal at day 4. In situ hybridization for TGF‐β1 performed on spinal cord sections 6 days after injection of cells localized TGF‐β1 mRNA to many nonneuronal cells with the typical morphology of microglia. In addition, TGF‐β1 mRNA was observed in the meninges, and massive accumulation of signal was seen over inflammatory cells infiltrating the nerve roots. Our data indicate that TGF‐β1 and ‐β3 are involved in regulating the glial response in EAN and that activated microglial cells might control their own activity state by expressing TGF‐β1. The expression of TGF‐β1 by infiltrating inflammatory cells in the nerve roots might represent an important endogenous mechanism to limit the extent of inflammation. © 1993 Wiley‐Liss, Inc.