bluuebunny/crossref_metadata_2025 huggingface dataset:
Aberrant localization of MAP5 immunoreactivity in the hippocampal formation in alzheimer's disease
Authors: Geddes J. W. , Lundgren K. , Kim Y. K.
Published: January 2005

Immunocytochemistry was used to examine MAP5 immunoreactivity in the hippocampal formation obtained postmortem from five elderly, normal individuals, six individuals with Alzheimer's disease (AD), and two “transition” cases that did not have a history of dementia but did exhibit significant AD pathology. In all of the cases examined, axonal staining was restricted to the mossy fibers and their terminal field in CA3 stratum lucidum. In control cases, MAP5 immunoreactivity was observed in the neuronal cytoplasm and the proximal portion of the apical dendrites of pyramidal and granule cells. In both AD and transition cases, increased intensity of immunostaining was observed in CA3 pyramidal, subicular, and dentate gyrus granule cell neurons. Within individual neurons, immunoreactivity filled the neuronal perikarya, including the nuclear region, and the apical dendrite. Punctate staining was observed in neuritic plaques, but neurofibrillary tangles and neuropil threads were not immunostained.

The increase and altered distribution of MAP5 immunoreactivity in both vulnerable and nonvulnerable neurons in AD may reflect an aberrant sprouting response. The increased expression of early cytoskeletal proteins may be tolerated in some regions such as CA3, but not in others including CA1 where the increased expression appear to precede aberrant phosphorylation, proteolysis, and incorporation of cytoskeletal proteins into AD pathology. Alternatively, the results could reflect sprouting in response to the neuronal loss and degeneration.
Local control of axonal properties by schwann cells: Neurofilaments and axonal transport in homologous and heterologous nerve grafts
Authors: De Waegh S. M. , Brady S. T.
Published: January 2005

A number of axonal properties, including slow axonal transport and neurofilament phosphorylation, are altered in a mutant mouse strain with a Schwann cell deficiency, the Trembler. The Trembler phenotype is associated with poor myelination and reduced axonal caliber in the peripheral nervous system, but the genetic lesion has not yet been identified. To determine whether changes in axonal properties resulted from a direct action of Schwann cells on the axon, a segment of sciatic nerve from myelin‐deficient Trembler mouse was grafted into the sciatic nerve of a normal mouse and normal axons were allowed to regenerate. Normal axons surrounded by Trembler Schwann cells are reduced in diameter, but resume their original diameter distal to the graft. Neurofilament transport was also affected locally in sciatic nerves with Trembler grafts into normal nerve. The velocity of neurofilament transport was not significantly different from controls in portions of the nerve proximal to the Trembler graft, but there was a reduction in neurofilament transport rates upon entering the Trembler graft. This was accompanied by an increase in the ratio of neurofilament over tubulin in the case of the Trembler graft, suggesting both a slowing of the neurofilament and an increase in the rate of tubulin transport. Using heterologous grafts of Trembler nerve segments into wild type nerves, Schwann cells were shown to locally influence axonal caliber, neurofilament organization, and slow axonal transport. These observations emphasize the importance of glial cells in modulating neuronal structure and functions, as well as focusing attention on the role of glia in the etiology of neuropathologies that alter the neuronal environment.
Interaction between cAMP elevation, identified growth factors, and serum components in regulating Schwann cell growth
Authors: Stewart H. J. S. , Eccleston P. A. , Jessen K. R. , Mirsky R.
Published: January 2005

Most previous studies on Schwann cell proliferation in vitro have used serum‐containing media. This complicates the analysis of agents required for cell division since serum contains an ill‐defined mixture of hormones and growth factors. Serum‐free medium has therefore been used to analyse the response of Schwann cell to previously identified Schwann cell mitogens.

Serum factors were not necessary for DNA synthesis in response to platelet‐derived growth factor, basic fibroblast growth factor, or glial growth factor, provided they were used in combination with forskolin to elevate intracellular cAMP. Transforming growth factor β1, a Schwann cell mitogen in serum, was not mitogenic under these conditions. Neither the growth factors nor forskolin were effective when used alone. Growth control was analysed further using long‐term cultured Schwann cells that had spontaneously immortalized. Measurements of endogenous cAMP levels in short‐ and long‐term Schwann cells revealed that long‐term cells had two to three times higher basal cAMP levels. As predicted by these findings, platelet‐derived growth factor, basic fibroblast growth factor, and glial growth factor stimulated DNA synthesis in long‐term cells without requiring costimulation by agents which elevate cAMP (while transforming growth factor β1 had no effect).

These results show first that in rat Schwann cells activation of at least two intracellular pathways, one of which is cAMP dependent, is required for stimulation of DNA synthesis, second that serum factors are not necessary for the synergy between cAMP and platelet‐derived growth factor, basic fibroblast growth factor, or glial growth factor, and third that the mitogenic stimulation by transforming growth factor β1 is more complex, requiring serum factors in addition to elevation of cAMP.
Correlation between morphological and biochemical effects of ethanol on neuroblast‐enriched cultures derived from three‐day‐old chick embryos
Authors: Kentroti S. , Vernadakis A.
Published: January 2005

We have shown that ethanol exposure during embryogenesis affects a variety of parameters of neuronal growth. In this study we examined the direct effects of ethanol exposure on developing neuroblasts in culture. Neuroblast‐enriched cultures derived from 3‐day‐old whole chick embryos were grown in the presence of ethanol at doses ranging from 12.5 to 50 mM from culture day 3–14. Cholinergic and GABAergic phenotypic expression were both significantly reduced following ethanol exposure as assessed by the activities of choline acetyltransferase and glutamate decarboxylase, respectively. Morphometric analysis of the growth patterns showed significant differences between control and ethanol‐treated cultures. Control cultures exhibited the characteristic pattern of growth consisting of neuronal aggregation with neuritic arborization, i.e., neuritic bundles and fasciculation. Cultures grown in ethanol from culture day 3 consisted of aggregates that measured significantly greater in size than those observed in control cultures. In addition, in ethanol‐treated cultures, the primary pattern of neuritic bundles was replaced by a complex network of individual neurites radiating from the central aggregate, forming a defined “neuritic field.” Morphometric analysis revealed that both neurite number and neurite length were significantly reduced in ethanol‐treated cultures. The biochemical data confirm earlier reports from this laboratory suggesting that ethanol exposure during early embryogenesis alters the normal neuronal pattern of phenotypic expression. In addition, we have presented evidence in this study that ethanol alters the morphological growth patterns of developing neurons. Although ethanol does not alter the ability of these cells to aggregate, there is a significant alteration in neuritic outgrowth. We conclude that the alterations in the growth patterns induced by ethanol hinder full neurotransmitter phenotypic expression.
Molecular cloning of NILE glycoprotein and evidence for its continued expression in mature rat CNS
Authors: Prince J. T. , Alberti L. , Healy P. A. , Nauman S. J. , Stallcup W. B.
Published: January 2005

The NILE glycoprotein is a rat neuronal cell adhesion molecule which has been reported to be very similar in structure, function, and distribution to the mouse L1 glycoprotein. Here we report the complete nucleotide sequence of the NILE message (5,208 nucleotides) and the deduced amino acid sequence of the NILE polypeptide (1,257 amino acids). The predicted NILE protein is 96% identical to L1 at the amino acid level, confirming that the two molecules are homologues. The sequence information shows that NILE is a transmembrane molecule with an extensive ectodomain and a much smaller cytoplasmic domain. The extracellular portion of the molecule contains six immunoglobulin C‐2 type domains followed by five fibronectin type III repeats. These two structural motifs are characteristic of several other cell adhesion molecules. The cytoplasmic tails of NILE and L1 are identical to each other and distinct from the cytoplasmic regions of all other cell adhesion molecules except Ng‐CAM and neuroglian. Several possible sites for phosphorylation are present in the cytoplasmic tail of NILE.

Antisera were produced against two NILE‐β‐galactosidase fusion proteins containing distinct segments of the NILE polypeptide: the cytoplasmic domain and the segment containing fibronectin type III repeats. Immunoblots with these antisera and Northern blots with a NILE cDNA probe indicate that NILE continues to be expressed in most areas of the mature rat brain. This contradicts previous immunofluorescence data, which suggested that NILE was substantially down‐regulated in maturing nerve fiber tracts. This raises the possibility that NILE could be masked in situ by interactions with other cell surface molecules.
A subpopulation of bone marrow‐derived macrophage‐like cells shares a unique ion channel pattern with microglia
Authors: Banati R. B. , Hoppe D. , Gottmann K. , Kreutzberg G. W. , Kettenmann H.
Published: January 2005

Rat microglia share a number of antigenic, functional, and morphological similarities with macrophages from other tissues, but are characterized by a distinctly different pattern of ion channels in the cellular membrane (Kettenmann et al., J Neurosci Res 26:278–287, 1990). Macrophages typically express outward and inward K+ currents. In contrast, microglia lack outward currents and only show inwardly rectifying K+ currents, regardless of the isolation or cultivation method employed for microglia. In this study we demonstrate that a subpopulation of bone marrow‐derived macrophage‐like cells possesses inward rectifier K+ currents, but no outward currents and thus with regard to the electrophysiological characteristics closely resembles microglia. A second population of bone marrow‐derived macrophage‐like cells shows the usual channel pattern described for other body macrophages. Our results strengthen the hypothesis that in the bone marrow distinct pools of precursor cells exist, possibly reflecting an early differential lineage determination for body and brain macrophages, i.e., microglia.
β‐Adrenergic treatment of C6 glioma cells produces opposite changes in c‐fos and c‐jun mRNA levels
Authors: Gubits R. M. , Yu H.
Published: January 2005

The AP1 transcriptional complex is a heterodimer composed of proteins encoded by the fos and jun proto‐oncogene families. Changes in the concentration and composition of AP1 occur after cells are perturbed in a variety of different ways (Curran, in Reddy et al., eds. “The Oncogene Handbook,” Amsterdam: Elsevier, pp 307–325, 1988; Sonnenberg et al., Neuron 3:359–365, 1989). Transient changes in AP1 conlent presumably result in altered expression of AP1‐regulated targel genes, that help to mediate the cell's long‐term response to changes in its environment. One factor that may be important in determining which target genes are regulated by AP1 in a given context is the identity of the jun family member present in the complex (Chiu et al., Cell 59:979–986, 1989; Schutte et al., Cell 59:987–997, 1989). Fos induction has been demonstrated after binding of β‐adrenergic ligands to their cell surface receptors (Barka et al., Mol Cell Biol 6:2984–2989, 1986; Gubits et al., Mol Brain Res 6:39–45, 1989; Arenander et al., J Neurosci Res 24:107–114, 1989; Mocchetti et al., Proc Natl Acad Sci USA 86:3891–3895, 1989). However, the response of the jun gene family to this treatment has not been reported. We have therefore examined the effect of β‐adrenergic receptor activation on the expression of c‐fos, c‐jun, and junB mRNA levels in C6 glioma cells. Our results indicate that c‐fos and junB mRNA levels are increased by 52‐ and 2.7‐fold, respectively, after 45 min of isoproterenol (IPR) treatment. Unexpectedly, we found a concomitant decrease in c‐jun mRNA levels of about 2.5‐fold. The changes in c‐fos and c‐jun mRNAs were found to be dose dependent and specifically mediated by β‐ rather than alpha‐adrenergic receptors. The alterations in AP1 composition implied by our findings suggest a role for this transcriptional complex in mediating late gene responses lo β‐adrenergic receptor activation.
Survival and proliferation in developing neuroblasts in cultures derived from embryos treated with ethanol during early neuroembryogenesis: Effects attenuated by somatostatin
Authors: Kentroti S. , Vernadakis A.
Published: January 2005

Ethamol exerts profound effects both in ovo and in culture on neuronal phenotypic expression. In order to define more directly the neuronal growth parameters sensitive to the neurotoxic effects of ethanol, embryos were exposed to ethanol (10 mg/50 μl/day) or saline (control) at embryonic days 1 and 2; neuron‐enriched cultures prepared from these treated embryos at 3 days of embryonic age. Cultures from both groups were labeled with [3H] thymidine and assessed for effects on neuronal survival and proliferation in culture. Treatment of embryos with ethanol in ovo resulted in a marked enhancement in normal neuronal death in culture with no significant effect on proliferation. Whereas somatostatin (SRIF) had no effect on normally occurring neuronal death in control cultures, addition of SRIF (100 nM) to the culture medium attenuated the ethanol‐induced neuronal cell death. Videometric analysis revealed that pretreatment of embryos with ethanol resulted in formation of more and larger aggregates as compared with controls, an effect that was augmented by addition of SRIF to the medium. The most profound effect of ethanol on growth patterns was observed in neurite outgrowth showing a marked reduction in both neurite number and length in embryos pretreated with ethanol. SRIF enhanced neurite outgrowth (neurite number and length) in cultures derived from ethanol‐treated embryos. These results suggest that the ethanol‐induced deficits in neuronal survival and morphology are reversible. We conclude that SRIF may enhance neuronal survival by promoting neuritic outgrowth, thus establishing the essential target cell contacts necessary for cell survival.
Schwann cell nerve growth factor receptor expression during initiation of remyelination
Authors: Fan X. , Gelman B. B.
Published: January 2005

Initiation of remyelination is a promising therapeutic strategy to treat patients with demyelinating diseases, but specific factors that control remyelination are not clear. We first reported that expression of nerve growth factor receptor (NGFR) was increased during initiation of remyelination (Fan and Gelman,Journal of Neuropathology and Experimental Neurology49:312, 1990). In this study, we characterized the timing and cellular localization of NGFR expression in a model of segmental demyelination and remyelination using immunohistochemistry and monoclonal antibody 192‐IgG, and compared it to an axonal neuropathy. At the onset of demyelination induced by tellurium (Te) poisoning, NGFR antigenicity was selectively expressed within and around demyelinating internodes in rat sciatic nerve. Dual fluorescence staining with myelin‐specific antigen showed that NGFR colocalized with demyelinated internodal units with relative specificity; Schwann cell S‐100 protein showed a concomitant down‐regulation in injured internodes. Peak expression of NGFR occurred during the transition between demyelination and remyelination (day 8 of Te), then declined exponentially. NGFR expression was most prominent in the cytoplasm of daughter Schwann cells as they established contact with denuded axons, and was sharply repressed as compact myelin began to accumulate. Rare colocalization with neurofilament antigens revealed intraaxonal deposits of NGFR in segmental demyelination. In the nerve crush model, Schwann cell NGFR expression was not segmentally distributed and was up‐regulated for a longer period of time. Our data establish that NGFR expression in the peripheral nervous system is not strictly linked to axon elongation, and that it probably functions during the initiation of myelination.
The structural integrity of glial scar tissue associated with a chronic spinal cord lesion can be altered by transplanted fetal spinal cord tissue
Authors: Houle J.
Published: January 2005

The potential for fetal spinal cord (FSC) tissue transplants to modify an established glial scar or to restrict the reformation of a scar following surgical manipulation of a chronic lesion site was studied in the injured rat spinal cord. Six to eight weeks after preparation of a hemisection lesion cavity, glial scar tissue was left intact in one group, whereas in a second group it was excised prior to transplantation of a suspension of FSC tissue. From the first group, examination of serial sections through the graft‐host interface that had been immunoreacted for glial fibrillary acidic protein (GFAP) demonstrated that in many cases the glial scar no longer was a continuous wall separating the two tissues. Quantitation of the area occupied by these discrete gaps in the scar provided an Index of Fusion, indicating the extent of direct contact between the transplant and host spinal cord. In some animals this constituted as much as 60% of the interface, while in others there were no breaks in the scar (0% fusion). Reinjury of the spinal cord lead to a rapid astrocytic response culminating in the re‐establishment of a dense matrix of glial cells and processes covered by a basal lamina. This reformed scar effectively isolated the spinal cord from the external environment of the cavity. When FSC tissue was transplanted after first removing scar tissue the continuity of reformed glial scarring at the graft‐host interface was altered. Distinct gaps in the scar appeared randomly along the interface. The mean Index of Fusion for animals receiving a moderate reinjury (removal of scar tissue only) was not as high as for those animals in which a more severe reinjury (expansion of the cavity by 0.5 mm) was performed before transplantation. The extent of graft‐host fusion was not significantly improved when scar tissue was removed prior to transplantation. These findings support the hypothesis that the presence of FSC tissue will have an effect on the persistence of glial scar tissue in a chronic lesion site as well as limit the extent to which a new scar is formed in response to a second injury to the spinal cord.
Substance P immunoreactivity of rat brain stem neurons in primary culture
Authors: Jacquin T. D. , Denizot J. P. , Denavit‐Saubie M.
Published: January 2005

Substance P (SP)‐ergic neurons from 16/17 day‐embryonic rat brain stem in primary culture were identified by immunocytochemistry using biotinylated avidine and phosphatase alcaline methods with affinity‐purified anti‐SP antibodies. An average of 84% of neurons contained SP from day 9 to day 21 after plating. These in vitro data show that SP‐containing neurons develop in our culture conditions. SP may act as a maturation factor as well as a neurotransmitter.
Loss and recovery of activities of α+ and α isozymes of (Na+ + K+)‐ATPase in cortical focal ischemia: GM1 ganglioside protects plasma membrane structure and function
Authors: Mahadik S. P. , Bharucha V. A. , Stadlin A. , Ortiz A. , Karpiak S. E.
Published: January 2005

Alterations in cellular membrane structure and the subsequent failure of its function after CNS ischemia were monitored by analyzing changes in the plasma membrane marker enzyme (Na+ + K+‐ATPase. The levels of two isozymes of Na+ + K+)‐ATPase, α+ and α, which have distinct cellular and anatomical distributions, were studied to determine if differential cellular damage occurs in primary and peri‐ischemic injury areas. The efficacy of monosialoganglioside (GM1) treatment was assessed, since this glycosphingolipid has been shown to reduce ischemic injury by protecting cell membrane structure/function. Using a rat model of cortical focal ischemia, levels of both ATPase isozyme activities were assayed in total membrane fractions from primary ischemic tissue (parietal cortex) and three peri‐ischemic tissue areas (frontal, occipital, and temporal cortex) at 1, 3, 5, 7, and 14 days after ischemia. No significant loss of either isozyme's activity occurred in any tissue area at 1 day after ischemia. At 5 dyas, in the primary ischemic area, both isozyme activity levels decreased by 70–75%. The α+ enzyme activity loss persisted up to 14 days, while a 17% recovery in α activity occurred. In the three peri‐ischemic tissue areas, enzyme activity losses ranged from 42%–59% at 3 days after ischemia. A complete restoration of both isozyme activities was seen at 14 days. After three days of GM1 ganglioside treatment there was no loss of total (Na+ + K+)‐ATPase activity in the three peri‐ischemic areas, and a significantly reduced loss in the primary infarct tissue. An autoradiographic analysis of brain coronal sections using 3H‐ouabain supports the enzymatic data and GM1 effects. Reductions in 3H‐ouabain binding in all cortical layers at 3 days after ischemia were visualized. GM1 treatment significantly reduced these 3H‐ouabain binding losses. In summary, time‐dependent quantitative changes in activity levels of ATPase isozymes (α+ and α) reflect the different degree of membrane damage that occurs in primary vs. peri‐ischemic tissues (e.g., irreversible vs. reversible membrane damage), and that ischemia affects cell membranes of all neural elements in a largely similar fashion. GM1 ganglioside was found to reduce plasma membrane damage in all CNS cell types. © 1992 Wiley‐Liss, Inc.
Secretion of apolipoprotein E by an astrocytoma cell line
Authors: Krul Elaine S. , Tang J.
Published: January 2005

Apolipoprotein (apo) E is a predominant protein in developing mammalian brain and in damaged peripheral nerve. Of particular interest is the observation that astrocytes in the central nervous system cease to produce apoE after nerve damage, whereas an increase in apoE production results after peripheral nerve injury. Differences in the response to injury with regard to the production of apoE may be related to dissimilarities in the abilities of the central and peripheral nervous systems to regenerate. As there are few data concerning the regulation of apoE gene expression in extrahepatic tissues, we employed a human astrocytoma cell line (CCF‐STTG1) as a model to study apoE production in astrocytes. CCF‐STTG1 cells secreted apoE constitutively in serumfree media. Cholesterol added to the media as cholesterol:phospholipid liposomes (2–100 μg/ml) or as human plasma LDL increased the amount of apoE secreted into the media, but had little or no effect on the relative abundance of apoE mRNA. By contrast, the commercially available triglyceride‐phospholipid emulsion Intralipid© added at dilutions of 1:50 to 1:500 caused a total inhibition of apoE secretion by the cells, but again, little change was noted in the relative abundance of apoE mRNA. Insulin (5 μg/ml) caused a 45–55% reduction in the amount of apoE secreted by the astrocytoma cells. Glucagon (5 μg/ml), on the other hand, did not increase apoE secretion, and apoE mRNA concentrations were not affected by either hormone treatment. ApoE was secreted from the astrocytoma cells associated with particles of plasma VLDL to IDL and HDL size. After feeding the cells with 20 μg/ml cholesterol as cholesterol:phospholipid liposomes, an increased proportion of apoE was secreted associated with the larger VLDL to IDL size particles, with a concomitant decrease in the proportion associated with the smaller HDL‐size particles. When cells were incubated with 5 μg/ml insulin, most of the apoE was associated with the HDL‐size particles. When cholesterol:phospholipid liposomes were added in the presence of insulin virtually all of the secreted apoE was found associated with the VLDL to IDL size particles. In summary, the regulation of apoE production in CCF‐STTG1 cells in many respects resembles that of other cells, including hepatocytes. However, it is clear that there remain to be identified cell specific factors which regulate apoE production in astrocytes. The CCF‐STTG1 cell line promises to provide a suitable model to investigate these questions. © 1992 Wiley‐Liss, Inc.
Laminin and its neurite outgrowth‐promoting domain in the brain in Alzheimer's disease and Down's syndrome patients
Authors: Murtomäki S. , Risteli J. , Risteli L. , Koivisto U.‐M. , Johansson S. , Liesi Päivi
Published: January 2005

Immunocytochemistry, radioimmunoassay, immunoblotting, Northern analysis, and polymerase chain reaction (PCR) technique were applied to investigate the distribution of laminin and its neurite outgrowth domain in brains of neuropathologically verified cases of Alzheimer's disease and Down's syndrome. New antibodies against a neurite outgrowth domain of laminin were characterized and were used in localization of this peptide antigen in the human brain. Laminin was found as large punctate deposits in all plaques in the affected brains. Laminin synthesis was increased as assessed by RNA blotting and immunoblotting, and glial cells were heavily immunoreactive with antibodies for a neurite outgrowth‐promoting peptide antigen of the B2 chain of laminin. This peptide antigen not only was produced by glial cells but also was deposited in the brain tissue. As this peptide antigen promotes neurite outgrowth at low concentrations, and is specifically neurotoxic at high concentrations, it may play a synergistic role with other molecules in inducing the sprouting and neurodegeneration occurring in brains of patients with either Alzheimer's disease or Down's syndrome. © 1992 Wiley‐Liss, Inc.
Voltage‐dependent potassium currents in cultured trout oligodendrocytes
Authors: Glassmeier G. , Jeserich G. , Krüppel T.
Published: January 2005

Ionic currents were recorded in cultured oligodendrocytes from the brain of trout using the whole‐cell configuration of the patch‐clamp technique. Outward currents were evoked at membrane potentials more positive than −40 mV, which could be separated into two components according to their kinetic parameters and their sensitivity to the holding potential: a fast inactivating current which was completely suppressed by 4‐aminopyridine and reduced by tetraethylammonium and a slow steady‐state conductance which was similarly sensitive to both potassium channel blockers. The current reversal potential was close to the potassium equilibrium potential. In contrast to mammalian oligodendrocytes but in similarity with cultured Schwann cells, trout oligodendrocytes did not exhibit any inwardly rectifying currents at hyperpolarized membrane potentials. © 1992 Wiley‐Liss, Inc.
EAE cerebrospinal fluid augments in vitro phagocytosis and metabolism of CNS myelin by macrophages
Authors: Sommer M. A. , Forno L. S. , Smith Marion E.
Published: January 2005

Previous studies from this laboratory have shown that CNS myelin is phagocytized and metabolized by cultured rat macrophages to a much larger extent when myelin is pretreated with serum containing antibodies to myelin constituents than when it is left untreated or pretreated with non‐specific serum. In this study the effect of cerebrospinal fluid (CSF) from rabbits with experimental allergic encephalomyelitis (EAE) in promoting myelin phagocytosis was examined. Fourteen rabbits were immunized with purified myelin in Freund's complete adjuvant, seven of which developed clinical EAE symptoms. Serum and CSF were collected from EAE and control rabbits, and the CSF was centrifuged to remove cells. Sera and CSF from these rabbits and from Freund's adjuvant‐immunized controls and untreated controls were measured for IgG content by radial diffusion assay, their myelin antibody characteristics were analyzed by immunoblots, and the ability of these serum and CSF samples to promote myelin phagocytosis when used for myelin opsonization was examined. The ability of a CSF sample to enhance radioactive myelin uptake and phagocytosis by cultured macrophages as measured by the appearance of radioactive cholesterol ester was linearly proportional to its total IgG titer, and correlated approximately both with clinical symptoms of the animal and the presence of antibody against the myelin constituents myelin basic protein, proteolipid protein, and galactocerebroside. The cholesterol esterification activities of EAE sera correlated to a lesser extent with IgG levels and clinical symptoms. These results suggest a possible role for CSF‐localized anti‐myelin antibody in demyelination in the CNS by macrophages in EAE, and they encourage further study of the role of CSF antibody in cell‐mediated demyelination in multiple sclerosis. © 1992 Wiley‐Liss, Inc.
Activity and distribution of phosphoinositidase C in rat sciatic nerve
Authors: Mathew J. , Date S. , Eichberg J.
Published: January 2005

The hydrolysis of phosphatidylinositol‐4,5‐bisphosphate (PIP2) by rat sciatic nerve cytosolic phosphoinositidase C [phosphoinositide‐specific phospholipase C (PIC)] was studied at neutral pH and at ionic concentrations that approximate intracellular conditions. The principal water‐soluble product formed was shown to be inositol trisphosphate by anion exchange chromatography. The maximum hydrolysis rate (2.5 nmol/min/mg protein) was achieved at < 100 nM Ca2+. Hydrolysis was markedly increased to 15 nmol/min/mg protein by inclusion of K+ in the reaction mixture. In the presence of 200 mM K+, the optimum Ca2+ was increased to approximately 600 nM. Higher Ca2+ concentrations progressively inhibited PIP2 hydrolysis. Mg2+ also inhibited the reaction, but the presence of equimolar amounts of ATP and Mg2+ had no effect. Appreciable degradation of phosphatidylinositol‐4‐phosphate (PIP) also occurred in the nanomolar Ca2+ range, whereas breakdown of phosphatidylinositol (PI) required millimolar Ca2+. The presence of PIP but not PI inhibited PIP2 hydrolysis. Upon subcellular fractionation of nerve, more than 50% of recovered PIC activity was in the cytosol and about 20% was located in a myelin‐enriched fraction. Using PIP2 as substrate, PIC activities in nerves from normal and streptozotocin‐induced diabetic animals were not different. However, the myelin‐associated enzyme from diabetic animals was more labile to freezing and thawing. © 1992 Wiley‐Liss, Inc.
Regional distribution and biochemical characteristics of high molecular weight tau in the nervous system
Authors: Taleghany N. , Oblinger M. M.
Published: January 2005

The present study examined the distribution of the high molecular weight (HMW) tau protein isoform in the nervous system by immunoblotting and immunohistochemistry. Some of the biochemical properties of this 110 kDa tau protein were explored, including its heat stability, phosphorylation and partitioning with cold/Ca2+ stable vs. soluble microtubules. Qualitative western blot analysis revealed that HMW tau is preferentially expressed in neurons with peripherally projecting axons. For example, this isotype was present in sciatic nerve, ventral and dorsal roots, trigeminal nerve, vagus nerve, dorsal root ganglia (DRG) and spinal cord, but was present in only trace amounts in CNS regions. Another tau isoform of slightly smaller size (90–100 kDa), termed mid‐molecular weight (MMW) tau, was present in abundant quantity in optic nerve samples and detectable in several other CNS regions, including hippocampus and cerebellum. The 110 kDa HMW tau as well as MMW tau and the other tau isoforms were found to be heat stable proteins. The HMW and MMW tau isoforms preferentially partitioned with the cold and Ca+2 insoluble tubulin fraction, but the association of HMW tau with stable microtubules was very susceptible to proteolysis. Dephosphorylation of fresh tissue with alkaline phosphatase produced no apparent shift in the mobility of HMW tau on SDS‐PAGE but did alter the mobility of other brain tau isoforms, including MMW tau. Immunocytochemical staining with tau‐1 antibody in the DRG, which contains HMW tau but no other tau isotypes, showed localization to mainly small neurons and was not altered by dephosphorylation of the histological sections. This suggests that HMW tau, which contains the tau‐1 epitope, is not phosphorylated in that site. © Wiley‐Liss, Inc.
Human serum stimulates alzheimer markers in cultured hippocampal neurons
Authors: Brewer G. J. , Ashford J. W.
Published: January 2005

The mechanism for promoting the distinct types of lesions in the Alzheimer disease (AD) brain and other changes outside the brain is unknown. We examined neurons in culture, unprotected by glia or a blood‐brain barrier, to determine if exposure to serum from Alzheimer patients would affect markers for Alzheimer brain lesions. Rat hippocampal neurons were first grown for 4 days in a new serum‐free culture medium, then exposed for 24 hr to human sera. Sera from 12 AD patients or their spouses increased four molecular markers characteristic of Alzheimer senile plaques and neurofibrillary tangles: Alz‐50, β‐amyloid (β/A4), MAP2, and ubiquitin, each with their expected cytologic distributions. Sera from ten young adults produced significantly less stimulation. By quantitative immunofluorescence, neuronal exposure to the elderly human sera produced 1.8‐ to 2.5‐fold increases in mean fluorescent area/cell for each of these four markers relative to no serum exposure. As controls, an unrelated neuronal marker, enolase, was unaffected and fetal bovine serum did not stimulate immunoreactivity. Neuron viability and somal area were unaffected at 24 hours. The MAP2 increases were dose dependent with negligible effect at 2% serum and maximum effect at 10% serum after 24 hr. The MAP2 increase was greater after 48 hr of exposure than 24 hr and negligible at 2 hr. This stimulation of AD markers by human serum suggests that the genesis of both neuronal plaques and tangles may arise from access of toxic serum factors to susceptible neurons and/or failure to detoxify these factors. © Wiley‐Liss, Inc.
Leukemia inhibitory factor promotes the neuronal development of spinal cord precursors from the neural tube
Authors: Richards L. J. , Kilpatrick T. J. , Bartlett P. F. , Murphy M.
Published: January 2005

Recent evidence from our laboratory has shown that leukemia inhibitory factor (LIF) can act early in peripheral nervous system development. We have investigated a potential role of LIF in the developing spinal cord. In explants and dissociated cultures of spinal cord primordium, LIF stimulated a profuse neurite outgrowth. To determine if these effects were related to neuronal differentiation, cells were plated at low cell density and stained for neurofilament. LIF stimulated an increase in the number of newly differentiated neurons, without inducing proliferation of the precursors. Given that LIF has previously reported effects as a cholinergic switching factor for sympathetic neurons, we investigated whether LIF had similar effects in these spinal cord cultures. LIF increased the number of cholinergic neurons in proportion to its overall effect on the stimulation of all neurofilament positive neurons in the culture. These data show that LIF stimulates the generation of spinal cord neurons from their precursors and further implicates a role for LIF in nervous system development. © 1992 Wiley‐Liss, Inc.
Differential regulation of the nerve growth factor and brain‐derived neurotrophic factor genes in L929 mouse fibroblasts
Authors: D'Mello S. R. , Jiang C. , Lamberti C. , Martin S. C. , Heinrich G.
Published: January 2005

Nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) are structurally related survival and differentiation factors for distinct sets of peripheral and central neurons. The regulation of NGF gene expression has been extensively studied in L929 mouse fibroblasts. L929 cells also express the BDNF gene. Northern blot hybridization analysis revealed 4 discrete BDNF mRNA species in L929 cells and rat hippocampus after induction of seizures with kainic acid. Serum as well as 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) stimulated NGF and all 4 BDNF mRNAs in L929 cells. Treatment with both agents induced NGF mRNA to a much larger extent than the BDNF mRNAs. The induction of the BDNF mRNAs was rapid, with nearly maximal levels by 1 hr. In contrast, NGF mRNA induction occurred later and peaked at 4–6 hr. Both NGF and BDNF mRNA induction were inhibited by actinomycin D. Cycloheximide, on the other hand, inhibited only NGF but not BDNF mRNA induction. Corticosterone rapidly decreased NGF mRNA but not the BDNF mRNAs, and had no effect on seizure‐induced NGF or BDNF mRNAs. Forskolin did not stimulate NGF or BDNF mRNAs. In contrast to NGF mRNA, forskolin did not interfere with the serum induction of BDNF mRNAs. These results demonstrate that 2 genes which encode closely related neurotrophic factors are differentially regulated in L929 cells. The molecular mechanisms which bring about this differential regulation remain to be elucidated. © 1992 Wiley‐Liss, Inc.
Biphasic changes in NCAM level after an NMDA lesion to the hippocampal formation: A quantitative dot‐immunobinding assay
Authors: Wang S. , Lees G. J. , Bock E. , Hamberger A. , Haglid K. G.
Published: January 2005

With a quantitative dot‐immunobinding assay, the time course changes of neuronal cell adhesion molecule (NCAM) concentrations and total tissue content were monitored in the rat hippocampus after a 40 nmol NMDA injection. A biphasic alteration was observed; a decrease occurred at day 3, an increase at day 30. The time course of changes differed from that of the glial fibrillary acidic protein (GFAP), a marker for reactive astroglial cell, but was similar to that for the markers of sprouting neurites, i.e., low (L) and high (H) molecular weight subunits of the neurofilament polypeptides. It is suggested that NCAM is implicated in the onset of neurite sprouting in the hippocampus after an excitotoxic trauma. © 1992 Wiley‐Liss, Inc.
Age‐related changes in rat brain monoamines release: Peculiarity of dopamine release
Authors: Cerrito F. , Aloisi G. , Arminio P.
Published: January 2005

Our aim has been to investigate the ability of the rat brain to retain its level of neurotransmitter release over life. We have investigated the neurotransmitter release from the rat brain synaptosomes prelabeled with 3H‐DA, 3H‐NA, or 3H‐5HT, and perfused with Krebs‐Ringer medium alone (basal release) or containing a high K+, calcium ionophore, tyramine or amphetamine (evoked release). Brain areas have been dissected of animals 45 days and 4, 6, and 11 months old. The results have shown a gradual reduction of the 3H‐NA release evoked by a high K+ from 45 days to 6 months, which is stabilized until 11 months of age. The reduction rate has been relatively different from the brain areas investigated (36% for the frontal cortex and 26% for the hippocampus and cerebellar cortex). A similar reduction has been seen with 3H‐5HT released from synaptosomes of the frontal cortex, hippocampus, and striatum. Surprisingly, the 3H‐DA release that was evoked by high K+ was greater in rats 11 months old than in younger rats; this effect has been seen in synaptosomes from the caudate and the frontal cortex. The calcium ionophore A23187 has shown a releasing picture similar to a high K+. When we analyzed a nonexocitotic, but probably carrier mediated, release (evoked by tyramine or amphetamine), there was reduced release of all of the above neurotransmitters from 45 days to 11 months of age. We presume that there have been adaptative changes in neurotransmitter evoked release due to changes in Ca++ utilization, as inferred from the results from calcium ionophore experiments and carrier performance. Aging seems to have preserved the extracellular neurotransmitter concentrations of Na and 5HT, compensating for the decreased carrier performance with a reduced depolarization rate of nerve terminals. DA homeostasis seems to have been altered with normal aging and may produce an elevation in extracellular DA and in its metabolite, yielding free radicals. This mechanism may play a role in degenerative changes associated with normal aging. © 1993 Wiley‐Liss, Inc.
Application of synthetic phospho‐ and unphospho‐ peptides to identify phosphorylation sites in a subregion of the tau molecule, which is modified in Alzheimer's disease
Authors: Liu W.‐K. , Moore W. T. , Williams R. T. , Hall F. L. , Yen Shu‐Hui
Published: January 2005

Phospho‐ and unphospho‐ peptides were used to define the essential sequence for a tau epitope, which is recognized by Tau‐1 antibody and phosphorylated in Alzheimer's disease (AD). The epitope was mapped within the amino acid residues 192–199 of tau and was phosphorylated by the p34cdc2/p58cyclin A proline directed kinase (PDPK), but not by purified mitogen activated protein kinase (p42mapk). Addition of phosphate to the last serine of the epitope was the most effective in abolishing the reactivity of the epitope to Tau‐1 antibody. Our results suggest that one and possibly more members of the PDPK family may play a role in the pathogenesis of AD. © 1993 Wiley‐Liss, Inc.
Isolation and age‐related characterization of mouse Schwann cells from dorsal root ganglion explants in type I collagen gels
Authors: Chi H. , Horie H. , Hikawa N. , Takenaka T.
Published: January 2005

A technique for isolation of adult Schwann cells (ScC) from dorsal root ganglia (DRG) is described. Decapsulated DRG explants embedded into type I collagen gels were cultured for 3 days in serum‐free medium during which ScC migrated from the explant. These explants were then grown in serum‐supplemented medium to allow ScC proliferation. On day 10 the number of ScC isolated from DRG explants per mouse was about 2.5 × 105, and the purity was greater than 95%. This culture system provided sufficient numbers of highly purified adult ScC in a shorter culture period (2–3 times) than other methods. We used ScC from this method to determine the age‐related changes in attachment, growth, and survival of ScC cultured in serum‐free medium. The attachment capacity of adult ScC on type I collagen or polylysine was similar to that of newborn ScC. However, the collagen promoted growth and survival of adult ScC but not that of neonatal ScC, indicating age‐related differences of ScC properties in vitro. © 1993 Wiley‐Liss, Inc.
Interleukin‐2 as a neurotrophic factor for supporting the survival of neurons cultured from various regions of fetal rat brain
Authors: Awatsuji H. , Furukawa Y. , Nakajima M. , Furukawa S. , Hayashi K.
Published: January 2005

Interleukin(IL)‐2 supported the survival and enhanced neurite extension of cultured hippocampal neurons prepared from embryonic 18‐day‐old rats. This neurotrophic effect was observed at concentrations of 2 to 200 U/ml, and almost all the neurons could survive for more than 2 days in the presence of 200 U/ml of IL‐2. This viability‐promoting effect of IL‐2 on the neurons was completely blocked with anti‐IL‐2 antibodies. IL‐2 also supported the survival of cultured cortical, striatal, and septal neurons. These results indicate that IL‐2 has a survival‐promoting effect on a wide variety of neurons. On the other hand, IL‐2 did not affect the choline acetyltransferase (ChAT) activity of striatal neurons, suggesting that this cytokine does not act as a differentiation factor for striatal cholinergic neurons. © 1993 Wiley‐Liss, Inc.
Identification of a collagen potentiated neurite promoting factor isolated from C6 glioma cells
Authors: Coyle D. E.
Published: January 2005

The C6 glial cell line has been used as a model cell system for the investigation of new glial produced neurotrophic and neurotropic molecules. By using the C6 cell line grown in a defined medium on collagen, this laboratory has isolated a distinct neurite promoting factor (NPF) that is potentiated by the presence of collagen (CPNPF). We have observed that C6 cells cultured in a defined medium on collagen (rat type‐I) slowed their growth rate and expressed an astrocytic‐ or oligodendrocytic‐like morphology. CPNPF, at this state of purity, appears to be a distinct NPF which induces neurite outgrowth (neurites of 1 or more somal diameters) in PC12 cells. These neurite promotion effects, however, appear to support the neuron morphology for only a short period (4 days) of time without the presence of neurotrophic factor (NTF). The neurite promoting activity is ineffective in inducing neurite outgrowth using mouse neuroblastoma cells (neuro‐2a). CPNPF appears to be a heat stable protein whose activity does not depend on the presence of intact collagen, heparin sulfate proteoglycan (HSPG), or chondroitin sulfate proteoglycan (CSPG). Exposure to dissociative conditions results in a loss of neurite promoting activity. CPNPF is not a glycoprotein that contains an accessible α‐D‐mannopyranosyl, α‐D‐glucopyranosyl, or a sterically related residue (hydroxyl groups in the C‐3,4, and 5 positions). Although these residues are not present on all glycoproteins, it does indicate that CPNPF is most likely not a glycoprotein. CPNPF activity is not blocked by neutralizing antibodies directed toward NGF, β‐FGF, IL‐1β, IL‐6, TGF‐β2, TGF‐β1.2, TGF‐β3, TGF‐β5, or EGF. CPNPF appears to either be oligomeric protein or a complex of proteins. On the basis of indirect evidence, it does not appear to be glial derived protease nexin‐I. The alteration in morphology of the C6 glial cell line by serum‐free conditions in the presence of collagen may have induced the production of a potentially new NPF not seen by previous investigators. © 1993 Wiley‐Liss, Inc.
p44mpk MAP kinase induces aizheimer type alterations in tau function and in primary hippocampal neurons
Authors: Lu Q. , Soria J. P. , Wood J. G.
Published: January 2005

Abnormally phosphorylated tau protein is a major component of the cytoskeletal pathology of Alzheimer's disease (AD) found in the neurofibrillary tangle (NFT) and neuritic plaque (NP). Identification of the kinase responsible for this phosphorylation has been difficult. In the test tube, several proline‐directed kinases, particularly mitogen‐activated protein (MAP) and cdc2 kinase, phosphorylate tau on sites that appear to mimic the abnormally phosphcrylated sites in AD. Important unanswered issues include: (1) whether this phosphorylation event occurs in the tightly regulated environment of a living cell; (2) whether this phosphorylation of tau affects its functional properties; and (3) what is the subcellular relationship of proline‐directed kinases and tau. We show here that tau can be phosphorylated in cultured hippocampal neurons by the MAP kinase p44mpk and phosphorylation of tau compromises its functional ability to assemble microtubules. We show further that MAP kinase copurifies with microtubule fractions where it is tyrosine phosphorylated and presumably active. These studies address and raise several important issues regarding the regulation of tau phosphorylation in normal and AD brain. © 1993 Wiley‐Liss, Inc.