myelin-basic-protein and Neuroblastoma

Pathological aggregation of the microtubule-associated protein tau and subsequent accumulation of neurofibrillary tangles (NFTs) or other tau-containing inclusions are defining histopathological features of many neurodegenerative diseases, which are collectively known as tauopathies. Due to conflicting results regarding a correlation between the presence of NFTs and disease progression, the mechanism linking pathological tau aggregation with cell death is poorly understood. An emerging view is that NFTs are not the toxic entity in tauopathies; rather, tau intermediates between monomers and NFTs are pathogenic. Several proteins associated with neurodegenerative diseases, such as β-amyloid (Aβ) and α-synuclein, have the tendency to form pore-like amyloid structures (annular protofibrils, APFs) that mimic the membrane-disrupting properties of pore-forming protein toxins. The present study examined the similarities of tau APFs with other tau amyloid species and showed for the first time the presence of tau APFs in brain tissue from patients with progressive supranuclear palsy (PSP) and dementia with Lewy bodies (DLB), as well as in the P301L mouse model, which overexpresses mutated tau. Furthermore, we found that APFs are preceded by tau oligomers and do not go on to form NFTs, evading fibrillar fate. Collectively, our results demonstrate that in vivo APF formation depends on mutations in tau, phosphorylation levels, and cell type. These findings establish the pathological significance of tau APFs in vivo and highlight their suitability as therapeutic targets for several neurodegenerative tauopathies.

Topics: Animals; Brain; Cell Line, Tumor; Cellular Structures; Enzyme-Linked Immunosorbent Assay; Humans; Immunoprecipitation; Lewy Body Disease; Mice; Mice, Transgenic; Mutation; Myelin Basic Protein; Neuroblastoma; Phosphorylation; Supranuclear Palsy, Progressive; tau Proteins

The expression level of hnRNP C1/C2 protein has been reported to be significantly decreased in the post-mortem brain of schizophrenic patients. In this study, we investigated whether overexpression of the hnRNP C variants hnRNP C1 and C2 changed the expression of myelination-related genes in the human neuroblastoma cell line SK-N-SH. In both hnRNP C1- and C2-overexpressing cells, the expression of quaking (QKI)-6 and QKI-7 significantly increased or decreased compared to the control, respectively. Intriguingly, QKI-5 and myelin basic protein were markedly up- or down-regulated by overexpressing hnRNP C2, respectively. Our findings are the first to demonstrate distinct functions of hnRNP C1 and C2, and may be helpful in understanding the functions of these molecules. These findings indicate that altered expression levels of hnRNP C in the brain of patients with schizophrenia could be involved in the pathophysiology of this disease through alteration of the QKI isoform and myelin basic protein expression.

Topics: Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoprotein Group C; Humans; Myelin Basic Protein; Neuroblastoma; Protein Isoforms; RNA-Binding Proteins

Activated microglia can influence the survival of neural cells through the release of cytotoxic factors. Here, we investigated the interaction between Toll-like receptor 4 (TLR4)-activated microglia and oligodendrocytes or their precursor cells (OPC). Primary rat or N9 microglial cells were activated by exposure to TLR4-specifc lipopolysaccharide (LPS), resulting in mitogen-activated protein kinase activation, increased CD68 and inducible nitric oxide synthase expression, and release of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6). Microglial conditioned medium (MGCM) from LPS-activated microglia attenuated primary OPC proliferation without inducing cell death. The microglial-induced inhibition of OPC proliferation was reversed by stimulating group III metabotropic glutamate receptors in microglia with the agonist L-AP4. In contrast to OPC, LPS-activated MGCM enhanced the survival of mature oligodendrocytes. Further investigation suggested that TNF and IL-6 released from TLR4-activated microglia might contribute to the effect of MGCM on OPC proliferation, insofar as TNF depletion of LPS-activated MGCM reduced the inhibition of OPC proliferation, and direct addition of TNF or IL-6 attenuated or increased proliferation, respectively. OPC themselves were also found to express proteins involved in TLR4 signalling, including TLR4, MyD88, and MAL. Although LPS stimulation of OPC did not induce proinflammatory cytokine release or affect their survival, it did trigger JNK phosphorylation, suggesting that TLR4 signalling in these cells is active. These findings suggest that OPC survival may be influenced not only by factors released from endotoxin-activated microglia but also through a direct response to endotoxins. This may have consequences for myelination under conditions in which microglial activation and cerebral infection are both implicated. , Inc.

Topics: Animals; Animals, Newborn; Bromodeoxyuridine; Cell Count; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cerebral Cortex; Culture Media, Conditioned; Cytokines; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Gangliosides; Gene Expression Regulation; Ki-67 Antigen; Lipopolysaccharides; Microglia; Myelin Basic Protein; Neuroblastoma; Oligodendroglia; Phosphorylation; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Stem Cells; Toll-Like Receptor 4

To determine if suppressing Nogo-A, an axonal inhibitory protein, will promote functional recovery in a murine model of multiple sclerosis (MS).. A small interfering RNA was developed to specifically suppress Nogo-A (siRNA-NogoA). The siRNA-NogoA silencing effect was evaluated in vitro and in vivo via immunohistochemistry. The siRNA was administered intravenously in 2 models of experimental autoimmune encephalomyelitis (EAE). Axonal repair was measured by upregulation of GAP43. Enzyme-linked immunosorbent assay, flow cytometry, and (3)H-thymidine incorporation were used to determine immunological changes in myelin-specific T cells in mice with EAE.. The siRNA-NogoA suppressed Nogo-A expression in vitro and in vivo. Systemic administration of siRNA-NogoA ameliorated EAE and promoted axonal repair, as demonstrated by enhanced GAP43+ axons in the lesions. Myelin-specific T-cell proliferation and cytokine production were unchanged in the siRNA-NogoA-treated mice.. Silencing Nogo-A in EAE promotes functional recovery. The therapeutic benefit appears to be mediated by axonal growth and repair, and is not attributable to changes in the encephalitogenic capacity of the myelin-specific T cells. Silencing Nogo-A may be a therapeutic option for MS patients to prevent permanent functional deficits caused by immune-mediated axonal damage.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; GAP-43 Protein; Gene Expression Regulation; Glycoproteins; Interferon-gamma; Interleukin-10; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin Basic Protein; Myelin Proteins; Myelin-Oligodendrocyte Glycoprotein; Neuroblastoma; Nogo Proteins; Peptide Fragments; RNA, Small Interfering; Spinal Cord; Tetradecanoylphorbol Acetate; Transfection

We show here that the choline transporter-like (CTL) family is more extensive than initially described with five genes in humans and complex alternative splicing. In adult rat tissues, CTL2-4 mRNAs are mainly detected in peripheral tissues, while CTL1 is widely expressed throughout the nervous system. During rat post-natal development, CTL1 is expressed in several subpopulations of neurones and in the white matter, where its spatio-temporal distribution profile recalls that of myelin basic protein, an oligodendrocyte marker. We identified two major rat splice variants of CTL1 (CTL1a and CTL1b) differing in their carboxy-terminal tails with both able to increase choline transport after transfection in neuroblastoma cells. In the developing brain, CTL1a is expressed in both neurones and oligodendroglial cells, whereas CTL1b is restricted to oligodendroglial cells. These findings suggest specific roles for CTL1 splice variants in both neuronal and oligodendrocyte physiology.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Animals, Newborn; Blotting, Northern; Brain; Cell Line, Tumor; Choline; Choline O-Acetyltransferase; DNA; Gene Expression Regulation, Developmental; Humans; In Situ Hybridization; Male; Membrane Transport Proteins; Mice; Molecular Sequence Data; Myelin Basic Protein; Neuroblastoma; Neurons; Oligodendroglia; Peripheral Nerves; Phylogeny; Protein Isoforms; Rats; Rats, Wistar; RNA; RNA, Messenger; Transfection; Tritium

The adhesion of eosinophils to nerve cells and the subsequent release of eosinophil products may contribute to the pathogenesis of conditions such as asthma and inflammatory bowel disease. In this study we have separately examined the consequences of eosinophil adhesion and degranulation for nerve cell morphology and development. Eosinophils induced neurite retraction of cultured guinea pig parasympathetic nerves and differentiated IMR32 cholinergic neuroblastoma cells. Inhibition of eosinophil adhesion to IMR32 cells attenuated this retraction. Eosinophil adhesion to IMR32 cells led to tyrosine phosphorylation of a number of nerve cell proteins, activation of p38 MAP kinase, and generation of neuronal reactive oxygen species (ROS). Inhibition of tyrosine kinases with genistein prevented both the generation of ROS in the nerve cells and neurite retraction. The p38 MAP kinase inhibitor SB-239063 prevented neurite retraction but had no effect on the induction of ROS. Thus eosinophils induced neurite retraction via two distinct pathways: by generation of tyrosine kinase-dependent ROS and by p38 MAP kinase. Eosinophils also prevented neurite outgrowth during differentiation of IMR32 cells. In contrast to their effect on neurite retraction, this effect was mimicked by medium containing products released from eosinophils and by eosinophil major basic protein. These results indicate that eosinophils modify the morphology of nerve cells by distinct mechanisms that involve adhesion and released proteins.

Topics: Animals; Cell Adhesion; Cell Degranulation; Cell Differentiation; Cell Line; Cellular Senescence; Enzyme Activation; Eosinophils; Guinea Pigs; Humans; In Vitro Techniques; Male; Mitogen-Activated Protein Kinases; Myelin Basic Protein; Neurites; Neuroblastoma; Neurons; p38 Mitogen-Activated Protein Kinases; Parasympathetic Nervous System; Polylysine; Protein-Tyrosine Kinases; Reactive Oxygen Species; Trachea

Neuroblastomas are neural crest-derived tumors that contain neuronal, melanocyte, and Schwann cell precursors. We examined the effects of treatment with gamma-interferon (gamma-IFN) and nerve growth factor (NGF), alone, and in combination, on these progenitor subpopulations in the human neuroblastoma cell line, SH-SY5Y. Using fluorescence-activated flow cytometry (FACS), changes in expression of three differentiation-specific or -associated marker proteins, the 200 kD neurofilament protein, the myelin basic protein, and the S-100 protein, were analyzed. Growth rates and morphological changes associated with each treatment over the 2-wk incubation period were noted. The greatest effects were observed with combined IFN + NGF treatment. These were significant increases in expression of all three proteins, distinctive morphological signs of differentiation, and extensive inhibition of proliferation compared to control cultures. Treatment with NGF alone resulted in increased neurofilament protein expression and in the length and number of neurite extensions, but there was no effect on the growth rate. IFN induced striking morphological changes, significant inhibition of growth, and changes in protein expression that correlated with neuronal to non-neuronal subpopulation shifts due to the death of differentiated cells. When treatment was discontinued after 15 d, the morphological changes induced by NGF were reversed within 2-3 d, while those induced by IFN +/- NGF were present up to 4 wk post-treatment. Small, neuroblastic colonies were observed throughout the treatment period and within 4-6 wk after the cessation of treatment this cell-type fully reconstituted the cultures suggesting the presence of a stem cell. Our results indicate that treatment with gamma-IFN +/- NGF can regulate growth and induce, either stem cells or progenitor neuronal, Schwann and melanocyte subpopulations in the SH-SY5Y cell line to irreversibly differentiate.

Topics: Cell Division; Flow Cytometry; Humans; Interferon-gamma; Melanocytes; Myelin Basic Protein; Nerve Growth Factors; Neural Crest; Neuroblastoma; Neurofilament Proteins; Neurons; S100 Proteins; Schwann Cells; Stem Cells; Tumor Cells, Cultured

We examined the effects of endogenous basic proteins rich in the amino acid L-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca2+ gradient by a Ca2+ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influx of extracellular Ca2+ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca2+ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism of L-arginine into L-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.

Topics: Amino Acid Oxidoreductases; Animals; Binding, Competitive; Calcium; Citrulline; Cyclic GMP; Cytosol; Histones; Myelin Basic Protein; Nerve Tissue Proteins; Neuroblastoma; Neurons; Nitric Oxide Synthase; Protamines; Rats; Stimulation, Chemical; Tumor Cells, Cultured

Protein phosphorylation and subsequent dephosphorylation was studied in digitonin-permeabilized neuroblastoma SH-SY5Y cells by measuring the incorporation of [32P]phosphate into myelin basic protein (MBP). 1,2-Dioctanoyl-sn-glycerol (DOG) and calcium synergistically induced phosphorylation of MBP, which was inhibited by the protein kinase C (PKC) pseudosubstrate peptide (PKC19-36). The phosphorylation increased for 10 min when a net dephosphorylation started to appear. The dephosphorylation was inhibited by okadaic acid. Regardless of calcium concentration, the presence of DOG was necessary for significant effects of okadaic acid on MBP phosphorylation. H7 and staurosporine dose-dependently inhibited the phosphorylation of MBP, induced by DOG and calcium in the presence of okadaic acid. Different PKC pseudosubstrate peptides were applied and all showed an inhibitory effect on the phosphorylation of MBP under these conditions. These results demonstrate the presence, in SH-SY5Y cells, of a protein phosphatase, possibly protein phosphatase 2A, with a high basal activity that counteracts PKC-induced phosphorylation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Amino Acid Sequence; Calcium; Cell Membrane Permeability; Diglycerides; Ethers, Cyclic; Humans; Isoenzymes; Isoquinolines; Molecular Sequence Data; Myelin Basic Protein; Neoplasm Proteins; Neuroblastoma; Okadaic Acid; Peptide Fragments; Phosphoprotein Phosphatases; Phosphorylation; Piperazines; Polymyxin B; Protein Kinase C; Protein Phosphatase 2; Protein Processing, Post-Translational; Signal Transduction; Staurosporine; Substrate Specificity; Tumor Cells, Cultured

We have studied transport and localization of MBP mRNA in oligodendrocytes in culture by microinjecting labeled mRNA into living cells and analyzing the intracellular distribution of the injected RNA by confocal microscopy. Injected mRNA initially appears dispersed in the perikaryon. Within minutes, the RNA forms granules which, in the case of MBP mRNA, are transported down the processes to the periphery of the cell where the distribution again becomes dispersed. In situ hybridization shows that endogenous MBP mRNA in oligodendrocytes also appears as granules in the perikaryon and processes and dispersed in the peripheral membranes. The granules are not released by extraction with non-ionic detergent, indicating that they are associated with the cytoskeletal matrix. Three dimensional visualization indicates that MBP mRNA granules are often aligned in tracks along microtubules traversing the cytoplasm and processes. Several distinct patterns of granule movement are observed. Granules in the processes undergo sustained directional movement with a velocity of approximately 0.2 micron/s. Granules at branch points undergo oscillatory motion with a mean displacement of 0.1 micron/s. Granules in the periphery of the cell circulate randomly with a mean displacement of approximately 1 micron/s. The results are discussed in terms of a multi-step pathway for transport and localization of MBP mRNA in oligodendrocytes. This work represents the first characterization of intracellular movement of mRNA in living cells, and the first description of the role of RNA granules in transport and localization of mRNA in cells.

Topics: Animals; Biological Transport; Cells, Cultured; Cytoplasmic Granules; Cytoskeleton; Immunohistochemistry; In Situ Hybridization; Mice; Microinjections; Myelin Basic Protein; Neuroblastoma; Oligodendroglia; RNA, Messenger; Time Factors; Tumor Cells, Cultured

Myelin basic protein (MBP) mRNAs are translocated from cell bodies into the slender processes connecting oligodendrocyte somas with the myelin sheath in vivo. This translocation was observed in mixed glial cultures prepared from newborn mouse brains and it occurred in approximately 25% of the cells expressing the gene. However, when "enriched" oligodendrocytes were prepared by shaking them free of other glial cells, MBP mRNA translocation occurred into the processes of essentially all of the cells. When enriched oligodendrocytes were plated back onto astrocytes, MBP mRNA was observed to be confined to the cell bodies of almost all the cells, indicating a marked inhibition of translocation of the mRNA. This inhibition of mRNA translocation did not appear to be mediated through soluble factors secreted by astrocytes or by "astromatrix," but rather through physical contact between the oligodendrocytes and astrocytes. Intact, but not necessarily live, astrocytes were required for the inhibition of mRNA translocation in the oligodendrocytes. Fibroblasts and a neuroblastoma cell line, SKN-SH, did not inhibit MBP mRNA translocation in oligodendrocytes suggesting that astrocyte surface-specific components might be involved in the interaction between astrocytes and oligodendrocytes in culture. These results suggest that contact between these two cell types can influence intramolecular events related to myelinogenesis.

Topics: Animals; Animals, Newborn; Astrocytes; Biological Transport; Cell Communication; Cells, Cultured; Fibroblasts; Mice; Mice, Inbred BALB C; Myelin Basic Protein; Neuroblastoma; Oligodendroglia; RNA, Messenger; Tumor Cells, Cultured

Human neuroblastoma (NB) cell lines have been suggested to represent a model of neural crest differentiation. The expression of several Schwann-cell-associated antigens was examined by flow cytometry and Northern blot analysis. Variable reactivity of the human NB cell lines was found in both the level and pattern of reactivity. Retinoic acid treatment of cell line SMS-KAN resulted in a neuron-like morphological differentiation and a decrease in several of the glial markers under study. Similarly, Northern blot analysis illustrated myelin-associated glycoprotein expression, and decreased expression of this message with retinoic acid treatment was consistent with the neuron-like morphological changes. Overall, human NB in vitro was found to be multipotential, but we have shown that it is capable of expressing several Schwann cell markers which are modulated during induced differentiation.

Topics: Biomarkers, Tumor; Blotting, Northern; Cell Line; Flow Cytometry; Gene Expression; Humans; Myelin Basic Protein; Myelin Proteins; Myelin-Associated Glycoprotein; Neuroblastoma; RNA; Schwann Cells; Tretinoin

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.

Topics: Acetophenones; Amino Acid Sequence; Arachidonic Acids; Calcium; Cell Membrane; Cell Membrane Permeability; Digitonin; Diglycerides; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Molecular Sequence Data; Myelin Basic Protein; Neuroblastoma; Phospholipases A; Phospholipases A2; Phosphorus; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Quinacrine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

To establish the neuroendocrine and neural features of peripheral neuroblastic tumors, a prospectively collected group of 12 neuroblastomas (NB), 2 ganglioneuroblastomas (GNB), and 4 ganglioneuromas (GN) was probed with a panel of monoclonal antibodies (MAbs) to neuroendocrine and neural antigens. All tumors expressed the pan-neuroendocrine markers synaptophysin and chromogranin A. They also showed extensive expression of neuronal antigens, ie, of each of the neurofilament (NF) triplet proteins and of the microtubule-associated proteins (MAPs) MAP2 and tau-protein. However, only in the GNBs and GNs was the pattern of NF phosphoisoforms relatively mature. In the latter tumors glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) could be demonstrated as well, suggesting the presence of nonmyelinating and myelinating Schwann cells, respectively. The glial markers did not colocalize with the neural markers. On the basis of these data, it was concluded that all peripheral neuroblastic tumors manifest molecular characteristics of neuroendocrine cells and of neurons. The latter were most developed in GNBs and GNs, in which they were accompanied by Schwann cell differentiation in a separate population of cells. The above-outlined neuronal profile of peripheral neuroblastic tumors, including NBs, distinguishes this group of tumors from the much-less neuronally differentiated primitive neuroectodermal tumors of the central nervous system.

Topics: Abdominal Neoplasms; Adrenal Gland Neoplasms; Adult; Antibodies, Monoclonal; Biomarkers, Tumor; Cell Differentiation; Cell Transformation, Neoplastic; Child; Child, Preschool; Chromogranin A; Chromogranins; Female; Ganglioneuroma; Glial Fibrillary Acidic Protein; Humans; Immune Sera; Immunohistochemistry; Infant; Intermediate Filament Proteins; Intermediate Filaments; Male; Membrane Proteins; Microtubule-Associated Proteins; Myelin Basic Protein; Nerve Tissue Proteins; Neuroblastoma; Neurons; Phosphorylation; Schwann Cells; Synaptophysin; tau Proteins