The Molecular Events Involved in Oligodendrocyte Precursor Cell Proliferation Induced by the Conditioned Medium from B104 Neuroblastoma Cells

The conditioned medium from B104 neuroblastoma cells (B104CM) induces proliferation of oligodendrocyte progenitor cells (OPCs) in vitro. However, the molecular events that occur during B104CM-induced proliferation of OPCs has not been well clarified. In the present study, using OPCs immunopanned from embryonic day 14 Sprague–Dawley rat spinal cords, we explored the activation of several signaling pathways and the expression of several important immediate early genes (IEGs) and cyclins in OPCs in response to B104CM. We found that B104CM can induce OPC proliferation through the activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2), but not PI3K or p38 MAPK signaling pathways in vitro. The IEGs involved in B104CM-induced OPC proliferation include c-fos, c-jun and Id2, but not c-myc, fyn, or p21. The cyclins D1, D2 and E are also involved in B104CM-stimulated proliferation of OPCs. The activation of Erk results in subsequent expression of IEGs (such as c-fos, c-jun and Id-2) and cyclins (including cyclin D1, D2 and E), which play key roles in cell cycle initiation and OPC proliferation. Collectively, these results suggest that the phosphorylation of Erk1/2 is an important molecular event during OPC proliferation induced by B104CM.     

E1B 55-Kilodalton-Associated Protein: a Cellular Protein with RNA-Binding Activity Implicated in Nucleocytoplasmic Transport of Adenovirus and Cellular mRNAs

The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.     

Transforming Growth Factor β1-Regulated Cell Proliferation and Expression of Neural Cell Adhesion Molecule in B104 Neuroblastoma Cells

The expression and activity of factors influencing early neuronal development are altered by ethanol. Such factors include growth factors, for example, platelet-derived growth factor and basic fibroblast growth factor (for cell proliferation), and cell adhesion molecules (for neuronal migration). One agent, transforming growth factor beta1 (TGFbeta1), may affect both events. We tested the hypothesis that ethanol alters myriad TGFbeta1-mediated activities [i.e., cell proliferation and neural cell adhesion molecule (N-CAM) expression] using B104 neuroblastoma cells. TGFbeta1 inhibited the proliferation of B104 cells as evidenced by decreases in cell number and [3H]thymidine ([3H]dT) incorporation. TGFbeta1 induced sustained activation of extracellular signal-regulated kinases (ERKs), which are part of the family of mitogen-activated protein kinases (MAPKs). Treatment with PD98059 (a MAPK kinase blocker) abolished TGFbeta1-regulated inhibition of [3H]dT incorporation. TGFbeta1-mediated growth inhibition was potentiated by ethanol exposure. Ethanol also produced prolonged activation of ERK, an effect that was partially eliminated by treatment with PD98059. On the other hand, TGFbeta1 up-regulated N-CAM expression, and this up-regulation was not affected by treatment with PD98059. Ethanol inhibited the TGFbeta1-induced up-regulation of N-CAM expression in a concentration-dependent manner. Thus, TGFbeta1 affects ERK-dependent cell proliferation and ERK-independent N-CAM expression in B104 cells. Both activities are sensitive to ethanol and may underlie the ethanol-induced alterations in the proliferation and migration of CNS neurons.     

From the Cell Membrane to the Nucleus: Unearthing Transport Mechanisms for Dynein

Mutations in the motor protein cytoplasmic dynein have been found to cause Charcot-Marie-Tooth disease, spinal muscular atrophy, and severe intellectual disabilities in humans. In mouse models, neurodegeneration is observed. We sought to develop a novel model which could incorporate the effects of mutations on distance travelled and velocity. A mechanical model for the dynein mediated transport of endosomes is derived from first principles and solved numerically. The effects of variations in model parameter values are analysed to find those that have a significant impact on velocity and distance travelled. The model successfully describes the processivity of dynein and matches qualitatively the velocity profiles observed in experiments.     

Contribution of G Protein Activation to Fluoride Stimulation of Phosphoinositide Hydrolysis in Human Neuroblastoma Cells

Abstract: To examine the possibility that NaF enhances phosphoinositide-specific phospholipase C (PIC) activity in neural tissues by a mechanism independent of a guanine nucleotide binding protein (G_p), we have evaluated the contribution of G_p activation to NaF-stimulated phosphoinositide hydrolysis in human SK-N-SH neuroblastoma cells. Addition of NaF to intact cells resulted in an increase in the release of inositol phosphates (450% of control values; EC₅₀ of ～ 8 mM). Inclusion of U-73122, an aminosteroid inhibitor of guanine nucleotide-regulated PIC activity in these cells, resulted in a dose-dependent inhibition of NaF-stimulated inositol lipid hydrolysis (IC₅₀ of ～ 3.5 μM). When added to digitonin-permeabilized cells, NaF or guanosine-5′-O-thiotriphosphate (GTPγS) resulted in a three- and sevenfold enhancement, respectively, of inositol phosphate release. In the combined presence of optimal concentrations of NaF and GTPγS, inositol phosphate release was less than additive, indicative of a common site of action. Inclusion of 2–5 mM concentrations of guanosine-5′-O-(2-thiodiphosphate) (GDPβS) fully blocked phosphoinositide hydrolysis elicited by GTPγS, whereas that induced by NaF was partially inhibited (65%). However, preincubation of the cells with GDPβS resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTPγS and NaF-stimulated inositol phosphate release were inhibited by inclusion of 10 μM U-73122 (54–71%). The presence of either NaF or GTPγS also resulted in a marked lowering of the Ca²⁺ requirement for activation of PIC in permeabilized cells. These results indicate that in SK-N-SH cells, little evidence exists for direct stimulation of PIC by NaF and that the majority of inositol phosphate release that occurs in the presence of NaF can be attributed to activation of G_p.     

A Novel, Drug-based, Cellular Assay for the Activity of Neurotoxic Mutants of the Prion Protein

In prion diseases, the infectious isoform of the prion protein (PrP^Sc) may subvert a normal, physiological activity of the cellular isoform (PrP^C). A deletion mutant of the prion protein (Δ105–125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP^C and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Δ105–125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Δ105–125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Δ105–125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.     

Two Types of Apoptotic Cell Death of Rat Central Nervous System-Derived Neuroblastoma B50 and B104 Cells: Apoptosis Induced During Proliferation and After Differentiation

Abstract: We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 m M DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.     

Protein Folding in the Cell: On the Mechanisms of Its Acceleration

The mechanisms responsible for protein folding in the cell can be divided in two groups. The ones in the first group would be those preventing the aggregation of unfolded polypeptide chains or of incompletely folded proteins, as well as the mechanisms which provide for the energy-consuming unfolding of incorrectly folded structures, giving them a chance to begin a new folding cycle. Mechanisms of this type do not affect the rate of folding (it occurs spontaneously), yet considerably increase the efficiency of the entire process. By contrast, the mechanisms belonging to second group actually accelerate protein folding by exerting a direct influence on the rate-limiting steps of the overall reaction. Although not a conventional one, such a classification helps define the topic of this review. Its main purpose is to discuss the ability of chaperonins (and that of some chaperones) to interact directly with substrate proteins in the course of their folding and thus accelerate the rate-limiting steps of that process. (Mechanisms of protein folding acceleration produced by the action of enzymes, e.g., peptidyl-prolyl cis/trans isomerase and protein disulfide isomerase, are not considered in this review.) Specific cases demonstrating an accelerated folding of some proteins encapsulated in the bacterial chaperonin GroEL cavity are considered, and the conditions favoring such acceleration are examined. Experimental data supporting the notion that the structure and functional properties of GroEL are not optimal for an effective folding of many of its substrate proteins is discussed. The current status of research on the mechanism behind the active participation of different subunits of eucaryotic cytosol chaperonin (CCT) in the final steps of the folding of actin and tubulin is reviewed. Particular attention is devoted to steric chaperones, which dramatically accelerate the formation of the native structure of their substrate proteins by stabilizing certain folding intermediates. The structural foundations underlying the effect of the subtilisin pro-domain on the folding of the mature enzyme are considered. The prospects of future studies into the mechanisms responsible for accelerating protein folding in the cell are commented upon.     

Role of the Disulfide Bond in Prion Protein Amyloid Formation: A Thermodynamic and Kinetic Analysis

Prion diseases are associated with the structural conversion of prion protein (PrP) to a β-sheet-rich aggregate, PrP^Sc. Previous studies have indicated that a reduction of the disulfide bond linking C179 and C214 of PrP yields an amyloidlike β-rich aggregate in vitro. To gain mechanistic insights into the reduction-induced aggregation, here I characterized how disulfide bond reduction modulates the protein folding/misfolding landscape of PrP, by examining 1) the equilibrium stabilities of the native (N) and aggregated states relative to the unfolded (U) state, 2) the transition barrier separating the U and aggregated states, and 3) the final structure of amyloidlike misfolded aggregates. Kinetic and thermodynamic experiments revealed that disulfide bond reduction decreases the equilibrium stabilities of both the N and aggregated states by ～3 kcal/mol, without changing either the amyloidlike aggregate structure, at least at the secondary structural level, or the transition barrier of aggregation. Therefore, disulfide bond reduction modulates the protein folding/misfolding landscape by entropically stabilizing disordered states, including the U and transition state of aggregation. This also indicates that the equilibrium stability of the N state, but not the transition barrier of aggregation, is the dominant factor determining the reduction-induced aggregation of PrP.     

Selective Hsp70-Dependent Docking of Hsp104 to Protein Aggregates Protects the Cell from the Toxicity of the Disaggregase

Hsp104 is a yeast chaperone that rescues misfolded proteins from aggregates associated with proteotoxic stress and aging. Hsp104 consists of N-terminal domain, regulatory M-domain and two ATPase domains, assembled into a spiral-shaped hexamer. Protein disaggregation involves polypeptide extraction from an aggregate and its translocation through the central channel. This process relies on Hsp104 cooperation with the Hsp70 chaperone, which also plays important role in regulation of the disaggregase. Although Hsp104 protein-unfolding activity enables cells to survive stress, when uncontrolled, it becomes toxic to the cell.In this work, we investigated the significance of the interaction between Hsp70 and the M-domain of Hsp104 for functioning of the disaggregation system. We identified phenylalanine at position 508 in Hsp104 to be the key site of interaction with Hsp70. Disruption of this site makes Hsp104 unable to bind protein aggregates and to confer tolerance in yeast cells. The use of this Hsp104 variant demonstrates that Hsp70 allows successful initiation of disaggregation only as long as it is able to interact with the disaggregase. As reported previously, this interaction causes release of the M-domain-driven repression of Hsp104. Now we reveal that, apart from this allosteric effect, the interaction between the chaperone partners itself contributes to effective initiation of disaggregation and plays important role in cell protection against Hsp104-induced toxicity. Interaction with Hsp70 shifts Hsp104 substrate specificity from non-aggregated, disordered substrates toward protein aggregates. Accordingly, Hsp70-mediated sequestering of the Hsp104 unfoldase in aggregates makes it less toxic and more productive.     

Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B

Hepatitis B virus (HBV) infection is one of the major problems that threatens global health. There have been many studies on HBV, but the relationship between HBV and host factors is largely unexplored and more studies are needed to clarify these interactions. Filamin B is an actin-binding protein that acts as a cytoskeleton protein, and it is involved in cell development and several signaling pathways. In this study, we showed that filamin B interacted with HBV core protein, and the interaction promoted HBV replication. The interaction between filamin B and core protein was observed in HEK 293T, Huh7 and HepG2 cell lines by co-immunoprecipitation and co-localization immnofluoresence. Overexpression of filamin B increased the levels of HBV total RNAs and pre-genome RNA (pgRNA), and improved the secretion level of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). In contrast, filamin B knockdown inhibited HBV replication, decreased the level of HBV total RNAs and pgRNA, and reduced the secretion level of HBsAg and HBeAg. In addition, we found that filamin B and core protein may interact with each other via four blocks of argentine residues at the C-terminus of core protein. In conclusion, we identify filamin B as a novel host factor that can interact with core protein to promote HBV replication in hepatocytes. Our study provides new insights into the relationship between HBV and host factors and may provide new strategies for the treatment of HBV infection.     

Organotypic Hippocampal Slice Cultures: A Model System to Study Basic Cellular and Molecular Mechanisms of Neuronal Cell Death,Neuroprotection, and Synaptic Plasticity

The hippocampus has become one of the most extensively studied areas of the mammalian brain, and its proper function is of utmost importance, particularly for learning and memory. The hippocampus is the most susceptible brain region for damage, and its impaired function has been documented in many human brain diseases, e.g. hypoxia, ischemia, and epilepsy regardless of the age of the affected patients. In addition to experimental in vivo models of these disorders, the investigation of basic anatomical, physiological, and molecular aspects requires an adequate experimental in vitro model, which should meet the requirements for well-preserved representation of various cell types, and functional information processing properties in the hippocampus. In this review, the characteristics of organotypic hippocampal slice cultures (OHCs) together with the main differences between the in vivo and in vitro preparations are first briefly outlined. Thereafter, the use of OHCs in studies focusing on neuron cell death and synaptic plasticity is discussed. Special issue dedicated to Dr. Simo S. Oja     

Selective vulnerability to neurodegenerative disease: the curious case of Prion Protein

The mechanisms underlying the selective targeting of specific brain regions by different neurodegenerative diseases is one of the most intriguing mysteries in medicine. For example, it is known that Alzheimer’s disease primarily affects parts of the brain that play a role in memory, whereas Parkinson’s disease predominantly affects parts of the brain that are involved in body movement. However, the reasons that other brain regions remain unaffected in these diseases are unknown. A better understanding of the phenomenon of selective vulnerability is required for the development of targeted therapeutic approaches that specifically protect affected neurons, thereby altering the disease course and preventing its progression. Prion diseases are a fascinating group of neurodegenerative diseases because they exhibit a wide phenotypic spectrum caused by different sequence perturbations in a single protein. The possible ways that mutations affecting this protein can cause several distinct neurodegenerative diseases are explored in this Review to highlight the complexity underlying selective vulnerability. The premise of this article is that selective vulnerability is determined by the interaction of specific protein conformers and region-specific microenvironments harboring unique combinations of subcellular components such as metals, chaperones and protein translation machinery. Given the abundance of potential contributory factors in the neurodegenerative process, a better understanding of how these factors interact will provide invaluable insight into disease mechanisms to guide therapeutic discovery.KEY WORDS: Huntington’s disease, Neurodegeneration, Spinocerebellar ataxia, Knock-in mice, Neuropathology, Prion diseases     

The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

PrP^Sc, a misfolded and aggregated form of the cellular prion protein PrP^C, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrP^C in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrP^C and PrP^Sc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrP^C. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrP^C. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrP^Sc. Other antibodies immunoprecipitate PrP^C, but not PrP^Sc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrP^C and PrP^Sc. Amino-proximal antibodies were found to react with repetitive PrP^C epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.     

In Silico Analysis of Prion Protein Mutants: A Comparative Study by Molecular Dynamics Approach

Polymorphisms in the human prion proteins lead to amino acid substitutions by the conversion of PrPC to PrPSc and amyloid formation, resulting in prion diseases such as familial Creutzfeldt–Jakob disease, Gerstmann–Straussler–Scheinker disease and fatal familial insomnia. Cation–π interaction is a non-covalent binding force that plays a significant role in protein stability. Here, we employ a novel approach by combining various in silico tools along with molecular dynamics simulation to provide structural and functional insight into the effect of mutation on the stability and activity of mutant prion proteins. We have investigated impressions of prevalent mutations including 1E1S, 1E1P, 1E1U, 1E1P, 1FKC and 2K1D on the human prion proteins and compared them with wild type. Structural analyses of the models were performed with the aid of molecular dynamics simulation methods. According to our results, frequently occurred mutations were observed in conserved sequences of human prion proteins and the most fluctuation values appear in the 2K1D mutant model at around helix 4 with residues ranging from 190 to 194. Our observations in this study could help to further understand the structural stability of prion proteins.     

Poly-N-Acetyllactosamine Glycans of Cellular Glycoproteins: Predominance of Linear Chains in Mouse Neuroblastoma and Rat Pheochromocytoma Cell Lines

To study the properties of protein-bound oligosaccharides in neuronally differentiating cells, two model systems were used: murine N1E-115 and N-18 neuroblastoma cells inducible by serum starvation and rat PC12 pheochromocytoma cells inducible by nerve growth factor. Glycopeptides were prepared from cells metabolically labeled with [3H]glucosamine and analyzed by gel filtration. The properties of the high-molecular-weight glycopeptides were studied using enzymatic digestion with neuraminidase and endo-beta-galactosidase. In contrast to other cell lines analyzed, the neuroblastoma and pheochromocytoma lines contained predominantly glycopeptides completely cleavable with endo-beta-galactosidase, which indicated that they were linear-type poly-N-acetyllactosamine glycans. The proportion of these linear chains in the high-molecular-weight fraction increased during neuronal differentiation in both cell systems. The linear nature of the glycans was also correlated with positive anti-i and negative anti-I reactivity of the cells in immunofluorescence microscopy. Specific cell surface labeling for poly-N-acetyllactosamine glycans and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several glycoprotein components, some of which showed changes during neuronal differentiation. The high proportion of linear poly-N-acetyllactosamine chains in these neuronal cell lines and its increase during neuronal differentiation suggests that these glycans may be a characteristic feature of neuronal or neuronally differentiating cells.     

Multi-Scale Modeling in Morphogenesis: A Critical Analysis of the Cellular Potts Model

Cellular Potts models (CPMs) are used as a modeling framework to elucidate mechanisms of biological development. They allow a spatial resolution below the cellular scale and are applied particularly when problems are studied where multiple spatial and temporal scales are involved. Despite the increasing usage of CPMs in theoretical biology, this model class has received little attention from mathematical theory. To narrow this gap, the CPMs are subjected to a theoretical study here. It is asked to which extent the updating rules establish an appropriate dynamical model of intercellular interactions and what the principal behavior at different time scales characterizes. It is shown that the longtime behavior of a CPM is degenerate in the sense that the cells consecutively die out, independent of the specific interdependence structure that characterizes the model. While CPMs are naturally defined on finite, spatially bounded lattices, possible extensions to spatially unbounded systems are explored to assess to which extent spatio-temporal limit procedures can be applied to describe the emergent behavior at the tissue scale. To elucidate the mechanistic structure of CPMs, the model class is integrated into a general multiscale framework. It is shown that the central role of the surface fluctuations, which subsume several cellular and intercellular factors, entails substantial limitations for a CPM''s exploitation both as a mechanistic and as a phenomenological model.