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991.
We have studied the role of different amino acids in the M2 transmembrane domain of the α7 neuronal nicotinic receptor by mutating residues that differ from the ones located at the same positions in other α (α2-α10) or β (β2-β4) subunits. Our aim was to investigate the contribution of these amino acids to the peculiar kinetic and inward rectification properties that differentiate the homomeric α7 receptor from other nicotinic receptors. Mutations of several residues strongly modified receptor function. We found that Thr245 had the most profound effect when mutated to serine, an amino acid present in all heteromeric receptors composed of α and β subunits, by dramatically increasing the maximal current, decreasing the decaying rate of the currents and decreasing receptor rectification. Some mutants also showed altered agonist-binding properties as revealed by shifts in the dose-response curves for acetylcholine. We conclude that residues in the M2 segment and flanking regions contribute to the unusual properties of the α7 receptor, especially to its characteristic fast kinetic behavior and strong inward rectification and furthermore to the potency of agonists. 相似文献
992.
Owczarek S Soroka V Kiryushko D Larsen MH Yuan Q Sandi C Berezin V Bock E 《Journal of neurochemistry》2011,117(6):984-994
Neuroplastin-65 (Np65) is a brain-specific cell adhesion molecule belonging to the immunoglobulin superfamily. Homophilic trans-interaction of Np65 mediates adhesion between cells and modulates synaptic plasticity. This interaction solely occurs through the first immunoglobulin (Ig) module of Np65, but the exact binding mechanism has not yet been elucidated. In this study, we identify the homophilic binding motif of Np65 and show that a synthetic peptide modeled after this motif, termed enplastin, binds to Np65. We demonstrate that both Np65- and enplastin-induced intracellular signaling depends on fibroblast growth factor receptor, p38 mitogen-activated protein kinase, Ca(2+) /calmodulin-dependent protein kinase, and cytoplasmic Ca(2+) concentration. In addition, we show that interference with Np65 homophilic binding by enplastin has an inhibitory effect on Np65-mediated neurite outgrowth in vitro and on the initial phase of spatial learning in rats. 相似文献
993.
Adam SobczakKatarzyna Debowska Magdalena BlazejczykMichael R. Kreutz Jacek Kuznicki Urszula Wojda 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2011,1813(5):1025-1037
Calmyrin1 (CaMy1) is an EF-hand Ca2+-binding protein expressed in several cell types, including brain neurons. Using a yeast two-hybrid screen of a human fetal brain cDNA library, we identified SCG10 protein (stathmin2) as a CaMy1 partner. SCG10 is a microtubule-destabilizing factor involved in neuronal growth during brain development. We found increased mRNA and protein levels of CaMy1 during neuronal development, which paralleled the changes in SCG10 levels. In developing primary rat hippocampal neurons in culture, CaMy1 and SCG10 colocalized in cell soma, neurites, and growth cones. Pull-down, coimmunoprecipitation, and proximity ligation assays demonstrated that the interaction between CaMy1 and SCG10 is direct and Ca2+-dependent in vivo and requires the C-terminal domain of CaMy1 (residues 99-192) and the N-terminal domain of SCG10 (residues 1-35). CaMy1 did not interact with stathmin1, a protein that is homologous with SCG10 but lacks the N-terminal domain characteristic of SCG10. CaMy1 interfered with SCG10 inhibitory activity in a microtubule polymerization assay. Moreover, CaMy1 overexpression inhibited SCG10-mediated neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. This CaMy1 activity did not occur when an N-terminally truncated SCG10 mutant unable to interact with CaMy1 was expressed. Altogether, these data suggest that CaMy1 via SCG10 couples Ca2+ signals with the dynamics of microtubules during neuronal outgrowth in the developing brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium. 相似文献
994.
995.
Matthew P. Taylor Radomir Kratchmarov Lynn W. Enquist 《Journal of visualized experiments : JoVE》2013,(78)
Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread. 相似文献
996.
Brain ischemia often results in neuronal necrosis, which may spread death to neighboring cells. However, the molecular events of neuronal necrosis and the mechanisms of this spreading death are poorly understood due to the limited genetic tools available for deciphering complicated responses in mammalian brains. Here, we engineered a Drosophila model of necrosis in a sub-population of neurons by expressing a leaky cation channel in the Drosophila eye. Expression of this channel caused necrosis in defined neurons as well as extensive spreading of cell death. Jun N-terminal kinase (JNK)-mediated, caspase-independent apoptosis was the primary mechanism of cell death in neurons, while caspase-dependent apoptosis was primarily involved in non-neuronal cell death. Furthermore, the JNK activation in surrounding neurons was triggered by reactive oxygen species (ROS) and Eiger (Drosophila tumor necrosis factor α (TNFα)) released from necrotic neurons. Because the Eiger/ROS/JNK signaling was also required for cell death induced by hypoxia and oxidative stress, our fly model of spreading death may be similar to brain ischemia in mammals. We performed large-scale genetic screens to search for novel genes functioning in necrosis and/or spreading death, from which we identified several classes of genes. Among them, Rho-associated kinase (ROCK) had been reported as a promising drug target for stroke treatment with undefined mechanisms. Our data indicate that ROCK and the related trafficking pathway genes regulate neuronal necrosis. We propose the suppression of the function of the trafficking system, ROS and cytokines, such as TNFα, as translational applications targeting necrosis and spreading death. 相似文献
997.
Highly conserved microRNA-9 (miR-9) has a critical role in various cellular processes including neurogenesis. However, its regulation by neurotropins that are known to mediate neurogenesis remains poorly defined. In this study, we identify platelet-derived growth factor-BB (PDGF-BB)-mediated upregulation of miR-9, which in turn downregulates its target gene monocyte chemotactic protein-induced protein 1 (MCPIP1), as a key player in modulating proliferation, neuronal differentiation as well as migration of neuronal progenitor cells (NPCs). Results indicate that miR-9-mediated NPC proliferation and neuronal differentiation involves signaling via the nuclear factor-kappa B (NF-κB) and cAMP response element-binding protein (CREB) pathways, and that NPC migration involves CREB but not the NF-κB signaling. These findings thus suggest that miR-9-mediated downregulation of MCPIP1 acts as a molecular switch regulation of neurogenesis. 相似文献
998.
Kumiko Sengoku Yuji Kurihara Fumio Nakamura Yukio Sasaki Yasufumi Sato Masayuki Yamane Masayuki Matsushita Angus C. Nairn Ken Takamatsu Yoshio Goshima Kohtaro Takei 《Developmental neurobiology》2013,73(3):230-246
Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry‐induced increase of phosphorylation of eukaryotic elongation factor‐2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2‐mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore‐assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin‐dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)‐evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP‐induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013 相似文献
999.
《朊病毒》2013,7(2):125-135
AbstractTransmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases caused by the misfolding of the cellular prion protein to an infectious form PrPSc. The intercellular transfer of PrPSc is a question of immediate interest as the cell-to-cell movement of the infectious particle causes the inexorable propagation of disease. We have previously identified tunneling nanotubes (TNTs) as one mechanism by which PrPSc can move between cells. Here we investigate further the details of this mechanism and show that PrPSc travels within TNTs in endolysosomal vesicles. Additionally we show that prion infection of CAD cells increases both the number of TNTs and intercellular transfer of membranous vesicles, thereby possibly playing an active role in its own intercellular transfer via TNTs. 相似文献
1000.
Sebastian Schildge Christian Bohrer Kristina Beck Christian Schachtrup 《Journal of visualized experiments : JoVE》2013,(71)
Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology. 相似文献