Primary Neurons (primary + neuron)

Distribution by Scientific Domains


Selected Abstracts


Involvement of astroglial ceramide in palmitic acid-induced Alzheimer-like changes in primary neurons

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2007
Sachin Patil
Abstract A high-fat diet has been shown to significantly increase the risk of the development of Alzheimer's disease (AD), a neurodegenerative disease histochemically characterized by the accumulation of amyloid beta (A,) protein in senile plaques and hyperphosphorylated tau in neurofibrillary tangles. Previously, we have shown that saturated free fatty acids (FFAs), palmitic and stearic acids, caused increased amyloidogenesis and tau hyperphosphorylaion in primary rat cortical neurons. These FFA-induced effects observed in neurons were found to be mediated by astroglial FFA metabolism. Therefore, in the present study we investigated the basic mechanism relating astroglial FFA metabolism and AD-like changes observed in neurons. We found that palmitic acid significantly increased de-novo synthesis of ceramide in astroglia, which in turn was involved in inducing both increased production of the A, protein and hyperphosphorylation of the tau protein. Increased amyloidogenesis and hyperphoshorylation of tau lead to formation of the two most important pathophysiological characteristics associated with AD, A, or senile plaques and neurofibrillary tangles, respectively. In addition to these pathophysiological changes, AD is also characterized by certain metabolic changes; abnormal cerebral glucose metabolism is one of the distinct characteristics of AD. In this context, we found that palmitic acid significantly decreased the levels of astroglial glucose transporter (GLUT1) and down-regulated glucose uptake and lactate release by astroglia. Our present data establish an underlying mechanism by which saturated fatty acids induce AD-associated pathophysiological as well as metabolic changes, placing ,astroglial fatty acid metabolism' at the center of the pathogenic cascade in AD. [source]


Regulation of the norepinephrine transporter by ,-synuclein-mediated interactions with microtubules

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007
Alexis M. Jeannotte
Abstract ,-Synuclein (,-Syn) regulates catecholaminergic neurotransmission. We demonstrate that ,-Syn regulates the activity and surface expression of the norepinephrine transporter (NET), depending on its expression levels. In cells co-transfected with NET and low amounts of ,-Syn, NET activity and cell surface expression were increased and protein interactions with ,-Syn decreased, compared with cells transfected with NET alone. Converse effects were observed at higher levels of ,-Syn expression. Treatment with nocodazole and other microtubule (MT) destabilizers abolished the expression-dependent bimodal regulation of NET by ,-Syn. At low ,-Syn levels, nocodazole had no effect on NET surface expression or protein interactions, while inducing increases in these measures at higher levels. Cells that were transfected with NET alone displayed no sensitivity to nocodazole, indicating that ,-Syn expression was necessary for the MT-dependent changes in NET activity. MT destabilizers also caused a significant increase in [3H]-NE uptake in brainstem primary neurons and synaptosomes from the frontal cortex, but not striatal synaptosomes. These findings suggest that the surface localization and activity of NET is modulated by ,-Syn in a manner that is both dependent on interactions with the MT cytoskeleton and varies across brain regions. [source]


Fractalkine reduces N -methyl- d -aspartate-induced calcium flux and apoptosis in human neurons through extracellular signal-regulated kinase activation

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2004
Kumaran Deiva
Abstract Our purpose was to investigate in human neurons the neuroprotective pathways induced by Fractalkine (FKN) against glutamate receptor-induced excitotoxicity. CX3CR1 and FKN are expressed constitutively in the tested human embryonic primary neurons and SK-N-SH, a human neuroblastoma cell line. Microfluorometry assay demonstrated that CX3CR1 was functional in 44% of primary neurons and in 70% of SK-N-SH. Fractalkine induced ERK1/2 phosphorylation within 1 min and Akt phosphorylation after 10 min, and both phosphorylation decreased after 20 min. No p38 and SAPK/JNK activation was observed after FKN treatment. Application of FKN triggered a 53% reduction of the NMDA-induced neuronal calcium influx, which was insensitive to pertussis toxin and LY294002 an inhibitor of Akt pathway, but abolished by PD98059, an ERK1/2 pathway inhibitor. Moreover, FKN significantly reduced neuronal NMDA-induced apoptosis, which was pertussis toxin insensitive and abolished in presence of PD98059 and LY294002. In conclusion, FKN protected human neurons from NMDA-mediated excitotoxicity in at least two ways with different kinetics: (i) an early ERK1/2 activation which reduced NMDA-mediated calcium flux; and (ii), a late Akt activation associated with the previously induced ERK1/2 activation. [source]


Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2004
Suling Zhao
Abstract We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3 -directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3,GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3,GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3,GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells. [source]


A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formation

GLIA, Issue 11 2007
Therese M. Roth
Abstract The neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. One known function of neurofibromin, the NF1 protein product, is to accelerate the slow intrinsic GTPase activity of Ras to increase the production of inactive rasGDP, with wide-ranging effects on p21ras pathways. Loss of neurofibromin in the autosomal dominant disorder NF1 is associated with tumors of the peripheral nervous system, particularly neurofibromas, benign lesions in which the major affected cell type is the Schwann cell (SC). NF1 is the most common cancer predisposition syndrome affecting the nervous system. We have developed an in vitro system for differentiating mouse embryonic stem cells (mESC) that are NF1 wild type (+/+), heterozygous (+/,), or null (,/,) into SC-like cells to study the role of NF1 in SC development and tumor formation. These mES-generated SC-like cells, regardless of their NF1 status, express SC markers correlated with their stage of maturation, including myelin proteins. They also support and preferentially direct neurite outgrowth from primary neurons. NF1 null and heterozygous SC-like cells proliferate at an accelerated rate compared to NF1 wild type; this growth advantage can be reverted to wild type levels using an inhibitor of MAP kinase kinase (Mek). The mESC of all NF1 types can also be differentiated into neuron-like cells. This novel model system provides an ideal paradigm for studies of the role of NF1 in cell growth and differentiation of the different cell types affected by NF1 in cells with differing levels of neurofibromin that are neither transformed nor malignant. © 2007 Wiley-Liss, Inc. [source]


Regulated interactions of the norepineprhine transporter by the actin and microtubule cytoskeletons

JOURNAL OF NEUROCHEMISTRY, Issue 5 2008
Alexis M. Jeannotte
Abstract One role of the actin cytoskeleton is to maintain the structural morphology and activity of the pre-synaptic terminal. We sought to determine if the actin cytoskeleton plays a role in regulating interactions between the norepinephrine transporter (NET) and alpha-Synuclein (,-Syn), two proteins expressed in the pre-synaptic terminal. In cells transfected with either 0.5 ,g/mL or 3 ,g/mL of ,-Syn and 1 ,g/mL of NET DNA, treatment with cytochalasin D, an actin depolymerizing agent, caused a dose-dependent decrease and increase, respectively, in [3H]-NE uptake. Protein interactions between NET, ,-actin, and ,-Syn were modified, along with levels of surface transporters. Treatment of primary brainstem neurons and frontal cortex synaptosomes with cytochalasin D caused a 115% and 28% increase, respectively, in NET activity. Depolymerization of both actin and microtubules did not alter NET activity in cells with 0.5 ,g/mL ,-Syn, but caused an increase in [3H]-NE uptake in cells transfected with 3 ,g/mL of ,-Syn and primary neurons. This is the first direct demonstration of NET activity being regulated via actin and modulated by interactions with ,-Syn. [source]


Partitioning of the plasma membrane Ca2+ -ATPase into lipid rafts in primary neurons: effects of cholesterol depletion

JOURNAL OF NEUROCHEMISTRY, Issue 2 2007
Lei Jiang
Abstract Spatial and temporal alterations in intracellular calcium [Ca2+]i play a pivotal role in a wide array of neuronal functions. Disruption in Ca2+ homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca2+ -ATPase (PMCA) is a high affinity Ca2+ transporter that plays a crucial role in the termination of [Ca2+]i signals and in the maintenance of low [Ca2+]i essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or ,lipid rafts' in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft-associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca2+ signaling in the central nervous system. [source]


A phosphatidylinositol transfer protein ,-dependent survival factor protects cultured primary neurons against serum deprivation-induced cell death

JOURNAL OF NEUROCHEMISTRY, Issue 3 2006
Hanneke Bunte
Abstract Selective neuronal loss is a prominent feature in both acute and chronic neurological disorders. Recently, a link between neurodegeneration and a deficiency in the lipid transport protein phosphatidylinositol transfer protein , (PI-TP,) has been demonstrated. In this context it may be of importance that fibroblasts overexpressing PI-TP, are known to produce and secrete bioactive survival factors that protect fibroblasts against UV-induced apoptosis. In the present study it was investigated whether the conditioned medium of cells overexpressing PI-TP, (CM,) has neuroprotective effects on primary neurons in culture. We show that CM, is capable of protecting primary, spinal cord-derived motor neurons from serum deprivation-induced cell death. Since the conditioned medium of wild-type cells was much less effective, we infer that the neuroprotective effect of CM, is linked (in part) to the PI-TP,-dependent production of arachidonic acid metabolites. The neuroprotective activity of CM, is partly inhibited by suramin, a broad-spectrum antagonist of G-protein coupled receptors. Western blot analysis shows that brain cortex and spinal cord express relatively high levels of PI-TP,, suggesting that the survival factor may be produced in neuronal tissue. We propose that the bioactive survival factor is implicated in neuronal survival. If so, PI-TP, could be a promising target to be evaluated in studies on the prevention and treatment of neurological disorders. [source]


ASPP2 inhibits APP-BP1-mediated NEDD8 conjugation to cullin-1 and decreases APP-BP1-induced cell proliferation and neuronal apoptosis

JOURNAL OF NEUROCHEMISTRY, Issue 3 2003
Yuzhi Chen
Abstract APP-BP1, first identified as a protein that interacts with the carboxyl (C) terminus of the amyloid precursor protein (APP), is one-half of the bipartite activating enzyme for the ubiquitin-like protein NEDD8. We report here that APP-BP1 also specifically interacts with apoptosis stimulating protein of p53 ASPP2 in non-transfected cells through the functional predominant N-terminal domain ASPP2(332,483). ASPP2 inhibits the ability of APP-BP1 to rescue the ts41 cell cycle mutation and inhibits APP-BP1 induced apoptosis in primary neurons. ASPP2 reduces the ability of NEDD8 to conjugate to Cullin-1, inhibits APP-BP1-dependent ts41 cell proliferation, and blocks the ability of APP-BP1 to cause apoptosis and to cause DNA synthesis in neurons. We also show that ASPP2 activates nuclear factor-,B (NF-,B) transcriptional activity, which seems to be inhibited by the neddylation pathway since the dominant negative NEDD8 activating enzyme causes enhanced NF-,B activity. Our data provide the first in vivo evidence that ASPP2 is a negative regulator of the neddylation pathway through specific interaction with APP-BP1 and suggest that dysfunction of the APP,BP1 interaction with APP may be one cause of Alzheimer's disease. [source]


Identification of a GM1/Sodium,Calcium exchanger complex in the nuclear envelope of non-neuronal cells

JOURNAL OF NEUROCHEMISTRY, Issue 2002
X. Xie
Our previous studies identified a Na,Ca exchanger (NCX) that is tightly associated with GM1 ganglioside and potentiated by it in the nuclear envelope (NE) of NG108-15 cells and primary neurons. The purpose of the present study was to explore whether this is a general phenomena or limited to neurons. Non-neuronal C6 (glioma), HeLa (Epithelial carcinoma) and NCTC (connective tissue) cell lines were used. Immunocytochemical staining with anti-NCX antibody and cholera toxin B subunit revealed that NCX and GM1 coexist in the nuclei from all 3 cell lines; in relation to plasma membrane, only HeLa cells showed staining for both NCX and GM1. Purified NE and non-nuclear membrane mixture (mainly plasma membrane) from the 3 cell lines were immunoprecipitated with a mouse monoclonal anti-NCX antibody and the precipitated proteins separated on SDS,PAGE. Analysis by immunoblot, showed that NCX is tightly associated with GM1 in the NE of all 3 cell lines. In contrast, NCX and the more loosely associated GM1 from plasma membrane of HeLa cells were separated by SDS,PAGE. Isolated nuclei from C6 cells were used for 45Ca2+ uptake experiments, which provided functional evidence that this exchanger protein is strongly potentiated by GM1. In similar experiments with Jurkat cells (T lymphocyte), no NCX was found. These results suggest a possible new and widely distributed mechanism for regulation of nuclear calcium by NCX in association with GM1. Acknowledgements:, supported by NIH grant NS 33912. [source]


Cyclic guanosine monophosphate signalling pathway plays a role in neural cell adhesion molecule-mediated neurite outgrowth and survival

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2007
Dorte Kornerup Ditlevsen
Abstract The neural cell adhesion molecule (NCAM) plays a crucial role in neuronal development, regeneration, and synaptic plasticity associated with learning and memory consolidation. Homophilic binding of NCAM leads to neurite extension and neuroprotection in various types of primary neurons through activation of a complex network of signalling cascades, including fibroblast growth factor receptor, Src-family kinases, the mitogen-activated protein kinase pathway, protein kinase C, phosphatidylinositol-3 kinase, and an increase in intracellular Ca2+. Here we present data indicating an involvement of cyclic GMP in NCAM-mediated neurite outgrowth in both hippocampal and dopaminergic neurons and in NCAM-mediated neuroprotection of dopaminergic neurons. In addition, evidence is presented suggesting that NCAM mediates activation of cGMP via synthesis of nitric oxide (NO) by NO synthase (NOS) and activation of soluble guanylyl cyclase by NO, leading to an increased synthesis of cGMP and activation by cGMP of protein kinase G. © 2007 Wiley-Liss, Inc. [source]


Pyruvate protection against ,-amyloid-induced neuronal death: Role of mitochondrial redox state

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2003
Gema Alvarez
Abstract The mechanism by which ,-amyloid protein (A,) causes degeneration in cultured neurons is not completely understood, but several lines of evidence suggest that A,-mediated neuronal death is associated with an enhanced production of reactive oxygen species (ROS) and oxidative damage. In the present study, we address whether supplementation of glucose-containing culture media with energy substrates, pyruvate plus malate (P/M), protects rat primary neurons from A,-induced degeneration and death. We found that P/M addition attenuated cell death evoked by ,-amyloid peptides (A,25,35 and A,1,40) after 24 hr treatment and that this effect was blocked by ,-ciano-3-hydroxycinnamate (CIN), suggesting that it requires mitochondrial pyruvate uptake. P/M supply to control and A,-treated neuronal cultures increases cellular reducing power, as indicated by the ability to reduce the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The early increases in ROS levels, measured by dichlorofluorescein (DCF) fluorescence, and caspase-3 activity that follow exposure to A, were notably reduced in the presence of P/M. These results place activation of caspase-3 most likely downstream of oxidative damage to the mitochondria and indicate that mitochondrial NAD(P) redox status plays a central role in the neuroprotective effect of pyruvate. Inhibition of respiratory chain complexes and mitochondrial uncoupling did not block the early increase in ROS levels, suggesting that A, could initiate oxidative stress by activating a source of ROS that is not accesible to the antioxidant defenses fueled by mitochondrial substrates. © 2003 Wiley-Liss, Inc. [source]


Sequential detergent fractionation of primary neurons for proteomics studies

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2008
Simonetta Bernocco Dr.
Abstract Proteomics studies employing primary neurons are difficult due to the neurons' characteristics. We have developed a detergent-based fractionation method which reduces complexity of the protein extracts, is sufficiently fast to allow differential proteomics analysis after treatments of neurons for short time periods, can be applied to small numbers of cells directly in culture plates, and allows differential extraction of proteins in a compartment-specific manner. The sequential use of detergent-containing buffers on neurons in culture plates yields four extracts enriched in cytosolic, membrane-bound or enclosed, nuclear, and cytoskeletal proteins. Fractionation of neurons was validated by comparison of the distribution of known subcellular marker proteins in the four extracts using Western blotting. Comparison of extracts by DIGE showed a clear difference in protein composition demonstrating significant variations with a fold change (FC) of at least 1.20 for 82% of the detected spots. Using proteins identified in these spots that could be assigned a subcellular localization based on descriptions in the Uniprot database, an extraction efficiency of 85% was calculated for cytosolic proteins in extract 1, 90% for membrane-bound and membrane-enclosed proteins in extract 2, 82% for nuclear proteins in extract 3 and 38% for cytoskeletal and RAFT proteins in extract 4. [source]


Synergistic neuroprotective effect via simian lentiviral vector-mediated simultaneous gene transfer of human pigment epithelium-derived factor and human fibroblast growth factor-2 in rodent models of retinitis pigmentosa

THE JOURNAL OF GENE MEDICINE, Issue 12 2008
Masanori Miyazaki
Abstract Background We previously demonstrated that a new lentiviral vector derived from nonpathogenic simian immunodeficiency virus (SIVagm) was efficient and safe for long-lasting retinal gene transfer, and that it provided the significant therapeutic effect of expressing human pigment epithelium-derived factor (hPEDF) in Royal College of Surgeons (RCS) rats. In the present study, to obtain a more pronounced outcome, we assessed the potential synergistic effect of the simultaneous gene transfer of hPEDF and human fibroblast growth factor-2 (hFGF-2) by improved third-generation SIV on RCS rats and retinal degeneration slow (rds) mice, because the former targets the primary neurons, including photoreceptor cells (PCs), whereas the latter is effective for targeting secondary neural cells, including Muller cells. Methods Vector solution (SIV-hPEDF, SIV-hFGF-2, a 1 : 1 mixture of SIV-hPEDF and SIV-hFGF-2, or SIV-enhanced green fluorescent protein) was injected into the peripheral subretinal space of 3-week-old RCS rats or rds mice. Histopathological and electroretinographic assessments were made at several points after gene transfer. Results Administration of SIV-hPEDF or SIV-hFGF-2 significantly delayed the histological PC degeneration and electrical deficit in RCS rats, and these delays were synergistically and significantly pronounced by SIV-hPEDF + SIV-hFGF-2 (1 : 1 mixture). In rds mice, functional therapeutic effects were observed even by SIV-PEDF, or SIV-FGF-2 alone and, moreover, both SIV-PEDF and SIV-FGF-2 showed higher therapeutic effects. Conclusions These synergistic rescues of retinitis pigmentosa (RP) model animals are the ,proof concept' that the ,dual' expression of hPEDF and hFGF-2 dramatically improved therapeutic efficacy by keeping lower titers. This strategy may contribute to safer and more effective gene therapy for RP. Copyright © 2008 John Wiley & Sons, Ltd. [source]


Lentiviral vectors for treating and modeling human CNS disorders

THE JOURNAL OF GENE MEDICINE, Issue 9 2004
Mimoun Azzouz
Abstract Vectors based on lentiviruses efficiently deliver genes into many different types of primary neurons from a broad range of species including man and the resulting gene expression is long term. These vectors are opening up new approaches for the treatment of neurological diseases such as Parkinson's disease (PD), Huntington's disease (HD), and motor neuron diseases (MNDs). Numerous animal studies have now been undertaken with these vectors and correction of disease models has been obtained. Lentiviral vectors also provide a new strategy for in vivo modeling of human diseases; for example, the lentiviral-mediated overexpression of mutated human ,-synuclein or huntingtin genes in basal ganglia induces neuronal pathology in animals resembling PD and HD in man. These vectors have been refined to a very high level and can be produced safely for the clinic. This review will describe the general features of lentiviral vectors with particular emphasis on vectors derived from the non-primate lentivirus, equine infectious anemia virus (EIAV). It will then describe some key examples of genetic correction and generation of genetic animal models of neurological diseases. The prospects for clinical application of lentiviral vectors for the treatment of PD and MNDs will also be outlined. Copyright © 2004 John Wiley & Sons, Ltd. [source]


Crystallization and preliminary X-ray crystallographic studies of human FAIM protein

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010
Guoming Li
Fas apoptosis inhibitory molecule (FAIM), an antagonist of Fas-induced cell death, is highly conserved and is broadly expressed in many tissues. It has been found that FAIM can stimulate neurite outgrowth in PC12 cells and primary neurons. However, the molecular mechanisms of action of FAIM are not understood in detail. Here, full-length human FAIM and two truncation constructs have successfully been cloned, expressed and purified in Escherichia coli. FAIM (1,90) was crystallized and diffracted to a resolution of 2.5,Å; the crystal belonged to space group P31, with unit-cell parameters a = b = 58.02, c = 71.11,Å, , = , = 90, , = 120°. There were two molecules in the asymmetric unit. [source]