Biomedical subjects

Georg C Terstappen

Publications and source records attributed to Georg C Terstappen.

18 recordsLinked to original sources

PubMed2006

Inhibition of Wnt signaling, modulation of Tau phosphorylation and induction of neuronal cell death by DKK1.

Expression of the Wnt antagonist Dickkopf-1 (DKK1) is induced during neurodegenerative processes associated with Alzheimer's Disease and brain ischemia. However, little is known about DKK1-mediated effects on neurons. We now describe that, in cultured neurons, DKK1 is able to inhibit canonical Wnt signaling, as assessed by TCF reporter assay and analysis of beta-catenin levels, and to elicit cell death associated with loss of BCL-2 expression, induction of BAX, and TAU hyperphosphorylation. Local infusion of DKK1 in rats caused neuronal cell death and astrocytosis in the CA1 region of the hippocampus and death of cholinergic neurons in the nucleus basalis magnocellularis. Both effects were reversed by systemic administration of lithium ions, which rescue the Wnt pathway by inhibiting glycogen synthase kinase-3beta. The demonstration that DKK1 inhibits Wnt signaling in neurons and causes neuronal death supports the hypothesis that inhibition of the canonical Wnt pathway contributes to the pathophysiology of neurodegenerative disorders.

Animals↗

PubMed2006

Detection of phosphorylation patterns in rat cortical neurons by combining phosphatase treatment and DIGE technology.

Although protein phosphorylation is probably the most studied post-translational modification occurring in cells, the number of proteins, which are the target of this modification, is still largely unknown. Increasing the coverage of the phosphoproteome as well as the detection of variation at the phosphorylation level would be very helpful for understanding the mechanisms of cell life and the modifications of the cell state leading to pathological conditions such as neurodegeneration. In order to further investigate variations occurring at the phosphorylation level, we have initiated the creation of a reference map of phosphorylated proteins in rat cortical neurons, employing a combination of phosphatase treatment and 2-DE/differential in gel electrophoresis technology. About 131 spots were recognized as phosphorylated proteins as they showed different migration behaviour after phosphatase treatment. The analysis of 42 selected spots was carried out by LC/MS/MS technology resulting in the identification of two new phosphoproteins.

Animals↗

PubMed2006

Inhibition of the canonical Wnt signaling pathway by apolipoprotein E4 in PC12 cells.

We examined the effect of the three human isoforms of apolipoprotein E (ApoE2, ApoE3, and ApoE4) on the canonical Wnt signaling pathway in undifferentiated PC12 cells. Addition of recombinant ApoE4 reduced Wingless-Int7a-stimulated gene expression at concentrations of 80 and 500 nm. Recombinant ApoE2 and ApoE3 were virtually inactive. Recombinant ApoE4 also inhibited Wnt signaling when combined with very low density lipoproteins (VLDLs) or in cells over-expressing the low density lipoprotein receptor-related protein, LRP6. In contrast, the enforced expression of LRP5 unmasked an inhibition by ApoE2 and ApoE3, which, however, were less effective than ApoE4 in inhibiting Wnt signaling. We also transfected PC12 cells with constructs encoding for the three human ApoE isoforms to examine whether endogenously expressed ApoE isoforms could modulate the Wnt pathway. Under these conditions, all three ApoE isoforms were able to inhibit Wnt signaling, although ApoE4 showed the greatest efficacy. Only the conditioned medium collected from cultures transfected with ApoE4 induced a significant inhibition of Wnt7a-stimulated gene expression, confirming that ApoE4 has an extracellular action that is not shared by the other ApoE isoforms. We conclude that ApoE4 behaves as an inhibitor of the canonical Wnt pathway in a context-independent manner.

Animals↗

PubMed2006

The Wnt signaling pathway as a target for the treatment of neurodegenerative disorders.

Although significant progress in understanding brain function has been made in the last 15 years, the unmet medical need for effective therapeutic treatment of devastating neurodegenerative disorders is still enormous and represents a formidable challenge at the beginning of the 21st century. With the recent accumulation of evidence that the Wnt signaling pathway might be impaired in such diseases, a new avenue for potential therapeutic intervention has been opened which comprises many putative drug targets.

Animals↗

PubMed2005

Differential inhibition of rat alpha3* and alpha7 nicotinic acetylcholine receptors by tetrandrine and closely related bis-benzylisoquinoline derivatives.

The patch-clamp technique was used to investigate the effects of bis-benzylisoquinoline alkaloids on two of the major neuronal nicotinic acetylcholine receptors (nAChRs), the alpha3-containing nAChR (alpha3*nAChR) endogenously expressed in PC12 cells and the rat alpha7-nAChR heterologously expressed in GH4C1 cells. Tetrandrine and hernandezine reversibly inhibited both receptors displaying half-maximal inhibitory concentrations (IC50) of 8.1 microM and 5.8 microM for alpha3*nAChR and 407.4 nM and 372.2 nM, respectively, for alpha7-nAChR. E6-berbamine completely inhibited the alpha3*nAChR with an IC50 of 5.1 microM, but only partially inhibited the alpha7-nAChR at concentrations up to 30 microM. Tetrandrine inhibition of alpha3*nAChR was functionally non-competitive. All three compounds displaced radiolabelled methyllycaconitine ([3H]-MLA) binding to alpha7-nAChR providing some evidence of competitive antagonism. The results demonstrate that these alkaloids are nAChRs antagonists, with tetrandrine and hernandezine displaying selectivity for one of the major neuronal subtype, the alpha7 nAChR. The different potencies and multiple modes of action on nAChRs may help to better understand the pharmacology of these receptors and to aid in novel drug design.

Alkaloids↗

PubMed2005

A continuous time-resolved fluorescence assay for identification of BACE1 inhibitors.

The aspartic protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) mediates the production of the neurotoxic amyloid beta peptide and is therefore considered an important drug target for treatment of Alzheimer's disease. We describe a new homogeneous time-resolved fluorescence quenching assay for the identification of BACE1 inhibitors that is characterized by minimal compound interference and allows both kinetic and end-point measurements. A fluorescent Eu-chelate as fluorescent donor, coupled to the N-terminus of a peptide containing the amyloid precursor protein Swedish mutation with a quenching molecule at the C-terminus as acceptor, is used as substrate. Upon peptide cleavage by BACE1, the energy transfer between donor and acceptor molecules is interrupted, leading to increased fluorescence emission of the donor. Compound interference, a common problem in fluorescence assays, is minimized with this technology because of the large Stoke's shift and the time-resolved fluorescence emission of the Eu-chelate. The assay reproduced IC50 values of known inhibitors and detected them also as hits in a screening campaign. A high signal-to-noise ratio of 289 and a Z' factor of 0.76 make this assay suitable for high-throughput screening.

Amyloid Precursor Protein Secretases↗

PubMed2004

Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is associated with neuronal degeneration in Alzheimer's brain.

We used primary cultures of cortical neurons to examine the relationship between beta-amyloid toxicity and hyperphosphorylation of the tau protein, the biochemical substrate for neurofibrillary tangles of Alzheimer's brain. Exposure of the cultures to beta-amyloid peptide (betaAP) induced the expression of the secreted glycoprotein Dickkopf-1 (DKK1). DKK1 negatively modulates the canonical Wnt signaling pathway, thus activating the tau-phosphorylating enzyme glycogen synthase kinase-3beta. DKK1 was induced at late times after betaAP exposure, and its expression was dependent on the tumor suppressing protein p53. The antisense induced knock-down of DKK1 attenuated neuronal apoptosis but nearly abolished the increase in tau phosphorylation in betaAP-treated neurons. DKK1 was also expressed by degenerating neurons in the brain from Alzheimer's patients, where it colocalized with neurofibrillary tangles and distrophic neurites. We conclude that induction of DKK1 contributes to the pathological cascade triggered by beta-amyloid and is critically involved in the process of tau phosphorylation.

Aged↗

PubMed2004

Nonradioactive rubidium ion efflux assay and its applications in drug discovery and development.

The recent sequencing of the human genome has created comprehensive information of all potential drug targets. Based on current estimations for the total number of genes, around 400 poreforming ion channel genes can be expected corresponding to about 1.3% of the human genome. Since many ion channels are involved in diseases and the currently marketed drugs act only on a small fraction of these pore-forming membrane proteins, there is a big opportunity for innovative ion channel drug discovery. In fact, recent advances in the development of functional ion channel assays are currently enabling a more systematic exploitation of this important target class. In particular, fluorescence-based methods, automated electrophysiology, and ion flux assays are most important in this regard. This article will briefly describe these methods focusing on the nonradioactive Rb(+) efflux assay that I developed in the early 1990s since it has found widespread application in drug discovery and development and greatly displaced (86)Rb(+) assays for the analysis of K(+) and nonselective cation channels in the pharmaceutical industry.

Animals↗

PubMed2003

The antidepressant fluoxetine blocks the human small conductance calcium-activated potassium channels SK1, SK2 and SK3.

The effects of fluoxetine (Prozac) on the activity of human small-conductance calcium-activated potassium (SK) channels were investigated utilizing a functional fluorescence assay with bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC(4)(3)). Fluoxetine blocked SK channels stably expressed in HEK 293 cells in a concentration-dependent manner displaying half-maximal inhibitory concentrations (IC(50)) of 9 microM for hSK1, 7 microM for hSK2 and 20 microM for hSK3. The block of hSK3 channels was confirmed by whole cell patch-clamp recordings of the recombinant cells and human TE 671 cells. Fluoxetine also inhibited [(125)I]apamin binding in a concentration-dependent manner displaying IC(50) values of 63 microM for hSK1, 148 microM for hSK2 and 295 microM for hSK3. These results provide new information concerning the mechanism of therapeutic and/or side effects of one of the most widely used antidepressant drugs.

Antidepressive Agents, Second-Generation↗

PubMed2003

Functional characterization of WNT7A signaling in PC12 cells: interaction with A FZD5 x LRP6 receptor complex and modulation by Dickkopf proteins.

WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased beta-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5.LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.

Animals↗

PubMed2003

The Wnt pathway, cell-cycle activation and beta-amyloid: novel therapeutic strategies in Alzheimer's disease?

Beta-amyloid protein (betaAP) is thought to cause neuronal loss in Alzheimer's disease (AD). Applied to neurons in culture, betaAP induces neuronal death and hyperphosphorylation of tau protein, which forms neurofibrillary tangles (NFTs) in AD brains. Neurons also undergo rapid apoptotic death following reactivation of a mitotic cycle. However, the molecular events that determine the fate of neurons challenged with betaAP (apoptotic death, formation of NFTs and survival) are unclear. We discuss a scenario for the pathogenesis of AD. This links betaAP-induced changes to the Wnt signaling pathway that promotes proliferation of progenitor cells and directs cells into a neuronal phenotype during brain development. We propose that betaAP-mediated facilitation of mitogenic Wnt signaling activates unscheduled mitosis in differentiated neurons. Furthermore, late downregulation of Wnt signaling by betaAP might lead to NFT formation. We propose that drugs that both inhibit the cell cycle and rescue Wnt activity could provide novel AD therapeutics.

Alzheimer Disease↗

PubMed2002

Pharmacological properties of rat alpha 7 nicotinic receptors expressed in native and recombinant cell systems.

The pharmacological properties of the rat alpha7 nicotinic acetylcholine receptor endogenously expressed in PC12 cells and recombinantly expressed in GH4C1 cells (alpha7-GH4C1 cells) were characterized and compared. Patch-clamp recordings demonstrated that activation by choline and block by methyllycaconitine and dihydro-beta-erythroidine were similar, but block by mecamylamine was different. Whereas in alpha7-GH4C1 cells the inhibition curve for mecamylamine was monophasic (IC(50) of 1.6 microM), it was biphasic in PC12 cells (IC(50) values of 341 nM and 9.6 microM). The same rank order of potency was obtained for various nicotinic agonists, while acetylcholine was 3.7-fold less potent and 1.5-fold more effective in PC12 cells. Dihydro-beta-erythroidine differentially blocked acetylcholine-evoked currents in both systems. Since reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed expression of alpha3, alpha4, alpha5, alpha7 and beta4 subunits in PC12 cells, whereas GH4C1 cells express only the beta4 subunit, our results suggest that more than one form of alpha7 containing heteromeric nicotinic receptors might be functionally expressed in PC12 cells.

Animals↗

PubMed2002

Pharmacological and molecular characterisation of SK3 channels in the TE671 human medulloblastoma cell line.

The expression of the small conductance calcium-activated potassium channels SK1, SK2 and SK3 was investigated in the TE671 human medulloblastoma cell line using RT-PCR and transcripts were detected only for SK3. Immunodetection experiments confirmed this result, demonstrating the presence of the SK3 protein. This potassium channel was characterised in TE671 cells using whole-cell patch-clamp recordings. Voltage steps to -100 mV from a holding potential of 0 mV in equimolar 140 mM intra- and extracellular K(+) (K(+)(in/out)) elicited an inward current. The reversal potential of this current shifted 56.6 mV per 10-fold increase in K(+)(out) thus suggesting K(+) selectivity. This current was dependent on the concentration of Ca(2+)(in) with an EC(50) of 104.2 nM. A pharmacological characterisation of this current revealed that it was not blocked by 1 microM charybdotoxin (ChTX), 0.3 microM iberiotoxin (IbTX) or 10 microM clotrimazole (CLT) and only modestly inhibited (<50%) by 30 nM scyllatoxin (ScTX), 200 microM dequalinium chloride (Deq) or 300 microM d-tubocurarine (d-TC). The non-selective SK blocker d-TC blocked the current with an IC(50) of 43.2 microM while apamin blocked the current to a much greater extent (87.8% at 1 microM) with an IC(50) of 4.3 nM. Furthermore, the current was significantly increased (132.6+/-5.2%, n=7) by 500 microM 1-ethyl-2-benzimidazolinone (EBIO). Collectively, these data demonstrate the presence of an endogenous SK3 channel in human TE671 cells.

Blotting, Western↗

PubMed2002

Molecular cloning and initial characterization of the MG61/PORC gene, the human homologue of the Drosophila segment polarity gene Porcupine.

Insect and vertebrate Porcupine genes encode multi-pass endoplasmic reticulum proteins involved in the processing of Wnt (wingless and int homologue) proteins, a class of secreted glycoprotein factors homologous to the Drosophila melanogaster segment polarity gene Wingless (Wg). Here we report the cloning of cDNAs encoding the human homologue of the Drosophila gene Porcupine (Porc), the characterization of its genomic structure and the quantitative analysis of its expression in a comprehensive panel of human tissues. The human Porcupine locus (MG61/PORC) spans 15 exons over approximately 12 kb of genomic sequence on Xp11.23. Real-time quantitative expression analysis reveals that MG61/PORC transcripts are expressed in multiple tissues, but are particularly abundant in the brain. Like its mouse and Xenopus homologues, MG61/PORC encodes four protein isoforms (A-D) generated through alternative splicing and expressed in a tissue-specific fashion. Finally, we present evidence indicating that MG61/PORC can influence the activity of a human Wnt7A expression construct in a T-cell factor-responsive reporter assay.

Acyltransferases↗

PubMed2002

Lithium induces gene expression through lymphoid enhancer-binding factor/T-cell factor responsive element in rat PC12 cells.

Lithium inhibits glycogen synthase kinase-3 (GSK-3), which leads to an increase of cytoplasmic beta-catenin levels. In some cell types, but not in others, activated beta-catenin interacts with members of the lymphoid enhancer-binding factor (LEF)/T-cell factor (TCF) family of transcription factors and induces gene expression. Lithium effect on LEF/TCF-mediated gene expression has never been evaluated in cells with a neuronal phenotype. We have constructed a LEF/TCF-dependent luciferase reporter gene to investigate lithium effects on transcription in PC12 cells. In transiently transfected PC12 cells, lithium induced a time-dependent increase in LEF/TCF-mediated luciferase activity. These results are consistent with the known inhibitory effects of lithium on GSK-3 and represent the first demonstration that a LEF/TCF responsive element also mediates lithium-induced gene expression in PC12 cells.

Animals↗

PubMed2001

Molecular cloning and characterization of the human diacylglycerol kinase beta (DGKbeta) gene: alternative splicing generates DGKbeta isotypes with different properties.

Diacylglycerol kinases are key modulators of levels of diacylglycerol, a second messenger involved in a variety of cellular responses to extracellular stimuli. A number of diacylglycerol kinases encoded by separate genes are present in mammalian genomes. We have cloned cDNAs encoding several isoforms of the human homologue of the rat diacylglycerol kinase beta gene and characterized two such isoforms that differ at their carboxyl terminus through alternative splicing and the usage of different polyadenylation signals. Quantitative analysis of gene expression in a panel of human tissue cDNAs revealed that transcripts corresponding to both isoforms are co-expressed in central nervous system tissues and in the uterus, with one variant being expressed at relatively higher levels. As green fluorescent protein fusions, the two isoforms displayed localization to different subcellular compartments, with one variant being associated with the plasma membrane, while the other isoform was predominantly localized within the cytoplasm. Differences were also observed in their subcellular localization in response to phorbol ester stimulation. Enzymatic assays demonstrated that the two isoforms display comparable diacylglycerol kinase activities. Therefore, the human diacylglycerol kinase beta gene can generate several enzyme isoforms, which can display different expression levels and subcellular localization but similar enzymatic activities in vitro.

Alternative Splicing↗

PubMedDate not supplied

Phosphoproteome analysis.

Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult task especially if quantitative comparisons have to be made. Several different experimental strategies can be employed to explore phosphoproteomes and this review will cover the most important ones such as incorporation of radiolabeled phosphate into proteins, application of specific antibodies against phosphorylated residues and direct staining of phosphorylated proteins in polyacrylamide gels. Moreover, methods to enrich phosphorylated proteins such as affinity chromatography (IMAC) and immunoprecipitation as well as mass spectrometry for identification of phosphorylated peptides and phosphorylation sites are also described.

Acrylic Resins↗

PubMedDate not supplied

A bioinformatics perspective on proteomics: data storage, analysis, and integration.

The field of proteomics is advancing rapidly as a result of powerful new technologies and proteomics experiments yield a vast and increasing amount of information. Data regarding protein occurrence, abundance, identity, sequence, structure, properties, and interactions need to be stored. Currently, a common standard has not yet been established and open access to results is needed for further development of robust analysis algorithms. Databases for proteomics will evolve from pure storage into knowledge resources, providing a repository for information (meta-data) which is mainly not stored in simple flat files. This review will shed light on recent steps towards the generation of a common standard in proteomics data storage and integration, but is not meant to be a comprehensive overview of all available databases and tools in the proteomics community.

Computational Biology↗