Biomedical subjects

Matthias Eder

Publications and source records attributed to Matthias Eder.

31 records · Page 2Linked to original sources

PubMed2003

CB1 cannabinoid receptors and on-demand defense against excitotoxicity.

Abnormally high spiking activity can damage neurons. Signaling systems to protect neurons from the consequences of abnormal discharge activity have been postulated. We generated conditional mutant mice that lack expression of the cannabinoid receptor type 1 in principal forebrain neurons but not in adjacent inhibitory interneurons. In mutant mice,the excitotoxin kainic acid (KA) induced excessive seizures in vivo. The threshold to KA-induced neuronal excitation in vitro was severely reduced in hippocampal pyramidal neurons of mutants. KA administration rapidly raised hippocampal levels of anandamide and induced protective mechanisms in wild-type principal hippocampal neurons. These protective mechanisms could not be triggered in mutant mice. The endogenous cannabinoid system thus provides on-demand protection against acute excitotoxicity in central nervous system neurons.

Animals↗

PubMed2003

Distribution and properties of functional postsynaptic kainate receptors on neocortical layer V pyramidal neurons.

The distribution of glutamate receptor subtypes on the surface of neurons is highly relevant for synaptic transmission and signal processing. In the present study we investigated the location and properties of functional kainate receptors (KARs) on the somatodendritic membrane of rat neocortical layer V pyramidal neurons. Infrared-guided laser stimulation was used to apply glutamate photolytically to the soma and various sites along the apical dendrite. Electrical currents, resulting from the activation of pharmacologically isolated KARs, were measured by whole-cell patch-clamp recording. In addition, KARs on somatic and dendritic outside-out patches were activated while still within the brain tissue. We found that functional KARs are located on the entire somatodendritic membrane that was examined. Fast kinetics, a linear I-V relationship, and a relatively high single-channel conductance are characteristic features of these receptors. We provide evidence that the unitary properties of somatic and dendritic KARs are identical. Regarding the subcellular distribution of KARs, our results indicate that the density of these receptors increases toward the distal dendrite. They are located mainly at extrasynaptic sites but also mediate fast synaptic signaling triggered by afferent stimulation. The differential distribution speaks in favor of a selective targeting of KARs on central neurons and may reflect a mechanism for a location-dependent regulation of synaptic efficacy. Furthermore, it is feasible to assume that extrasynaptic KARs could be activated by a "spillover" of synaptically released glutamate, ambient glutamate in the CSF, or glutamate released from adjacent astrocytes.

Animals↗

PubMed2003

Activation of mGlu receptors induces LTD without affecting postsynaptic sensitivity of CA1 neurons in rat hippocampal slices.

Two forms of long-term depression (LTD) of excitatory synaptic transmission have been identified in the mammalian CNS, which are induced by the synaptic activation of N-methyl-D-aspartate (NMDA) and metabotropic glutamate (mGlu) receptors, respectively. The mGlu receptor-dependent form of LTD can be activated by application of 3,5-dihydroxyphenylglycine (DHPG), a group I selective mGlu receptor agonist. DHPG-induced LTD is increasingly being used to investigate the mechanisms of mGlu receptor-dependent LTD. However, recent experiments have argued for both a pre- and postsynaptic locus of expression of DHPG-induced LTD. In the present study we report that DHPG-induced LTD is not associated with changes in the sensitivity of CA1 neurons to bath applied AMPA. Furthermore, in contrast to homosynaptic LTD, DHPG-induced LTD is also not associated with changes in sensitivity to focally uncaged L-glutamate. These data do not support the notion that DHPG-induced LTD requires a modification of AMPA receptors, such as their internalisation, but are compatible with a presynaptic mechanism of expression.

Animals↗

PubMed2003

Inhibition of GM-CSF receptor function by stable RNA interference in a NOD/SCID mouse hematopoietic stem cell transplantation model.

RNA interference (RNAi) describes a highly conserved mechanism of sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA (dsRNA). Whereas RNAi is applied to study gene function in different organisms and in variant cell types, little is known about RNAi in human hematopoietic stem and progenitor cells and their myeloid progeny. To address this issue, short hairpin RNAs (shRNA) were designed to target the common beta-chain of the human receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 (betaGMR). These receptors regulate proliferation, survival, differentiation, and functional activity of hematopoietic cells. In addition to markedly inhibiting mRNA and protein expression, anti-beta-GMR shRNAs were also found to inhibit receptor function in a cell culture model. Furthermore, lentiviral gene transfer of shRNA expression cassettes into primary normal CD34+ cells selectively inhibited colony formation of transduced progenitors when stimulated with GM-CSF/IL-3 but not when stimulated with cytokines that do not signal via beta-GMR. Finally, anti-beta-GMR shRNAs had no detectable effect on engraftment or lineage composition of lentivirally transduced human CD34+ cells transplanted into NOD/SCID mice. However, the growth defect of transduced colony-forming cells under stimulation with GM-CSF/IL-3 remains unchanged in bone marrow cells harvested from individual NOD/SCID mice 6 weeks after transplantation. These data indicate that lentiviral gene transfer of shRNA expression cassettes may be used to induce long-term RNAi in human hematopoietic stem and progenitor cells for functional genetics and potential therapeutic intervention.

Animals↗

PubMed2003

Gene silencing mediated by small interfering RNAs in mammalian cells.

RNA interference (RNAi) as a protecting mechanism against invasion by foreign genes was first described in C. elegans and has subsequently been demonstrated in diverse eukaryotes such as insects, plants, fungi and vertebrates. RNAi is the mechanism of sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNAs (dsRNA) homologous to the gene being suppressed. dsRNAs are processed by Dicer, a cellular ribonuclease III, to generate duplexes of about 21 nt with 3'-overhangs (small interfering RNA, siRNA) which mediate sequence-specific mRNA degradation. In mammalian cells siRNA molecules are capable of specifically silencing gene expression without induction of the unspecific interferon response pathway. Thus, siRNAs have become a new and powerful alternative to other genetic tools such as antisense oligonucleotides and ribozymes to analyze loss-of-function phenotypes. Application of siRNA duplexes to interfere with the expression of a specific gene requires knowledge of target accessibility, highly effective delivery of siRNAs into target cells and for some applications long-term siRNA expression. Effective strategies to deliver siRNAs to target cells in cell culture include transduction by physical or chemical transfection. An alternative strategy uses the endogenous expression of siRNAs by various Pol III promoter expression cassettes that allow transcription of functional siRNAs or their precursors. This review summarizes some genetic and biochemical aspects of RNAi, the delivery and application of siRNAs to target cells, the kinetics of RNAi and the utility of siRNAs as analytical and potential therapeutic tools.

Animals↗

PubMed2002

Specific inhibition of bcr-abl gene expression by small interfering RNA.

Small interfering RNAs (siRNAs) were designed to target the bcr-abl oncogene, which causes chronic myeloid leukemia (CML) and bcr-abl-positive acute lymphoblastic leukemia (ALL). Chemically synthesized anti-bcr-abl siRNAs were selected using reporter gene constructs and were found to reduce bcr-abl mRNA up to 87% in bcr-abl-positive cell lines and in primary cells from CML patients. This mRNA reduction was specific for bcr-abl because c-abl and c-bcr mRNA levels remained unaffected. Furthermore, protein expression of BCR-ABL and of laminA/C was reduced by specific siRNAs up to 80% in bcr-abl-positive and normal CD34(+) cells, respectively. Finally, anti-bcr-abl siRNA inhibited BCR-ABL-dependent, but not cytokine-dependent, proliferation in a bcr-abl-positive cell line. These data demonstrate that siRNA can specifically and efficiently interfere with the expression of an oncogenic fusion gene in hematopoietic cells.

Animals↗

PubMed2002

Neocortical long-term potentiation and long-term depression: site of expression investigated by infrared-guided laser stimulation.

The synaptic site of expression of long-term potentiation (LTP) and long-term depression (LTD) is still a matter of debate. To address the question of presynaptic versus postsynaptic expression of neocortical LTP and LTD in a direct approach, we measured the glutamate sensitivity of apical dendrites of layer 5 pyramidal neurons during LTP and LTD. We used infrared-guided laser stimulation to release glutamate from its "caged" form with high spatial and temporal resolution. Responses to photolytically released glutamate and synaptically evoked EPSPs were recorded with patch-clamp pipettes from the neuronal somata. LTP and LTD could be induced by electrical stimulation at the same synapses in succession. The NMDA receptor-dependent LTD was accompanied by a decrease in the dendritic glutamate sensitivity, suggesting a postsynaptic expression of neocortical LTD. In contrast, LTP was never accompanied by a change in the dendritic glutamate sensitivity. A possible explanation for this finding is a presynaptic expression of neocortical LTP. Another set of experiments corroborated these results: Photolytic application of glutamate with a frequency of 5 Hz caused a long-lasting Ca2+ and NMDA receptor-dependent decrease in the dendritic glutamate sensitivity. In contrast, LTP of dendritic glutamate sensitivity was never induced by photostimulation, despite several experimental modifications to prevent washout of the induction mechanism and to induce a stronger postsynaptic Ca2+ influx. In conclusion, our findings provide strong evidence for a postsynaptic expression of neocortical LTD and favor a primarily presynaptic locus of neocortical LTP.

Animals↗

PubMed2002

Infrared-guided laser stimulation of neurons in brain slices.

Infrared-guided laser stimulation is a new technique that allows precise and rapid stimulation of visualized neurons in brain slices. Infrared imaging of neurons with a new contrast system is combined with the photolytic release of caged neurotransmitters by an ultraviolet (UV) laser. Addition of caged neurotransmitters to the superfusion medium of neurons in brain slices allows local excitation in the micrometer range with a focused spot of UV light. In this way, the distribution of glutamate or gamma-aminobutyric acid (GABA) receptors on neuronal dendrites can be mapped. Furthermore, this technique can be used to map the connectivity of neuronal networks through the controlled stimulation of neighboring neurons. Because the laser stimulation can be performed much faster than can paired recording, it is also possible to search for synaptic connections between distant neurons that have a low probability of connectivity.

Animals↗

PubMed2002

Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells.

This study reports a lentiviral gene transfer protocol for efficient transduction of adult human peripheral blood (PB)-derived CD34+ NOD/SCID-repopulating cells (SRCs) using vesicular stomatitis virus-G protein (VSV-G)-pseudotyped lentiviruses encoding for enhanced green fluorescence protein (eGFP). Lentiviral stocks were concentrated by anion exchange chromatography, and transduction was performed under serum-free conditions at a multiplicity of infection (MOI) between 3 and 50. Similar transduction efficiencies were achieved in the presence and absence of cytokines. Transduction of PB-derived CD34+ cells at a MOI of 3 resulted in gene transfer efficiencies into SRCs of 9.2% and 12.0% in the absence and presence of cytokines, respectively. Using improved lentiviral vectors, transduction frequency varied between 42.0% (MOI 10) and 36.0% (MOI 50) with multilineage transgene expression within SRC-derived myeloid and lymphoid cells. The protocol described can be adapted for clinical application of lentiviral gene transfer into PB-derived CD34+ cells from adult patients.

Adult↗

PubMed2002

Gene transfer into hematopoietic stem cells using lentiviral vectors.

Gene transfer into hematopoietic cells is currently being used to modulate immune responses, to protect hematopoietic cells against cytotoxic drugs or viral genes, and to restore gene deficiencies due to either inborn genetic defects or acquired loss of regular gene function. In particular, gene addition strategies for inherited severe combined immunodeficiencies (SCID) due to adenosine deaminase (ADA) deficiency or defects of the interleukin-2 receptor gamma-chain represent potentially curative strategies based on gene transfer into hematopoietic cells using recombinant retroviral vectors. Since long-term correction of genetic defects in hematopoietic cells often requires transduction of hematopoietic stem cells, an effective gene transfer into stem cells with efficient long-term and multi-lineage transgene expression is the desired goal for these therapeutic strategies. However, gene transfer strategies with retroviral vectors unable to integrate into non-cycling cells are limited by the quiescent state of the stem cells that have to be stimulated by cytokines to induce cell cycle progression. To circumvent these barriers, lentiviral vector systems based on HIV-1 have recently been developed which are able to deliver and express genes in non-dividing cells both in vitro and in vivo. This review outlines the development and improvement of lentivirus-based gene transfer protocols and discusses the use of lentiviral vectors in preclinical gene therapy studies.

Adenosine Deaminase↗

PubMed2002

Diagnostic value of procalcitonin serum levels in comparison with C-reactive protein in allogeneic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Infections represent the major complications following allogeneic stem cell transplantation (SCT). A promising marker for a more specific and early detection of bacterial or fungal infections is procalcitonin (PCT). DESIGN AND METHODS: Maximum values (m) and increase (Delta) of PCT and C-reactive protein (CRP) were prospectively analyzed during 214 clinical events in a cohort of 61 patients undergoing allogeneic SCT. Systemic reactions during bacterial or fungal infections were classified according to the ACCP/SCCM criteria. RESULTS: mPCT and mCRP (normal <0.5 microg/L and <5 mg/L, respectively) levels were high during bacterial and fungal infections (median 2.3 microg/L and 188 mg/L), moderately elevated during fever of unknown origin (median 1.5 microg/L and 82 mg/L) and low during clinical events for which there was no evidence of bacterial or fungal infections (median 0.4 microg/L and 55 mg/L). The area under the receiver operator characteristic (ROC) curve was 0.70 for mPCT, 0.76 for mCRP, 0.76 for DeltaPCT and 0.83 for DeltaCRP. Cut-off concentrations for optimum prediction of bacterial or fungal infection were: mPCT > 1 microg/L, mCRP > 100 mg/L, DeltaPCT > 1 microg/L and DeltaCRP > 50 mg/L. An increase of PCT during a bacterial or fungal infection was usually detected 1 day after the onset of fever, while the rise of CRP occurred 1 day before. mPCT was strongly correlated with the severity of systemic reaction during infection (sepsis vs severe sepsis/septic shock: p=0.0002). INTERPRETATION AND CONCLUSIONS: The diagnostic value of PCT was not superior to that of CRP in the detection of bacterial or fungal infections after allogeneic SCT. However, PCT assays may be useful in studies which compare the severity of infectious complications.

Adolescent↗

PubMedDate not supplied

Activation of the cannabinoid receptor type 1 decreases glutamatergic and GABAergic synaptic transmission in the lateral amygdala of the mouse.

The endogenous cannabinoid system has been shown recently to play a crucial role in the extinction of aversive memories. As the amygdala is presumably involved in this process, we investigated the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN-2) on synaptic transmission in the lateral amygdala (LA) of wild-type and cannabinoid receptor type 1 (CB1)-deficient mice. Extracellular field potential recordings and patch-clamp experiments were performed in an in vitro slice preparation. We found that WIN-2 reduces basal synaptic transmission and pharmacologically isolated AMPA receptor- and GABA(A) receptor-mediated postsynaptic currents in wild-type, but not in CB1-deficient mice. These results indicate that, in the LA, cannabinoids modulate both excitatory and inhibitory synaptic transmission via CB1. WIN-2-induced changes of paired-pulse ratio and of spontaneous and miniature postsynaptic currents suggest a presynaptic site of action. Inhibition of G(i/o) proteins and blockade of voltage-dependent and G protein-gated inwardly rectifying K(+) channels inhibited WIN-2 action on basal synaptic transmission. In contrast, modulation of the adenylyl cyclase-protein kinase A pathway, and blockade of presynaptic N- and P/Q- or of postsynaptic L- and R/T-type voltage-gated Ca(2+) channels did not affect WIN-2 effects. Our results indicate that the mechanisms underlying cannabinoid action in the LA partly resemble those observed in the nucleus accumbens and differ from those described for the hippocampus.

Amygdala↗

PubMedDate not supplied

Modulation of gene expression by lentiviral-mediated delivery of small interfering RNA.

RNA interference (RNAi) mediates sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA. RNAi is an effective tool for functional genomics in C. elegans, whereas biochemical characteristics of RNAi hamper its use in mammalian cells. We here analyze kinetic and quantitative aspects of mammalian RNAi in different cell lines using novel lentiviral constructs with double H1-shRNA expression cassettes located in the U3 region of the LTR. Using enhanced green fluorescence protein (EGFP) as a target gene for RNAi and red fluorescence protein (RFP) as surrogate marker for intracellular siRNA expression, we show that long-term siRNA expression mediates stable RNAi. Furthermore, RNAi-induced gene silencing varies from minimal to complete loss-of-function phenotypes within homogeneous and between different cell populations. Interestingly, the extent of gene silencing correlates to lentiviral integrations as well as siRNA expression levels in target cells. Finally, we demonstrate functional gene silencing of a cytokine receptor gene in normal CD34+ hematopoietic progenitor cells. These data suggest that the use of suitable lentiviral constructs to quantify siRNA expression in living cells may allow analysis of loss-of-function phenotypes in a dose-dependent manner in a wide variety of target cells.

Animals↗