Synaptogenesis of hippocampal neurons in primary cell culture.

Hippocampal neurons in dissociated cell culture are one of the most extensively used model systems in the field of molecular and cellular neurobiology. Only limited data are however available on the normal time frame of synaptogenesis, synapse number and ultrastructure of excitatory synapses during early development in culture. Therefore, we analyzed the synaptic ultrastructure and morphology and the localization of presynaptic (Bassoon) and postsynaptic (ProSAP1/Shank2) marker proteins in cultures established from rat embryos at embryonic day 19, after 3, 7, 10, 14, and 21 days in culture. First excitatory synapses were identified at day 7 with a clearly defined postsynaptic density and presynaptically localized synaptic vesicles. Mature synapses on dendritic spines were seen from day 10 onward, and the number of synapses steeply increased in the third week. Fenestrated or multiple synapses were found after 14 or 21 days, respectively. So-called dense-core vesicles, responsible for the transport of proteins to the active zone of the presynaptic specialization, were seen on cultivation day 3 and 7 and could be detected in axons and especially in the presynaptic subcompartments. The expression and localization of the presynaptic protein Bassoon and of the postsynaptic molecule ProSAP1/Shank2 was found to correlate nicely with the ultrastructural results. This regular pattern of development and maturation of excitatory synapses in hippocampal culture starting from day 7 in culture should ease the comparison of synapse number and morphology of synaptic contacts in this widely used model system.

Efficient targeting of proteins to post-synaptic densities of excitatory synapses using a novel pSDTarget vector system.

Pre- and post-synaptic targeting of synaptic molecules is depending upon specific targeting signals that are encoded within defined regions of the respective protein. For the post-synaptic scaffolding proteins of excitatory synapses, ProSAP1/Shank2 and ProSAP2/Shank3 this targeting information is located within about 460aa of the C-terminus. We found the C-terminal targeting signal to be bipartite composed of a 135aa stretch and the SAM (sterile alpha motif) domain embedding a relatively large variable spacer region. Based on this we developed a new GFP vector system called pSDTarget to easily clone proteins of interest as GFP fusion proteins flanked by a bipartite targeting signal for post-synaptic densities (PSDs) of excitatory synapses. The targeting signal has been derived from the PSD scaffolding protein ProSAP1/Shank2. In hippocampal neuron culture we could effectively localize and attach i.e. Glutathion-S-transferase (GST) at PSDs of excitatory synapses already during early synaptogenesis. Moreover, Gephyrin, an important scaffold molecule of inhibitory post-synapses was succesfully targeted to excitatory synapses followed by the subsequent recruitment of GABAergic receptors leading to hybrid synaptic contacts. In light of the role of specific protein domains for plastic changes of the post-synaptic compartment or investigations focusing on synaptogenesis, signalling and/or transsynaptic crosstalk this vector system provides a powerful and innovative tool for the functional analysis of molecular mechanisms and structural changes in a small but well defined neuronal compartment.

HCMV-infection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia.

BACKGROUND: The impact of infections with the human cytomegalovirus (HCMV) for the development of atherosclerosis and restenosis is still unclear. Both a clear correlation and no correlation at all have been reported in clinical, mostly serological studies. In our study we employed a human non-injury ex vivo organ culture model to investigate the effect of an in vitro permissive HCMV-infection on cell proliferation and neointimal hyperplasia for a period of 56 days. RESULTS: During routine-nephrectomies parts of renal arteries from 71 patients were obtained and prepared as human organ cultures. Cell free HCMV infection was performed with the fibroblast adapted HCMV strain AD169, the endotheliotropic strain TB40E, and a clinical isolate (AN 365). After 3, 7, 14, 21, 28, 35, and 56 days in culture staining of HCMV-antigens was carried out and reactive cell proliferation and neointimal thickening were analysed. Successful HCMV-infection was accomplished with all three virus strains studied. During the first 21 days in organ culture no cell proliferation or neointimal hyperplasia was detected. At day 35 and day 56 moderate cell proliferation and neointimal hyperplasia was found both in HCMV-infected segments and mock infected controls. Neointimal hyperplasia in productively HCMV-infected segments was lower than in non infected at day 35 and day 56, but relatively higher after infection with the endotheliotropic TB40E in comparison with the two other strains. CONCLUSION: The data do not support the hypothesis that HCMV-infection triggers restenosis via a stimulatory effect on cell proliferation and neointimal hyperplasia in comparison to non infected controls. Interestingly however, even after lytic infection, a virus strain specific difference was observed.

Formation of cellular projections in neural progenitor cells depends on SK3 channel activity.

Ion channels are potent modulators for developmental processes in progenitor cells. In a screening approach for different ion channels in neural progenitor cells (NPCs) we observed a 1-ethyl-2-benzimidazolinone (1-EBIO) activated inward current, which could be blocked by scyllatoxin (ScTX, IC50=2+/- 0.3 nmol/L). This initial evidence for the expression of the small conductance Ca2+ activated K+-channel SK3 was confirmed by the detection of SK3 transcripts and protein in NPCs. Interestingly, SK3 proteins were highly expressed in non-differentiated NPCs with a focused localization in lamellipodia as well as filopodial structures. The activation of SK3 channels using 1-EBIO lead to an immediate filopodial sprouting and the translocation of the protein into these novel filopodial protrusions. Both effects could be prevented by the pre-incubation of NPCs with ScTX. Our study gives first evidence that the formation and prolongation of filopodia in NPCs is, at least in part, effectively induced and regulated by SK3 channels.

Maturation of synaptic contacts in differentiating neural stem cells.

NSCs are found in the developing brain, as well as in the adult brain. They are self-renewing cells that maintain the capacity to differentiate into all major brain-specific cell types, such as glial cells and neurons. However, it is still unclear whether these cells are capable of gaining full functionality, which is one of the major prerequisites for NSC-based cell replacement strategies of neurological diseases. The ability to establish and maintain polarized excitatory synaptic contacts would be one of the basic requirements for intercellular communication and functional integration into existing neuronal networks. In primary cultures of hippocampal neurons, it has already been shown that synaptogenesis is characterized by a well-ordered, time-dependent targeting and recruitment of pre- and postsynaptic proteins. In this study, we investigated the expression and localization of important pre- and postsynaptic proteins, including Bassoon and synaptophysin, as well as proteins of the ProSAP/Shank family, in differentiating rat fetal mesencephalic NSCs. Moreover, we analyzed the ultrastructural features of neuronal cell-cell contacts during synaptogenesis. We show that NSCs express and localize cytoskeletal and scaffolding molecules of the pre- and postsynaptic specializations in a well-defined temporal order, leading to mature synaptic contacts after 14 days of differentiation. The temporal and spatial pattern of synaptic maturation is comparable to synaptogenesis of hippocampal neurons grown in primary culture. Therefore, with respect to the general ability to create mature synaptic contacts, NSCs seem to be well equipped to potentially compensate for lost or injured brain tissue. Disclosure of potential conflicts of interest is found at the end of this article.

Abelson interacting protein 1 (Abi-1) is essential for dendrite morphogenesis and synapse formation.

Synaptogenesis and synaptic plasticity depend crucially on the dynamic and locally specific regulation of the actin cytoskeleton. We identified an important component for controlled actin assembly, abelson interacting protein-1 (Abi-1), as a binding partner for the postsynaptic density (PSD) protein ProSAP2/Shank3. During early neuronal development, Abi-1 is localized in neurites and growth cones; at later stages, the protein is enriched in dendritic spines and PSDs, as are components of a trimeric complex consisting of Abi-1, Eps8 and Sos-1. Abi-1 translocates upon NMDA application from PSDs to nuclei. Nuclear entry depends on abelson kinase activity. Abi-1 co-immunoprecipitates with the transcription factor complex of Myc/Max proteins and enhances E-box-regulated gene transcription. Downregulation of Abi-1 by small interfering RNA results in excessive dendrite branching, immature spine and synapse morphology and a reduction of synapses, whereas overexpression of Abi-1 has the opposite effect. Data show that Abi-1 can act as a specific synapto-nuclear messenger and is essentially involved in dendrite and synapse formation.

Human cytomegalovirus-induced reduction of extracellular matrix proteins in vascular smooth muscle cell cultures: a pathomechanism in vasculopathies?

Human cytomegalovirus (HCMV) infection appears to be linked to the pathogenesis of atherosclerosis. An association between HCMV infection and an enhanced restenosis rate as well as the induction of vasculopathies after solid organ transplantation has been documented. Knowledge of the cellular and molecular basis of these findings is limited, however. By Northern blot and RT-PCR analysis of human foreskin fibroblasts (HFF) and human coronary artery smooth muscle cells (SMC), we identified extracellular matrix (ECM) genes that were downregulated after HCMV infection, including collagen type I and fibronectin. Quantitative immunoassays showed a significant reduction of soluble collagen type I and fibronectin proteins in supernatants of both cell types. This was shown to be a direct effect of HCMV infection and not due to a response to interferons released from infected cells, since neutralization of alpha and beta interferon activity could not block virus-induced downregulation of matrix proteins. As the amount of ECM depends on both synthesis and degradation, we also assessed the influence of HCMV on the activity of matrix metalloproteinases (MMP). Interestingly, a significant difference in virus-induced matrix degradation could be shown between the two cell types. HCMV upregulated MMP-2 protein and activity in SMC but not in HFF. Thus, HCMV infection of SMC reduces ECM dramatically by inducing two independent mechanisms that influence synthesis as well as degradation of ECM. These may represent molecular mechanisms for HCMV-induced pathogenesis of inflammatory vasculopathies and may facilitate dissemination of HCMV by promoting the detachment of infected cells in vivo.

An architectural framework that may lie at the core of the postsynaptic density.

The postsynaptic density (PSD) is a complex assembly of proteins associated with the postsynaptic membrane that organizes neurotransmitter receptors, signaling pathways, and regulatory elements within a cytoskeletal matrix. Here we show that the sterile alpha motif domain of rat Shank3/ProSAP2, a master scaffolding protein located deep within the PSD, can form large sheets composed of helical fibers stacked side by side. Zn2+, which is found in high concentrations in the PSD, binds tightly to Shank3 and may regulate assembly. Sheets of the Shank protein could form a platform for the construction of the PSD complex.

The human cytomegalovirus UL78 gene is highly conserved among clinical isolates, but is dispensable for replication in fibroblasts and a renal artery organ-culture system.

The human cytomegalovirus (HCMV) UL78 ORF is considered to encode a seven-transmembrane receptor. However, neither the gene nor the UL78 protein has been characterized so far. The objective of this study was to investigate the UL78 gene and to clarify whether it is essential for replication. UL78 transcription was activated early after infection, was inhibited by cycloheximide but not by phosphonoacetic acid, and resulted in a 1.7 kb mRNA. Later in the replication cycle, a second mRNA of 4 kb evolved, comprising the UL77 and UL78 ORFs. The 5' end of the UL78 mRNA initiated 48 bp upstream of the translation start and the polyadenylated tail started 268 bp downstream of the UL78 translation stop codon within the UL79 ORF. By using bacterial artificial chromosome technology, a recombinant HCMV lacking most of the UL78 coding region was constructed. Successful reconstitution of the UL78-deficient virus proved that the gene was not essential for virus replication in fibroblasts. The deletion also did not reduce virus replication in ex vivo-cultured sections of human renal arteries. Analysis of viral proteins at different stages of the replication cycle confirmed these results. Among clinical HCMV isolates, the predicted UL78 protein was highly conserved. However, an accumulation of different single mutations could be found in the N-terminal region and at the very end of the C terminus. Due to the absence of an in vivo HCMV model, the role of UL78 in the pathogenesis of HCMV infection in humans remains unclear.

Reconstitution of CD40 and CD80 in dendritic cells generated from blasts of patients with acute myeloid leukemia.

Acute myeloid leukemia (AML) is a clonal disease of hematopoiesis with poor clinical outcome despite recent improvements in chemotherapy and stem cell transplantation regimens. Immunotherapy with dendritic cells (DCs) eliciting specific T cell responses to leukemia-associated antigens (LAAs) might be a therapeutic option. DCs must express HLA class I/II molecules and the costimulatory molecules CD40, CD80 and CD86 to effectively activate T cells for the subsequent lysis of leukemic blasts. The expression of these antigens on DCs generated from 15 AML patients (AML-DCs) and on DCs generated from 15 healthy volunteers (HV-DCs) was analyzed by FACS. All DCs displayed the typical morphology and tested negative for B, T and NK cell markers. The sustained mRNA expression of LAAs such as PRAME, RHAMM or WT-1 proved that the AML-DCs originated from AML blasts. Compared with AML blasts, the expression of CD40, CD80, CD86 and HLA-DR was upregulated during DC culture to a median of 80-98% on AML-DCs. HLA-ABC was preserved on AML-DCs (median 95%). Expression of CD40, CD80 and CD83 remained lower on AML-DCs than on HV-DCs. AML-DCs express at least one LAA and strongly express HLA and costimulatory molecules, the prerequisites for eliciting T cell responses. AML-DCs may play a role in vaccine-based immunotherapies for AML patients.

Human cytomegalovirus infection in human renal arteries in vitro.

Studies with animal cytomegaloviruses, epidemiological data from humans as well as in vitro studies suggest the involvement of the human cytomegalovirus (HCMV) in the development of atherosclerosis. Cell culture systems are insufficient for examination of the entire pathogenetic process and a satisfactory animal model for HCMV is not available. An organ culture model was established for HCMV infection of human renal arteries in vitro. After infection with three representative HCMV strains, infectious virus was recovered from supernatants until 144 days post-infection with a peak around day 30 due to a long-lasting productive HCMV infection in still vital cells. Differences in cell tropism and kinetics of infection were identified between the HCMV strains. Specifically, differences in infecting endothelial cells and virus penetration into the lamina media were observed. In infected artery segments, but also in some non-infected arteries from seropositive donors, HCMV DNA could be localized by in situ PCR. Nevertheless, HCMV early antigen was detected by immunohistochemistry exclusively in artery segments infected in vitro. The new organ culture model will permit the study of functional and molecular consequences of HCMV infection in a more physiological micro-environment.