Bianca Vaida

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.

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.

Comparison between Human Cytomegalovirus pUL97 and Murine Cytomegalovirus (MCMV) pM97 Expressed by MCMV and Vaccinia Virus: pM97 Does Not Confer Ganciclovir Sensitivity

ABSTRACT The UL97 protein (pUL97) of human cytomegalovirus (HCMV) is a protein kinase that also phosphorylates ganciclovir (GCV), but its biological function is not yet clear. The M97 protein (pM97) of mouse cytomegalovirus (MCMV) is the homolog of pUL97. First, we studied the consequences of genetic replacement of M97 by UL97. Using the infectious bacterial plasmid clone of the full-length MCMV genome (M. Wagner, S. Jonjic, U. H. Koszinowski, and M. Messerle, J. Virol. 73:7056–7060, 1999), we replaced the M97 gene with the UL97 gene and constructed an MCMV M97 deletion mutant and a revertant virus. In addition, pUL97 and pM97 were expressed by recombinant vaccinia virus to compare both for known functions. Remarkably, pM97 proved not to be the reason for the GCV sensitivity of MCMV. When expressed by the recombinant MCMV, however, pUL97 was phosphorylated and endowed MCMV with the capacity to phosphorylate GCV, thereby rendering MCMV more susceptible to GCV. We found that deletion of pM97, although it is not essential for MCMV replication, severely affected virus growth. This growth deficit was only partially amended by pUL97 expression. When expressed by recombinant vaccinia viruses, both proteins were phosphorylated and supported phosphorylation of GCV, but pUL97 was about 10 times more effective than pM97. One hint of the functional differences between the proteins was provided by the finding that pUL97 accumulates in the nucleus, whereas pM97 is predominantly located in the cytoplasm of infected cells. In vivo testing revealed that the UL97-MCMV recombinant should allow evaluation of novel antiviral drugs targeted to the UL97 protein of HCMV in mice.

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.

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.

Altered Expression of Extracellular Matrix in Human-Cytomegalovirus-Infected Cells and a Human Artery Organ Culture Model to Study Its Biological Relevance

The influence of human cytomegalovirus (HCMV) on the transcription of 11 selected, representative extracellular matrix genes was investigated in cell culture. Northern blot hybridization indicated the downregulation of all mRNAs investigated. Based on our results and the known repression of other extracellular matrix transcripts and the β-actin transcription during HCMV infection, we suggest that one molecular mechanism contributing to the cytopathic effect may be the transcriptional downregulation of genes encoding proteins involved in cell structure and intercellular connection. To further study the biological relevance of this and other pathogenetic mechanisms, we established a human renal artery organ culture system and characterized this new infection model for HCMV. Our model is a new suitable system for the investigation of molecular as well as functional consequences of HCMV infection in a more physiological microenvironment.

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

BackgroundThe 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.ResultsDuring 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.ConclusionThe 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.

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.