Michela Visintin

The Neuronal Microtubule-Associated Protein Tau Is a Substrate for Caspase-3 and an Effector of Apoptosis

We have identified a class of tau fragments inducing apoptosis in different cellular contexts, including a human teratocarcinoma‐derived cell line (NT2 cells) representing committed human neuronal precursors. We have found a transition point inside the tau molecule beyond which the fragments lose their ability to induce apoptosis. This transition point is located around one of the putative caspase‐3 cleavage sites. This is the only site that can be effectively used by caspase‐3 in vitro, releasing the C‐terminal 19 amino acids of tau. These results establish tau as a substrate for an apoptotic protease that turns tau itself into an effector of apoptosis. Accordingly, tau may be involved in a self‐propagating process like what has been predicted for the pathogenesis of different neurodegenerative disorders.

Intracellular antibodies for proteomics

Abstract The intracellular antibody technology has many applications for proteomics studies. The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen–antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology. We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naı̈ve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins. The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.

Intracellular Single-Chain Variable Fragments Directed to the Src Homology 2 Domains of Syk Partially Inhibit FcεRI Signaling in the RBL-2H3 Cell Line

Intracellular expression of Ab fragments has been efficiently used to inactivate therapeutic targets, oncogene products, and to induce viral resistance in plants. Ab fragments expressed in the appropriate cell compartment may also help to elucidate the functions of a protein of interest. We report in this study the successful targeting of the protein tyrosine kinase Syk in the RBL-2H3 rat basophilic leukemia cell line. We isolated from a phage display library human single-chain variable fragments (scFv) directed against the portion of Syk containing the Src homology 2 domains and the linker region that separates them. Among them, two scFv named G4G11 and G4E4 exhibited the best binding to Syk in vivo in a yeast two-hybrid selection system. Stable transfectants of RBL-2H3 cells expressing cytosolic G4G11 and G4E4 were established. Immunoprecipitation experiments showed that intracellular G4G11 and G4E4 bind to Syk, but do not inhibit the activation of Syk following FcεRI aggregation, suggesting that the scFv do not affect the recruitment of Syk to the receptor. Nevertheless, FcεRI-mediated calcium mobilization and the release of inflammatory mediators are inhibited, and are consistent with a defect in Bruton’s tyrosine kinase and phospholipase C-γ2 tyrosine phosphorylation and activation. Interestingly, FcεRI-induced mitogen-activated protein kinase phosphorylation is not altered, suggesting that intracellular G4G11 and G4E4 do not prevent the coupling of Syk to the Ras pathway, but they selectively inhibit the pathway involving phospholipase C-γ2 activation.

Gephyrin selective intrabodies as a new strategy for studying inhibitory receptor clustering.

The microtubule-binding protein gephyrin is known to play a pivotal role in targeting and clustering postsynaptic inhibitory receptors. Here, the Intracellular Antibodies Capture Technology (IATC) was used to select two single-chain antibody fragments or intrabodies, which, fused to nuclear localization signals (NLS), were able to efficiently and selectively remove gephyrin from glycine receptor (GlyR) clusters. Co-transfection of NLS-tagged individual intrabodies with gephyrin-enhanced green fluorescent protein (EGFP) in HEK 293 cells revealed a partial relocalization of gephyrin aggregates onto the nucleus or in the perinuclear area. When expressed in cultured neurons, these intrabodies caused a significant reduction in the number of immunoreactive GlyR clusters, which was associated with a decrease in the peak amplitude of glycine-evoked whole cell currents as assessed with electrophysiological experiments. Hampering protein function at a posttranslational level may represent an attractive alternative for interfering with gephyrin function in a more spatially localized manner.

Direct intracellular selection and biochemical characterization of a recombinant anti-proNGF single chain antibody fragment

proNGF, the precursor of the neurotrophin NGF, is widely expressed in central and peripheral nervous system. Its physiological functions are still largely unknown, although it emerged from studies in the last decade that proNGF has additional and distinct functions with respect to NGF, besides acting chaperone-like for NGF folding during its biogenesis. The regulation of proNGF/NGF ratio represents a crucial process for homeostasis of brain and other tissues, and understanding the molecular aspects of these differences is important. We report the selection and characterization of a recombinant monoclonal anti-proNGF antibody in single chain Fv fragment (scFv) format. The selection exploited the Intracellular Antibody Capture Technology (IACT), starting from a naïve mouse SPLINT (Single Pot Library of INTracellular antibodies) library. This antibody (scFv FPro10) was expressed recombinantly in Escherichia coli, was proven to be highly soluble and stable, and thoroughly characterized from the biochemical-biophysical point of view. scFv FPro10 displays high affinity and specificity for proNGF, showing no cross-reactivity with other pro-neurotrophins. A structural model was obtained by SAXS. scFv FPro10 represents a new tool to be exploited for the selective immunoanalysis of proNGF, both in vitro and in vivo, and might help in understanding the molecular function of proNGF in neurodegeneration.

Selecting Intracellular Antibodies Using the Two-Hybrid System

The ectopic expression of functional antibodies can be utilized to specifically interfere with the function of selected antigens in cells, tissues or in organisms [1]. The efficacy of the ectopic antibody expression has been already demonstrated in several cases and its use has potential applications to disease therapy and in functional genomic studies.

P2-380: Novel recombinant antibodies against AD biomarkers linked to neurotrophin signalling as potential diagnostic tools

achieve a uniform quality level for this study, the dataset was reduced to 224 subjects from 6 centers. A supervised fully connected ANN was implemented with a single layer of hidden units and 30 inputs (3 modalities, each with their respective 3x3 neighborhood and spatial information). The output comprised of four probability maps GM, WM, CSF and ARWMC, but only the ARWMC output is assessed here. Before segmentation all data were pre-processed for head motion and intensity variations. The ANN was trained with an ROI dataset drawn on 6 subjects from 4 centers. For segmentation, the optimal threshold of the ARWMC probability map was determined using the Index of Similarity describing the overlap between ANN and manual ROI’s. Here a threshold of 90% was found to be optimal. Results: From each of the 6 center the correlation coefficient between ANN and manually determined were generally high: 0.97, 0.97, 0.87, 0.88, 0.73 and 0.98. The correlation of the combined set was 0.89. The two first centers, with a correlation coefficient of 0.97, had two subjects in the training set which may explain their better performance. Deviating quality in the FLAIR image contributes to the lowest correlation in a center. In other cases a systematic bias was present in some anatomical regions (e.g. septum pellucidum, where lesion were not drawn manually) or regarding lacunar infarcts. Conclusion: In this multi-center investigation of ARWMC segmentation a high average correlation of 0.89 was found. Improvements may be gained from representing more centres in the training set. The results demonstrate the generalizability of ANN methods, and underline the importance of rigorous standardization of MRI quality in multi-center studies.

P4-323: Intracellularly selected recombinant antibodies targeting beta-amyloid oligomers

Giovanni Meli, Michela Visintin, Isabella Cannistraci, Daniela Avossa, Anita Westlind-Danielsson, Antonino Cattaneo, International School for Advanced Studies (SISSA), Trieste, Italy; European Brain Research Institute (EBRI), Roma, Italy; Lay Line Genomics S.p.A., Roma, Italy; Department of Neurochemistry and Neurotoxicology, The Arrhenius Laboratories for Natural Sciences, Stockholm, Sweden. Contact e-mail: meli@sissa.it

The neuronal microtubule associated protein tau is a substrate for caspase-3 and an effector of apoptosis

We have identified a class of tau fragments inducing apoptosis in different cellular contexts, including a human teratocarcinoma-derived cell line (NT2 cells) representing committed human neuronal precursors. We have found a transition point inside the tau molecule beyond which the fragments lose their ability to induce apoptosis. This transition point is located around one of the putative caspase-3 cleavage sites. This is the only site that can be effectively used by caspase-3 in vitro, releasing the C-terminal 19 amino acids of tau. These results establish tau as a substrate for an apoptotic protease that turns tau itself into an effector of apoptosis. Accordingly, tau may be involved in a self-propagating process like what has been predicted for the pathogenesis of different neurodegenerative disorders.