Identification and characterization of nanobodies targeting the EphA4 receptor

The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. Weidentified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb39 andNb53 inhibited ephrin-induced phosphorylationoftheEphA4proteininacell-basedassay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3. Our results demonstrate the potential of Nanobodies to target the ligand-binding domain of EphA4. These Nanobodiesmaydeservefurtherevaluationaspotentialtherapeutics in disorders in which EphA4-mediated signaling plays a role

Microglial activation arises after aggregation of phosphorylated-tau in a neuron-specific P301S tauopathy mouse model

Alzheimer's disease, progressive supranuclear palsy and frontotemporal dementia are characterized by neuronal expression of aberrant tau protein, tau hyperphosphorylation (pTAU), tau aggregation and neurofibrillary tangle formation sequentially culminating into neuronal cell death, a process termed tauopathy. Our aim was to address at which tauopathy stage neuroinflammation starts and to study the related microglial phenotype. We used Thy1-hTau.P301S (PS) mice expressing human tau with a P301S mutation specifically in neurons. Significant levels of cortical pTAU were present from 2 months onwards. Dystrophic morphological complexity of cortical microglia arose after pTAU accumulation concomitant with increased microglial lysosomal volumes and a significant loss of homeostatic marker Tmem119. Interestingly, we detected increases in neuronal pTAU and postsynaptic structures in the lysosomes of PS microglia. Moreover, the overall cortical postsynaptic density was decreased in 6-month-old PS mice. Together, our results indicate that microglia adopt a pTAU-associated phenotype, and are morphologically and functionally distinct from wild-type microglia after neuronal pTAU accumulation has initiated

Regional vulnerability and spreading of hyperphosphorylated tau in seeded mouse brain

We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool

Heparanase activates the syndecan-syntenin-ALIX exosome pathway

Exosomes are secreted vesicles of endosomal origin involved in signaling processes. We recently showed that the syndecan heparan sulfate proteoglycans control the biogenesis of exosomes through their interaction with syntenin-1 and the endosomal-sorting complex required for transport accessory component ALIX. Here we investigated the role of heparanase, the only mammalian enzyme able to cleave heparan sulfate internally, in the syndecan-syntenin-ALIX exosome biogenesis pathway. We show that heparanase stimulates the exosomal secretion of syntenin-1, syndecan and certain other exosomal cargo, such as CD63, in a concentration-dependent manner. In contrast, exosomal CD9, CD81 and flotillin-1 are not affected. Conversely, reduction of endogenous heparanase reduces the secretion of syntenin-1-containing exosomes. The ability of heparanase to stimulate exosome production depends on syntenin-1 and ALIX. Syndecans, but not glypicans, support exosome biogenesis in heparanase-exposed cells. Finally, heparanase stimulates intraluminal budding of syndecan and syntenin-1 in endosomes, depending on the syntenin-ALIX interaction. Taken together, our findings identify heparanase as a modulator of the syndecan-syntenin-ALIX pathway, fostering endosomal membrane budding and the biogenesis of exosomes by trimming the heparan sulfate chains on syndecans. In addition, our data suggest that this mechanism controls the selection of specific cargo to exosomes.Cell Research advance online publication 3 March 2015; doi:10.1038/cr.2015.29.status: publishe

Biochemical characterization of malate synthase G of P. aeruginosa

ABSTRACT: BACKGROUND: Malate synthase catalyzes the second step of the glyoxylate bypass, the condensation of acetyl coenzyme A and glyoxylate to form malate and coenzyme A (CoA). In several microorganisms, the glyoxylate bypass is of general importance to microbial pathogenesis. The predicted malate synthase G of Pseudomonas aeruginosa has also been implicated in virulence of this opportunistic pathogen. RESULTS: Here, we report the verification of the malate synthase activity of this predicted protein and its recombinant production in E. coli, purification and biochemical characterization. The malate synthase G of P. aeruginosa PAO1 has a temperature and pH optimum of 37.5 degrees C and 8.5, respectively. Although displaying normal thermal stability, the enzyme was stable up to incubation at pH 11. The following kinetic parameters of P. aeruginosa PAO1 malate synthase G were obtained: Km glyoxylate (70 microM), Km acetyl CoA (12 microM) and Vmax (16.5 micromol/minutes/millig enzyme). In addition, deletion of the corresponding gene showed that it is a prerequisite for growth on acetate as sole carbon source. CONCLUSIONS: The implication of the glyoxylate bypass in the pathology of various microorganisms makes malate synthase G an attractive new target for antibacterial therapy. The purification procedure and biochemical characterization assist in the development of antibacterial components directed against this target in P. aeruginosa.status: publishe

A procedure for systematic identification of bacteriophage–host interactions of P. aeruginosa phages

AbstractImmediately after bacteriophage infection, phage early proteins establish optimal conditions for phage infection, often through a direct interaction with host-cell proteins. We implemented a yeast two-hybrid approach for Pseudomonas aeruginosa phages as a first step in the analysis of these – often uncharacterized – proteins. A 24-fold redundant prey library of P. aeruginosa PAO1 (7.32×106 independent clones), was screened against early proteins (gp1 to 9) of ϕKMV, a P. aeruginosa-infecting member of the Podoviridae; interactions were verified using an independent in vitro assay. None resembles previously known bacteriophage–host interactions, as the three identified target malate synthase G, a regulator of a secretion system and a regulator of nitrogen assimilation. Although at least two-bacteriophage infections are non-essential to ϕKMV infection, their disruption has an influence on infection efficiency. This methodology allows systematic analysis of phage proteins and is applicable as an interaction analysis tool for P. aeruginosa

Syntenin controls migration, growth, proliferation, and cell cycle progression in cancer cells

The scaffold protein syntenin abounds during fetal life where it is important for developmental movements. In human adulthood, syntenin gain-of-function is increasingly associated with various cancers and poor prognosis. Depending on the cancer model analyzed, syntenin affects various signaling pathways. We previously have shown that syntenin allows syndecan heparan sulfate proteoglycans to escape degradation. This indicates that syntenin has the potential to support sustained signaling of a plethora of growth factors and adhesion molecules. Here, we aim to clarify the impact of syntenin loss-of-function on cancer cell migration, growth, and proliferation, using cells from various cancer types and syntenin shRNA and siRNA silencing approaches. We observed decreased migration, growth, and proliferation of the mouse melanoma cell line B16F10, the human colon cancer cell line HT29 and the human breast cancer cell line MCF7. We further documented that syntenin controls the presence of active β1 integrin at the cell membrane and G1/S cell cycle transition as well as the expression levels of CDK4, Cyclin D2, and Retinoblastoma proteins. These data confirm that syntenin supports the migration and growth of tumor cells, independently of their origin, and further highlight the attractiveness of syntenin as potential therapeutic target.status: publishe

The genome and structural Proteome of YuA, a new Pseudomonas aeruginosa phage resembling M6

Pseudomonas aeruginosa phage YuA (Siphoviridae) was isolated from a pond near Moscow, Russia. It has an elongated head, encapsulating a circularly permuted genome of 58,663 bp, and a flexible, noncontractile tail, which is terminally and subterminally decorated with short fibers. The YuA genome is neither Mu- nor lambda-like and encodes 78 gene products that cluster in three major regions involved in (i) DNA metabolism and replication, (ii) host interaction, and (iii) phage particle formation and host lysis. At the protein level, YuA displays significant homology with phages M6, phi JL001, 73, B3, DMS3, and D3112. Eighteen YuA proteins were identified as part of the phage particle by mass spectrometry analysis. Five different bacteria] promoters were experimentally identified using a promoter trap assay, three of which have a sigma(54)-specific binding site and regulate transcription in the genome region involved in phage particle formation and host lysis. The dependency of these promoters on the host sigma(54) factor was confirmed by analysis of an rpoN mutant strain of P. aeruginosa PAO1. At the DNA level, YuA is 91% identical to the recently (July 2007) annotated phage M6 of the Lindberg typing set. Despite this level of DNA homology throughout the genome, both phages combined have 15 unique genes that do not occur in the other phage. The genome organization of both phages differs substantially from those of the other known Pseudomonas-infecting Siphoviridae, delineating them as a distinct genus within this family.status: publishe