Quantitative live cell imaging of a tauopathy model enables the identification of a polypharmacological drug candidate that restores physiological microtubule interaction.

Tauopathies such as Alzheimer's disease are characterized by aggregation and increased phosphorylation of the microtubule-associated protein tau. Tau's pathological changes are closely linked to neurodegeneration, making tau a prime candidate for intervention. We developed an approach to monitor pathological changes of aggregation-prone human tau in living neurons. We identified 2-phenyloxazole (PHOX) derivatives as putative polypharmacological small molecules that interact with tau and modulate tau kinases. We found that PHOX15 inhibits tau aggregation, restores tau's physiological microtubule interaction, and reduces tau phosphorylation at disease-relevant sites. Molecular dynamics simulations highlight cryptic channel-like pockets crossing tau protofilaments and suggest that PHOX15 binding reduces the protofilament's ability to adopt a PHF-like conformation by modifying a key glycine triad. Our data demonstrate that live-cell imaging of a tauopathy model enables screening of compounds that modulate tau-microtubule interaction and allows identification of a promising polypharmacological drug candidate that simultaneously inhibits tau aggregation and reduces tau phosphorylation.

Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol.

The high pathogenicity of the Ebola virus reflects multiple concurrent processes on infection. Among other important determinants, Ebola fusogenic glycoprotein (GP) has been associated with the detachment of infected cells and eventually leads to vascular leakage and haemorrhagic fever. Here we report that the membrane-anchored GP is sufficient to induce the detachment of adherent cells. The results show that the detachment induced through either full-length GP1,2 or the subunit GP2 depends on cholesterol and the structure of the transmembrane domain. These data reveal a novel molecular mechanism in which GP regulates Ebola virus assembly and suggest that cholesterol-reducing agents could be useful as therapeutics to counteract GP-mediated cell detachment.

Proliferation and cilia dynamics in neural stem cells prospectively isolated from the SEZ.

Neural stem cells (NSCs) generate new neurons in vivo and in vitro throughout adulthood and therefore are physiologically and clinically relevant. Unveiling the mechanisms regulating the lineage progression from NSCs to newborn neurons is critical for the transition from basic research to clinical application. However, the direct analysis of NSCs and their progeny is still elusive due to the problematic identification of the cells. We here describe the isolation of highly purified genetically unaltered NSCs and transit-amplifying precursors (TAPs) from the adult subependymal zone (SEZ). Using this approach we show that a primary cilium and high levels of epidermal growth factor receptor (EGFR) at the cell membrane characterize quiescent and cycling NSCs, respectively. However, we also observed non-ciliated quiescent NSCs and NSCs progressing into the cell cycle without up-regulating EGFR expression. Thus, the existence of NSCs displaying distinct molecular and structural conformations provides more flexibility to the regulation of quiescence and cell cycle progression.

Virus-Like Particles Harboring CCL19, IL-2 and HPV16 E7 Elicit Protective T Cell Responses in HLA-A2 Transgenic Mice.

Infection by high-risk genotypes of human papillomaviruses (HR-HPVs) is the cause of cancer of the uterine cervix. Although prophylactic vaccines directed against the two most prevalent HR-HPV types (HPV16 and 18) have been commercialized recently, there is a need for effective therapeutic vaccines against HR-HPVs. We have tested in mice a chimeric protein composed of the hepatitis B small surface antigen (HBsAg(S)) flanked at its N-terminus by chemokine CC ligand 19/macrophage inflammatory protein-3ß (CCL19/MIP-3ß), and at the C-terminus by interleukin 2 (IL-2) and an artificial HPV16 E7 polytope. This protein is assembled into nanoparticles and both CCL19 and IL-2 conserve their functionality. HLA-A2 (AAD) transgenic mice immunized with a plasmid encoding this protein mounted specific T cell responses against E7 without the need of an adjuvant. Furthermore, vaccination prevented the development of tumors after implantation of the E6/E7-expressing TC-1/A2 tumor cell line. Our results suggest that vaccines based on HBsAg(S) nanoparticles carrying short E7 epitopes and immune-stimulatory domains might be of therapeutic value in the treatment of patients suffering from cervical pre-cancer or cancer lesions caused by HR-HPVs.

Nuclear factor erythroid-derived 2 (Nfe2) regulates JunD DNA-binding activity via acetylation: a novel mechanism regulating trophoblast differentiation.

We recently demonstrated that the bZip transcription factor nuclear factor erythroid-derived 2 (Nfe2) represses protein acetylation and expression of the transcription factor glial cell missing 1 (Gcm1) in trophoblast cells, preventing excess syncytiotrophoblast formation and permitting normal placental vascularization and embryonic growth. However, the Gcm1 promoter lacks a Nfe2-binding site and hence the mechanisms linking Nfe2 and Gcm1 expression remained unknown. Here we show that Nfe2 represses JunD DNA-binding activity to the Gcm1 promoter during syncytiotrophoblast differentiation. Interventional studies using knockdown and knockin approaches show that enhanced JunD DNA-binding activity is required for increased expression of Gcm1 and syncytiotrophoblast formation as well as impaired placental vascularization and reduced growth of Nfe2(-/-) embryos. Induction of Gcm1 expression requires binding of JunD to the -1441 site within the Gcm1 promoter, which is distinct from the -1314 site previously shown to induce Gcm1 expression by other bZip transcription factors. Nfe2 modulates JunD binding to the Gcm1 promoter via acetylation, as reducing JunD acetylation using the histone acetyltransferase inhibitor curcumin reverses the increased JunD DNA-binding activity observed in the absence of Nfe2. This identifies a novel mechanism through which bZip transcription factors interact. Within the placenta this interaction regulates Gcm1 expression, syncytiotrophoblast formation, placental vascularization, and embryonic growth.

p45NF-E2 represses Gcm1 in trophoblast cells to regulate syncytium formation, placental vascularization and embryonic growth.

Absence of the leucine zipper transcription factor p45NF-E2 results in thrombocytopenia, impaired placental vascularization and intrauterine growth restriction (IUGR) in mice. The mechanism underlying the p45NF-E2-dependent placental defect and IUGR remains unknown. Here, we show that the placental defect and IUGR of p45NF-E2 (Nfe2) null mouse embryos is unrelated to thrombocytopenia, establishing that embryonic platelets and platelet-released mediators are dispensable for placentation. Rather, p45NF-E2, which was hitherto thought to be specific to hematopoietic cells, is expressed in trophoblast cells, where it is required for normal syncytiotrophoblast formation, placental vascularization and embryonic growth. Expression of p45NF-E2 in labyrinthine trophoblast cells colocalizes with that of Gcm1, a transcription factor crucial for syncytiotrophoblast formation. In the absence of p45NF-E2, the width of syncytiotrophoblast layer 2 and the expression of Gcm1 and Gcm1-dependent genes (Synb and Cebpa) are increased. In vitro, p45NF-E2 deficiency results in spontaneous syncytiotrophoblast formation, which can be reversed by Gcm1 knockdown. Increased Gcm1 expression in the absence of p45NF-E2 is dependent on enhanced protein acetylation, including post-translational modification of Gcm1. Increasing and inhibiting acetylation in the placenta of wild-type control embryos phenocopies and corrects, respectively, the changes observed in p45NF-E2-deficient embryos. These studies identify a novel function of p45NF-E2 during placental development: in trophoblast cells, p45NF-E2 represses Gcm1 and syncytiotrophoblast formation via acetylation.

Cell surface tetraspanin Tspan8 contributes to molecular pathways of exosome-induced endothelial cell activation.

Tumor-derived exosomes containing the tetraspanin Tspan8 can efficiently induce angiogenesis in tumors and tumor-free tissues. However, little information exists on exosome-endothelial cell (EC) interactions or the proangiogenic role of tetraspanins, which are a constitutive component of exosomes. In this study, we used a rat adenocarcinoma model (AS-Tspan8) to explore the effects of exosomal Tspan8 on angiogenesis. Tspan8 contributed to a selective recruitment of proteins and mRNA into exosomes, including CD106 and CD49d, which were implicated in exosome-EC binding and EC internalization. We found that EC internalized Tspan8-CD49d complex-containing exosomes. Exosome uptake induced vascular endothelial growth factor (VEGF)-independent regulation of several angiogenesis-related genes, including von Willebrand factor, Tspan8, chemokines CXCL5 and MIF, chemokine receptor CCR1, and, together with VEGF, VEGF receptor 2. EC uptake of Tspan8-CD49d complex-containing exosomes was accompanied by enhanced EC proliferation, migration, sprouting, and maturation of EC progenitors. Unraveling these new pathways of exosome-initiated EC regulation could provide new options for therapeutic interference with tumor-induced angiogenesis.

Distinct roles of myosin Va in membrane remodeling and exocytosis of secretory granules.

Hormone- and neuropeptide-containing secretory granules (SGs) of neuroendocrine PC12 cells are formed at the trans- Golgi network as immature SGs. These intermediates are converted to mature SGs in a complex maturation process, including matrix condensation, processing of cargo proteins and removal of proteins and membrane in clathrin-coated vesicles. The resulting mature SGs undergo Ca2+-dependent exocytosis upon an appropriate stimulus. We here show that the motor protein myosin Va is implicated in a maturation step of SGs, their binding to F-actin and their stimulated exocytosis. Interference with myosin Va function blocked the removal of the transmembrane protein furin from maturing SGs without affecting condensation and processing of proteins of the SG lumen. Furthermore, the ATP-inhibited binding of SGs to F-actin decreased with progressive maturation and upon interference with myosin Va function. Moreover, the expression of a dominant-negative myosin Va-tail or shRNA-based downregulation of myosin Va interfered with stimulated exocytosis of SGs. In summary,our data suggest an essential function of myosin Va in the membrane remodeling of SGs during maturation and a role in their exocytosis.

Expression of distinct splice variants of the stem cell marker prominin-1 (CD133) in glial cells.

Prominin-1 (CD133) is a cholesterol-interacting pentaspan membrane glycoprotein specifically associated with plasma membrane protrusions. Prominin-1 is expressed by various stem and progenitor cells, notably neuroepithelial progenitors found in the developing embryonic brain. Here, we further investigated its expression in the murine brain. Biochemical analyses of brain membranes at early stages of development revealed the expression of two distinct splice variants of prominin-1, s1 and s3, which have different cytoplasmic C-terminal domains. The relative abundance of the s3 variant increased toward adulthood, whereas the opposite was observed for the s1 variant. Our combined in situ hybridization and immunohistochemistry revealed the expression of prominin-1 in a subpopulation of Olig-2-positive oligodendroglial cells present within white matter tracts of postnatal and adult brain. Furthermore, immunohistological and biochemical characterization suggested strongly that the s3 variant is a novel component of myelin. Consistent with this, the expression of prominin-1.s3 was significantly reduced in the brain of myelin-deficient mice. Finally, oligodendrocytes expressed selectively the s3 variant whereas GFAP-positive astrocytes expressed the s1 variant in primary glial cell cultures derived from embryonic brains. Collectively, our data demonstrate a complex expression pattern of prominin-1 molecules in developing adult brain. Given that prominin-1 is thought to act as an organizer of plasma membrane protrusions, they further suggest that a specific prominin-1 splice variant might play a role in morphogenesis and/or maintenance of the myelin sheath.

Prospective isolation of late development multipotent precursors whose migration is promoted by EGFR.

A simple procedure to isolate neural stem cells would greatly facilitate direct studies of their properties. Here, we exploited the increase in EGF receptor (EGFR) levels, that occurs in late development stem cells or in younger precursors upon exposure to FGF-2, to isolate cells expressing high levels of EGFR (EGFR(high)) from the developing and the adult brain. Independently of age and region of isolation, EGFR(high) cells were highly enriched in multipotent precursors and displayed similar antigenic characteristics, with the exception of GFAP and Lex/SSEA-1 that were mainly expressed in adult EGFR(high) cells. EGFR levels did not correlate with neurogenic potential, indicating that the increase in EGFR expression does not directly affect differentiation. Instead, in the brain, many EGFR(high) precursors showed tangential orientation and, whether isolated from the cortex or striatum, EGFR(high) precursors displayed characteristics of cells originating from the ventral GZ such as expression Dlx and Mash-1 and the ability to generate GABAergic neurons and oligodendrocytes. Moreover, migration of EGFR(high) cells on telencephalic slices required EGFR activity. Thus, the developmentally regulated increase in EGFR levels may affect tangential migration of multipotent precursors. In addition, it can be used as a marker to effectively isolate telencephalic multipotent precursors from embryonic and adult tissue.

Identification of novel Prominin-1/CD133 splice variants with alternative C-termini and their expression in epididymis and testis.

Prominin-1/CD133 is a five-membrane-span glycoprotein that is thought to act as an organizer of plasma-membrane protrusions. Here, we report the molecular and cell-biological characterization of four novel prominin-1 splice variants isolated from a mouse testis cDNA library and referred to as prominin-1.s3 to prominin-1.s6. Compared with kidney-derived prominin-1.s1, the s3, s4 and s5 variants contain a distinct cytoplasmic C-terminal domain. The s4 and s5 variants bear, in addition, two and one inframe deletion(s), respectively, in the extracellular domains. The s6 variant displays a truncated C-terminal domain caused by a premature termination resulting from intron retention. Upon their ectopic expression in Chinese hamster ovary cells, the s3 and s6 variants were found to be concentrated in plasma-membrane protrusions, whereas the s4 and s5 variants did not reach the cell surface. Biochemical analyses suggest that most of the prominin-1 in the adult male reproductive system is expressed as the s6 variant. Immunohistological and electron microscopic analyses show that prominin-1 is: (1) confined to the apical surface of the epithelium all along the epididymal duct, with the exception of the initial segment; (2) concentrated in stereocilia of the epididymal duct epithelium; and (3) found on the tail of developing spermatozoa in seminiferous tubules. Our data suggest that prominin-1 is involved in the formation and/or stabilization of epididymal stereocilia and the tail of spermatozoa, and hence might play a dual role in the biogenesis of spermatozoa.

Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells.

Neuroendocrine secretory granules, the storage organelles for neuropeptides and hormones, are formed at the trans-Golgi network, stored inside the cell and exocytosed upon stimulation. Previously, we have reported that newly formed secretory granules of PC12 cells are transported in a microtubule-dependent manner from the trans-Golgi network to the F-actin-rich cell cortex, where they undergo short directed movements and exhibit a homogeneous distribution. Here we provide morphological and biochemical evidence that myosin Va is associated with secretory granules. Expression of a dominant-negative tail domain of myosin Va in PC12 cells led to an extensive clustering of secretory granules close to the cell periphery, a loss of their cortical restriction and a strong reduction in their motility in the actin cortex. Based on this data we propose a model that implies a dual transport system for secretory granules: after microtubule-dependent delivery to the cell periphery, secretory granules exhibit a myosin Va-dependent transport leading to their restriction and even dispersal in the F-actin-rich cortex of PC12 cells.

AN2, the mouse homologue of NG2, is a surface antigen on glial precursor cells implicated in control of cell migration.

Molecular studies have demonstrated that the murine AN2 antigen is the mouse homologue of the rat NG2 and human MCSP protein. The molecule is a single-pass transmembrane protein which carries a variable number of glyco- and glycosaminoglycan chains according to cell type and developmental stage. AN2/NG2 has two extracellular Laminin G-like domains which are classically involved in cell adhesion and recognition. It possesses a single PDZ binding motif in the short intracellular tail. The AN2/NG2 antigen is expressed by glial progenitor cells in developing and adult CNS and also by immature Schwann cells. Antibodies against AN2/NG2 inhibit the migration of antigen-positive cells in in vitro assays, suggesting that the molecule plays a role in migration. Many AN2/NG2-positive cells surround synapses in the developing and adult brain. A recently identified intracellular partner of AN2/NG2 is the glutamate receptor interacting protein GRIP, which binds to the GluRB subunit of the AMPA subclass of glutamate receptors. The AN2/NG2 protein may position AMPA receptors on perisynaptic glial cells towards active synapses by binding to a neuronal receptor. Many highly migratory neural tumors including melanomas express AN2/NG2. In the demyelinating disease Multiple Sclerosis, some patients synthesise antibodies against the protein. Such antibodies may play a pathological role by inhibiting the migration of oligodendrocyte progenitor cells to demyelinated axons thus blocking remyelination, as well as possibly interfering with glial neuronal signalling at synapses and nodes of Ranvier.