Wnt5a Regulates Midbrain Dopaminergic Axon Growth and Guidance

During development, precise temporal and spatial gradients are responsible for guiding axons to their appropriate targets. Within the developing ventral midbrain (VM) the cues that guide dopaminergic (DA) axons to their forebrain targets remain to be fully elucidated. Wnts are morphogens that have been identified as axon guidance molecules. Several Wnts are expressed in the VM where they regulate the birth of DA neurons. Here, we describe that a precise temporo-spatial expression of Wnt5a accompanies the development of nigrostriatal projections by VM DA neurons. In mice at E11.5, Wnt5a is expressed in the VM where it was found to promote DA neurite and axonal growth in VM primary cultures. By E14.5, when DA axons are approaching their striatal target, Wnt5a causes DA neurite retraction in primary cultures. Co-culture of VM explants with Wnt5a-overexpressing cell aggregates revealed that Wnt5a is capable of repelling DA neurites. Antagonism experiments revealed that the effects of Wnt5a are mediated by the Frizzled receptors and by the small GTPase, Rac1 (a component of the non-canonical Wnt planar cell polarity pathway). Moreover, the effects were specific as they could be blocked by Wnt5a antibody, sFRPs and RYK-Fc. The importance of Wnt5a in DA axon morphogenesis was further verified in Wnt5a−/− mice, where fasciculation of the medial forebrain bundle (MFB) as well as the density of DA neurites in the MFB and striatal terminals were disrupted. Thus, our results identify a novel role of Wnt5a in DA axon growth and guidance

Sustained proliferation in cancer: mechanisms and novel therapeutic targets

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression

Ryk, a receptor regulating Wnt5a-mediated neurogenesis and axon morphogenesis of ventral midbrain dopaminergic neurons

Ryk is an atypical transmembrane receptor tyrosine kinase that has been shown to play multiple roles in development through the modulation of Wnt signaling. Within the developing ventral midbrain (VM), Wnts have been shown to contribute to the proliferation, differentiation, and connectivity of dopamine (DA) neurons; however, the Wnt-related receptors regulating these events remain less well described. In light of the established roles of Wnt5a in dopaminergic development (regulating DA differentiation as well as axonal growth and repulsion), and its interaction with Ryk elsewhere within the central nervous system, we investigated the potential role of Ryk in VM development. Here we show temporal and spatial expression of Ryk within the VM, suggestive of a role in DA neurogenesis and axonal plasticity. In VM primary cultures, we show that the effects of Wnt5a on VM progenitor proliferation, DA differentiation, and DA axonal connectivity can be inhibited using an Ryk-blocking antibody. In support, Ryk knockout mice showed reduced VM progenitors and DA precursor populations, resulting in a significant decrease in DA cells. However, Ryk⁻/⁻ mice displayed no defects in DA axonal growth, guidance, or fasciculation of the MFB, suggesting other receptors may be involved and/or compensate for the loss of this receptor. These findings identify for the first time Ryk as an important receptor for midbrain DA development.13 page(s

Identification of a novel glucose transporter-like protein - GLUT-12

Facilitative glucose transporters exhibit variable hexose affinity and tissue-specific expression. These characteristics contribute to specialized metabolic properties of cells. Here we describe the characterization of a novel glucose transporter-like molecule, GLUT-12. GLUT-12 was identified in MCF-7 breast cancer cells by homology to the insulin-regulatable glucose transporter GLUT-4. The GLUT-12 cDNA encodes 617 amino acids, which possess features essential for sugar transport. Di-leucine motifs are present in NH and COOH termini at positions similar to the GLUT-4 FQQI and LL targeting motifs. GLUT-12 exhibits 29% amino acid identity with GLUT-4 and 40% to the recently described GLUT-10. Like GLUT-10, a large extracellular domain is predicted between transmembrane domains 9 and 10. Genomic organization of GLUT-12 is highly conserved with GLUT-10 but distinct from GLUTs 1-5. Immunofluorescence showed that, in the absence of insulin, GLUT-12 is localized to the perinuclear region in MCF-7 cells. Immunoblotting demonstrated GLUT-12 expression in skeletal muscle, adipose tissue, and small intestine. Thus GLUT-12 is potentially part of a second insulin-responsive glucose transport system

Generation of mouse MAbs to the RYK extracellular region and epitope mapping.

<p>(A) Flow cytometry using purified mouse anti-RYK MAbs 1B4, 1G8, 5E3 and 6G1 on 293-EBNA cells stably expressing pVITRO3-mcs (empty vector control; V) or hRYKFCT (RYK). All antibodies detected RYK in hRYKFCT-transfected but not vector-transfected cells. (B) Schematic of the mouse Ryk fusion proteins used in this study. EC, extracellular region; WD, WIF domain. (C) Western blot analysis of purified mouse Ryk fusion proteins using mouse anti-RYK MAbs 1B4 and 6G1. The pattern of binding was the same for both antibodies. The presence of all the fusion proteins was confirmed by stripping the membrane and reprobing with rabbit anti-Ryk^EC polyclonal antibody. Molecular mass standards are shown at left in kDa. IB, immunoblot. (D) ELISA results using mouse anti-RYK MAbs 1B4 and 6G1 on an immobilized peptide library of the entire human RYK extracellular region. Peptides 3 to 37: RYK WIF domain; peptide 47: FLAG epitope (incubated with mouse anti-FLAG M2 MAb; positive control); well 48: empty (negative control). The MAbs were used at 2 µg/mL. All antibodies bound to the same epitope, in peptides 40−42. The location of the epitope is shown schematically (bottom). Epitopes for the 1G8 and 5E3 antibodies were identical (data not shown). OD, optical density.</p

Characterization of the human inhibitory anti-RYK IgG_1κ antibody (RWD1).

<p>(A) RWD1 IPs from lysates of HEK293T cells transiently transfected with vectors encoding human RYK domain swap derivatives (lanes 1–5) were immunoblotted (IB) with anti-FLAG antibody. Lysate input (lanes 6–9) is shown at right. Molecular mass standards are shown at left in kDa. WIF1-WD, WIF domain from human WIF1; ROR2-CRD, CRD from human ROR2; H, heavy chain; L, light chain. (B) Anti-FLAG IPs were immunoblotted (IB) with an anti-Myc antibody to detect Wnt binding to hRYK.Fc. Molecular mass standards are shown at left in kDa. (C) ELISA analysis of immobilized RWD1 probed with hRYK.Fc. Increased hRYK.Fc binding to the antibody was observed with higher concentrations of either immobilized RWD1 (left panel) or soluble hRYK.Fc (right panel). Results represent the mean±standard deviation of two or three independent experiments. IgG, human IgG; OD, optical density. (D) HEK293T cells transiently transfected with the plasmids indicated (at right) were fixed as shown (above), paraffin-embedded and subjected to IHC using RWD1 biotinylated on <i>N</i>-glycan chains. Bar in panel (iv) represents 50 µm. NBF, neutral-buffered formalin; PFA, paraformaldehyde. (E) Example of IHC on a tumor from a formalin-fixed and paraffin-embedded human breast cancer tissue microarray stained with bRWD1 (upper panel) or human MAb isotype control (bIgG_1κ; lower panel). Open arrow, RYK on a cancer cell; filled arrow, RYK on tumor stroma; arrowhead, RYK-positive cancer cell nucleus; b, biotinylated on <i>N</i>-glycan chains. Bar represents 50 µm.</p

RWD1 inhibits Wnt signaling and Ryk function in neurons.

<p>(A) Western blot analysis of lysates from SN4741 cells treated with Wnt5a (300 ng/mL) or vehicle (PBS; C) and with human IgG (50 µg/mL) or RWD1 (50 µg/mL). P-Dvl2, phosphorylated Dvl2. Molecular mass standards are shown at left in kDa. The three lanes were non-consecutive but on the one membrane. The normalized ratio P-Dvl2/total Dvl2 (corrected for β-actin) is shown beneath each lane. (B) Quantification of neurite growth from E15.5 mouse cortical neurons treated with human IgG (50 µg/mL), RWD1 (50 µg/mL), Wnt5a (300 ng/ml) and/or vehicle (PBS; C). Results represent the mean±SEM of four independent experiments. *P&lt;0.05; **P&lt;0.01; ***P&lt;0.001.</p

Proteolytic processing of the mouse Ryk extracellular region.

<p>(A) Proteolysis of Ryk in mammalian cell lines. Cells were transiently transfected with pcDNA3.mM2RFCT and lysed 48 h later. Anti-FLAG immunoprecipitates (IP) were immunoblotted (IB) with rabbit anti-Ryk^IC polyclonal antibody. Molecular mass standards are shown at left in kDa. MEFs, mouse embryonic fibroblasts; RDTI.2, Ryk-deficient large T antigen-immortalized fibroblasts derived from a <i>Ryk</i>^−/− embryo. (B) Consensus cleavage sites in the mouse Ryk extracellular region for PC1, PC2, furin, PC4, PC5, PACE4 and PC7 (single-letter amino acid code; basic residues conforming to the consensus in blue; residues numbered according to NCBI Reference Sequence NP_038677.3; (K/R)X_n(K/R)↓, where X is any residue, n = 0, 2, 4 or 6 and the downward arrow represents cleavage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075447#pone.0075447-Seidah1" target="_blank">[53]</a>). The furin cleavage site in the mouse c-Met extracellular domain is shown for comparison. (C) COS-7 cells were transiently transfected with plasmids encoding mM2RFCT or the derivatives V594A; K186Q (monobasic, MB); KK181→QQ181 (dibasic, DB); KRRK176→QQQQ176 (tetrabasic, TB); and QQQQ176;QQ181;Q186 (compound mutant; CM). The α₁-PDX.FLAG protein (p54, p57 and p64 isoforms indicated) was expressed to inhibit endogenous furin. Mouse c-Met.FLAG was expressed as a positive control for inhibition of furin. The location and identity of Ryk-CTF was confirmed using anti-Ryk^IC polyclonal antibody (bottom panel). Molecular mass standards are shown at left in kDa. (D) Transiently transfected COS-7 cells were treated with potential activators of receptor shedding for 30 min. Anti-Myc IPs were prepared from the conditioned medium and immunoblotted with an anti-Myc antibody. Molecular mass standards are shown at left in kDa. V, empty vector (pcDNA3)-transfected cells; PBSS, phosphate-buffered saline with calcium and magnesium used as diluent for pervanadate. (E) Transiently transfected COS-7 cells were pre-treated with protease inhibitors, then shedding was activated with TFP (100 µM, 30 min). Anti-Myc IPs were analyzed as in (C). Molecular mass standards are shown at left in kDa. (F) Model for sequential proteolysis of Ryk. The metalloprotease-mediated cleavage in step (ii) has constitutive and inducible components. Green, WIF domain; gold, PTK domain; red, transmembrane helix; Ryk-FL, full-length uncleaved Ryk; Ryk-CTF, Ryk carboxyl-terminal fragment; Ryk-ICD, Ryk intracellular domain fragment; Rβ, predicted membrane-associated peptide analogous to amyloid-β peptide.</p