Lentiviral Vectors to Probe and Manipulate the Wnt Signaling Pathway

Background: The Wnt signaling pathway plays key roles in development, adult tissue homeostasis and stem cell maintenance. Further understanding of the function of Wnt signaling in specific cell types could benefit from lentiviral vectors expressing reporters for the Wnt pathway or vectors interfering with signaling. Methodology/Principal Findings: We have developed a set of fluorescent and luminescent lentiviral vectors that report Wnt signaling activity and discriminate between negative and uninfected cells. These vectors possess a 7xTcf-eGFP or 7xTcf-FFluc (Firefly Luciferase) reporter cassette followed by either an SV40-mCherry or SV40-Puro R (puromycin N-acetyltransferase) selection cassette. We have also constructed a vector that allows drug-based selection of cells with activated Wnt signaling by placing Puro R under the control of the 7xTcf promoter. Lastly, we have expressed dominantnegative Tcf4 (dnTcf4) or constitutively active beta-catenin (b-catenin 4A) from the hEF1a promoter in a SV40-Puro R or SV40mCherry backbone to create vectors that inhibit or activate the Wnt signaling pathway. These vectors will be made available to the scientific community through Addgene. Conclusions: These novel lentiviruses are efficient tools to probe and manipulate Wnt signaling. The use of a selection cassette in Wnt-reporter viruses enables discriminating between uninfected and non-responsive cells, an important requirement for experiments where selection of clones is not possible. The use of a chemiluminescent readout enable

Amyloid-beta oligomerization is associated with the generation of a typical peptide fragment fingerprint

Amyloid-beta (A beta) peptide oligomerization plays a central role in the pathogenesis of Alzheimer's disease (AD), and A beta oligomers are collectively considered an appealing therapeutic target for the treatment of AD. However, the molecular mechanisms leading to the pathologic accumulation of oligomers are unclear, and the exact structural composition of oligomers is being debated. Using targeted and quantitative mass spectrometry, we reveal site-specific A beta autocleavage during the early phase of aggregation, producing a typical A beta fragment signature and that truncated A beta peptides can form stable oligomeric complexes with full-length A beta peptide. We show that the use of novel anti-A beta antibodies raised against these truncated A beta isoforms allows for monitoring and targeting the accumulation of truncated A beta. fragments. Antibody-enabled screening of transgenic models of AD as well as human postmortem brain tissue and cerebrospinal fluid revealed that aggregation-associated A beta cleavage is a highly relevant clinical feature of AD. (C) 2016 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved

Lentiviruses that inhibit or activate the Wnt signaling pathway.

<p>To measure Wnt signaling activity in HEK293T and HEK293A, cells were transfected with the reporter construct pMegaTOPFlash prior to Wnt3A addition. A) HEK293T cells were infected with the hEF1α-dnTcf4//SV40-mCherry (“EdTC”) virus, and mCherry-expressing cells were selected by fluorescence-activated cell sorting. Induction of a Wnt reporter by purified Wnt3A protein (500 ng/ml final) was impaired in EdTC-infected cells compared to mock-infected cells (top). All selected cells were expressing mCherry (bottom). B) Mouse LS/L or HEK293A cells were infected with the hEF1α-dnTcf4//SV40-Puro^R (“EdTP”) virus and selected with 1 µg/ml puromycin. Puromycin selection led to enrichment of LS/L cells with an impaired response to Wnt3A-conditioned medium (left, compare “+” with “−” puromycin). Induction of a Wnt reporter by purified Wnt3A protein (500 ng/ml final) was also impaired in EdTP-infected HEK293A cells compared to mock-infected cells (right). C) HEK293T cells were infected with the hEF1α-β-catenin^4A//SV40-mCherry (“EβC”) virus at different multiplicities of infection (1, 0.3 and 0.1 relative concentration of virus per cell). Increasing the number of infected cells (as seen by the amount of mCherry-positive cells in a confluent field, right) led to increasing activation of the Wnt signaling pathway in absence of any exogenous Wnt source (left). D) Mouse LS/L and HEK293A cells were infected with the hEF1α-β-catenin^4A//SV40-Puro^R (“EβP”) virus and selected with 1 µg/ml puromycin. Selection with puromycin led to the enrichment of cells with constitutive activation of the Wnt signaling pathway (left, compare “−” with “Puro”). Infection of HEK293A with the EβP virus similarly led to ligand-independent activation of the Wnt signaling pathway (right). In panels A and B, activity is relative to vehicle-treated cells. In panels C and D, activity is relative to mock-infected cells.</p

Luminescent and drug-resistant reporters for the Wnt signaling pathway.

<p>A) HEK293T cells were infected with the 7xTcf-FFluc (“7TF”) virus and responded to purified Wnt3A protein in a dose-dependent manner. B) HEK293A cells were infected with the 7xTcf-FFluc//SV40-mCherry (“7TFC”) virus and responded similarly to purified Wnt3A protein (left). Cells expressed mCherry in a constitutive manner (right). C) HEK293A cells were infected with the 7xTcf-FFluc//SV40-Puro^R (“7TFP”) virus and also responded to purified Wnt3A protein in a dose-dependent manner (left). Selection of 7TFP-infected HEK293T cells with puromycin (1 µg/ml) allowed for enrichment of Wnt-responding cells, resulting in increased luciferase signal upon addition of conditioned medium (right, compare “Puro” with “−”). D) Adipose-derived mesenchymal cells (“ADMCs”) were infected with the 7xTcf-Puro^R (7TP) lentivirus. Addition of purified Wnt3A protein (500 ng/ml final, “Wnt3A”) allowed selection of Wnt-responding cells with puromycin (1 µg/ml), while cells incubated in absence of Wnt3A (“vhc”) were all killed by the drug treatment. For all graphs, activity is relative to vehicle- or mock-treated cells.</p

Selectable lentiviruses with Wnt-induced eGFP.

<p>A) Schematic description of the 7xTcf-eGFP//SV40-mCherry (7TGC) and 7xTcf-eGFP//SV40-Puro^R (7TGP) lentiviruses. LTR: Long Terminal Repeat, ¬: packaging signal, RRE: Rev Response Element, cPPT: central PolyPurine Tract, WPRE: Woodchuck hepatitis Post-transcriptional Regulatory Element, dPPT: distal PolyPurine Tract, SIN: Self Inactivated (LTR). B) HEK293 or mouse L cells were infected with the 7TGC lentivirus. All infected cells expressed mCherry and addition of purified Wnt3A protein (500 ng/ml final, “Wnt3A”) led to expression of eGFP. In absence of Wnt signal (“vhc”), eGFP was not expressed. C) HEK293T cells were infected with the 7TGP lentivirus and either selected in 1 µg/ml puromycin (“7TGP+puro”) or grown in absence of drug (“7TGP”). Expression of eGFP was induced by Wnt3A-conditioned medium (“Wnt3A”) and Wnt-responsive cells were strongly enriched after puromycin.</p

Nodal.Gdf1 Heterodimers with Bound Prodomains Enable Serum-independent Nodal Signaling and Endoderm Differentiation

The TGF beta family member Nodal is central to control pluripotent stem cell fate, but its use as a stem cell differentiation factor is limited by low specific activity. During development, Nodal depends on growth and differentiation factor (Gdf)-1 and on the shared co-receptor Cryptic to specify visceral left-right axis asymmetry. We therefore asked whether the functionality of Nodal can be augmented by Gdf1. Because Nodal and Gdf1 coimmunoprecipitate each other, they were predicted to form heterodimers, possibly to facilitate diffusion or to increase the affinity for signaling receptors. Here, we report that Gdf1 suppresses an unexpected dependence of Nodal on serum proteins and that it is critically required for non-autonomous signaling in cells expressing Cryptic. Nodal, Gdf1, and their cleaved propeptides copurified as a heterodimeric low molecular weight complex that stimulated Activin receptor (Acvr) signaling far more potently than Nodal alone. Although heterodimerization with Gdf1 did not increase binding of Nodal to Fc fusions of co-receptors or Acvr extracellular domains, it was essential for soluble Acvr2 to inhibit Nodal signaling. This implies that Gdf1 potentiates Nodal activity by stabilizing a low molecular weight fraction that is susceptible to neutralization by soluble Acvr2. Finally, in differentiating human ES cells, endodermal markers were more efficiently induced by Nodal.Gdf1 than by Nodal, suggesting that Nodal.Gdf1 is an attractive new reagent to direct stem cell differentiation