WNT5A signaling contributes to Aβ-induced neuroinflammation and neurotoxicity

Neurodegenration is a pathological hallmark of Alzheimer's disease (AD), but the underlying molecular mechanism remains elusive. Here, we present evidence that reveals a crucial role of Wnt5a signaling in this process. We showed that Wnt5a and its receptor Frizzled-5 (Fz5) were up-regulated in the AD mouse brain, and that beta-amyloid peptide (Aβ), a major constituent of amyloid plaques, stimulated Wnt5a and Fz5 expression in primary cortical cultures; these observations indicate that Wnt5a signaling could be aberrantly activated during AD pathogenesis. In support of such a possibility, we observed that inhibition of Wnt5a signaling attenuated while activation of Wnt5a signaling enhanced Aβ-evoked neurotoxicity, suggesting a role of Wnt5a signaling in AD-related neurodegeneration. Furthermore, we also demonstrated that Aβ-induced neurotoxicity depends on inflammatory processes, and that activation of Wnt5a signaling elicited the expression of proinflammatory cytokines IL-1β and TNF-α whereas inhibition of Wnt5a signaling attenuated the Aβ-induced expression of the cytokines in cortical cultures. Our findings collectively suggest that aberrantly up-regulated Wnt5a signaling is a crucial pathological step that contributes to AD-related neurodegeneration by regulating neuroinflammation.     

Reconstitution of a Frizzled8��Wnt3a��LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Wnt/β-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/β-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD·Wnt3a·LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth β-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/β-catenin signaling by Dkk1.     

Receptor tyrosine kinases activate canonical WNT/β-catenin signaling via MAP kinase/LRP6 pathway and direct β-catenin phosphorylation

Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling.     

Discovery and structure–activity relationship of a novel spirocarbamate series of NPY Y5 antagonists

A novel series of trans-8-aminomethyl-1-oxa-3-azaspiro[4.5]decan-2-one derivatives was identified with potent NPY Y5 antagonist activity. Optimization of the original lead furnished compounds 23p and 23u, which combine sub-nanomolar Y5 activity with metabolic stability, oral bioavailability, brain penetration and strong preclinical profile for development. Both compounds significantly inhibited the food intake induced by a Y5 selective agonist with minimal effective doses of 3 mg/kg po.     

Synthesis and Biological Activity of a Bis-Substituted 3′-Deoxyadenosine Analog of 2–5A

Abstract

The 5′-monophosphate, p5′(3′dA)2′p5′A2′5′(3′dA), was synthesized and found to bind to the 2–5A-dependent endonuclease of mouse L cells only two-three times less effectively than the parent p5′A2′p5′A2′p5′A. When evaluated for its ability to activate the 2–5A-dependent endonuclease, ppp5′(3′dA)2′p5′A2′p5′(3′dA) was found to be fifty times more effective than ppp5′A2′p5′(3′dA)2′p5′A and ten times less effective than 2–5A as an endonuclease activator     

WNT7A regulates tumor growth and progression in ovarian cancer through the WNT/β-catenin pathway

Abnormal activation the WNT/β-catenin signaling pathway has been associated with ovarian carcinomas, but a specific WNT ligand and pertinent downstream mechanisms are not fully understood. In this study, we found abundant WNT7A in the epithelium of serous ovarian carcinomas, but not detected in borderline and benign tumors, normal ovary, or endometrioid carcinomas. To characterize the role of WNT7A in ovarian tumor growth and progression, nude mice were injected either intraperitoneally or subcutaneously with WNT7A knocked down SKOV3.ip1 and overexpressed SKOV3 cells. In the intraperitoneal group, mice receiving SKOV3.ip1 cells with reduced WNT7A expression developed significantly fewer tumor lesions. Gross and histologic examination revealed greatly reduced invasion of WNT7A knockdown cells into intestinal mesentery and serosa compared with the control cells. Tumor growth was regulated by loss or overexpression of WNT7A in mice receiving subcutaneous injection as well. In vitro analysis of cell function revealed that cell proliferation, adhesion, and invasion were regulated by WNT7A. The activity of the T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter was stimulated by overexpression of WNT7A in ovarian cancer cells. Cotransfection with WNT7A and FZD5 receptor further increased activity, and this effect was inhibited by cotransfection with SFRP2 or dominant negative TCF4. Overexpression of WNT7A stimulated matrix metalloproteinase 7 (MMP7) promoter, and mutation of TCF-binding sites in MMP7 promoter confirmed that activation of MMP7 promoter by WNT7A was mediated by β-catenin/TCF signaling. Collectively, these results suggest that reexpression of WNT7A during malignant transformation of ovarian epithelial cells plays a critical role in ovarian cancer progression mediated by WNT/β-catenin signaling pathway.     

Dating and localizing an invasion from post-introduction data and a coupled reaction–diffusion–absorption model

Journal of Mathematical Biology - Invasion of new territories by alien organisms is of primary concern for environmental and health agencies and has been a core topic in mathematical modeling, in...     

Recognition and processing of double-stranded DNA by ExoX,a distributive 3′–5′ exonuclease

Members of the DnaQ superfamily are major 3′–5′ exonucleases that degrade either only single-stranded DNA (ssDNA) or both ssDNA and double-stranded DNA (dsDNA). However, the mechanism by which dsDNA is recognized and digested remains unclear. Exonuclease X (ExoX) is a distributive DnaQ exonuclease that cleaves both ssDNA and dsDNA substrates. Here, we report the crystal structures of Escherichia coli ExoX in complex with three different dsDNA substrates: 3′ overhanging dsDNA, blunt-ended dsDNA and 3′ recessed mismatch-containing dsDNA. In these structures, ExoX binds to dsDNA via both a conserved substrate strand-interacting site and a previously uncharacterized complementary strand-interacting motif. When ExoX complexes with blunt-ended dsDNA or 5′ overhanging dsDNA, a ‘wedge’ composed of Leu12 and Gln13 penetrates between the first two base pairs to break the 3′ terminal base pair and facilitates precise feeding of the 3′ terminus of the substrate strand into the ExoX cleavage active site. Site-directed mutagenesis showed that the complementary strand-binding site and the wedge of ExoX are dsDNA specific. Together with the results of structural comparisons, our data support a mechanism by which normal and mismatched dsDNA are recognized and digested by E. coli ExoX. The crystal structures also provide insight into the structural framework of the different substrate specificities of the DnaQ family members.     

A selection system to study C5a–C5a-receptor interactions: Phage display of a novel C5a anaphylatoxin,Fos-C5aAla27

Binding and effector domains of the human anaphylatoxin C5a have been determined by either site directed mutagenesis or synthetic peptide studies. However, the lack of specific selection methods, which allow direct investigation of C5a–C5a-receptor interaction made these studies laborious. To overcome these limitations we have constructed a novel Fos-C5a expressed on the tip of a filamentous phage. To guarantee for a free C-terminus which is required for C5a activity C5a cDNA was cloned into the phagemid vector pJuFo. Helper phage infection of pJuFo-C5a transformed cells resulted in a mutant phage displaying Fos-C5a on its surface. However studies with Bt₂cAMP differentiated U937 cells revealed that phage displayed Fos-C5a is functional inactive. Subsequently we replaced a nonconserved cysteine residue at position 27 by alanine and obtained Fos-C5a^Ala27. Both the purified and the phage displayed Fos-C5a^Ala27 proteins were functional active and induced enzyme release from differentiated U937 cells. In addition, purified Fos-C5a^Ala27 exhibited the same binding profile as compared to rhC5a. Fos-C5a^Ala27 displaying phages were mixed with phage harboring only the pJuFo plasmid at a ratio of 10⁶. After four successive rounds of panning on differentiated U937 cells Fos-C5a^Ala27 phages were enriched to 100% as shown by C5a-specific ELISA. We expect this approach to prove helpful for studying C5a–C5a-receptor interactions, i.e. to screen C5a libraries for high affinity binders with agonistic or antagonistic properties directly on cells.     

Mendelian segregation: a choice between “order” and “chaos”

Research supported in part by a US-Israel Binational Science Foundation grant 85-00021 and by NIH grants GM 28016 and GM 10452     

“Canadian Experience” discourse and anti-racialism in a “post-racial” society

“Canadian Experience” is a paradox for many immigrants in Canada and contributes to their exclusion from the labour market. Through an analysis of Canadian English print media, from 2006 to 2011, we illustrate how “Canadian Experience” discourse places the responsibility of immigrant labour market integration on immigrants themselves and constructs their experiences of exclusion as non-racial. This is theorized as a “post-racial” strategy that relies on anti-racialism (avoidance of racial references) to deny the existence and effects of racism, thereby allowing the Canadian public to maintain its façade of innocence but perpetuates “racism without racists”. The discourse de-historicizes postcolonial racial hierarchy and promotes a de-racialized neo-liberal model for immigrant inclusion. This has implications for anti-racism and settlement service provision.     

Gut–organ axis: a microbial outreach and networking

Human gut microbiota (GM) includes a complex and dynamic population of microorganisms that are crucial for well-being and survival of the organism. It has been reported as diverse and relatively stable with shared core microbiota, including Bacteroidetes and Firmicutes as the major dominants. They are the key regulators of body homeostasis, involving both intestinal and extra-intestinal effects by influencing many physiological functions such as metabolism, maintenance of barrier homeostasis, inflammation and hematopoiesis. Any alteration in GM community structures not only trigger gut disorders but also influence other organs and cause associated diseases. In recent past, the GM has been defined as a ‘vital organ’ with its involvement with other organs; thus, establishing a link or a bi- or multidirectional communication axis between the organs via neural, endocrine, immune, humoral and metabolic pathways. Alterations in GM have been linked to several diseases known to humans; although the exact interaction mechanism between the gut and the organs is yet to be defined. In this review, the bidirectional relationship between the gut and the vital human organs was envisaged and discussed under several headings. Furthermore, several disease symptoms were also revisited to redefine the communication network between the gut microbes and the associated organs.     

Determination and pharmacokinetics of a furosemide–amiloride drug combination

The study presents an accurate and precise HPLC assay for the determination of furosemide and amiloride in human specimens. Both drugs were extracted from human plasma with ethyl acetate; furosemide was extracted at pH 1 and amiloride at pH 12. While chromatographic separation conditions, i.e., column, mobile phase and flow-rate were the same for both investigated drugs, furosemide was detected using a UV absorbance detector, whereas amiloride, because of its very low therapeutic range, was detected with a spectrofluorimetric detector. The linearity of the furosemide and amiloride assays were confirmed over the range of 30–3000 ng/ml and 0.5–30 ng/ml, respectively. These concentrations correspond well with the therapeutic ranges of both drugs. The extraction recoveries, depending on concentration, exceed 80% for furosemide and 74% for amiloride. The reported methods were applied to pharmacokinetic investigations of the two compounds taken in form of a drug combination.     

Heterozygosity–behavior and heterozygosity–fitness correlations in a salamander with limited dispersal

Inbreeding and genetic drift can decrease genetic heterozygosity, and this low heterozygosity can depress fitness, resulting in heterozygosity–fitness correlations (HFCs). HFCs are typically small in magnitude, a result often attributed to power of the analyses. Animal behaviors often affect fitness and are often heritable to some degree. We hypothesized that heterozygosity influences behavior, which, in turn, potentially influences fitness. Specifically, in red-backed salamanders, Plethodon cinereus, which have limited dispersal and the potential for inbreeding, we tested whether heterozygosity, as estimated from six microsatellite loci, affected home range size, juvenile growth, and survival. We found that salamanders with higher heterozygosity had larger home ranges and grew faster, which is indicative of reproductive success. However, we found no effects of heterozygosity on survival. We conclude that, because activity in P. cinereus is tightly linked to food uptake and mass gain, heterozygosity influences growth via effects on foraging behavior. Future research should investigate how the relationship between heterozygosity, behavior, and fitness may be affected or mediated by endocrine or immune systems.