Clostridium difficile toxin A attenuates Wnt/ß-catenin signaling in intestinal epithelial cells.

Clostridium difficile toxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. In this study, we evaluated the effects of TcdA on the Wnt/ß-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50, and 100 ng/ml of TcdA for 24 h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of ß-catenin and Western blotting for ß-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our Western blot analysis showed a decrease in the ß-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/ß-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3ß inhibitor), or constitutively active ß-catenin, as determined by a TCF reporter assay. Furthermore, preincubation of RKO cells with TcdA for 12 h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA is important for TcdA antiproliferative effects.

Targeting the neurotoxic species in Alzheimer's disease: inhibitors of Abeta oligomerization.

In the past two decades, a large body of evidence has established a causative role for the beta-amyloid peptide (Abeta) in Alzheimer's disease (AD). However, recent debate has focused on whether amyloid fibrils or soluble oligomers of Abeta are the main neurotoxic species that contribute to neurodegeneration and dementia. Considerable early evidence has indicated that amyloid fibrils are toxic, but some recent studies support the notion that Abeta oligomers are the primary neurotoxins. While this crucial aspect of AD pathogenesis remains controversial, effective therapeutic strategies should ideally target both oligomeric and fibrillar species of Abeta. Here, we describe the anti-amyloidogenic and neuroprotective actions of some di- and tri-substituted aromatic compounds. Inhibition of the formation of soluble Abeta oligomers was monitored using a specific antibody-based assay that discriminates between Abeta oligomers and monomers. Thioflavin T and electron microscopy were used to screen for inhibitors of fibril formation. Taken together, these results led to the identification of compounds that more effectively block Abeta oligomerization than fibrillization. It is significant that such compounds completely blocked the neurotoxicity of Abeta to rat hippocampal neurons in culture. These findings provide a basis for the development of novel small molecule Abeta inhibitors with potential applications in AD.

Chordin-like CR domains and the regulation of evolutionarily conserved extracellular signaling systems.

In fruit flies as well as in humans the Short gastrulation (Sog)/Chordin protein functions as an antagonist of the signaling of decapentaplegic (Dpp)/bone morphogenetic protein (BMP) in the extracellular space. Such antagonism inhibits Dpp/BMP signaling by blocking its binding to the receptor. Modulation of Dpp/BMP signaling is phylogenetically conserved and is a key step for the establishment of the dorso-ventral axis in vertebrates and invertebrates. Molecular studies have shown that the inhibitory activity of Chordin on BMP resides in specific cysteine-rich (CR) domains. Interestingly, Chordin-like CR domains are present in a growing number of extracellular proteins, several of which appear to be involved in BMP signaling regulation. We review here the conservation of the Chordin and Sog proteins, and in particular their functional domain, the CR domain. We discuss how the study of CR domains may provide a general mechanism for the regulation of growth factor signaling in the extracellular space.

Concentration-dependent actions of glial chondroitin sulfate on the neuritic growth of midbrain neurons.

Astrocytes located in two distinct regions of midbrain differ in their neuritic growth support abilities. Midbrain neurons cultured onto astrocyte monolayers from the lateral (L) region develop long and branched neurites while neurons cultured onto astrocyte monolayers from the medial (M) region develop short or no neurites. The extracellular matrix of these astrocytes has an important role in promoting or inhibiting the growth of these neurons. Differences on the compartmental distribution, as well as on the concentration of GAGs of L and M astrocytes, may be related to their differential capacity of supporting neuritic growth. Indeed, enzymatic digestion of heparan sulfate (HS) and chondroitin sulfate (CS) chains also pointed to an important function for GAGs on axon navigation. In order to better characterize the role of CS on the growth of midbrain neurites, we treated L and M astrocyte monolayers with 1 mM of beta-D-xyloside. Under these conditions, astrocytes oversynthesized and secreted CS protein-free chains to the culture medium. M astrocytes had a significant reduction in their neuritic growth-inhibiting ability after xyloside treatment, suggesting a promoting role for soluble CS in neuritic growth. Chondroitin 4-sulfate (CS-4) added in different concentrations to M astrocyte cultures turned this glia into a permissive substrate, acting in a linear way as far as the largest neurite was concerned. However, a U-shaped dose-effect curve on neurite growth resulted from the similar treatment of L astrocytes. These results suggest that glial CS-4 could be involved in the neurite growth modulating properties of midbrain neurons in a complex concentration-dependent way.

Wnt stabilization of ß-catenin reveals principles for morphogen receptor-scaffold assemblies.

Wnt signaling stabilizes ß-catenin through the LRP6 receptor signaling complex, which antagonizes the ß-catenin destruction complex. The Axin scaffold and associated glycogen synthase kinase-3 (GSK3) have central roles in both assemblies, but the transduction mechanism from the receptor to the destruction complex is contentious. We report that Wnt signaling is governed by phosphorylation regulation of the Axin scaffolding function. Phosphorylation by GSK3 kept Axin activated ("open") for ß-catenin interaction and poised for engagement of LRP6. Formation of the Wnt-induced LRP6-Axin signaling complex promoted Axin dephosphorylation by protein phosphatase-1 and inactivated ("closed") Axin through an intramolecular interaction. Inactivation of Axin diminished its association with ß-catenin and LRP6, thereby inhibiting ß-catenin phosphorylation and enabling activated LRP6 to selectively recruit active Axin for inactivation reiteratively. Our findings reveal mechanisms for scaffold regulation and morphogen signaling.

Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6.

The Wnt/beta-catenin signaling pathway plays essential roles in cell proliferation and differentiation, and deregulated beta-catenin protein levels lead to many types of human cancers. On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6. As a result, beta-catenin phosphorylation by GSK3 is inhibited and beta-catenin protein is stabilized. However, how LRP6 phosphorylation and the ensuing LRP6-Axin interaction lead to the inhibition of beta-catenin phosphorylation by GSK3 is not fully understood. In this study, we reconstituted Axin-dependent beta-catenin phosphorylation by GSK3 and CK1 in vitro using recombinant proteins, and found that the phosphorylated PPPSPXS peptides directly inhibit beta-catenin phosphorylation by GSK3 in a sequence and phosphorylation-dependent manner. This inhibitory effect of phosphorylated PPPSPXS motifs is direct and specific for GSK3 phosphorylation of beta-catenin at Ser33/Ser37/Thr41 but not for CK1 phosphorylation of beta-catenin at Ser45, and is independent of Axin function. We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/beta-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo. Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of beta-catenin. This model provides a possible mechanism to account, in part, for inhibition of beta-catenin phosphorylation by Wnt-activated LRP6.

Amyloid-beta binds to the extracellular cysteine-rich domain of Frizzled and inhibits Wnt/beta-catenin signaling.

The amyloid-beta peptide (Abeta) plays a major role in neuronal dysfunction and neurotoxicity in Alzheimer disease. However, the signal transduction mechanisms involved in Abeta-induced neuronal dysfunction remain to be fully elucidated. A major current unknown is the identity of the protein receptor(s) involved in neuronal Abeta binding. Using phage display of peptide libraries, we have identified a number of peptides that bind Abeta and are homologous to neuronal receptors putatively involved in Abeta interactions. We report here on a cysteine-linked cyclic heptapeptide (denominated cSP5) that binds Abeta with high affinity and is homologous to the extracellular cysteine-rich domain of several members of the Frizzled (Fz) family of Wnt receptors. Based on this homology, we investigated the interaction between Abeta and Fz. The results show that Abeta binds to the Fz cysteine-rich domain at or in close proximity to the Wnt-binding site and inhibits the canonical Wnt signaling pathway. Interestingly, the cSP5 peptide completely blocks Abeta binding to Fz and prevents inhibition of Wnt signaling. These results indicate that the Abeta-binding site in Fz is homologous to cSP5 and that this is a relevant target for Abeta-instigated neurotoxicity. Furthermore, they suggest that blocking the interaction of Abeta with Fz might lead to novel therapeutic approaches to prevent neuronal dysfunction in Alzheimer disease.

Modulators of axonal growth and guidance at the brain midline with special reference to glial heparan sulfate proteoglycans

Bilaterally symmetric organisms need to exchange information between the left and right sides of their bodies to integrate sensory input and to coordinate motor control. Thus, an important choice point for developing axons is the Central Nervous System (CNS) midline. Crossing of this choice point is influenced by highly conserved, soluble or membrane-bound molecules such as the L1 subfamily, laminin, netrins, slits, semaphorins, Eph-receptors and ephrins, etc. Furthermore, there is much circumstantial evidence for a role of proteoglycans (PGs) or their glycosaminoglycan (GAG) moieties on axonal growth and guidance, most of which was derived from simplified models. A model of intermediate complexity is that of cocultures of young neurons and astroglial carpets (confluent cultures) obtained from medial and lateral sectors of the embryonic rodent midbrain soon after formation of its commissures. Neurite production in these cocultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exerted an inhibitory or non-permissive effect on neuritic growth that was correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment with GAG lyases shows minor effects of CS and discloses a major inhibitory or non-permissive role for HS. The results are discussed in terms of available knowledge on the binding of HSPGs to interative proteins and underscore the importance of understanding glial polysaccharide arrays in addition to its protein complement for a better understanding of neuron-glial interactions

Heterogeneity of median and lateral midbrain radial glia and astrocytes

In the developing mammalian midbrain, radial glial cells are divided into median formations and lateral radial systems with differential properties including rate and timing of cell proliferation, expression of cytoskeletal and calcium-binding proteins, storage of glycogen and relations to afferent fiber systems. To test hypothesis that radial glial cells of median and lateral midbrain sectors and/or their derivatives are heterogeneous in their relations with local neurons, an in vitro system has been developed and has also been characterized in terms of extracellular matrix (ECM) components. Confluent astrocyte cultures, derived from median (M) or lateral (L) embryonic mouse midbrain sectors, were used as substrates for culturing dissociated cells from median (m) or lateral (l) sectors of embryonic midbrains. In spite of the morphological invariance of glial substrates at confluency, cells that were plated onto these substrates and that were immunoreactive for neuronal markers (MAP2, polysialylated N-CAM or betaIII tubulin) showed differences in the aggregation of somata and in the length, caliber and branching of neurites. These differences, which depend mostly on the sector of origin of astrocytes (L: permissive, M: non-permissive for neuronal growth), suggest that the substrates may differ in adhesiveness and/or their carrying of growth-promoting vs. growth-interfering molecules. Indeed, L and M cultures differ in laminin deposition patterns (L: fibrillar, M: punctate pattern). Furthermore, sulfated glycosaminoglycans (s-GAGs) isolated from the pericellular (P), intracellular (I) and extracellular (E) compartments of these sectoral cultures also showed correlations with the ability to support neurite growth. The total amount of s-GAGs in M cultures was twice that in L cultures and was particularly high in the P compartment, with about 3 times as much heparan sulfate (HS) and about 15 times as much chondroitin sulfate (CS) in this fraction of M than in the corresponding compartment of L glia. Our results indicate that cultured astrocytes have heterogeneous properties including different organizatio of their extracellular matrix that reflect the roles played by their parent radial glia in regions favorable to axonal growth or barrier regions of the developing brain.

Effects of the thyroid hormone (T3) on astrocytes [correction of effecs]

Thyroid hormones have profound effects on growth and development. In the brain L-3,5,3'- triiodothyronine (T3), the bioactive hormone, is involved with the harmonious development acting in neuronal and glial cell differentiation. T3 acts on the cells by interacting with nuclear receptors that can regulate the expression of several genes. Astrocytes also show receptors to the hormone. We reported herein data on the effects of T3 on astrocytes. We have verified that T3 has a morphological effect on cultured cortical astrocytes with rearrangement of GFAP filaments, and induces proliferation in the cultured cerebellar astrocytes of newborn rats. We discuss here the effects of T3 on astrocytes, considering the possibility that thyroid hormone prepares the astrocytes to interact with neurons.