Genome-wide identification of new Wnt/beta-catenin target genes in the human genome using CART method.

BACKGROUND: The importance of in silico predictions for understanding cellular processes is now widely accepted, and a variety of algorithms useful for studying different biological features have been designed. In particular, the prediction of cis regulatory modules in non-coding human genome regions represents a major challenge for understanding gene regulation in several diseases. Recently, studies of the Wnt signaling pathway revealed a connection with neurodegenerative diseases such as Alzheimer's. In this article, we construct a classification tool that uses the transcription factor binding site motifs composition of some gene promoters to identify new Wnt/beta-catenin pathway target genes potentially involved in brain diseases. RESULTS: In this study, we propose 89 new Wnt/beta-catenin pathway target genes predicted in silico by using a method based on multiple Classification and Regression Tree (CART) analysis. We used as decision variables the presence of transcription factor binding site motifs in the upstream region of each gene. This prediction was validated by RT-qPCR in a sample of 9 genes. As expected, LEF1, a member of the T-cell factor/lymphoid enhancer-binding factor family (TCF/LEF1), was relevant for the classification algorithm and, remarkably, other factors related directly or indirectly to the inflammatory response and amyloidogenic processes also appeared to be relevant for the classification. Among the 89 new Wnt/beta-catenin pathway targets, we found a group expressed in brain tissue that could be involved in diverse responses to neurodegenerative diseases, like Alzheimer's disease (AD). These genes represent new candidates to protect cells against amyloid beta toxicity, in agreement with the proposed neuroprotective role of the Wnt signaling pathway. CONCLUSIONS: Our multiple CART strategy proved to be an effective tool to identify new Wnt/beta-catenin pathway targets based on the study of their regulatory regions in the human genome. In particular, several of these genes represent a new group of transcriptional dependent targets of the canonical Wnt pathway. The functions of these genes indicate that they are involved in pathophysiology related to Alzheimer's disease or other brain disorders.

Wnt signaling in neuroprotection and stem cell differentiation.

In the past several years, we postulated that the loss of Wnt signaling was implicated in the pathology of Alzheimer's disease (AD). Since then, our lab and other groups have confirmed the involvement of the Wnt signaling in some aspects of AD. So far, we have demonstrated that activation of Wnt signaling protects neurons against neurotoxic injuries, including both amyloid-beta (Abeta) fibrils and Abeta oligomers by using either lithium, an inhibitor of the glycogen-synthase-kinase-3beta (GSK-3beta), or different Wnt ligands. Also, we have found that several molecules which activate well known neurotransmitter systems and other signaling system, are able by crosstalk to activate Wnt/beta-catenin signaling in order to protect neurons against both Abeta fibrils or Abeta oligomers. In particular, the activation of non-canonical Wnt signaling was able to protect postsynaptic regions and dendritic spines against Abeta oligomers. Furthermore Wnt signaling ligands also affect stem cells, and they are also involved in cell fate decision during neurogenesis and embryonic development as well as in adult stem cells differentiation in the nervous system. The Wnt signaling plays a key role modulating their cell differentiation or proliferation states. Altogether, these findings in both stem cell biology and neuroprotection, may introduce new approaches in the treatment of neurodegenerative diseases, including drug screening and therapies against neurodegenerative diseases which activates the Wnt signaling pathway.

Calcium/calmodulin-dependent protein kinase type IV is a target gene of the Wnt/beta-catenin signaling pathway.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. The expression of CaMKIV and the activation of CREB regulated genes are involved in memory and neuronal survival. We report here that: (a) a bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes, being CaMKIV a very interesting candidate; (b) CaMKIV promoter contains TCF/LEF transcription motifs similar to those present in Wnt target genes; (c) biochemical studies indicate that lithium and the canonical ligand Wnt-3a induce CaMKIV mRNA and protein expression levels in rat hippocampal neurons as well as CaMKIV promoter activity; (d) treatment of hippocampal neurons with Wnt-3a increases the binding of beta-catenin to the CaMKIV promoter: (e) In vivo activation of the Wnt signaling improve spatial memory impairment and restores the expression of CaMKIV in a mice double transgenic model for Alzheimer's disease which shows decreased levels of the kinase. We conclude that CaMKIV is regulated by the Wnt signaling pathway and that its expression could play a role in the neuroprotective function of the Wnt signaling against the Alzheimer's amyloid peptide.

Wnt-7a induces presynaptic colocalization of alpha 7-nicotinic acetylcholine receptors and adenomatous polyposis coli in hippocampal neurons.

Nicotinic acetylcholine receptors (nAChRs) contribute significantly to hippocampal function. Alpha7-nAChRs are present in presynaptic sites in hippocampal neurons and may influence transmitter release, but the factors that determine their presynaptic localization are unknown. We report here that Wnt-7a, a ligand active in the canonical Wnt signaling pathway, induces dissociation of the adenomatous polyposis coli (APC) protein from the beta-catenin cytoplasmic complex and the interaction of APC with alpha7-nAChRs in hippocampal neurons. Interestingly, Wnt-7a induces the relocalization of APC to membranes, clustering of APC in neurites, and coclustering of APC with different, presynaptic protein markers. Wnt-7a also increases the number and size of coclusters of alpha7-nAChRs and APC in presynaptic terminals. These short-term changes in alpha7-nAChRs occur in the few minutes after ligand exposure and involve translocation to the plasma membrane without affecting total receptor levels. Longer-term exposure to Wnt-7a increases nAChR alpha7 subunit levels in an APC-independent manner and increases clusters of alpha7-nAChRs in neurites via an APC-dependent process. Together, these results demonstrate that stimulation through the canonical Wnt pathway regulates the presynaptic localization of APC and alpha7-nAChRs with APC serving as an intermediary in the alpha7-nAChR relocalization process. Modulation by Wnt signaling may be essential for alpha7-nAChR expression and function in synapses.

Heparin activates Wnt signaling for neuronal morphogenesis.

Wnt factors are secreted ligands that affect different aspects of the nervous system behavior like neurodevelopment, synaptogenesis and neurodegeneration. In different model systems, Wnt signaling has been demonstrated to be regulated by heparan sulfate proteoglycans (HSPGs). Whether HSPGs modulate Wnt signaling in the context of neuronal behavior is currently unknown. Here we demonstrate that activation of Wnt signaling with the endogenous ligand Wnt-7a results in an increased of neurite outgrowth in the neuroblastoma N2a cell line. Interestingly, heparin induces glycogen synthase kinase-3beta (GSK-3beta) inhibition, beta-catenin stabilization and morphological differentiation in both N2a cells and in rat primary hippocampal neuronal cultures. We also show that heparin modulates Wnt-3a-induced stabilization of beta-catenin. Several extracellular matrix and membrane-attached HSPGs were found to be expressed in both in vitro neuronal models. Changes in the expression of specific HSPGs were observed upon differentiation of N2a cells. Taken together, our findings suggest that HSPGs may modulate canonical Wnt signaling for neuronal morphogenesis.

Beta-amyloid oligomers affect the structure and function of the postsynaptic region: role of the Wnt signaling pathway.

BACKGROUND: Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the growing population of elderly people. Synaptic dysfunction is an early manifestation of AD. The cellular mechanism by which beta-amyloid peptide (Abeta) affects synapses remains unclear. Abeta oligomers target synapses in cultured rat hippocampal neurons suggesting that they play a key role in the regulation of synapses. OBJECTIVE: The aim of this work is to study the effect of Abeta oligomers on the central synapses and the possible role of the Wnt signaling pathway in preventing the Abeta effects. METHODS: We used rat hippocampal neurons, immunofluorescence and western blot procedures to detect synaptic proteins. RESULTS: Abeta oligomers induced a reduction of the postsynaptic density protein 95 (PSD-95) and the NMDA glutamate receptors. We found that Wnt-5a, a noncanonical Wnt ligand, prevents the decrease triggered by Abeta oligomers in the glutamate receptor and PSD-95. CONCLUSION: Altogether, our results suggest that Abeta oligomers decrease the synaptic responses by affecting the postsynaptic region at different levels. The Wnt signaling activation prevents synaptic damage induced by Abeta, which raises the possibility of a new therapeutic intervention for the treatment of synaptic changes observed in AD.

Acetylcholinesterase interaction with Alzheimer amyloid beta.

Acetylcholinesterase (AChE) is an enzyme involved in cholinergic and non-cholinergic functions in both the central and peripheral nervous system, most of the AChE is found as a tetrameric form bound to neuronal membranes. Early cytochemical studies have demonstrated that the AChE associated with senile plaques differs enzymatically from the AChE associated with neurons in several respects. Biochemical studies indicated that AChE induces amyloid fibril formation and form highly toxic AChE-Abeta complexes. A 3.5 kDa peptide containing a tryptophan of the enzyme peripheral binding site (PAS) mimics the effect of the whole enzyme on amyloid formation. The neurotoxicity induced by AChE-Abeta complexes indicated that they trigger more neurodegeneration than those of the Abeta peptide alone, both in vitro (hippocampal neurons) and in vivo (rats injected in the dorsal hippocampus as a model of Alzheimer). The fact that AChE is able to accelerate amyloid formation and that such effect is sensitive to drugs that block PAS of the enzyme, suggests that specific and new AChE inhibitors may well provide an attractive possibility for treating Alzheimer's disease.

Structure and function of amyloid in Alzheimer's disease.

This review is focused on the structure and function of Alzheimer's amyloid deposits. Amyloid formation is a process in which normal well-folded cellular proteins undergo a self-assembly process that leads to the formation of large and ordered protein structures. Amyloid deposition, oligomerization, and higher order polymerization, and the structure adopted by these assemblies, as well as their functional relationship with cell biology are underscored. Numerous efforts have been directed to elucidate these issues and their relation with senile dementia. Significant advances made in the last decade in amyloid structure, dynamics and cell biology are summarized and discussed. The mechanism of amyloid neurotoxicity is discussed with emphasis on the Wnt signaling pathway. This review is focused on Alzheimer's amyloid fibrils in general and has been divided into two parts dealing with the structure and function of amyloid.

Acetylcholinesterase-amyloid-beta-peptide interaction: effect of Congo Red and the role of the Wnt pathway.

The cholinergic system impairment observed in Alzheimer's disease (AD) patients leads to the cognitive, global and behavioral dysfunction commonly associated with dementia. The only treatment for AD has been the use of inhibitors of acetylcholinesterase (AChE) (E.C. 3.1.1.7), which is one of the several proteins associated with amyloid plaque deposits. Recently, novel dual inhibitors of AChE have been developed that target both the active site of the enzyme as well as the peripheral anionic site (PAS). Such inhibitors prevent the aggregation of amyloid-beta-peptide (Abeta) into Alzheimer's fibrils. The incorporation of AChE, as a "chaperone" into amyloid aggregates results in the modification of the biochemical properties of the enzyme, including: sensitivity to low pH, inhibition at high substrate concentration, and increases of the Abeta neurotoxicity. Congo Red dye stabilizes the Abeta monomer, is able to inhibit oligomerization, and inhibits the binding of AChE to Abeta. However no effect of Congo Red on the binding of AChE to the Abeta preformed fibrils was observed. These studies suggest that different interactions between Abeta soluble-AChE and Abeta fibrils-AChE take place during the association between them. Docking studies were performed to evaluate the binding of Congo Red to Abeta in order to identify putative binding sites in the Abeta monomer that might interact with AChE. The binding site involves a region between residues 12 and 16. Finally, recent studies are consistent with the idea that a attenuating beta-catenin loss of function of Wnt signaling components may play a role in the progression of neurodegenerative disease, such as AD, providing a connection between AChE-Abeta neurotoxicity and the Wnt signal transduction pathway.

An overview of the current and novel drugs for Alzheimer's disease with particular reference to anti-cholinesterase compounds.

Several cellular processes could be targeted if the complex nature of Alzheimer's disease (AD) was already understood. Most of AD treatments have been focused on the inhibition of acetylcholinesterase (AChE) in order to raise the levels of its substrate, i.e. the neurotransmitter acetylcholine (ACh), to augment cognitive functions of affected patients. Effectiveness in AChE inhibition and side-effect issues of clinical (tacrine, donepezil, galanthamine and rivastigmine) as well as of novel inhibitors is reviewed here. Novel design methods for the inhibition of AChE include the use of in silico tools to predict the interactions between AChE and the desired compound, both at the active site of the enzyme, responsible of hydrolysing ACh and with the peripheral anionic site (PAS), which has been described as a promoting agent of the amyloid beta-peptide (A beta) aggregation present in the senile plaques of the brain of AD individuals.

Acetylcholinesterase (AChE)--amyloid-beta-peptide complexes in Alzheimer's disease. the Wnt signaling pathway.

Alzheimer's disease (AD) is characterized by selective neuronal cell death, which is probably caused by amyloid beta-peptide (Abeta) oligomers and fibrils. We have found that acetylcholinesterase (AChE), a senile plaque component, increases amyloid fibril assembly with the formation of highly toxic complexes (Abeta-AChE). The neurotoxic effect induced by Abeta-AChE complexes was higher than that induced by the Abeta peptide alone as shown both in vitro (hippocampal neurons) and in vivo (rats injected with Abeta peptide in the dorsal hippocampus). Interestingly, treatment with Abeta-AChE complexes decreases the cytoplasmic beta-catenin level, a key component of Wnt signaling. Conversely, the activation of this signaling pathway by Wnt-3a promotes neuronal survival and rescues changes in Wnt components (activation or subcellular localization). Moreover Frzb-1, a Wnt antagonist reverses the Wnt-3a neuroprotection effect against Abeta neurotoxicity. Compounds that mimic the Wnt signaling or modulate the cross-talking with this pathway could be used as neuroprotective agents for therapeutic strategies in AD patients.

An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from Penicillium purpurogenum.

The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 A resolution (PDB 1G66). The enzyme possesses the alpha/beta hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.

Synaptotoxicity in Alzheimer's disease: the Wnt signaling pathway as a molecular target.

Recent evidence supports a role of the Wnt pathway in neurodegenerative disorders such as Alzheimer's disease (AD). A relationship between amyloid-beta-peptide (Abeta)-induced neurotoxicity and a decrease in the cytoplasmatic levels of beta-catenin has been proposed. Also, the inhibition of glycogen synthase kinase (GSK-3beta), a central modulator of the pathway, protects rat hippocampal neurons from Abeta-induced damage. Interestingly, during the progression of AD, it has been described that active GSK-3beta is found in neuronal cell bodies and neurites, co-localizing with pre-neurofibrillary tangles observed in disease brains. Since Abeta oligomers are associated with the post-synaptic region and we have found that the non-canonical Wnt signaling modulates PSD-95 and glutamate receptors, we propose that the synaptic target for Abeta oligomers in AD is the postsynaptic region and at the molecular level is the non-canonical Wnt signaling pathway. Altogether, our evidence suggests that a sustained loss of Wnt signaling function may be involved in the Abeta-dependent neurodegeneration observed in AD brains and that the activation of this signaling pathway could be of therapeutic interest in AD.