Publications by authors named "Claudia Gaspar"

32 Publications

Impairment of adenosinergic system in Rett syndrome: Novel therapeutic target to boost BDNF signalling.

Neurobiol Dis 2020 11 14;145:105043. Epub 2020 Aug 14.

Instituto de Farmacologia e Neurociências, Faculdade de Medicina e Instituto de Medicina Molecular - João Lobo Antunes, Universidade de Lisboa, Lisboa, Portugal. Electronic address:

Rett syndrome (RTT; OMIM#312750) is mainly caused by mutations in the X-linked MECP2 gene (methyl-CpG-binding protein 2 gene; OMIM*300005), which leads to impairments in the brain-derived neurotrophic factor (BDNF) signalling. The boost of BDNF mediated effects would be a significant breakthrough but it has been hampered by the difficulty to administer BDNF to the central nervous system. Adenosine, an endogenous neuromodulator, may accomplish that role since through AR it potentiates BDNF synaptic actions in healthy animals. We thus characterized several hallmarks of the adenosinergic and BDNF signalling in RTT and explored whether AR activation could boost BDNF actions. For this study, the RTT animal model, the Mecp2 knockout (Mecp2) (B6.129P2 (C)-Mecp2tm1.1Bird/J) mouse was used. Whenever possible, parallel data was also obtained from post-mortem brain samples from one RTT patient. Ex vivo extracellular recordings of field excitatory post-synaptic potentials in CA1 hippocampal area were performed to evaluate synaptic transmission and long-term potentiation (LTP). RT-PCR was used to assess mRNA levels and Western Blot or radioligand binding assays were performed to evaluate protein levels. Changes in cortical and hippocampal adenosine content were assessed by liquid chromatography with diode array detection (LC/DAD). Hippocampal ex vivo experiments revealed that the facilitatory actions of BDNF upon LTP is absent in Mecp2 mice and that TrkB full-length (TrkB-FL) receptor levels are significantly decreased. Extracts of the hippocampus and cortex of Mecp2 mice revealed less adenosine amount as well as less AR protein levels when compared to WT littermates, which may partially explain the deficits in adenosinergic tonus in these animals. Remarkably, the lack of BDNF effect on hippocampal LTP in Mecp2 mice was overcome by selective activation of AR with CGS21680. Overall, in Mecp2 mice there is an impairment on adenosinergic system and BDNF signalling. These findings set the stage for adenosine-based pharmacological therapeutic strategies for RTT, highlighting AR as a therapeutic target in this devastating pathology.
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http://dx.doi.org/10.1016/j.nbd.2020.105043DOI Listing
November 2020

Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient-specific neural cells.

Biotechnol J 2015 Oct 30;10(10):1578-88. Epub 2015 Jun 30.

Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.

Standardization of culture methods for human pluripotent stem cell (PSC) neural differentiation can greatly contribute to the development of novel clinical advancements through the comprehension of neurodevelopmental diseases. Here, we report an approach that reproduces neural commitment from human induced pluripotent stem cells using dual-SMAD inhibition under defined conditions in a vitronectin-based monolayer system. By employing this method it was possible to obtain neurons derived from both control and Rett syndrome patients' pluripotent cells. During differentiation mutated cells displayed alterations in the number of neuronal projections, and production of Tuj1 and MAP2-positive neurons. Although investigation of a broader number of patients would be required, these observations are in accordance with previous studies showing impaired differentiation of these cells. Consequently, our experimental methodology was proved useful not only for the generation of neural cells, but also made possible to compare neural differentiation behavior of different cell lines under defined culture conditions. This study thus expects to contribute with an optimized approach to study the neural commitment of human PSCs, and to produce patient-specific neural cells that can be used to gain a better understanding of disease mechanisms.
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http://dx.doi.org/10.1002/biot.201400751DOI Listing
October 2015

Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo.

PLoS Genet 2015 Jun 26;11(6):e1005328. Epub 2015 Jun 26.

Institut für Molekularbiologie OE5250, Medizinische Hochschule Hannover, Hannover, Germany.

Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool). In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity) raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt) and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1Dll4ki), we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM) where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1Dll4ki/+ mice, the Dll1Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4.
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http://dx.doi.org/10.1371/journal.pgen.1005328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4482573PMC
June 2015

Cancer stemness in Wnt-driven mammary tumorigenesis.

Carcinogenesis 2014 Jan 16;35(1):2-13. Epub 2013 Aug 16.

Department of Pathology, Josephine Nefkens Institute, Erasmus MC 3000 CA Rotterdam, The Netherlands and.

Wnt signaling plays a central role in mammary stem cell (MaSC) homeostasis and in breast cancer. In particular, epigenetic alterations at different members of the Wnt pathway have been identified among triple-negative, basal-like breast cancers. Previously, we developed a mouse model for metaplastic breast adenocarcinoma, a subtype of triple-negative breast cancer, by targeting a hypomorphic mutations in the endogenous Apc gene (Apc (1572T/+)). Here, by employing the CD24 and CD29 cell surface antigens, we have identified a subpopulation of mammary cancer stem cells (MaCSCs) from Apc (1572T/+) capable of self-renewal and differentiation both in vivo and in vitro. Moreover, immunohistochemical analysis of micro- and macrolung metastases and preliminary intravenous transplantation assays suggest that the MaCSCs underlie metastasis at distant organ sites. Expression profiling of the normal and tumor cell subpopulations encompassing MaSCs and CSCs revealed that the normal stem cell compartment is more similar to tumor cells than to their own differentiated progenies. Accordingly, Wnt signaling appears to be active in both the normal and cancer stem cell compartments, although at different levels. By comparing normal with cancer mouse mammary compartments, we identified a MaCSC gene signature able to predict outcome in breast cancer in man. Overall, our data indicate that constitutive Wnt signaling activation affects self-renewal and differentiation of MaSCs leading to metaplasia and basal-like adenocarcinomas.
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http://dx.doi.org/10.1093/carcin/bgt279DOI Listing
January 2014

Wnt signaling regulates the lineage differentiation potential of mouse embryonic stem cells through Tcf3 down-regulation.

PLoS Genet 2013 May 2;9(5):e1003424. Epub 2013 May 2.

Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.
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http://dx.doi.org/10.1371/journal.pgen.1003424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3642041PMC
May 2013

Following butter flavour deterioration with an acoustic wave sensor.

Talanta 2012 Sep 26;99:904-8. Epub 2012 Jul 26.

CESAM & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal.

Off-flavours develop naturally in butter and the process is accelerated by heat. An acoustic wave sensor was used to detect the aroma compounds evolved from heated butter and the results have shown that registered marked changes were coincident to odour changes detected by sensory analysis. The flavour compounds have also been analysed by GC/MS for identification. The response of the sensor was fully characterized in terms of the sensitivity to each of the identified compounds, and sensitivities of the system SPME/sensor were compared with the sensitivities of the system SPME/GC/MS. It was found that the sensor analytical system was more sensitive to methylketones than to fatty acids. The SPME/GC/MS system also showed the highest sensitivity to 2-heptanone, followed by 2-nonanone, but third place was occupied by undecanone and butanoic acid, to which the sensor showed moderate sensitivity. 2-heptanone was found to be an appropriate model compound to follow odour changes till the 500 h, and the lower sensitivity of the sensor to butanoic acid showed to be a positive characteristic, as saturation was prevented, and other more subtle changes in the flavour could be perceived.
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http://dx.doi.org/10.1016/j.talanta.2012.07.055DOI Listing
September 2012

Expanded ATXN3 frameshifting events are toxic in Drosophila and mammalian neuron models.

Hum Mol Genet 2012 May 14;21(10):2211-8. Epub 2012 Feb 14.

Center of Excellence in Neuroscience of the Université de Montréal (CENUM), Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM), Montréal, Québec, Canada.

Spinocerebellar ataxia type 3 is caused by the expansion of the coding CAG repeat in the ATXN3 gene. Interestingly, a -1 bp frameshift occurring within an (exp)CAG repeat would henceforth lead to translation from a GCA frame, generating polyalanine stretches instead of polyglutamine. Our results show that transgenic expression of (exp)CAG ATXN3 led to -1 frameshifting events, which have deleterious effects in Drosophila and mammalian neurons. Conversely, transgenic expression of polyglutamine-encoding (exp)CAA ATXN3 was not toxic. Furthermore, (exp)CAG ATXN3 mRNA does not contribute per se to the toxicity observed in our models. Our observations indicate that expanded polyglutamine tracts in Drosophila and mouse neurons are insufficient for the development of a phenotype. Hence, we propose that -1 ribosomal frameshifting contributes to the toxicity associated with (exp)CAG repeats.
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http://dx.doi.org/10.1093/hmg/dds036DOI Listing
May 2012

Restless legs syndrome-associated MEIS1 risk variant influences iron homeostasis.

Ann Neurol 2011 Jul 27;70(1):170-5. Epub 2011 Jun 27.

Centre of Excellence in Neuromics, CHUM Research Centre, University of Montreal, Montreal, Quebec, Canada.

Restless legs syndrome (RLS) is a frequent sleep disorder that is linked to disturbed iron homeostasis. Genetic studies identified MEIS1 as an RLS-predisposing gene, where the RLS risk haplotype is associated with decreased MEIS1 mRNA and protein expression. We show here that RNA interference treatment of the MEIS1 worm orthologue increases ferritin expression in Caenorhabditis elegans and that the RLS-associated haplotype leads to increased expression of ferritin and DMT1 in RLS brain tissues. Additionally, human cells cultured under iron-deficient conditions show reduced MEIS1 expression. Our data establish a link between the RLS MEIS1 gene and iron metabolism.
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http://dx.doi.org/10.1002/ana.22435DOI Listing
July 2011

Transit defect of potassium-chloride Co-transporter 3 is a major pathogenic mechanism in hereditary motor and sensory neuropathy with agenesis of the corpus callosum.

J Biol Chem 2011 Aug 31;286(32):28456-65. Epub 2011 May 31.

Centre of Excellence in Neuromics, University of Montreal, Centre Hospitalier de l'Université de Montréal-Research Center, Montreal, Quebec H2L 4M1, Canada.

Missense and protein-truncating mutations of the human potassium-chloride co-transporter 3 gene (KCC3) cause hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), which is a severe neurodegenerative disease characterized by axonal dysfunction and neurodevelopmental defects. We previously reported that KCC3-truncating mutations disrupt brain-type creatine kinase-dependent activation of the co-transporter through the loss of its last 140 amino acids. Here, we report a novel and more distal HMSN/ACC-truncating mutation (3402C → T; R1134X) that eliminates only the last 17 residues of the protein. This small truncation disrupts the interaction with brain-type creatine kinase in mammalian cells but also affects plasma membrane localization of the mutant transporter. Although it is not truncated, the previously reported HMSN/ACC-causing 619C → T (R207C) missense mutation also leads to KCC3 loss of function in Xenopus oocyte flux assay. Immunodetection in Xenopus oocytes and in mammalian cultured cells revealed a decreased amount of R207C at the plasma membrane, with significant retention of the mutant proteins in the endoplasmic reticulum. In mammalian cells, curcumin partially corrected these mutant protein mislocalizations, with more protein reaching the plasma membrane. These findings suggest that mis-trafficking of mutant protein is an important pathophysiological feature of HMSN/ACC causative KCC3 mutations.
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http://dx.doi.org/10.1074/jbc.M111.226894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3151088PMC
August 2011

Two Notch ligands, Dll1 and Jag1, are differently restricted in their range of action to control neurogenesis in the mammalian spinal cord.

PLoS One 2010 Nov 24;5(11):e15515. Epub 2010 Nov 24.

Faculdade de Medicina de Lisboa, Instituto de Medicina Molecular, Lisboa, Portugal.

Background: Notch signalling regulates neuronal differentiation in the vertebrate nervous system. In addition to a widespread function in maintaining neural progenitors, Notch signalling has also been involved in specific neuronal fate decisions. These functions are likely mediated by distinct Notch ligands, which show restricted expression patterns in the developing nervous system. Two ligands, in particular, are expressed in non-overlapping complementary domains of the embryonic spinal cord, with Jag1 being restricted to the V1 and dI6 progenitor domains, while Dll1 is expressed in the remaining domains. However, the specific contribution of different ligands to regulate neurogenesis in vertebrate embryos is still poorly understood.

Methodology/principal Findings: In this work, we investigated the role of Jag1 and Dll1 during spinal cord neurogenesis, using conditional knockout mice where the two genes are deleted in the neuroepithelium, singly or in combination. Our analysis showed that Jag1 deletion leads to a modest increase in V1 interneurons, while dI6 neurogenesis was unaltered. This mild Jag1 phenotype contrasts with the strong neurogenic phenotype detected in Dll1 mutants and led us to hypothesize that neighbouring Dll1-expressing cells signal to V1 and dI6 progenitors and restore neurogenesis in the absence of Jag1. Analysis of double Dll1;Jag1 mutant embryos revealed a stronger increase in V1-derived interneurons and overproduction of dI6 interneurons. In the presence of a functional Dll1 allele, V1 neurogenesis is restored to the levels detected in single Jag1 mutants, while dI6 neurogenesis returns to normal, thereby confirming that Dll1-mediated signalling compensates for Jag1 deletion in V1 and dI6 domains.

Conclusions/significance: Our results reveal that Dll1 and Jag1 are functionally equivalent in controlling the rate of neurogenesis within their expression domains. However, Jag1 can only activate Notch signalling within the V1 and dI6 domains, whereas Dll1 can signal to neural progenitors both inside and outside its domains of expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015515PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2991363PMC
November 2010

Family study of restless legs syndrome in Quebec, Canada: clinical characterization of 671 familial cases.

Arch Neurol 2010 May;67(5):617-22

Center of Excellence in Neuromics, Research Centre of the University of Montreal Hospital Centre, and the Department of Medicine, University of Montreal, Montréal, Québec Canada.

Objectives: To fully ascertain the familial aggregation of restless legs syndrome (RLS) and to characterize the clinical features of familial RLS (fRLS) cases.

Design: A case series survey with a high response rate.

Setting: Academic research center.

Participants: Consecutive RLS probands (n = 249) were followed up in a specialized sleep center for 15 years. A total of 671 cases of fRLS met the current standard diagnostic criteria, including 192 probands characterized using multidimensional clinical assessments and 479 affected family members assessed by their responses to a structured questionnaire telephone diagnostic interview.

Main Outcome Measures: Sibling and offspring relative risk ratio and clinical and genetic features of patients with fRLS and families.

Results: Our data showed that RLS aggregates in families with a familial rate of 77%, a sibling relative risk of 3.6 (95% confidence interval, 2.8-4.4), and an offspring relative risk of 1.8 (1.0-2.7). Familial RLS is a chronic disorder with a mean (SD) disease duration of 24 (16) years and a wide range of age of onset (mean [SD], 28 [15] years), with most family members having early-onset disease but mild to moderate RLS symptoms. Our clinical data also indicated that fRLS is more prominent among women who also had increased incidence of anemia/iron deficiency, arthritis, and number of pregnancies. Pregnancy-related RLS seems to be a characteristic feature of fRLS, and afflicted women tend to have a much younger age of onset.

Conclusions: Restless legs syndrome significantly aggregated in families with variable phenotypic expressivity, and the siblings of severely affected individuals have an increased risk of developing the disease.
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http://dx.doi.org/10.1001/archneurol.2010.67DOI Listing
May 2010

Genome-wide TDT analysis in French-Canadian families with Tourette syndrome.

Can J Neurol Sci 2010 Jan;37(1):110-2

The Centre of Excellence in Neuromics, CHUM Research Center Department of Medicine, University of Montreal, Montreal, Quebec, Canada.

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http://dx.doi.org/10.1017/s0317167100009744DOI Listing
January 2010

Association of intronic variants of the BTBD9 gene with Tourette syndrome.

Arch Neurol 2009 Oct;66(10):1267-72

Sainte Justine Hospital Research Center and the Center of Excellence in Neuromics, University of Montreal, Montreal, QC H2L 4M1, Canada.

Objective: To test the association between Tourette syndrome (TS) and genetic variants in genomic loci MEIS1, MAP2K5/LBXCOR1, and BTBD9, for which genome-wide association studies in restless legs syndrome and periodic limb movements during sleep revealed common risk variants.

Design: Case-control association study.

Setting: Movement disorder clinic in Montreal. Subjects We typed 14 single-nucleotide polymorphisms spanning the 3 genomic loci in 298 TS trios, 322 TS cases (including 298 probands from the cohort of TS trios), and 290 control subjects.

Main Outcome Measures: Clinical diagnosis of TS, obsessive-compulsive disorder, and attention-deficit disorder.

Results: The study provided 3 single-nucleotide polymorphisms within BTBD9 associated with TS (chi(2) = 8.02 [P = .005] for rs9357271), with the risk alleles for restless legs syndrome and periodic limb movements during sleep overrepresented in the TS cohort. We stratified our group of patients with TS according to presence or absence of obsessive-compulsive disorder and/or attention-deficit disorder and found that variants in BTBD9 were strongly associated with TS without obsessive-compulsive disorder (chi(2) = 12.95 [P < .001] for rs9357271). Furthermore, allele frequency of rs9357271 inversely correlated with severity of obsessive-compulsive disorder as measured by the Yale-Brown Obsessive Compulsive Scale score.

Conclusion: Variants in BTBD9 that predispose to restless legs syndrome and periodic limb movements during sleep are also associated with TS, particularly TS without obsessive-compulsive disorder.
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http://dx.doi.org/10.1001/archneurol.2009.213DOI Listing
October 2009

A targeted constitutive mutation in the APC tumor suppressor gene underlies mammary but not intestinal tumorigenesis.

PLoS Genet 2009 Jul 3;5(7):e1000547. Epub 2009 Jul 3.

Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/beta-catenin signaling. Notably, genotype-phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/beta-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/beta-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc(+/1572T) mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc(+/1572T) mice suggests that specific dosages of Wnt/beta-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.
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http://dx.doi.org/10.1371/journal.pgen.1000547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2697381PMC
July 2009

MEIS1 intronic risk haplotype associated with restless legs syndrome affects its mRNA and protein expression levels.

Hum Mol Genet 2009 Mar 6;18(6):1065-74. Epub 2009 Jan 6.

Centre of Excellence in Neuromics of University of Montreal, CHUM Research Center, University of Montreal, Montréal, Québec, Canada.

Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible urge to move the legs at night, which is often accompanied by unpleasant sensations. A recent genomewide association study identified an association between RLS and intronic markers from the MEIS1 gene. Comparative genomic analysis indicates that MEIS1 is the only gene encompassed in this evolutionarily conserved chromosomal segment, i.e. a conservation synteny block, from mammals to fish. We carried out a series of experiments to delineate the role of MEIS1 in RLS pathogenesis and the underlying genetic mechanism. We sequenced all 13 MEIS1 exons and their splice junctions in 285 RLS probands with confirmed clinical diagnosis and did not identify any causative coding or exon-intron junction mutations. We found no evidence of structural variation or disease-associated haplotype differential splicing. However, sequencing of conserved regions of MEIS1 introns 8 and 9 identified a novel single nucleotide polymorphism (C13B_2) significantly associated with RLS (allelic association, P = 1.81E-07). We detected a significant decrease in MEIS1 mRNA expression by quantitative real-time polymerase chain reaction in lymphoblastoid cell lines (LCLs) and brain tissues from RLS patients homozygous for the intronic RLS risk haplotype, compared with those homozygous for the non-risk haplotype. Finally, we found significantly decreased MEIS1 protein levels in the same batch of LCLs and brain tissues from the homozygous carriers of the risk haplotype, compared with the homozygous non-carriers. Therefore, these data suggest that reduced expression of the MEIS1 gene, possibly through intronic cis-regulatory element(s), predisposes to RLS.
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http://dx.doi.org/10.1093/hmg/ddn443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722232PMC
March 2009

Smad4 haploinsufficiency: a matter of dosage.

Pathogenetics 2008 Nov 3;1(1). Epub 2008 Nov 3.

Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

Background: The inactivation of tumor suppressor genes follows Alfred Knudson's 'two-hit' model: both alleles need to be inactivated by independent mutation events to trigger tumor formation. However, in a minority of tumor suppressor genes a single hit is sufficient to initiate tumorigenesis notwithstanding the presence of the wild-type allele, a condition known as haploinsufficiency. The SMAD4 gene is an intracellular mediator of the TGF-beta and BMP signal transduction pathways and a tumor suppressor involved in pancreatic and colorectal tumorigenesis. In Smad4-mutant mouse models, haploinsufficiency characterizes the development of gastrointestinal polyps with initial retention of the wild-type allele and protein expression within the nascent tumors and in their direct microenvironment. Similarly, germline SMAD4 mutations are responsible for a subset of patients affected by juvenile polyposis syndrome, an autosomal dominant intestinal cancer syndrome. To date, the molecular and cellular consequences of SMAD4 haploinsufficiency on TGF-beta and BMP signaling and on genome-wide gene expression have not been investigated.

Results: Here we show that, similar to previous observations in Smad4-mutant mouse models, haploinsufficiency characterizes a substantial fraction of the juvenile polyps arising in patients with germline SMAD4 mutations. Also, mouse embryonic and intestinal cells heterozygous for a targeted Smad4 null mutation are characterized by a corresponding 50% reduction of the Smad4 protein levels. Reporter assays revealed that mouse Smad4+/- cells exert intermediate inhibitory effects on both TGF-beta and BMP signaling. Genome-wide expression profiling analysis of Smad4+/- and Smad4-/- cells pinpointed a subset of dosage-dependent transcriptional target genes encompassing, among others, members of the TGF-beta and Wnt signaling pathways. These SMAD4 dosage-dependent transcriptional changes were confirmed and validated in a subset of target genes in intestinal tissues from juvenile polyposis syndrome patients.

Conclusion: Smad4 haploinsufficiency is sufficient to significantly inhibit both TGF-beta and BMP signal transduction and results in the differential expression of a broad subset of target genes likely to underlie tumor formation both from the mesenchymal and epithelial compartments. The results of our study, performed in normal rather than tumor cells where additional (epi-) genetic alterations may confound the analysis, are relevant for our understanding and elucidation of the initial steps underlying SMAD4-driven intestinal tumorigenesis.
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http://dx.doi.org/10.1186/1755-8417-1-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2580039PMC
November 2008

Autosomal-dominant locus for Restless Legs Syndrome in French-Canadians on chromosome 16p12.1.

Mov Disord 2009 Jan;24(1):40-50

Center of Excellence in Neuromics, CHUM Research Center-Notre Dame Hospital, Quebec, Canada.

We describe an autosomal-dominant locus for Restless Legs Syndrome (RLS) in a French-Canadian (FC) pedigree. Genome-wide microsatellite scan and linkage analysis were used in this study. The locus maps to chromosome 16p12.1 and spans 1.18 Mega bases. The maximum multipoint LOD scores are of 3.5 over the total of 10 markers. Evidence for the same locus was also found in a smaller FC pedigree sime095. The analysis of the sequence of 8 annotated genes within the region did not reveal any pathogenic mutations. Copy number variation and karyotype analyses did not reveal any chromosomal abnormality in the region. Further analyses of the region are necessary to find the genetic cause of RLS in this family.
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http://dx.doi.org/10.1002/mds.22263DOI Listing
January 2009

HMSN/ACC truncation mutations disrupt brain-type creatine kinase-dependant activation of K+/Cl- co-transporter 3.

Hum Mol Genet 2008 Sep 19;17(17):2703-11. Epub 2008 Jun 19.

Department of Medicine, Centre of Excellence in Neuromics, CHUM Research Centre, University of Montreal, Montreal, QC, Canada.

The potassium-chloride co-transporter 3 (KCC3) is mutated in hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC); however, the molecular mechanisms of HMSN/ACC pathogenesis and the exact role of KCC3 in the development of the nervous system remain poorly understood. The functional regulation of this transporter by protein partners is also largely unknown. Using a yeast two-hybrid approach, we discovered that the C-terminal domain (CTD) of KCC3, which is lost in most HMSN/ACC-causing mutations, directly interacts with brain-specific creatine kinase (CK-B), an ATP-generating enzyme that is also a partner of KCC2. The interaction of KCC3 with CK-B was further confirmed by in vitro glutathione S-transferase pull-down assay, followed by sequencing of the pulled-down complexes. In transfected cultured cells, immunofluorescence labeling showed that CK-B co-localizes with wild-type KCC3, whereas the kinase fails to interact with the inactive truncated KCC3. Finally, CK-B's inhibition by DNFB results in reduction of activity of KCC3 in functional assays using Xenopus laevis oocytes. This physical and functional association between the co-transporter and CK-B is, therefore, the first protein-protein interaction identified to be potentially involved in the pathophysiology of HMSN/ACC.
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http://dx.doi.org/10.1093/hmg/ddn172DOI Listing
September 2008

cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo.

Proc Natl Acad Sci U S A 2008 May 19;105(20):7281-6. Epub 2008 May 19.

Department of Tissue Regeneration, Institute for Biomedical Technology, University of Twente, 7500 AE, Enschede, The Netherlands.

Tissue engineering of large bone defects is approached through implantation of autologous osteogenic cells, generally referred to as multipotent stromal cells or mesenchymal stem cells (MSCs). Animal-derived MSCs successfully bridge large bone defects, but models for ectopic bone formation as well as recent clinical trials demonstrate that bone formation by human MSCs (hMSCs) is inadequate. The expansion phase presents an attractive window to direct hMSCs by pharmacological manipulation, even though no profound effect on bone formation in vivo has been described so far using this approach. We report that activation of protein kinase A elicits an immediate response through induction of genes such as ID2 and FosB, followed by sustained secretion of bone-related cytokines such as BMP-2, IGF-1, and IL-11. As a consequence, PKA activation results in robust in vivo bone formation by hMSCs derived from orthopedic patients.
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http://dx.doi.org/10.1073/pnas.0711190105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2387183PMC
May 2008

Cross-species comparison of human and mouse intestinal polyps reveals conserved mechanisms in adenomatous polyposis coli (APC)-driven tumorigenesis.

Am J Pathol 2008 May 10;172(5):1363-80. Epub 2008 Apr 10.

Dept. of Pathology, Erasmus MC, PO Box 2040, 3000CA Rotterdam, The Netherlands.

Expression profiling is a well established tool for the genome-wide analysis of human cancers. However, the high sensitivity of this approach combined with the well known cellular and molecular heterogeneity of cancer often result in extremely complex expression signatures that are difficult to interpret functionally. The majority of sporadic colorectal cancers are triggered by mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, leading to the constitutive activation of the Wnt/beta-catenin signaling pathway and formation of adenomas. Despite this common genetic basis, colorectal cancers are very heterogeneous in their degree of differentiation, growth rate, and malignancy potential. Here, we applied a cross-species comparison of expression profiles of intestinal polyps derived from hereditary colorectal cancer patients carrying APC germline mutations and from mice carrying a targeted inactivating mutation in the mouse homologue Apc. This comparative approach resulted in the establishment of a conserved signature of 166 genes that were differentially expressed between adenomas and normal intestinal mucosa in both species. Functional analyses of the conserved genes revealed a general increase in cell proliferation and the activation of the Wnt/beta-catenin signaling pathway. Moreover, the conserved signature was able to resolve expression profiles from hereditary polyposis patients carrying APC germline mutations from those with bi-allelic inactivation of the MYH gene, supporting the usefulness of such comparisons to discriminate among patients with distinct genetic defects.
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http://dx.doi.org/10.2353/ajpath.2008.070851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2329845PMC
May 2008

Asian origin for the worldwide-spread mutational event in Machado-Joseph disease.

Arch Neurol 2007 Oct;64(10):1502-8

Instituto de Patologia e Imunologia Molecular da Universidade do Porto and Faculdade de Ciências, University of Porto, Porto, Portugal.

Background: Machado-Joseph disease is the most frequent dominant ataxia worldwide. Despite its frequency and presence in many populations, only 2 founder mutations have been suggested to explain its current geographic distribution.

Objectives: To trace back in history the main mutational events in Machado-Joseph disease, we aimed to assess ancestral haplotypes and population backgrounds, to date the mutations, and to trace the routes and time of introduction of the founder haplotypes in different populations.

Design, Setting, And Participants: We studied 264 families with Machado-Joseph disease from 20 different populations. Six intragenic single-nucleotide polymorphisms were used to determine ancestral mutational events; 4 flanking short tandem repeats were used to construct extended haplotypes and measure accumulation of genetic diversity over time within each lineage.

Results: The worldwide-spread lineage, TTACAC, had its highest diversity in the Japanese population, where we identified the ancestral short tandem repeat-based haplotype. Accumulated variability suggested a postneolithic mutation, about 5774 +/- 1116 years old, with more recent introductions in North America, Germany, France, Portugal, and Brazil. As to the second mutational event, in the GTGGCA lineage, only 7 families (of 71 families) did not have Portuguese ancestry, although gene diversity was again smaller in Portuguese families (0.44) than in non-Portuguese families (0.93).

Conclusions: The worldwide-spread mutation may have first occurred in Asia and later been diffused throughout Europe, with a founder effect accounting for its high prevalence in Portugal; the other Machado-Joseph disease lineage is more recent, about 1416 +/- 434 years old, and its dispersion may be explained mainly by recent Portuguese emigration.
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http://dx.doi.org/10.1001/archneur.64.10.1502DOI Listing
October 2007

Genetic association studies of neurotensin gene and restless legs syndrome in French Canadians.

Sleep Med 2008 Mar 17;9(3):273-82. Epub 2007 Jul 17.

Center for the Study of Brain Diseases, CHUM Research Center - Notre Dame Hospital, University of Montreal, Bureau Y-3616-2, 1560, rue Sherbrooke Est, Montréal, Que., Canada H2L 4MI.

Background And Purpose: The neurotensin gene (NTS), a known dopamine modulator, is located within the candidate region for the first genetic locus of restless legs syndrome (RLS1) on chromosome 12q. Though no causative mutation was found in selected patients in a previous mutation analysis, the involvement of NTS in RLS cannot be completely excluded as a potential positional and functional candidate gene. The purpose of the current study is to further explore the NTS gene for potential functional variant(s) in its entire genomic and potential regulatory regions and their possible association with RLS symptoms.

Methods And Subjects: We resequenced the coding regions and sequenced all the intronic and potential regulatory regions of the NTS gene in additional patients and controls. We carried out full scale gene-based case-control and family-based genetic association studies using the sequence variants detected during mutational analysis.

Results: No coding or variants in regulatory and intronic regions compatible with a deleterious mutation were detected. Seven polymorphisms with elevated allele frequencies in the Caucasian population did not show association with RLS in two independent case-control groups and 110 RLS families.

Conclusion: The NTS gene on chromosome 12q is most unlikely to play a direct role in RLS etiology.
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http://dx.doi.org/10.1016/j.sleep.2007.03.020DOI Listing
March 2008

Molecular genetic studies of DMT1 on 12q in French-Canadian restless legs syndrome patients and families.

Am J Med Genet B Neuropsychiatr Genet 2007 Oct;144B(7):911-7

Laboratoire d'étude des maladies du cerveau, Centre de recherche du CHUM, Hôpital Notre-Dame, Université de Montréal, 1560 rue Sherbrooke Est, Montréal, Québec, Canada.

Converging evidence from clinical observations, brain imaging and pathological findings strongly indicate impaired brain iron regulation in restless legs syndrome (RLS). Animal models with mutation in (DMT1) divalent metal transporter 1 gene, an important brain iron transporter, demonstrate a similar iron deficiency profile as found in RLS brain. The human DMT1 gene, mapped to chromosome 12q near the RLS1 locus, qualifies as an excellent functional and possible positional candidate for RLS. DMT1 protein levels were assessed in lymphoblastoid cell lines from RLS patients and controls. Linkage analyses were carried out with markers flanking and within the DMT1 gene. Selected patient samples from RLS families with compatible linkage to the RLS1 locus on 12q were fully sequenced in both the coding regions and the long stretches of UTR sequences. Finally, selected sequence variants were further studied in case/control and family-based association tests. A clinical association of anemia and RLS was further confirmed in this study. There was no detectable difference in DMT1 protein levels between RLS patient lymphoblastoid cell lines and normal controls. Non-parametric linkage analyses failed to identify any significant linkage signals within the DMT1 gene region. Sequencing of selected patients did not detect any sequence variant(s) compatible with DMT1 harboring RLS causative mutation(s). Further studies did not find any association between ten SNPs, spanning the whole DMT1 gene region, and RLS affection status. Finally, two DMT1 intronic SNPs showed positive association with RLS in patients with a history of anemia, when compared to RLS patients without anemia.
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http://dx.doi.org/10.1002/ajmg.b.30528DOI Listing
October 2007

APC and oncogenic KRAS are synergistic in enhancing Wnt signaling in intestinal tumor formation and progression.

Gastroenterology 2006 Oct 16;131(4):1096-109. Epub 2006 Aug 16.

UMR144/Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France.

Background & Aims: Synchronous activation of the Wnt signaling pathway, mostly because of loss of function of the APC tumor suppressor, and of the oncogenic KRAS-signaling pathway is very frequent in colorectal cancer and is associated with poor prognosis.

Methods: We have generated a compound transgenic mouse model, KRAS(V12G)/Apc(+/1638N), to recapitulate the human disease and compared it with single transgenic littermates.

Results: Compound mutant mice are characterized by a 10-fold increase in tumor multiplicity and by accelerated tumor progression, resulting in strongly enhanced morbidity and mortality. Tumors from compound mutant mice proliferate faster and show decreased levels of apoptosis. Several lines of evidence indicate that the observed increase in tumor multiplicity and malignant transformation is caused by the synergistic activation of Wnt signaling in cells with oncogenic KRAS and loss-of-function Apc mutations. Activated KRAS is known to induce tyrosine phosphorylation of beta-catenin, leading to its release from E-cadherin at the adherens junction. This results in an increased beta-catenin pool in the cytoplasma, its subsequent translocation to the nucleus, and the transcriptional activation of Wnt downstream target genes. Accordingly, intestinal tumors from KRAS(V12G)/Apc(+/1638N) mice show a significant increase in cells with nuclear accumulation of beta-catenin when compared with Apc(+/1638N) animals. Moreover, Apc/KRAS-mutant embryonic stem cells show a significantly enhanced beta-catenin/T-cell factor-mediated transcriptional activation, accompanied by increased beta-catenin nuclear localization.

Conclusions: This KRAS-induced increase in Wnt/beta-catenin signaling may enhance the plasticity and self-renewal capacity of the tumor, thus resulting in the drastically augmented tumor multiplicity and malignant behavior in compound mutant animals.
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http://dx.doi.org/10.1053/j.gastro.2006.08.011DOI Listing
October 2006

Genetics of familial and sporadic amyotrophic lateral sclerosis.

Biochim Biophys Acta 2006 Nov-Dec;1762(11-12):956-72. Epub 2006 Feb 10.

Center for the Study of Brain Diseases, CHUM Research Center, Notre Dame Hospital, J.A. de Sève Pavillion, Room Y-3633, 1560, Sherbrooke Street East, Montreal, QC, Canada H2L 4M1.

Diseases affecting motor neurons, such as amyotrophic lateral sclerosis (Lou Gerhig's disease), hereditary spastic paraplegia and spinal bulbar muscular atrophy (Kennedy's disease) are a heterogeneous group of chronic progressive diseases and are among the most puzzling yet untreatable illnesses. Over the last decade, identification of mutations in genes predisposing to these disorders has provided the means to better understand their pathogenesis. The discovery 13 years ago of SOD1 mutations linked to ALS, which account for less than 2% of total cases, had a major impact in the field. However, despite intensive research effort, the pathways leading to the specific motor neurons degeneration in the presence of SOD1 mutations have not been fully identified. This review provides an overview of the genetics of both familial and sporadic forms of ALS.
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http://dx.doi.org/10.1016/j.bbadis.2006.01.004DOI Listing
January 2007

Ribosomal frameshifting on MJD-1 transcripts with long CAG tracts.

Hum Mol Genet 2005 Sep 8;14(18):2649-60. Epub 2005 Aug 8.

Department of Medicine and Research Center, Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, Suite Y3616-2, 1560 Sherbrooke Street East, Montreal, Quebec H2L 4M1, Canada.

The expanded CAG tract diseases are a heterogeneous group of late-onset neurodegenerative disorders characterized by the accumulation of insoluble protein material and premature neuronal cell death. Recent work has provided support for several mechanisms that may account for neurodegeneration, but no unifying mechanism has emerged. We have previously demonstrated that in SCA3, the expanded CAG tract in the MJD-1 transcript is prone to frameshifting, which may lead to the production of polyalanine-containing proteins. To further examine the occurrence of frameshifting and understand its mechanism and possible role in pathogenesis, a cellular model was established. We show that this phenomenon results from ribosomal slippage to the -1 frame exclusively, that ribosomal frameshifting depends on the presence of long CAG tracts and that polyalanine-frameshifted proteins may enhance polyglutamine-associated toxicity, possibly contributing to pathogenesis. Finally, we present evidence that anisomycin, a ribosome-interacting drug that reduces -1 frameshifting, also reduces toxicity, suggesting a new therapeutic opportunity for these disorders.
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http://dx.doi.org/10.1093/hmg/ddi299DOI Listing
September 2005

Transgenic expression of an expanded (GCG)13 repeat PABPN1 leads to weakness and coordination defects in mice.

Neurobiol Dis 2005 Apr;18(3):528-36

McGill University Health Center Research Institute and the Centre for Research in Neurosciences, McGill University, 1650 Cedar Avenue (L7-224), Montréal, Québec, Canada, H3G 1A4.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder caused by a (GCG)n trinucleotide repeat expansion in the poly(A) binding protein nuclear-1 (PABPN1) gene, which in turn leads to an expanded polyalanine tract in the protein. We generated transgenic mice expressing either the wild type or the expanded form of human PABPN1, and transgenic animals with the expanded form showed clear signs of abnormal limb clasping, muscle weakness, coordination deficits, and peripheral nerves alterations. Analysis of mitotic and postmitotic tissues in those transgenic animals revealed ubiquitinated PABPN1-positive intranuclear inclusions (INIs) in neuronal cells. This latter observation led us to test and confirm the presence of similar INIs in postmortem brain sections from an OPMD patient. Our results indicate that expanded PABPN1, presumably via the toxic effects of its polyalanine tract, can lead to inclusion formation and neurodegeneration in both the mouse and the human.
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http://dx.doi.org/10.1016/j.nbd.2004.09.021DOI Listing
April 2005

Morphological changes in tumour type after radiotherapy are accompanied by changes in gene expression profile but not in clinical behaviour.

J Pathol 2004 Oct;204(2):183-92

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

The morphological features of neoplastic cells, combined with a stromal reaction, determine the presence of cancer at the microscopic level. Malignant tumours arise through a series of genetic alterations, but these do not entirely explain invasive and metastatic behaviour and correlate only weakly with morphological changes. In order to understand the relationship between the morphology of cancer tissue, gene expression, and clinical behaviour, a study of radiation-induced mucinous rectal carcinoma was performed. Short-term radiotherapy of rectal carcinoma results in an increased incidence of mucinous carcinoma. A cohort of rectal carcinomas (n = 1304), from patients who participated in a randomized radiotherapy trial, was evaluated for the presence and amount of a mucinous component. The results were compared with data from the pre-irradiation biopsies and revealed the presence of two distinct classes of mucinous carcinoma in the radiotherapy group, namely pre-existing (un-induced; n = 24) and induced mucinous carcinoma (n = 29). Clinical data, pathological parameters, and immunohistochemical data from these patients and their tumours showed that induced mucinous carcinomas were more comparable to typical adenocarcinomas than to pre-existing mucinous carcinomas. The prognosis of patients with induced mucinous carcinoma was significantly better than that of patients with pre-existing mucinous carcinomas (91.2% versus 39.3% recurrence-free interval at 2 years, p = 0.02). Gene expression profiles of the different groups of mucinous carcinomas and adenocarcinomas were analysed using Affymetrix Human Cancer Chips. Surprisingly, despite the difference in prognosis, the expression profile of radiation-induced mucinous carcinomas was very closely related to that of their un-induced counterparts. It is shown in the present study that radiation therapy of rectal cancer leads within a few days to substantial changes in both morphology and expression profile. However, the morphology of the pre-therapy biopsy predicts patient survival far better than post-therapy expression profiles. It is concluded that tumour morphology equates to expression profile, but that external factors might influence both, leading to sub-optimal prognostication.
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http://dx.doi.org/10.1002/path.1621DOI Listing
October 2004

APC dosage effects in tumorigenesis and stem cell differentiation.

Int J Dev Biol 2004 ;48(5-6):377-86

Dept. of Pathology, Josephine Nefkens Institute, Erasmus University Medical Center, 3000 DR Rotterdam, The Netherlands.

It is well established that concentration gradients of signaling molecules (the so-called "morphogens") organize and pattern tissues in developing animals. In particular, studies in Drosophila and different vertebrates have shown that gradients of the Wnt, Hedgehog (Hh) and transforming growth factor-beta (TGF-beta) families of morphogens play critical roles in limb patterning. Morphogens are often expressed in organizing centres and can act over a long range to coordinate the patterning of an entire field of cells. These observations imply that exposure to different concentrations of these diffusible factors may trigger differential cellular responses. In order to study these dosage-dependent Wnt/beta-catenin signaling effects, we have generated several hypomorphic mutant alleles at the mouse Apc locus and studied their cellular and phenotypic outcomes in stem cell renewal and differentiation, and in tumorigenesis. The results clearly show that Apc mutations differentially affect the capacity of stem cells to differentiate in a dosage-dependent fashion. Likewise, different Apc mutations (and the corresponding Wnt signaling dosages) confer different degrees of susceptibility to tumorigenesis in the corresponding mouse models. These results have implications for the understanding of the molecular and cellular basis of tumor initiation by defects in the Wnt pathway. We propose a model in which adult somatic stem cell compartments are characterized by tissue-specific beta-catenin threshold levels for cell proliferation, differentiation and apoptosis. Different APC mutations will result in different levels of beta-catenin signaling, thus conferring different degrees of tumor susceptibility in different tissues. Hence, beta-catenin dosage-dependent effects may not only explain how a single pathway is involved in the development and homeostasis of different tissues, but also its pleiotrophic role in tumorigenesis.
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http://dx.doi.org/10.1387/ijdb.041807cgDOI Listing
February 2005

Wnt signaling inhibits osteogenic differentiation of human mesenchymal stem cells.

Bone 2004 May;34(5):818-26

Institute for Biomedical Technology, University of Twente, Enschede, The Netherlands.

Human mesenchymal stem cells (hMSCs) from the bone marrow represent a potential source of pluripotent cells for autologous bone tissue engineering. We previously discovered that over activation of the Wnt signal transduction pathway by either lithium or Wnt3A stimulates hMSC proliferation while retaining pluripotency. Release of Wnt3A or lithium from porous calcium phosphate scaffolds, which we use for bone tissue engineering, could provide a mitogenic stimulus to implanted hMSCs. To define the proper release profile, we first assessed the effect of Wnt over activation on osteogenic differentiation of hMSCs. Here, we report that both lithium and Wnt3A strongly inhibit dexamethasone-induced expression of the osteogenic marker alkaline phosphatase (ALP). Moreover, lithium partly inhibited mineralization of hMSCs whereas Wnt3A completely blocked it. Time course analysis during osteogenic differentiation revealed that 4 days of Wnt3A exposure before the onset of mineralization is sufficient to block mineralization completely. Gene expression profiling in Wnt3A and lithium-exposed hMSCs showed that many osteogenic and chondrogenic markers, normally expressed in proliferating hMSCs, are downregulated upon Wnt stimulation. We conclude that Wnt signaling inhibits dexamethasone-induced osteogenesis in hMSCs. In future studies, we will try to limit release of lithium or Wnt3A from calcium phosphate scaffolds to the proliferative phase of osteogenesis.
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http://dx.doi.org/10.1016/j.bone.2004.01.016DOI Listing
May 2004