Novel role of the synaptic scaffold protein Dlgap4 in ventricular surface integrity and neuronal migration during cortical development.

Subcortical heterotopias are malformations associated with epilepsy and intellectual disability, characterized by the presence of ectopic neurons in the white matter. Mouse and human heterotopia mutations were identified in the microtubule-binding protein Echinoderm microtubule-associated protein-like 1, EML1. Further exploring pathological mechanisms, we identified a patient with an EML1-like phenotype and a novel genetic variation in DLGAP4. The protein belongs to a membrane-associated guanylate kinase family known to function in glutamate synapses. We showed that DLGAP4 is strongly expressed in the mouse ventricular zone (VZ) from early corticogenesis, and interacts with key VZ proteins including EML1. In utero electroporation of Dlgap4 knockdown (KD) and overexpression constructs revealed a ventricular surface phenotype including changes in progenitor cell dynamics, morphology, proliferation and neuronal migration defects. The Dlgap4 KD phenotype was rescued by wild-type but not mutant DLGAP4. Dlgap4 is required for the organization of radial glial cell adherens junction components and actin cytoskeleton dynamics at the apical domain, as well as during neuronal migration. Finally, Dlgap4 heterozygous knockout (KO) mice also show developmental defects in the dorsal telencephalon. We hence identify a synapse-related scaffold protein with pleiotropic functions, influencing the integrity of the developing cerebral cortex.

Mutations in the Heterotopia Gene Eml1/EML1 Severely Disrupt the Formation of Primary Cilia.

Apical radial glia (aRGs) are predominant progenitors during corticogenesis. Perturbing their function leads to cortical malformations, including subcortical heterotopia (SH), characterized by the presence of neurons below the cortex. EML1/Eml1 mutations lead to SH in patients, as well as to heterotopic cortex (HeCo) mutant mice. In HeCo mice, some aRGs are abnormally positioned away from the ventricular zone (VZ). Thus, unraveling EML1/Eml1 function will clarify mechanisms maintaining aRGs in the VZ. We pinpoint an unknown EML1/Eml1 function in primary cilium formation. In HeCo aRGs, cilia are shorter, less numerous, and often found aberrantly oriented within vesicles. Patient fibroblasts and human cortical progenitors show similar defects. EML1 interacts with RPGRIP1L, a ciliary protein, and RPGRIP1L mutations were revealed in a heterotopia patient. We also identify Golgi apparatus abnormalities in EML1/Eml1 mutant cells, potentially upstream of the cilia phenotype. We thus reveal primary cilia mechanisms impacting aRG dynamics in physiological and pathological conditions.

Eml1 loss impairs apical progenitor spindle length and soma shape in the developing cerebral cortex.

The ventricular zone (VZ) of the developing cerebral cortex is a pseudostratified epithelium that contains progenitors undergoing precisely regulated divisions at its most apical side, the ventricular lining (VL). Mitotic perturbations can contribute to pathological mechanisms leading to cortical malformations. The HeCo mutant mouse exhibits subcortical band heterotopia (SBH), likely to be initiated by progenitor delamination from the VZ early during corticogenesis. The causes for this are however, currently unknown. Eml1, a microtubule (MT)-associated protein of the EMAP family, is impaired in these mice. We first show that MT dynamics are perturbed in mutant progenitor cells in vitro. These may influence interphase and mitotic MT mechanisms and indeed, centrosome and primary cilia were altered and spindles were found to be abnormally long in HeCo progenitors. Consistently, MT and spindle length regulators were identified in EML1 pulldowns from embryonic brain extracts. Finally, we found that mitotic cell shape is also abnormal in the mutant VZ. These previously unidentified VZ characteristics suggest altered cell constraints which may contribute to cell delamination.

The transcription factor HNF-4α: a key factor of the intestinal uptake of fatty acids in mouse.

With an excessive postprandial accumulation of intestine-derived, triglyceride-rich lipoproteins being a risk factor of cardiovascular diseases, it is essential to characterize the mechanisms controlling the intestinal absorption of dietary lipids. Our aim was to investigate the role of the transcription factor hepatocyte nuclear factor (HNF)-4α in this process. We used transgenic mice with a specific and inducible intestinal knockout of Hnf-4α gene. One hour after a lipid bolus, in the presence of the lipase inhibitor tyloxapol, lower amounts of triglycerides were found in both plasma and intestinal epithelium of the intestine-specific Hnf-4α knockout (Hnf-4α(intΔ)) mice compared with the Hnf-4α(loxP/loxP) control mice. These discrepancies were due to a net decrease of the intestinal uptake of fatty acid in Hnf-4α(intΔ) mice compared with Hnf-4α(loxP/loxP) mice, as assessed by the amount of radioactivity that was recovered in intestine and plasma after gavage with labeled triolein or oleic acid, or in intestinal epithelial cells isolated from jejunum after a supply of labeled oleic acid-containing micelles. This decreased fatty acid uptake was associated with significant lower levels of the fatty acid transport protein-4 mRNA and protein along the intestinal tract and with a lower acyl-CoA synthetase activity in Hnf-4α(intΔ) mice compared with the control mice. We conclude that the transcription factor HNF-4α is a key factor of the intestinal absorption of dietary lipids, which controls this process as early as in the initial step of fatty acid uptake by enterocytes.

GLUT2 accumulation in enterocyte apical and intracellular membranes: a study in morbidly obese human subjects and ob/ob and high fat-fed mice.

OBJECTIVE: In healthy rodents, intestinal sugar absorption in response to sugar-rich meals and insulin is regulated by GLUT2 in enterocyte plasma membranes. Loss of insulin action maintains apical GLUT2 location. In human enterocytes, apical GLUT2 location has not been reported but may be revealed under conditions of insulin resistance. RESEARCH DESIGN AND METHODS: Subcellular location of GLUT2 in jejunal enterocytes was analyzed by confocal and electron microscopy imaging and Western blot in 62 well-phenotyped morbidly obese subjects and 7 lean human subjects. GLUT2 locations were assayed in ob/ob and ob/+ mice receiving oral metformin or in high-fat low-carbohydrate diet-fed C57Bl/6 mice. Glucose absorption and secretion were respectively estimated by oral glucose tolerance test and secretion of [U-(14)C]-3-O-methyl glucose into lumen. RESULTS: In human enterocytes, GLUT2 was consistently located in basolateral membranes. Apical GLUT2 location was absent in lean subjects but was observed in 76% of obese subjects and correlated with insulin resistance and glycemia. In addition, intracellular accumulation of GLUT2 with early endosome antigen 1 (EEA1) was associated with reduced MGAT4a activity (glycosylation) in 39% of obese subjects on a low-carbohydrate/high-fat diet. Mice on a low-carbohydrate/high-fat diet for 12 months also exhibited endosomal GLUT2 accumulation and reduced glucose absorption. In ob/ob mice, metformin promoted apical GLUT2 and improved glucose homeostasis. Apical GLUT2 in fasting hyperglycemic ob/ob mice tripled glucose release into intestinal lumen. CONCLUSIONS: In morbidly obese insulin-resistant subjects, GLUT2 was accumulated in apical and/or endosomal membranes of enterocytes. Functionally, apical GLUT2 favored and endosomal GLUT2 reduced glucose transepithelial exchanges. Thus, altered GLUT2 locations in enterocytes are a sign of intestinal adaptations to human metabolic pathology.

Hepatocyte nuclear factor 4alpha, a key factor for homeostasis, cell architecture, and barrier function of the adult intestinal epithelium.

Hepatocyte nuclear factor 4alpha (HNF-4alpha) is a transcription factor which is highly expressed in the intestinal epithelium from duodenum to colon and from crypt to villus. The homeostasis of this constantly renewing epithelium relies on an integrated control of proliferation, differentiation, and apoptosis, as well as on the functional architecture of the epithelial cells. In order to determine the consequences of HNF-4alpha loss in the adult intestinal epithelium, we used a tamoxifen-inducible Cre-loxP system to inactivate the Hnf-4a gene. In the intestines of adult mice, loss of HNF-4alpha led to an increased proliferation in crypts and to an increased expression of several genes controlled by the Wnt/beta-catenin system. This control of the Wnt/beta-catenin signaling pathway by HNF-4alpha was confirmed in vitro. Cell lineage was affected, as indicated by an increased number of goblet cells and an impairment of enterocyte and enteroendocrine cell maturation. In the absence of HNF-4alpha, cell-cell junctions were destabilized and paracellular intestinal permeability increased. Our results showed that HNF-4alpha modulates Wnt/beta-catenin signaling and controls intestinal epithelium homeostasis, cell function, and cell architecture. This study indicates that HNF-4alpha regulates the intestinal balance between proliferation and differentiation, and we hypothesize that it might act as a tumor suppressor.

No somatic genetic change in the paxillin gene in nonsmall-cell lung cancer.

The paxillin gene (PXN) encodes a focal adhesion associated protein that could be involved in the progression of lung cancer through its interactions with the actin cytoskeleton and key signal transduction oncogenes. PXN mutations and PXN amplifications were recently identified in nonsmall-cell lung cancer (NSCLC) and amplifications were associated with MET increased copy number. The description of tumors with two to three mutations in the PXN gene and the overrepresentation of GC to AT transitions were unexpected and needed confirmation. The aim of this study was to validate the incidence of PXN somatic alterations in NSCLC and to correlate them to other common genetic alterations. PXN mutations and copy number changes at PXN, EGFR, and MET loci were analyzed on DNAs from frozen tumor samples (n = 159) that had been previously screened for mutations at EGFR, KRAS, BRAF, ERBB2, STK11, PIK3CA, and TP53. We found PXN polymorphisms including nonsynonymous ones but no PXN amplification and only 1/159 (<1%) somatic tumor mutation F416L. In conclusion, we do not deny the possible involvement of PXN in cancer but our findings do not support a major role for PXN somatic changes in lung carcinogenesis.

Loss of sugar detection by GLUT2 affects glucose homeostasis in mice.

BACKGROUND: Mammals must sense the amount of sugar available to them and respond appropriately. For many years attention has focused on intracellular glucose sensing derived from glucose metabolism. Here, we studied the detection of extracellular glucose concentrations in vivo by invalidating the transduction pathway downstream from the transporter-detector GLUT2 and measured the physiological impact of this pathway. METHODOLOGY/PRINCIPAL FINDINGS: We produced mice that ubiquitously express the largest cytoplasmic loop of GLUT2, blocking glucose-mediated gene expression in vitro without affecting glucose metabolism. Impairment of GLUT2-mediated sugar detection transiently protected transgenic mice against starvation and streptozotocin-induced diabetes, suggesting that both low- and high-glucose concentrations were not detected. Transgenic mice favored lipid oxidation, and oral glucose was slowly cleared from blood due to low insulin production, despite massive urinary glucose excretion. Kidney adaptation was characterized by a lower rate of glucose reabsorption, whereas pancreatic adaptation was associated with a larger number of small islets. CONCLUSIONS/SIGNIFICANCE: Molecular invalidation of sugar sensing in GLUT2-loop transgenic mice changed multiple aspects of glucose homeostasis, highlighting by a top-down approach, the role of membrane glucose receptors as potential therapeutic targets.

Molecular characterization of four ADAMTS13 mutations responsible for congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome).

ADAMTS13 mutations S203P, R268P, R507Q and A596V were previously identified in French patients with hereditary thrombotic thrombocytopenic purpura (TTP) (Upshaw-Schulman syndrome). Mutated recombinant (r) ADAMTS13 were transiently expressed in COS-7 cells and characterized in comparison with wild-type (WT) rADAMTS13. ADAMTS13 antigen was qualitatively and quantitatively estimated by electrophoretic analysis and ELISA. Enzymatic activity was qualitatively and quantitatively estimated using GST-VWF73, FRETS-VWF73 fragments and full-length rVWF-WT as substrates. The four mutants and rADAMTS13-WT were present within the cells. Secretion level of rADAMTS13-WT reached 1,200 ng/ml. The four mutations strongly altered the secretion and biological activity of rADAMTS13. The percentage secretion was 21, 38 and 17% for rADAMTS13-S203P, -R268P and -A596V compared with rADAMTS13-WT. rADAMTS13-R507Q concentration was under the detection limit of the assay. In the four cases, no enzymatic activity was detected. After concentration, we confirmed that mutations S203P and R268P totally abolished the proteolytic activity of ADAMTS13. Due to the very low protease concentration, activity of rADAMTS13-R507Q was below the threshold of the assays. rADAMTS13-A596V had no proteolytic activity towards the full-length rVWF-WT whereas it exhibited a decreased specific activity of about 30% of that of rADAMTS13-WT towards FRETS-VWF73 fragment. Binding study of mutated rADAMTS13-S203P, -R268P and -A596V showed that the three mutations strongly decreased the interaction of ADAMTS13 with VWF. In conclusion, the four mutations, which led to a secretion defect, a loss of enzymatic activity and a decreased binding to the substrate, are responsible for the hereditary TTP in patients.

Prognostic significance of MMP-1 and MMP-3 functional promoter polymorphisms in colorectal cancer.

PURPOSE: Matrix metalloproteinase (MMP) belongs to a large group of proteases capable of breaking essentially all components of the extracellular matrix. They are implicated in all steps of tumorogenesis, cancer invasion, and metastasis. Among them, metalloproteinase type 1 (MMP-1) is implicated in tumor invasion and metastasis in different types of cancers including colorectal cancer in which its expression was correlated with poor prognosis. A polymorphism in the promoter region of the MMP-1 gene leads to a variation of its level of transcription. STUDY DESIGN: MMP-1 -1607ins/delG and MMP-3 - 1612 ins/delA promoter polymorphisms were genotyped by multiplex PCR from 201 colorectal cancer patients. The median follow-up of patients was 30 months. The MMP genotypes were correlated to clinical outcome. RESULTS: Patients with the -1607insG/-1607insG MMP-1 genotype had significantly worse specific survival than the others in the whole series (P < 0.04), in stage I to III patients (P < 0.001), and in patients stage I and II (P < 0.01). In multivariate analysis, MMP-1-1607insG allele showed to be an independent poor prognostic factor after adjustment on stage, age, and the use of adjuvant chemotherapy. MMP-3 polymorphism was not associated with survival. CONCLUSIONS: In the subgroups of nondistant metatastic patients (stages I and II, and stages I-III), an inverse relation between the number of MMP-1-1607insG allele and survival was observed suggesting a gene dosage effect. Our results are consistent with the importance of MMP-1-1607ins/delG functional polymorphism in regulating transcription level and with the relationship between MMP-1 expression and cancer invasion, metastasis, and prognosis.

Single nucleotide polymorphisms in MMP1 and MMP3 gene promoters as risk factor in head and neck squamous cell carcinoma.

Matrix metalloproteinase (MMP) 1 and 3 genes play an important role in initiating tumor growth and promoting cell spread. Since MMP1 and MMP3 transcription levels can be modulated by promoter polymorphism, we investigated the impact of different genotypes on the occurrence of head and neck cancer in a Caucasian case control study. DNA was extracted from 126 male head and neck cancer patients and from 249 male hospitalized-based controls. Genotyping was carried out using PCR multiplex allowing the co-amplification of MMAP1-1607 bp and MMP3-1171 bp polymorphisms. PCR products were separated on a capillary electrophoresis. The MMP1-2G and the MMP3-6A allele frequencies were significantly lower in cases than in controls. In particular homozygous 2G/2G individuals were at lower risk of cancer than the 1G/1G carriers (OR= 0.37 95%CI [0.19-0.71], p=0.003). MMP1 and MMP3 polymorphisms were in moderate linkage disequilibrium in cases and controls (D'=0.41 and D'=0.46). Haplotype frequencies distribution derived from these 2 polymorphisms was significantly different between cases and controls (p=0.01). The haplotype analysis suggested an implication of both MMP1 and MMP3 polymorphisms in the head and neck squamous cell carcinoma susceptibility. Indeed, the presence of the MMP1-2G and MMP3-6A alleles seemed to be associated with decreased risk of head and neck squamous cell carcinoma but mainly when they were carried by the same haplotype. By comparison to the 1G-5A haplotype, the 2G-6A haplotype was associated with a lower risk of head and neck squamous cell carcinoma (OR=0.52 95%CI [0.34-0.80], p=0.003).

Myeloperoxidase: new polymorphisms and relation with lung cancer risk.

Myeloperoxidase (MPO) is an haemoprotein that metabolizes tobacco smoke procarcinogens such as benzo(a)pyrene and aromatic amines into highly reactive intermediates. A polymorphism in the MPO promoter (-463G>A), resulting in lower expression, was previously associated with a decrease in lung cancer risk. In this study, the MPO gene was analysed for new polymorphisms by denaturing high-performance liquid chromatography and sequencing on a panel of 48 subjects. Subsequently, the association between newly described polymorphisms, the -463G>A single nucleotide polymorphism (SNP) and lung cancer risk was investigated in a case-control study comprising 245 Caucasian cases and 249 Caucasian controls. Genetic polymorphisms were found in exons 2 (V53F), 6 (M251T), 7 (A332V), 11 (I642L) and 12 (I717V), with allelic frequencies of 6.0%, 1.8%, 1.8%, 0.2% and 1.8%, respectively. A new polymorphism located in the intron 11 3' splice site was found (0.4%). The known -463G>A polymorphism (24.0%) and four new polymorphisms in promoter region were also detected. No correlation between these new polymorphisms and lung cancer risk was found. For the -463G>A polymorphism, no modification of lung cancer risk was observed for either the G/A genotype [odds ratio (OR) 1.19; 95% confidence interval (CI) 0.8-1.8] or the A/A genotype (OR 0.97; 95% CI 0.4-2.6). Linkage analysis showed a strong disequilibrium between -463G>A polymorphism and exonic SNPs. However, the distribution of reconstructed haplotypes did not differ significantly between cases and controls. Further studies need to be performed to investigate the role of these polymorphisms in others diseases.

Delineation and candidate gene mutation screening of the 18q22 minimal region of deletion in head and neck squamous cell carcinoma.

The 18q chromosome arm is frequently lost in advanced head and neck squamous cell carcinoma. Twenty-four microsatellite markers located on chromosome 18q were genotyped in 145 primary tumors and 10 cell lines in order to identify putative tumor suppressor genes implicated in tumor progression. Two different minimal common regions of loss (MCRL) were identified at 18q22 and 18q23 respectively. To refine and delineate boundaries of an homozygous deletion found in one cell line, 44 extra markers located at 18q22 were analysed and the homozygous deletion was precisely defined within a critical region of 4.9 Mb. Four known genes (CDH7, CDH19, DNAM-1, FLJ23594) located in this critical region and two EST clusters (Hs.96900, Hs.98628) were selected for further investigations. For these six genes, genomic structures were established, somatic mutations were screened in 20 HNSCC and 10 cell lines and transcription levels were determined in eight cell lines. No somatic mutations were found in any of the candidate genes analysed (57 coding exons). However, differential transcription levels were observed for CDH19 and Hs.96900 in head and neck cancer cell lines supporting their putative involvement through down regulation mechanisms in head and neck cancer progression.

Long-term results of TPMT activity monitoring in azathioprine-treated renal allograft recipients.

Thiopurine methyltransferase (TPMT) is implicated in the metabolism of azathioprine. The consequences of differential TPMT activity induction by azathioprine on the long-term results after renal transplantation were investigated. The erythrocyte TPMT activity in 82 patients on days 0, 7, and 30 was prospectively evaluated. Because various patterns of TPMT activity variation were noted, the population was subsequently divided between inductors (n = 47) and noninductors (n = 35). Data regarding patient and graft survival and acute rejection episodes were collected. Renal allograft assessment was performed at 3 mo and 2 yr to evaluate the renal function and the histologic lesions on routine biopsies. Data regarding azathioprine-related toxicity also were collected. In a subgroup of patients (n = 19), azathioprine blood levels were determined at day 7 and day 30. The graft survival censoring death was statistically improved in TPMT inductor patients when compared with non-TPMT inductors (P < 0.05). Among TPMT inductors, an acute rejection episode was observed in 34% of the patients versus 69% among non-TPMT inductors (P = 0.002). At 3 mo, serum creatinine was significantly lower among TPMT inductors when compared with non-TPMT inductors (123.1 +/- 7.6 and 161.4 +/- 13.9 micromol/L, respectively; P = 0.01). On routine allograft biopsies at 2 yr (n = 61), grade 2 or 3 chronic lesions were present in 19% versus 25%, respectively (P = NS). At days 7 and 30, the azathioprine blood levels were higher among patients who experienced acute rejection (P < 0.02). TPMT activity induction was observed in 57% of renal transplant recipients who received azathioprine. This induction was associated with better graft outcome. The appropriate conversion from azathioprine, which is a pro-drug, into 6-mercaptopurine could explain both better graft outcome and TPMT induction. Assessing the ability of azathioprine metabolism at an individualized level before transplantation may allow a more accurate choice among the different immunosuppressive treatments.