-
[show abstract]
[hide abstract]
ABSTRACT: In the respiratory system, Na+ absorption and Cl- secretion are balanced in order to maintain an appropriate airway surface fluid (ASF) volume and ensure an efficient mucociliary clearance. In cystic fibrosis (CF), this equilibrium is disrupted by mutations in the CFTR gene resulting in the absence of functional CFTR-dependent Cl- secretion. The consequences of defective Cl- transport are worsened by the persistence of Na+ absorption which contributes to airway surface dehydration. We asked whether a normal ASF can be restored equally by recovering Cl- secretion from mutated CFTR or by reducing Na+ absorption. This is highly relevant in the selection of the best strategy for the treatment of CF patients. We have analysed the ASF thickness of primary cultured bronchial CF and non-CF epithelia after silencing the epithelial Na+ channel (ENaC) with specific siRNAs and after pharmacological stimulation of CFTR. Our results indicate that i) single siRNAs complementary to ENaC subunits are enough to reduce ENaC transcripts, Na+ channel activity and fluid transport but only silencing both alpha and beta ENaC subunits at the same time leads to an increase of the ASF (from nearly 7 µm to more than 9 µm); ii) ASF thickness obtained in this way is about half that measured after maximal CFTR stimulation on non-CF epithelia (10-14 µm); iii) pharmacological rescue of mutant CFTR increases ASF to the same extent as ENaC silencing. Our results indicate that CFTR rescue and ENaC silencing both produce a significant and long-lasting increase of the airway hydration in vitro.
American Journal of Respiratory Cell and Molecular Biology 04/2013; · 5.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: TMEM16A and TMEM16B proteins are Ca2+ activated Cl- channels (CaCCs) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a ten-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimeric proteins in which different domains of the TMEM16A protein were replaced by equivalent domains of TMEM16B. Replacement of the N-terminus, transmenbrane domains 1 and 2 (TMD1-2), the first intracellular loop, and TMD3-4 did not change channel properties. Instead, replacement of the intracellular loop 3, decreased the apparent Ca2+ affinity by nearly eight-fold with respect to wild type TMEM16A. In contrast, the membrane currents derived from chimeras having the TMD7-8 or the carboxy-terminus of TMEM16B showed faster activation and deactivation rates without a change in Ca2+-sensitivity. Significantly accelerated kinetics were also found when the entire carboxy-terminus of the TMEM16A protein (77 amino acid residues) was deleted. Our findings indicate that the third intracellular loop of TMEM16A and TMEM16B protein is the site involved in Ca2+-sensitivity, whereas the carboxy-terminal part, including transmembrane domains 7 and 8, affect the rate of transition between the open and the closed state.
Biochemical Journal 04/2013; · 4.90 Impact Factor
-
Marta Rusmini,
Paola Griseri,
Francesca Lantieri,
Ivana Matera,
Kelly L Hudspeth,
Alessandra Roberto,
Joanna Mikulak,
Stefano Avanzini,
Valentina Rossi,
Girolamo Mattioli,
Vincenzo Jasonni, Roberto Ravazzolo,
William J Pavan,
Alessio Pini-Prato,
Isabella Ceccherini,
Domenico Mavilio
[show abstract]
[hide abstract]
ABSTRACT: Hirschsprung disease (HSCR) is a rare congenital anomaly characterized by the absence of enteric ganglia in the distal intestinal tract. While classified as a multigenic disorder, the altered function of the RET tyrosine kinase receptor is responsible for the majority of the pathogenesis of HSCR. Recent evidence demonstrate a strong association between RET and the homeostasis of immune system. Here, we utilize a unique cohort of fifty HSCR patients to fully characterize the expression of RET receptor on both innate (monocytes and Natural Killer lymphocytes) and adaptive (B and T lymphocytes) human peripheral blood mononuclear cells (PBMCs) and to explore the role of RET signaling in the immune system. We show that the increased expression of RET receptor on immune cell subsets from HSCR individuals correlates with the presence of loss-of-function RET mutations. Moreover, we demonstrate that the engagement of RET on PBMCs induces the modulation of several inflammatory genes. In particular, RET stimulation with glial-cell line derived neurotrophic factor family (GDNF) and glycosyl-phosphatidylinositol membrane anchored co-receptor a1 (GFRa1) trigger the up-modulation of genes encoding either for chemokines (CCL20, CCL2, CCL3, CCL4, CCL7, CXCL1) and cytokines (IL-1b, IL-6 and IL-8) and the down-regulation of chemokine/cytokine receptors (CCR2 and IL8-Ra). Although at different levels, the modulation of these ''RET-dependent genes'' occurs in both healthy donors and HSCR patients. We also describe another set of genes that, independently from RET stimulation, are differently regulated in healthy donors versus HSCR patients. Among these ''RET-independent genes'', there are CSF-1R, IL1-R1, IL1-R2 and TGFb-1, whose levels of transcripts were lower in HSCR patients compared to healthy donors, thus suggesting aberrancies of inflammatory responses at mucosal level. Overall our results demonstrate that immune system actively participates in the physiopathology of HSCR disease by modulating inflammatory programs that are either dependent or independent from RET signaling. This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
PLoS ONE 03/2013; 8(3):e59066. · 4.09 Impact Factor
-
Ivana Matera,
Marco Musso,
Paola Griseri,
Marta Rusmini,
Marco Di Duca,
Man-Ting So,
Domenico Mavilio,
Xiaoping Miao,
Paul Hk Tam, Roberto Ravazzolo,
Isabella Ceccherini,
Merce Garcia-Barcelo
[show abstract]
[hide abstract]
ABSTRACT: RET common variants are associated with Hirschsprung disease (HSCR; colon aganglionosis), a congenital defect of the enteric nervous system. We analysed a well-known HSCR-associated RET haplotype that encompasses linked alleles in coding and non-coding/regulatory sequences. This risk-haplotype correlates with reduced level of RET expression when compared to the wild-type counterpart. As allele-specific expression (ASE) contributes to phenotypic variability in health and disease, we investigated whether RET ASE could contribute to the overall reduction of RET mRNA detected in carriers. We tested heterozygous neuroblastoma cell-lines, ganglionic gut tissues (18 HSCR and 14 non-HSCR individuals) and peripheral blood mononuclear cells (PBMCs; 16 HSCR and 14 non-HSCR individuals). Analysis of the data generated by SNaPshot and Pyrosequencing revealed that the RET risk-haplotype is significantly more expressed in gut than in PBMCs (p = 0.0045). No ASE difference was detected between patients and controls, irrespective of the sample type. Comparison of total RET expression levels between gut samples with and without ASE, correlated reduced RET expression with preferential transcription from the RET risk-haplotype. Non-random RET ASE occurs in ganglionic gut regardless of the disease status. RET ASE should not be excluded as a disease mechanism acting during development.
Human Mutation 02/2013; · 5.69 Impact Factor
-
Marta Rusmini,
Paola Griseri,
Francesca Lantieri,
Ivana Matera,
Kelly L Hudspeth,
Alessandra Roberto,
Joanna Mikulak,
Stefano Avanzini,
Valentina Rossi,
Girolamo Mattioli,
Vincenzo Jasonni, Roberto Ravazzolo,
William J Pavan,
Alessio Pini-Prato,
Isabella Ceccherini,
Domenico Mavilio
PLoS ONE 01/2013; 8(4). · 4.09 Impact Factor
-
Tiziana Bachetti,
Sabrina Chiesa,
Patrizio Castagnola,
Daniele Bani,
Eleonora Di Zanni,
Alessia Omenetti,
Andrea D'Osualdo,
Alessandro Fraldi,
Andrea Ballabio, Roberto Ravazzolo,
Alberto Martini,
Marco Gattorno,
Isabella Ceccherini
[show abstract]
[hide abstract]
ABSTRACT: OBJECTIVES: Tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is caused by TNFRSF1A mutations, known to induce intracellular retention of the TNFα receptor 1 (TNFR1) protein, defective TNFα-induced apoptosis, and production of reactive oxygen species. As downregulation of autophagy, the main cellular pathway involved in insoluble aggregate elimination, has been observed to increase the inflammatory response, we investigated whether it plays a role in TRAPS pathogenesis. METHODS: The possible link between TNFRSF1A mutations and inflammation in TRAPS was studied in HEK-293T cells, transfected with expression constructs for wild-type and mutant TNFR1 proteins, and in monocytes derived from patients with TRAPS, by investigating autophagy function, NF-κB activation and interleukin (IL)-1β secretion. RESULTS: We found that autophagy is responsible for clearance of wild-type TNFR1, but when TNFR1 is mutated, the autophagy process is defective, probably accounting for mutant TNFR1 accumulation as well as TRAPS-associated induction of NF-κB activity and excessive IL-1β secretion, leading to chronic inflammation. Autophagy inhibition due to TNFR1 mutant proteins can be reversed, as demonstrated by the effects of the antibiotic geldanamycin, which was found to rescue the membrane localisation of mutant TNFR1 proteins, reduce their accumulation and counteract the increased inflammation by decreasing IL-1β secretion. CONCLUSIONS: Autophagy appears to be an important mechanism in the pathogenesis of TRAPS, an observation that provides a rationale for the most effective therapy in this autoinflammatory disorder. Our findings also suggest that autophagy could be proposed as a novel therapeutic target for TRAPS and possibly other similar diseases.
Annals of the rheumatic diseases 10/2012; · 8.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: : The Poland anomaly (PA) comprises unilateral absence or hypoplasia of the pectoralis major muscle and a variable degree of ipsilateral hand and upper limb anomalies. Various hand and upper limb anomalies classifications in PA have been previously published. In this work, a new classification of hand and upper limb anomalies in PA is proposed, on the basis of the clinical and instrumental evaluation of 175 patients.
: The patients have been followed by a multidisciplinary approach, consisting in orthopaedic, surgical, and genetic evaluation and chest, upper limb, and ultrasound examination of major and minor pectoralis muscles, heart, and kidney.
: Hand and upper limb anomalies were classified in 8 groups on the basis of the clinical degree of severity and on the basis of the presence of coexisting associated anomalies. Data regarding the sex and laterality, previously reported in the medical literature, were confirmed by our analysis. Etiopathogenetic mechanisms leading to the anomaly are discussed.
: The proposed classification is derived from the observation of the widest group of patients described in the medical literature. Our proposal could help in the management of patients affected by Poland syndrome and in understanding etiological and pathologic aspects of the disease.
: IV.
Journal of pediatric orthopedics 10/2012; 32(7):727-31. · 1.23 Impact Factor
-
Paolo Scudieri,
Emanuela Caci,
Silvia Bruno,
Loretta Ferrera,
Marco Schiavon,
Elvira Sondo,
Valeria Tomati,
Ambra Gianotti,
Olga Zegarra-Moran,
Nicoletta Pedemonte,
Federico Rea, Roberto Ravazzolo,
Luis J V Galietta
[show abstract]
[hide abstract]
ABSTRACT: The TMEM16A protein has a potential role as a Ca(2+)-activated Cl(-) channel (CaCC) in airway epithelia where it may be important in the homeostasis of the airway surface fluid. We investigated the function and expression of TMEM16A in primary human bronchial epithelial cells and in a bronchial cell line (CFBE41o-). Under resting conditions, TMEM16A protein expression was relatively low. However, TMEM16A silencing with siRNAs caused a marked inhibition of CaCC activity thus demonstrating that a low TMEM16A expression is sufficient to support Ca(2+)-dependent Cl(-) transport. Following treatment for 24-72 hours with interleukin-4 (IL-4), a cytokine that induces mucous cell metaplasia, TMEM16A protein expression was strongly increased in approximately 50% of primary bronchial epithelial cells, with a specific localization in the apical membrane. IL-4 treatment also increased the percentage of cells expressing MUC5AC, a marker of goblet cells. Interestingly, MUC5AC was detected specifically in cells expressing TMEM16A. In particular, MUC5AC was found in 15% and 60% of TMEM16A-positive cells when epithelia were treated with IL-4 for 24 or 72 hours, respectively. In contrast, ciliated cells showed expression of the CFTR Cl(-) channel but not of TMEM16A. Our results indicate that TMEM16A protein is responsible for CaCC activity in airway epithelial cells, particularly in cells treated with IL-4, and that TMEM16A upregulation by IL-4 appears as an early event of goblet cell differentiation. These findings suggest that TMEM16A expression is particularly required under conditions of mucus hypersecretion to ensure adequate secretion of electrolytes and water.
The Journal of Physiology 09/2012; · 4.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, the only members of group I mGlu receptors, are implicated in synaptic plasticity and mechanisms of feedback control of glutamate release. They exhibit nearly complementary distributions throughout the central nervous system, well evident in the cerebellum, where mGlu1 receptor is most intensely expressed while mGlu5 receptor is not. Despite their different distribution, they show a similar subcellular localization and use common transducing pathways. We recently described the Grm1(crv4) mouse with motor coordination deficits and renal anomalies caused by a spontaneous mutation inactivating the mGlu1 receptor. To define the neuropathological mechanisms in these mice, we evaluated expression and function of the mGlu5 receptor in cerebral and cerebellar cortices. Western blot and immunofluorescence analyses showed mGlu5 receptor overexpression. Quantitative reverse transcriptase-polymerase chain reaction results indicated that the up-regulation is already evident at RNA level. Functional studies confirmed an enhanced glutamate release from cortical cerebral and cerebellar synaptosomes when compared with wild-type that is abolished by the mGlu5 receptor-specific inhibitor, 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP). Finally, acute MPEP treatment of Grm1(crv4/crv4) mice induced an evident although incomplete improvement of motor coordination, suggesting that mGlu5 receptors enhanced activity worsens, instead of improving, the motor-coordination defects in the Grm1(crv4/crv4) mice.
Cerebral Cortex 07/2012; · 6.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Heterozygous mutations of the GFAP gene are responsible for Alexander disease, a neurodegenerative disorder characterized by intracytoplasmic Rosenthal fibers (RFs) in dystrophic astrocytes. In vivo and in vitro models have shown co-localization of mutant GFAP proteins with the small heat shock proteins (sHSPs) HSP27 and alphaB-crystallin, ubiquitin and proteasome components. Results reported by several recent studies agree on ascribing an altered cytoskeletal pattern to mutant GFAP proteins, an effect which induces mutant proteins accumulation, leading to impaired proteasome function and autophagy induction. On the basis of the protective role shown by both these small heat shock proteins (sHSPs), and on the already well established neuroprotective effects of curcumin in several diseases, we have investigated the effects of this compound in an in vitro model of Alexander disease, consisting in U251-MG astrocytoma cells transiently transfected with a construct encoding for GFAP carrying the p.R239C mutation in frame with the reporter green fluorescent protein (GFP). In particular, depending on the dose used, we have observed that curcumin is able to induce both HSP27 and alphaB-crystallin, to reduce expression of both RNA and protein of endogenous GFAP, to induce autophagy and, finally, to rescue the filamentous organization of the GFAP mutant protein, thus suggesting a role of this spice in counteracting the pathogenic effects of GFAP mutations.
Experimental Cell Research 06/2012; 318(15):1844-54. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We propose a method for the characterization of the local intrinsic curvature of adsorbed DNA molecules. It relies on a novel statistical chain descriptor, namely the ensemble averaged product of curvatures for two nanosized segments, symmetrically placed on the contour of atomic force microscopy imaged chains. We demonstrate by theoretical arguments and experimental investigation of representative samples that the fine mapping of the average product along the molecular backbone generates a characteristic pattern of variation that effectively highlights all pairs of DNA tracts with large intrinsic curvature. The centrosymmetric character of the chain descriptor enables targetting strands with unknown orientation. This overcomes a remarkable limitation of the current experimental strategies that estimate curvature maps solely from the trajectories of end-labeled molecules or palindromes. As a consequence our approach paves the way for a reliable, unbiased, label-free comparative analysis of bent duplexes, aimed to detect local conformational changes of physical or biological relevance in large sample numbers. Notably, such an assay is virtually inaccessible to the automated intrinsic curvature computation algorithms proposed so far. We foresee several challenging applications, including the validation of DNA adsorption and bending models by experiments and the discrimination of specimens for genetic screening purposes.
Nucleic Acids Research 03/2012; 40(11):e84. · 8.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The ACVR1/Alk-2 gene, encoding a BMP type I receptor, is mutated in Fibrodysplasia Ossificans Progressiva, a severe form of heterotopic ossification. Regulation of ACVR1/Alk-2 expression, still poorly understood, is likely to be controlled by transcriptional and post-transcriptional mechanisms. In our work, we focused on the functional role of the 3'UTR region of the gene and on microRNAs as possible modulators of the ACVR1/Alk-2 expression.
The ACVR1/Alk-2 3'UTR region consists of a 1.1 kb sequence harboring several putative, well-conserved binding sites for miRNAs in its proximal half, and AU-rich elements in the distal one, as assessed by bioinformatic analysis. The functional role of this region was tested in presence of transcription inhibitors and in transfection experiments in different cell lines, with a ACVR1/Alk-2-3'UTR reporter construct. By this transfection-based approach, we have also verified that three microRNAs, among those predicted to target ACVR1/Alk-2 gene by in silico analysis, can bind its 3'UTR sequence thereby modulating its expression.
In this work we demonstrated that the ACVR1/Alk-2 transcript is unstable in presence of inhibitors of transcription. Functional analysis of the 3'UTR region by Luciferase reporter assays showed that it plays an inhibitory role on ACVR1/Alk-2 gene expression. Moreover, we found that specific miRNAs are involved in modulating ACVR1/Alk-2 gene expression as suggested by binding sites prediction in its 3'UTR sequence. In particular, we found that mir148b and mir365 were able to down-regulate ACVR1/Alk-2 expression, whereas mir26a showed a positive effect on its mRNA. Our data contribute to elucidate some of the mechanisms intervening in the modulation of ACVR1/Alk-2 expression. Considering that no specific and effective treatment of FOP is available, clarifying the basic mechanisms of the ACVR1/Alk-2 gene biology may provide means to develop innovative therapeutics approaches.
PLoS ONE 01/2012; 7(12):e50958. · 4.09 Impact Factor
-
Anwar Baban,
Michele Torre,
Sara Costanzo,
Stefania Gimelli,
Sebastiano Bianca,
Maria Teresa Divizia,
Filippo Maria Sénès,
Livia Garavelli,
Francesca Rivieri,
Margherita Lerone,
Maura Valle, Roberto Ravazzolo,
Maria Grazia Calevo
[show abstract]
[hide abstract]
ABSTRACT: Poland anomaly (PA) is a pectoral muscle hypoplasia/aplasia variably associated with ipsilateral thoracic (TA) and/or upper limb anomalies (ULA). PA is usually sporadic and sometimes familial, making recurrence risk an issue in genetic counseling. Multidisciplinary evaluation of 240 PA patients was carried out, including physical examination of patients and their parents in 190 PA (subjects of the study). Familial conditions were classified into three groups. Group1: true familial PA (F-PA): pectoral muscle defects with familial recurrence: 8(4.2%). Group2: familial Poland-like anomaly families (F-PLA): PA index case and ≥1 relative(s) showing normal pectoral muscles but ULA and/or TA common in PA: 16(8.4%). Group3: sporadic PA (S-PA): 166(87.4%). F-PA indicated a stronger male (87.5%) and left side (62.5%) prevalence, but fewer ULA (37.5%) compared to the other two groups. Maternal transmission (6/8) was more common in F-PA. Statistical significance was not reached due to the small number of F-PA and F-PLA. Karyotyping and array-comparative genomic hybridization were performed in 13 families. Three maternally inherited copy number variants were identified in three patients: 1p31.1 deletion, Xp11.22 duplication, and 16q23.1 duplication. Interestingly, the proband's mother carrying the 16q23.1 duplication displayed moderate breast and areola asymmetry, but normal pectoral muscles on ultrasound. Though there is no recent review discussing recurrence of PA, we reviewed 31 published PA families. On the basis of our study and previous reports, familial PA is not uncommon. Nonetheless, no information can be derived either regarding a molecular basis or clinical tools with which to identify cases with recurrence risk. © 2011 Wiley Periodicals, Inc.
American Journal of Medical Genetics Part A 11/2011; · 2.39 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Heterozygous in frame duplications of the PHOX2B gene, leading to polyalanine (polyAla) expansions ranging from +5 to +13 residues of a 20-alanine stretch, have been identified in the vast majority of patients affected with Congenital Central Hypoventilation Syndrome (CCHS), a rare neurocristopathy characterized by absence of adequate autonomic control of respiration with decreased sensitivity to hypoxia and hypercapnia. Ventilatory supports such as tracheostomy, nasal mask or diaphragm pacing represent the only options available for affected. We have already shown that the severity of the CCHS phenotype correlates with the length of polyAla expansions, ultimately leading to formation of toxic intracytoplasmic aggregates and impaired PHOX2B mediated transactivation of target gene promoters, such as DBH. At present, there is no specific treatment to reduce cell aggregates and to ameliorate patients' respiration. In this work, we have undertaken in vitro analyses aimed at assessing the effects of molecules on the cellular response to polyAla PHOX2B aggregates. In particular, we tested 17-AAG, ibuprofen, 4-PBA, curcumin, trehalose, congo red and chrysamine G for their ability to i) recover the nuclear localisation of polyAla expanded PHOX2B, ii) rescue of PHOX2B mediated transactivation of the DBH promoter, and iii) clearance of PHOX2B (+13 Ala) aggregates. Our data have suggested that 17-AAG and curcumin are effective in vitro in both rescuing the nuclear localization and transactivation activity of PHOX2B carrying the largest expansion of polyAla and promoting the clearance of aggregates of these mutant proteins inducing molecular mechanisms such as ubiquitin-proteasome (UPS), autophagy and heat shock protein (HSP) systems.
Neurobiology of Disease 09/2011; 45(1):508-18. · 5.40 Impact Factor
-
Sébastien Jacquemont,
Alexandre Reymond,
Flore Zufferey,
Louise Harewood,
Robin G Walters,
Zoltán Kutalik,
Danielle Martinet,
Yiping Shen,
Armand Valsesia,
Noam D Beckmann, [......],
Joris Andrieux,
Xavier Estivill,
James F Gusella,
Omar Gustafsson,
Andres Metspalu,
Stephen W Scherer,
Kari Stefansson,
Alexandra I F Blakemore,
Jacques S Beckmann,
Philippe Froguel
[show abstract]
[hide abstract]
ABSTRACT: Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ≤ 18.5 kg per m(2) in adults and ≤ -2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ∼600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance.
Nature 08/2011; 478(7367):97-102. · 36.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca(2+)-activated Cl(-) channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A(0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A(0) expression is associated with Ca(2+)-activated Cl(-) channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16(abc) (total length: 982 amino acids), TMEM16A(0) completely lacks voltage-dependent activation. Furthermore, TMEM16A(0) and TMEM16A(abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A(0) has the basic domains required for anion transport and Ca(2+)-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.
Biochimica et Biophysica Acta 05/2011; 1808(9):2214-23. · 4.66 Impact Factor
-
Min Li,
Silvia Armelloni,
Masami Ikehata,
Alessandro Corbelli,
Marzia Pesaresi,
Novella Calvaresi,
Laura Giardino,
Deborah Mattinzoli,
Francesca Nisticò,
Serena Andreoni,
Aldamaria Puliti, Roberto Ravazzolo,
Gianluigi Forloni,
Piergiorgio Messa,
Maria Pia Rastaldi
[show abstract]
[hide abstract]
ABSTRACT: Nephrin is an immunoglobulin-like adhesion molecule first discovered as a major component of the podocyte slit diaphragm, where its integrity is essential to the function of the glomerular filtration barrier. Outside the kidney, nephrin has been shown in other restricted locations, most notably in the central nervous system (CNS) of embryonic and newborn rodents. With the aim of better characterizing nephrin expression and its role in the CNS of adult rodents, we studied its expression pattern and possible binding partners in CNS tissues and cultured neuronal cells and compared these data to those obtained in control renal tissues and podocyte cell cultures. Our results show that, besides a number of locations already found in embryos and newborns, endogenous nephrin in adult rodent CNS extends to the pons and corpus callosum and is expressed by granule cells and Purkinje cells of the cerebellum, with a characteristic alternating expression pattern. In primary neuronal cells we find nephrin expression close to synaptic proteins and demonstrate that nephrin co-immunoprecipitates with Fyn kinase, glutamate receptors and the scaffolding molecule PSD95, an assembly that is reminiscent of those made by synaptic adhesion molecules. This role seems to be confirmed by our findings of impaired maturation and reduced glutamate exocytosis occurring in Neuro2A cells upon nephrin silencing. Of note, we disclose that the very same nephrin interactions occur in renal glomeruli and cultured podocytes, supporting our hypothesis that podocytes organize and use similar molecular intercellular signalling modules to those used by neuronal cells.
The Journal of Pathology 04/2011; 225(1):118-28. · 6.32 Impact Factor
-
Aldamaria Puliti,
Pia Irene Anna Rossi,
Gianluca Caridi,
Alessandro Corbelli,
Masami Ikehata,
Silvia Armelloni,
Min Li,
Cristina Zennaro,
Valerio Conti,
Carlotta Maria Vaccari,
Michela Cassanello,
Maria Grazia Calevo,
Laura Emionite, Roberto Ravazzolo,
Maria Pia Rastaldi
[show abstract]
[hide abstract]
ABSTRACT: The metabotropic glutamate (mGlu) receptor 1 (GRM1) has been shown to play an important role in neuronal cells by triggering, through calcium release from intracellular stores, various signaling pathways that finally modulate neuron excitability, synaptic plasticity, and mechanisms of feedback regulation of neurotransmitter release. Herein, we show that Grm1 is expressed in glomerular podocytes and that a glomerular phenotype is exhibited by Grm1(crv4) mice carrying a spontaneous recessive inactivating mutation of the gene. Homozygous Grm1(crv4/crv4) and, to a lesser extent, heterozygous mice show albuminuria, podocyte foot process effacement, and reduced levels of nephrin and other proteins known to contribute to the maintenance of podocyte cell structure. Overall, the present data extend the role of mGlu1 receptor to the glomerular filtration barrier. The regulatory action of mGlu1 receptor in dendritic spine morphology and in the control of glutamate release is well acknowledged in neuronal cells. Analogously, we speculate that mGlu1 receptor may regulate foot process morphology and intercellular signaling in the podocyte.
American Journal Of Pathology 03/2011; 178(3):1257-69. · 4.89 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Heterozygous trinucleotide in frame duplications, leading to expansions of variable lengths of a 20-alanine stretch (polyAla), is the most frequent PHOX2B variant associated with congenital central hypoventilation syndrome (CCHS), a rare neurocristopathy characterized by defective response of the autonomic nervous system to hypoxia and hypercapnia. Sequencing analysis has shown that the vast majority of polyAla expansions arise de novo; while in about 10% of cases, mutations are inherited by one parent who carries either constitutive or somatic mutations. To investigate transmission of PHOX2B mutant alleles from asymptomatic individuals, we have reassessed 44 parental pairs, previously resulted not to carry any mutation, by coupling amplification with FAM-tagged primers and capillary electrophoresis. Low levels of somatic mosaicism were shown in five parents previously undetected, thus increasing the inherited occurrence of the disease from 10% to 25% of the cases. Analysis of the technical detection limits has confirmed a power of resolution much higher for the "FAM" protocol than for the "sequencing" method. These observations are going to have relevant implications on how the carrier status of asymptomatic parents should be assessed and on successive genetic counseling to CCHS families.
Journal of Molecular Medicine 02/2011; 89(5):505-13. · 4.67 Impact Factor
-
Sergio Crovella,
Ludovica Segat,
Annalisa Amato,
Emmanouil Athanasakis,
Valentino Bezzerri,
Cesare Braggion,
Rosaria Casciaro,
Giuseppe Castaldo,
Carla Colombo,
Angela Elvira Covone, [......],
Francesca Pardo,
Serena Quattrucci,
Valeria Raia, Roberto Ravazzolo,
Manuela Seia,
Valentino Stanzial,
Lisa Termini,
Laura Zazzeron,
Giulio Cabrini,
Paolo Gasparini
[show abstract]
[hide abstract]
ABSTRACT: The identification of cystic fibrosis (CF) patients who are at greater risk of lung damage could be clinically valuable. Thus, we attempted to replicate previous findings and verify the possible association between three single nucleotide polymorphisms (SNPs c.-52G>A, c.-44C>G and c.-20G>A) in the 5' untranslated region (5' UTR) of the β defensin 1 (DEFB1) gene and the CF pulmonary phenotype.
Genomic DNA from 92 Italian CF patients enrolled in different regional CF centres was extracted from peripheral blood and genotyped for DEFB1 SNPs using TaqMan(®) allele specific probes. In order to avoid genetic confounding causes that can account for CF phenotype variability, all patients were homozygous for the F508del CFTR mutation, and were then classified on the basis of clinical and functional data as mild lung phenotype (Mp, n=50) or severe lung phenotype patients (Sp, n=42).
For the c.-20G>A SNP, the frequency of the A allele, as well as the AA genotype, were significantly more frequent in Mp than in Sp patients, and thus this was associated with a protective effect against severe pulmonary disease (OR=0.48 and 0.28, respectively). The effect of the c.-20G>A A allele is consistent with a recessive model, and the protective effect against Sp is exerted only when it is present in homozygosis. For the other two SNPs, no differences were observed as allelic and genotypic frequency in the two subgroups of CF patients.
Our results, although necessary to be confirmed in larger and multiethnic populations, reinforce DEFB1 as a candidate modifier gene of the CF pulmonary phenotype.
Clinical Chemistry and Laboratory Medicine 11/2010; 49(1):49-54. · 2.15 Impact Factor
