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1.
Proc Natl Acad Sci U S A ; 119(43): e2205277119, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36252012

RESUMO

Mucins are the main macrocomponents of the mucus layer that protects the digestive tract from pathogens. Fucosylation of mucins increases mucus viscoelasticity and its resistance to shear stress. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here, we show that levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients. Recapitulating this FUT8 overexpression in mucin-producing HT29-18N2 colonic cell line increases delivery of MUC1 to the plasma membrane and extracellular release of MUC2 and MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to removal from the cell surface than mucins secreted by FUT8-depleted cells (FUT8 KD). FUT8 KD causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. These data fit well with the Fut8-/- mice phenotype, which are protected from UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins. Together, our data reveal that FUT8 modifies the biophysical properties of mucus by controlling levels of cell surface MUC1 and quantity and quality of secreted MUC2 and MUC5AC. We suggest that these changes in mucus viscoelasticity likely facilitate bacterial-epithelial interactions leading to inflammation and UC progression.


Assuntos
Colite Ulcerativa , Fucosiltransferases , Animais , Camundongos , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Fucosiltransferases/genética , Inflamação , Mucina-2/genética , Mucina-2/metabolismo , Células HT29
2.
J Biol Chem ; 294(3): 816-826, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30482841

RESUMO

Regulated mucin secretion is essential for the formation of the mucus layer that protects the underlying epithelial cells from foreign particles. Alterations in the quantity or quality of secreted mucins are therefore detrimental to airway and colon physiology. Based on various biochemical assays in several human cell lines, we report here that Na+/Ca2+ exchanger 2 (NCX2) works in conjunction with transient receptor potential cation channel subfamily M member 4 (TRPM4), and perhaps TRPM5, Na+ channels to control Ca2+-mediated secretion of both mucin 2 (MUC2) and MUC5AC from HT29-18N2 colonic cancer cells. Differentiated normal bronchial epithelial (NHBE) cells and tracheal cells from patients with cystic fibrosis (CFT1-LC3) expressed only TRPM4 and all three isoforms of NCXs. Blocking the activity of TRPM4 or NCX proteins abrogated MUC5AC secretion from NHBE and CFT1-LC3 cells. Altogether, our findings reveal that NCX and TRPM4/TRPM5 are both required for mucin secretion. We therefore propose that these two proteins could be potential pharmacological targets to control mucus-related pathologies such as cystic fibrosis.


Assuntos
Cálcio/metabolismo , Células Caliciformes/metabolismo , Mucina-5AC/metabolismo , Mucina-2/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Canais de Cátion TRPM/metabolismo , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Células Caliciformes/patologia , Humanos , Mucina-5AC/genética , Mucina-2/genética , Trocador de Sódio e Cálcio/genética , Canais de Cátion TRPM/genética
3.
Elife ; 122023 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-36961129

RESUMO

We show that TANGO2 in mammalian cells localizes predominantly to mitochondria and partially at mitochondria sites juxtaposed to lipid droplets (LDs) and the endoplasmic reticulum. HepG2 cells and fibroblasts of patients lacking TANGO2 exhibit enlarged LDs. Quantitative lipidomics revealed a marked increase in lysophosphatidic acid (LPA) and a concomitant decrease in its biosynthetic precursor phosphatidic acid (PA). These changes were exacerbated in nutrient-starved cells. Based on our data, we suggest that TANGO2 function is linked to acyl-CoA metabolism, which is necessary for the acylation of LPA to generate PA. The defect in acyl-CoA availability impacts the metabolism of many other fatty acids, generates high levels of reactive oxygen species, and promotes lipid peroxidation. We suggest that the increased size of LDs is a combination of enrichment in peroxidized lipids and a defect in their catabolism. Our findings help explain the physiological consequence of mutations in TANGO2 that induce acute metabolic crises, including rhabdomyolysis, cardiomyopathy, and cardiac arrhythmias, often leading to fatality upon starvation and stress.


Assuntos
Ácidos Graxos , Metabolismo dos Lipídeos , Animais , Humanos , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Fibroblastos/metabolismo , Homeostase , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Mamíferos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte Vesicular/metabolismo
4.
Nat Commun ; 14(1): 3710, 2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37349283

RESUMO

Agonist-mediated stimulated pathway of mucin and insulin release are biphasic in which rapid fusion of pre-docked granules is followed by slow docking and fusion of granules from the reserve pool. Here, based on a cell-culture system, we show that plasma membrane-located tetraspanin-8 sequesters syntaxin-2 to control mucin release. Tetraspanin-8 affects fusion of granules during the second phase of stimulated mucin release. The tetraspanin-8/syntaxin-2 complex does not contain VAMP-8, which functions with syntaxin-2 to mediate granule fusion. We suggest that by sequestering syntaxin-2, tetraspanin-8 prevents docking of granules from the reserve pool. In the absence of tetraspanin-8, more syntaxin-2 is available for docking and fusion of granules and thus doubles the quantities of mucins secreted. This principle also applies to insulin release and we suggest a cell type specific Tetraspanin/Syntaxin combination is a general mechanism regulating the fusion of dense core granules.


Assuntos
Ilhotas Pancreáticas , Sintaxina 1/metabolismo , Ilhotas Pancreáticas/metabolismo , Secreção de Insulina , Exocitose/fisiologia , Insulina/metabolismo , Mucinas/metabolismo , Grânulos Citoplasmáticos/metabolismo
5.
J Neurosci ; 31(5): 1885-94, 2011 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-21289198

RESUMO

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.


Assuntos
Álcalis/farmacologia , Córtex Cerebral/metabolismo , Fibroblastos/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neurônios/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Amiodarona/farmacologia , Animais , Animais Recém-Nascidos , Proteína 5 Relacionada à Autofagia , Bepridil/farmacologia , Northern Blotting , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Cloroquina/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/efeitos dos fármacos , Degeneração Lobar Frontotemporal/tratamento farmacológico , Degeneração Lobar Frontotemporal/genética , Granulinas , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Macrolídeos/farmacologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Mutação , Neurônios/efeitos dos fármacos , Progranulinas , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiazóis/farmacologia
6.
Ann Neurol ; 65(5): 603-9, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19288468

RESUMO

OBJECTIVE: Mutations that lead to a loss of progranulin (PGRN) explain a considerable portion of the occurrence of frontotemporal lobar degeneration. We tested a biomarker allowing rapid detection of a loss of PGRN. METHODS: We used an enzyme-linked immunosorbent assay to measure in serum the PGRN protein levels of six affected and eight unaffected carriers from within an extended Belgian founder family segregating the null mutation IVS1+5G>C. Further, we measured serum PGRN levels in 2 patients with another null mutation (a Met1 and a frameshift mutation), in 4 patients carrying a predicted pathogenic missense mutation and in 5 patients carrying a benign missense polymorphism, in 9 unaffected noncarrier relatives, and in 22 community controls. RESULTS: Serum PGRN levels were reduced in both affected and unaffected null mutation carriers compared with noncarrier relatives (p(exact) < 0.0001), and allowed perfect discrimination between carriers and noncarriers (sensitivity: 1.0; 1 - specificity: 0.0). Serum PGRN levels in Cys139Arg and Arg564Cys mutation carriers were significantly lower than in controls, but greater than in null mutation carriers, fitting the hypothesis of partial loss of function caused by these missense mutations. As expected, levels for carriers of benign missense polymorphisms were not significantly different from controls. INTERPRETATION: Our results indicate that the serum PGRN level is a reliable biomarker for diagnosing and early detection of frontotemporal lobar degeneration caused by PGRN null mutations, and provided the first in vivo evidence that at least some missense mutations in PGRN may lead to a (partial) loss of PGRN.


Assuntos
Biomarcadores/sangue , Demência/sangue , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Idoso , Idoso de 80 Anos ou mais , Arginina/genética , Cisteína/genética , Demência/genética , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , Progranulinas
7.
J Cell Biol ; 219(4)2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32328640

RESUMO

Nutrient deprivation triggers the release of signal-sequence-lacking Acb1 and the antioxidant superoxide dismutase 1 (SOD1). We now report that secreted SOD1 is functionally active and accompanied by export of other antioxidant enzymes such as thioredoxins (Trx1 and Trx2) and peroxiredoxin Ahp1 in a Grh1-dependent manner. Our data reveal that starvation leads to production of nontoxic levels of reactive oxygen species (ROS). Treatment of cells with N-acetylcysteine (NAC), which sequesters ROS, prevents antioxidants and Acb1 secretion. Starved cells lacking Grh1 are metabolically active, but defective in their ability to regrow upon return to growth conditions. Treatment with NAC restored the Grh1-dependent effect of starvation on cell growth. In sum, starvation triggers ROS production and cells respond by secreting antioxidants and the lipogenic signaling protein Acb1. We suggest that starvation-specific unconventional secretion of antioxidants and Acb1-like activities maintain cells in a form necessary for growth upon their eventual return to normal conditions.


Assuntos
Antioxidantes/metabolismo , Proteínas de Transporte/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo
8.
Hum Mutat ; 30(4): E570-4, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19191332

RESUMO

A non-synonymous polymorphism, rs2986017 (p.P86L), in the newly characterized calcium homeostasis modulator 1 (CALHM1) gene located in the Alzheimer dementia (AD) linkage region on 10q24.33, was reported to increase risk of AD, and affect calcium homeostasis and amyloid beta accumulation. We aimed to investigate the association between this functional polymorphism and AD in an independent study population. We genotyped rs2986017 in 362 Belgian AD patients and 519 ethnically matched control individuals. We found no evidence of association between rs2986017 and risk of disease, nor did we find an effect on onset age. Despite its functional properties, our study suggests the polymorphism does not contribute significantly to AD risk in the Belgian population.


Assuntos
Doença de Alzheimer/genética , Canais de Cálcio/genética , Glicoproteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico , Bélgica , Demência Vascular/diagnóstico , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Modelos Logísticos , Masculino , Doenças Neurodegenerativas/diagnóstico , Razão de Chances , Fatores de Risco , Análise de Sequência de DNA
9.
Hum Mutat ; 30(2): E338-44, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18853460

RESUMO

GRB-associated binding protein 2 (GAB2) was recently reported to be a modifier of late-onset Alzheimer dementia (AD) risk in carriers of the APOE epsilon4 allele in a genome-wide association analysis. We aimed to investigate this association in a well-characterized Belgian late-onset AD patient/control group: 528 Belgian AD patients (mean onset age 79.0+/-5.2 years, 70.2% females) and 601 ethnically matched control individuals (mean age 61.9+/-15.3 years, 57.1% females) were genotyped for 10 SNPs across the GAB2 locus. For 2 SNPs the most common genotype was associated with risk for AD, with the most significant result for rs4945261 [OR 1.49 (95%CI 1.04-2.15)]. After stratification by presence or absence of APOE epsilon4 these associations were present in APOE epsilon4 carriers only. When assessing the effect of APOE and rs4945261 in one model, rs4945261 did not show a main effect, but the joint risk effect of rs4945261-GG and APOE epsilon4 on AD was significant (OR 3.87, 95%CI 2.66-5.63; p=1.0E-12), with a deviation of 1.87 from the multiplicative model of interaction. Haplotype analyses showed evidence of association in the total (global p(sim) 0.04) and APOE epsilon4+ (global p(sim) 0.02) but not in the APOE epsilon4 - group (global p(sim) 0.6). The association was driven by a higher frequency of the major haplotype in patients. Our data independently replicate an association between GAB2 and late-onset AD, which appears to be limited to APOE epsilon4 carriers.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/genética , Predisposição Genética para Doença , Mutação/genética , Idoso , Apolipoproteína E4/genética , Bélgica , Estudos de Casos e Controles , Feminino , Frequência do Gene , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , População Branca/genética
10.
Hum Mutat ; 30(8): 1207-13, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19462468

RESUMO

Expression levels of the amyloid precursor protein (APP) and beta-site amyloid (Abeta) cleaving enzyme 1 (BACE1) have been implicated in Alzheimer disease (AD) progression. In a well-characterized Belgian group of 358 AD patients and 462 controls, we examined whether genetic variability in microRNA (miRNA) binding sites of APP and BACE1 or in associated miRNAs influenced risk for AD. Direct sequencing identified six variants in the 3' untranslated region (UTR) of APP and 29 variants in the 3' UTR of BACE1, of which few variants were restricted to patients: in APP; 4 variants in 6 patients ( approximately 2%) and in BACE1; 7 variants in 11 patients ( approximately 3.5%). Further genetic screening of the miR-29 cluster encoding the miR-29a/b-1 genes showed 10 variants in close proximity of this cluster. Association studies using all common variants detected in the 3' UTR of BACE1 and the miR-29 gene cluster did not identify an association with AD risk. However, we did observe statistical interaction between rs535860 (BACE1 3' UTR) and rs34772568 (near miR29a; odds ratio [OR](interaction), 0.4; 95% confidence interval [CI], 0.17-0.96; P=0.033). While the exact role of the patient-specific miRNA variants within the 3' UTR region of APP and BACE1 demands further analyses, this study does not support a major contribution of miRNA genetic variability to AD pathogenesis.


Assuntos
Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Predisposição Genética para Doença , Variação Genética , MicroRNAs/genética , Regiões 3' não Traduzidas , Idoso , Sequência de Bases , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
11.
Hum Mutat ; 30(7): 1054-61, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19405094

RESUMO

The relative contribution of simple mutations and copy number variations (CNVs) in SNCA, PARK2, PINK1, PARK7, and LRRK2 to the genetic etiology of Parkinson disease (PD) is still unclear because most studies did not completely analyze each gene. In a large group of Belgian PD patients (N = 310) and control individuals (N = 270), we determined the mutation frequency of both simple mutations and CNVs in these five PD genes, using direct sequencing, multiplex amplicon quantification (MAQ), and real-time PCR assays. Overall, we identified 14 novel heterozygous variants, of which 11 were absent in control individuals. We observed eight PARK2 (multiple) exon multiplications in PD patients and one exon deletion in a control individual. Furthermore, we identified one SNCA whole-gene duplication. The PARK2 and LRRK2 mutation frequencies in Belgian PD patients were similar to those reported in other studies. However, at this stage the true pathogenic nature of some heterozygous mutations in recessive genes remains elusive. Furthermore, though mutations is SNCA, PINK1, and PARK7 are rare, our identification of a SNCA duplication confirmed that screening of these genes remains meaningful.


Assuntos
Dosagem de Genes , Mutação , Doença de Parkinson/genética , Bélgica/epidemiologia , Estudos de Casos e Controles , Análise Mutacional de DNA , Frequência do Gene , Genética Populacional , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Proteínas Oncogênicas/genética , Proteína Desglicase DJ-1 , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , alfa-Sinucleína/genética
12.
Dev Cell ; 49(1): 145-155.e4, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30880003

RESUMO

Signal-sequence-lacking interleukin (IL)-1ß, is cleaved by caspase-1 to mature mIL-1ß, which is secreted, without entering the endoplasmic reticulum. We report that macrophages of GRASP55-/- mice are defective in mIL-1ß secretion and retain it as intracellular aggregates. Intriguingly, GRASP55-/- macrophages are defective in the IRE1α branch of the unfolded protein response. This finding fits well with our data that inhibition of IRE1α also impairs mIL-1ß secretion and causes its accumulation in intracellular aggregates. PERK inhibition, on the other hand, controls caspase-1-mediated conversion of proIL-1ß to mIL-1ß. These findings reveal translation-independent functions of PERK and IRE1α: PERK controls the production of mIL-1ß, which is then followed by GRASP55 and IRE1α activity to keep mIL-1ß in a secretion-competent form.


Assuntos
Endorribonucleases/genética , Proteínas da Matriz do Complexo de Golgi/genética , Interleucina-1beta/genética , Agregados Proteicos/genética , Proteínas Serina-Treonina Quinases/genética , eIF-2 Quinase/genética , Animais , Caspase 1/genética , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/antagonistas & inibidores
13.
J Cell Biol ; 218(11): 3861-3879, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31488582

RESUMO

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathway function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to Golgi membranes. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos
14.
Hum Mutat ; 29(5): 769-70, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18407551

RESUMO

SORL1 has recently been identified as a major genetic contributor to increased risk for late-onset Alzheimer disease (AD). Here we aimed at replicating this finding in a large, well-characterized group of 550 Belgian late-onset AD patients and 637 healthy control individuals using a gene-wide genotyping approach across the SORL1 locus. We observed significant associations, both for individual SNPs (SNPs 6, 8, 9, 10 and 27; p-values ranging from 0.001 to 0.040) and 3-SNP haplotypes (SNPs 5-6-7 and SNPs 25-26-27; p-values ranging from 0.008 to 0.035). Moreover, the associations at SNP 8, 9 and 10 represented a direct replication of the initial association data. Two signals in distinct regions of the gene were shown to be mutually independent, supporting allelic heterogeneity at the SORL1 locus in the Belgian population. Our findings confirm that genetic variants in SORL1 may be important risk factors for late-onset AD.


Assuntos
Idade de Início , Doença de Alzheimer/genética , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Idoso , Alelos , Bélgica , Estudos de Casos e Controles , Feminino , Heterogeneidade Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de Risco
15.
Brain ; 130(Pt 9): 2320-6, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17586559

RESUMO

The CSF biomarkers beta-amyloid peptide (Abeta(1-42)), total tau protein (T-tau) and tau phosphorylated at threonine 181 (P-tau(181P)) were determined in autopsy-confirmed Alzheimer's disease patients in order to study possible associations with the epsilon4 allele of APOE and density and spread of plaques (SP) and tangles (NFT). CSF levels of Abeta(1-42), T-tau and P-tau(181P) were determined in 50 Alzheimer's disease patients using commercially available single parameter ELISA kits (INNOTEST(R)). Genomic DNA was extracted from whole blood and the APOE genotype was determined using standard methods. Tangle burden was assessed by means of Braak's NFT stages (I-VI), whereas the plaque burden was assessed by means of Braak's SP stages (A-C). CSF biomarker levels were not different when comparing epsilon4 carriers (n = 21) and non-carriers (n = 29) (P > 0.05 for all comparisons). No significant correlations between the number of epsilon4 alleles (0, 1 or 2) and CSF levels of Abeta(1-42) (Spearman Rank Order: r = -0.057, P = 0.695), T-tau (r = 0.104, P = 0.472) and P-tau(181P) (r = 0.062, P = 0.668) were found. Braak's SP (Abeta(1-42): r = -0.155, P = 0.280; T-tau: r = -0.044, P = 0.763; P-tau(181P): r = -0.010, P = 0.947) and NFT (Abeta(1-42): r = -0.145, P = 0.315; T-tau: r = 0.117, P = 0.415; P-tau(181P): r = 0.150, P = 0.296) stages were not significantly correlated with CSF biomarker levels. In conclusion, CSF levels of Abeta(1-42), T-tau and P-tau(181P) were not associated with epsilon4, tangle or plaque burden in 50 autopsy-confirmed Alzheimer's disease patients. In the light of future biomarker applications like monitoring of disease progression and as allocortical neuropathological changes significantly contribute to clinical symptoms, the concept of in vivo surrogate biomarkers should be further explored.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Apolipoproteína E4/genética , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Biomarcadores/líquido cefalorraquidiano , Encéfalo/patologia , Progressão da Doença , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Índice de Gravidade de Doença
16.
Elife ; 72018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30272559

RESUMO

Regulated mucin secretion from specialized goblet cells by exogenous agonist-dependent (stimulated) and -independent (baseline) manner is essential for the function of the epithelial lining. Over extended periods, baseline release of mucin can exceed quantities released by stimulated secretion, yet its regulation remains poorly characterized. We have discovered that ryanodine receptor-dependent intracellular Ca2+ oscillations effect the dissociation of the Ca2+-binding protein, KChIP3, encoded by KCNIP3 gene, from mature mucin-filled secretory granules, allowing for their exocytosis. Increased Ca2+ oscillations, or depleting KChIP3, lead to mucin hypersecretion in a human differentiated colonic cell line, an effect reproduced in the colon of Kcnip3-/- mice. Conversely, overexpressing KChIP3 or abrogating its Ca2+-sensing ability, increases KChIP3 association with granules, and inhibits baseline secretion. KChIP3 therefore emerges as the high-affinity Ca2+ sensor that negatively regulates baseline mucin secretion. We suggest KChIP3 marks mature, primed mucin granules, and functions as a Ca2+ oscillation-dependent brake to control baseline secretion. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Colo/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Mucina-5AC/metabolismo , Animais , Células Caliciformes/metabolismo , Células HEK293 , Células HT29 , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Mucina-5AC/genética , Vesículas Secretórias/metabolismo
17.
Hum Mutat ; 28(4): 416, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17345602

RESUMO

Null mutations in the progranulin gene (GRN, PGRN) were recently identified as the causal mechanism underlying frontotemporal dementia (FTD) with ubiquitin-positive brain pathology linked to chromosome 17 (FTDU-17). In a Belgian and French FTD series comprising 332 patients, we reported 13 PGRN null mutations which were mainly nonsense and frameshift mutations resulting in premature stop codons. Here we report in the same patient series three missense mutations of which two (c.743C>T, p.Pro248Leu and c.1294C>T, p.Arg432Cys) were predicted in silico to severely affect protein folding and/or processing leading to PGRN protein haploinsufficiency. In addition, we observed three sequence variations in the 5' regulatory region that might potentially affect PGRN transcription activity. Our findings extend the mutation spectrum in PGRN leading to loss of functional PGRN as the basis for FTD.


Assuntos
Demência/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mutação de Sentido Incorreto , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Estudos de Casos e Controles , Sequência Conservada , Análise Mutacional de DNA , Demência/metabolismo , Demência/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Dados de Sequência Molecular , Progranulinas , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína
18.
Arch Neurol ; 64(10): 1436-46, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17923627

RESUMO

BACKGROUND: Progranulin gene (PGRN) haploinsufficiency was recently associated with ubiquitin-positive frontotemporal lobar degeneration linked to chromosome 17q21 (FTLDU-17). OBJECTIVE: To assess whether PGRN genetic variability contributed to other common neurodegenerative brain diseases, such as Alzheimer disease (AD) or Parkinson disease (PD). DESIGN: Mutation analysis of PGRN. SETTING: Memory Clinic of the Middelheim General Hospital. Patients We analyzed 666 Belgian patients with AD and 255 with PD. MAIN OUTCOME MEASURES: Results of PGRN sequencing, PGRN transcript analysis, short tandem repeat genotyping, and neuropathologic analysis. RESULTS: We identified 2 patients with AD and 1 patient with PD who carried the null mutation IVS0 + 5G>C, which we reported earlier in an extensively characterized Belgian founder family, DR8, segregating FTLDU. Postmortem pathologic diagnosis of the patient with PD revealed both FTLDU and Lewy body pathologic features. In addition, we identified in PGRN only 1 other null mutation, the nonsense mutation p.Arg535X, in 1 patient with probable AD. However, in vitro analysis predicted a PGRN C-truncated protein, although it remains to be elucidated if this shortened transcript leads to haploinsufficiency. CONCLUSIONS: Our mutation data indicated that null mutations are rare in patients with AD (3/666 = 0.45%) and PD (1/255 = 0.39%). Also, AD and PD clinical diagnoses in patients who carry PGRN null mutations likely result from etiologic heterogeneity rather than PGRN haploinsufficiency.


Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Doença de Parkinson/diagnóstico , Doença de Parkinson/genética , Fatores Etários , Idoso , Doença de Alzheimer/epidemiologia , Autopsia , Bélgica/epidemiologia , Encéfalo/patologia , Cromossomos Humanos Par 17/genética , Códon sem Sentido/genética , Análise Mutacional de DNA , Feminino , Efeito Fundador , Variação Genética , Genótipo , Heterozigoto , Humanos , Imuno-Histoquímica , Doença por Corpos de Lewy/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Doença de Parkinson/epidemiologia , Linhagem , Progranulinas , Sequências de Repetição em Tandem/genética , Ubiquitina/metabolismo
19.
Brain ; 129(Pt 11): 2977-83, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16921174

RESUMO

We assessed the impact of amyloid precursor protein (APP) gene locus duplications in early onset Alzheimer's disease in a Dutch population-based sample. Using real-time PCR and an in-house-developed multiplex amplicon quantification assay, we identified a genomic APP duplication in 1 out of 10 multigenerational families segregating early onset Alzheimer's disease. In this family, cerebral amyloid angiopathy (CAA) coincided with this disease. The duplicated genomic region included no other genes than APP and extended maximally over 0.7 Mb. In a sample of 65 familial early onset patients, we observed the same APP genomic duplication in one patient (1.7%), while in 36 isolated patients duplications in the APP locus were absent. This indicated that APP locus duplications explained <2% of familial, non-autosomal dominant Alzheimer's disease and are an infrequent cause of de novo mutation. Our findings corroborated a recent French study, and indicated that investigating genomic duplications in the APP locus in families segregating Alzheimer's disease and CAA should be considered.


Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Angiopatia Amiloide Cerebral/genética , Duplicação Gênica , Adulto , Idade de Início , Idoso , Doença de Alzheimer/complicações , Angiopatia Amiloide Cerebral/complicações , Feminino , Genes Dominantes , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Linhagem , Reação em Cadeia da Polimerase/métodos
20.
Brain ; 129(Pt 11): 2984-91, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16931535

RESUMO

It is well established that Alzheimer's disease causing mutations in APP, PSEN1 and PSEN2 lead to a relative increased production of Abeta42, thereby fostering its deposition in plaques. Recently others and we showed that amyloid precursor protein (APP) overproduction, either as a result of genomic locus duplication or altered regulatory sequences in the APP promoter region, leads to early-onset disease. Here, we have expanded our study of genetic variability in the APP promoter to a large group of well-documented Belgian patients (n = 750, mean onset age = 75.0 +/- 8.6, range = 37-96). We identified three different APP promoter mutations (-369C-->G, -534G-->A and -479C-->T) in seven patients. In patients with onset < or =70 years (n = 204), we identified one patient carrying the London APP V717I mutation while no patients carried an APP locus duplication, indicating that APP promoter mutations (n = 2) were more frequently associated with increased risk for early-onset Alzheimer's disease. The two mutations (-369C-->G and -534G-->A) increasing APP promoter activity by nearly 2-fold and mimicking an APP duplication, appeared in probands of families with multiple patients with dementia. The -479C-->T mutation that increased APP expression only mildly (1.2-fold), was observed in four patients with onset ages ranging from 62 to 79 years (mean 71.5 years), suggesting that its contribution to disease risk is more pronounced at later age due to modulating factors. In conclusion, we provided evidence that mutations in APP regulatory sequences are more frequent than APP coding mutations, and that increased APP transcriptional activity constitutes a risk factor for Alzheimer's disease with onset ages inversely correlated with levels of APP expression.


Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Variação Genética , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Regiões Promotoras Genéticas , Fatores de Risco , Transcrição Gênica
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