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1.
Front Neurosci ; 14: 578993, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33122994

RESUMO

Alpha-Synuclein (α-Syn) is a central protein in the pathogenesis of synucleinopathies, a group of neurodegenerative disorders including Parkinson's disease (PD). Although its role in neurotransmission is well established, the precise role of this protein in disease pathogenesis is still not fully understood. It is, however, widely regarded to be associated with the misfolding and accumulation of toxic intracellular aggregates. In fact, α-Syn is the most abundant protein component of Lewy bodies and Lewy neurites, which are also characterized by a high lipid content. Lipids, the main constituents of cellular membranes, have been implicated in many aspects of PD-related processes. α-Syn interacts with membrane phospholipids and free fatty acids via its N-terminal domain, and altered lipid-protein complexes might enhance both its binding to synaptic and mitochondrial membranes and its oligomerization. Several studies have highlighted a specific interaction of α-Syn with the phospholipid cardiolipin (CL), a major constituent of mitochondrial membranes. By interacting with CL, α-Syn is able to disrupt mitochondrial membrane integrity, leading to mitochondrial dysfunction. Additionally, externalized CL is able to facilitate the refolding of toxic α-Syn species at the outer mitochondrial membrane. In this review, we discuss how α-Syn/lipid interactions, in particular the α-Syn/CL interaction at the mitochondrial membrane, may affect α-Syn aggregation and mitochondrial dysfunction and may thus represent an important mechanism in the pathogenesis of PD.

2.
Nat Hum Behav ; 2020 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-32989287

RESUMO

Handedness has been extensively studied because of its relationship with language and the over-representation of left-handers in some neurodevelopmental disorders. Using data from the UK Biobank, 23andMe and the International Handedness Consortium, we conducted a genome-wide association meta-analysis of handedness (N = 1,766,671). We found 41 loci associated (P < 5 × 10-8) with left-handedness and 7 associated with ambidexterity. Tissue-enrichment analysis implicated the CNS in the aetiology of handedness. Pathways including regulation of microtubules and brain morphology were also highlighted. We found suggestive positive genetic correlations between left-handedness and neuropsychiatric traits, including schizophrenia and bipolar disorder. Furthermore, the genetic correlation between left-handedness and ambidexterity is low (rG = 0.26), which implies that these traits are largely influenced by different genetic mechanisms. Our findings suggest that handedness is highly polygenic and that the genetic variants that predispose to left-handedness may underlie part of the association with some psychiatric disorders.

3.
Cell Death Discov ; 6: 45, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32550012

RESUMO

The Parkinson's disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a crucial modulator of the autophagy-lysosome pathway, but unclarity exists on the precise mechanics of its role and the direction of this modulation. In particular, LRRK2 is involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and a significant proportion of LRRK2 patients presenting Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to obtain a comprehensive knowledge on LRRK2-associated pathology. Here, we investigated a G2019S-LRRK2 recombinant cell line exhibiting accumulation of endogenous, phosphorylated alpha-synuclein. We found that G2019S-LRRK2 leads to accumulation of LC3 and abnormalities in lysosome morphology and proteolytic activity in a kinase-dependent fashion, but independent from constitutively active Rab10. Notably, LRRK2 inhibition was ineffective upon upstream blockade of autophagosome-lysosome fusion events, highlighting this step as critical for alpha-synuclein clearance.

4.
Dis Model Mech ; 13(7)2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32471864

RESUMO

The identification of genetic variants that predispose individuals to cardiovascular disease and a better understanding of their targets would be highly advantageous. Genome-wide association studies have identified variants that associate with QT-interval length (a measure of myocardial repolarization). Three of the strongest associating variants (single-nucleotide polymorphisms) are located in the putative promotor region of CNOT1, a gene encoding the central CNOT1 subunit of CCR4-NOT: a multifunctional, conserved complex regulating gene expression and mRNA stability and turnover. We isolated the minimum fragment of the CNOT1 promoter containing all three variants from individuals homozygous for the QT risk alleles and demonstrated that the haplotype associating with longer QT interval caused reduced reporter expression in a cardiac cell line, suggesting that reduced CNOT1 expression might contribute to abnormal QT intervals. Systematic siRNA-mediated knockdown of CCR4-NOT components in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) revealed that silencing CNOT1 and other CCR4-NOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, the cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes in vivo (CNOT1/N ot1 and CNOT7/8/Pop2) was either lethal or resulted in dilated cardiomyopathy, reduced contractility or a propensity for arrhythmia. Silencing CNOT2/Not2, CNOT4/ N ot4 and CNOT6/6L/twin also affected cardiac chamber size and contractility. Developmental studies suggested that CNOT1/Not1 and CNOT7/8/Pop2 are required during cardiac remodeling from larval to adult stages. To summarize, we have demonstrated how disease-associated genes identified by GWAS can be investigated by combining human cardiomyocyte cell-based and whole-organism in vivo heart models. Our results also suggest a potential link of CNOT1 and CNOT7/8 to QT alterations and further establish a crucial role of the CCR4-NOT complex in heart development and function.This article has an associated First Person interview with the first author of the paper.

5.
Nat Commun ; 11(1): 2542, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439900

RESUMO

The electrocardiographic PR interval reflects atrioventricular conduction, and is associated with conduction abnormalities, pacemaker implantation, atrial fibrillation (AF), and cardiovascular mortality. Here we report a multi-ancestry (N = 293,051) genome-wide association meta-analysis for the PR interval, discovering 202 loci of which 141 have not previously been reported. Variants at identified loci increase the percentage of heritability explained, from 33.5% to 62.6%. We observe enrichment for cardiac muscle developmental/contractile and cytoskeletal genes, highlighting key regulation processes for atrioventricular conduction. Additionally, 8 loci not previously reported harbor genes underlying inherited arrhythmic syndromes and/or cardiomyopathies suggesting a role for these genes in cardiovascular pathology in the general population. We show that polygenic predisposition to PR interval duration is an endophenotype for cardiovascular disease, including distal conduction disease, AF, and atrioventricular pre-excitation. These findings advance our understanding of the polygenic basis of cardiac conduction, and the genetic relationship between PR interval duration and cardiovascular disease.


Assuntos
Arritmias Cardíacas/genética , Eletrocardiografia , Loci Gênicos/genética , Predisposição Genética para Doença/genética , Arritmias Cardíacas/fisiopatologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/fisiopatologia , Endofenótipos , Feminino , Expressão Gênica , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Herança Multifatorial , Locos de Características Quantitativas/genética
6.
Stem Cell Res ; 41: 101624, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31715428

RESUMO

Mutations in the PRKN gene, encoding parkin, are the most frequent known cause of recessive Parkinson's disease (PD). We report the generation of an induced pluripotent stem cell (iPSC) line of a patient carrying a homozygous deletion of exon 3 in the PRKN gene. Skin fibroblasts were reprogrammed using non-integrating episomal plasmids. The generated cell line (EURACi005-A; iPS-2011) exhibits expression of pluripotency markers, the potential to differentiate into all three germ layers, and a stable karyotype. This iPSC line provides a valuable resource for further research on the pathomechanism and drug testing for PRKN-linked PD.


Assuntos
Técnicas de Cultura de Células/métodos , Linhagem Celular/patologia , Éxons/genética , Células-Tronco Pluripotentes Induzidas/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Deleção de Sequência/genética , Ubiquitina-Proteína Ligases/genética , Sequência de Bases , Feminino , Homozigoto , Humanos , Reprodutibilidade dos Testes
7.
Stem Cell Res ; 41: 101656, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31733438

RESUMO

Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.


Assuntos
Sistemas CRISPR-Cas , Neurônios Dopaminérgicos/metabolismo , Edição de Genes , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/biossíntese , Células-Tronco Pluripotentes Induzidas/metabolismo , Reação em Cadeia da Polimerase , Transgenes , Linhagem Celular , Neurônios Dopaminérgicos/citologia , Proteínas de Fluorescência Verde/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Microscopia de Fluorescência
8.
Sci Rep ; 9(1): 11995, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427613

RESUMO

Familial hypercholesterolemia (FH) is characterised by elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and a substantial risk for cardiovascular disease. The autosomal-dominant FH is mostly caused by mutations in LDLR (low density lipoprotein receptor), APOB (apolipoprotein B), and PCSK9 (proprotein convertase subtilisin/kexin). Recently, STAP1 has been suggested as a fourth causative gene. We analyzed STAP1 in 75 hypercholesterolemic patients from Berlin, Germany, who are negative for mutations in canonical FH genes. In 10 patients with negative family history, we additionally screened for disease causing variants in LDLRAP1 (low density lipoprotein receptor adaptor protein 1), associated with autosomal-recessive hypercholesterolemia. We identified one STAP1 variant predicted to be disease causing. To evaluate association of serum lipid levels and STAP1 carrier status, we analyzed 20 individuals from a population based cohort, the Cooperative Health Research in South Tyrol (CHRIS) study, carrying rare STAP1 variants. Out of the same cohort we randomly selected 100 non-carriers as control. In the Berlin FH cohort STAP1 variants were rare. In the CHRIS cohort, we obtained no statistically significant differences between carriers and non-carriers of STAP1 variants with respect to lipid traits. Until such an association has been verified in more individuals with genetic variants in STAP1, we cannot estimate whether STAP1 generally is a causative gene for FH.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/etiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Feminino , Estudos de Associação Genética/métodos , Humanos , Metabolismo dos Lipídeos , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Análise de Sequência de DNA
9.
Circ Genom Precis Med ; 12(7): e002384, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31306056

RESUMO

BACKGROUND: Lipids are increasingly involved in cardiovascular risk prediction as potential proarrhythmic influencers. However, knowledge is limited about the specific mechanisms connecting lipid alterations with atrial conduction. METHODS: To shed light on this issue, we conducted a broad assessment of 151 sphingo- and phospholipids, measured using mass spectrometry, for association with atrial conduction, measured by P wave duration (PWD) from standard electrocardiograms, in the MICROS study (Microisolates in South Tyrol) (n=839). Causal pathways involving lipidomics, body mass index (BMI), and PWD were assessed using 2-sample Mendelian randomization analyses based on published genome-wide association studies of lipidomics (n=4034) and BMI (n=734 481), and genetic association analysis of PWD in 5 population-based studies (n=24 236). RESULTS: We identified an association with relative phosphatidylcholine 38:3 (%PC 38:3) concentration, which was replicated in the ORCADES (Orkney Complex Disease Study; n=951), with a pooled association across studies of 2.59 (95% CI, 1.3-3.9; P=1.1×10-4) ms PWD per mol% increase. While being independent of cholesterol, triglycerides, and glucose levels, the %PC 38:3-PWD association was mediated by BMI. Results supported a causal effect of BMI on both PWD ( P=8.3×10-5) and %PC 38:3 ( P=0.014). CONCLUSIONS: Increased %PC 38:3 levels are consistently associated with longer PWD, partly because of the confounding effect of BMI. The causal effect of BMI on PWD reinforces evidence of BMI's involvement into atrial electrical activity.


Assuntos
Artérias/fisiopatologia , Índice de Massa Corporal , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Lipídeos/química , Adulto , Idoso , Artérias/metabolismo , Eletrocardiografia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Metabolismo dos Lipídeos , Lipidômica , Masculino , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Fatores de Risco
10.
Kidney Int ; 96(2): 480-488, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31248648

RESUMO

Damage of mitochondrial DNA (mtDNA) with reduction in copy number has been proposed as a biomarker for mitochondrial dysfunction and oxidative stress. Chronic kidney disease (CKD) is associated with increased mortality and risk of cardiovascular disease, but the underlying mechanisms remain incompletely understood. Here we investigated the prognostic role of mtDNA copy number for cause-specific mortality in 4812 patients from the German Chronic Kidney Disease study, an ongoing prospective observational national cohort study of patients with CKD stage G3 and A1-3 or G1-2 with overt proteinuria (A3) at enrollment. MtDNA was quantified in whole blood using a plasmid-normalized PCR-based assay. At baseline, 1235 patients had prevalent cardiovascular disease. These patients had a significantly lower mtDNA copy number than patients without cardiovascular disease (fully-adjusted model: odds ratio 1.03, 95% confidence interval [CI] 1.01-1.05 per 10 mtDNA copies decrease). After four years of follow-up, we observed a significant inverse association between mtDNA copy number and all-cause mortality, adjusted for kidney function and cardiovascular disease risk factors (hazard ratio 1.37, 95% CI 1.09-1.73 for quartile 1 compared to quartiles 2-4). When grouped by causes of death, estimates pointed in the same direction for all causes but in a fully-adjusted model decreased copy numbers were significantly lower only in infection-related death (hazard ratio 1.82, 95% CI 1.08-3.08). A similar association was observed for hospitalizations due to infections in 644 patients (hazard ratio 1.19, 95% CI 1.00-1.42 in the fully-adjusted model). Thus, our data support a role of mitochondrial dysfunction in increased cardiovascular disease and mortality risks as well as susceptibility to infections in patients with CKD.


Assuntos
Doenças Cardiovasculares/epidemiologia , Variações do Número de Cópias de DNA , DNA Mitocondrial/genética , Infecções/epidemiologia , Insuficiência Renal Crônica/mortalidade , Idoso , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/terapia , Causas de Morte , DNA Mitocondrial/sangue , Feminino , Seguimentos , Alemanha/epidemiologia , Hospitalização/estatística & dados numéricos , Humanos , Infecções/sangue , Infecções/etiologia , Infecções/terapia , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/patologia , Estresse Oxidativo/genética , Prevalência , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/genética , Fatores de Risco
11.
NPJ Parkinsons Dis ; 5: 5, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30963107

RESUMO

Modeling Parkinson's disease (PD) using advanced experimental in vitro models is a powerful tool to study disease mechanisms and to elucidate unexplored aspects of this neurodegenerative disorder. Here, we demonstrate that three-dimensional (3D) differentiation of expandable midbrain floor plate neural progenitor cells (mfNPCs) leads to organoids that resemble key features of the human midbrain. These organoids are composed of midbrain dopaminergic neurons (mDANs), which produce and secrete dopamine. Midbrain-specific organoids derived from PD patients carrying the LRRK2-G2019S mutation recapitulate disease-relevant phenotypes. Automated high-content image analysis shows a decrease in the number and complexity of mDANs in LRRK2-G2019S compared to control organoids. The floor plate marker FOXA2, required for mDAN generation, increases in PD patient-derived midbrain organoids, suggesting a neurodevelopmental defect in mDANs expressing LRRK2-G2019S. Thus, we provide a robust method to reproducibly generate 3D human midbrain organoids containing mDANs to investigate PD-relevant patho-mechanisms.

12.
Seizure ; 66: 81-85, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30818181

RESUMO

PURPOSE: Mutations in SZT2 have been previously reported in several cases of early onset epilepsy and intellectual disability. In this study we investigate potential causal mutations in two male siblings affected by early onset epilepsy, intellectual disability and macrocephaly. METHODS: We use family-based whole-exome sequencing to identify candidate variants. RESULTS: We report the identification of two potential causal SZT2 mutations in compound heterozygous state. We observe considerable differences in the clinical phenotype severity of the two affected individuals. The cerebral MRI revealed no abnormalities in the older affected brother, while in the youngest one it revealed a right frontal polymicrogiria. Moreover, while good seizure control was achieved in the older affected individual the younger brother is affected by pharmacoresistant epilepsy, progressive spastic paraplegia, cortical myoclonus and a more severe intellectual disability. We also analyzed the relative location of the reported pathogenic mutations in the SZT2 protein. CONCLUSION: Variable phenotypic expressivity is observed for this condition, while the location and type of mutations in SZT2 also has a potential impact on epilepsy severity. These findings extend our knowledge of epileptogenic conditions related to SZT2 and mTOR signaling.


Assuntos
Epilepsia/genética , Saúde da Família , Deficiência Intelectual/genética , Megalencefalia/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Adulto , Análise Mutacional de DNA , Epilepsia/complicações , Epilepsia/diagnóstico por imagem , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/diagnóstico por imagem , Estudos Longitudinais , Imagem por Ressonância Magnética , Masculino , Megalencefalia/complicações , Megalencefalia/diagnóstico por imagem , Sequenciamento Completo do Exoma , Adulto Jovem
13.
Int J Mol Sci ; 20(3)2019 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-30754623

RESUMO

Mutations in the PRKN gene (encoding parkin) have been linked to the most frequent known cause of recessive Parkinson's disease (PD), and parkin dysfunction represents a risk factor for sporadic PD. Parkin is widely neuroprotective through different cellular pathways, as it protects dopaminergic neurons from apoptosis in a series of cellular and animal models of PD. The mitochondrial protein apoptosis-inducing factor (AIF) is an important cell death effector, which, upon cellular stress in many paradigms, is redistributed from the mitochondria to the nucleus to function as a proapoptotic factor, mostly independent of caspase activity, while in normal mitochondria it functions as an antiapoptotic factor. AIF is known to participate in dopaminergic neuron loss in experimental PD models and in patients with PD. We, therefore, investigated possible crosstalk between parkin and AIF. By using immunoprecipitation and proximity ligation assays, we demonstrated a physical interaction between the two proteins. Nuclear AIF translocation was significantly reduced by parkin expression in neuroblastoma SH-SY5Y cells after exposure to an apoptogenic stimulus. These results were confirmed in primary murine cortical neurons, which showed a higher nuclear translocation of AIF in parkin-deficient neurons upon an excitotoxic stimulus. Our results indicate that the interaction of parkin with AIF interferes with the nuclear translocation of AIF, which might contribute to the neuroprotective activity of parkin.


Assuntos
Fator de Indução de Apoptose/metabolismo , Neurônios/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Ligação Proteica , Transporte Proteico , Ubiquitina-Proteína Ligases/genética
15.
Neurobiol Dis ; 121: 34-46, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30236862

RESUMO

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. PGC-1α, encoded by PPARGC1A, is a transcriptional co-activator that has been implicated in the pathogenesis of neurodegenerative disorders. We recently discovered multiple new PPARGC1A transcripts that initiate from a novel promoter located far upstream of the reference gene promoter, are CNS-specific and are more abundant than reference gene transcripts in whole brain. These CNS-specific transcripts encode two main full-length and several truncated isoforms via alternative splicing. Truncated CNS-isoforms include 17 kDa proteins that lack the second LXXLL motif serving as an interaction site for several nuclear receptors. We now determined expression levels of CNS- and reference gene transcripts in 5 brain regions of 21, 8, and 13 deceased subjects with idiopathic PD, Lewy body dementia and controls without neurodegenerative disorders, respectively. We observed reductions of CNS-specific transcripts (encoding full-length isoforms) only in the substantia nigra pars compacta of PD and Lewy body dementia. However, in the substantia nigra and globus pallidus of PD cases we found an up-regulation of transcripts encoding the 17 kDa proteins that inhibited the co-activation of several transcription factors by full-length PGC-1α proteins in transfection assays. In two established animal models of PD, the PPARGC1A expression profiles differed from the profile in human PD in that the levels of CNS- and reference gene transcripts were decreased in several brain regions. Furthermore, we identified haplotypes in the CNS-specific region of PPARGC1A that appeared protective for PD in a clinical cohort and a post-mortem sample (P = .0002). Thus, functional and genetic studies support a role of the CNS-specific PPARGC1A locus in PD.


Assuntos
Encéfalo/metabolismo , Doença de Parkinson/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Loci Gênicos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/genética
16.
Nat Commun ; 9(1): 4455, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30367059

RESUMO

Thyroid dysfunction is an important public health problem, which affects 10% of the general population and increases the risk of cardiovascular morbidity and mortality. Many aspects of thyroid hormone regulation have only partly been elucidated, including its transport, metabolism, and genetic determinants. Here we report a large meta-analysis of genome-wide association studies for thyroid function and dysfunction, testing 8 million genetic variants in up to 72,167 individuals. One-hundred-and-nine independent genetic variants are associated with these traits. A genetic risk score, calculated to assess their combined effects on clinical end points, shows significant associations with increased risk of both overt (Graves' disease) and subclinical thyroid disease, as well as clinical complications. By functional follow-up on selected signals, we identify a novel thyroid hormone transporter (SLC17A4) and a metabolizing enzyme (AADAT). Together, these results provide new knowledge about thyroid hormone physiology and disease, opening new possibilities for therapeutic targets.


Assuntos
2-Aminoadipato Transaminase/metabolismo , Regulação da Expressão Gênica/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo I/metabolismo , Hormônios Tireóideos/genética , Tireotropina/metabolismo , 2-Aminoadipato Transaminase/genética , Animais , Transporte Biológico , Células COS , Chlorocebus aethiops , Grupo com Ancestrais do Continente Europeu , Estudo de Associação Genômica Ampla , Humanos , Hipertireoidismo/genética , Hipertireoidismo/fisiopatologia , Hipotireoidismo/genética , Hipotireoidismo/fisiopatologia , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Proteínas Cotransportadoras de Sódio-Fosfato Tipo I/genética , Glândula Tireoide/metabolismo , Glândula Tireoide/fisiopatologia , Hormônios Tireóideos/metabolismo
17.
Sci Rep ; 8(1): 15347, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30337569

RESUMO

Alterations of mitochondrial DNA (mtDNA) copy number have been associated with a wide variety of phenotypes and diseases. Unfortunately, the literature provides scarce methodical information about duplex targeting of nuclear and mtDNA that meets the quality criteria for qPCR. Therefore, we established a method for mtDNA copy number quantification using a quantitative PCR assay that allows for simultaneous targeting of a single copy nuclear gene (beta-2-microglobulin) and the t-RNALeu gene on the mtDNA. We include a plasmid containing both targets in order to normalize against differences in emission intensities of the fluorescent dyes Yakima Yellow and FAM. Applying the plasmid calibrator on an internal control reduced the intra-assay variability from 21% (uncorrected) to 7% (plasmid-corrected). Moreover, we noted that DNA samples isolated with different methods revealed different numbers of mtDNA copies, thus highlighting an important influence of the pre-analytical procedures. In summary, we developed a precise assay for mitochondrial copy number detection relative to nuclear DNA. Our method is applicable to comparative mitochondrial DNA copy number studies since the use of the dual insert plasmid allows correcting for the unequal emission intensities of the different fluorescent labels of the two targets.


Assuntos
Variações do Número de Cópias de DNA , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Plasmídeos/genética , Estudos de Casos e Controles , Estudos de Coortes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mitocôndrias/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos
18.
Clin Chim Acta ; 486: 320-328, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30114408

RESUMO

BACKGROUND: Plasma and serum are the most widely used matrices in clinical studies. However, some variability in absolute concentrations of metabolites are likely to be observed in these collection tubes matrices. METHODS: We analyzed 189 metabolites using the same protocol for quantitative targeted metabolomics (LC-MS/MS AbsoluteIDQ p180 Kit Biocrates) in three types of samples, serum, plasma EDTA and citrate, of 80 subjects from the Cooperative Health Research In South Tyrol cohort (40 healthy elderly and 40 healthy young). RESULTS: The concentration levels were higher in serum than citrate and EDTA, in particular for amino acids and biogenic amines. The average Pearson's correlation coefficients were however always higher than 0.7 for these two classes of metabolites. We could also demonstrate that blank EDTA vacutainer tubes contain a significant amount of sarcosine. Finally, we compared the metabolome of young people against elderly subjects and found that the highest number of metabolites significantly changing with age was detected in serum. CONCLUSION: Serum samples provide higher sensitivity for biomarker discovery studies. Due to the presence of spurious amount of sarcosine in vacutainer EDTA tubes, plasma EDTA is not suitable for studies requiring accurate quantification of sarcosine.


Assuntos
Coleta de Amostras Sanguíneas , Contaminação de Equipamentos , Metabolômica , Sarcosina/análise , Sarcosina/sangue , Aminas/metabolismo , Biomarcadores/sangue , Cromatografia Líquida , Ácido Cítrico/química , Ácido Edético/química , Humanos , Espectrometria de Massas em Tandem
19.
Mol Neurodegener ; 13(1): 40, 2018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-30071902

RESUMO

The combination of genetics and genomics in Parkinson´s disease has recently begun to unveil molecular mechanisms possibly underlying disease onset and progression. In particular, catabolic processes such as autophagy have been increasingly gaining relevance as post-mortem evidence and experimental models suggested a participation in neurodegeneration and alpha-synuclein Lewy body pathology. In addition, familial Parkinson´s disease linked to LRRK2 and alpha-synuclein provided stronger correlation between etiology and alterations in autophagy. More detailed cellular pathways are proposed and genetic risk factors that associate with idiopathic Parkinson´s disease provide further clues in dissecting contributions of single players. Nevertheless, the fine-tuning of these processes remains elusive, as the initial stages of the pathways are not yet clarified.In this review, we collect literature evidence pointing to autophagy as the common, downstream target of Parkinsonian dysfunctions and augment current knowledge on the factors that direct the subsequent steps. Cell and molecular biology evidence indicate that p38 signaling underlies neurodegeneration and autoptic observations suggest a participation in neuropathology. Moreover, alpha-synuclein and LRRK2 also appear involved in the p38 pathway with additional roles in the regulation of GTPase signaling. Small GTPases are critical modulators of p38 activation and thus, their functional interaction with aSyn and LRRK2 could explain much of the detailed mechanics of autophagy in Parkinson´s disease.We propose a novel hypothesis for a more comprehensive working model where autophagy is controlled by upstream pathways, such as GTPase-p38, that have been so far underexplored in this context. In addition, etiological factors (LRRK2, alpha-synuclein) and risk loci might also combine in this common mechanism, providing a powerful experimental setting to dissect the cause of both familial and idiopathic disease.


Assuntos
Autofagia/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Doença de Parkinson/fisiopatologia , Humanos , Doença de Parkinson/etiologia , Doença de Parkinson/genética
20.
J Pain ; 19(12): 1424-1434, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30017960

RESUMO

The self-reported Pain Sensitivity Questionnaire (PSQ) is a valid supplement to experimental pain testing. However, the latent constructs determining the originally proposed 1 general score (PSQ-total) and 2 subscores (PSQ-moderate and PSQ-minor) have not been consistently investigated in population-based studies or between genders. Based on a single construct hypothesized by expert knowledge or alternative constructs upon empirical evidence, PSQ structures were explored and confirmed among 4,820 participants aged 18 to 93 years of the Cooperative Health Research In South Tyrol (CHRIS) study. By exploratory factor analysis, we identified 3 alternative sets of PSQ imagined painful situations comprising 14, 10, and 9 items, which displayed simple structures of the rotated factor loadings of direct interpretation. In confirmatory analysis (CFA) of 1 latent factor, the 10-item set yielded acceptable goodness-of-fit overall, better fit than the alternative sets and consistent structural properties between genders. Separate analyses based on 14- and 9-item sets returned considerable correlations between 2 latent constructs. In higher-order CFA with each set, 1 first-order general factor explained a large part of the variances of 2 second-order factors. One dominant construct consistently describes the factorial structure of the PSQ. Averaging across the 10-item set, the PSQ-short score represents a structurally robust, gender-consistent, and practical measure of general pain sensitivity. PERSPECTIVE: One dominant latent construct of general pain sensitivity consistently determines responses to the self-reported PSQ. The PSQ-short score maintains similar psychometric properties to the PSQ-total and between genders. This measure is attractive for large-scale research and clinical screening of pain sensitivity.


Assuntos
Medição da Dor , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Limiar da Dor , Psicometria , Reprodutibilidade dos Testes , Fatores Sexuais , Inquéritos e Questionários , Adulto Jovem
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