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1.
J Vasc Surg ; 68(6S): 201S-207S, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29804740

RESUMO

OBJECTIVE: The main objective of this study was to define a role of sphingosine-1-phosphate receptor 1 (S1PR1) in the arterial injury response of a human artery. The hypotheses were tested that injury induces an expansion of S1PR1-positive cells and that these cells accumulate toward the lumen because they follow the sphingosine-1-phosphate gradient from arterial wall tissue (low) to plasma (high). METHODS: A humanized rat model was used in which denuded human internal mammary artery (IMA) was implanted into the position of the abdominal aorta of immunosuppressed Rowett nude rats. This injury model is characterized by medial as well as intimal hyperplasia, whereby intimal cells are of human origin. At 7, 14, and 28 days after implantation, grafts were harvested and processed for fluorescent immunostaining for S1PR1 and smooth muscle α-actin. Nuclei were stained with 4',6-diamidine-2'-phenylindole dihydrochloride. Using digitally reconstructed, complete cross sections of grafts, intimal and medial areas were measured, whereby the medial area had virtually been divided into an outer (toward adventitia) and inner (toward lumen) layer. The fraction of S1PR1-positive cells was determined in each layer by counting S1PR1-positive and S1PR1-negative cells. RESULTS: The fraction of S1PR1-postive cells in naive IMA is 58.9% ± 6.0% (mean ± standard deviation). At day 28 after implantation, 81.6% ± 4.4% of medial cells were scored S1PR1 positive (P < .01). At day 14, the ratio between S1PR1-positive and S1PR1-negative cells was significantly higher in the lumen-oriented inner layer (9.3 ± 2.1 vs 6.0 ± 1.0; P < .01). Cells appearing in the intima at day 7 and day 14 were almost all S1PR1 positive. At day 28, however, about one-third of intimal cells were scored S1PR1 negative. CONCLUSIONS: From these data, we conclude that denudation of IMA specifically induces the expansion of S1PR1-positive cells. Based on the nonrandom distribution of S1PR1-positive cells, we consider the possibility that much like lymphocytes, S1PR1-positive smooth muscle cells also use S1PR1 to recognize the sphingosine-1-phosphate gradient from tissue (low) to plasma (high) and so migrate out of the media toward the intima of the injured IMA.


Assuntos
Aorta Abdominal/cirurgia , Oclusão de Enxerto Vascular/metabolismo , Artéria Torácica Interna/transplante , Músculo Liso Vascular/transplante , Miócitos de Músculo Liso/transplante , Neointima , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/patologia , Humanos , Lisofosfolipídeos/metabolismo , Masculino , Artéria Torácica Interna/metabolismo , Artéria Torácica Interna/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Ratos Nus , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Fatores de Tempo
2.
PLoS One ; 11(12): e0168302, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27973607

RESUMO

BACKGROUND AND OBJECTIVES: Atherosclerotic changes of arteries are the leading cause for deaths in cardiovascular disease and greatly impair patient's quality of life. Sphingosine-1-phosphate (S1P) is a signaling sphingolipid that regulates potentially pro-as well as anti-atherogenic processes. Here, we investigate whether serum-S1P concentrations are associated with peripheral artery disease (PAD) and carotid stenosis (CS). METHODS AND RESULTS: Serum was sampled from blood donors (controls, N = 174) and from atherosclerotic patients (N = 132) who presented to the hospital with either clinically relevant PAD (N = 102) or CS (N = 30). From all subjects, serum-S1P was measured by mass spectrometry and blood parameters were determined by routine laboratory assays. When compared to controls, atherosclerotic patients before invasive treatment to restore blood flow showed significantly lower serum-S1P levels. This difference cannot be explained by risk factors for atherosclerosis (old age, male gender, hypertension, hypercholesteremia, obesity, diabetes or smoking) or comorbidities (Chronic obstructive pulmonary disease, kidney insufficiency or arrhythmia). Receiver operating characteristic curves suggest that S1P has more power to indicate atherosclerosis (PAD and CS) than high density lipoprotein-cholesterol (HDL-C). In 35 patients, serum-S1P was measured again between one and six months after treatment. In this group, serum-S1P concentrations rose after treatment independent of whether patients had PAD or CS, or whether they underwent open or endovascular surgery. Post-treatment S1P levels were highly associated to platelet numbers measured pre-treatment. CONCLUSIONS: Our study shows that PAD and CS in humans is associated with decreased serum-S1P concentrations and that S1P may possess higher accuracy to indicate these diseases than HDL-C.


Assuntos
Aterosclerose/sangue , Lisofosfolipídeos/sangue , Esfingosina/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Coagulação Sanguínea , Estenose das Carótidas/sangue , Estudos de Casos e Controles , HDL-Colesterol/metabolismo , Estudos de Coortes , Feminino , Humanos , Lipoproteínas HDL/sangue , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/sangue , Prognóstico , Curva ROC , Análise de Regressão , Fatores de Risco , Transdução de Sinais , Esfingosina/sangue , Adulto Jovem
3.
Tissue Cell ; 47(3): 266-72, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25890870

RESUMO

The vascular endothelium as well as subendothelium are objects of many researches as it is directly involved in a multiplicity of physiological and pathological settings. Detailed study of endothelial function became feasible with the development of techniques to culture endothelial cells (EC) in vitro. Limitations of this approach have become apparent with the realization that cell culture dedifferentiate with time and do not exhibit properties of intact tissue. Here we describe the development of a novel ex vivo tissue model to study cell-vascular wall interactions by using isolated mouse aorta patches. Validation of this model was performed by demonstrating cell attachment and changes in cell shape typical for cell spreading during adhesion. A major advantage of this model is that cell-endothelium interaction and its molecular backgrounds can now be studied more feasibly on an intact and native tissue.


Assuntos
Aorta/fisiologia , Adesão Celular/fisiologia , Desdiferenciação Celular/fisiologia , Endotélio Vascular/citologia , Animais , Aorta/citologia , Comunicação Celular/fisiologia , Técnicas de Cultura de Células , Forma Celular/fisiologia , Endotélio Vascular/fisiologia , Camundongos
4.
PLoS One ; 10(1): e0117154, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25565633

RESUMO

Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.


Assuntos
Príons/metabolismo , Scrapie/transmissão , Animais , Bioensaio , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Bovinos , Linhagem Celular , Encefalopatia Espongiforme Bovina/metabolismo , Encefalopatia Espongiforme Bovina/patologia , Encefalopatia Espongiforme Bovina/transmissão , Endopeptidase K/metabolismo , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPSc/análise , Proteínas PrPSc/metabolismo , Scrapie/metabolismo , Scrapie/patologia , Ovinos
5.
Biochem Biophys Res Commun ; 445(1): 23-9, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24502948

RESUMO

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc(C) into a disease-associated isoform PrP(Sc). PrP(Sc) accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP(Sc) species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP(Sc) formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge). We could detect six different classes of highly potent inhibitors of PrP(Sc) propagation in vitro and identified piperazine derivatives as a new inhibitory lead structure, which increased incubation time of scrapie infected mice.


Assuntos
Encéfalo/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas PrPSc/metabolismo , Scrapie/prevenção & controle , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Injeções Intraperitoneais , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Piperazina , Piperazinas/administração & dosagem , Piperazinas/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Scrapie/metabolismo
6.
PLoS One ; 8(12): e82255, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24367508

RESUMO

Alzheimer's disease (AD) is the most common form of dementia in the elderly with progressive cognitive decline and memory loss. According to the amyloid-hypothesis, AD is caused by generation and subsequent cerebral deposition of ß-amyloid (Aß). Aß is generated through sequential cleavage of the transmembrane Amyloid-Precursor-Protein (APP) by two endoproteinases termed beta- and gamma-secretase. Increased APP-expression caused by APP gene dosage effects is a risk factor for the development of AD. Here we carried out a large scale screen for novel compounds aimed at decreasing APP-expression. For this we developed a screening system employing a cell culture model of AD. A total of 10,000 substances selected for their ability of drug-likeness and chemical diversity were tested for their potential to decrease APP-expression resulting in reduced Aß-levels. Positive compounds were further evaluated for their effect at lower concentrations, absence of cytotoxicity and specificity. The six most promising compounds were characterized and structure function relationships were established. The novel compounds presented here provide valuable information for the development of causal therapies for AD.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Linhagem Celular , Sobrevivência Celular/fisiologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Acta Neuropathol ; 125(6): 795-813, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23604588

RESUMO

In neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and prion diseases, deposits of aggregated disease-specific proteins are found. Oligomeric aggregates are presumed to be the key neurotoxic agent. Here we describe the novel oligomer modulator anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole], an aggregation inhibitor we developed based on a systematic high-throughput screening campaign combined with medicinal chemistry optimization. In vitro, anle138b blocked the formation of pathological aggregates of prion protein (PrP(Sc)) and of α-synuclein (α-syn), which is deposited in PD and other synucleinopathies such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Notably, anle138b strongly inhibited all prion strains tested including BSE-derived and human prions. Anle138b showed structure-dependent binding to pathological aggregates and strongly inhibited formation of pathological oligomers in vitro and in vivo both for prion protein and α-synuclein. Both in mouse models of prion disease and in three different PD mouse models, anle138b strongly inhibited oligomer accumulation, neuronal degeneration, and disease progression in vivo. Anle138b had no detectable toxicity at therapeutic doses and an excellent oral bioavailability and blood-brain-barrier penetration. Our findings indicate that oligomer modulators provide a new approach for disease-modifying therapy in these diseases, for which only symptomatic treatment is available so far. Moreover, our findings suggest that pathological oligomers in neurodegenerative diseases share structural features, although the main protein component is disease-specific, indicating that compounds such as anle138b that modulate oligomer formation by targeting structure-dependent epitopes can have a broad spectrum of activity in the treatment of different protein aggregation diseases.


Assuntos
Encéfalo/efeitos dos fármacos , Doença de Parkinson/terapia , Doenças Priônicas/terapia , Príons/efeitos dos fármacos , Pirazóis/agonistas , Pirimidinas/agonistas , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Técnicas de Cultura de Células , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doenças Priônicas/etiologia , Doenças Priônicas/metabolismo , Príons/metabolismo , Rotenona/farmacologia , alfa-Sinucleína/farmacologia
8.
PLoS One ; 6(9): e24624, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21931781

RESUMO

The cellular prion protein (PrP(C)) plays a fundamental role in prion disease. PrP(C) is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrP(C) is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrP(C) and also replaced the GPI-anchor of PrP(C) by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrP(C) in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrP(C). Exchange of the PrP(C) GPI-anchor for the one of Thy-1 redirects PrP(C) to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrP(C), with the GPI-anchor being dominant over N-glycans.


Assuntos
Glicosilfosfatidilinositóis/metabolismo , Polissacarídeos/metabolismo , Proteínas PrPC/metabolismo , Animais , Biotinilação , Linhagem Celular , Cães , Camundongos , Microscopia Confocal , Transporte Proteico
9.
ChemMedChem ; 6(10): 1928-37, 2011 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-21755599

RESUMO

Transmissible spongiform encephalopathies (TSE) or prion diseases belong to a category of fatal and so far untreatable neurodegenerative conditions. All prion diseases are characterized by both degeneration in the central nervous system (CNS) in humans and animals and the deposition and accumulation of Proteinase K-resistant prion protein (PrP(res)). Until now, no pharmaceutical product has been available to cure these diseases or to alleviate their associated symptoms. Here, a cell-culture screening system is described that allows for the large-scale analysis of the PrP(res) inhibitory potential of a library of compounds and the identification of structural motifs leading potent compounds able to cause PrP(res) clearance at the cellular level. Based on different scrapie-infected cell lines, 10,000 substances were tested, out of which 530 potential inhibitors were identified. After re-screening and validation using a series of dilutions, 14 compounds were identified as the most effective. These 14 compounds were then used for therapeutic studies in a mouse bioassay to test and verify their in vivo potency. Two compounds exhibited therapeutic potential in the mouse model by significantly extending the survival time of intracerebrally infected mice, when treated 90 days after infection with scrapie.


Assuntos
Ensaios de Triagem em Larga Escala , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Camundongos , Proteínas PrPSc/metabolismo , Scrapie/tratamento farmacológico
10.
Antimicrob Agents Chemother ; 55(10): 4774-81, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21746938

RESUMO

Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative disorders that can be transmitted by natural infection or inoculation. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. The emergence of a variant form of CJD (vCJD), which has been associated with BSE, produced strong pressure to search for effective treatments with new drugs. Up to now, however, TSEs have proved incurable, although many efforts have been made both in vitro and in vivo to search for potent therapeutic and prophylactic compounds. For this purpose, we analyzed a compound library consisting of 10,000 compounds with a cell-based high-throughput screening assay dealing with scrapie-infected scrapie mouse brain and ScN(2)A cells and identified a new class of inhibitors consisting of 3,5-diphenylpyrazole (DPP) derivatives. The most effective DPP derivative showed half-maximal inhibition of PrP(Sc) formation at concentrations (IC(50)) of 0.6 and 1.2 µM, respectively. This compound was subsequently subjected to a number of animal experiments using scrapie-infected wild-type C57BL/6 and transgenic Tga20 mice. The DPP derivative induced a significant increase of incubation time both in therapeutic and prophylactic experiments. The onset of the prion disease was delayed by 37 days after intraperitoneal and 42 days after oral application, respectively. In summary, we demonstrate a high in vitro efficiency of DPP derivatives against prion infections that was substantiated in vivo for one of these compounds. These results indicate that the novel class of DPP compounds should comprise excellent candidates for future therapeutic studies.


Assuntos
Proteínas PrPSc/metabolismo , Pirazóis/uso terapêutico , Scrapie/tratamento farmacológico , Animais , Ensaios de Triagem em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenóis/farmacologia , Fenóis/uso terapêutico , Fenóis/toxicidade , Pirazóis/efeitos adversos , Pirazóis/farmacologia , Pirazóis/toxicidade , Scrapie/mortalidade , Scrapie/prevenção & controle
11.
PLoS One ; 5(11): e13906, 2010 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-21085647

RESUMO

Prion diseases are transmissible fatal neurodegenerative disorders affecting humans and animals. A central step in disease progression is the accumulation of a misfolded form (PrP(Sc)) of the host encoded prion protein (PrP(C)) in neuronal and non-neuronal tissues. The involvement of peripheral tissues in preclinical states increases the risk of accidental transmission. On the other hand, detection of PrP(Sc) in non-neuronal easy-accessible compartments such as muscle may offer a novel diagnostic tool. Primate models have proven invaluable to investigate prion diseases. We have studied the deposition of PrP(Sc) in muscle and central nervous system of rhesus monkeys challenged with sporadic Creutzfeldt-Jakob disease (sCJD), variant CJD (vCJD) and bovine spongiform encephalopathy (BSE) in preclinical and clinical stage using biochemical and morphological methods. Here, we show the preclinical presence of PrP(Sc) in muscle and central nervous system of rhesus monkeys experimentally infected with vCJD.


Assuntos
Músculos/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Macaca mulatta , Miocárdio/metabolismo , Doenças Priônicas/patologia , Língua/metabolismo
12.
Infect Disord Drug Targets ; 9(1): 40-7, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19200014

RESUMO

Until now it is still not clear which structural elements of the prion protein (PrP) are involved in its conversion process. Characterisation of these essential regions would help to understand the conversion process itself and might help to develop specific therapeutic approaches to inhibit PrP(res) formation by dominant inhibitory mutations. To address this important question 33 evenly spaced insertion mutants were generated spanning the entire sequence of the murine 3F4-tagged PrP. The mutants were expressed by retroviral transduction in three different scrapie infected cell lines (ScN2a; SMB[RC040]; SMB[22F]). The convertibility was affected not only by introducing the insertion in the putatively refolded region (aa100-170), but also in the C-terminus of PrP (up to aa214). Moreover, dominant inhibitory effects on conversion were observed for PrP-mutants at four distinguished regions (aa100-112; aa130-154; aa166-172, aa196-200). Computer based structural analysis revealed that these segments were organized in two structurally clearly separated regions supporting the idea that they could function as protein-protein interaction sites which are necessary during seed formation.


Assuntos
Regulação da Expressão Gênica , Mutagênese Insercional , Proteínas PrPSc/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Glicosilação , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas PrPSc/química , Proteínas PrPSc/genética , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico
13.
Dev Biol ; 321(2): 434-43, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18652818

RESUMO

The neuronal protein 25 (NP25), a member of the calponin (CaP) protein family, has previously been identified as neuron-specific protein in the adult rat brain. Here, we show an early onset of NP25 expression in the chick embryo neural tube. NP25 represents, together with NeuroM, one of the earliest markers for postmitotic neurons. To elucidate its function in the developing nervous system, NP25 was overexpressed in E5 and E9 sensory neurons, E7 sympathetic neurons and PC12 cells that show different endogenous NP25 expression levels. Whereas E5 and E9 sensory neurons and PC12 cells, which express low endogenous levels of NP25, responded by enhanced neurite outgrowth, a reduction of neurite length was observed in sympathetic neurons, which already express high endogenous levels of NP25. Knockdown of NP25 in sensory neurons using NP25 siRNA resulted in shorter neurites, whereas reduction of NP25 expression in sympathetic neurons led to increased neurite length. These results suggest a dynamic function for NP25 in the regulation of neurite growth, with an optimal level of NP25 required for maximal growth.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Tubo Neural/metabolismo , Neuritos/fisiologia , Neurônios/metabolismo , Animais , Bromodesoxiuridina , Proteínas de Ligação ao Cálcio/genética , Embrião de Galinha , Hibridização In Situ , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Neuritos/metabolismo , Células PC12 , Interferência de RNA , Ratos , Calponinas
14.
Neurodegener Dis ; 5(6): 347-54, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18349519

RESUMO

BACKGROUND: Alzheimer's disease (AD) and prion diseases such as sporadic Creutzfeldt-Jakob disease (sCJD) share common features concerning their molecular pathogenesis and neuropathological presentation and the coexistence of AD and CJD in patients suggest an association between the deposition of the proteolytically processed form of the amyloid precursor protein, beta-amyloid (Abeta), which deposits in AD, and the abnormal form of the prion protein, PrP(Sc), which deposits in sCJD. METHODS: We have characterized sCJD patients (n = 14), AD patients (n = 5) and nondemented controls (n = 5) with respect to the deposition of PrP(Sc) and Abeta morphologically, biochemically and genetically and correlated these findings to clinical data. RESULTS: sCJD-diseased individuals with abundant deposits of Abeta present with a specific clinicopathological profile, defined by higher age at disease onset, long disease duration, a genetic profile and only minimal amounts of PrP(Sc) in the cerebellum. CONCLUSION: The co-occurrence of pathological changes typical for sCJD and AD in combination with the inverse association between accumulation of Abeta and PrP(Sc) in a subgroup of sCJD patients is indicative of common pathways involved in the generation or clearance of Abeta and PrP(Sc) in a subgroup of sCJD patients.


Assuntos
Peptídeos beta-Amiloides/análise , Química Encefálica , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Fragmentos de Peptídeos/análise , Proteínas PrPSc/análise , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/genética , Western Blotting , Síndrome de Creutzfeldt-Jakob/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Lobo Frontal/química , Lobo Frontal/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Presenilina-1/análise , Presenilina-1/genética , Presenilina-2/análise , Presenilina-2/genética , Proteínas Priônicas , Príons/análise , Príons/genética , Nexinas de Proteases , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/genética
15.
Proc Natl Acad Sci U S A ; 104(45): 17861-6, 2007 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-17971443

RESUMO

During development, coordinate regulation of cell cycle exit and differentiation of neuronal precursors is essential for generation of appropriate number of neurons and proper wiring of neuronal circuits. BM88 is a neuronal protein associated in vivo with terminal neuron-generating divisions, marking the exit of proliferative cells from the cell cycle. Here, we provide functional evidence that BM88 is sufficient to initiate the differentiation of spinal cord neural precursors toward acquisition of generic neuronal and subtype-specific traits. Gain-of-function approaches show that BM88 negatively regulates proliferation of neuronal precursors, driving them to prematurely exit the cell cycle, down-regulate Notch1, and commit to a neuronal differentiation pathway. The combined effect on proliferation and differentiation results in precocious induction of neurogenesis and generation of postmitotic neurons within the ventricular zone. The dual action of BM88 is not recapitulated by the cell cycle inhibitor p27Kip1, suggesting that cell cycle exit does not induce differentiation by default. Mechanistically, induction of endogenous BM88 by forced expression of the proneural gene Mash1 indicates that BM88 is part of the differentiation program activated by proneural genes. Furthermore, BM88 gene silencing conferred by small interfering RNA in spinal cord neural progenitor cells enhances cell cycle progression and impairs neuronal differentiation. Our results implicate BM88 in the synchronization of cell cycle exit and differentiation of neuronal precursors in the developing nervous system.


Assuntos
Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Animais , Ciclo Celular , Diferenciação Celular , Eletroporação , Embrião de Mamíferos , Hibridização In Situ , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Células-Tronco/citologia , Células-Tronco/fisiologia
16.
J Biol Chem ; 282(26): 18702-10, 2007 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-17468101

RESUMO

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.


Assuntos
Técnicas de Cultura de Células/métodos , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Scrapie/fisiopatologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Meios de Cultura/metabolismo , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Expressão Gênica , Hipocampo/citologia , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas PrPSc/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Retroviridae/genética , Scrapie/patologia , Scrapie/transmissão , Ovinos , Transdução Genética
17.
Vaccine ; 25(30): 5631-6, 2007 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-17391814

RESUMO

Prion diseases are a heterogeneous group of disorders with an invariably fatal disease course. Although various etiologies have been proposed it is apparent that at least a subset of these diseases are of infectious nature. An essential part of the infectious agent, termed the prion, is mainly composed of an abnormal isoform (PrP(Sc)) of a host-encoded normal cellular protein (PrP(C)). The molecular details of the pathophysiology of this group of diseases are unclear but the conversion of PrP(C) to PrP(Sc) plays a fundamental role. In all human prion diseases, PrP(Sc) is deposited in the central nervous system. These disorders include sporadic, genetic and acquired Creutzfeldt-Jakob disease. The molecular classification of human prion diseases is important in order to understand underlying disease mechanisms and for the development of novel therapy protocols. Current classification systems are based on the assessment of clinical presentation, genetic investigations, neuropathological findings and biochemical analysis of PrP(Sc).


Assuntos
Doenças Priônicas/patologia , Doenças Priônicas/fisiopatologia , Príons/classificação , Príons/genética , Sistema Nervoso Central/patologia , Humanos , Doenças Priônicas/diagnóstico , Doenças Priônicas/genética , Príons/isolamento & purificação
18.
Development ; 133(1): 141-50, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16319110

RESUMO

Sympathetic neurons are generated through a succession of differentiation steps that initially lead to noradrenergic neurons innervating different peripheral target tissues. Specific targets, like sweat glands in rodent footpads, induce a change from noradrenergic to cholinergic transmitter phenotype. Here, we show that cytokines acting through the gp 130 receptor are present in sweat glands. Selective elimination of the gp 130 receptor in sympathetic neurons prevents the acquisition of cholinergic and peptidergic features (VAChT, ChT1, VIP) without affecting other properties of sweat gland innervation. The vast majority of cholinergic neurons in the stellate ganglion, generated postnatally, are absent in gp 130-deficient mice. These results demonstrate an essential role of gp 130-signaling in the target-dependent specification of the cholinergic neurotransmitter phenotype.


Assuntos
Fibras Adrenérgicas/metabolismo , Diferenciação Celular/fisiologia , Receptor gp130 de Citocina/metabolismo , Citocinas/metabolismo , Transdução de Sinais/fisiologia , Glândulas Sudoríparas/embriologia , Glândulas Sudoríparas/inervação , Animais , Pesos e Medidas Corporais , Células Cultivadas , Primers do DNA , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Gânglio Estrelado/citologia , Gânglio Estrelado/metabolismo , Glândulas Sudoríparas/anatomia & histologia , Glândulas Sudoríparas/metabolismo
19.
Development ; 129(6): 1387-96, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11880348

RESUMO

Sympathetic ganglia are composed of noradrenergic and cholinergic neurons. Cholinergic sympathetic neurons are characterized by the expression of choline acetyl transferase (ChAT), vesicular acetylcholine transporter (VAChT) and the vasoactive intestinal peptide (VIP). To investigate the role of cytokine growth factor family members in the development of cholinergic sympathetic neurons, we interfered in vivo with the function of the subclass of cytokine receptors that contains LIFRbeta as essential receptor subunit. Expression of LIFRbeta antisense RNA interfered with LIFRbeta expression and strongly reduced the developmental induction of VIP expression. By contrast, ganglion size and the number of ChAT-positive cells were not reduced. These results demonstrate a physiological role of cytokines acting through LIFRbeta-containing receptors in the control of VIP expression in sympathetic neurons.


Assuntos
Citocinas/metabolismo , Proteínas de Membrana Transportadoras , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Sistema Nervoso Simpático/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Proteínas de Transporte/metabolismo , Embrião de Galinha , Colina/fisiologia , Colina O-Acetiltransferase/metabolismo , Clonagem Molecular , Receptor gp130 de Citocina , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Receptores de OSM-LIF , Alinhamento de Sequência , Transdução de Sinais , Proteínas Vesiculares de Transporte de Acetilcolina
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