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1.
J Chromatogr A ; 1711: 464454, 2023 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-37871502

RESUMO

Phthalates are mainly used as plasticizers in polyvinyl chloride (PVC). However, prolonged exposure to phthalates poses considerable risks to human health. Consequently, the utilization of phthalates in consumer products is subject to regulations, with a defined threshold of 0.1 %. In this study, we developed an accurate and simultaneous method for determination of 11 representative phthalates and a non-phthalate plasticizer (di(2-ethylhexyl) terephthalate, DEHT) in PVC as a higher-order reference method. Homogeneously prepared PVC samples, each containing approximately 0.1 % of the target plasticizer compounds, were analyzed using gas chromatography-mass spectrometry (GC-MS) with deuterium-labeled phthalates and DEHT. The developed method could effectively separate and quantify all target plasticizers without interference with each other and potential overlap between the isomeric forms of phthalates, di-isodecyl phthalate, and di-isononyl phthalate. The developed method has high-order metrological quality, exhibiting exceptional selectivity, accuracy, repeatability (≤ 2.17 %), reproducibility (≤ 2.16 %), and relative expanded uncertainty (≤ 5.6 %). This analytical method is thus suitable for accurately assessing the target plasticizer levels in PVC products for ensuring compliance with the established 0.1 % threshold. This method was successfully applied to quantify twelve distinct plasticizers in PVC products obtained from the Korean market, validating its effectiveness and reliability in real-world scenarios.


Assuntos
Dietilexilftalato , Ácidos Ftálicos , Humanos , Plastificantes/análise , Cloreto de Polivinila/química , Reprodutibilidade dos Testes , Ácidos Ftálicos/análise , Espectrometria de Massas , Cromatografia Gasosa-Espectrometria de Massas/métodos , Isótopos , Dietilexilftalato/análise
2.
Cell ; 185(4): 712-728.e14, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35063084

RESUMO

Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.


Assuntos
Mitocôndrias/metabolismo , Degeneração Neural/metabolismo , Mapas de Interação de Proteínas , Sinapses/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Aminoácidos/metabolismo , Biotinilação , Encéfalo/metabolismo , Encéfalo/patologia , Núcleo Celular/metabolismo , Progressão da Doença , Metabolismo Energético , Demência Frontotemporal/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Degeneração Neural/patologia , Neurônios/metabolismo , Ligação Proteica , Domínios Proteicos , Proteômica , Índice de Gravidade de Doença , Frações Subcelulares/metabolismo , Tauopatias/genética , Proteínas tau/química
4.
Sci Rep ; 11(1): 6079, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727605

RESUMO

Mutations in the GBA1 gene encoding glucocerebrosidase (GCase) are linked to Gaucher (GD) and Parkinson's Disease (PD). Since some GD and PD patients develop ocular phenotypes, we determined whether ocular phenotypes might result from impaired GCase activity and the corresponding accumulation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph) in the Gba1D409V/D409V knock-in (Gba KI/KI; "KI") mouse. Gba KI mice developed age-dependent pupil dilation deficits to an anti-muscarinic agent; histologically, the iris covered the anterior part of the lens with adhesions between the iris and the anterior surface of the lens (posterior synechia). This may prevent pupil dilation in general, beyond an un-responsiveness of the iris to anti-muscarinics. Gba KI mice displayed atrophy and pigment dispersion of the iris, and occlusion of the iridocorneal angle by pigment-laden cells, reminiscent of secondary open angle glaucoma. Gba KI mice showed progressive thinning of the retina consistent with retinal degeneration. GluSph levels were increased in the anterior and posterior segments of the eye, suggesting that accumulation of lipids in the eye may contribute to degeneration in this compartment. We conclude that the Gba KI model provides robust and reproducible eye phenotypes which may be used to test for efficacy and establish biomarkers for GBA1-related therapies.


Assuntos
Doença de Gaucher , Glaucoma de Ângulo Aberto , Glucosilceramidase , Mutação de Sentido Incorreto , Doença de Parkinson , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Doença de Gaucher/enzimologia , Doença de Gaucher/genética , Doença de Gaucher/patologia , Técnicas de Introdução de Genes , Glaucoma de Ângulo Aberto/enzimologia , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/patologia , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/patologia
5.
Ann Rehabil Med ; 43(1): 81-86, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30852874

RESUMO

OBJECTIVE: To find out whether levels of fibrin degradation products (FDP) and D-dimer are increased in breast cancer-related lymphedema (BCRL) as in many vascular diseases. FDP and D-dimer have been used in blood tests to help differentiate deep vein thrombosis in the diagnosis of lymphedema. Levels of FDP and D-dimer are often elevated in patients with BCRL. METHODS: Patients with BCRL (group I), non-lymphedema after breast cancer treatment (group II), and deep venous thrombosis (group III) from January 2012 to December 2016 were enrolled. Levels of FDP and D-dimer were measured in all groups and compared among groups. RESULTS: Mean values of FDP and D-dimer of group I were 5.614±12.387 and 1.179±2.408 µg/µL, respectively. These were significantly higher than their upper normal limits set in our institution. Levels of FDP or D-dimer were not significantly different between group I and group II. However, values of FDP and D-dimer in group III were significantly higher than those in group I. CONCLUSION: Values of FDP and D-dimer were much higher in patients with thrombotic disease than those in patients with lymphedema. Thus, FDP and D-dimer can be used to differentiate between DVT and lymphedema. However, elevated levels of FDP or D-dimer cannot indicate the occurrence of lymphedema.

6.
J Neurosci ; 38(15): 3680-3688, 2018 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-29540553

RESUMO

Hyperacetylation of tau has been implicated in neurodegeneration and cognitive decline in tauopathy brains. The nicotinamide adenosine dinucleotide-dependent class-III protein deacetylase SIRT1 is one of the major enzymes involved in removal of acetyl groups from tau in vitro However, whether SIRT1 regulates acetylation of pathogenic tau and ameliorates tau-mediated pathogenesis remains unclear. Here, we report deacetylating activity of SIRT1 for acetylated Lys174 (K174) of tau in tauP301S transgenic mice with a brain-specific SIRT1 deletion. We show that SIRT1 deficiency leads to exacerbation of premature mortality, synapse loss, and behavioral disinhibition in tauP301S transgenic mice of both sexes. By contrast, SIRT1 overexpression by stereotaxic delivery of adeno-associated virus that encodes SIRT1 into the hippocampus reduces acetylated K174 tau. Furthermore, SIRT1 overexpression significantly attenuates the spread of tau pathology into anatomically connected brain regions of tauP301S transgenic mice of both sexes. These findings suggest the functional importance of SIRT1 in regulating pathogenic tau acetylation and in suppressing the spread of tau pathology in vivoSIGNIFICANCE STATEMENT In neurodegenerative disorders with inclusions of microtubule-associated protein tau, aberrant lysine acetylation of tau plays critical roles in promoting tau accumulation and toxicity. Identifying strategies to deacetylate tau could interfere with disease progression; however, little is known about how pathogenic tau is deacetylated in vivo Here we show that the protein deacetylase SIRT1 reduces tau acetylation in a mouse model of neurodegeneration. SIRT1 deficiency in the brain aggravates synapse loss and behavioral disinhibition, and SIRT1 overexpression ameliorates propagation of tau pathology.


Assuntos
Sirtuína 1/metabolismo , Tauopatias/metabolismo , Proteínas tau/metabolismo , Acetilação , Animais , Feminino , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Masculino , Aprendizagem em Labirinto , Camundongos , Sirtuína 1/genética , Transmissão Sináptica , Tauopatias/patologia , Tauopatias/fisiopatologia
7.
Ann Rehabil Med ; 42(6): 798-803, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30613072

RESUMO

OBJECTIVE: To investigate the relationship between peak cough flow (PCF), pulmonary function tests (PFT), and severity of dysphagia in patients with ischemic stroke. METHODS: This study included patients diagnosed with ischemic stroke, who underwent videofluoroscopic swallowing study (VFSS), PCF and PFT from March 2016 to February 2017. The dysphagia severity was assessed using the videofluoroscopic dysphagia scale (VDS). Correlation analysis of VDS, PFT and PCF was performed. Patients were divided into three groups based on VDS score. One-way ANOVA of VDS was performed to analyze PCF, forced vital capacity (FVC), forced expiratory volume in one second (FEV1), and age among the different groups. RESULTS: The correlation coefficients of VDS and PCF, VDS and FVC, and VDS and FEV1 were -0.836, -0.508, and -0.430, respectively, all of which were statistically significant at the level of p<0.001. The one-way ANOVA indicated statistically significant differences in PCF, FVC, FEV1, and age among the VDS groups. Statistically significant differences in VDS and age were observed between aspiration pneumoia and non-aspiration pneumonia groups. CONCLUSION: Coughing is a useful factor in evaluating the risk of aspiration in dysphagia patients. Evaluation of respiratory and coughing function should be conducted during the swallowing assessment of patients with ischemic stroke.

8.
Alzheimers Dement (N Y) ; 3(4): 507-512, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29124108

RESUMO

INTRODUCTION: Frontotemporal lobar degeneration-causing mutations in the progranulin (GRN) gene reduce progranulin protein (PGRN) levels, suggesting that restoring PGRN in mutation carriers may be therapeutic. Nimodipine, a Food and Drug Administration-approved blood-brain barrier-penetrant calcium channel blocker, increased PGRN levels in PGRN-deficient murine models. We sought to assess safety and tolerability of oral nimodipine in human GRN mutation carriers. METHODS: We performed an open-label, 8-week, dose-finding, phase 1 clinical trial in eight GRN mutation carriers to assess the safety and tolerability of nimodipine and assayed fluid and radiologic markers to investigate therapeutic endpoints. RESULTS: There were no serious adverse events; however, PGRN concentrations (cerebrospinal fluid and plasma) did not change significantly following treatment (percent changes of -5.2 ± 10.9% in plasma and -10.2 ± 7.8% in cerebrospinal fluid). Measurable atrophy within the left middle frontal gyrus was observed over an 8-week period. DISCUSSION: While well tolerated, nimodipine treatment did not alter PGRN concentrations or secondary outcomes.

9.
J Biol Chem ; 292(47): 19209-19225, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-28972160

RESUMO

The ubiquitin-proteasome system (UPS) is responsible for most selective protein degradation in eukaryotes and regulates numerous cellular processes, including cell cycle control and protein quality control. A component of this system, the deubiquitinating enzyme USP14, associates with the proteasome where it can rescue substrates from degradation by removal of the ubiquitin tag. We previously found that a small-molecule inhibitor of USP14, known as IU1, can increase the rate of degradation of a subset of proteasome substrates. We report here the synthesis and characterization of 87 variants of IU1, which resulted in the identification of a 10-fold more potent USP14 inhibitor that retains specificity for USP14. The capacity of this compound, IU1-47, to enhance protein degradation in cells was tested using as a reporter the microtubule-associated protein tau, which has been implicated in many neurodegenerative diseases. Using primary neuronal cultures, IU1-47 was found to accelerate the rate of degradation of wild-type tau, the pathological tau mutants P301L and P301S, and the A152T tau variant. We also report that a specific residue in tau, lysine 174, is critical for the IU1-47-mediated tau degradation by the proteasome. Finally, we show that IU1-47 stimulates autophagic flux in primary neurons. In summary, these findings provide a powerful research tool for investigating the complex biology of USP14.


Assuntos
Embrião de Mamíferos/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Neurônios/metabolismo , Pirróis/farmacologia , Ubiquitina Tiolesterase/fisiologia , Proteínas tau/metabolismo , Animais , Células Cultivadas , Citoplasma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Pirróis/síntese química , Ratos Sprague-Dawley , Ubiquitina/metabolismo , Ubiquitinação
10.
Neuron ; 90(2): 245-60, 2016 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-27041503

RESUMO

Tau toxicity has been implicated in the emergence of synaptic dysfunction in Alzheimer's disease (AD), but the mechanism by which tau alters synapse physiology and leads to cognitive decline is unclear. Here we report abnormal acetylation of K274 and K281 on tau, identified in AD brains, promotes memory loss and disrupts synaptic plasticity by reducing postsynaptic KIdney/BRAin (KIBRA) protein, a memory-associated protein. Transgenic mice expressing human tau with lysine-to-glutamine mutations to mimic K274 and K281 acetylation (tauKQ) exhibit AD-related memory deficits and impaired hippocampal long-term potentiation (LTP). TauKQ reduces synaptic KIBRA levels and disrupts activity-induced postsynaptic actin remodeling and AMPA receptor insertion. The LTP deficit was rescued by promoting actin polymerization or by KIBRA expression. In AD patients with dementia, we found enhanced tau acetylation is linked to loss of KIBRA. These findings suggest a novel mechanism by which pathogenic tau causes synaptic dysfunction and cognitive decline in AD pathogenesis.


Assuntos
Actinas/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Transtornos da Memória/fisiopatologia , Plasticidade Neuronal/fisiologia , Receptores de AMPA/metabolismo , Transdução de Sinais , Proteínas tau/metabolismo , Acetilação , Doença de Alzheimer/metabolismo , Animais , Hipocampo/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Potenciação de Longa Duração/genética , Potenciação de Longa Duração/fisiologia , Transtornos da Memória/genética , Camundongos , Camundongos Transgênicos , Fosfoproteínas , Cultura Primária de Células , Proteínas tau/genética
11.
Nat Med ; 21(10): 1154-62, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26390242

RESUMO

Tauopathies, including frontotemporal dementia (FTD) and Alzheimer's disease (AD), are neurodegenerative diseases in which tau fibrils accumulate. Recent evidence supports soluble tau species as the major toxic species. How soluble tau accumulates and causes neurodegeneration remains unclear. Here we identify tau acetylation at Lys174 (K174) as an early change in AD brains and a critical determinant in tau homeostasis and toxicity in mice. The acetyl-mimicking mutant K174Q slows tau turnover and induces cognitive deficits in vivo. Acetyltransferase p300-induced tau acetylation is inhibited by salsalate and salicylate, which enhance tau turnover and reduce tau levels. In the PS19 transgenic mouse model of FTD, administration of salsalate after disease onset inhibited p300 activity, lowered levels of total tau and tau acetylated at K174, rescued tau-induced memory deficits and prevented hippocampal atrophy. The tau-lowering and protective effects of salsalate were diminished in neurons expressing K174Q tau. Targeting tau acetylation could be a new therapeutic strategy against human tauopathies.


Assuntos
Transtornos Cognitivos/fisiopatologia , Doenças Neurodegenerativas/fisiopatologia , Proteínas tau/fisiologia , Acetilação , Animais , Comportamento Animal , Humanos , Camundongos , Proteínas tau/metabolismo
12.
Neuron ; 84(2): 416-31, 2014 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-25374362

RESUMO

Synaptic vesicle docking, priming, and fusion at active zones are orchestrated by a complex molecular machinery. We employed hippocampal organotypic slice cultures from mice lacking key presynaptic proteins, cryofixation, and three-dimensional electron tomography to study the mechanism of synaptic vesicle docking in the same experimental setting, with high precision, and in a near-native state. We dissected previously indistinguishable, sequential steps in synaptic vesicle active zone recruitment (tethering) and membrane attachment (docking) and found that vesicle docking requires Munc13/CAPS family priming proteins and all three neuronal SNAREs, but not Synaptotagmin-1 or Complexins. Our data indicate that membrane-attached vesicles comprise the readily releasable pool of fusion-competent vesicles and that synaptic vesicle docking, priming, and trans-SNARE complex assembly are the respective morphological, functional, and molecular manifestations of the same process, which operates downstream of vesicle tethering by active zone components.


Assuntos
Proteínas SNARE/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Hipocampo/metabolismo , Fusão de Membrana/fisiologia , Camundongos , Neurônios/metabolismo , Neurônios/ultraestrutura , Sinapses/ultraestrutura
13.
Nat Med ; 20(10): 1157-64, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25261995

RESUMO

Haploinsufficiency of the progranulin (PGRN) gene (GRN) causes familial frontotemporal lobar degeneration (FTLD) and modulates an innate immune response in humans and in mouse models. GRN polymorphism may be linked to late-onset Alzheimer's disease (AD). However, the role of PGRN in AD pathogenesis is unknown. Here we show that PGRN inhibits amyloid ß (Aß) deposition. Selectively reducing microglial expression of PGRN in AD mouse models impaired phagocytosis, increased plaque load threefold and exacerbated cognitive deficits. Lentivirus-mediated PGRN overexpression lowered plaque load in AD mice with aggressive amyloid plaque pathology. Aß plaque load correlated negatively with levels of hippocampal PGRN, showing the dose-dependent inhibitory effects of PGRN on plaque deposition. PGRN also protected against Aß toxicity. Lentivirus-mediated PGRN overexpression prevented spatial memory deficits and hippocampal neuronal loss in AD mice. The protective effects of PGRN against Aß deposition and toxicity have important therapeutic implications. We propose enhancing PGRN as a potential treatment for PGRN-deficient FTLD and AD.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cognição/fisiologia , Modelos Animais de Doenças , Feminino , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/terapia , Regulação da Expressão Gênica , Granulinas , Humanos , Imunidade Inata/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Fagocitose , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Progranulinas , Ratos , Regulação para Cima
14.
J Neurosci ; 33(42): 16698-714, 2013 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-24133272

RESUMO

Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms.


Assuntos
Exocitose/fisiologia , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Sintaxina 1/metabolismo , Animais , Linhagem Celular , Hipocampo/citologia , Hipocampo/metabolismo , Fusão de Membrana/fisiologia , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Vesículas Sinápticas/genética , Sintaxina 1/genética
15.
Front Aging Neurosci ; 5: 53, 2013 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-24093018

RESUMO

Silent information regulator 2 proteins (sirtuins or SIRTs) are a group of deacetylases (or deacylases) whose activities are dependent on and regulated by nicotinamide adenine dinucleotide (NAD(+)). Compelling evidence supports that sirtuins play major roles in many aspects of physiology, especially in pathways related to aging - the predominant and unifying risk factor for neurodegenerative diseases. In this review, we highlight the molecular mechanisms underlying the protective effects of sirtuins in neurodegenerative diseases, focusing on protein homeostasis, neural plasticity, mitochondrial function, and sustained chronic inflammation. We will also examine the potential and challenges of targeting sirtuin pathways to block these pathogenic pathways.

16.
J Clin Invest ; 122(11): 3955-9, 2012 11.
Artigo em Inglês | MEDLINE | ID: mdl-23041626

RESUMO

Progranulin (PGRN) is a widely expressed secreted protein that is linked to inflammation. In humans, PGRN haploinsufficiency is a major inherited cause of frontotemporal dementia (FTD), but how PGRN deficiency causes neurodegeneration is unknown. Here we show that loss of PGRN results in increased neuron loss in response to injury in the CNS. When exposed acutely to 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydrophine (MPTP), mice lacking PGRN (Grn⁻/⁻) showed more neuron loss and increased microgliosis compared with wild-type mice. The exacerbated neuron loss was due not to selective vulnerability of Grn⁻/⁻ neurons to MPTP, but rather to an increased microglial inflammatory response. Consistent with this, conditional mutants lacking PGRN in microglia exhibited MPTP-induced phenotypes similar to Grn⁻/⁻ mice. Selective depletion of PGRN from microglia in mixed cortical cultures resulted in increased death of wild-type neurons in the absence of injury. Furthermore, Grn⁻/⁻ microglia treated with LPS/IFN-γ exhibited an amplified inflammatory response, and conditioned media from these microglia promoted death of cultured neurons. Our results indicate that PGRN deficiency leads to dysregulated microglial activation and thereby contributes to increased neuron loss with injury. These findings suggest that PGRN deficiency may cause increased neuron loss in other forms of CNS injury accompanied by neuroinflammation.


Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Intoxicação por MPTP/metabolismo , Microglia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurotoxinas/efeitos adversos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Granulinas , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Intoxicação por MPTP/genética , Intoxicação por MPTP/patologia , Camundongos , Camundongos Knockout , Microglia/patologia , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Neurotoxinas/farmacologia , Progranulinas
17.
Neuron ; 67(6): 953-66, 2010 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-20869593

RESUMO

Neurodegenerative tauopathies characterized by hyperphosphorylated tau include frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease (AD). Reducing tau levels improves cognitive function in mouse models of AD and FTDP-17, but the mechanisms regulating the turnover of pathogenic tau are unknown. We found that tau is acetylated and that tau acetylation prevents degradation of phosphorylated tau (p-tau). We generated two antibodies specific for acetylated tau and showed that tau acetylation is elevated in patients at early and moderate Braak stages of tauopathy. Histone acetyltransferase p300 was involved in tau acetylation and the class III protein deacetylase SIRT1 in deacetylation. Deleting SIRT1 enhanced levels of acetylated-tau and pathogenic forms of p-tau, probably by blocking proteasome-mediated degradation. Inhibiting p300 with a small molecule promoted tau deacetylation and eliminated p-tau associated with tauopathy. Modulating tau acetylation could be a new therapeutic strategy to reduce tau-mediated neurodegeneration.


Assuntos
Tauopatias/etiologia , Tauopatias/metabolismo , Proteínas tau/metabolismo , Acetilação/efeitos dos fármacos , Análise de Variância , Animais , Animais Recém-Nascidos , Carbazóis/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Cicloeximida/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoprecipitação/métodos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Sprague-Dawley , Sirtuína 1/genética , Sirtuína 1/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tauopatias/genética , Fatores de Tempo , Transfecção/métodos , Ubiquitinação/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/fisiologia , Proteínas tau/genética
18.
Science ; 321(5895): 1507-10, 2008 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-18703708

RESUMO

During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a "closed" conformation outside the SNARE complex and an "open" conformation in the SNARE complex. Although SNARE complexes containing open syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed syntaxin-1 is unknown. We generated knockin/knockout mice that expressed only open syntaxin-1B. Syntaxin-1B(Open) mice were viable but succumbed to generalized seizures at 2 to 3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1B(Open) synapses, and the size of the readily releasable vesicle pool was decreased; however, the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.


Assuntos
Vesículas Sinápticas/fisiologia , Sintaxina 1/química , Sintaxina 1/metabolismo , Animais , Cálcio/metabolismo , Epilepsia/etiologia , Potenciais Pós-Sinápticos Excitadores , Fusão de Membrana , Camundongos , Camundongos Knockout , Proteínas Munc18/metabolismo , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas SNARE/metabolismo , Sacarose/metabolismo , Sinapses/fisiologia , Vesículas Sinápticas/ultraestrutura , Sintaxina 1/genética
19.
Proc Natl Acad Sci U S A ; 104(10): 3823-8, 2007 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-17360437

RESUMO

C(2) domains are autonomously folded protein modules that generally act as Ca(2+)- and phospholipid-binding domains and/or as protein-protein interaction domains. We now report the primary structures and biochemical properties of a family of evolutionarily conserved mammalian proteins, referred to as E-Syts, for extended synaptotagmin-like proteins. E-Syts contain an N-terminal transmembrane region, a central juxtamembranous domain that is conserved from yeast to human, and five (E-Syt1) or three (E-Syt2 and E-Syt3) C-terminal C(2) domains. Only the first E-Syt C(2) domain, the C(2)A domain, includes the complete sequence motif that is required for Ca(2+) binding in C(2) domains. Recombinant protein fragments of E-Syt2 that include the first C(2) domain are capable of Ca(2+)-dependent phospholipid binding at micromolar concentrations of free Ca(2+), suggesting that E-Syts bind Ca(2+) through their first C(2) domain in a phospholipid complex. E-Syts are ubiquitously expressed, but enriched in brain. Expression of myc-tagged E-Syt proteins in transfected cells demonstrated localization to intracellular membranes for E-Syt1 and to plasma membranes for E-Syt2 and E-Syt3. Structure/function studies showed that the plasma-membrane localization of E-Syt2 and E-Syt3 was directed by their C-terminal C(2)C domains. This result reveals an unexpected mechanism by which the C(2)C domains of E-Syt2 and E-Syt3 functions as a targeting motif that localizes these proteins into the plasma membrane independent of their transmembrane region. Viewed together, our findings suggest that E-Syts function as Ca(2+)-regulated intrinsic membrane proteins with multiple C(2) domains, expanding the repertoire of such proteins to a fourth class beyond synaptotagmins, ferlins, and MCTPs (multiple C(2) domain and transmembrane region proteins).


Assuntos
Cálcio/metabolismo , Sinaptotagminas/fisiologia , Motivos de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Sequência Conservada , Evolução Molecular , Exocitose , Humanos , Dados de Sequência Molecular , Família Multigênica , Fosfolipídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Sinaptotagminas/química
20.
Proc Natl Acad Sci U S A ; 100(1): 32-7, 2003 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-12506202

RESUMO

Sec1Munc18-like (SM) proteins functionally interact with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) in membrane fusion, but the mechanisms of these interactions differ. In vertebrates, SM proteins that mediate exocytosis (Munc18-1, 18-2, and 18c) bind to the closed conformation of syntaxins 1-4, which requires the N-terminal H(abc) domains and SNARE motifs of these syntaxins. In contrast, SM proteins that mediate Golgi and endoplasmic reticulum fusion (Sly1 and Vps45) bind only to short N-terminal sequences of syntaxins 5, 16, or 18, independently of their H(abc) domains and SNARE motifs. We now show that Munc18-1, Sly1, and Vps45 interact with cognate syntaxins via similar, autonomously folded N-terminal domains, but the syntaxin 5-binding surface of the Sly1 N-terminal domain is opposite to the syntaxin 1-binding surface of the Munc18-1 N-terminal domain. In transfected cells, the N-terminal domain of Sly1 specifically disrupts the structure of the Golgi complex, supporting the notion that the interaction of Sly1 with syntaxin 5 is essential for fusion. These data, together with previous results, suggest that a relatively small N-terminal domain of SM proteins is dedicated to mechanistically distinct interactions with SNAREs, leaving the remaining large parts of SM proteins free to execute their as yet unknown function as effector domains.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Animais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Chlorocebus aethiops , Clonagem Molecular , Escherichia coli/genética , Modelos Moleculares , Proteínas Munc18 , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusão/metabolismo , Proteínas SNARE , Proteínas de Saccharomyces cerevisiae/metabolismo , Transfecção , Células Vero
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