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1.
Biosci Biotechnol Biochem ; 84(1): 1-16, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31538538

RESUMO

Recent investigations suggest that soluble oligomeric amyloid ß (Aß) species may be involved in early onset of Alzheimer's disease (AD). Using systematic proline replacement, solid-state NMR, and ESR, we identified a toxic turn at position 22 and 23 of Aß42, the most potent neurotoxic Aß species. Through radicalization, the toxic turn can induce formation of the C-terminal hydrophobic core to obtain putative Aß42 dimers and trimers. Synthesized dimer and trimer models showed that the C-terminal hydrophobic core plays a critical role in the formation of high molecular weight oligomers with neurotoxicity. Accordingly, an anti-toxic turn antibody (24B3) that selectively recognizes a toxic dimer model of E22P-Aß42 was developed. Sandwich enzyme-linked immunosorbent assay with 24B3 and 82E1 detected a significantly higher ratio of Aß42 with a toxic turn to total Aß42 in cerebrospinal fluid of AD patients compared with controls, suggesting that 24B3 could be useful for early onset of AD diagnosis.


Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais/uso terapêutico , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fragmentos de Peptídeos/imunologia , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/química , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Modelos Moleculares , Emaranhados Neurofibrilares/química , Fragmentos de Peptídeos/química , Placa Amiloide/química , Prolina/química , Agregados Proteicos , Agregação Patológica de Proteínas , Estrutura Terciária de Proteína
2.
Biosens Bioelectron ; 133: 183-191, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30928737

RESUMO

Alzheimer's disease (AD) is a chronic central neurodegenerative disease. The pathological features of AD are the extracellular deposition of senile plaques formed by amyloid-ß oligomers (AßOs) and the intracellular accumulation of neurofibrillary tangles. However, due to the lack of effective method and experimental models to study the cognitive decline, communication at cell resolution and the implementation of interventions, the diagnosis and treatment on AD still progress slowly. In this paper, we established a pathological model of AD in vitro based on AßOs-induced hippocampal neuronal network chip for multi-site dynamic analysis of the neuronal electrical activity and network connection. The multiple characteristic parameters, including positive and negative spike intervals, firing rate and peak-to-peak values, were extracted through the analysis of spike signals, and two firing patterns from the interneurons and pyramidal neurons were recorded. The spatial firing patterns mapping and cross-correlation between channels were performed to validate the degeneration of neuronal network connectivity. Moreover, an electrical stimulation with frequency at 40 Hz was exerted to preliminarily explore the therapeutic effect on the pathological model of AD. This neuronal network chip enables the implementation of AD models in vitro for studying basic mechanisms of neurodegeneration within networks and for the parallel testing of various potential therapies. It can be a novel technique in the research of AD pathological model in vitro.


Assuntos
Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/isolamento & purificação , Técnicas Biossensoriais , Sistemas Microeletromecânicos/métodos , Peptídeos beta-Amiloides/química , Estimulação Elétrica , Eletrólitos/química , Hipocampo/química , Hipocampo/efeitos da radiação , Humanos , Interneurônios/química , Interneurônios/efeitos da radiação , Dispositivos Lab-On-A-Chip , Rede Nervosa/química , Rede Nervosa/efeitos da radiação , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/efeitos da radiação , Células Piramidais/química , Células Piramidais/efeitos da radiação
3.
J Mol Biol ; 430(21): 4119-4131, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30121297

RESUMO

Alzheimer's disease is a tauopathy characterized by pathological fibrillization of tau protein to form the paired helical filaments (PHFs), which constitute neurofibrillary tangles. The methylthioninium (MT) moiety reverses the proteolytic stability of the PHF core and is in clinical development for treatment of Alzheimer's disease in a stable reduced form as leuco-MT. It has been hypothesized that MT acts via oxidation of cysteine residues, which is incompatible with activity in the predominantly reducing environment of living cells. We have shown recently that the PHF-core tau unit assembles spontaneously in vitro to form PHF-like filaments. Here we describe studies using circular dichroism, SDS-PAGE, transmission electron microscopy and site-directed mutagenesis to elucidate the mechanism of action of the MT moiety. We show that MT inhibitory activity is optimal in reducing conditions, that the active moiety is the reduced leuco-MT form of the molecule and that its mechanism of action is cysteine independent.


Assuntos
Cisteína/metabolismo , Azul de Metileno/análogos & derivados , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Heparina/química , Humanos , Azul de Metileno/química , Estrutura Molecular , Emaranhados Neurofibrilares/ultraestrutura , Proteínas Recombinantes , Análise Espectral
4.
Methods Mol Biol ; 1779: 99-111, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29886530

RESUMO

Deposition of Tau aggregates in patient's brains is a hallmark of several neurodegenerative diseases collectively called Tauopathies. One of the most studied Tauopathies is Alzheimer disease (AD) in which Tau protein aggregates into filaments and coalesces into neurofibrillary tangles. The distribution of Tau filaments is a reliable indicator of the clinical stages of AD (Braak stages), but intermediate oligomeric assemblies of Tau are considered to be more directly toxic to neurons than late stage filaments. Studying the elusive role of Tau oligomers has been difficult because of their dynamic nature and paucity of methods to purify them in vitro. In this chapter, we describe methods to purify Tau oligomers to near homogeneity and to characterize them by hydrophobic interaction chromatography and biophysical methods such as fluorescence spectrophotometry, dynamic light scattering, atomic force microscopy, and others. Functional characterization includes the assessment of synapses and toxicity assays which show that oligomers can damage synapses locally but show little toxicity to neurons globally.


Assuntos
Doença de Alzheimer/metabolismo , Proteínas tau/química , Proteínas tau/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Difusão Dinâmica da Luz , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Emaranhados Neurofibrilares/química , Multimerização Proteica
5.
J Biol Chem ; 293(7): 2408-2421, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29259137

RESUMO

The accumulation of aberrantly aggregated MAPT (microtubule-associated protein Tau) defines a spectrum of tauopathies, including Alzheimer's disease. Mutations in the MAPT gene cause frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), characterized by neuronal pathological Tau inclusions in the form of neurofibrillary tangles and Pick bodies and in some cases glial Tau pathology. Increasing evidence points to the importance of prion-like seeding as a mechanism for the pathological spread in tauopathy and other neurodegenerative diseases. Herein, using a cell culture model, we examined a multitude of genetic FTDP-17 Tau variants for their ability to be seeded by exogenous Tau fibrils. Our findings revealed stark differences between FTDP-17 Tau variants in their ability to be seeded, with variants at Pro301 and Ser320 showing robust aggregation with seeding. Similarly, we elucidated the importance of certain Tau protein regions and unique residues, including the role of Pro301 in inhibiting Tau aggregation. We also revealed potential barriers in cross-seeding between three-repeat and four-repeat Tau isoforms. Overall, these differences alluded to potential mechanistic differences between wildtype and FTDP-17 Tau variants, as well as different Tau isoforms, in influencing Tau aggregation. Furthermore, by combining two FTDP-17 Tau variants (either P301L or P301S with S320F), we generated aggressive models of tauopathy that do not require exogenous seeding. These models will allow for rapid screening of potential therapeutics to alleviate Tau aggregation without the need for exogenous Tau fibrils. Together, these studies provide novel insights in the molecular determinants that modulate Tau aggregation.


Assuntos
Tauopatias/metabolismo , Proteínas tau/metabolismo , Motivos de Aminoácidos , Humanos , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/genética , Emaranhados Neurofibrilares/metabolismo , Príons/química , Príons/genética , Príons/metabolismo , Agregados Proteicos , Tauopatias/genética , Proteínas tau/química , Proteínas tau/genética
6.
J Biol Chem ; 293(8): 2888-2902, 2018 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-29282295

RESUMO

Amyloid-ß (Aß) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aß and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aß and hIAPP, termed Aß(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aß and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aß(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aß and hIAPP.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Modelos Moleculares , Emaranhados Neurofibrilares/metabolismo , Fragmentos de Peptídeos/metabolismo , Substituição de Aminoácidos , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Animais , Linhagem Celular Tumoral , Biologia Computacional , Cristalografia por Raios X , Desenho de Drogas , Células HEK293 , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/ultraestrutura , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/efeitos dos fármacos , Emaranhados Neurofibrilares/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Nootrópicos/química , Nootrópicos/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Agregação Patológica de Proteínas/prevenção & controle , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
7.
Sci Rep ; 7(1): 15603, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29142266

RESUMO

Neuritic plaques and neurofibrillary tangles are crucial morphological criteria for the definite diagnosis of Alzheimer's disease. We evaluated 12 unstained frontal cortex and hippocampus samples from 3 brain donors with Alzheimer's disease and 1 control with hyperspectral Raman microscopy on samples of 30 × 30 µm. Data matrices of 64 × 64 pixels were used to quantify different tissue components including proteins, lipids, water and beta-sheets for imaging at 0.47 µm spatial resolution. Hierarchical cluster analysis was performed to visualize regions with high Raman spectral similarities. The Raman images of proteins, lipids, water and beta-sheets matched with classical brain morphology. Protein content was 2.0 times, the beta-sheet content 5.6 times and Raman broad-band autofluorescence was 2.4 times higher inside the plaques and tangles than in the surrounding tissue. The lipid content was practically equal inside and outside. Broad-band autofluorescence showed some correlation with protein content and a better correlation with beta-sheet content. Hyperspectral Raman imaging combined with hierarchical cluster analysis allows for the identification of neuritic plaques and neurofibrillary tangles in unstained, label-free slices of human Alzheimer's disease brain tissue. It permits simultaneous quantification and distinction of several tissue components such as proteins, lipids, water and beta-sheets.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Emaranhados Neurofibrilares/química , Placa Amiloide/química , Idoso , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/patologia , Análise por Conglomerados , Feminino , Hipocampo/diagnóstico por imagem , Hipocampo/metabolismo , Humanos , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/patologia , Microscopia Óptica não Linear , Placa Amiloide/patologia , Proteínas tau/química , Proteínas tau/metabolismo
8.
ACS Chem Neurosci ; 8(7): 1459-1464, 2017 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-28453930

RESUMO

The antioxidant activity of selenium, which is mainly conferred by its incorporation into dedicated selenoproteins, has been suggested as a possible neuroprotective approach for mitigating neuronal loss in Alzheimer's disease. However, there is inconsistent information with respect to selenium levels in the Alzheimer's disease brain. We examined the concentration and cellular compartmentalization of selenium in the temporal cortex of Alzheimer's disease and control brain tissue. We found that Alzheimer's disease was associated with decreased selenium concentration in both soluble (i.e., cytosolic) and insoluble (i.e., plaques and tangles) fractions of brain homogenates. The presence of the APOE ε4 allele correlated with lower total selenium levels in the temporal cortex and a higher concentration of soluble selenium. Additionally, we found that age significantly contributed to lower selenium concentrations in the peripheral membrane-bound and vesicular fractions. Our findings suggest a relevant interaction between APOE ε4 and selenium delivery into brain, and show changes in cellular selenium distribution in the Alzheimer's disease brain.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Apolipoproteína E4/genética , Química Encefálica/genética , Selênio/análise , Lobo Temporal/química , Idoso , Envelhecimento/genética , Envelhecimento/metabolismo , Citosol/química , Feminino , Heterozigoto , Humanos , Masculino , Espectrometria de Massas , Emaranhados Neurofibrilares/química , Placa Amiloide/química
9.
J Recept Signal Transduct Res ; 36(5): 445-58, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27305963

RESUMO

Glycogen synthase kinase-3ß (GSK-3ß) is a serine/threonine kinase which has attracted significant attention during recent years in drug design studies. The deregulation of GSK-3ß increased the loss of hippocampal neurons by triggering apoptosis-mediating production of neurofibrillary tangles and alleviates memory deficits in Alzheimer's disease (AD). Given its role in the formation of neurofibrillary tangles leading to AD, it has been a major therapeutic target for intervention in AD, hence was targeted in the present study. Twenty crystal structures were refined to generate pharmacophore models based on energy involvement in binding co-crystal ligands. Four common e-pharmacophore models were optimized from the 20 pharmacophore models. Shape-based screening of four e-pharmacophore models against nine established small molecule databases using Phase v3.9 had resulted in 1800 compounds having similar pharmacophore features. Rigid receptor docking (RRD) was performed for 1800 compounds and 20 co-crystal ligands with GSK-3ß to generate dock complexes. Interactions of the best scoring lead obtained through RRD were further studied with quantum polarized ligand docking (QPLD), induced fit docking (IFD) and molecular mechanics/generalized Born surface area. Comparing the obtained leads to 20 co-crystal ligands resulted in 18 leads among them, lead1 had the lowest docking score, lower binding free energy and better binding orientation toward GSK-3ß. The 50 ns MD simulations run confirmed the stable nature of GSK-3ß-lead1 docking complex. The results from RRD, QPLD, IFD and MD simulations confirmed that lead1 might be used as a potent antagonist for GSK-3ß.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta/química , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Doença de Alzheimer/patologia , Apoptose/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Drogas , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Humanos , Ligantes , Memória/efeitos dos fármacos , Simulação de Acoplamento Molecular , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/efeitos dos fármacos , Emaranhados Neurofibrilares/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ligação Proteica , Bibliotecas de Moléculas Pequenas/uso terapêutico , Interface Usuário-Computador
10.
ACS Chem Neurosci ; 7(7): 995-1003, 2016 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-27225823

RESUMO

The formation of tau aggregates is strongly linked to the neurodegenerative process in tauopathies such as Alzheimer's disease (AD). Yet only a few molecules have shown to efficiently prevent the in vitro formation of those aggregates, and the identification of such molecules is still an ongoing interest in a therapeutic context. Herein, we report the in vitro evaluation of a series of aurones against the fibrillation of the tau-derived hexapeptide AcPHF6 model. Using thioflavin T-based fluorescence assays, circular dichroism and atomic force microscopy, we showed that aurones are capable of efficiently interacting with the tau-derived hexapeptide. Importantly, this work reveals a significant activity observed for polyhydroxylated aurones. In particular, aurone 23 displayed an almost complete inhibition of fibers formation as shown by AFM at a peptide/inhibitor 1:1 ratio. It is similar to that observed for myricetin, a polyphenolic compound, well-known to prevent the in vitro elongation of tau fibers. Moreover, a tetrahydroxylated isomer, compound 24, was shown as a chemical probe of fibers rather than an inhibitor. Consequently, these results highlight aurones as a new promising scaffold to interfere with tau aggregation for both treatment and diagnosis of AD.


Assuntos
Benzofuranos/química , Benzofuranos/síntese química , Modelos Químicos , Emaranhados Neurofibrilares/metabolismo , Peptídeos/síntese química , Proteínas tau/química , Dicroísmo Circular/métodos , Fluorescência , Humanos , Microscopia de Força Atômica , Emaranhados Neurofibrilares/química , Peptídeos/química , Proteínas tau/metabolismo
13.
Adv Exp Med Biol ; 821: 11-7, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25416106

RESUMO

Neuropathological hallmarks of Alzheimer's disease (AD) include tangles (NFT) and beta amyloid (Aß) plaques. Despite numerous neuropathological studies that assessed the relationship of cognitive decline with neuropathologic lesions, their correlation still remains unclear. NFTs and Aß plaques have been widely implicated and described in normal aging. The number of NFTs in the CA1 and the entorhinal cortex seems to be more closely related to cognitive status, compared to the amyloid load whose role still remains controversial in the AD. In this review, we refer to our main studies performed in Geneva during the past two decades attempting to assess the correlation of pathology with clinical expression. The theory of cognitive reserve has been proposed for further understanding of interindividual differences in terms of compensation despite the presence of pathological lesions. The increasing prevalence of the AD, the limitations of actual treatments, as well as the high public cost reflect the imperative need for better therapeutic and early diagnosis strategies in the future.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Emaranhados Neurofibrilares/metabolismo , Placa Amiloide/metabolismo , Fatores Etários , Idoso , Envelhecimento/patologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Encéfalo/patologia , Química Encefálica , Humanos , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/patologia , Placa Amiloide/patologia , Índice de Gravidade de Doença
14.
Int J Clin Exp Pathol ; 7(3): 938-47, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24696712

RESUMO

Vascular dementia (VaD) is the second most common form of dementia and is currently defined as a cerebral vessel vascular disease leading to ischemic episodes. Apolipoprotein E (apoE) gene polymorphism has been proposed as a risk factor for VaD, however, to date there are few documented post-mortem studies on apoE pathology in the VaD brain. To investigate a potential role for the apoE protein, we analyzed seven confirmed cases of VaD by immunohistochemistry utilizing an antibody that specifically detects the amino-terminal fragment of apoE. Application of this antibody, termed N-terminal, apoE cleavage fragment (nApoECF) revealed consistent labeling within neurofibrillary tangles (NFTs), blood vessels, and reactive astrocytes. Labeling occurred in VaD cases that had confirmed APOE genotypes of 3/3, 3/4, and 4/4, with respect to NFTs, staining of the nApoECF co-localized with PHF-1 and was predominantly localized to large, stellate neurons in layer II of the entorhinal cortex. Quantitative analysis indicated that approximately 38.4% of all identified NFTs contained the amino-terminal fragment of apoE. Collectively, these data support a role for the proteolytic cleavage of apoE in the VaD and support previous reports that APOE polymorphism is significantly associated with susceptibility in this disease.


Assuntos
Apolipoproteínas E/metabolismo , Demência Vascular/patologia , Emaranhados Neurofibrilares/patologia , Idoso , Idoso de 80 Anos ou mais , Demência Vascular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/metabolismo
15.
Pathol Biol (Paris) ; 62(3): 162-8, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24698014

RESUMO

Prion protein and prion-like proteins share a number of characteristics. From the molecular point of view, they are constitutive proteins that aggregate following conformational changes into insoluble particles. These particles escape the cellular clearance machinery and amplify by recruiting the soluble for of their constituting proteins. The resulting protein aggregates are responsible for a number of neurodegenerative diseases such as Creutzfeldt-Jacob, Alzheimer, Parkinson and Huntington diseases. In addition, there are increasing evidences supporting the inter-cellular trafficking of these aggregates, meaning that they are "transmissible" between cells. There are also evidences that brain homogenates from individuals developing Alzheimer and Parkinson diseases propagate the disease in recipient model animals in a manner similar to brain extracts of patients developing Creutzfeldt-Jacob's disease. Thus, the propagation of protein aggregates from cell to cell may be a generic phenomenon that contributes to the evolution of neurodegenerative diseases, which has important consequences on human health issues. Moreover, although the distribution of protein aggregates is characteristic for each disease, new evidences indicate the possibility of overlaps and crosstalk between the different disorders. Despite the increasing evidences that support prion or prion-like propagation of protein aggregates, there are many unanswered questions regarding the mechanisms of toxicity and this is a field of intensive research nowadays.


Assuntos
Proteínas do Tecido Nervoso/química , Doenças Neurodegenerativas/metabolismo , Doenças Priônicas/metabolismo , Agregação Patológica de Proteínas/metabolismo , Envelhecimento , Doença de Alzheimer/prevenção & controle , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Autofagia , Biopolímeros , Ensaios Clínicos Fase II como Assunto , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Endocitose , Humanos , Corpos de Inclusão/química , Corpos de Inclusão/patologia , Camundongos , Doenças Neurodegenerativas/patologia , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/patologia , Placa Amiloide/química , Placa Amiloide/patologia , Polissacarídeos/uso terapêutico , Doenças Priônicas/patologia , Doenças Priônicas/veterinária , Príons/química , Agregação Patológica de Proteínas/patologia , Conformação Proteica , Solubilidade
16.
Zhonghua Bing Li Xue Za Zhi ; 43(10): 651-6, 2014 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-25567589

RESUMO

OBJECTIVE: To recognize relationship of protein related neurodegeneration abnormal aggregation in the aged brains with their cognitive and motor functions. METHODS: Brain tissues from the consecutive autopsy cases of the aged from January 2005 to December 2006 in PLA General Hospital were carried out for immunohistochemical staining with beta amyloid, tau, α-synuclein and ubiquitin antibodies. The consortium to establish a registry for Alzheimer's disease (CERAD) was used to semi-quantitatively analyze Aß positive core plaques density and Braak staging for tau positive neurofibrillary tangles (NFTs) and α-synuclein positive Lewy bodies. In addition, Aß positive cerebral amyloid angiopathy (CAA), neuritic plaques and various ubiquitin positive structures were also observed. The relationship of these protein abnormal depositions in the aged brains with cognitive and motor functions were analyzed. RESULTS: In brain tissues of 16 consecutive autopsy cases of the aged from 78 to 95 years, there were 13 cases with Aß positive core plaques, their density was 2 cases with sparse, 2 cases with moderate and 9 cases with frequent, respectively, according to CREAD.Eight cases with Aß positive CAA were found, including 6 cases of mild CAA and 2 cases of severe CAA. There were 12 cases with tau positive NFTs, including 6 cases with Braak stageI-II, 4 cases with stage III-IV and 2 cases with stage V-VI. There were 5 cases with frequent Aß core plaques, meanwhile existing numerous tau/ubiquitin positive neuritic plaques and Braak stage IV-VI of tau positive NFTs, all of them presented cognitive dysfunction. Among 4 other cases with frequent Aß core plaques, only one case coexisted α-synuclein positive Lewy bodies showed moderate cognitive impairment, remaining 3 cases did not present cognitive dysfunction. There were 4 cases with α-synuclein positive Lewy bodies in the brainstem, and all of these cases presented parkinsonian motor dysfunction. 13 cases with ubiquitin positive structures were found. CONCLUSIONS: Beta amyloid protein positive deposit in the aged brain is an important marker of normal brain aging and cognitive impairment; frequent Aß core plaques in the neocortex plus Braak IV and above tau positive NFTs are closely related to cognitive dysfunction of Alzheimer's disease; α-synuclein positive Lewy bodies in the brainstem is one of the important pathological markers of parkinsonian motor disorders; ubiquitin deposition involves the development of some characteristic structures of several neurodegenerative diseases.


Assuntos
Doença de Alzheimer/metabolismo , Química Encefálica , Encéfalo/patologia , Emaranhados Neurofibrilares/química , Idoso , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/análise , Autopsia , Angiopatia Amiloide Cerebral , Humanos , Emaranhados Neurofibrilares/patologia , Placa Amiloide , Ubiquitina/análise , alfa-Sinucleína/análise , Proteínas tau/análise
17.
ACS Chem Neurosci ; 4(11): 1488-500, 2013 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-24007594

RESUMO

Recent epidemiological data have shown that patients suffering from Type 2 Diabetes Mellitus have an increased risk to develop Alzheimer's disease and vice versa. A possible explanation is the cross-sequence interaction between Aß and amylin. Because the resulting amyloid oligomers are difficult to probe in experiments, we investigate stability and conformational changes of Aß-amylin heteroassemblies through molecular dynamics simulations. We find that Aß is a good template for the growth of amylin and vice versa. We see water molecules permeate the ß-strand-turn-ß-strand motif pore of the oligomers, supporting a commonly accepted mechanism for toxicity of ß-rich amyloid oligomers. Aiming for a better understanding of the physical mechanisms of cross-seeding and cell toxicity of amylin and Aß aggregates, our simulations also allow us to identify targets for the rational design of inhibitors against toxic fibril-like oligomers of Aß and amylin oligomers.


Assuntos
Peptídeos beta-Amiloides/efeitos adversos , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/efeitos adversos , Emaranhados Neurofibrilares/química , Motivos de Aminoácidos/efeitos dos fármacos , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/patologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Polipeptídeo Amiloide das Ilhotas Pancreáticas/biossíntese , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Modelos Químicos , Simulação de Dinâmica Molecular , Emaranhados Neurofibrilares/efeitos dos fármacos , Emaranhados Neurofibrilares/patologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Proteínas tau/química , Proteínas tau/metabolismo
19.
Brain Res ; 1475: 106-15, 2012 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-22902767

RESUMO

Although the risk factor for harboring the apolipoprotein E4 (apoE4) allele in late-onset Alzheimer's disease (AD) is well known, the mechanism by which apoE4 contributes to AD pathogenesis has yet to be clarified. Preferential cleavage of the ApoE4 isoform relative to other polymorphic forms appears to be significant, as the resulting fragments are associated with hallmarks of AD. To examine the possible role of apoE4 proteolysis in AD, we designed a site-directed antibody directed at position D172, which would yield a predicted amino-terminal fragment previously identified in AD brain extracts. Western blot analysis utilizing this novel antibody, termed the amino-terminal apoE4 cleavage fragment (nApoE4CF) Ab, consistently identified the predicted amino-terminal fragment (∼18kDa) in several commercially available forms of human recombinant apoE4 purified from E. coli. Mass spectrometry confirmed the identity of this 18kDa fragment as being an amino-terminal fragment of apoE4. Immunohistochemical experiments indicated the nApoE4CF Ab specifically labeled neurofibrillary tangles (NFTs) in AD frontal cortex sections that colocalized with the mature tangle marker PHF-1. Taken together, these results suggest a novel cleavage event of apoE4, generating an amino-terminal fragment that localizes within NFTs of the AD brain.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Apolipoproteína E4/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/genética , Sequência de Aminoácidos , Apolipoproteína E4/genética , Humanos , Dados de Sequência Molecular , Emaranhados Neurofibrilares/química , Fragmentos de Peptídeos/genética
20.
J Am Chem Soc ; 134(34): 13982-9, 2012 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-22862303

RESUMO

One of the hallmarks of Alzheimer's disease is the self-assembly of the microtubule-associated protein tau into fibers termed "paired helical filaments" (PHFs). However, the structural basis of PHF assembly at atomic detail is largely unknown. Here, we applied solid-state nuclear magnetic resonance (ssNMR) spectroscopy to investigate in vitro assembled PHFs from a truncated three-repeat tau isoform (K19) that represents the core of PHFs. We found that the rigid core of the fibrils is formed by amino acids V306 to S324, only 18 out of 99 residues, and comprises three ß-strands connected by two short kinks. The first ß-strand is formed by the well-studied hexapeptide motif VQIVYK that is known to self-aggregate in a steric zipper arrangement. Results on mixed [(15)N:(13)C]-labeled K19 fibrils show that ß-strands are stacked in a parallel, in-register manner. Disulfide bridges formed between C322 residues of different molecules lead to a disturbance of the ß-sheet structure, and polymorphism in ssNMR spectra is observed. In particular, residues K321-S324 exhibit two sets of resonances. Experiments on K19 C322A PHFs further confirm the influence of disulfide bond formation on the core structure. Our structural data are supported by H/D exchange NMR measurements on K19 as well as a truncated four-repeat isoform of tau (K18). Site-directed mutagenesis studies show that single-point mutations within the three different ß-strands result in a significant loss of PHF aggregation efficiency, highlighting the importance of the ß-structure-rich regions for tau aggregation.


Assuntos
Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/genética , Proteínas tau/química , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/ultraestrutura , Ressonância Magnética Nuclear Biomolecular , Mutação Puntual , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Estrutura Secundária de Proteína , Proteínas tau/metabolismo , Proteínas tau/ultraestrutura
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