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1.
Sci Rep ; 12(1): 15287, 2022 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-36088484

RESUMO

Strong evidence demonstrates a significant association between cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD). For this reason, interest in understanding the underlying vascular pathologies that contribute to AD remain. CAA research has primarily focused on arterioles and capillaries, overlooking the draining venules. Therefore, this study sought to examine venular amyloid pathology and its relationship to arteriolar amyloidosis throughout AD progression in the TgF344-AD rat model. Antibodies targeting the amyloid-beta peptide (Aß) sequence suggest morphological differences between arteriolar and venular amyloid. Mass spectrometric analyses of isolated cortical parenchymal plaques, arteriolar and venular amyloid demonstrated presence of Aß in all three samples, as well as proteins known to be associated with AD. Histopathological analysis indicates a significant age effect for both arteriolar and venular amyloid accumulation, with accumulation initiated in the somatosensory cortex followed by the motor and cingulate cortex. Lastly, significant arteriolar amyloid accumulates relative to venular amyloid deposition in AD progression. Overall, understanding venular and arteriolar amyloid pathology provides insight into the complex connection between CAA and AD.


Assuntos
Doença de Alzheimer , Angiopatia Amiloide Cerebral , Doença de Alzheimer/metabolismo , Amiloide , Animais , Angiopatia Amiloide Cerebral/patologia , Ratos , Ratos Endogâmicos F344 , Ratos Transgênicos , Vênulas/metabolismo
2.
Cereb Cortex ; 30(2): 563-574, 2020 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-31188425

RESUMO

Disruption of attention is an early and disabling symptom of Alzheimer's disease (AD). The underlying cellular mechanisms are poorly understood and treatment options for patients are limited. These early attention deficits are evident in the TgCRND8 mouse, a well-established murine model of AD that recapitulates several features of the disease. Here, we report severe impairment of the nicotinic receptor-mediated excitation of prefrontal attentional circuitry in TgCRND8 mice relative to wild-type littermate controls. We demonstrate that this impairment can be remedied by apamin, a bee venom neurotoxin peptide that acts as a selective antagonist to the SK family of calcium-sensitive potassium channels. We probe this seeming upregulation of calcium-sensitive inhibition and find that the attenuated nicotinic firing rates in TgCRND8 attention circuits are mediated neither by greater cellular calcium signals nor by elevated SK channel expression. Instead, we find that TgCRND8 mice show enhanced functional coupling of nicotinic calcium signals to inhibition. This SK-mediated inhibition exerts a powerful negative feedback on nicotinic excitation, dampening attention-relevant signaling in the TgCRND8 brain. These mechanistic findings identify a new cellular target involved in the modulation of attention and a novel therapeutic target for early attention deficits in AD.


Assuntos
Doença de Alzheimer/fisiopatologia , Apamina/administração & dosagem , Córtex Pré-Frontal/fisiologia , Células Piramidais/fisiologia , Receptores Nicotínicos/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores
3.
J Neurosci Methods ; 328: 108424, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494186

RESUMO

BACKGROUND: The evaluation of histological tissue samples plays a crucial role in deciphering preclinical disease and injury mechanisms. High-resolution images can be obtained quickly however data acquisition are often bottlenecked by manual analysis methodologies. NEW METHOD: We describe and validate a pipeline for a novel machine learning-based analytical method, using the Opera High-Content Screening system and Harmony software, allowing for detailed image analysis of cellular markers in histological samples. RESULTS: To validate the machine learning pipeline, analyses of single proteins in mouse brain sections were utilized. To demonstrate adaptability of the pipeline for multiple cell types and epitopes, the percent brain coverage of microglial cells, identified by ionized calcium binding adaptors molecule 1 (Iba1), and of astrocytes, by glial fibrillary acidic protein (GFAP) demonstrated no significant differences between automated and manual analyses protocols. Further to examine the robustness of this protocol for multiple proteins simultaneously labeling of rat brain sections were utilized; co-localization of astrocytic endfeet on blood vessels, using aquaporin-4 and tomato lectin respectively, were efficiently identified and quantified by the novel pipeline and were not significantly different between the two analyses protocols. Comparison with Existing Methods: The automated platform maintained the sensitivity and accuracy of manual analysis, while accomplishing the analyses in 1/200th of the time. CONCLUSIONS: We demonstrate the benefits and potential of adapting an automated high-throughput machine-learning analytical approach for the analysis ofin situ tissue samples, show effectiveness across different animal models, while reducing analysis time and increasing productivity.


Assuntos
Astrócitos , Imunofluorescência/métodos , Técnicas Histológicas/métodos , Processamento de Imagem Assistida por Computador/métodos , Microglia , Neurociências/métodos , Aprendizado de Máquina Supervisionado , Animais , Animais Geneticamente Modificados , Imunofluorescência/normas , Técnicas Histológicas/normas , Processamento de Imagem Assistida por Computador/normas , Camundongos , Neurociências/normas , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
4.
ACS Chem Neurosci ; 3(3): 167-77, 2012 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-22860186

RESUMO

To identify a lead skeleton structure for optimization of scyllo-inositol-based inhibitors of amyloid-beta peptide (Aß) aggregation, we have synthesized aldoxime, hydroxamate, carbamate, and amide linked scyllo-inositol derivatives. These structures represent backbones that can be readily expanded into a wide array of derivatives. They also provide conservative modifications of the scyllo-inositol backbone, as they maintain the display of the equatorial polar atoms, preserving the stereochemical requirement necessary for maximum inhibition of Aß(1-42) fiber formation. In addition, a reliable work plan for screening derivatives was developed in order to preferentially identify a backbone(s) structure that prevents fibrillogenesis and stabilizes nontoxic small molecular weight oligomers, as we have previously reported for scyllo-inositol. In the present studies, we have adapted a high throughput ELISA-based oligomerization assay followed by atomic force microscopy to validate the results screen compounds. The lead compounds were then tested for toxicity and ability to rescue Aß(1-42) induced toxicity in vitro and the affinity of the compounds for Aß(1-42) compared by mass spectrometry. The data to suggest that compounds must maintain a planar conformation to exhibit activity similar to scyllo-inositol and that the oxime derivative represents the lead backbone for future development.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Inositol/farmacologia , Oximas/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Amiloide/química , Amiloide/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Inositol/química , Oximas/química , Conformação Proteica/efeitos dos fármacos
5.
Pharmacopsychiatry ; 36 Suppl 2: S130-5, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14574627

RESUMO

One of the major pathological features of Alzheimer's disease is the presence of extracellular amyloid plaques that are predominantly composed of the amyloid-beta peptide (Abeta). Characterisation of plaques demonstrated the predominance of two peptides differing at the carboxyl terminus by 2 hydrophobic amino acids, Abeta40 and Abeta42. Diffuse plaques associated with AD are composed predominantly of Abeta42, whereas senile plaques contain both Abeta40 and Abeta42. Recently, it has been suggested that diffuse plaque formation is initiated as a plasma membrane bound Abeta species and that Abeta42 is the critical component. In order to investigate this hypothesis, we have examined Abeta40/42-lipid interactions using in situ atomic force microscopy, electron microscopy and fluorescence anisotropy. While the association of Abeta42 with planar bilayers resulted in peptide aggregation but no fibre formation, this was not the case for Abeta40 where we observed preferential fibre formation. Cholesterol, a key membrane component and modulating factor in AD, is inversely correlated with the extent of Abeta40/42-bilayer interaction. These results were confirmed using fluorescence anisotropy to evaluate the effect of Abeta on membrane fluidity and fluorimetry to confirm membrane integrity. Our results suggest that the enhanced amyloidogenic properties of Abeta42 are not correlated with fibril formation but aggregation on bilayer surfaces.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Química Encefálica , Colesterol/metabolismo , Bicamadas Lipídicas/metabolismo , Emaranhados Neurofibrilares/metabolismo , Fragmentos de Peptídeos/metabolismo , beta-Ciclodextrinas , Peptídeos beta-Amiloides/química , Animais , Encéfalo/metabolismo , Ciclodextrinas/farmacologia , Di-Hidropiridinas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Fluidez de Membrana , Microscopia de Força Atômica/métodos , Fragmentos de Peptídeos/química , Espectrometria de Fluorescência/métodos , Fatores de Tempo
6.
Ann N Y Acad Sci ; 977: 376-83, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12480775

RESUMO

One of the major pathological features of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques that are predominantly composed of the amyloid-beta peptide (Abeta). Characterization of plaques demonstrated the predominance of two peptides differing at the carboxyl terminus by two hydrophobic amino acids, Abeta40 and Abeta42. Diffuse plaques associated with AD are composed predominantly of Abeta42, whereas senile plaques contain both Abeta40 and Abeta42. Recently, it has been suggested that diffuse plaque formation is initiated as a plasma membrane-bound Abeta species and that Abeta42 is the critical component. In order to investigate this hypothesis, we have examined Abeta40/42-lipid interactions using in situ atomic force microscopy, electron microscopy, and fluorescence anisotropy. While the association of Abeta42 with planar bilayers resulted in peptide aggregation, but no fiber formation, this was not the case for Abeta40, where we observed preferential fiber formation. Cholesterol, a key membrane component and modulating factor in AD, is inversely correlated with the extent of Abeta40/42-bilayer interaction. These results were confirmed using fluorescence anisotropy to evaluate the effect of Abeta on membrane fluidity and fluorimetry to confirm membrane integrity. Our results suggest that the enhanced amyloidogenic properties of Abeta42 are not correlated with fibril formation, but with aggregation on bilayer surfaces.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/patologia , Colesterol/metabolismo , Humanos , Lipídeos de Membrana/metabolismo , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia
7.
Nat Med ; 8(11): 1263-9, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12379850

RESUMO

Immunization of transgenic mouse models of Alzheimer disease using amyloid-beta peptide (Abeta) reduces both the Alzheimer disease-like neuropathology and the spatial memory impairments of these mice. However, a therapeutic trial of immunization with Abeta42 in humans was discontinued because a few patients developed significant meningo-encephalitic cellular inflammatory reactions. Here we show that beneficial effects in mice arise from antibodies selectively directed against residues 4-10 of Abeta42, and that these antibodies inhibit both Abeta fibrillogenesis and cytotoxicity without eliciting an inflammatory response. These findings provide the basis for improved immunization antigens as well as attempts to design small-molecule mimics as alternative therapies.


Assuntos
Vacinas contra Alzheimer/uso terapêutico , Peptídeos beta-Amiloides/antagonistas & inibidores , Anticorpos/uso terapêutico , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Animais , Anticorpos/imunologia , Encéfalo/imunologia , Encéfalo/metabolismo , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular
8.
Amyloid ; 8 Suppl 1: 10-9, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11676284

RESUMO

Amyloid plaques are the principal features of Alzheimers disease (AD) pathology and are considered to be a major factor in the disease process. These fibrillar deposits are composed primarily of the 40-42 residue amyloid-beta (Abeta) peptide which is a proteolytic product of a larger membrane precursor protein. Electron microscopy and X-ray diffraction have revealed that the mature amyloid fibrils are assembled as a highly beta-sheet polymer that has a well-defined protofilament quaternary structure. This organization is observed for amyloid fibrils from a wide variety of disorders and appears to represent a structural superfamily. Amyloid plaques also contain a number of other components such as proteoglycans that contain highly sulfated glycosaminoglycan (GAG) chains. These amyloid-associated elements may contribute to the aggregation and/or stabilization of Abeta as insoluble fibrils. We have recently developed an aggressive model for Abeta plaque formation in transgenic mice that exhibits an "early-onset" phenotype. Immunocytochemistry has demonstrated that even with this rapid progression, Abeta deposits within the neuropil and cerebrovascular system all co-localize with heparan sulfate proteoglycans (HSPG). These findings indicate a number of structural features that can be targeted as potential sites for the development of amyloid inhibitors. In addition, the use of small compounds that interfere with the proteoglycan-amyloid pathway may be effective therapeutic agents that can be assessed through the use of these transgenic models.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Animais , Modelos Animais de Doenças , Hipocampo/metabolismo , Humanos , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Força Atômica , Estrutura Molecular , Proteoglicanas/metabolismo , Difração de Raios X
9.
J Environ Pathol Toxicol Oncol ; 20 Suppl 1: 1-14, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11570667

RESUMO

Previous studies have determined that alpha-quartz (crystalline silica) can cause pulmonary inflammation, damage, and fibrosis. However, the temporal relationship between silica inhalation and pulmonary inflammation, damage, and fibrosis has not been fully examined. To address this gap in our knowledge of silica-induced pulmonary fibrosis, a chronic inhalation study using rats was designed. Specifically, rats were exposed to a silica aerosol (15 mg/m3 silica, 6 h/d, 5 d/wk, 116 d), and measurements of pulmonary inflammation, damage, and fibrosis were monitored throughout the study. We report (1) data demonstrating that the silica aerosol generation and exposure system produced a consistent silica aerosol of respirable size particles; (2) the time course of silica deposition in the lung; (3) calculations that demonstrate that the rats were not in pulmonary overload; (4) histopathological data demonstrating time-dependent enhancement of silica-induced alveolitis, epithelial hypertrophy and hyperplasia, alveolar lipoproteinosis, and pulmonary fibrosis in the absence of overload; and (5) biochemical data documenting the development of lipidosis, lung damage, and fibrosis.


Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Pulmão/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Dióxido de Silício/toxicidade , Administração por Inalação , Animais , Carga Corporal (Radioterapia) , Lavagem Broncoalveolar , Lipidoses/induzido quimicamente , Pulmão/patologia , Masculino , Tamanho da Partícula , Fibrose Pulmonar/patologia , Ratos , Ratos Endogâmicos F344 , Dióxido de Silício/administração & dosagem , Organismos Livres de Patógenos Específicos , Fatores de Tempo
10.
J Mol Biol ; 311(4): 723-34, 2001 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-11518526

RESUMO

Recent studies have suggested that cholesterol, an important determinant of the physical state of biological membranes, plays a significant role in the development of Alzheimer's disease. We have employed in situ scanning probe microscopy, fluorescence anisotropy, and electron microscopy to investigate how cholesterol levels within total brain lipid bilayers effect amyloid beta-peptide (Abeta)-assembly. Fluorescence anisotropy measurements revealed that the relative fluidity of the total brain lipid membranes was influenced by the level of cholesterol and the addition of Abeta40 resulted in a decrease in the overall vesicle fluidity. In situ scanning probe microscopy performed on supported planar bilayers of total brain lipid revealed a correlation between membrane fluidity, as influenced by cholesterol level, and the extent of Abeta-insertion and subsequent fibrillogenesis. These observations were consistent with fluorescence microscopy studies of PC-12 and SH-SY5Y cell lines exposed to exogenous Abeta, which revealed an inverse correlation between membrane cholesterol level, and Abeta-cell surface binding and subsequent cell death. These results collectively suggest that Abeta-cell surface interactions are mediated by cellular cholesterol levels, the distribution of cholesterol throughout the cell, and membrane fluidity.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Colesterol/metabolismo , Fluidez de Membrana , beta-Ciclodextrinas , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Ciclodextrinas/farmacologia , Polarização de Fluorescência , Lipossomos/química , Lipossomos/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Microscopia de Força Atômica , Microscopia Eletrônica , Células PC12 , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Ratos
11.
J Biol Chem ; 276(36): 33561-8, 2001 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-11438533

RESUMO

Alzheimer's disease pathology has demonstrated amyloid plaque formation associated with plasma membranes and the presence of intracellular amyloid-beta (A beta) accumulation in specific vesicular compartments. This suggests that lipid composition in different compartments may play a role in A beta aggregation. To test this hypothesis, we have isolated cellular membranes from human brain to evaluate A beta 40/42-lipid interactions. Plasma, endosomal, lysosomal, and Golgi membranes were isolated using sucrose gradients. Electron microscopy demonstrated that A beta fibrillogenesis is accelerated in the presence of plasma and endosomal and lysosomal membranes with plasma membranes inducing an enhanced surface organization. Alternatively, interaction of A beta with Golgi membranes fails to progress to fibril formation, suggesting that A beta-Golgi head group interaction stabilizes A beta. Fluorescence spectroscopy using the environment-sensitive probes 1,6-diphenyl-1,3,5-hexatriene, laurdan, N-epsilon-dansyl-L-lysine, and merocyanine 540 demonstrated variations in the inherent lipid properties at the level of the fatty acyl chains, glycerol backbone, and head groups, respectively. Addition of A beta 40/42 to the plasma and endosomal and lysosomal membranes decreases the fluidity not only of the fatty acyl chains but also the head group space, consistent with A beta insertion into the bilayer. In contrast, the Golgi bilayer fluidity is increased by A beta 40/42 binding which appears to result from lipid head group interactions and the production of interfacial packing defects.


Assuntos
Peptídeos beta-Amiloides/química , Membrana Celular/química , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Anisotropia , Encéfalo/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Dimerização , Endossomos/química , Endossomos/metabolismo , Corantes Fluorescentes/farmacologia , Complexo de Golgi/metabolismo , Humanos , Lipídeos/química , Lisossomos/metabolismo , Masculino , Microscopia Eletrônica , Modelos Químicos , Peptídeos/química , Fosfolipídeos/química , Ligação Proteica , Pirimidinonas/farmacologia , Espectrometria de Fluorescência , Espectrofotometria
12.
Biophys J ; 80(3): 1359-71, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11222297

RESUMO

Identifying the mechanisms responsible for the assembly of proteins into higher-order structures is fundamental to structural biology and understanding specific disease pathways. The amyloid-beta (Abeta) peptide is illustrative in this regard as fibrillar deposits of Abeta are characteristic of Alzheimer's disease. Because Abeta includes portions of the extracellular and transmembrane domains of the amyloid precursor protein, it is crucial to understand how this peptide interacts with cell membranes and specifically the role of membrane structure and composition on Abeta assembly and cytotoxicity. We describe the results of a combined circular dichroism spectroscopy, electron microscopy, and in situ tapping mode atomic force microscopy (TMAFM) study of the interaction of soluble monomeric Abeta with planar bilayers of total brain lipid extract. In situ extended-duration TMAFM provided evidence of membrane disruption via fibril growth of initially monomeric Abeta1-40 peptide within the total brain lipid bilayers. In contrast, the truncated Abeta1-28 peptide, which lacks the anchoring transmembrane domain found in Abeta1-40, self-associates within the lipid headgroups but does not undergo fibrillogenesis. These observations suggest that the fibrillogenic properties of Abeta peptide are in part a consequence of membrane composition, peptide sequence, and mode of assembly within the membrane.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/ultraestrutura , Dimiristoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/ultraestrutura , Química Encefálica , Dicroísmo Circular , Humanos , Microfibrilas/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica , Modelos Moleculares , Estrutura Secundária de Proteína
13.
J Biol Chem ; 276(9): 6412-9, 2001 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-11106653

RESUMO

In Alzheimer's disease, the major pathological features are diffuse and senile plaques that are primarily composed of the amyloid-beta (A beta) peptide. It has been proposed that proteoglycans and glycosaminoglycans (GAG) facilitate amyloid fibril formation and/or stabilize the plaque aggregates. To develop effective therapeutics based on A beta-GAG interactions, understanding the A beta binding motif on the GAG chain is imperative. Using electron microscopy, fluorescence spectroscopy, and competitive inhibition ELISAs, we have evaluated the ability of chondroitin sulfate-derived monosaccharides and disaccharides to induce the structural changes in A beta that are associated with GAG interactions. Our results demonstrate that the disaccharides GalNAc-4-sulfate(4S), Delta UA-GalNAc-6-sulfate(6S), and Delta UA-GalNAc-4,6-sulfate(4S,6S), the iduronic acid-2-sulfate analogues, and the monosaccharides d-GalNAc-4S, d-GalNAc-6S, and d-GalNAc-4S,6S, but not d-GalNAc, d-GlcNAc, or Delta UA-GalNAc, induce the fibrillar features of A beta-GAG interactions. The binding affinities of all chondroitin sulfate-derived saccharides mimic those of the intact GAG chains. The sulfated monosaccharides and disaccharides compete with the intact chondroitin sulfate and heparin GAGs for A beta binding, as illustrated by competitive inhibition ELISAs. Therefore, the development of therapeutics based on the model of A beta-chondroitin sulfate binding may lead to effective inhibitors of the GAG-induced amyloid formation that is observed in vitro.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Sulfatos de Condroitina/metabolismo , Dissacarídeos/metabolismo , Glicosaminoglicanos/metabolismo , Monossacarídeos/metabolismo , Peptídeos beta-Amiloides/química , Sítios de Ligação , Heparitina Sulfato/metabolismo , Humanos
14.
Eur J Biochem ; 267(21): 6353-61, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11029577

RESUMO

One of the major clinical features of Alzheimer's disease is the presence of extracellular amyloid plaques that are associated with glycosaminoglycan-containing proteoglycans. It has been proposed that proteoglycans and glycosaminoglycans facilitate amyloid fibril formation and/or stabilize these aggregates. Characterization of proteoglycan-protein interactions has suggested that basic amino acids in a specific conformation are necessary for glycosaminoglycan binding. Amyloid-beta peptide (Abeta) has a cluster of basic amino acids at the N-terminus (residues 13-16, His-His-Gln-Lys), which are considered critical for glycosaminoglycan interactions. To understand the molecular recognition of glycosaminoglycans by Abeta, we have examined a series of synthetic peptides with systematic alanine substitutions. These include: His13-->Ala, His14-->Ala, Lys16-->Ala, His13His14Lys16-->Ala and Arg5His6-->Ala. Alanine substitutions result in differences in both the secondary and fibrous structure of Abeta1-28 as determined by circular dichroism spectroscopy and electron microscopy. The results demonstrate that the His-His-Gln-Lys region of Abeta, and in particular His13, is an important structural domain, as Ala substitution produces a dysfunctional folding mutant. Interaction of the substituted peptides with heparin and chondroitin sulfate glycosaminoglycans demonstrate that although electrostatic interactions contribute to binding, nonionic interactions such as hydrogen bonding and van der Waals packing play a role in glycosaminoglycan-induced Abeta folding and aggregation.


Assuntos
Doença de Alzheimer/metabolismo , Substituição de Aminoácidos/genética , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Glicosaminoglicanos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Alanina/genética , Alanina/metabolismo , Doença de Alzheimer/genética , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/ultraestrutura , Sulfatos de Condroitina/metabolismo , Dicroísmo Circular , Dermatan Sulfato/metabolismo , Glicosaminoglicanos/ultraestrutura , Heparina/metabolismo , Humanos , Ligação de Hidrogênio , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/ultraestrutura , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Eletricidade Estática
15.
Eur J Biochem ; 267(22): 6692-8, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11054124

RESUMO

The amyloid beta-peptide (Abeta) is a principal component of insoluble amyloid plaques which are characteristic neuropathological features of Alzheimer's disease. Abeta also exists as a normal soluble protein that undergoes a pathogenic transition to an aggregated, fibrous form. This transition can be affected by extraneous proteinaceous and nonproteinaceous elements, such as zinc ions, which may promote aggregation and/or stabilization of the fibrils. Protein chelation of zinc is typically mediated by histidines, cysteines and carboxylates. Previous studies have demonstrated that the Abeta-Zn2+ binding site is localized within residues 6-28 and that histidines may serve as the principal sites of interaction. To localize key residues within this region, a series of Abeta peptides (residues 1-28) were synthesized that contained systematic His/Ala substitutions. Circular dichroism and electron microscopy were used to monitor the effects of Zn2+ on the peptide beta-sheet conformation and fibril aggregation. Our results indicate that substitution of either His13 or His14 but not His6 eliminates the zinc-mediated effects. These observations indicate a specific zinc binding site within Abeta that involves these central histidine residues.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/química , Zinco/metabolismo , Substituição de Aminoácidos , Peptídeos beta-Amiloides/ultraestrutura , Sítios de Ligação , Dicroísmo Circular , Cisteína , Histidina , Microfibrilas/efeitos dos fármacos , Microfibrilas/ultraestrutura , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Estrutura Secundária de Proteína , Zinco/farmacologia
16.
J Biol Chem ; 275(44): 34328-34, 2000 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-10915790

RESUMO

With the discovery of missense mutations (A53T and A30P) in alpha-synuclein (alpha-Syn) in several families with early onset familial Parkinson's disease, alpha-Syn aggregation and fibril formation have been thought to play a role in the pathogenesis of alpha-synucleinopathies, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. As previous reports have suggested that alpha-Syn plays a role in lipid transport and synaptic membrane biogenesis, we investigated whether alpha-Syn binds to a specific lipid ligand using thin layer chromatography overlay and examined the changes in its secondary structure using circular dichroism spectroscopy. alpha-Syn was found to bind to acidic phospholipid vesicles and this binding was significantly augmented by the presence of phosphatidylethanolamine, a neutral phospholipid. We further examined the interaction of alpha-Syn with lipids by in situ atomic force microscopy. The association of soluble wild-type alpha-Syn with planar lipid bilayers resulted in extensive bilayer disruption and the formation of amorphous aggregates and small fibrils. The A53T mutant alpha-Syn disrupted the lipid bilayers in a similar fashion but at a slower rate. These results suggest that alpha-Syn membrane interactions are physiologically important and the lipid composition of the cellular membranes may affect these interactions in vivo.


Assuntos
Metabolismo dos Lipídeos , Proteínas do Tecido Nervoso/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Encéfalo/metabolismo , Membrana Celular/metabolismo , Cromatografia em Camada Fina , Dicroísmo Circular , Humanos , Bicamadas Lipídicas , Masculino , Microscopia de Força Atômica , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Sinucleínas , alfa-Sinucleína
17.
J Struct Biol ; 130(2-3): 259-70, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10940230

RESUMO

Amyloid formation is a key pathological feature of Alzheimer's disease and is considered to be a major contributing factor to neurodegeneration and clinical dementia. Amyloid is found as both diffuse and senile plaques in the parenchyma of the brain and is composed primarily of the 40- to 42-residue amyloid-beta (Abeta) peptides. The characteristic amyloid fiber exhibits a high beta-sheet content and may be generated in vitro by the nucleation-dependent self-association of the Abeta peptide and an associated conformational transition from random to beta-conformation. Growth of the fibrils occurs by assembly of the Abeta seeds into intermediate protofibrils, which in turn self-associate to form mature fibers. This multistep process may be influenced at various stages by factors that either promote or inhibit Abeta fiber formation and aggregation. Identification of these factors and understanding the driving forces behind these interactions as well as the structural motifs necessary for these interactions will help to elucidate potential sites that may be targeted to prevent amyloid formation and its associated toxicity. This review will discuss some of the modulating factors that have been identified to date and their role in fibrillogenesis.


Assuntos
Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/ultraestrutura , Peptídeos beta-Amiloides/química , Dimerização , Humanos , Metabolismo dos Lipídeos , Lipídeos/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas/metabolismo , Proteínas/farmacologia
18.
J Biol Chem ; 275(24): 18495-502, 2000 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-10764800

RESUMO

Inositol has 8 stereoisomers, four of which are physiologically active. myo-Inositol is the most abundant isomer in the brain and more recently shown that epi- and scyllo-inositol are also present. myo-Inositol complexes with Abeta42 in vitro to form a small stable micelle. The ability of inositol stereoisomers to interact with and stabilize small Abeta complexes was addressed. Circular dichroism spectroscopy demonstrated that epi- and scyllo- but not chiro-inositol were able to induce a structural transition from random to beta-structure in Abeta42. Alternatively, none of the stereoisomers were able to induce a structural transition in Abeta40. Electron microscopy demonstrated that inositol stabilizes small aggregates of Abeta42. We demonstrate that inositol-Abeta interactions result in a complex that is non-toxic to nerve growth factor-differentiated PC-12 cells and primary human neuronal cultures. The attenuation of toxicity is the result of Abeta-inositol interaction, as inositol uptake inhibitors had no effect on neuronal survival. The use of inositol stereoisomers allowed us to elucidate an important structure-activity relationship between Abeta and inositol. Inositol stereoisomers are naturally occurring molecules that readily cross the blood-brain barrier and may represent a viable treatment for AD through the complexation of Abeta and attenuation of Abeta neurotoxic effects.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Inositol/metabolismo , Peptídeos beta-Amiloides/química , Animais , Dicroísmo Circular , Humanos , Inositol/química , Microscopia Eletrônica , Modelos Químicos , Neurônios/metabolismo , Células PC12 , Conformação Proteica , Ratos , Estereoisomerismo
19.
Nature ; 408(6815): 979-82, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11140685

RESUMO

Much evidence indicates that abnormal processing and extracellular deposition of amyloid-beta peptide (A beta), a proteolytic derivative of the beta-amyloid precursor protein (betaAPP), is central to the pathogenesis of Alzheimer's disease (reviewed in ref. 1). In the PDAPP transgenic mouse model of Alzheimer's disease, immunization with A beta causes a marked reduction in burden of the brain amyloid. Evidence that A beta immunization also reduces cognitive dysfunction in murine models of Alzheimer's disease would support the hypothesis that abnormal A beta processing is essential to the pathogenesis of Alzheimer's disease, and would encourage the development of other strategies directed at the 'amyloid cascade'. Here we show that A beta immunization reduces both deposition of cerebral fibrillar A beta and cognitive dysfunction in the TgCRND8 murine model of Alzheimer's disease without, however, altering total levels of A beta in the brain. This implies that either a approximately 50% reduction in dense-cored A beta plaques is sufficient to affect cognition, or that vaccination may modulate the activity/abundance of a small subpopulation of especially toxic A beta species.


Assuntos
Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Vacinação , Doença de Alzheimer/patologia , Amiloide/administração & dosagem , Animais , Cricetinae , Modelos Animais de Doenças , Hipocampo/patologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Aprendizagem em Labirinto , Mesocricetus , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Placa Amiloide
20.
Drug News Perspect ; 13(5): 281-8, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12937642

RESUMO

The molecular and cellular processes that lead to the production of the amyloid beta (A beta) peptide and some of the processes associated with A beta fibrillogenesis and neurotoxicity have recently been elucidated. Experimental results have suggested that abnormalities in the processing of the beta-amyloid precursor protein (beta APP) are central to the pathogenesis of Alzheimer's disease (AD). beta APP processing includes two mutually exclusive proteolytic cleavage pathways, one involving the putative gamma-secretase enzyme, the identity of which remains unknown. Recent evidence has suggested the presenilin 1 and presenilin 2 genes are necessary for gamma-secretase activities. Another gene associated with susceptibility to AD is the apolipoprotein E (APOE) gene. Given the important role that abnormal processing of beta APP plays in the genesis of AD, most current efforts are directed at either modulating A beta peptide production or inhibiting its ability to aggregate into fibrils and cause neurotoxicity. To inhibit A beta production, one strategy might be to inhibit either beta-secretase or gamma-secretase. Several approaches to the inhibition of A beta aggregation are under investigation.

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