RESUMO
Non-fibrillar soluble oligomeric forms of amyloid-ß peptide (oAß) and tau proteins are likely to play a major role in Alzheimer's disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAß initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aß, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oAß levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oAß to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairment. These findings provide a novel view of the effects of tau and Aß on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with Aß and tau pathology.
Assuntos
Potenciação de Longa Duração , Memória , Agregados Proteicos , Agregação Patológica de Proteínas , Multimerização Proteica , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Espaço Extracelular/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Neurônios/metabolismo , Proteínas tau/químicaRESUMO
Increasing evidence points to the cytotoxicity of islet amyloid polypeptide (IAPP) aggregates as a major contributor to the loss of ß-cell mass in type 2 diabetes. Prevention of IAPP formation represents a potential treatment to increase ß-cell survival and function. The IAPP inhibitory peptide, D-ANFLVH, has been previously shown to prevent islet amyloid accumulation in cultured human islets. To assess its activity in vivo, D-ANFLVH was administered by intraperitoneal injection into a human IAPP transgenic mouse model, which replicates type 2 diabetes islet amyloid pathology. The peptide was a potent inhibitor of islet amyloid deposition, resulting in reduced islet cell apoptosis and preservation of ß-cell area leading to improved glucose tolerance. These findings provide support for a key role of islet amyloid in ß-cell survival and validate the application of anti-amyloid compounds as therapeutic strategies to maintain normal insulin secretion in patients with type 2 diabetes.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Homeostase/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 2/sangue , Modelos Animais de Doenças , Células Secretoras de Insulina/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/uso terapêutico , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Camundongos TransgênicosRESUMO
Islet transplantation is a promising therapy for patients with diabetes, but its long-term success is limited by many factors, including the formation of islet amyloid deposits. Heparin is employed in clinical islet transplantation to reduce clotting but also promotes fibrillization of amyloidogenic proteins. We hypothesized that heparin treatment of islets during pre-transplant culture may enhance amyloid formation leading to beta cell loss and graft dysfunction. Heparin promoted the fibrillization of human islet amyloid polypeptide (IAPP) and enhanced its toxicity to INS-1 beta cells. Heparin increased amyloid deposition in cultured human islets, but surprisingly decreased islet cell apoptosis. Treatment of human islets with heparin prior to transplantation increased the likelihood of graft failure. Removal of islet heparan sulfate glycosaminoglycans, which localize with islet amyloid deposits in type 2 diabetes, by heparinase treatment decreased amyloid deposition and protected against islet cell death. These findings raise the possibility that pretransplant treatment of human islets with heparin could potentiate IAPP aggregation and amyloid formation and may be detrimental to subsequent graft function.
Assuntos
Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Heparina Liase/farmacologia , Heparina/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Amiloide/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/cirurgia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Rejeição de Enxerto/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/citologia , Transplante das Ilhotas Pancreáticas/métodos , Camundongos Endogâmicos NOD , Camundongos SCID , Estreptozocina/efeitos adversosRESUMO
Inherited variants in multiple different genes are associated with increased risk for Alzheimer's disease (AD). In many of these genes, the inherited variants alter some aspect of the production or clearance of the neurotoxic amyloid ß-peptide (Aß). Thus missense, splice site or duplication mutants in the presenilin 1 (PS1), presenilin 2 (PS2) or the amyloid precursor protein (APP) genes, which alter the levels or shift the balance of Aß produced, are associated with rare, highly penetrant autosomal dominant forms of Familial Alzheimer's Disease (FAD). Similarly, the more prevalent late-onset forms of AD are associated with both coding and non-coding variants in genes such as SORL1, PICALM and ABCA7 that affect the production and clearance of Aß. This review summarises some of the recent molecular and structural work on the role of these genes and the proteins coded by them in the biology of Aß. We also briefly outline how the emerging knowledge about the pathways involved in Aß generation and clearance can be potentially targeted therapeutically. This article is part of Special Issue entitled "Neuronal Protein".
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Transdução de Sinais/fisiologia , Pesquisa Translacional Biomédica , Doença de Alzheimer/genética , Animais , HumanosRESUMO
Murine models of Alzheimer's disease (AD) have been used to draw associations between atrophy of neural tissue and underlying pathology. In this study, the early-onset TgCRND8 mouse model of AD and littermate controls were scanned longitudinally with in vivo manganese-enhanced MRI (MEMRI) before and after the onset of amyloid plaque deposition at 12 weeks of age. Separate cohorts of mice were scanned at 1 week (ex vivo imaging) and 4 weeks (MEMRI) of age to investigate early life alterations in the brain. Contrary to our expectations, differences in neuroanatomy were found in early post-natal life, preceding plaque deposition by as much as 11 weeks. Many of these differences remained at all imaging time points, suggesting that they were programmed early in life and were unaffected by the onset of pathology. Furthermore, rather than showing atrophy, many regions of the TgCRND8 brain grew at a faster rate compared to controls. These regions contained the greatest density of amyloid plaques and reactive astrocytes. Our findings suggest that pathological processes as well as an alteration in brain development influence the TgCRND8 neuroanatomy throughout the lifespan.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Atrofia , Encéfalo/metabolismo , Modelos Animais de Doenças , Imageamento por Ressonância Magnética/métodos , Camundongos Transgênicos , Placa Amiloide/metabolismo , Placa Amiloide/patologiaRESUMO
The amyloid precursor protein (APP) and amyloid-ß (Aß) peptide play central roles in the pathology and etiology of Alzheimer's disease. Amyloid-induced impairments in neurogenesis have been investigated in several transgenic mouse models but the mechanism of action remains to be conclusively demonstrated. The changes in neurogenesis during this transition of increasing Aß levels and plaque formation were investigated in the present study. We found that the proliferation of newborn cell in the dentate gyrus was enhanced prior to elevations in soluble Aß production as well as amyloid deposition in 5-week-old TgCRND8 mice, which are well-established Alzheimer's disease models, compared to non-transgenic (Non-Tg) mice. The number of BrdU-positive cells remained higher in TgCRND8 vs Non-Tg mice for a period of 8weeks. The numbers of BrdU/NeuN-positive cells were not significantly different in TgCRND8 compared to Non-Tg mice. A significant decrease in BrdU/GFAP but not in BrdU/S100ß was found in Tg vs Non-Tg at 6-weeks of age. In addition, a unique observation was made using isolated neuroprogenitor cells from TgCRND8 mice which were found to be less viable in culture and produced substantial amounts of secreted Aß peptides. This suggests that the proliferation of neural progenitors in vivo may be modulated by high levels of APP expression and the resulting Aß generated directly by the progenitor cells. These findings indicate that cell proliferation is increased prior to Aß deposition and that cell viability is decreased in TgCRND8 mice over time.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Hipocampo/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Contagem de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA , Giro Denteado/fisiologia , Proteína Glial Fibrilar Ácida , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/fisiologia , Neurônios/fisiologia , Proteínas Nucleares/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismoRESUMO
The presenilin complex is composed of four core proteins (presenilin 1 or presenilin 2, APH1, nicastrin, and PEN2). Several endogenous proteins have been reported to selectively modulate the function of the presenilin complexes; these include transmembrane trafficking protein, 21-KD (TMP21), CD147 antigen (basigin), the γ-secretase-activating protein (gSAP), and the orphan G-protein-coupled receptor 3. Because the structure and assembly of these complexes underlies their activity, this review will discuss current work on the assembly of the complex and on presenilin-interacting proteins that regulate secretase activity.
Assuntos
Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilina-1/química , Presenilina-2/química , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/fisiologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Endopeptidases , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/fisiologia , Presenilina-1/fisiologia , Presenilina-2/fisiologiaRESUMO
Islet transplantation is a promising treatment for diabetes but long-term success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.
Assuntos
Amiloide/metabolismo , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Sequência de Aminoácidos , Amiloide/química , Amiloide/fisiologia , Animais , Dicroísmo Circular , Rejeição de Enxerto , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Camundongos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , SuínosRESUMO
Type 2 diabetes is a progressive disease characterised by islet amyloid deposits in the majority of patients. Amyloid formation is considered a significant factor in deterioration of islet function and reduction in beta cell mass, and involves aggregation of monomers of the normally soluble beta cell peptide, human islet amyloid polypeptide (hIAPP) into oligomers, fibrils and, ultimately, mature amyloid deposits. Despite extensive in vitro studies, the process of hIAPP aggregation in vivo is poorly understood, though it is widely reported to promote cytotoxicity. Recently, studies have suggested that only the early stages of fibril assembly, and in particular small hIAPP oligomers, are responsible for beta cell cytotoxicity. This challenges the prior concept that newly formed fibrils and/or mature fibrillar amyloid are cytotoxic. Herein, evidence both for and against the toxic hIAPP oligomer hypothesis is presented; from this, it is apparent that what exactly causes beta cell death when hIAPP aggregates remains debatable. Moreover, substantially more work with more specific reagents and techniques than are currently available will be required to identify conclusively the toxic species resulting from hIAPP aggregation. Keeping an open mind on the nature of the cytotoxic insult has implications for therapeutic developments and clinical care in type 2 diabetes.
Assuntos
Amiloidose/patologia , Diabetes Mellitus Tipo 2/etiologia , Células Secretoras de Insulina/patologia , Morte Celular , Diabetes Mellitus Tipo 2/patologia , Humanos , Pancreatopatias/etiologia , Pancreatopatias/patologiaRESUMO
Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-beta protein (Abeta) that is central to the pathogenesis of Alzheimer's disease. PS1 regulates the intramembranous proteolysis of a 99-amino-acid C-terminal fragment of the amyloid precursor protein (APP-C99), a cleavage event that releases Abeta following a reaction catalyzed by an enzyme termed 'gamma-secretase'. The molecular mechanism of PS1-mediated, gamma-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the gamma-secretase protease or whether instead PS1 mediates gamma-secretase activity indirectly, perhaps by regulating the trafficking or presentation of substrates to the 'authentic' protease, which may be a molecule distinct from PS1. To address this issue, the baculovirus expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively active mutant form of PS1 lacking exon 9 (PS1DeltaE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Abeta-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1DeltaE9 mutation. The addition to APP-C99-infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. These results suggest that PS1DeltaE9 molecules expressed in Sf9 cells retain the ability to modulate Abeta levels. Baculoviral-expressed PS1DeltaE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting gamma-secretase activity from its individual purified component parts.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Baculoviridae/genética , Éxons/genética , Proteínas de Membrana/genética , Deleção de Sequência , Peptídeos beta-Amiloides/metabolismo , Animais , Linhagem Celular , Humanos , Presenilina-1 , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecçãoRESUMO
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 MolecularRESUMO
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 XRESUMO
Amyloid proteins and peptides comprise a diverse group of molecules that vary both in size and amino-acid sequence, yet assemble into amyloid fibrils that have a common core structure. Kinetic studies of amyloid fibrillogenesis have revealed that certain amyloid proteins form oligomeric intermediates prior to fibril formation. We have investigated fibril formation with a peptide corresponding to residues 195-213 of the human prion protein. Through a combination of kinetic and equilibrium studies, we have found that the fibrillogenesis of this peptide proceeds as an all-or-none reaction where oligomeric intermediates are not stably populated. This variation in whether oligomeric intermediates are stably populated during fibril formation indicates that amyloid proteins assemble into a common fibrillar structure; however, they do so through different pathways.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Príons/química , Príons/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Transferência de Energia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Microscopia Eletrônica , Dados de Sequência Molecular , Naftalenossulfonatos , Nefelometria e Turbidimetria , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Espectrometria de FluorescênciaRESUMO
The presenilins and nicastrin, a type 1 transmembrane glycoprotein, form high molecular weight complexes that are involved in cleaving the beta-amyloid precursor protein (betaAPP) and Notch in their transmembrane domains. The former process (termed gamma-secretase cleavage) generates amyloid beta-peptide (Abeta), which is involved in the pathogenesis of Alzheimer's disease. The latter process (termed S3-site cleavage) generates Notch intracellular domain (NICD), which is involved in intercellular signalling. Nicastrin binds both full-length betaAPP and the substrates of gamma-secretase (C99- and C83-betaAPP fragments), and modulates the activity of gamma-secretase. Although absence of the Caenorhabditis elegans nicastrin homologue (aph-2) is known to cause an embryonic-lethal glp-1 phenotype, the role of nicastrin in this process has not been explored. Here we report that nicastrin binds to membrane-tethered forms of Notch (substrates for S3-site cleavage of Notch), and that, although mutations in the conserved 312-369 domain of nicastrin strongly modulate gamma-secretase, they only weakly modulate the S3-site cleavage of Notch. Thus, nicastrin has a similar role in processing Notch and betaAPP, but the 312-369 domain may have differential effects on these activities. In addition, we report that the Notch and betaAPP pathways do not significantly compete with each other.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidases , Sítios de Ligação/fisiologia , Membrana Celular/ultraestrutura , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Mutação/fisiologia , Estrutura Terciária de Proteína/fisiologia , Receptores Notch , TransfecçãoRESUMO
Missense mutations in presenilin 1 (PS1) and presenilin 2 (PS2) are associated with early-onset familial Alzheimer's disease which displays an accelerated deposition of amyloid plaques and neurofibrillary tangles. Presenilins are multi-spanning transmembrane proteins which localize primarily to the endoplasmic reticulum and the Golgi compartments. We have previously demonstrated that PS1 exists as a high-molecular-mass complex that is likely to contain several functional ligands. Potential binding proteins were screened by the yeast two-hybrid system using the cytoplasmically orientated PS1 loop domain which was shown to interact strongly with members of the armadillo family of proteins, including beta-catenin, p0071 and a novel neuron-specific plakophilin-related armadillo protein (NPRAP). Armadillo proteins can have dual functions that encompass the stabilization of cellular junctions/synapses and the mediation of signal transduction pathways. Our observations suggest that PS1 may contribute to both aspects of armadillo-related pathways involving neurite outgrowth and nuclear translocation of beta-catenin upon activation of the wingless (Wnt) pathway. Alzheimer's disease (AD)-related presenilin mutations exhibit a dominant gain of aberrant function resulting in the prevention of beta-catenin translocation following Wnt signalling. These findings indicate a functional role for PS1 in signalling and suggest that mistrafficking of selected presenilin ligands may be a potential mechanism in the genesis of AD.
Assuntos
Doença de Alzheimer/etiologia , Proteínas de Membrana/fisiologia , Transativadores , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Animais , Proteínas do Citoesqueleto/fisiologia , Humanos , Substâncias Macromoleculares , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Modelos Neurológicos , Estrutura Molecular , Mutação de Sentido Incorreto , Presenilina-1 , Presenilina-2 , Processamento de Proteína Pós-Traducional , Transdução de Sinais , beta CateninaRESUMO
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 , EspectrofotometriaRESUMO
Human islet amyloid polypeptide (hIAPP) accumulates as pancreatic amyloid in type 2 diabetes and readily forms fibrils in vitro. Investigations into the mechanism of hIAPP fibril formation have focused largely on residues 20 to 29, which are considered to comprise a primary amyloidogenic domain. In rodents, proline substitutions within this region and the subsequent beta-sheet disruption, prevents fibril formation. An additional amyloidogenic fragment within the C-terminal sequence, residues 30 to 37, has been identified recently. We have extended these observations by examining a series of overlapping peptide fragments from the human and rodent sequences. Using protein spectroscopy (CD/FTIR), electron microscopy and X-ray diffraction, a previously unrecognised amyloidogenic domain was localised within residues 8 to 20. Synthetic peptides corresponding to this region exhibited a transition from random coil to beta-sheet conformation and assembled into fibrils having a typical amyloid-like morphology. The comparable rat 8-20 sequence, which contains a single His18Arg substitution, was also capable of assembling into amyloid-like fibrils. Examination of peptide fragments corresponding to residues 1 to 13 revealed that the immediate N-terminal region is likely to have only a modulating influence on fibril formation or conformational conversion. The contributions of charged residues as they relate to the amyloid-forming 8-20 sequence were also investigated using IAPP fragments and by assessing the effects of pH and counterions. The identification of these principal amyloidogenic sequences and the effects of associated factors provide details on the IAPP aggregation pathway and structure of the peptide in its fibrillar state.
Assuntos
Amiloide/química , Amiloide/metabolismo , Amiloidose/metabolismo , Ilhotas Pancreáticas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Amiloide/genética , Amiloide/ultraestrutura , Amiloidose/complicações , Animais , Benzotiazóis , Dicroísmo Circular , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Ilhotas Pancreáticas/patologia , Microscopia Eletrônica , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Espectroscopia de Infravermelho com Transformada de Fourier , Tiazóis , Difração de Raios XRESUMO
Both APP and PS-1 are causal genes for early-onset familial Alzheimer's disease (AD) and their mutation effects on cerebral Abeta deposition in the senile plaques were examined in human brains of 29 familial AD (23 PS-1, 6 APP) cases and 14 sporadic AD cases in terms of Abeta40 and Abeta42. Abeta isoform data were evaluated using repeated measures analysis of variance which adjusted for within-subject measurement variation and confounding effects of individual APP and PS-1 mutations, age at onset, duration of illness and APOE genotype. We observed that mutations in both APP and PS-1 were associated with a significant increase of Abeta42 in plaques as been documented previously. In comparison to sporadic AD cases, both APP717 and PS-1 mutation cases had an increased density (measured as the number of plaques/mm(2)) and area (%) of Abeta42 plaques. However, we found an unexpected differential effect of PS-1 but not APP717 mutation cases. At least some of PS-1 but not APP717 mutation cases had the significant increase of density and area of Abeta40-plaques as compared to sporadic AD independently of APOE genotype. Our results suggest that PS-1 mutations affect cerebral accumulation of Abeta burden in a different fashion from APP717 mutations in their familial AD brains.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Proteínas de Membrana/genética , Mutação/genética , Placa Amiloide/genética , Placa Amiloide/patologia , Adulto , Idade de Início , Idoso , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/genética , Contagem de Células , Feminino , Genótipo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Presenilina-1RESUMO
The zinc-binding protein S100beta has been identified as an interacting partner with the microtubule-associated protein tau. Both proteins are individually affected in Alzheimer's disease (AD). S100beta, is overexpressed in the disease, whereas hyperphosphorylated tau constitutes the primary component of neurofibrillary tangles. In this study, we examine factors that modulate their binding and the potential role the complex may play in AD pathogenesis. Zinc was identified as a critical component in the binding process and a primary modulator of S100beta-associated cellular responses. Abnormally phosphorylated tau extracted from AD tissue displayed a dramatically reduced capacity to bind S100beta, which was restored by pretreatment with alkaline phosphatase. In differentiated SH-SY5Y cells, exogenous S100beta was internalized and colocalized with tau consistent with an intracellular association. This was enhanced by the addition of zinc and eliminated by divalent metal chelators. S100beta uptake was also accompanied by extensive neurite outgrowth that may be mediated by its interaction with tau. S100beta-tau binding may represent a key pathway for neurite development, possibly through S100beta modulation of tau phosphorylation and/or functional stabilization of microtubules and process formation. S100beta-tau interaction may be disrupted by hyperphosphorylation and/or imbalances in zinc metabolism, and this may contribute to the neurite dystrophy associated with AD.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas S100 , Zinco/metabolismo , Proteínas tau/metabolismo , Encéfalo/metabolismo , Imunofluorescência , Humanos , Fosforilação , Subunidade beta da Proteína Ligante de Cálcio S100RESUMO
Metallothionein III (MT-III) is a functionally distinct member of the metallothionein family that displays neuroinhibitory activity and is involved in the repair of neuronal damage. Altered expression levels of MT-III have been observed in Alzheimer's disease (AD) which has led to suggestions that it could be a mitigating factor in AD-related neuronal dysfunction. However, conflicting results have been reported on this issue which may be due to methodological differences and/or sampling size. In the current study, we have assessed MT-III expression in a large number of AD cases through the quantification of mRNA as well as by immunohistochemistry and Western blotting using an MT-III specific antibody. The results of this comprehensive study indicate that the mononucleosome DNA encoding MT-III is occluded preventing transcription and that message levels are reduced by approximately 30%. In addition, protein levels were specifically decreased by approximately 55% in temporal cortex. These data support the conclusion that MT-III is significantly downregulated in AD and may contribute to the loss of its protective effects and/or repair functions that lead to an exacerbation of the pathogenic processes.