RESUMO
Amyloid-ß (Aß) aggregation intermediates, including oligomers and protofibrils (PFs), have attracted attention as neurotoxic aggregates in Alzheimer's disease. However, due to the complexity of the aggregation pathway, the structural dynamics of aggregation intermediates and how drugs act on them have not been clarified. Here we used high-speed atomic force microscopy to observe the structural dynamics of Aß42 PF at the single-molecule level and the effect of lecanemab, an anti-Aß PF antibody with the positive results from Phase 3 Clarity AD. PF was found to be a curved nodal structure with stable binding angle between individual nodes. PF was also a dynamic structure that associates with other PF molecules and undergoes intramolecular cleavage. Lecanemab remained stable in binding to PFs and to globular oligomers, inhibiting the formation of large aggregates. These results provide direct evidence for a mechanism by which antibody drugs interfere with the Aß aggregation process.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Microscopia de Força Atômica , Fragmentos de PeptídeosRESUMO
Oligomers of the amyloid ß-protein (Aß) have been implicated in the pathogenesis of Alzheimer's disease (AD) through their toxicity towards neurons. Understanding the process of oligomerization may contribute to the development of therapeutic agents, but this has been difficult due to the complexity of oligomerization and the metastability of the oligomers thus formed. To understand the kinetics of oligomer formation, and how that relates to the progression of AD, we developed models of the oligomerization process. Here, we use experimental data from cell viability assays and proxies for rate constants involved in monomer-dimer-trimer kinetics to develop a simple mathematical model linking Aß assembly to oligomer-induced neuronal degeneration. This model recapitulates the rapid growth of disease incidence with age. It does so through incorporation of age-dependent changes in rates of Aß monomer production and elimination. The model also describes clinical progression in genetic forms of AD (e.g., Down's syndrome), changes in hippocampal volume, AD risk after traumatic brain injury, and spatial spreading of the disease due to foci in which Aß production is elevated. Continued incorporation of clinical and basic science data into the current model will make it an increasingly relevant model system for doing theoretical calculations that are not feasible in biological systems. In addition, terms in the model that have particularly large effects are likely to be especially useful therapeutic targets.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Modelos Biológicos , Idoso , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Lesões Encefálicas Traumáticas , Biologia Computacional , Demência , Feminino , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Neurônios/metabolismo , Multimerização ProteicaRESUMO
Targeted overexpression of angiotensin-converting enzyme (ACE), an amyloid-ß protein degrading enzyme, to brain resident microglia and peripheral myelomonocytes (ACE10 model) substantially diminished Alzheimer's-like disease in double-transgenic APPSWE/PS1ΔE9 (AD+) mice. In this study, we explored the impact of selective and transient angiotensin-converting enzyme overexpression on macrophage behaviour and the relative contribution of bone marrow-derived ACE10 macrophages, but not microglia, in attenuating disease progression. To this end, two in vivo approaches were applied in AD+ mice: (i) ACE10/GFP+ bone marrow transplantation with head shielding; and (ii) adoptive transfer of CD115+-ACE10/GFP+ monocytes to the peripheral blood. Extensive in vitro studies were further undertaken to establish the unique ACE10-macrophage phenotype(s) in response to amyloid-ß1-42 fibrils and oligomers. The combined in vivo approaches showed that increased cerebral infiltration of ACE10 as compared to wild-type monocytes (â¼3-fold increase; P < 0.05) led to reductions in cerebral soluble amyloid-ß1-42, vascular and parenchymal amyloid-ß deposits, and astrocytosis (31%, 47-80%, and 33%, respectively; P < 0.05-0.0001). ACE10 macrophages surrounded brain and retinal amyloid-ß plaques and expressed 3.2-fold higher insulin-like growth factor-1 (P < 0.01) and â¼60% lower tumour necrosis factor-α (P < 0.05). Importantly, blood enrichment with CD115+-ACE10 monocytes in symptomatic AD+ mice resulted in pronounced synaptic and cognitive preservation (P < 0.05-0.001). In vitro analysis of macrophage response to well-defined amyloid-ß1-42 conformers (fibrils, prion rod-like structures, and stabilized soluble oligomers) revealed extensive resistance to amyloid-ß1-42 species by ACE10 macrophages. They exhibited 2-5-fold increased surface binding to amyloid-ß conformers as well as substantially more effective amyloid-ß1-42 uptake, at least 8-fold higher than those of wild-type macrophages (P < 0.0001), which were associated with enhanced expression of surface scavenger receptors (i.e. CD36, scavenger receptor class A member 1, triggering receptor expressed on myeloid cells 2, CD163; P < 0.05-0.0001), endosomal processing (P < 0.05-0.0001), and â¼80% increased extracellular degradation of amyloid-ß1-42 (P < 0.001). Beneficial ACE10 phenotype was reversed by the angiotensin-converting enzyme inhibitor (lisinopril) and thus was dependent on angiotensin-converting enzyme catalytic activity. Further, ACE10 macrophages presented distinct anti-inflammatory (low inducible nitric oxide synthase and lower tumour necrosis factor-α), pro-healing immune profiles (high insulin-like growth factor-1, elongated cell morphology), even following exposure to Alzheimer's-related amyloid-ß1-42 oligomers. Overall, we provide the first evidence for therapeutic roles of angiotensin-converting enzyme-overexpressing macrophages in preserving synapses and cognition, attenuating neuropathology and neuroinflammation, and enhancing resistance to defined pathognomonic amyloid-ß forms.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Placa Amiloide/metabolismo , Transferência Adotiva , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Transplante de Medula Óssea , Modelos Animais de Doenças , Técnicas In Vitro , Fator de Crescimento Insulin-Like I/metabolismo , Lisinopril/farmacologia , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Microglia/patologia , Monócitos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Peptidil Dipeptidase A/metabolismo , Placa Amiloide/patologia , Presenilina-1/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Amyloid ß-protein (Aß) molecules tend to aggregate and subsequently form low MW (LMW) oligomers, high MW (HMW) aggregates such as protofibrils, and ultimately fibrils. These Aß species can generally form amyloid plaques implicated in the neurodegeneration of Alzheimer disease (AD), but therapies designed to reduce plaque load have not demonstrated clinical efficacy. Recent evidence implicates amyloid oligomers in AD neuropathology, but the precise mechanisms are uncertain. We examined the mechanisms of neuronal dysfunction from HMW-Aß1-42 exposure by measuring membrane integrity, reactive oxygen species (ROS) generation, membrane lipid peroxidation, membrane fluidity, intracellular calcium regulation, passive membrane electrophysiological properties, and long-term potentiation (LTP). HMW-Aß1-42 disturbed membrane integrity by inducing ROS generation and lipid peroxidation, resulting in decreased membrane fluidity, intracellular calcium dysregulation, depolarization, and impaired LTP. The damaging effects of HMW-Aß1-42 were significantly greater than those of LMW-Aß1-42. Therapeutic reduction of HMW-Aß1-42 may prevent AD progression by ameliorating direct neuronal membrane damage.-Yasumoto, T., Takamura, Y., Tsuji, M., Watanabe-Nakayama, T., Imamura, K., Inoue, H., Nakamura, S., Inoue, T., Kimura, A., Yano, S., Nishijo, H., Kiuchi, Y., Teplow, D. B., Ono, K. High molecular weight amyloid ß1-42 oligomers induce neurotoxicity via plasma membrane damage.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Cálcio/metabolismo , Linhagem Celular Tumoral , Eletrofisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Fluidez de Membrana , Microscopia de Força Atômica , Peso Molecular , Técnicas de Patch-Clamp , Espécies Reativas de Oxigênio/metabolismoRESUMO
Aggregation of amyloidogenic proteins into insoluble amyloid fibrils is implicated in various neurodegenerative diseases. This process involves protein assembly into oligomeric intermediates and fibrils with highly polymorphic molecular structures. These structural differences may be responsible for different disease presentations. For this reason, elucidation of the structural features and assembly kinetics of amyloidogenic proteins has been an area of intense study. We report here the results of high-speed atomic force microscopy (HS-AFM) studies of fibril formation and elongation by the 42-residue form of the amyloid ß-protein (Aß1-42), a key pathogenetic agent of Alzheimer's disease. Our data demonstrate two different growth modes of Aß1-42, one producing straight fibrils and the other producing spiral fibrils. Each mode depends on initial fibril nucleus structure, but switching from one growth mode to another was occasionally observed, suggesting that fibril end structure fluctuated between the two growth modes. This switching phenomenon was affected by buffer salt composition. Our findings indicate that polymorphism in fibril structure can occur after fibril nucleation and is affected by relatively modest changes in environmental conditions.
Assuntos
Peptídeos beta-Amiloides/química , Amiloide/ultraestrutura , Fragmentos de Peptídeos/química , Amiloide/química , Peptídeos beta-Amiloides/síntese química , Soluções Tampão , Humanos , Microscopia de Força Atômica , Fragmentos de Peptídeos/síntese química , Cloreto de Potássio/química , Conformação Proteica , Cloreto de Sódio/química , Soluções , Propriedades de SuperfícieRESUMO
Osteopontin (OPN), a matricellular immunomodulatory cytokine highly expressed by myelomonocytic cells, is known to regulate immune cell migration, communication, and response to brain injury. Enhanced cerebral recruitment of monocytes achieved through glatiramer acetate (GA) immunization or peripheral blood enrichment with bone marrow (BM)-derived CD115+ monocytes (MoBM) curbs amyloid ß-protein (Aß) neuropathology and preserves cognitive function in murine models of Alzheimer's disease (ADtg mice). To elucidate the beneficial mechanisms of these immunomodulatory approaches in AD, we focused on the potential role of OPN in macrophage-mediated Aß clearance. Here, we found extensive OPN upregulation along with reduction of vascular and parenchymal Aß burden in cortices and hippocampi of GA-immunized ADtg mice. Treatment combining GA with blood-grafted MoBM further increased OPN levels surrounding residual Aß plaques. In brains from AD patients and ADtg mice, OPN was also elevated and predominantly expressed by infiltrating GFP+- or Iba1+-CD45high monocyte-derived macrophages engulfing Aß plaques. Following GA immunization, we detected a significant increase in a subpopulation of inflammatory blood monocytes (CD115+CD11b+Ly6Chigh) expressing OPN, and subsequently, an elevated population of OPN-expressing CD11b+Ly6C+CD45high monocyte/macrophages in the brains of these ADtg mice. Correlogram analyses indicate a strong linear correlation between cerebral OPN levels and macrophage infiltration, as well as a tight inverse relation between OPN and Aß-plaque burden. In vitro studies corroborate in vivo findings by showing that GA directly upregulates OPN expression in BM-derived macrophages (MФBM). Further, OPN promotes a phenotypic shift that is highly phagocytic (increased uptake of Aß fibrils and surface scavenger receptors) and anti-inflammatory (altered cell morphology, reduced iNOS, and elevated IL-10 and Aß-degrading enzyme MMP-9). Inhibition of OPN expression in MФBM, either by siRNA, knockout (KOOPN), or minocycline, impairs uptake of Aß fibrils and hinders GA's neuroprotective effects on macrophage immunological profile. Addition of human recombinant OPN reverses the impaired Aß phagocytosis in KOOPN-MФBM. This study demonstrates that OPN has an essential role in modulating macrophage immunological profile and their ability to resist pathogenic forms of Aß.
Assuntos
Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Osteopontina/metabolismo , Animais , Encéfalo/irrigação sanguínea , Modelos Animais de Doenças , Encefalite/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/metabolismo , Fagocitose , Regulação para CimaRESUMO
Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid ß-protein (Aß) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aß oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na(+)/K(+)-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aß-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/toxicidade , ATPase Trocadora de Sódio-Potássio/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Espectrometria de Massas , Modelos Biológicos , Modelos Moleculares , Imagem Molecular , Dados de Sequência Molecular , Peso Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/metabolismo , Agregados Proteicos , Ligação Proteica/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
Amyloid ß-protein (Aß) assembly is a seminal process in Alzheimer's disease. Elucidating the mechanistic features of this process is thought to be vital for the design and targeting of therapeutic agents. Computational studies of the most pathologic form of Aß, the 42-residue Aß42 peptide, have suggested that hydrogen bonding involving Ser26 may be particularly important in organizing a monomer folding nucleus and in subsequent peptide assembly. To study this question, we experimentally determined structure-activity relationships among Aß42 peptides in which Ser26 was replaced with Gly, Ala, α-aminobutryic acid (Abu), or Cys. We observed that aliphatic substitutions (Ala and Abu) produced substantially increased rates of formation of ß-sheet, hydrophobic surface, and fibrils, and higher levels of cellular toxicity. Replacement of the Ser hydroxyl group with a sulfhydryl moiety (Cys) did not have these effects. Instead, this peptide behaved like native Aß42, even though the hydropathy of Cys was similar to that of Abu and very different from that of Ser. We conclude that H bonding of Ser26 is the factor most important in its contribution to Aß42 conformation, assembly, and subsequent toxicity.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Ligação de Hidrogênio , Conformação Proteica , Dobramento de ProteínaRESUMO
The neuropathology of Alzheimer's disease (AD) includes amyloid plaque formation by the amyloid ß-protein (Aß) and intracellular paired helical filament formation by tau protein. These neuropathogenetic features correlate with disease progression and have been revealed in brains of AD patients using positron emission tomography (PET). One of the most useful positron emission tomography imaging agents has been Pittsburgh Compound-B (PiB). However, since its introduction in 2002, substantial evidence has accumulated suggesting that Aß oligomerization and protofibril formation, rather than fibril formation per se, may be the more important pathogenetic event in AD. Detecting protofibrils and oligomeric forms of Aß thus may be of value. We report here the results of experiments to determine whether PiB binds to oligomers or protofibrils formed by Aß40 and Aß42. We observed strong binding to Aß42 fibrils, significant binding to protofibrils, and weaker binding to Aß42 oligomers. PiB also binds Aß40 fibrils, but its binding to Aß40 protofibrils and oligomers is substantially lower than for that observed for Aß42.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Compostos de Anilina/farmacologia , Encéfalo/metabolismo , Tiazóis/farmacologia , Proteínas tau/metabolismo , Encéfalo/efeitos dos fármacos , Progressão da Doença , Humanos , Emaranhados Neurofibrilares/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Ligação ProteicaRESUMO
Amyloid-ß (Aß) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aß and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aß and p-tau in large populations of individual synaptic terminals. Soluble Aß oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aß was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aß oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aß-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aß drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/análise , Sinapses/patologia , Proteínas tau/análise , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Masculino , Microscopia Confocal , Fosforilação , Ratos , Ratos TransgênicosRESUMO
Evidence suggests that amyloid ß-protein (Aß) oligomers may be seminal pathogenic agents in Alzheimer's disease (AD). If so, developing oligomer-targeted therapeutics requires an understanding of oligomer structure. This has been difficult due to the instability of these non-covalently associated Aß assemblies. We previously used rapid, zero-length, in situ chemical cross-linking to stabilize oligomers of Aß40. These enabled us to isolate pure, stable populations of dimers, trimers, and tetramers and to determine their structure-activity relationships. However, equivalent methods applied to Aß42 did not produce stable oligomers. We report here that the use of an Aß42 homologue, [F10, Y42]Aß42, coupled with sequential denaturation/dissociation and gel electrophoresis procedures, provides the means to produce highly pure, stable populations of oligomers of sizes ranging from dimer through dodecamer that are suitable for structure-activity relationship determination.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , HumanosRESUMO
A key pathogenic agent in Alzheimer's disease (AD) is the amyloid ß-protein (Aß), which self-assembles into a variety of neurotoxic structures. Establishing structure-activity relationships for these assemblies, which is critical for proper therapeutic target identification and design, requires aggregation and neurotoxicity experiments that are properly controlled with respect to the Aß peptide itself. "Reverse" Aß or non-Aß peptides suffer from the fact that their biophysical properties are too similar or dissimilar, respectively, to those of native Aß for them to be appropriate controls. For this reason, we used simple protein design principles to create scrambled Aß peptides predicted to behave distinctly from native Aß. We showed that our prediction was true by monitoring secondary structure dynamics with thioflavin T fluorescence and circular dichroism spectroscopy, determining oligomer size distributions, and assaying neurotoxic activity. We then demonstrated the utility of the scrambled Aß peptides by using them to control experiments examining the effects of Aß monomers, dimers, higher-order oligomers, and fibrils on gene expression in primary rat hippocampal neurons. Significant changes in gene expression were observed for all peptide assemblies, but fibrils induced the largest changes. Weighted gene co-expression network analysis revealed two predominant gene modules related to Aß treatment. Many genes within these modules were associated with inflammatory signaling pathways.
Assuntos
Peptídeos beta-Amiloides/química , Biopolímeros/química , Expressão Gênica , Animais , Dicroísmo Circular , Feminino , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Evidence suggests that oligomers of the 42-residue form of the amyloid ß-protein (Aß), Aß42, play a critical role in the etiology of Alzheimer's disease (AD). Here we use high resolution atomic force microscopy to directly image populations of small oligomers of Aß42 that occur at the earliest stages of aggregation. We observe features that can be attributed to a monomer and to relatively small oligomers, including dimers, hexamers, and dodecamers. We discovered that Aß42 hexamers and dodecamers quickly become the dominant oligomers after peptide solubilization, even at low (1 µM) concentrations and short (5 min) incubation times. Soon after (≥10 min), dodecamers are observed to seed the formation of extended, linear preprotofibrillar ß-sheet structures. The preprotofibrils are a single Aß42 layer in height and can extend several hundred nanometers in length. To our knowledge this is the first report of structures of this type. In each instance the preprotofibril is associated off center with a single layer of a dodecamer. Protofibril formation continues at longer times, but is accompanied by the formation of large, globular aggregates. Aß40, by contrast, does not significantly form the hexamer or dodecamer but instead produces a mixture of smaller oligomers. These species lead to the formation of a branched chain-like network rather than discrete structures.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Biopolímeros/metabolismo , Humanos , Microscopia de Força AtômicaRESUMO
In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer's disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid ß-protein peptide (Aß), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aß peptides can form oligomeric structures resembling cylindrins and ß-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aß fragments, including Aß(24-34), Aß(25-35) and Aß(26-36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt ß-barrel structures and that ß-barrels can be formed by folding an out-of-register ß-sheet, a common type of structure found in amyloid proteins.
Assuntos
Peptídeos beta-Amiloides/química , Proteínas Sanguíneas/química , Sequência de Aminoácidos , Microscopia Eletrônica de Transmissão , Conformação Proteica , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A popular working hypothesis of Alzheimer's disease causation is amyloid ß-protein oligomers are the key neuropathogenetic agents. Rigorously elucidating the role of oligomers requires the production of stable oligomers of each size. We previously used zero-length photochemical cross-linking to allow stabilization, isolation, and determination of structure-activity relationships of pure populations of Aß40 dimers, trimers, and tetramers. We also attempted to study Aß42 but found that Aß42 oligomers subjected to the same procedures were not completely stable. On the basis of the fact that Tyr is a critical residue in cross-linking chemistry, we reasoned that the chemical accessibility of Tyr10 in Aß42 must differ from that in Aß40. We thus chemically synthesized four singly substituted Tyr variants that placed the Tyr in different positions across the Aß42 sequence. We then studied the stability of the resulting cross-linked oligomers as well as procedures for fractionating the oligomers to obtain pure populations of different sizes. We found that [Phe(10),Tyr(42)]Aß42 produced stable oligomers yielding highly pure populations of dimers through heptamers. This provides the means to establish formal structure-activity relationships of these important Aß42 assemblies. In addition, we were able to analyze the dissociation patterns of non-cross-linked oligomers to produce a model for oligomer formation. This work is relevant to the determination of structure-activity relationships that have the potential to provide mechanistic insights into disease pathogenesis.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/etiologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Reagentes de Ligações Cruzadas , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Agregação Patológica de Proteínas/genética , Engenharia de Proteínas , Estabilidade Proteica , Estrutura Quaternária de ProteínaRESUMO
Epidemiological evidence that red wine consumption negatively correlates with risk of Alzheimer's disease has led to experimental studies demonstrating that grape seed extracts inhibit the aggregation and oligomerization of Aß in vitro and ameliorate neuropathology and behavioral deficits in a mouse model of Alzheimer's disease. The active agent in the extracts is a mixed population of polyphenolic compounds. To evaluate the relative potency of each of these compounds, HPLC was used to fractionate the mixture into monomers, dimers, and oligomers. Each fraction was analyzed for its effect on Aß conformational dynamics (circular dichroism), oligomerization (zero-length photochemical cross-linking), aggregation kinetics (Thioflavin T fluorescence), and morphology (electron microscopy). The relative activities of each fraction were determined on the basis of molar concentration (mol/L) or mass concentration (g/L). When molar concentration, the number concentration of each polyphenolic compound, was considered, the oligomer fraction was the most potent inhibitor of Aß oligomerization and aggregation. However, when mass concentration, the number concentration of phenolic groups, was considered, monomers were the most potent inhibitors. To understand these ostensibly contradictory results, a model of polyphenol:Aß complexation was developed. This model, which was found to be consistent with published X-ray crystallographic studies, offers an explanation for the effects of functional group polyvalency on inhibitor activity. Our data emphasize the importance of an in-depth understanding of the mechanism(s) underlying 'concentration dependence' in inhibitor systems involving polyfunctional agents.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/biossíntese , Polifenóis/farmacologia , Vitis/química , Peptídeos beta-Amiloides/ultraestrutura , Animais , Benzotiazóis , Dicroísmo Circular , Reagentes de Ligações Cruzadas , Corantes Fluorescentes , Camundongos , Modelos Moleculares , Peso Molecular , Emaranhados Neurofibrilares/patologia , Polifenóis/química , Conformação Proteica , Sementes/química , Relação Estrutura-Atividade , TiazóisRESUMO
While polyphenolic compounds have many health benefits, the potential development of polyphenols for the prevention/treatment of neurological disorders is largely hindered by their complexity as well as by limited knowledge regarding their bioavailability, metabolism, and bioactivity, especially in the brain. We recently demonstrated that dietary supplementation with a specific grape-derived polyphenolic preparation (GP) significantly improves cognitive function in a mouse model of Alzheimer's disease (AD). GP is comprised of the proanthocyanidin (PAC) catechin and epicatechin in monomeric (Mo), oligomeric, and polymeric forms. In this study, we report that following oral administration of the independent GP forms, only Mo is able to improve cognitive function and only Mo metabolites can selectively reach and accumulate in the brain at a concentration of â¼400 nM. Most importantly, we report for the first time that a biosynthetic epicatechin metabolite, 3'-O-methyl-epicatechin-5-O-ß-glucuronide (3'-O-Me-EC-Gluc), one of the PAC metabolites identified in the brain following Mo treatment, promotes basal synaptic transmission and long-term potentiation at physiologically relevant concentrations in hippocampus slices through mechanisms associated with cAMP response element binding protein (CREB) signaling. Our studies suggest that select brain-targeted PAC metabolites benefit cognition by improving synaptic plasticity in the brain, and provide impetus to develop 3'-O-Me-EC-Gluc and other brain-targeted PAC metabolites to promote learning and memory in AD and other forms of dementia.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Antioxidantes/uso terapêutico , Proantocianidinas/uso terapêutico , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Animais , Antioxidantes/metabolismo , Disponibilidade Biológica , Biotransformação , Western Blotting , Cromatografia Líquida de Alta Pressão , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Dieta , Sistemas de Liberação de Medicamentos , Feminino , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Polifenóis/química , Polifenóis/farmacologia , Proantocianidinas/farmacocinética , Proantocianidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/efeitos dos fármacos , Vitis/químicaRESUMO
Cerebral deposition of amyloid ß protein (Aß) is an invariant feature of Alzheimer disease (AD), and epidemiological evidence suggests that moderate consumption of foods enriched with phenolic compounds reduce the incidence of AD. We reported previously that the phenolic compounds myricetin (Myr) and rosmarinic acid (RA) inhibited Aß aggregation in vitro and in vivo. To elucidate a mechanistic basis for these results, we analyzed the effects of five phenolic compounds in the Aß aggregation process and in oligomer-induced synaptic toxicities. We now report that the phenolic compounds blocked Aß oligomerization, and Myr promoted significant NMR chemical shift changes of monomeric Aß. Both Myr and RA reduced cellular toxicity and synaptic dysfunction of the Aß oligomers. These results suggest that Myr and RA may play key roles in blocking the toxicity and early assembly processes associated with Aß through different binding.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Antioxidantes/farmacologia , Cinamatos/farmacologia , Depsídeos/farmacologia , Flavonoides/farmacologia , Multimerização Proteica/efeitos dos fármacos , Sinapses/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Sinapses/patologia , Ácido RosmarínicoRESUMO
Gamma-secretase and signal peptide peptidase (SPP) are unusual GxGD aspartyl proteases, which mediate intramembrane proteolysis. In addition to SPP, a family of SPP-like proteins (SPPLs) of unknown function has been identified. We demonstrate that SPPL2b utilizes multiple intramembrane cleavages to liberate the intracellular domain of tumor necrosis factor alpha (TNFalpha) into the cytosol and the carboxy-terminal counterpart into the extracellular space. These findings suggest common principles for regulated intramembrane proteolysis by GxGD aspartyl proteases.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Endopeptidases/metabolismo , Membranas Intracelulares/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases/genética , Sítios de Ligação/genética , Linhagem Celular , Citosol/química , Citosol/enzimologia , Citosol/metabolismo , Humanos , Dados de Sequência Molecular , Mutação/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
This review examines the role of angiotensin-converting enzyme (ACE) in the context of Alzheimer's disease (AD) and its potential therapeutic value. ACE is known to degrade the neurotoxic 42-residue long alloform of amyloid ß-protein (Aß42), a peptide strongly associated with AD. Previous studies in mice, demonstrated that targeted overexpression of ACE in CD115+ myelomonocytic cells (ACE10 models) improved their immune responses to effectively reduce viral and bacterial infection, tumor growth, and atherosclerotic plaque. We further demonstrated that introducing ACE10 myelomonocytes (microglia and peripheral monocytes) into the double transgenic APPSWE/PS1ΔE9 murine model of AD (AD+ mice), diminished neuropathology and enhanced the cognitive functions. These beneficial effects were dependent on ACE catalytic activity and vanished when ACE was pharmacologically blocked. Moreover, we revealed that the therapeutic effects in AD+ mice can be achieved by enhancing ACE expression in bone marrow (BM)-derived CD115+ monocytes alone, without targeting central nervous system (CNS) resident microglia. Following blood enrichment with CD115+ ACE10-monocytes versus wild-type (WT) monocytes, AD+ mice had reduced cerebral vascular and parenchymal Aß burden, limited microgliosis and astrogliosis, as well as improved synaptic and cognitive preservation. CD115+ ACE10-versus WT-monocyte-derived macrophages (Mo/MΦ) were recruited in higher numbers to the brains of AD+ mice, homing to Aß plaque lesions and exhibiting a highly Aß-phagocytic and anti-inflammatory phenotype (reduced TNFα/iNOS and increased MMP-9/IGF-1). Moreover, BM-derived ACE10-Mo/MΦ cultures had enhanced capability to phagocytose Aß42 fibrils, prion-rod-like, and soluble oligomeric forms that was associated with elongated cell morphology and expression of surface scavenger receptors (i.e., CD36, Scara-1). This review explores the emerging evidence behind the role of ACE in AD, the neuroprotective properties of monocytes overexpressing ACE and the therapeutic potential for exploiting this natural mechanism for ameliorating AD pathogenesis.