RESUMO
The aggregation of amyloid-ß (Aß) is postulated to be the crucial event in Alzheimer's disease (AD). In particular, small neurotoxic Aß oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized d-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aß. The underlying hypothesis is that ligands bind monomeric Aß and stabilize these species within the various equilibria with Aß assemblies, leading ultimately to the elimination of Aß oligomers. One of the hereby identified d-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aß monomers with micromolar affinities; (iii) eliminate Aß oligomers; (iv) reduce Aß-induced cytotoxicity; and (v) disassemble preformed Aß aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aß monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD.
Assuntos
Peptídeos beta-Amiloides/química , Oligopeptídeos/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , RatosRESUMO
One of the characteristic pathological hallmarks of Alzheimer's disease (AD) is neuritic plaques. The sequence of events leading to deposition of amyloid-ß (Aß) peptides in plaques is not clear. Here we investigate the effects of D3, an Aß oligomer directed D-enantiomeric peptide that was obtained from a mirror image phage display selection against monomeric or small oligomeric forms of Aß42, on Aß deposition in aged AßPP/PS1 double transgenic AD-model mice. Using Alzet minipumps, we infused the brains of these AD model mice for 8 weeks with FITC-labeled D3, and examined the subsequent changes in pathology and cognitive deficits. Initial cognitive deficits are similar comparing control and D3-FITC-treated mice, but the treated mice show a significant improvement on the last day of testing. Further, we show that there is a substantial reduction in the amount of amyloid deposits in the animals treated with D3-FITC, compared to the control mice. Finally, the amount of activated microglia and astrocytes surrounding Aß deposits is dramatically reduced in the D3-FITC-treated mice. Our findings demonstrate that treatments with the high affinity Aß42 oligomer binding D-enantiomeric peptide D3 significantly decrease Aß deposits and the associated inflammatory response, and improve cognition even when applied only at late stages and high age. Together, this suggests that the treatment reduces the level of Aß peptide in the brains of AßPP/PS1 mice, possibly by increasing Aß outflow from the brain. In conclusion, treatments with this D-peptide have great potential to be successful in AD patients.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/genética , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/patologia , Oligopeptídeos/administração & dosagem , Placa Amiloide/tratamento farmacológico , Placa Amiloide/patologia , Presenilina-1/genética , Envelhecimento/genética , Envelhecimento/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Animais , Transtornos Cognitivos/genética , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/patologia , Infusões Intraventriculares , Masculino , Camundongos , Camundongos Transgênicos , Oligopeptídeos/genética , Placa Amiloide/genética , Resultado do TratamentoRESUMO
Alzheimer's disease (AD) is a devastating disease affecting predominantly the aging population. One of the characteristic pathological hallmarks of AD are neuritic plaques, consisting of amyloid-ß peptide (Aß). While there has been some advancement in diagnostic classification of AD patients according to their clinical severity, no fully reliable method for pre-symptomatic diagnosis of AD is available. To enable such early diagnosis, which will allow the initiation of treatments early in the disease progress, neuroimaging tools are under development, making use of Aß-binding ligands that can visualize amyloid plaques in the living brain. Here we investigate the properties of a newly designed series of D-enantiomeric peptides which are derivatives of ACI-80, formerly called D1, which was developed to specifically bind aggregated Aß1-42. We describe ACI-80 derivatives with increased stability and Aß binding properties, which were characterized using surface plasmon resonance and enzyme-linked immunosorbent assays. The specific interactions of the lead compounds with amyloid plaques were validated by ex vivo immunochemistry in transgenic mouse models of AD. The novel compounds showed increased binding affinity and are promising candidates for further development into in vivo imaging compounds.
Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Amiloide/química , Diagnóstico por Imagem/métodos , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Ligantes , Camundongos , Neuritos/metabolismo , Neuritos/patologia , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligação ProteicaRESUMO
One of the two characteristic pathological hallmarks of Alzheimer's disease (AD) are neuritic plaques. The sequence of events leading to the extracellular deposition of amyloid ß (Aß) peptides in plaques or in diffuse deposits is not clear. Here we investigate the relationship between aggregation and deposition of Aß by using peptides that bind to Aß as antifibrillization treatments in APP/PS1 double transgenic AD-model mice. Using Alzet minipumps, we infused the brain of these AD-model mice for 4 weeks with one of the three small D-amino acid peptides (i.e., D1, D3, or D3-FITC) that were designed to bind specifically to Aß42, and examined the subsequent improvement in cognitive deficits after 3 weeks and analyzed amyloid deposition in the brain following the behavioral analysis. Cognitive deficits are similar comparing control and D3-treated mice, but D1-treated mice are slightly, but significantly, impaired. In contrast, there is a substantial improvement in the cognitive deficits in the animals treated with D3-FITC, compared to the other mice. In contrast, we show that there is a substantial reduction in the amount of amyloid deposits in the animals treated with D3, compared to the other groups of mice. Furthermore, the amount of activated microglia and astrocytes surrounding Aß deposits is dramatically reduced in both the D3- and D3-FITC-treated mice. Our findings demonstrate that treatments with a high-affinity Aß-42-binding D-amino acid peptide significantly decrease Aß deposits and the associated inflammatory response. Together, this suggests that aggregation likely plays an important role in the deposition of Aß protein in APP/PS1 transgenic mice and that antiaggregation treatments with D-peptides may be successful in AD patients.
Assuntos
Peptídeos beta-Amiloides/química , Amiloide/metabolismo , Inflamação/prevenção & controle , Fragmentos de Peptídeos/uso terapêutico , Animais , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/químicaRESUMO
A key feature of Alzheimer disease (AD) is the pathologic self-association of the amyloid-ß (Aß) peptide, leading to the formation of diffusible toxic Aß oligomers and extracellular amyloid plaques. Next to extracellular Aß, intraneuronal Aß has important pathological functions in AD. Agents that specifically interfere with the oligomerization processes either outside or inside of neurons are highly desired for the elucidation of the pathologic mechanisms of AD and might even pave the way for new AD gene therapeutic approaches. Here, we characterize the Aß binding peptide L3 and its influence on Aß oligomerization in vitro. Preliminary studies in cell culture demonstrate that stably expressed L3 reduces cell toxicity of externally added Aß in neuroblastoma cells.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/química , Terapia Genética/métodos , Animais , Benzotiazóis , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração , Modelos Animais de Doenças , Humanos , Camundongos , Nefelometria e Turbidimetria , Neurônios/metabolismo , Peptídeos/química , Análise de Sequência de DNA , Tiazóis/químicaRESUMO
Alzheimer's disease (AD) is the most common cause of dementia in elderly people, and age is the major nongenetic risk factor for sporadic AD. A hallmark of AD is the accumulation of amyloid in the brain, which is composed mainly of the amyloid beta-peptide (Aß) in the form of oligomers and fibrils. However, how aging induces Aß aggregation is not yet fully determined. Some residues in the Aß sequence seem to promote Aß-induced toxicity in association with age-dependent risk factors for AD, such as (i) increased GM1 brain membrane content, (ii) altered lipid domain in brain membrane, (iii) oxidative stress. However, the role of Aß sequence in promoting aggregation following interaction with the plasma membrane is not yet demonstrated. As Tyr10 is implicated in the induction of oxidative stress and stabilization of Aß aggregation, we substituted Tyr 10 with a synthetic amino acid that abolishes Aß-induced oxidative stress and shows an accelerated interaction with GM1. This variant peptide shows impaired aggregation properties and increased affinity for GM1. It has a dominant negative effect on amyloidogenesis in vitro, in cellulo, and in isolated synaptosomes. The present study shed new light in the understanding of Aß-membrane interactions in Aß-induced neurotoxicity. It demonstrates the relevance of Aß sequence in (i) Aß-membrane interaction, underlining the role of age-dependent enhanced GM1 content in promoting Aß aggregation, (ii) Aß aggregation, and (iii) Aß-induced oxidative stress. Our results open the way for the design of peptides aimed to inhibit Aß aggregation and neurotoxicity.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/genética , Humanos , Estresse Oxidativo/fisiologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Tirosina/químicaRESUMO
Aging is the most significant risk factor for Alzheimer disease (AD). The pathological hallmark of AD is the accumulation of aggregated amyloid-beta (Abeta) forms and insoluble plaques, mainly composed of Abeta, in the brain of the patient. Recently, we reported on the selection of D-enantiomeric, Abeta-binding peptides D1 and D3. D1 was selected against aggregated Abeta species to address diagnosis by in vivo imaging of amyloid plaques, whereas D3 was selected using low-molecular-weight Abeta species, therefore addressing therapeutical studies. Here, we use a surface plasmon resonance method to confirm that both peptides show the desired binding specificities.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/análise , Amiloide/química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/efeitos dos fármacos , Humanos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/química , Placa Amiloide/efeitos dos fármacos , Placa Amiloide/metabolismo , Ligação Proteica , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Multimerização Proteica/efeitos dos fármacos , Especificidade por Substrato , Ressonância de Plasmônio de SuperfícieRESUMO
Several lines of evidence suggest that the amyloid-ß-peptide (Aß) plays a central role in the pathogenesis of Alzheimer's disease (AD). Not only Aß fibrils but also small soluble Aß oligomers in particular are suspected to be the major toxic species responsible for disease development and progression. The present study reports on in vitro and in vivo properties of the Aß targeting d-enantiomeric amino acid peptide D3. We show that next to plaque load and inflammation reduction, oral application of the peptide improved the cognitive performance of AD transgenic mice. In addition, we provide in vitro data elucidating the potential mechanism underlying the observed in vivo activity of D3. These data suggest that D3 precipitates toxic Aß species and converts them into nonamyloidogenic, nonfibrillar, and nontoxic aggregates without increasing the concentration of monomeric Aß. Thus, D3 exerts an interesting and novel mechanism of action that abolishes toxic Aß oligomers and thereby supports their decisive role in AD development and progression.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Modelos Animais de Doenças , Aprendizagem em Labirinto/efeitos dos fármacos , Oligopeptídeos/administração & dosagem , Administração Oral , Doença de Alzheimer/psicologia , Animais , Cognição/efeitos dos fármacos , Cognição/fisiologia , Feminino , Humanos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Oligopeptídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Estereoisomerismo , Resultado do TratamentoRESUMO
The immunophilin-like FKBP42 TWISTED DWARF1 (TWD1) has been shown to control plant development via the positive modulation of ABCB/P-glycoprotein (PGP)-mediated transport of the plant hormone auxin. TWD1 functionally interacts with two closely related proteins, ABCB1/PGP1 and ABCB19/PGP19/MDR1, both of which exhibit the ability to bind to and be inhibited by the synthetic auxin transport inhibitor N-1-naphylphtalamic acid (NPA). They are also inhibited by flavonoid compounds, which are suspected modulators of auxin transport. The mechanisms by which flavonoids and NPA interfere with auxin efflux components are unclear. We report here the specific disruption of PGP1-TWD1 interaction by NPA and flavonoids using bioluminescence resonance energy transfer with flavonoids functioning as a classical established inhibitor of mammalian and plant PGPs. Accordingly, TWD1 was shown to mediate modulation of PGP1 efflux activity by these auxin transport inhibitors. NPA bound to both PGP1 and TWD1 but was excluded from the PGP1-TWD1 complex expressed in yeast, suggesting a transient mode of action in planta. As a consequence, auxin fluxes and gravitropism in twd1 roots are less affected by NPA treatment, whereas TWD1 gain-of-function promotes root bending. Our data support a novel model for the mode of drug-mediated P-glycoprotein regulation mediated via protein-protein interaction with immunophilin-like TWD1.