The aggregation of amyloid-β (Aβ) is postulated to be the crucial event in Alzheimer’s disease (AD). In particular, small neurotoxic Aβ 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 Aβ. The underlying hypothesis is that ligands bind monomeric Aβ and stabilize these species within the various equilibria with Aβ assemblies, leading ultimately to the elimination of Aβ 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 Aβ monomers with micromolar affinities; (iii) eliminate Aβ oligomers; (iv) reduce Aβ-induced cytotoxicity; and (v) disassemble preformed Aβ aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aβ monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD.

Alzheimer's disease (AD) is the most prominent neurodegenerative disease affecting more than 24 million people worldwide. Currently, it is the sixth-leading cause of death, but until now there is no causal therapy available. The amyloid-beta (Aβ) peptide plays an important role in the pathology of the disease. Especially the soluble, most harmful neurotoxic oligomers of Aβ are discussed to be responsible for the development and progression of the disease. In our group we identified the D-enantiomeric peptide D3 via mirror image phage display, which reduces the formation of Aβ oligomers in vitro [1-3]. In vivo, D3 treatment of tg AD mice yielded improved cognitive abilities and reduced Aβ plaque load compared to untreated mice [4]. Via peptide microarrays we were able to identify derivatives of D3, of which one of them is named DB3. This peptide was characterized via different biophysical and biochemical methods to prove its possible therapeutical abilities. Additionally, we further optimized DB3 and characterized this derivative in vitro, too. We could show that DB3 and the optimized version of the peptide (DB3o) inhibit Aβ1-42 aggregation. As already described for D3 [3], DB3 reduced the content of Aβ oligomers and lead to the formation of bigger aggregates. Thereby formed Aβ-DB3-coaggregates feature amorphe structures and do not show any seeding potential. DB3o has the same properties, but is already effective at lower concentrations. Both substances seem to disassemble preformed Aβ protofibrils.

It is widely believed that Alzheimer's disease pathogenesis is driven by the production and deposition of the amyloid-β peptide (Aβ) in the brain. In this study, we employ a combination of in silico and in vitro approaches to investigate the inhibitory properties of selected arginine-rich D-enantiomeric peptides (D-peptides) against amyloid aggregation. The D-peptides include D3, a 12-residue peptide with anti-amyloid potencies demonstrated in vitro and in vivo, RD2, a scrambled sequence of D3, as well as truncated RD2 variants. Using a global optimization method together with binding free energy calculations followed by molecular dynamics simulations, we perform a detailed analysis of D-peptide binding to Aβ monomer and a fibrillar Aβ structure. Results obtained from both molecular simulations and surface plasmon resonance experiments reveal a strong binding of D3 and RD2 to Aβ, leading to a significant reduction in the amount of β structures in both monomer and fibril, which was also demonstrated in Thioflavin T assays. The binding of the D-peptides to Aβ is driven by electrostatic interactions, mostly involving the D-arginine residues and Glu11, Glu22 and Asp23 of Aβ. Furthermore, we show that the anti-amyloid activities of the D-peptides depend on the length and sequence of the Dpeptide, its ability to form multiple weak hydrophobic interactions with Aβ, as well as the Aβ oligomer size.

One of the characteristic pathological hallmarks of Alzheimer's disease (AD) is neuritic plaques. The sequence of events leading to deposition of amyloid-β (Aβ) peptides in plaques is not clear. Here we investigate the effects of D3, an Aβ oligomer directed D-enantiomeric peptide that was obtained from a mirror image phage display selection against monomeric or small oligomeric forms of Aβ42, on Aβ deposition in aged AβPP/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 Aβ deposits is dramatically reduced in the D3-FITC-treated mice. Our findings demonstrate that treatments with the high affinity Aβ42 oligomer binding D-enantiomeric peptide D3 significantly decrease Aβ 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 Aβ peptide in the brains of AβPP/PS1 mice, possibly by increasing Aβ outflow from the brain. In conclusion, treatments with this D-peptide have great potential to be successful in AD patients.

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 (Aβ) in the form of oligomers and fibrils. However, how aging induces Aβ aggregation is not yet fully determined. Some residues in the Aβ sequence seem to promote Aβ-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 Aβ 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 Aβ aggregation, we substituted Tyr 10 with a synthetic amino acid that abolishes Aβ-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 Aβ-membrane interactions in Aβ-induced neurotoxicity. It demonstrates the relevance of Aβ sequence in (i) Aβ-membrane interaction, underlining the role of age-dependent enhanced GM1 content in promoting Aβ aggregation, (ii) Aβ aggregation, and (iii) Aβ-induced oxidative stress. Our results open the way for the design of peptides aimed to inhibit Aβ aggregation and neurotoxicity.

A key feature of Alzheimer disease (AD) is the pathologic self-association of the amyloid-β (Aβ) peptide, leading to the formation of diffusible toxic Aβ oligomers and extracellular amyloid plaques. Next to extracellular Aβ, intraneuronal Aβ 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 Aβ binding peptide L3 and its influence on Aβ oligomerization in vitro. Preliminary studies in cell culture demonstrate that stably expressed L3 reduces cell toxicity of externally added Aβ in neuroblastoma cells.

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 β (Aβ) peptides in plaques or in diffuse deposits is not clear. Here we investigate the relationship between aggregation and deposition of Aβ by using peptides that bind to Aβ 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 Aβ42, 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 Aβ deposits is dramatically reduced in both the D3- and D3-FITC-treated mice. Our findings demonstrate that treatments with a high-affinity Aβ-42-binding d-amino acid peptide significantly decrease Aβ deposits and the associated inflammatory response. Together, this suggests that aggregation likely plays an important role in the deposition of Aβ protein in APP/PS1 transgenic mice and that antiaggregation treatments with d-peptides may be successful in AD patients.

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-β peptide (Aβ). 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 Aβ-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 Aβ1-42. We describe ACI-80 derivatives with increased stability and Aβ 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.

Alzheimer's disease (AD) is the most common cause of dementia in elderly people and age is the major non-genetic risk factor for sporadic AD. A hallmark of AD is the accumulation of amyloid in the brain, which is composed mainly by the amyloid beta-peptide (Ab) in the form of oligomers and fibrils. However, how aging induces Ab aggregation is not yet fully determined. Tyr 10 in Ab sequences seems to be associated to the 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 Tyr 10 in Ab aggregation following interaction with the plasma membrane is not yet demonstrated. We decided to explore the function of Tyr10 in Ab aggregation in vitro and in cellulo. To elucidate the role of Tyr 10 in Ab aggregation and toxicity, we substituted in Ab1-42 sequence Tyr10 with the synthetic amino acid para-amino-Phenylalanine. We analyzed the aggregation properties of wild-type and mutant Ab in vitro and in cellulo. Moreover, we analyzed their uptake in neuronal cells. The Ab mutant shows impaired aggregation properties and sensibly increased affinity for GM1 in vitro. It has a dominant negative effect on amyloidogenesis in vitro and in cellulo, where it impairs the aggregation of wild-type Ab. Moreover, this mutant does not induce oxidative stress and it blocks wild-type Ab -induced cell death. The present study shed new light in the understanding of Ab-membrane interactions in Ab-induced neurotoxicity. It demonstrates the relevance of Tyr 10 in (i) Ab-membrane interaction, (ii) Ab aggregation, (iii) Ab-induced oxidative stress. Furthermore, our results open the way for the design of peptides aimed to inhibit Ab aggregation and neurotoxicity.

Alzheimer's disease is incurable, only palliative therapies are available. The amyloid-ß-peptide (Aß) is suggested as the major causative agent. Driven by its amyloidogenic properties Aß forms several species of ß-sheet-rich aggregates. Less the insoluble fibrils, but especially the soluble Aß oligomers are discussed as most harmful neurotoxic species, responsible for disease development and progression. Aß-targeting D-peptides like D3 and ß-sheet-breaking organic aminopyrazoles were developed by mirror-image phage display and rational drug design, respectively. D3 was extensively characterized using biophysical/biochemical methods and already successfully tested regarding his therapeutic properties in transgenic AD mice. Aminopyrazoles are well-known for their capability to disturb ß-sheets. By combining a D3 and an aminopyrazole trimer, we have developed a novel Aß-targeting hybrid compound with superior in vitro properties. Here, we report on in vivo, in vitro and in silico properties of the Aß-targeting D-enantiomeric amino acid peptide D3 as well as on synergistic properties of the novel hybrid compound JM169. In vitro, D3 specifically precipitates toxic Aß species and converts them into non-amyloidogenic, non-fibrillar and non-toxic amorphous aggregates. Oral application of D3 reduced Aß plaque load, inflammation and improves the cognitive performance of AD transgenic mice. Additionally we show the reduction of naturally secreted Aß oligomers and Aß caused impairment of long-term potentiation in hippocampal slices by the D3-aminopyrazole-hybrid JM169. The D-peptide D3 modulates the oligomerization of Aß42 in vitro and exerts interesting therapeutic features. Furthermore, D3 was covalently linked to an aminopyrazole to design an entire different substance class with novel synergistic properties in targeting Aß.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid beta (Abeta) peptides in amyloid deposits in the cerebral tissue. Current evidence indicates that intraneuronal accumulation of Abeta is an early pathological biomarker for the onset of AD and may contribute to a cascade of neurodegenerative events. Many reports found strong evidence that intermediates in the aggregation process called ‘oligomers’ are the principal pathogenic species that drive neuronal dysfunction rather than the large amyloid aggregates. There are studies suggesting that cellular membranes play a major role in Abeta oligomerization process and that Abeta uptake in neuronal cells is responsible for Abeta-induced neuronal dysfunction and toxicity. We investigate the link between in vitro amyloid structure and the capability of Abeta peptides to be internalized in neuronal cells and induce toxicity. We analyzed in vitro the aggregation properties of an Abeta mutant with impaired aggregation properties by dynamic light scattering (DLS) and thioflavin-T binding. We also measured its capability to alter the aggregation properties of wild-type Abeta. Next, we analyzed the uptake and aggregation of this Abeta mutant in neuronal cells and its capability to alter the uptake of wild-type Abeta. By confocal imaging and Congo red staining, we analyzed the binding at the plasma membrane, internalization and further elongation of wild-type oligomers (Aβ42) compared to the mutant Abeta oligomers We found that adhesion at the plasma membrane of Abeta peptides is inversely correlated with their capability to elongate. These results were finally correlated with the capability of mutant and wild-type Abeta to induce cell toxicity. These studies give insight on the structural basis for Abeta-induced neuronal toxicity and open the way for new therapeutic strategies aimed at selecting Abeta mutants that alter the uptake and the toxicity induced by wild-type Abeta.

Today, only palliative therapies for Alzheimer's disease (AD) are available. Several lines of evidence suggest that the amyloid-β-peptide (Aβ) plays a central role in the pathogenesis of AD. Not only Aβ fibrils, but also small soluble Aβ oligomers in particular are suspected to be the major toxic species responsible for disease development and progression. We successfully tested Aβ binding D-peptides in AD transgenic mouse models regarding their therapeutic properties. Additionally, we used several biophysical and biochemical methods for the elucidation of the possible mechanism. The present study reports on in vitro and in vivo properties of the Aβ 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 a novel potential mechanism underlying the observed in vivo activity of D3. The D-enantiomeric peptide D3 is able to modulate Aβ42 oligomerization in vitro. Regardless of its in vivo mechanism of action, D3 exerts therapeutically interesting activities.

More than the sum of its parts: Novel hybrid compounds consisting of an organic β-sheet-breaking moiety and a signaling, D-enantiomeric Aβ-recognizing peptide moiety have been designed (see picture). The compounds, which were chemically synthesized and characterized by several techniques, combine rational design and drug selection from libraries and inhibit Aβ oligomerization and Aβ-induced synaptic pathology. Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Several lines of evidence suggest that the amyloid-β-peptide (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). Not only Aβ fibrils but also small soluble Aβ 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 Aβ 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 Aβ species and converts them into nonamyloidogenic, nonfibrillar, and nontoxic aggregates without increasing the concentration of monomeric Aβ. Thus, D3 exerts an interesting and novel mechanism of action that abolishes toxic Aβ oligomers and thereby supports their decisive role in AD development and progression.

Mehr als die Summe der Teile: Neuartige Hybridsubstanzen bestehen aus einem organischen, β-Faltblatt-brechenden Teil und einem die Zielsubstanz (Aβ) erkennenden, D-enantiomeren Peptidteil (siehe Bild). Die Substanzen wurden chemisch synthetisiert und mit verschiedenen Techniken charakterisiert. Sie vereinen rationales Design mit Substanzselektion aus Bibliotheken und inhibieren die Aβ-Oligomeraggregation und Aβ-induzierte synaptische Pathologie. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Aging is the most significant risk factor for Alzheimer disease (AD). The pathological hallmark of AD is the accumulation of aggregated amyloid-beta (A beta) forms and insoluble plaques, mainly composed of A beta, in the brain of the patient. Recently, we reported on the selection of D-enantiomeric, A beta-binding peptides D1 and D3. D1 was selected against aggregated A beta species to address diagnosis by in vivo imaging of amyloid plaques, whereas D3 was selected using low-molecular-weight A beta species, therefore addressing therapeutical studies. Here, we use a surface plasmon resonance method to confirm that both peptides show the desired binding specificities.