Christina Dammers

Purification and Characterization of Recombinant N-Terminally Pyroglutamate-Modified Amyloid-β Variants and Structural Analysis by Solution NMR Spectroscopy.

Alzheimer’s disease (AD) is the leading cause of dementia in the elderly and is characterized by memory loss and cognitive decline. Pathological hallmark of AD brains are intracellular neurofibrillary tangles and extracellular amyloid plaques. The major component of these plaques is the highly heterogeneous amyloid-β (Aβ) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-β (pEAβ) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated Aβ. Compared to unmodified Aβ, pEAβ is known to show increased hydrophobicity, higher toxicity, faster aggregation and β-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEAβ isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEAβ and comparison to its non-modified variant has so far been seriously hampered by the lack of highly pure recombinant and isotope-enriched protein. Here we present, to our knowledge, for the first time a reproducible protocol for the production of pEAβ from a recombinant precursor expressed in E. coli in natural isotope abundance as well as in uniformly [U-15N]- or [U-13C, 15N]-labeled form, with yields of up to 15 mg/l E. coli culture broth. The chemical state of the purified protein was evaluated by RP-HPLC and formation of pyroglutamate was verified by mass spectroscopy. The recombinant pyroglutamate-modified Aβ peptides showed characteristic sigmoidal aggregation kinetics as monitored by thioflavin-T assays. The quality and quantity of produced pEAβ40 and pEAβ42 allowed us to perform heteronuclear multidimensional NMR spectroscopy in solution and to sequence-specifically assign the backbone resonances under near-physiological conditions. Our results suggest that the presented method will be useful in obtaining cost-effective high-quality recombinant pEAβ40 and pEAβ42 for further physiological and biochemical studies.

Structural Analysis and Aggregation Propensity of Pyroglutamate Aβ(3-40) in Aqueous Trifluoroethanol

A hallmark of Alzheimer’s disease (AD) is the accumulation of extracellular amyloid-β (Aβ) plaques in the brains of patients. N-terminally truncated pyroglutamate-modified Aβ (pEAβ) has been described as a major compound of Aβ species in senile plaques. pEAβ is more resistant to degradation, shows higher toxicity and has increased aggregation propensity and β-sheet stabilization compared to non-modified Aβ. Here we characterized recombinant pEAβ(3–40) in aqueous trifluoroethanol (TFE) solution regarding its aggregation propensity and structural changes in comparison to its non-pyroglutamate-modified variant Aβ(1–40). Secondary structure analysis by circular dichroism spectroscopy suggests that pEAβ(3–40) shows an increased tendency to form β-sheet-rich structures in 20% TFE containing solutions where Aβ(1–40) forms α-helices. Aggregation kinetics of pEAβ(3–40) in the presence of 20% TFE monitored by thioflavin-T (ThT) assay showed a typical sigmoidal aggregation in contrast to Aβ(1–40), which lacks ThT positive structures under the same conditions. Transmission electron microscopy confirms that pEAβ(3–40) aggregated to large fibrils and high molecular weight aggregates in spite of the presence of the helix stabilizing co-solvent TFE. High resolution NMR spectroscopy of recombinantly produced and uniformly isotope labeled [U-15N]-pEAβ(3–40) in TFE containing solutions indicates that the pyroglutamate formation affects significantly the N-terminal region, which in turn leads to decreased monomer stability and increased aggregation propensity.

Pyroglutamate-Modified Amyloid-β(3–42) Shows α-Helical Intermediates before Amyloid Formation

Pyroglutamate-modified amyloid-β (pEAβ) has been described as a relevant Aβ species in Alzheimers-disease-affected brains, with pEAβ (3-42) as a dominant isoform. Aβ (1-40) and Aβ (1-42) have been well characterized under various solution conditions, including aqueous solutions containing trifluoroethanol (TFE). To characterize structural properties of pEAβ (3-42) possibly underlying its drastically increased aggregation propensity compared to Aβ (1-42), we started our studies in various TFE-water mixtures and found striking differences between the two Aβ species. Soluble pEAβ (3-42) has an increased tendency to form β-sheet-rich structures compared to Aβ (1-42), as indicated by circular dichroism spectroscopy data. Kinetic assays monitored by thioflavin-T show drastically accelerated aggregation leading to large fibrils visualized by electron microscopy of pEAβ (3-42) in contrast to Aβ (1-42). NMR spectroscopy was performed for backbone and side-chain chemical-shift assignments of monomeric pEAβ (3-42) in 40% TFE solution. Although the difference between pEAβ (3-42) and Aβ (1-42) is purely N-terminal, it has a significant impact on the chemical environment of >20% of the total amino acid residues, as revealed by their NMR chemical-shift differences. Freshly dissolved pEAβ (3-42) contains two α-helical regions connected by a flexible linker, whereas the N-terminus remains unstructured. We found that these α-helices act as a transient intermediate to β-sheet and fibril formation of pEAβ (3-42).

Selection and characterization of tau binding D-enantiomeric peptides with potential for therapy of Alzheimer disease

A variety of neurodegenerative disorders, including Alzheimer disease (AD), are associated with neurofibrillary tangles composed of the tau protein, as well as toxic tau oligomers. Inhibitors of pathological tau aggregation, interrupting tau self-assembly, might be useful for the development of therapeutics. Employing mirror image phage display with a large peptide library (over 109 different peptides), we have identified tau fibril binding peptides consisting of d-enantiomeric amino acids. d-enantiomeric peptides are extremely protease stable and not or less immunogenic than l-peptides, and the suitability of d-peptides for in vivo applications have already been demonstrated. Phage display selections were performed using fibrils of the d-enantiomeric hexapeptide VQIVYK, representing residues 306 to 311 of the tau protein, as a target. VQIVYK has been demonstrated to be important for fibril formation of the full lengths protein and forms fibrils by itself. Here, we report on d-enantiomeric peptides, which bind to VQIVYK, tau isoforms like tau3RD (K19) as well as to full lengths tau fibrils, and modulate the aggregation of the respective tau form. The peptides are able to penetrate cells and might be interesting for therapeutic and diagnostic applications in AD research.

Aβ oligomer eliminating compounds interfere successfully with pEAβ(3-42) induced motor neurodegenerative phenotype in transgenic mice

Currently, there are no causative or disease modifying treatments available for Alzheimers disease (AD). Previously, it has been shown that D3, a small, fully d-enantiomeric peptide is able to eliminate low molecular weight Aβ oligomers in vitro, enhance cognition and reduce plaque load in AD transgenic mice. To further characterise the therapeutic potential of D3 towards N-terminally truncated and pyroglutamated Aβ (pEAβ(3-42)) we tested D3 and its head-to-tail tandem derivative D3D3 both in vitro and in vivo in the new mouse model TBA2.1. These mice produce human pEAβ(3-42) leading to a strong, early onset motor neurodegenerative phenotype. In the present study, we were able to demonstrate 1) strong binding affinity of both D3 and D3D3 to pEAβ(3-42) in comparison to Aβ(1-42) and 2) increased affinity of the tandem derivative D3D3 in comparison to D3. Subsequently we tested the therapeutic potentials of both peptides in the TBA2.1 animal model. Truly therapeutic, non-preventive treatment with D3 and D3D3 clearly slowed the progression of the neurodegenerative TBA2.1 phenotype, indicating the strong therapeutic potential of both peptides against pEAβ(3-42) induced neurodegeneration.

PEASS(3-42) INCREASES AGGREGATION PROPENSITY OF ASS(1-42) BY ACCELERATING PRIMARY AND SECONDARY PATHWAYS

Background: A variety of amyloid-b (Ab) peptides differing in length and modification have been discovered with pyroglutamate-modified Ab variants (pEAb) as a significant proportion. pEAb was shown to be more likely to form b-sheet structures and to have a dramatically enhanced aggregation propensity compared to non-modified Ab, suggesting its role in inducing and facilitating Ab oligomerization and accumulation. Despite this potential importance, pEAb aggregationmechanism and the influence on the kinetics of other Ab variants have not yet been elucidated. Methods:Aggregation kinetics of recombinant pEAb(3-42) and Ab(1-42) monomers were analysed at various concentrations and mixing ratios by thioflavin-T assays. Both seeded and nonseeded experiments were performed, in order to be able to investigate the effects of monomeric and aggregated pEAb on the aggregation of Ab(1-42), and vice versa. Results:pEAb(3-42) was found to aggregate much faster than Ab(1-42) and critical concentrations above which aggregation was observed were drastically decreased by one order of magnitude. Mixtures of both peptides, at concentrations where either species alone did not display aggregation, were found to form amyloid fibrils. The addition of pEAb(3-42) monomers and fibrils was shown to notably accelerate Ab(1-42) aggregation. In contrast, the aggregation kinetics of pEAb(3-42) was slowed down in the presence of monomers as well as fibrils of Ab(1-42). Conclusions:We elucidated the co-aggregation mechanism of Ab(1-42) with themore toxic and aggregation prone variant pEAb(3-42). The presence of small amounts of pEAb(3-42) monomers increases primary nucleation of Ab(1-42) fibrils and serve as a highly catalytic surface for secondary nucleation and elongation of non-truncated Ab species. Ab(1-42) monomers drastically decelerate pEAb(3-42) primary and secondary nucleation but do not decrease pEAb(3-42) elongation rate. Ab(1-42) fibrils are not suitable as templates for the addition of monomeric pEAb(3-42). In fact, high concentrations of fibrillary Ab(1-42) can prevent pEAb(3-42) aggregation, presumably due to non-reactive binding of pEAb monomer to Ab(1-42) fibril surfaces. In concentrations where both species do not aggregate as homofibrils, mixtures of pEAb(3-42) and Ab(1-42) display aggregation suggesting crossprimary nucleation. Thus, pEAb(3-42) catalyzes aggregating of Ab(1-42) affecting all reaction processes while Ab(1-42) dramatically slows down pEAb(3-42) primary nucleation and secondary pathways by non-reactive surface binding.