Pyroglutamate-Modified Amyloid-ß(3-42) Shows α-Helical Intermediates before Amyloid Formation.

Pyroglutamate-modified amyloid-ß (pEAß) has been described as a relevant Aß species in Alzheimer's-disease-affected brains, with pEAß (3-42) as a dominant isoform. Aß (1-40) and Aß (1-42) have been well characterized under various solution conditions, including aqueous solutions containing trifluoroethanol (TFE). To characterize structural properties of pEAß (3-42) possibly underlying its drastically increased aggregation propensity compared to Aß (1-42), we started our studies in various TFE-water mixtures and found striking differences between the two Aß species. Soluble pEAß (3-42) has an increased tendency to form ß-sheet-rich structures compared to Aß (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 pEAß (3-42) in contrast to Aß (1-42). NMR spectroscopy was performed for backbone and side-chain chemical-shift assignments of monomeric pEAß (3-42) in 40% TFE solution. Although the difference between pEAß (3-42) and Aß (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 pEAß (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 ß-sheet and fibril formation of pEAß (3-42).

High-Affinity Binding of Monomeric but Not Oligomeric Amyloid-ß to Ganglioside GM1 Containing Nanodiscs.

The interaction of the amyloid-ß protein (Aß) with neuronal cell membranes plays a crucial role in Alzheimer's disease. Aß undergoes structural changes upon binding to ganglioside GM1 containing membranes leading to altered molecular characteristics of the protein. The physiological role of the Aß interaction with the ganglioside GM1 is still unclear. In order to further elucidate the molecular requirements of Aß membrane binding, we tested different nanodiscs varying in their lipid composition, regarding the charge of the headgroups as well as ganglioside GM1 concentration. Nanodiscs are excellent model membrane systems for studying protein membrane interactions, and we show here their suitability to investigate the membrane interaction of Aß. In particular, we set out to investigate whether the binding activity of GM1 to Aß is specific for the assembly state of Aß and compared the binding affinities of monomeric with oligomeric Aß. Using fluorescence titration experiments, we demonstrate high-affinity binding of Aß(1-40) to GM1 containing nanodiscs, with dissociation constants, KD, in the range from 25 to 41 nM, in a GM1 concentration-dependent manner. Biolayer interferometry experiments confirmed the high-affinity binding of monomeric Aß(1-40) (KD of 24 nM to 49 nM) as well as of Aß(1-42) (KD of 30 nM) to GM1 containing nanodiscs, and no binding to phospholipid containing nanodiscs. Interestingly, and in contrast to monomeric Aß, neither oligomeric Aß(1-40) nor oligomeric Aß(1-42) binds to GM1 nanodiscs. To the best of our knowledge, this is the first report of a loss of function for monomeric Aß upon aggregation.

Aß oligomer eliminating compounds interfere successfully with pEAß(3-42) induced motor neurodegenerative phenotype in transgenic mice.

Currently, there are no causative or disease modifying treatments available for Alzheimer's disease (AD). Previously, it has been shown that D3, a small, fully d-enantiomeric peptide is able to eliminate low molecular weight Aß 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 Aß (pEAß(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 pEAß(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 pEAß(3-42) in comparison to Aß(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 pEAß(3-42) induced neurodegeneration.

Selection and Characterization of Tau Binding á´ -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.

Purification and Characterization of Recombinant N-Terminally Pyroglutamate-Modified Amyloid-ß 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-ß (Aß) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-ß (pEAß) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated Aß. Compared to unmodified Aß, pEAß is known to show increased hydrophobicity, higher toxicity, faster aggregation and ß-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEAß isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEAß 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 pEAß 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 Aß peptides showed characteristic sigmoidal aggregation kinetics as monitored by thioflavin-T assays. The quality and quantity of produced pEAß40 and pEAß42 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 pEAß40 and pEAß42 for further physiological and biochemical studies.

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

A hallmark of Alzheimer's disease (AD) is the accumulation of extracellular amyloid-ß (Aß) plaques in the brains of patients. N-terminally truncated pyroglutamate-modified Aß (pEAß) has been described as a major compound of Aß species in senile plaques. pEAß is more resistant to degradation, shows higher toxicity and has increased aggregation propensity and ß-sheet stabilization compared to non-modified Aß. Here we characterized recombinant pEAß(3-40) in aqueous trifluoroethanol (TFE) solution regarding its aggregation propensity and structural changes in comparison to its non-pyroglutamate-modified variant Aß(1-40). Secondary structure analysis by circular dichroism spectroscopy suggests that pEAß(3-40) shows an increased tendency to form ß-sheet-rich structures in 20% TFE containing solutions where Aß(1-40) forms α-helices. Aggregation kinetics of pEAß(3-40) in the presence of 20% TFE monitored by thioflavin-T (ThT) assay showed a typical sigmoidal aggregation in contrast to Aß(1-40), which lacks ThT positive structures under the same conditions. Transmission electron microscopy confirms that pEAß(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]-pEAß(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.