Early stage ß-amyloid-membrane interactions modulate lipid dynamics and influence structural interfaces and fibrillation.

Molecular interactions between ß-amyloid (Aß) peptide and membranes contribute to the neuronal toxicity of Aß and the pathology of Alzheimer's disease. Neuronal plasma membranes serve as biologically relevant environments for the Aß aggregation process as well as affect the structural polymorphisms of Aß aggregates. However, the nature of these interactions is unknown. Here, we utilized solid-state NMR spectroscopy to explore the site-specific interactions between Aß peptides and lipids in synaptic plasma membranes at the membrane-associated nucleation stage. The key results show that different segments in the hydrophobic sequence of Aß initiate membrane binding and interstrand assembling. We demonstrate early stage Aß-lipid interactions modulate lipid dynamics, leading to more rapid lipid headgroup motion and reduced lateral diffusive motion. These early events influence the structural polymorphisms of yielded membrane-associated Aß fibrils with distinct C-terminal quaternary interface structure compared to fibrils grown in aqueous solutions. Based on our results, we propose a schematic mechanism by which Aß-lipid interactions drive membrane-associated nucleation processes, providing molecular insights into the early events of fibrillation in biological environments.

Application of DNP-enhanced solid-state NMR to studies of amyloid-ß peptide interaction with lipid membranes.

The cellular membrane disruption induced by the aggregation of Aß peptide has been proposed as a plausible cause of neuronal cell death during Alzheimer's disease. The molecular-level details of the Aß interaction with cellular membranes were previously probed using solid state NMR (ssNMR), however, due to the limited sensitivity of the latter, studies were limited to samples with high Aß-to-lipid ratio. The dynamic nuclear polarization (DNP) is a technique for increasing the sensitivity of NMR. In this work we demonstrate the feasibility of DNP-enhanced ssNMR studies of Aß40 peptide interacting with various model liposomes: (1) a mixture of zwitterionic 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG); (2) a mixture of POPC, POPG, cholesterol, sphingomyelin and ganglioside GM1; (3) the synaptic plasma membrane vesicles (SPMVs) extracted from rat brain tissues. In addition, DNP-ssNMR was applied to capturing changes in Aß40 conformation taking place upon the peptide insertion into POPG liposomes. The signal enhancements under conditions of DNP allow carrying out informative 2D ssNMR experiments with about 0.25 mg of Aß40 peptides (i.e. reaching Aß40-to-lipid ratio of 1:200). In the studied liposome models, the 13C NMR chemical shifts at many 13C-labelled sites of Aß40 are characteristic of ß-sheets. In addition, in POPG liposomes the peptide forms hydrophobic contacts F19-L34 and F19-I32. Both the chemical shifts and hydrophobic contacts of Aß40 in POPG remain the same before and after 8 h of incubation. This suggests that conformation at the 13C-labelled sites of the peptide is similar before and after the insertion process. Overall, our results demonstrate that DNP helps to overcome the sensitivity limitation of ssNMR, and thereby expand the applicability of ssNMR for charactering the Aß peptide interacting with lipids.

Fibrillization of 40-Residue ß-Amyloid Peptides in Membrane-Like Environments Leads to Different Fibril Structures and Reduced Molecular Polymorphisms.

The molecular-level polymorphism in ß-Amyloid (Aß) fibrils have recently been considered as a pathologically relevant factor in Alzheimer's disease (AD). Studies showed that the structural deviations in human-brain-seeded Aß fibrils potentially correlated with the clinical histories of AD patients. For the 40-residue Aß (Aß40) fibrils derived from human brain tissues, a predominant molecular structure was proposed based on solid-state nuclear magnetic resonance (ssNMR) spectroscopy. However, previous studies have shown that the molecular structures of Aß 40 fibrils were sensitive to their growth conditions in aqueous environments. We show in this work that biological membranes and their phospholipid bilayer mimics serve as environmental factors to reduce the structural heterogeneity in Aß40 fibrils. Fibrillization in the presence of membranes leads to fibril structures that are significantly different to the Aß40 fibrils grown in aqueous solutions. Fibrils grown from multiple types of membranes, including the biological membranes extracted from the rats' synaptosomes, shared similar ssNMR spectral features. Our studies emphasize the biological relevance of membranes in Aß40 fibril structures and fibrillization processes.

The on-fibrillation-pathway membrane content leakage and off-fibrillation-pathway lipid mixing induced by 40-residue ß-amyloid peptides in biologically relevant model liposomes.

Disruption of the synaptic plasma membrane (SPM) induced by the aggregation of ß-amyloid (Aß) peptides has been considered as a potential mechanism for the neurotoxicity of Aß in Alzheimer's disease (AD). However, the molecular basis of such membrane disruption process remains unclear, mainly because of the severe systematic heterogeneity problem that prevents the high-resolution studies. Our previous studies using a two-component phosphatidylcholine (PC)/phosphatidylglycerol (PG) model liposome showed the presence of Aß-induced membrane disruptions that were either on the pathway or off the pathway of fibril formation. The present study focuses on a more biologically relevant model membrane with compositions that mimic the outer leaflet of SPMs. The main findings are: (1) the two competing membrane disruption effects discovered in PC/PG liposomes and their general peptide-to-lipid-molar-ratio dependence persist in the more complicated membrane models; (2) the SPM-mimic membrane promotes the formation of certain "on-fibrillation-pathway" intermediates with higher α-helical structural population, which lead to more rapid and significant of membrane content leakage; (3) although the "on-fibrillation-pathway" intermediate structures show dependence on membrane compositions, there seems to be a common final fibril structure grown from different liposomes, suggesting that there may be a predominant fibril structure for 40-residue Aß (i.e. Aß40) peptides in biologically-relevant membranes. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.

Solid-State-NMR-Structure-Based Inhibitor Design to Achieve Selective Inhibition of the Parallel-in-Register ß-Sheet versus Antiparallel Iowa Mutant ß-Amyloid Fibrils.

Solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been widely applied to characterize the high-resolution structures of ß-amyloid (Aß) fibrils. While these structures provide crucial molecular insights on the deposition of amyloid plaques in Alzheimer's diseases (AD), ssNMR structures have been rarely used so far as the basis for designing inhibitors. It remains a challenge because the ssNMR-based Aß fibril structures were usually obtained with sparsely isotope-labeled peptides with limited experimental constraints, where the structural models, especially the side-chain coordinates, showed restricted precision. However, these structural models often possess a higher accuracy within the hydrophobic core regions with more well-defined experimental data, which provide potential targets for the molecular design. This work presents an ssNMR-based molecular design to achieve selective inhibition of a particular type of Aß fibrillar structure, which was formed with the Iowa mutant of Aß with parallel-in-register ß-sheet hydrophobic core. The results show that short peptides that mimic the C-terminal ß-strands of the fibril may have a preference in binding to the parallel Aß fibrils rather than the antiparallel fibrils, mainly due to the differences in the high-resolution structures in the fibril elongation interfaces. The Iowa mutant Aß fibrils are utilized in this work mainly as a model to demonstrate the feasibility of the strategy because it is relatively straightforward to distinguish the parallel and antiparallel fibril structures using ssNMR. Our results suggest that it is potentially feasible to design structure-selective inhibitors and/or diagnostic agents to Aß fibrils using ssNMR-based structural models.

Distinct Membrane Disruption Pathways Are Induced by 40-Residue ß-Amyloid Peptides.

Cellular membrane disruption induced by ß-amyloid (Aß) peptides has been considered one of the major pathological mechanisms for Alzheimer disease. Mechanistic studies of the membrane disruption process at a high-resolution level, on the other hand, are hindered by the co-existence of multiple possible pathways, even in simplified model systems such as the phospholipid liposome. Therefore, separation of these pathways is crucial to achieve an in-depth understanding of the Aß-induced membrane disruption process. This study, which utilized a combination of multiple biophysical techniques, shows that the peptide-to-lipid (P:L) molar ratio is an important factor that regulates the selection of dominant membrane disruption pathways in the presence of 40-residue Aß peptides in liposomes. Three distinct pathways (fibrillation with membrane content leakage, vesicle fusion, and lipid uptake through a temporarily stable ionic channel) become dominant in model liposome systems under specific conditions. These individual systems are characterized by both the initial states of Aß peptides and the P:L molar ratio. Our results demonstrated the possibility to generate simplified Aß-membrane model systems with a homogeneous membrane disruption pathway, which will benefit high-resolution mechanistic studies in the future. Fundamentally, the possibility of pathway selection controlled by P:L suggests that the driving forces for Aß aggregation and Aß-membrane interactions may be similar at the molecular level.