Extraction of In-Cell ß-Amyloid Fibrillar Aggregates for Studying Molecular-Level Structural Propagations Using Solid-State NMR Spectroscopy.

Molecular-level structural polymorphisms of ß-amyloid (Aß) fibrils have recently been recognized as pathologically significant. High-resolution solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been utilized to study these structural polymorphisms, particularly in ex-vivo fibrils seeded from amyloid extracts of post-mortem brain tissues of Alzheimer's disease (AD) patients. One unaddressed question in current ex-vivo seeding protocol is whether fibrillation from exogenous monomeric Aß peptides, added to the extracted seeds, can be quantitatively suppressed. Addressing this issue is critical because uncontrolled fibrillation could introduce biased molecular structural polymorphisms in the resulting fibrils. Here, we present a workflow to optimize the key parameters of ex-vivo seeding protocols, focusing on the quantification of amyloid extraction and the selection of exogenous monomeric Aß concentrations to minimize nonseeded fibrillation. We validate this workflow using three structurally different 40-residue Aß (Aß40) fibrillar seeds, demonstrating their ability to propagate their structural features to exogenous wild-type Aß40.

Modulation of aggregation and structural polymorphisms of ß-amyloid fibrils in cellular environments by pyroglutamate-3 variant cross-seeding.

Amyloidogenic deposition of ß-amyloid (Aß) peptides in human brain involves not only the wild-type Aß (wt-Aß) sequences, but also posttranslationally modified Aß (PTM-Aß) variants. Recent studies hypothesizes that the PTM-Aß variants may trigger the deposition of wt-Aß, which underlies the pathology of Sporadic Alzheimer's disease. Among PTM-Aß variants, the pyroglutamate-3-Aß (pyroE3-Aß) has attracted much attention because of their significant abundances and broad distributions in senile plaques and dispersible and soluble oligomers. pyroE3-specific antibodies are being tested as potential anti-Aß drugs in clinical trials. However, evidence that support the triggering effect of pyroE3-Aß on wt-Aß in cells remain lacking, which diminishes its pathological relevance. We show here that cross-seeding with pyroE3-Aß40 leads to accelerated extracellular and intracellular aggregation of wt-Aß40 in different neuronal cells. Cytotoxicity levels are elevated through the cross-seeded aggregation, comparing with the self-seeded aggregation of wt-Aß40 or the static presence of pyroE3-Aß40 seeds. For the extracellular deposition in mouse neuroblastoma Neuro2a (N2a) cells, the cytotoxicity elevation correlates positively with the seeding efficiency. Besides aggregation rates, cross-seeding with pyroE3-Aß40 also modulates the molecular level structural polymorphisms of the resultant wt-Aß40 fibrils. Using solid-state nuclear magnetic resonance (ssNMR) spectroscopy, we identified key structural differences between the parent pyroE3/ΔE3 and wt-Aß40 fibrils within their fibrillar cores. Structural propagation from seeds to daughter fibrils is demonstrated to be more pronounced in the extracellular seeding in N2a cells by comparing the ssNMR spectra from different seeded wt-Aß40 fibrils, but less significant in the intracellular seeding process in human neuroblastoma SH-SY5Y cells.

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.

Modulation of Lipid Dynamics in the ß-Amyloid Aggregates Induced Membrane Fragmentation.

Nonspecific membrane disruption is considered a plausible mechanism for the cytotoxicity induced by ß-amyloid (Aß) aggregates. In scenarios of high local Aß concentrations, a two-step membrane fragmentation model has been proposed. Initially, membrane-embedded Aß oligomeric aggregates form, followed by membrane fragmentation. However, the key molecular-level interactions between Aß oligomeric aggregates and lipids that drive the second-stage membrane fragmentation remain unclear. This study monitors the time-dependent changes in lipid dynamics and water accessibility of model liposomes during Aß-induced membrane fragmentation. Our results indicate that lipid dynamics on the nanosecond to microsecond time scale undergo rapid acceleration upon initial incubation with membrane-incorporated Aß oligomeric aggregates, followed by a slow deceleration process. Concurrently, lipid headgroups become less accessible to water. Both observations suggest a carpet-like mechanism of membrane disruption for the Aß-induced membrane fragmentation process.

In-cell 31P solid-state NMR measurements of the lipid dynamics and influence of exogeneous ß-amyloid peptides on live neuroblastoma neuro-2a cells.

Non-specific disruption of cellular membranes induced by aggregation of exogeneous ß-amyloid (Aß) peptides is considered a viable pathological mechanism in Alzheimer's disease (AD). The solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been widely applied in model liposomes to provide important insights on the molecular interactions between membranes and Aß aggregates. Yet, the feasibility of in-cell ssNMR spectroscopy to probe Aß-membrane interactions in native cellular environments has rarely been tested. Here we report the application of in-cell31P ssNMR spectroscopy on live mouse neuroblastoma Neuro-2a (N2a) cells under moderate magic angle spinning (MAS) conditions. Both cell viability and cytoplasmic membrane integrity are retained for up to six hours under 5 kHz MAS frequency at 277 K, which allow applications of direct-polarization 31P spectroscopy and 31P spin-spin (T2) relaxation measurements. The 31P T2 relaxation time constant of N2a cells is significantly increased compared with the model liposome prepared with comparable major phospholipid compositions. With the addition of 5 µM 40-residue Aß (Aß1-40) peptides, the 31P T2 relaxation is instantly accelerated. This work demonstrates the feasibility of using in-cell31P ssNMR to investigate the Aß-membrane interactions in the biologically relevant cellular system.