Structural characterization of amyloid aggregates with spatially resolved infrared spectroscopy.

The self-assembly of proteins and peptides into ordered structures called amyloid fibrils is a hallmark of numerous diseases, impacting the brain, heart, and other organs. The structure of amyloid aggregates is central to their function and thus has been extensively studied. However, the structural heterogeneities between aggregates as they evolve throughout the aggregation pathway are still not well understood. Conventional biophysical spectroscopic methods are bulk techniques and only report on the average structural parameters. Understanding the structure of individual aggregate species in a heterogeneous ensemble necessitates spatial resolution on the length scale of the aggregates. Recent technological advances have led to augmentation of infrared (IR) spectroscopy with imaging modalities, wherein the photothermal response of the sample upon vibrational excitation is leveraged to provide spatial resolution beyond the diffraction limit. These combined approaches are ideally suited to map out the structural heterogeneity of amyloid ensembles. AFM-IR, which integrates IR spectroscopy with atomic force microscopy enables identification of the structural facets the oligomers and fibrils at individual aggregate level with nanoscale resolution. These capabilities can be extended to chemical mapping in diseased tissue specimens with submicron resolution using optical photothermal microscopy, which combines IR spectroscopy with optical imaging. This book chapter provides the basic premise of these novel techniques and provides the typical methodology for using these approaches for amyloid structure determination. Detailed procedures pertaining to sample preparation and data acquisition and analysis are discussed and the aggregation of the amyloid ß peptide is provided as a case study to provide the reader the experimental parameters necessary to use these techniques to complement their research efforts.

Colocalization of ß-Sheets and Carotenoids in Aß Plaques Revealed with Multimodal Spatially Resolved Vibrational Spectroscopy.

The aggregation of amyloid ß(Aß) peptides is at the heart of Alzheimer's disease development and progression. As a result, amyloid aggregates have been studied extensively in vitro, and detailed structural information on fibrillar amyloid aggregates is available. However, forwarding these structural models to amyloid plaques in the human brain is still a major challenge. The chemistry of amyloid plaques, particularly in terms of the protein secondary structure and associated chemical moieties, remains poorly understood. In this report, we use Raman microspectroscopy to identify the presence of carotenoids in amyloid plaques and demonstrate that the abundance of carotenoids is correlated with the overall protein secondary structure of plaques, specifically to the population of ß-sheets. While the association of carotenoids with plaques has been previously identified, their correlation with the ß structure has never been identified. To further validate these findings, we have used optical photothermal infrared (O-PTIR) spectroscopy, which is a spatially resolved technique that yields complementary infrared contrast to Raman. O-PTIR unequivocally demonstrates the presence of elevated ß-sheets in carotenoid-containing plaques and the lack of ß structure in noncarotenoid plaques. Our findings underscore the potential link between anti-inflammatory species as carotenoids to specific secondary structural motifs within Aß plaques and highlight the possible role of chemically distinct plaques in neuroinflammation, which can uncover new mechanistic insights and lead to new therapeutic strategies for AD.

Intermediate Antiparallel Fibrils in Aß40 Dutch Mutant Aggregation: Insights from Nanoscale Infrared Spectroscopy.

Cerebral amyloid angiopathy (CAA), which involves amyloid deposition in blood vessels leading to fatal cerebral hemorrhage and recurring strokes, is present in the majority Alzheimer's disease (AD) cases. Familial mutations in the amyloid ß peptide are correlated to higher risks of CAA and are mostly comprised of mutations at residues 22 and 23. While the structure of the wild-type Aß peptide has been investigated in great detail, less is known about the structure of mutants involved in CAA and evolutions thereof. This is particularly true for mutations at residue 22, for which detailed molecular structures, as typically determined from Nuclear Magnetic Resonance (NMR) spectroscopy or electron microscopy, do not exist. In this report, we have used nanoscale infrared (IR) spectroscopy augmented with atomic force microscopy (AFM-IR) to investigate structural evolution of the Aß Dutch mutant (E22Q) at the single aggregate level. We show that in the oligomeric stage, the structural ensemble is distinctly bimodal, with the two subtypes differing with respect to population of parallel ß sheets. Fibrils on the other hand are structurally homogeneous, with early-stage fibrils distinctly antiparallel in character, which develop parallel ß sheets upon maturation. Furthermore, the antiparallel structure is found to be a persistent feature across different stages of aggregation.

ß-Carotene, a Potent Amyloid Aggregation Inhibitor, Promotes Disordered Aß Fibrillar Structure.

The aggregation of amyloid beta (Aß) into fibrillar aggregates is a key feature of Alzheimer's disease (AD) pathology. ß-carotene and related compounds have been shown to associate with amyloid aggregates and have direct impact on the formation of amyloid fibrils. However, the precise effect of ß-carotene on the structure of amyloid aggregates is not known, which poses a limitation towards developing it as a potential AD therapeutic. In this report, we use nanoscale AFM-IR spectroscopy to probe the structure of Aß oligomers and fibrils at the single aggregate level and demonstrate that the main effect of ß-carotene towards modulating Aß aggregation is not to inhibit fibril formation but to alter the secondary structure of the fibrils and promote fibrils that lack the characteristic ordered beta structure.

Nanoscale Infrared Spectroscopy Identifies Parallel to Antiparallel ß-Sheet Transformation of Aß Fibrils.

Spontaneous aggregation of amyloid beta (Aß) proteins leading to the formation of oligomers and eventually into fibrils has been identified as a key pathological signature of Alzheimer's disease. The structure of late-stage aggregates have been studied in depth by conventional structural biology techniques, including nuclear magnetic resonance, X-ray crystallography, and infrared spectroscopy; however, the structure of early-stage aggregates is less known due to their transient nature. As a result, the structural evolution of amyloid aggregates from early oligomers to mature fibrils is still not fully understood. Here, we have applied atomic force microscopy-infrared nanospectroscopy to investigate the aggregation of Aß 16-22, which spans the amyloidogenic core of the Aß peptide. Our results demonstrate that Aß 16-22 involves a structural transition from oligomers with parallel ß-sheets to antiparallel fibrils through disordered and possibly helical intermediate fibril structures, contrary to the known aggregation pathway of full-length Aß.

Nanoscale Infrared Spectroscopy Identifies Structural Heterogeneity in Individual Amyloid Fibrils and Prefibrillar Aggregates.

Amyloid plaques are one of the central manifestations of Alzheimer's disease pathology. Aggregation of the amyloid beta (Aß) protein from amorphous oligomeric species to mature fibrils has been extensively studied. However, structural heterogeneities in prefibrillar species, and how that affects the structure of later-stage aggregates are not yet well understood. The integration of infrared spectroscopy with atomic force microscopy (AFM-IR) allows for identifying the signatures of individual nanoscale aggregates by spatially resolving spectra. We use AFM-IR to demonstrate that amyloid oligomers exhibit significant structural variations as evidenced in their infrared spectra. This heterogeneity is transmitted to and retained in protofibrils and fibrils. We show that amyloid fibrils do not always conform to their putative ordered structure and structurally different domains exist in the same fibril. We further demonstrate that these structural heterogeneities manifest themselves as a lack of ß sheet structure in amyloid plaques in Alzheimer's tissue using infrared imaging.