Label-Free Infrared Spectroscopic Imaging Reveals Heterogeneity of β-Sheet Aggregates in Alzheimer’s Disease

Abstract

The aggregation of the amyloid beta (Aβ) protein into plaques is a pathological feature of Alzheimer's disease (AD). While amyloid aggregates have been extensively studied in vitro, their structural aspects and associated chemistry in the brain are not fully understood. In this report, we demonstrate, using infrared spectroscopic imaging, that Aβ plaques exhibit significant heterogeneities in terms of their secondary structure and phospholipid content. We show that the capabilities of discrete frequency infrared imaging (DFIR) are ideally suited for characterization of amyloid deposits in brain tissues and employ DFIR to identify nonplaque β-sheet aggregates distributed throughout brain tissues. We further demonstrate that phospholipid-rich β-sheet deposits exist outside of plaques in all diseased tissues, indicating their potential clinical significance. This is the very first application of DFIR toward a characterization of protein aggregates in an AD brain and provides a rapid, label-free approach that allows us to uncover β-sheet heterogeneities in the AD, which may be significant for targeted therapeutic strategies in the future.

Structural heterogeneity of amyloid aggregates identified by spatially resolved nanoscale infrared spectroscopy

“…For visualization of the beta sheet content of the plaques, we plot the spatial map of the ratio of the spectral intensity at 1628cm -1 normalized to the intensity at 1660cm -1 in Fig. 5C-D, which has been demonstrated to be reflective of the beta sheet content in amide-I spectra 47, 49 . It can be clearly seen that the plaques, while expected to contain fibrils with parallel beta sheet structure, exhibit very little beta sheet signature as evidenced by lack of bright pixels within the plaque boundary.…”

“…These tissues were deparaffinized in hexane for 24 prior to imaging. The microscope design is based upon work published by Bhargava and coworkers 50 , and has been described in detail elsewhere 47, 50 . The microscope uses a tunable Quantum Cascade Laser (Block Engineering) as the illumination source and a TE cooled mercury cadmium telluride (MCT) detector (Boston Electronics).…”

“…AFM is not a technique well-suited for tissue imaging, and hence extension of AFM-IR towards characterizing plaques is experimentally challenging. Infrared confocal imaging, which integrates infrared spectroscopy with optical microscopy, is an approach ideal for tissue applications and allows for mapping the chemistry of amyloid aggregates in AD tissues while providing the same molecular contrast [47][48][49] . We have recently demonstrated the capabilities of infrared imaging towards identifying the chemical heterogeneities of plaques 47 , wherein we demonstrated that amyloid plaques of the same morphology can vary significantly in terms of their beta sheet content.…”

“…Infrared confocal imaging, which integrates infrared spectroscopy with optical microscopy, is an approach ideal for tissue applications and allows for mapping the chemistry of amyloid aggregates in AD tissues while providing the same molecular contrast [47][48][49] . We have recently demonstrated the capabilities of infrared imaging towards identifying the chemical heterogeneities of plaques 47 , wherein we demonstrated that amyloid plaques of the same morphology can vary significantly in terms of their beta sheet content. The heterogeneity in the amyloid aggregates observed in this study are in excellent agreement with our prior work and provide a molecular-level explanation of the plaque chemical variations.…”

Effect of Cholesterol Molecules on Aβ1-42 Wild-Type and Mutants Trimers

“…Indeed, it was shown that free cholesterol has an inhibitory effect on the aggregation of the 37-residue amylin protein, which has two hydrophobic regions, LANFLV and FGAIL, separated by HSSNN, in both solutions and on model membranes [62]. Clearly, a better understanding of the interactions of free cholesterols or free phospholipids on amyloid aggregates in the brain either by computational [63] or experimental [25,64] means must be further explored. To this end, we are coupling a coarse-grained protein force field in an aqueous solution [65,66] with coarse-grained cholesterols and phospholipids [67] to explore larger aggregates and the impact of other disease-causing and disease-protecting mutations [68][69][70][71].…”