Heterotypic Seeding Generates Mixed Amyloid Polymorphs.

Aggregation of the amyloid ß (Aß) peptide into fibrils represents one of the major biochemical pathways underlying the development of Alzheimer's disease (AD). Extensive studies have been carried out to understand the role of fibrillar seeds on the overall kinetics of amyloid aggregation. However, the precise effect of seeds that are structurally or sequentially different from Aß on the structure of the resulting amyloid aggregates is yet to be fully understood. In this work, we use nanoscale infrared spectroscopy to probe the spectral facets of individual aggregates formed by aggregating Aß42 with antiparallel fibrillar seeds of Aß (16-22) and E22Q Aß (1-40) Dutch mutant and demonstrate that Aß can form heterotypic or mixed polymorphs that deviate significantly from its expected parallel cross ß structure. We further show that formation of heterotypic aggregates is not limited to coaggregation of Aß and its isomers, and that the former can form heterotypic fibrils with alpha synuclein and brain protein lysates. These findings highlight the complexity of Aß aggregation in AD and underscore the need to explore how Aß interacts with other brain components, which is crucial for developing better therapeutic strategies for AD.

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.

α-Synuclein emulsifies TDP-43 prion-like domain-RNA liquid droplets to promote heterotypic amyloid fibrils.

Many neurodegenerative diseases including frontotemporal lobar degeneration (FTLD), Lewy body disease (LBD), multiple system atrophy (MSA), etc., show colocalized deposits of TDP-43 and α-synuclein (αS) aggregates. To understand whether these colocalizations are driven by specific molecular interactions between the two proteins, we previously showed that the prion-like C-terminal domain of TDP-43 (TDP-43PrLD) and αS synergistically interact to form neurotoxic heterotypic amyloids in homogeneous buffer conditions. However, it remains unclear if αS can modulate TDP-43 present within liquid droplets and biomolecular condensates called stress granules (SGs). Here, using cell culture and in vitro TDP-43PrLD - RNA liquid droplets as models along with microscopy, nanoscale AFM-IR spectroscopy, and biophysical analyses, we uncover the interactions of αS with phase-separated droplets. We learn that αS acts as a Pickering agent by forming clusters on the surface of TDP-43PrLD - RNA droplets. The aggregates of αS on these clusters emulsify the droplets by nucleating the formation of heterotypic TDP-43PrLD amyloid fibrils, structures of which are distinct from those derived from homogenous solutions. Together, these results reveal an intriguing property of αS to act as a Pickering agent while interacting with SGs and unmask the hitherto unknown role of αS in modulating TDP-43 proteinopathies.

Stearic Acid as an Atomic Layer Deposition Inhibitor: Spectroscopic Insights from AFM-IR.

Modern-day chip manufacturing requires precision in placing chip materials on complex and patterned structures. Area-selective atomic layer deposition (AS-ALD) is a self-aligned manufacturing technique with high precision and control, which offers cost effectiveness compared to the traditional patterning techniques. Self-assembled monolayers (SAMs) have been explored as an avenue for realizing AS-ALD, wherein surface-active sites are modified in a specific pattern via SAMs that are inert to metal deposition, enabling ALD nucleation on the substrate selectively. However, key limitations have limited the potential of AS-ALD as a patterning method. The choice of molecules for ALD blocking SAMs is sparse; furthermore, deficiency in the proper understanding of the SAM chemistry and its changes upon metal layer deposition further adds to the challenges. In this work, we have addressed the above challenges by using nanoscale infrared spectroscopy to investigate the potential of stearic acid (SA) as an ALD inhibiting SAM. We show that SA monolayers on Co and Cu substrates can inhibit ZnO ALD growth on par with other commonly used SAMs, which demonstrates its viability towards AS-ALD. We complement these measurements with AFM-IR, which is a surface-sensitive spatially resolved technique, to obtain spectral insights into the ALD-treated SAMs. The significant insight obtained from AFM-IR is that SA SAMs do not desorb or degrade with ALD, but rather undergo a change in substrate coordination modes, which can affect ALD growth on substrates.

Intermediate Antiparallel ß Structure in Amyloid ß Plaques Revealed by Infrared Spectroscopic Imaging.

Aggregation of amyloid ß (Aß) peptides into extracellular plaques is a hallmark of the molecular pathology of Alzheimer's disease (AD). Amyloid aggregates have been extensively studied in vitro, and it is well-known that mature amyloid fibrils contain an ordered parallel ß structure. The structural evolution from unaggregated peptide to fibrils can be mediated through intermediate structures that deviate significantly from mature fibrils, such as antiparallel ß-sheets. However, it is currently unknown if these intermediate structures exist in plaques, which limits the translation of findings from in vitro structural characterizations of amyloid aggregates to AD. This arises from the inability to extend common structural biology techniques to ex vivo tissue measurements. Here we report the use of infrared (IR) imaging, wherein we can spatially localize plaques and probe their protein structural distributions with the molecular sensitivity of IR spectroscopy. Analyzing individual plaques in AD tissues, we demonstrate that fibrillar amyloid plaques exhibit antiparallel ß-sheet signatures, thus providing a direct connection between in vitro structures and amyloid aggregates in the AD brain. We further validate results with IR imaging of in vitro aggregates and show that the antiparallel ß-sheet structure is a distinct structural facet of amyloid fibrils.

α-Synuclein emulsifies TDP-43 prion-like domain - RNA liquid droplets to promote heterotypic amyloid fibrils.

Many neurodegenerative diseases including frontotemporal lobar degeneration (FTLD), Lewy body disease (LBD), multiple system atrophy (MSA), etc., show colocalized deposits of TDP-43 and α-synuclein (αS) aggregates. To understand whether these colocalizations are driven by specific molecular interactions between the two proteins, we previously showed that the prion-like C-terminal domain of TDP-43 (TDP-43PrLD) and αS synergistically interact to form neurotoxic heterotypic amyloids in homogeneous buffer conditions. However, it remains unclear whether and how αS modulates TDP-43 present within liquid droplets and biomolecular condensates called stress granules (SGs). Here, using cell culture and in vitro TDP-43PrLD - RNA liquid droplets as models along with microscopy, nanoscale spatially-resolved spectroscopy, and other biophysical analyses, we uncover the interactions of αS with phase-separated droplets. We learn that αS acts as a Pickering agent by forming clusters on the surface of TDP-43PrLD - RNA droplets and emulsifying them. The 'hardening' of the droplets that follow by αS aggregates on the periphery, nucleates the formation of heterotypic TDP-43PrLD amyloid fibrils with structures distinct from those derived from homogenous solutions. Together, these results reveal an intriguing property of αS as a Pickering agent in interacting with SGs and unmask the hitherto unknown role of αS in modulating TDP-43 proteinopathies.

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.

Intermediate antiparallel beta structure in amyloid plaques revealed by infrared spectroscopic imaging.

Aggregation of amyloid beta (Aß) peptides into extracellular plaques is a hallmark of the molecular pathology of Alzheimer's disease (AD). Amyloid aggregates have been extensively studied in-vitro, and it is well known that mature amyloid fibrils contain an ordered parallel ß structure. The structural evolution from unaggregated peptide to fibrils can be mediated through intermediate structures that deviate significantly from mature fibrils, such as antiparallel ß-sheets. However, it is currently unknown if these intermediate structures exist in plaques, which limits the translation of findings from in-vitro structural characterizations of amyloid aggregates to AD. This arises from the inability to extend common structural biology techniques to ex-vivo tissue measurements. Here we report the use of infrared (IR) imaging, wherein we can spatially localize plaques and probe their protein structural distributions with the molecular sensitivity of IR spectroscopy. Analyzing individual plaques in AD tissues, we demonstrate that fibrillar amyloid plaques exhibit antiparallel ß-sheet signatures, thus providing a direct connection between in-vitro structures and amyloid aggregates in AD brain. We further validate results with IR imaging of in-vitro aggregates and show that antiparallel ß-sheet structure is a distinct structural facet of amyloid fibrils.

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.

Intermediate antiparallel fibrils in Aß40 Dutch mutant aggregation: nanoscale insights from AFM-IR.

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 cases. Familial mutations in the amyloid ß peptide is 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 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 anti parallel 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.

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.

Structurally Distinct Polymorphs of Tau Aggregates Revealed by Nanoscale Infrared Spectroscopy.

Aggregation of the tau protein plays a central role in several neurodegenerative diseases collectively known as tauopathies, including Alzheimer's and Parkinson's disease. Tau misfolds into fibrillar ß sheet structures that constitute the paired helical filaments found in neurofibrillary tangles. It is known that there can be significant structural heterogeneities in tau aggregates associated with different diseases. However, while structures of mature fibrils have been studied, the structural distributions in early-stage tau aggregates is not well-understood. In the present study, we use atomic force microscopy-IR to investigate nanoscale spectra of individual tau fibrils at different stages of aggregation and demonstrate the presence of multiple fibrillar polymorphs that exhibit different secondary structures. We further show that mature fibrils contain significant amounts of antiparallel ß sheets. Our results are the very first application of nanoscale infrared spectroscopy to tau aggregates and underscore the promise of spatially resolved infrared spectroscopy for investigating protein aggregation.

Label-Free Infrared Spectroscopic Imaging Reveals Heterogeneity of ß-Sheet Aggregates in Alzheimer's Disease.

The aggregation of the amyloid beta (Aß) 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 Aß 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 ß-sheet aggregates distributed throughout brain tissues. We further demonstrate that phospholipid-rich ß-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 ß-sheet heterogeneities in the AD, which may be significant for targeted therapeutic strategies in the future.

Heterogeneous Amyloid ß-Sheet Polymorphs Identified on Hydrogen Bond Promoting Surfaces Using 2D SFG Spectroscopy.

Two-dimensional sum-frequency generation spectroscopy (2D SFG) is used to study the structures of the pentapeptide FGAIL on hydrogen bond promoting surfaces. FGAIL is the most amyloidogenic portion of the human islet amyloid polypeptide (hIAPP or amylin). In the presence of a pure gold surface, FGAIL does not form ordered structures. When the gold is coated with a self-assembled monolayer of mercaptobenzoic acid (MBA), 2D SFG spectra reveal features associated with ß-sheets. Also observed are cross peaks between the FGAIL peptides and the carboxylic acid groups of the MBA monolayer, indicating that the peptides are in close contact with the surface headgroups. In the second set of samples, FGAIL peptides chemically ligated to the MBA monolayer also exhibited ß-sheet features but with a much simpler spectrum. From simulations of the experiments, we conclude that the hydrogen bond promoting surface catalyzes the formation of both parallel and antiparallel ß-sheet structures with several different orientations. When ligated, parallel sheets with only a single orientation are the primary structure. Thus, this hydrogen bond promoting surface creates a heterogeneous distribution of polymorph structures, consistent with a concentration effect that allows nucleation of many different amyloid seeding structures. A single well-defined seed favors one polymorph over the others, showing that the concentrating influence of a membrane can be counterbalanced by factors that favor directed fiber growth. These experiments lay the foundation for the measurement and interpretation of ß-sheet structures with heterodyne-detected 2D SFG spectroscopy. The results of this model system suggest that a heterogeneous distribution of polymorphs found in nature are an indication of nonselective amyloid aggregation whereas a narrow distribution of polymorph structures is consistent with a specific protein or lipid interaction that directs fiber growth.

Mapping Solvation Environments in Porous Metal-Organic Frameworks with Infrared Chemical Imaging.

We report here the first mesoscale characterization of solvent environments in the metal-organic framework (MOF) Cu3(BTC)2 using infrared imaging. Two characteristic populations of the MOF structures corresponding to the carboxylate binding to the Cu(II) (metal) ions were observed, which reflect a regular solvated MOF structure with axial solvents in the binuclear copper paddlewheel and an unsolvated defect mode that lacks axial solvent coordination. Infrared imaging also shows strong correlation between solvent localization and the spatial distribution of the solvated population within the MOF. This is a vital result as any remnant solvent molecules adsorbed inside of MOFs can render them less effective. We propose fast IR imaging as a potential characterization technique that can measure adsorbate and defect distributions in MOFs.

Watching Proteins Wiggle: Mapping Structures with Two-Dimensional Infrared Spectroscopy.

Proteins exhibit structural fluctuations over decades of time scales. From the picosecond side chain motions to aggregates that form over the course of minutes, characterizing protein structure over these vast lengths of time is important to understanding their function. In the past 15 years, two-dimensional infrared spectroscopy (2D IR) has been established as a versatile tool that can uniquely probe proteins structures on many time scales. In this review, we present some of the basic principles behind 2D IR and show how they have, and can, impact the field of protein biophysics. We highlight experiments in which 2D IR spectroscopy has provided structural and dynamical data that would be difficult to obtain with more standard structural biology techniques. We also highlight technological developments in 2D IR that continue to expand the scope of scientific problems that can be accessed in the biomedical sciences.

Spatially Resolved Two-Dimensional Infrared Spectroscopy via Wide-Field Microscopy.

We report the first wide-field microscope for measuring two-dimensional infrared (2D IR) spectroscopic images. We concurrently collect more than 16 000 2D IR spectra, made possible by a new focal plane array detector and mid-IR pulse shaping, to generate hyperspectral images with multiple frequency dimensions and diffraction-limited spatial resolution. Both frequency axes of the spectra are collected in the time domain by scanning two pairs of femtosecond pulses using a dual acousto-optic modulator pulse shaper. The technique is demonstrated by imaging a mixture of metal carbonyl absorbed polystyrene beads. The differences in image formation between FTIR and 2D IR microscopy are also explored by imaging a patterned USAF test target. We find that our 2D IR microscope has diffraction-limited spatial resolution and enhanced contrast compared to FTIR microscopy because of the nonlinear scaling of the 2D IR signal to the absorptivity coefficient for the vibrational modes. Images generated using off-diagonal peaks, created from vibrational anharmonicities, improve the molecular discrimination and eliminate noise. Two-dimensional wide-field IR microscopy provides information on vibrational lifetimes, molecular couplings, transition dipole orientations, and many other quantities that can be used for creating image contrast to help disentangle and interpret complex and heterogeneous samples. Such experiments made possible could include the study of amyloid proteins in tissues, protein folding in heterogeneous environments, and structural dynamics in devices employing mid-IR materials.

Experimental implementations of 2D IR spectroscopy through a horizontal pulse shaper design and a focal plane array detector.

Aided by advances in optical engineering, two-dimensional infrared spectroscopy (2D IR) has developed into a promising method for probing structural dynamics in biophysics and material science. We report two new advances for 2D IR spectrometers. First, we report a fully reflective and totally horizontal pulse shaper, which significantly simplifies alignment. Second, we demonstrate the applicability of mid-IR focal plane arrays (FPAs) as suitable detectors in 2D IR experiments. FPAs have more pixels than conventional linear arrays and can be used to multiplex optical detection. We simultaneously measure the spectra of a reference beam, which improves the signal-to-noise by a factor of 4; and two additional beams that are orthogonally polarized probe pulses for 2D IR anisotropy experiments.