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.

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.

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.

Impact of HMGB1 binding on the structural alterations of platinum drug-treated single dsDNA molecule.

High mobility group B1 (HMGB1) is an architectural protein that recognizes the DNA damage sites formed by the platinum anticancer drugs. However, the impact of HMGB1 binding on the structural alterations of the platinum drug-treated single dsDNA molecules has remained largely unknown. Herein, the structural alterations induced by the platinum drugs, the mononuclear cisplatin and it's analog the trinuclear BBR3464, have been probed in presence of HMGB1, by atomic force microscopy (AFM) and AFM-based force spectroscopy. It is observed that the drug-induced DNA loop formation enhanced upon HMGB1 binding, most likely as a result of HMGB1-induced increase in DNA conformational flexibility that allowed the drug-binding sites to come close and form double adducts, thereby resulting in enhanced loop formation via inter-helix cross-linking. Since HMGB1 enhances DNA flexibility, the near-reversible structural transitions as observed in the force-extension curves (for 1 h drug treatment), generally occurred at lower forces in presence of HMGB1. The DNA structural integrity was largely lost after 24 h drug treatment as no reversible transition could be observed. The Young's modulus of the dsDNA molecules, as estimated from the force-extension analysis, increased upon drug treatment, due to formation of the drug-induced covalent cross-links and consequent reduction in DNA flexibility. The Young's modulus increased further in presence of HMGB1 due to HMGB1-induced enhancement in DNA flexibility that could ease formation of the drug-induced covalent cross-links. To our knowledge, this is the first report that shows an increase in the stiffness of the platinum drug-treated DNA molecules in presence of HMGB1.

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.

Free Cholesterol Accelerates Aß Self-Assembly on Membranes at Physiological Concentration.

The effects of membranes on the early-stage aggregation of amyloid ß (Aß) have come to light as potential mechanisms by which neurotoxic species are formed in Alzheimer's disease. We have shown that direct Aß-membrane interactions dramatically enhance the Aß aggregation, allowing for oligomer assembly at physiologically low concentrations of the monomer. Membrane composition is also a crucial factor in this process. Our results showed that apart from phospholipids composition, cholesterol in membranes significantly enhances the aggregation kinetics. It has been reported that free cholesterol is present in plaques. Here we report that free cholesterol, along with its presence inside the membrane, further accelerate the aggregation process by producing aggregates more rapidly and of significantly larger sizes. These aggregates, which are formed on the lipid bilayer, are able to dissociate from the surface and accumulate in the bulk solution; the presence of free cholesterol accelerates this dissociation as well. All-atom molecular dynamics simulations show that cholesterol binds Aß monomers and significantly changes the conformational sampling of Aß monomer; more than doubling the fraction of low-energy conformations compared to those in the absence of cholesterol, which can contribute to the aggregation process. The results indicate that Aß-lipid interaction is an important factor in the disease prone amyloid assembly process.

Topographically smooth and stable supported lipid bilayer for high-resolution AFM studies.

The cellular membrane has been identified to play a critical role in various biological processes including the assembly of biological systems. Membranes are complex, primarily two-dimensional assemblies with varied lipid compositions depending on the particular region of the cell. Supported lipid bilayers are considered as appropriate models for physio-chemical studies of membranes including numerous single molecule techniques. Atomic force microscopy (AFM) as a topographic technique is a fully appropriate single molecule technique capable of direct observation of molecular processes on membranes. However, reliable experimental AFM studies require the preparation of the bilayer with a sub-nanometer smooth morphology, which remains stable over long-time observation. Here we present the methodology, which allows one to prepare a smooth, stable, structurally homogeneous lipid bilayer without the presence of any trapped vesicles. We described the application of such lipid bilayers to probe time-dependent early stages of aggregation of monomeric amyloid proteins. Importantly, the proposed methodology can be extended to bilayers with various compositions, by incorporating different lipids for on-membrane aggregation study including cholesterol. Furthermore, this methodology development allowed us to monitor the aggregation of amyloid protein at its physiologically relevant low protein concentration. The flexibility of altering the membrane composition allows to identify the specific role of a particular lipid towards the aggregation kinetics, revealing the plausible mechanism of disease development.

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.

Cholesterol in Membranes Facilitates Aggregation of Amyloid ß Protein at Physiologically Relevant Concentrations.

The formation of amyloid ß (1-42) (Aß42) oligomers is considered to be a critical step in the development of Alzheimer's disease (AD). However, the mechanism underlying this process at physiologically low concentrations of Aß42 remains unclear. We have previously shown that oligomers assemble at such low Aß42 monomer concentrations in vitro on phospholipid membranes. We hypothesized that membrane composition is the factor controlling the aggregation process. Accumulation of cholesterol in membranes is associated with AD development, suggesting that insertion of cholesterol into membranes may initiate the Aß42 aggregation, regardless of a low monomer concentration. We used atomic force microscopy (AFM) to test the hypothesis and directly visualize the aggregation process of Aß42 on the surface of a lipid bilayer depending on the cholesterol presence. Time-lapse AFM imaging unambiguously demonstrates that cholesterol in the lipid bilayer significantly enhances the aggregation process of Aß42 at nanomolar monomer concentration. Quantitative analysis of the AFM data shows that both the number of Aß42 oligomers and their sizes grow when cholesterol is present. Importantly, the aggregation process is dynamic, so the aggregates assembled on the membrane can dissociate from the bilayer surface into the bulk solution. Computational modeling demonstrated that the lipid bilayer containing cholesterol had an elevated affinity to Aß42. Moreover, monomers adopted the aggregation-prone conformations present in amyloid fibrils. The results lead to the model for the on-surface aggregation process in which the self-assembly of Aß oligomers is controlled by the lipid composition of cellular membranes.

Two C-terminal sequence variations determine differential neurotoxicity between human and mouse α-synuclein.

BACKGROUND: α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. METHODS: Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey's multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn's multiple comparisons test or a two-tailed Mann-Whitney test. RESULTS: Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. CONCLUSIONS: Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.

Interaction of Aß42 with Membranes Triggers the Self-Assembly into Oligomers.

The self-assembly of amyloid ß (Aß) proteins into oligomers is the major pathogenic event leading to Alzheimer's disease (AD). Typical in vitro experiments require high protein concentrations, whereas the physiological concentration of Aß is in the picomolar to low nanomolar range. This complicates the translation of results obtained in vitro to understanding the aggregation process in vivo. Here, we demonstrate that Aß42 self-assembles into aggregates on membrane bilayers at low nanomolar concentrations - a pathway in which the membrane plays the role of a catalyst. Additionally, physiological ionic conditions (150 mM NaCl) significantly enhance on-membrane aggregation, leading to the rapid formation of oligomers. The self-assembly process is reversible, so assembled aggregates can dissociate from the membrane surface into the bulk solution to further participate in the aggregation process. Molecular dynamics simulations demonstrate that the transient membrane-Aß interaction dramatically changes the protein conformation, facilitating the assembly of dimers. The results indicate peptide-membrane interaction is the critical step towards oligomer formation at physiologically low protein concentrations.

Molecular Model for the Surface-Catalyzed Protein Self-Assembly.

The importance of cell surfaces in the self-assembly of proteins is widely accepted. One biologically significant event is the assembly of amyloidogenic proteins into aggregates, which leads to neurodegenerative disorders like Alzheimer's and Parkinson's diseases. The interaction of amyloidogenic proteins with cellular membranes appears to dramatically facilitate the aggregation process. Recent findings indicate that, in the presence of surfaces, aggregation occurs at physiologically low concentrations, suggesting that interaction with surfaces plays a critical role in the disease-prone aggregation process. However, the molecular mechanisms behind the on-surface aggregation process remain unclear. Here, we provide a theoretical model that offers a molecular explanation. According to this model, monomers transiently immobilized to surfaces increase the local monomer protein concentration and thus work as nuclei to dramatically accelerate the entire aggregation process. This physical-chemical theory was verified by experimental studies, using mica surfaces, to examine the aggregation kinetics of amyloidogenic α-synuclein protein and non-amyloidogenic cytosine deaminase APOBEC3G.

Free-Energy-Based Gene Mutation Detection Using LNA Probes.

We have developed a label-free approach for direct detection of gene mutations using free-energy values that are derived from single-molecule force spectroscopy (SMFS)-based nucleic acid unbinding experiments. From the duplex unbinding force values acquired by SMFS, the force-loading-rate-independent Gibbs free-energy values were derived using Jarzinsky's equality treatment. Because it provides molecule-by-molecule information, this approach is a major shift compared to the earlier reports on label-free detection of DNA sequences, which are mostly based on ensemble level data. We tested our approach in the disease model framework of multiple drug-resistant tuberculosis using the nuclease-resistant and conformationally rigid locked nucleic acid probes that are a robust and efficient alternative to the DNA probes. All of the major mutations in Mycobacterium tuberculosis (MTB), as relevant to MTB's resistance to the first-line anti-TB drugs rifampicin and isoniazid, could be identified, and the wild type could be discriminated from the most prevalent mutation and the most prevalent mutation from the less occurring ones. Our approach could also identify DNA sequences (45 mer), having overhang stretches at different positions with respect to the complementary stretch. Probably for the first time, the findings show that free-energy-based detection of gene mutations is possible at molecular resolution.