Studies Of Amyloid Formation Research Articles

Raman spectroscopy in the study of amyloid formation and phase separation.

Neurodegenerative diseases, such as Alzheimer's and Parkinson's, share a common pathological feature of amyloid structure accumulation. However, the structure-function relationship between these well-ordered, β-sheet-rich, filamentous protein deposits and disease etiology remains to be defined. Recently, an emerging hypothesis has linked phase separation, a process involved in the formation of protein condensates, to amyloid formation, suggesting that liquid protein droplets serve as loci for amyloid initiation. To elucidate how these processes contribute to disease progression, tools that can directly report on protein secondary structural changes are needed. Here, we review recent studies that have demonstrated Raman spectroscopy as a powerful vibrational technique for interrogating amyloid structures; one that offers sensitivity from the global secondary structural level to specific residues. This probe-free technique is further enhanced via coupling to a microscope, which affords structural data with spatial resolution, known as Raman spectral imaging (RSI). In vitro and in cellulo applications of RSI are discussed, highlighting studies of protein droplet aging, cellular internalization of fibrils, and Raman imaging of intracellular water. Collectively, utilization of the myriad Raman spectroscopic methods will contribute to a deeper understanding of protein conformational dynamics in the complex cellular milieu and offer potential clinical diagnostic capabilities for protein misfolding and aggregation processes in disease states.

Segmental 13C-Labeling for Raman Studies of α-Synuclein Amyloid Structure in Cells

Parkinson's disease (PD) is a prevalent age-related neurodegenerative disease associated with the aggregation of the neuronal protein α-synuclein (α-syn) into β-sheet-rich fibrils, called amyloid. Natively, α-syn is an intrinsically disordered protein, and it is thought conformational changes in α-syn are linked to its pathogenicity. Thus, the ability to determine conformational changes of α-syn from soluble, unstructured monomers to β-sheet-rich aggregates is critical for understanding the role of amyloid fibrils in disease progression. In this work, we use Raman microspectroscopy to study the aggregation of segmentally 13C-labeled α-syn in real-time. Native chemical ligation is used to link a uniformly 13C-labeled N-terminal portion of 1–86 to its C-terminal 12C-complement of 87–140. This ligated construct, 13C1-8612C87-140-α-syn, yields two distinct amide-I bands for observing region-specific conformational changes, as isotopic labels shift the carbonyl stretching frequencies to two spectrally-resolvable energies. Transmission electron microscopy shows fibril morphologies for 13C1-8612C87-140-α-syn are indistinguishable to those formed by full length 13C- and 12C-α-syn, indicating chemical ligation is not perturbative to amyloid formation. Raman kinetic data show the disordered-to-β-sheet conformational change is first observed for the N-terminal followed by the C-terminal region. 13C1-8612C87-140-α-syn fibrils are then passively fed to cell cultures and probed by Raman microspectroscopy to gain structural insights into region-specific intracellular α-syn conformations. Cell viability assays are also used to understand how amyloid structures influence cell health. This work demonstrates coupling strategic isotopic-labeling with Raman microspectroscopy can reveal both molecular and mechanistic insights into α-syn amyloid formation in vitro and in cellular environments. We anticipate this strategy will be broadly applicable to other studies of amyloid formation.

Parameter Distribution Analysis of Tau Fragment K18 Fibrillization

Neural amyloid deposits of microtubule-associated protein tau are implicated in a number of neurodegenerative disorders, notably Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE). Fibrillization of tau, and of amyloid-forming proteins in general, appears to involve nucleation-dependent polymerization wherein small concentrations of “nuclei” form initially, followed by the rapid, highly favorable addition of further monomer to nuclei/fibril ends. Secondary nucleation, the formation of nuclei from fibrillar material, is a particularly important determinant of amyloid formation kinetics. When the reaction is monitored, this type of behavior results in highly cooperative, sigmoidal fibrillization curves. A number of small molecules derived from natural products have been shown to inhibit tau amyloid formation, but our understanding of their mechanistic effects is largely empirical. A thorough investigation of the kinetic and structural effects of these compounds could aid in the rational design of more potent, specific inhibitors.Toward this end, we are utilizing a combination of fluorescence spectroscopy, mathematical modeling and numerical simulation to evaluate the heparin-induced fibrillization of a fragment of tau, K18. This strategy enables us to examine entire distributions of model parameter values that describe the data with comparable accuracy, as opposed to the conventional approach of identifying a single “best-fit” set of parameters. Both for experimental K18 fibrillization timecourses and for simulated sets of test data, the parameter distribution approach appears to better reflect the true experimental uncertainties involved in studies of amyloid formation than conventional least-squares fitting. These parameter distributions are sensitive to relatively small changes in the underlying kinetic rates, and we discuss how they can be used to assign detailed effects to known small molecule inhibitors of tau amyloid formation in an effort to generate more detailed models for their mechanisms of action.

Extensive amyloid formation in transplanted microencapsulated mouse and human islets

Objective: Deposition of cell toxic islet amyloid is a frequent finding in type 2 diabetes and also in transplanted human islets, where it is a possible explanation for their long-term failure. One suggested reason for amyloid in transplanted islets is that their low vascular density results in a disturbed local clearance of islet amyloid polypeptide (IAPP). To test this hypothesis we analysed accumulation of amyloid in microencapsulated islets, which exemplify a non-vascularised islet graft. Methods: Isolated islets from human or transgenic mice expressing human IAPP were microencapsulated in alginate and cultured in vitro or transplanted under the kidney capsule of normoglycemic nude mice. The degree of amyloid was determined after Congo red staining and subcellular alterations were analysed with electron microscopy. Results: Insulin and IAPP secretion from transgenic mouse islets were markedly increased during stimulation with glucose after one week of culture, but encapsulated islets in general released less insulin. Amyloid was detected after both one and three weeks of culture in the transgenic mouse islets and the encapsulated islets were most affected. After transplantation, electron microscopy displayed both intra- and extracellular amyloid in microencapsulated as well as in non-encapsulated human and transgenic mouse islet grafts. However, amyloid was more frequent in the encapsulated grafts. Conclusion: Micro-encapsulation of pancreatic islets might serve as an important tool for studies of amyloid formation under enhanced circumstances.

2DIR spectroscopy of human amylin fibrils reflects stable β-sheet structure.

The aggregation of human amylin to form amyloid contributes to islet β-cell dysfunction in type 2 diabetes. Studies of amyloid formation have been hindered by the low structural resolution or relatively modest time resolution of standard methods. Two-dimensional infrared (2DIR) spectroscopy, with its sensitivity to protein secondary structures and its intrinsic fast time resolution, is capable of capturing structural changes during the aggregation process. Moreover, isotope labeling enables the measurement of residue-specific information. The diagonal line widths of 2DIR spectra contain information about dynamics and structural heterogeneity of the system. We illustrate the power of a combined atomistic molecular dynamics simulation and theoretical and experimental 2DIR approach by analyzing the variation in diagonal line widths of individual amide I modes in a series of labeled samples of amylin amyloid fibrils. The theoretical and experimental 2DIR line widths suggest a "W" pattern, as a function of residue number. We show that large line widths result from substantial structural disorder and that this pattern is indicative of the stable secondary structure of the two β-sheet regions. This work provides a protocol for bridging MD simulation and 2DIR experiments for future aggregation studies.

Ester to Amide Switch Peptides Provide a Simple Method for Preparing Monomeric Islet Amyloid Polypeptide under Physiologically Relevant Conditions and Facilitate Investigations of Amyloid Formation

A major issue in studies of amyloid formation is the difficulty of preparing the polypeptide of interest in an initially monomeric state under physiologically relevant conditions. This is particularly problematic for polypeptides which are natively unfolded in their unaggregated state, and perhaps the most challenging such system is islet amyloid polypeptide (Amylin), the causative agent of amyloid formation in type-2 diabetes. Preparation of islet amyloid polypeptide with the Ser-19 Ser-20 amide bond replaced by an ester circumvents these problems. The modified peptide is unstructured and monomeric at slightly acidic pH's as judged by analytical ultracentrifugation, gel filtration, dynamic light scattering, and CD. A rapid pH jump leads to deprotonation of the Ser-20 amide group, and a subsequent rapid O to N acyl shift regenerates normal human islet amyloid polypeptide. The half time, t(1/2), for the conversion to normal islet amyloid polypeptide is 70 s at pH 7.4. The amyloid fibrils which are formed by the regenerated islet amyloid polypeptide are indistinguishable from those formed by the wild type polypeptide. The approach allows studies of amyloid formation by islet amyloid polypeptide to be carried out from a well-defined, physiologically relevant starting state in the absence of denaturants or organic cosolvents.

Microscopic Factors that Control β-Sheet Registry in Amyloid Fibrils Formed by Fragment 11–25 of Amyloid β Peptide: Insights from Computer Simulations

Short fragments of amyloidogenic proteins are widely used as model systems in studies of amyloid formation. Fragment 11–25 of the amyloid β protein involved in Alzheimer's disease (Aβ11–25) was recently shown to form amyloid fibrils composed of anti-parallel β-sheets. Interestingly, fibrils grown under neutral and acidic conditions were seen to possess different registries of their inter-β-strand hydrogen bonds. In an effort to explain the microscopic origin of this pH dependence, we studied Aβ11–25 fibrils using methods of theoretical modeling. Several structural models were built for fibrils at low and neutral pH levels and these were examined in short molecular dynamics simulations in explicit water. The models that displayed the lowest free energy, as estimated using an implicit solvent model, were selected as representative of the true fibrillar structure. It was shown that the registry of these models agrees well with the experimental results. At neutral pH, the main contribution to the free energy difference between the two registries comes from the electrostatic interactions. The charge group of the carboxy terminus makes a large contribution to these interactions and thus appears to have a critical role in determining the registry.