Prion Protein Octarepeat Domain Forms Transient ß-Sheet Structures upon Residue-Specific Binding to Cu(II) and Zn(II) Ions.

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in PrPC misfolding. PrPC is a combined Cu(II) and Zn(II) metal-binding protein, where the main metal-binding site is located in the octarepeat (OR) region. Thus, the biological function of PrPC may involve the transport of divalent metal ions across membranes or buffering concentrations of divalent metal ions in the synaptic cleft. Recent studies have shown that an excess of Cu(II) ions can result in PrPC instability, oligomerization, and/or neuroinflammation. Here, we have used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region of PrPC. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Binding of the first metal ion results in a structural transition from the polyproline II helix to the ß-turn structure, while the binding of additional metal ions induces the formation of ß-sheet structures. Fluorescence spectroscopy data indicate that the OR region can bind both Cu(II) and Zn(II) ions at neutral pH, but under acidic conditions, it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of either metal ion to the OR region results in the formation of ß-hairpin structures. As the formation of ß-sheet structures can be a first step toward amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSE diseases.

Metals in ALS TDP-43 Pathology.

Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease and similar neurodegenerative disorders take their toll on patients, caregivers and society. A common denominator for these disorders is the accumulation of aggregated proteins in nerve cells, yet the triggers for these aggregation processes are currently unknown. In ALS, protein aggregation has been described for the SOD1, C9orf72, FUS and TDP-43 proteins. The latter is a nuclear protein normally binding to both DNA and RNA, contributing to gene expression and mRNA life cycle regulation. TDP-43 seems to have a specific role in ALS pathogenesis, and ubiquitinated and hyperphosphorylated cytoplasmic inclusions of aggregated TDP-43 are present in nerve cells in almost all sporadic ALS cases. ALS pathology appears to include metal imbalances, and environmental metal exposure is a known risk factor in ALS. However, studies on metal-to-TDP-43 interactions are scarce, even though this protein seems to have the capacity to bind to metals. This review discusses the possible role of metals in TDP-43 aggregation, with respect to ALS pathology.

Effects of in vivo conditions on amyloid aggregation.

One of the grand challenges of biophysical chemistry is to understand the principles that govern protein misfolding and aggregation, which is a highly complex process that is sensitive to initial conditions, operates on a huge range of length- and timescales, and has products that range from protein dimers to macroscopic amyloid fibrils. Aberrant aggregation is associated with more than 25 diseases, which include Alzheimer's, Parkinson's, Huntington's, and type II diabetes. Amyloid aggregation has been extensively studied in the test tube, therefore under conditions that are far from physiological relevance. Hence, there is dire need to extend these investigations to in vivo conditions where amyloid formation is affected by a myriad of biochemical interactions. As a hallmark of neurodegenerative diseases, these interactions need to be understood in detail to develop novel therapeutic interventions, as millions of people globally suffer from neurodegenerative disorders and type II diabetes. The aim of this review is to document the progress in the research on amyloid formation from a physicochemical perspective with a special focus on the physiological factors influencing the aggregation of the amyloid-ß peptide, the islet amyloid polypeptide, α-synuclein, and the hungingtin protein.

Copper ions induce dityrosine-linked dimers in human but not in murine islet amyloid polypeptide (IAPP/amylin).

Dysregulation and aggregation of the peptide hormone IAPP (islet amyloid polypeptide, a.k.a. amylin) into soluble oligomers that appear to be cell-toxic is a known aspect of diabetes mellitus (DM) Type 2 pathology. IAPP aggregation is influenced by several factors including interactions with metal ions such as Cu(II). Because Cu(II) ions are redox-active they may contribute to metal-catalyzed formation of oxidative tyrosyl radicals, which can generate dityrosine cross-links. Here, we show that such a process, which involves Cu(II) ions bound to the IAPP peptide together with H2O2, can induce formation of large amounts of IAPP dimers connected by covalent dityrosine cross-links. This cross-linking is less pronounced at low pH and for murine IAPP, likely due to less efficient Cu(II) binding. Whether IAPP can carry out its hormonal function as a cross-linked dimer is unknown. As dityrosine concentrations are higher in blood plasma of DM Type 2 patients - arguably due to disease-related oxidative stress - and as dimer formation is the first step in protein aggregation, generation of dityrosine-linked dimers may be an important factor in IAPP aggregation and thus relevant for DM Type 2 progression.

Metal binding to the amyloid-ß peptides in the presence of biomembranes: potential mechanisms of cell toxicity.

The amyloid-ß (Aß) peptides are key molecules in Alzheimer's disease (AD) pathology. They interact with cellular membranes, and can bind metal ions outside the membrane. Certain oligomeric Aß aggregates are known to induce membrane perturbations and the structure of these oligomers-and their membrane-perturbing effects-can be modulated by metal ion binding. If the bound metal ions are redox active, as e.g., Cu and Fe ions are, they will generate harmful reactive oxygen species (ROS) just outside the membrane surface. Thus, the membrane damage incurred by toxic Aß oligomers is likely aggravated when redox-active metal ions are present. The combined interactions between Aß oligomers, metal ions, and biomembranes may be responsible for at least some of the neuronal death in AD patients.

The Neuronal Tau Protein Blocks in Vitro Fibrillation of the Amyloid-ß (Aß) Peptide at the Oligomeric Stage.

In Alzheimer's disease, amyloid-ß (Aß) plaques and tau neurofibrillary tangles are the two pathological hallmarks. The co-occurrence and combined reciprocal pathological effects of Aß and tau protein aggregation have been observed in animal models of the disease. However, the molecular mechanism of their interaction remain unknown. Using a variety of biophysical measurements, we here show that the native full-length tau protein solubilizes the Aß40 peptide and prevents its fibrillation. The tau protein delays the amyloid fibrillation of the Aß40 peptide at substoichiometric ratios, showing different binding affinities toward the different stages of the aggregated Aß40 peptides. The Aß monomer structure remains random coil in the presence of tau, as observed by nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy and photoinduced cross-linking methods. We propose a potential interaction mechanism for the influence of tau on Aß fibrillation.

Co-aggregation of pro-inflammatory S100A9 with α-synuclein in Parkinson's disease: ex vivo and in vitro studies.

BACKGROUND: Chronic neuroinflammation is a hallmark of Parkinson's disease (PD) pathophysiology, associated with increased levels of pro-inflammatory factors in PD brain tissues. The pro-inflammatory mediator and highly amyloidogenic protein S100A9 is involved in the amyloid-neuroinflammatory cascade in Alzheimer's disease. This is the first report on the co-aggregation of α-synuclein (α-syn) and S100A9 both in vitro and ex vivo in PD brain. METHODS: Single and sequential immunohistochemistry, immunofluorescence, scanning electron and atomic force (AFM) microscopies were used to analyze the ex vivo PD brain tissues for S100A9 and α-syn location and aggregation. In vitro studies revealing S100A9 and α-syn interaction and co-aggregation were conducted by NMR, circular dichroism, Thioflavin-T fluorescence, AFM, and surface plasmon resonance methods. RESULTS: Co-localized and co-aggregated S100A9 and α-syn were found in 20% Lewy bodies and 77% neuronal cells in the substantia nigra; both proteins were also observed in Lewy bodies in PD frontal lobe (Braak stages 4-6). Lewy bodies were characterized by ca. 10-23 µm outer diameter, with S100A9 and α-syn being co-localized in the same lamellar structures. S100A9 was also detected in neurons and blood vessels of the aged patients without PD, but in much lesser extent. In vitro S100A9 and α-syn were shown to interact with each other via the α-syn C-terminus with an apparent dissociation constant of ca. 5 µM. Their co-aggregation occurred significantly faster and led to formation of larger amyloid aggregates than the self-assembly of individual proteins. S100A9 amyloid oligomers were more toxic than those of α-syn, while co-aggregation of both proteins mitigated the cytotoxicity of S100A9 oligomers. CONCLUSIONS: We suggest that sustained neuroinflammation promoting the spread of amyloidogenic S100A9 in the brain tissues may trigger the amyloid cascade involving α-syn and S100A9 and leading to PD, similar to the effect of S100A9 and Aß co-aggregation in Alzheimer's disease. The finding of S100A9 involvement in PD may open a new avenue for therapeutic interventions targeting S100A9 and preventing its amyloid self-assembly in affected brain tissues.

Cross-interactions between the Alzheimer Disease Amyloid-ß Peptide and Other Amyloid Proteins: A Further Aspect of the Amyloid Cascade Hypothesis.

Many protein folding diseases are intimately associated with accumulation of amyloid aggregates. The amyloid materials formed by different proteins/peptides share many structural similarities, despite sometimes large amino acid sequence differences. Some amyloid diseases constitute risk factors for others, and the progression of one amyloid disease may affect the progression of another. These connections are arguably related to amyloid aggregates of one protein being able to directly nucleate amyloid formation of another, different protein: the amyloid cross-interaction. Here, we discuss such cross-interactions between the Alzheimer disease amyloid-ß (Aß) peptide and other amyloid proteins in the context of what is known from in vitro and in vivo experiments, and of what might be learned from clinical studies. The aim is to clarify potential molecular associations between different amyloid diseases. We argue that the amyloid cascade hypothesis in Alzheimer disease should be expanded to include cross-interactions between Aß and other amyloid proteins.

Ancient water bottle use and polycyclic aromatic hydrocarbon (PAH) exposure among California Indians: a prehistoric health risk assessment.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are the main toxic compounds in natural bitumen, a fossil material used by modern and ancient societies around the world. The adverse health effects of PAHs on modern humans are well established, but their health impacts on past populations are unclear. It has previously been suggested that a prehistoric health decline among the native people living on the California Channel Islands may have been related to PAH exposure. Here, we assess the potential health risks of PAH exposure from the use and manufacture of bitumen-coated water bottles by ancient California Indian societies. METHODS: We replicated prehistoric bitumen-coated water bottles with traditional materials and techniques of California Indians, based on ethnographic and archaeological evidence. In order to estimate PAH exposure related to water bottle manufacture and use, we conducted controlled experiments to measure PAH contamination 1) in air during the manufacturing process and 2) in water and olive oil stored in a completed bottle for varying periods of time. Samples were analyzed with gas chromatography/mass spectrometry (GC/MS) for concentrations of the 16 PAHs identified by the US Environmental Protection Agency (EPA) as priority pollutants. RESULTS: Eight PAHs were detected in concentrations of 1-10 µg/m3 in air during bottle production and 50-900 ng/L in water after 2 months of storage, ranging from two-ring (naphthalene and methylnaphthalene) to four-ring (fluoranthene) molecules. All 16 PAHs analyzed were detected in olive oil after 2 days (2 to 35 µg/kg), 2 weeks (3 to 66 µg/kg), and 2 months (5 to 140 µg/kg) of storage. CONCLUSIONS: For ancient California Indians, water stored in bitumen-coated water bottles was not a significant source of PAH exposure, but production of such bottles could have resulted in harmful airborne PAH exposure.

Self-Assembled Cyclic d,l-α-Peptides as Generic Conformational Inhibitors of the α-Synuclein Aggregation and Toxicity: In Vitro and Mechanistic Studies.

Many peptides and proteins with large sequences and structural differences self-assemble into disease-causing amyloids that share very similar biochemical and biophysical characteristics, which may contribute to their cross-interaction. Here, we demonstrate how the self-assembled, cyclic d,l-α-peptide CP-2, which has similar structural and functional properties to those of amyloids, acts as a generic inhibitor of the Parkinson's disease associated α-synuclein (α-syn) aggregation to toxic oligomers by an "off-pathway" mechanism. We show that CP-2 interacts with the N-terminal and the non-amyloid-ß component region of α-syn, which are responsible for α-syn's membrane intercalation and self-assembly, thus changing the overall conformation of α-syn. CP-2 also remodels α-syn fibrils to nontoxic amorphous species and permeates cells through endosomes/lysosomes to reduce the accumulation and toxicity of intracellular α-syn in neuronal cells overexpressing α-syn. Our studies suggest that targeting the common structural conformation of amyloids may be a promising approach for developing new therapeutics for amyloidogenic diseases.

Non-chaperone proteins can inhibit aggregation and cytotoxicity of Alzheimer amyloid ß peptide.

Many factors are known to influence the oligomerization, fibrillation, and amyloid formation of the Aß peptide that is associated with Alzheimer disease. Other proteins that are present when Aß peptides deposit in vivo are likely to have an effect on these aggregation processes. To separate specific versus broad spectrum effects of proteins on Aß aggregation, we tested a series of proteins not reported to have chaperone activity: catalase, pyruvate kinase, albumin, lysozyme, α-lactalbumin, and ß-lactoglobulin. All tested proteins suppressed the fibrillation of Alzheimer Aß(1-40) peptide at substoichiometric ratios, albeit some more effectively than others. All proteins bound non-specifically to Aß, stabilized its random coils, and reduced its cytotoxicity. Surprisingly, pyruvate kinase and catalase were at least as effective as known chaperones in inhibiting Aß aggregation. We propose general mechanisms for the broad-spectrum inhibition Aß fibrillation by proteins. The mechanisms we discuss are significant for prognostics and perhaps even for prevention and treatment of Alzheimer disease.

Evaluating sexual dimorphism in the human mastoid process: A viewpoint on the methodology.

The mastoid process is one of the most sexually dimorphic features in the human skull, and is therefore often used to identify the sex of skeletons. Numerous techniques for assessing variation in the size and shape of the mastoid process have been proposed and implemented in osteological research, but its complex form still presents difficulties for consistent and effective analysis. In this article, we compare the different techniques and variables that have been used to define, measure, and visually score sexual dimorphism in the mastoid process. We argue that the current protocols fail to capture the full morphological range of this bony projection, and suggest ways of improving and standardizing them, regarding both traditional and 3D-based approaches.

Alzheimer peptides aggregate into transient nanoglobules that nucleate fibrils.

Protein/peptide oligomerization, cross-ß strand fibrillation, and amyloid deposition play a critical role in many diseases, but despite extensive biophysical characterization, the structural and dynamic details of oligomerization and fibrillation of amyloidic peptides/proteins remain to be fully clarified. Here, we simultaneously monitored the atomic, molecular, and mesoscopic states of aggregating Alzheimer's amyloid ß (Aß) peptides over time, using a slow aggregation protocol and a fast aggregation protocol, and determined the cytotoxicity of the intermediate states. We show that in the early stage of fast fibrillation (the lag phase) the Aß peptides coalesced into apparently unstructured globules (15-200 nm in diameter), which slowly grew larger. Then a sharp transition occurred, characterized by the first appearance of single fibrillar structures of approximately ≥100 nm. These fibrils emerged from the globules. Simultaneously, an increase was observed for the cross-ß strand conformation that is characteristic of the fibrils that constitute mature amyloid. The number and size of single fibrils rapidly increased. Eventually, the fibrils coalesced into mature amyloid. Samples from the early lag phase of slow fibrillation conditions were especially toxic to cells, and this toxicity sharply decreased when fibrils formed and matured into amyloid. Our results suggest that the formation of fibrils may protect cells by reducing the toxic structures that appear in the early lag phase of fibrillation.

The role of pro-inflammatory S100A9 in Alzheimer's disease amyloid-neuroinflammatory cascade.

Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflammation-related neurodegeneration. Here, we provide insights into S100A9 specific mechanisms of action in Alzheimer's disease (AD). Due to its inherent amyloidogenicity S100A9 contributes to amyloid plaque formation together with Aß. In traumatic brain injury (TBI) S100A9 itself rapidly forms amyloid plaques, which were reactive with oligomer-specific antibodies, but not with Aß and amyloid fibrillar antibodies. They may serve as precursor-plaques for AD, implicating TBI as an AD risk factor. S100A9 was observed in some hippocampal and cortical neurons in TBI, AD and non-demented aging. In vitro S100A9 forms neurotoxic linear and annular amyloids resembling Aß protofilaments. S100A9 amyloid cytotoxicity and native S100A9 pro-inflammatory signaling can be mitigated by its co-aggregation with Aß, which results in a variety of micron-scale amyloid complexes. NMR and molecular docking demonstrated transient interactions between native S100A9 and Aß. Thus, abundantly present in AD brain pro-inflammatory S100A9, possessing also intrinsic amyloidogenic properties and ability to modulate Aß aggregation, can serve as a link between the AD amyloid and neuroinflammatory cascades and as a prospective therapeutic target.

The hairpin conformation of the amyloid ß peptide is an important structural motif along the aggregation pathway.

The amyloid ß (Aß) peptides are 39-42 residue-long peptides found in the senile plaques in the brains of Alzheimer's disease (AD) patients. These peptides self-aggregate in aqueous solution, going from soluble and mainly unstructured monomers to insoluble ordered fibrils. The aggregation process(es) are strongly influenced by environmental conditions. Several lines of evidence indicate that the neurotoxic species are the intermediate oligomeric states appearing along the aggregation pathways. This minireview summarizes recent findings, mainly based on solution and solid-state NMR experiments and electron microscopy, which investigate the molecular structures and characteristics of the Aß peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the Aß peptides in most of the described structures. There are certain variations in different hairpin conformations, for example regarding H-bonding partners, which could be one reason for the molecular heterogeneity observed in the aggregated systems. Interacting hairpins are the building blocks of the insoluble fibrils, again with variations in how hairpins are organized in the cross-section of the fibril, perpendicular to the fibril axis. The secondary structure propensities can be seen already in peptide monomers in solution. Unfortunately, detailed structural information about the intermediate oligomeric states is presently not available. In the review, special attention is given to metal ion interactions, particularly the binding constants and ligand structures of Aß complexes with Cu(II) and Zn(II), since these ions affect the aggregation process(es) and are considered to be involved in the molecular mechanisms underlying AD pathology.

Endogenous polyamines reduce the toxicity of soluble aß peptide aggregates associated with Alzheimer's disease.

Polyamines promote the formation of the Aß peptide amyloid fibers that are a hallmark of Alzheimer's disease. Here we show that polyamines interact with nonaggregated Aß peptides, thereby reducing the peptide's hydrophobic surface. We characterized the associated conformational change through NMR titrations and molecular dynamics simulations. We found that even low concentrations of spermine, sperimidine, and putrescine fully protected SH-SY5Y (a neuronal cell model) against the most toxic conformational species of Aß, even at an Aß oligomer concentration that would otherwise kill half of the cells or even more. These observations lead us to conclude that polyamines interfere with the more toxic prefibrillar conformations and might protect cells by promoting the structural transition of Aß toward its less toxic fibrillar state that we reported previously. Since polyamines are present in brain fluid at the concentrations where we observed all these effects, their activity needs to be taken into account in understanding the molecular processes related to the development of Alzheimer's disease.

Hereditary Transthyretin Amyloidosis (hATTR) with Polyneuropathy Clusters Are Located in Ancient Mining Districts: A Possible Geochemical Origin of the Disease.

Hereditary transthyretin amyloidosis (hATTR) with polyneuropathy (formerly known as Familial Amyloid Polyneuropathy (FAP)) is an endemic amyloidosis involving the harmful aggregation of proteins, most commonly transthyretin (TTR) but sometimes also apolipoprotein A-1 or gelsolin. hATTR appears to be transmitted as an autosomal dominant trait. Over 100 point mutations have been identified, with the Val30Met substitution being the most common. Yet, the mechanism of pathogenesis and the overall origin of hATTR remain unclear. Here, we argue that hATTR could be related to harmful metal exposure. hATTR incidence is unevenly distributed globally, and the three largest defined clusters exist in Japan, Portugal, and Sweden. All three disease regions are also ancient mining districts with associated metal contamination of the local environment. There are two main mechanisms for how harmful metals, after uptake into tissues and body fluids, could induce hATTR. First, the metals could directly influence the expression, function, and/or aggregation of the proteins involved in hATTR pathology. Such metal-protein interactions might constitute molecular targets for anti-hATTR drug design. Second, metal exposure could induce hATTR -associated genetic mutations, which may have happened several generations ago. These two mechanisms can occur in parallel. In conclusion, the possibility that hATTR could be related to metal exposure in geochemically defined regions deserves further attention.

In vitro and mechanistic studies of an antiamyloidogenic self-assembled cyclic D,L-α-peptide architecture.

Misfolding of the Aß protein and its subsequent aggregation into toxic oligomers are related to Alzheimer's disease. Although peptides of various sequences can self-assemble into amyloid structures, these structures share common three-dimensional features that may promote their cross-reaction. Given the significant similarities between amyloids and the architecture of self-assembled cyclic D,L-α-peptide, we hypothesized that the latter may bind and stabilize a nontoxic form of Aß, thereby preventing its aggregation into toxic forms. By screening a focused library of six-residue cyclic D,L-α-peptides and optimizing the activity of a lead peptide, we found one cyclic D,L-α-peptide (CP-2) that interacts strongly with Aß and inhibits its aggregation. In transmission electron microscopy, optimized thioflavin T and cell survival assays, CP-2 inhibits the formation of Aß aggregates, entirely disassembles preformed aggregated and fibrillar Aß, and protects rat pheochromocytoma PC12 cells from Aß toxicity, without inducing any toxicity by itself. Using various immunoassays, circular dichroism spectroscopy, photoinduced cross-linking of unmodified proteins (PICUP) combined with SDS/PAGE, and NMR, we probed the mechanisms underlying CP-2's antiamyloidogenic activity. NMR spectroscopy indicates that CP-2 interacts with Aß through its self-assembled conformation and induces weak secondary structure in Aß. Upon coincubation, CP-2 changes the aggregation pathway of Aß and alters its oligomer distribution by stabilizing small oligomers (1-3 mers). Our results support studies suggesting that toxic early oligomeric states of Aß may be composed of antiparallel ß-peptide structures and that the interaction of Aß with CP-2 promotes formation of more benign parallel ß-structures. Further studies will show whether these kinds of abiotic cyclic D,L-α-peptides are also beneficial as an intervention in related in vivo models.

Inhibiting and reversing amyloid-ß peptide (1-40) fibril formation with gramicidin S and engineered analogues.

In Alzheimer's disease, amyloid-ß (Aß) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aß peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aß amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a ß-sheet conformation, binds to the Aß peptide hairpin-stacked fibril through ß-sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four-times higher than that of the natural product was identified.