Expanding assay range to accommodate a monoclonal antibody therapeutic quantification in blood and cerebrospinal fluid.

Antibody therapeutic levels in neurodegenerative diseases are often measured in both serum and cerebrospinal fluid (CSF). Due to 0.1% drug partition from serum to CSF and the higher sensitivity needs, usually two different assays are required. The different Gyrolab Bioaffy compact discs can extend the dynamic range of assays. Here, an assay was developed and adapted on two different Gyrolab Bioaffy compact discs (200 and 4000 nl) to achieve the required sensitivity and assay dynamic range needed for the measurement of drug in both serum and CSF. This was accomplished by using the same critical reagents with minimal assay development to transition from a serum to a CSF assay.

An Enantiomeric Fragment Pair (EFP) Approach for the Study of Cellular Uptake of Intrinsically Disordered Proteins.

The study of intrinsically disordered and amyloidogenic proteins poses a major challenge to researchers due to the propensity of the system to aggregate and to form amyloid fibrils and deposits. This intrinsic nature limits the way amyloids can be studied and increases the level of complexity of the techniques needed to study the system of interest. Recent reports suggest that cellular recognition and internalization of pre-fibrillary species of amyloidogenic peptides and proteins may initiate some of its toxic actions. Therefore, developing novels tools to facilitate the understanding and determination of the interactions between intrinsically disordered proteins and the cellular membrane is becoming increasingly valuable. Here, we present and propose an approach for the study of the interactions of intrinsically disordered proteins with the cellular surface based on the use of enantiomeric fragment pairs (EFPs). By following a stepwise methodology in which the amyloidogenic peptide or protein is fragmented into specific segments, we show how this approach can be exploited to differentiate between different types of cellular uptake, to determine the degree of receptor-mediated cellular internalization of intrinsically disordered peptides and proteins, and to pinpoint the specific regions within the amino acid sequence responsible for the cellular recognition. Adopting this approach overcomes aggregation-related challenges and offers a particularly well-suited platform for the elucidation of receptor-intermediated recognition, uptake, and toxicity.

Hollow Gold Nanosphere Templated Synthesis of PEGylated Hollow Gold Nanostars and Use for SERS Detection of Amyloid Beta in Solution.

Hollow gold nanospheres (HGNs) have been used as the template for seed-mediated growth of multibranched hollow gold nanostars (HNS). The HGNs were synthesized via anerobic reduction of cobalt chloride to cobalt nanoparticles and then formation of a gold shell via galvanic replacement followed by the oxidation of the cobalt core. We obtained control of the inner core size of the HGNs by increasing the size of the sacrificial cobalt core and by varying the ratio of B(OH)3/BH4 using boric acid rather than 48 h aged borohydride. We synthesized the HNS by reducing Au3+ ions in the presence of Ag+ ions using ascorbic acid, creating a spiky morphology that varied with the Au3+/Ag+ ratio. A broadly tunable localized surface plasmon resonance was achieved through control of both the inner core and the spike length. Amyloid beta (Aß) was conjugated to the HNS by using a heterobifunctional PEG linker and identified by the vibrational modes associated with the conjugated ring phenylalanine side chain. A bicinchoninic acid assay was used to determine the concentration of Aß conjugated to HNS as 20 nM, which is below the level of Aß that negatively affects long-term potentiation. Both the core size and spike length were shown to affect the optical properties of the resulting nanostructures. This HGN templated method introduced a new parameter for enhancing the plasmonic properties of gold nanostars, namely, the addition of a hollow core. Hollow gold nanostars are highly desirable for a wide range of applications, including high sensitivity disease detection and monitoring.

Constraints on the Structure of Fibrils Formed by a Racemic Mixture of Amyloid-ß Peptides from Solid-State NMR, Electron Microscopy, and Theory.

Previous studies have shown that racemic mixtures of 40- and 42-residue amyloid-ß peptides (d,l-Aß40 and d,l-Aß42) form amyloid fibrils with accelerated kinetics and enhanced stability relative to their homochiral counterparts (l-Aß40 and l-Aß42), suggesting a "chiral inactivation" approach to abrogating the neurotoxicity of Aß oligomers (Aß-CI). Here we report a structural study of d,l-Aß40 fibrils, using electron microscopy, solid-state nuclear magnetic resonance (NMR), and density functional theory (DFT) calculations. Two- and three-dimensional solid-state NMR spectra indicate molecular conformations in d,l-Aß40 fibrils that resemble those in known l-Aß40 fibril structures. However, quantitative measurements of 13C-13C and 15N-13C distances in selectively labeled d,l-Aß40 fibril samples indicate a qualitatively different supramolecular structure. While cross-ß structures in mature l-Aß40 fibrils are comprised of in-register, parallel ß-sheets, our data indicate antiparallel ß-sheets in d,l-Aß40 fibrils, with alternation of d and l molecules along the fibril growth direction, i.e., antiparallel "rippled sheet" structures. The solid-state NMR data suggest the coexistence of d,l-Aß40 fibril polymorphs with three different registries of intermolecular hydrogen bonds within the antiparallel rippled sheets. DFT calculations support an energetic preference for antiparallel alignments of the ß-strand segments identified by solid-state NMR. These results provide insight into the structural basis for Aß-CI and establish the importance of rippled sheets in self-assembly of full-length, naturally occurring amyloidogenic peptides.

Understanding and controlling amyloid aggregation with chirality.

Amyloid aggregation and human disease are inextricably linked. Examples include Alzheimer disease, Parkinson disease, and type II diabetes. While seminal advances on the mechanistic understanding of these diseases have been made over the last decades, controlling amyloid fibril formation still represents a challenge, and it is a subject of active research. In this regard, chiral modifications have increasingly been proved to offer a particularly well-suited approach toward accessing to previously unknown aggregation pathways and to provide with novel insights on the biological mechanisms of action of amyloidogenic peptides and proteins. Here, we summarize recent advances on how the use of mirror-image peptides/proteins and d-amino acid incorporations have helped modulate amyloid aggregation, offered new mechanistic tools to study cellular interactions, and allowed us to identify key positions within the peptide/protein sequence that influence amyloid fibril growth and toxicity.

Evidence for aggregation-independent, PrPC-mediated Aß cellular internalization.

Evidence linking amyloid beta (Aß) cellular uptake and toxicity has burgeoned, and mechanisms underlying this association are subjects of active research. Two major, interconnected questions are whether Aß uptake is aggregation-dependent and whether it is sequence-specific. We recently reported that the neuronal uptake of Aß depends significantly on peptide chirality, suggesting that the process is predominantly receptor-mediated. Over the past decade, the cellular prion protein (PrPC) has emerged as an important mediator of Aß-induced toxicity and of neuronal Aß internalization. Here, we report that the soluble, nonfibrillizing Aß (1-30) peptide recapitulates full-length Aß stereoselective cellular uptake, allowing us to decouple aggregation from cellular, receptor-mediated internalization. Moreover, we found that Aß (1-30) uptake is also dependent on PrPC expression. NMR-based molecular-level characterization identified the docking site on PrPC that underlies the stereoselective binding of Aß (1-30). Our findings therefore identify a specific sequence within Aß that is responsible for the recognition of the peptide by PrPC, as well as PrPC-dependent cellular uptake. Further uptake stereodifferentiation in PrPC-free cells points toward additional receptor-mediated interactions as likely contributors for Aß cellular internalization. Taken together, our results highlight the potential of targeting cellular surface receptors to inhibit Aß cellular uptake as an alternative route for future therapeutic development for Alzheimer's disease.

Assessing Reproducibility in Amyloid ß Research: Impact of Aß Sources on Experimental Outcomes.

The difficulty of synthesizing and purifying the amyloid ß (Aß) peptide, combined with its high aggregation propensity and low solubility under physiological conditions, leads to a wide variety of experimental results from kinetic assays to biological activity. Thus, it becomes challenging to reproduce outcomes, and this limits our ability to rely on reported results as the foundation for new research. This article examines variability of the Aß peptide from different sources, comparing purity, and oligomer and fibril formation propensity side by side. The results highlight the importance of performing rigorous controls so that meaningful biophysical, biochemical, and neurobiological results can be obtained to improve our understanding on Aß.

A Focused Chiral Mutant Library of the Amyloid ß 42 Central Electrostatic Cluster as a Tool To Stabilize Aggregation Intermediates.

Amyloidogenic peptides and proteins aggregate into fibrillary structures that are usually deposited in tissues and organs and are often involved in the development of diseases. In contrast to native structured proteins, amyloids do not follow a defined energy landscape toward the fibrillary state and often generate a vast population of aggregation intermediates that are transient and exceedingly difficult to study. Here, we employ chiral editing as a tool to study the aggregation mechanism of the Amyloid ß (Aß) 42 peptide, whose aggregation intermediates are thought to be one of the main driving forces in Alzheimer's disease (AD). Through the design of a focused chiral mutant library (FCML) of 16 chiral Aß42 variants, we identified several point D-substitutions that allowed us to modulate the aggregation propensity and the biological activity of the peptide. Surprisingly, the reduced propensity toward aggregation and the stabilization of oligomeric intermediates did not always correlate with an increase in toxicity. In the present study, we show how chiral editing can be a powerful tool to trap and stabilize Aß42 conformers that might otherwise be too transient and dynamic to study, and we identify sites within the Aß42 sequence that could be potential targets for therapeutic intervention.

A Facile Method for the Separation of Methionine Sulfoxide Diastereomers, Structural Assignment, and DFT Analysis.

Methionine (Met) oxidation is an important biological redox node, with hundreds if not thousands of protein targets. The process yields methionine oxide (MetO). It renders the sulfur chiral, producing two distinct, diastereomerically related products. Despite the biological significance of Met oxidation, a reliable protocol to separate the resultant MetO diastereomers is currently lacking. This hampers our ability to make peptides and proteins that contain stereochemically defined MetO to then study their structural and functional properties. We have developed a facile method that uses supercritical CO2 chromatography and allows obtaining both diastereomers in purities exceeding 99 %. 1 H NMR spectra were correlated with X-ray structural information. The stereochemical interconversion barrier at sulfur was calculated as 45.2 kcal mol-1 , highlighting the remarkable stereochemical stability of MetO sulfur chirality. Our protocol should open the road to synthesis and study of a wide variety of stereochemically defined MetO-containing proteins and peptides.

Trapping and Characterization of Nontoxic Aß42 Aggregation Intermediates.

Amyloid ß (Aß) 42 is an aggregation-prone peptide and the believed seminal etiological agent of Alzheimer's disease (AD). Intermediates of Aß42 aggregation, commonly referred to as diffusible oligomers, are considered to be among the most toxic forms of the peptide. Here, we studied the effect of the age-related epimerization of Ser26 (i.e., S26s chiral edit) in Aß42 and discovered that this subtle molecular change led to reduced fibril formation propensity. Surprisingly, the resultant soluble aggregates were nontoxic. To gain insight into the structural changes that occurred in the peptide upon S26s substitution, the system was probed using an array of biophysical and biochemical methods. These experiments consistently pointed to the stabilization of aggregation intermediates in the Aß42-S26s system. To better understand the changes arising as a consequence of the S26s substitution, molecular level structural studies were performed. Using a combined nuclear magnetic resonance (NMR)- and density functional theory (DFT)-computational approach, we found that the S26s chiral edit induced only local structural changes in the Gly25-Ser26-Asn27 region. Interestingly, these subtle changes enabled the formation of an intramolecular Ser26-Asn27 H-bond, which disrupted the ability of Asn27 to engage in the fibrillogenic side chain-to-side chain H-bonding pattern. This reveals that intermolecular stabilizing interactions between Asn27 side chains are a key element controlling Aß42 aggregation and toxicity.

A DFT-Assisted Topological Analysis of Four Polymorphic, S-Shaped Aß42 Fibril Structures.

Amyloid ß 42 (Aß42) is an inherently disordered peptide, whose toxic actions are believed to play important roles in the etiology of Alzheimer's disease. Four fibril structures of the peptide that display broadly similar characteristics were recently published, but a systematic comparison of these structures is lacking. In this paper, a topological framework was created to enable such understanding and produced new insights into subtle structural elements that underlie the overall structural diversity. A DFT-based analysis illuminated some of the energetic differences that arise as a consequence.

Suppression of Oligomer Formation and Formation of Non-Toxic Fibrils upon Addition of Mirror-Image Aß42 to the Natural l-Enantiomer.

Racemates often have lower solubility than enantiopure compounds, and the mixing of enantiomers can enhance the aggregation propensity of peptides. Amyloid beta (Aß) 42 is an aggregation-prone peptide that is believed to play a key role in Alzheimer's disease. Soluble Aß42 aggregation intermediates (oligomers) have emerged as being particularly neurotoxic. We hypothesized that the addition of mirror-image d-Aß42 should reduce the concentration of toxic oligomers formed from natural l-Aß42. We synthesized l- and D-Aß42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogues of the enantiomers showed their colocalization in racemic fibrils. Owing to the enhanced fibril formation propensity, racemic Aß42 was less prone to form soluble oligomers. This resulted in the protection of cells from the toxicity of l-Aß42 at concentrations up to 50 µm. The mixing of Aß42 enantiomers thus accelerates the formation of non-toxic fibrils.

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (Aß), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (Aß42) and amyloid beta 40 (Aß40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (>95%) Aß42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the Aß40 peptide with identical success and without the need to alter the method.

Using chiral peptide substitutions to probe the structure function relationship of a key residue of Aß42.

Amyloid beta-protein 42 plays an important role in the onset and progression of Alzheimer's disease. Familial mutations have identified the glutamate residue 22 as a hotspot with regard to peptide neurotoxicity. We introduce an approach to study the influence of systematic sidechain modification at this residue, employing chirality as a structural probe. Circular dichroism experiments reveal that charge-preserving alterations of the amino acid sidechain attenuate the characteristic random coil to ß-sheet transition associated with the wildtype peptide. Removal of the negative charge from residue 22, a trait observed with all known familial mutations at this residue, gives rise to a peptide with limited random coil propensity and high ß-sheet characteristics. Our approach can be extended to other residues of Aß, as well as further amyloidogenic peptides.

Introduction of d-Glutamate at a Critical Residue of Aß42 Stabilizes a Prefibrillary Aggregate with Enhanced Toxicity.

The amyloid beta peptide 42 (Aß42) is an aggregation-prone peptide that plays a pivotal role in Alzheimer's disease. We report that a subtle perturbation to the peptide through a single chirality change at glutamate 22 leads to a pronounced delay in the ß-sheet adoption of the peptide. This was accompanied by an attenuated propensity of the peptide to form fibrils, which was correlated with changes at the level of the fibrillary architecture. Strikingly, the incorporation of d-glutamate was found to stabilize a soluble, ordered macromolecular assembly with enhanced cytotoxicity to PC12 cells, highlighting the importance of advanced prefibrillary Aß aggregates in neurotoxicity.