A Potent Sybody Selectively Inhibits α-Synuclein Amyloid Formation by Binding to the P1 Region.

Increasing research efforts focus on exploiting antibodies to inhibit the amyloid formation of neurodegenerative proteins. Nevertheless, it is challenging to discover antibodies that inhibit this process in a specific manner. Using ribosome display, we screened for synthetic single-domain antibodies, i.e., sybodies, of the P1 region of α-synuclein (residues 36-42), a protein that forms amyloid in Parkinson's disease and multiple-system atrophy. Hits were assessed for direct binding to a P1 peptide and the inhibition of amyloid formation. We discovered a sybody, named αSP1, that inhibits amyloid formation of α-synuclein at substoichiometric concentrations in a specific manner, even within highly crowded heterogeneous mixtures. Fluorescence resonance energy transfer-based binding assays and seeding experiments with and without αSP1 further demonstrate the importance of the P1 region for both primary and secondary nucleation mechanisms of amyloid assembly.

A single-domain antibody detects and neutralises toxic Aß42 oligomers in the Alzheimer's disease CSF.

BACKGROUND: Amyloid-ß42 (Aß42) aggregation consists of a complex chain of nucleation events producing soluble oligomeric intermediates, which are considered the major neurotoxic agents in Alzheimer's disease (AD). Cerebral lesions in the brain of AD patients start to develop 20 years before symptom onset; however, no preventive strategies, effective treatments, or specific and sensitive diagnostic tests to identify people with early-stage AD are currently available. In addition, the isolation and characterisation of neurotoxic Aß42 oligomers are particularly difficult because of their transient and heterogeneous nature. To overcome this challenge, a rationally designed method generated a single-domain antibody (sdAb), named DesAb-O, targeting Aß42 oligomers. METHODS: We investigated the ability of DesAb-O to selectively detect preformed Aß42 oligomers both in vitro and in cultured neuronal cells, by using dot-blot, ELISA immunoassay and super-resolution STED microscopy, and to counteract the toxicity induced by the oligomers, monitoring their interaction with neuronal membrane and the resulting mitochondrial impairment. We then applied this approach to CSF samples (CSFs) from AD patients as compared to age-matched control subjects. RESULTS: DesAb-O was found to selectively detect synthetic Aß42 oligomers both in vitro and in cultured cells, and to neutralise their associated neuronal dysfunction. DesAb-O can also identify Aß42 oligomers present in the CSFs of AD patients with respect to healthy individuals, and completely prevent cell dysfunction induced by the administration of CSFs to neuronal cells. CONCLUSIONS: Taken together, our data indicate a promising method for the improvement of an early diagnosis of AD and for the generation of novel therapeutic approaches based on sdAbs for the treatment of AD and other devastating neurodegenerative conditions.

The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC)n in C9orf72. Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD.

Site-Specific 68Ga Radiolabeling of Trastuzumab Fab via Methionine for ImmunoPET Imaging.

Bioconjugates of antibodies and their derivatives radiolabeled with ß+-emitting radionuclides can be utilized for diagnostic PET imaging. Site-specific attachment of radioactive cargo to antibody delivery vectors provides homogeneous, well-defined immunoconjugates. Recent studies have demonstrated the utility of oxaziridine chemistry for site-specific labeling of methionine residues. Herein, we applied this approach to site-specifically radiolabel trastuzumab-derived Fab immunoconjugates with 68Ga, which can be used for in vivo PET imaging of HER2-positive breast cancer tumors. Initially, a reactive azide was introduced to a single solvent-accessible methionine residue in both the wild-type Fab and an engineered derivative containing methionine residue M74, utilizing the principles of oxaziridine chemistry. Subsequently, these conjugates were functionalized with a modified DFO chelator incorporating dibenzocyclooctyne. The resulting DFO-WT and DFO-M74 conjugates were radiolabeled with generator-produced [68Ga]Ga3+, to yield the novel PET radiotracers, [68Ga]Ga-DFO-WT and [68Ga]Ga-DFO-M74. In vitro and in vivo studies demonstrated that [68Ga]Ga-DFO-M74 exhibited a higher affinity for HER2 receptors. Biodistribution studies in mice bearing orthotopic HER2-positive breast tumors revealed a higher uptake of [68Ga]Ga-DFO-M74 in the tumor tissue, accompanied by rapid renal clearance, enabling clear delineation of tumors using PET imaging. Conversely, [68Ga]Ga-DFO-WT exhibited lower uptake and inferior image contrast compared to [68Ga]Ga-DFO-M74. Overall, the results demonstrate that the highly facile methionine-oxaziridine modification approach can be simply applied to the synthesis of stable and site-specifically modified radiolabeled antibody-chelator conjugates with favorable pharmacokinetics for PET imaging.

Natural Inhibitors of Amyloid Aggregation.

Amyloid aggregates are diverse proteinaceous assemblies, including one or more protein species, wherein the molecules interact according to characteristic patterns [...].

Unveiling the Rational Development of Stimuli-Responsive Silk Fibroin-Based Ionogel Formulations.

We present an approach for the rational development of stimuli-responsive ionogels which can be formulated for precise control of multiple unique ionogel features and fill niche pharmaceutical applications. Ionogels are captivating materials, exhibiting self-healing characteristics, tunable mechanical and structural properties, high thermal stability, and electroconductivity. However, the majority of ionogels developed require complex chemistry, exhibit high viscosity, poor biocompatibility, and low biodegradability. In our work, we overcome these limitations. We employ a facile production process and strategically integrate silk fibroin, the biocompatible ionic liquids (ILs) choline acetate ([Cho][OAc]), choline dihydrogen phosphate ([Cho][DHP]), and choline chloride ([Cho][Cl]), traditional pharmaceutical excipients, and the model antiepileptic drug phenobarbital. In the absence of ILs, we failed to observe gel formation; yet in the presence of ILs, thermoresponsive ionogels formed. Systems were assessed via visual tests, transmission electron microscopy, confocal reflection microscopy, dynamic light scattering, zeta potential and rheology measurements. We formed diverse ionogels of strengths ranging between 18 and 642 Pa. Under 25 °C storage, formulations containing polyvinylpyrrolidone (PVP) showed an ionogel formation period ranging over 14 days, increasing in the order of [Cho][DHP], [Cho][OAc], and [Cho][Cl]. Formulations lacking PVP showed an ionogel formation period ranging over 32 days, increasing in the order of [Cho][OAc], [Cho][DHP] and [Cho][Cl]. By heating from 25 to 60 °C, immediately following preparation, thermoresponsive ionogels formed below 41 °C in the absence of PVP. Based on our experimental results and density functional theory calculations, we attribute ionogel formation to macromolecular crowding and confinement effects, further enhanced upon PVP inclusion. Holistically, applying our rational development strategy enables the production of ionogels of tunable physicochemical and rheological properties, enhanced drug solubility, and structural and energetic stability. We believe our rational development approach will advance the design of biomaterials and smart platforms for diverse drug delivery applications.

α-Synuclein Aggregation Is Triggered by Oligomeric Amyloid-ß 42 via Heterogeneous Primary Nucleation.

An increasing number of cases where amyloids of different proteins are found in the same patient are being reported. This observation complicates diagnosis and clinical intervention. Amyloids of the amyloid-ß peptide or the protein α-synuclein are traditionally considered hallmarks of Alzheimer's and Parkinson's diseases, respectively. However, the co-occurrence of amyloids of these proteins has also been reported in patients diagnosed with either disease. Here, we show that soluble species containing amyloid-ß can induce the aggregation of α-synuclein. Fibrils formed under these conditions are solely composed of α-synuclein to which amyloid-ß can be found associated but not as part of the core of the fibrils. Importantly, by global kinetic analysis, we found that the aggregation of α-synuclein under these conditions occurs via heterogeneous primary nucleation, triggered by soluble aggregates containing amyloid-ß.

Galectin-3 shapes toxic alpha-synuclein strains in Parkinson's disease.

Parkinson's Disease (PD) is a neurodegenerative and progressive disorder characterised by intracytoplasmic inclusions called Lewy bodies (LB) and degeneration of dopaminergic neurons in the substantia nigra (SN). Aggregated α-synuclein (αSYN) is known to be the main component of the LB. It has also been reported to interact with several proteins and organelles. Galectin-3 (GAL3) is known to have a detrimental function in neurodegenerative diseases. It is a galactose-binding protein without known catalytic activity and is expressed mainly by activated microglial cells in the central nervous system (CNS). GAL3 has been previously found in the outer layer of the LB in post-mortem brains. However, the role of GAL3 in PD is yet to be elucidated. In post-mortem samples, we identified an association between GAL3 and LB in all the PD subjects studied. GAL3 was linked to less αSYN in the LB outer layer and other αSYN deposits, including pale bodies. GAL3 was also associated with disrupted lysosomes. In vitro studies demonstrate that exogenous recombinant Gal3 is internalised by neuronal cell lines and primary neurons where it interacts with endogenous αSyn fibrils. In addition, aggregation experiments show that Gal3 affects spatial propagation and the stability of pre-formed αSyn fibrils resulting in short, amorphous toxic strains. To further investigate these observations in vivo, we take advantage of WT and Gal3KO mice subjected to intranigral injection of adenovirus overexpressing human αSyn as a PD model. In line with our in vitro studies, under these conditions, genetic deletion of GAL3 leads to increased intracellular αSyn accumulation within dopaminergic neurons and remarkably preserved dopaminergic integrity and motor function. Overall, our data suggest a prominent role for GAL3 in the aggregation process of αSYN and LB formation, leading to the production of short species to the detriment of larger strains which triggers neuronal degeneration in a mouse model of PD.

An antibody scanning method for the detection of α-synuclein oligomers in the serum of Parkinson's disease patients.

Misfolded α-synuclein oligomers are closely implicated in the pathology of Parkinson's disease and related synucleinopathies. The elusive nature of these aberrant assemblies makes it challenging to develop quantitative methods to detect them and modify their behavior. Existing detection methods use antibodies to bind α-synuclein aggregates in biofluids, although it remains challenging to raise antibodies against α-synuclein oligomers. To address this problem, we used an antibody scanning approach in which we designed a panel of 9 single-domain epitope-specific antibodies against α-synuclein. We screened these antibodies for their ability to inhibit the aggregation process of α-synuclein, finding that they affected the generation of α-synuclein oligomers to different extents. We then used these antibodies to investigate the size distribution and morphology of soluble α-synuclein aggregates in serum and cerebrospinal fluid samples from Parkinson's disease patients. Our results indicate that the approach that we present offers a promising route for the development of antibodies to characterize soluble α-synuclein aggregates in biofluids.

A Facile Method to Produce N-Terminally Truncated α-Synuclein.

α-Synuclein is a key protein of the nervous system, which regulates the release and recycling of neurotransmitters in the synapses. It is also involved in several neurodegenerative conditions, including Parkinson's disease and Multiple System Atrophy, where it forms toxic aggregates. The N-terminus of α-synuclein is of particular interest as it has been linked to both the physiological and pathological functions of the protein and undergoes post-translational modification. One such modification, N-terminal truncation, affects the aggregation propensity of the protein in vitro and is also found in aggregates from patients' brains. To date, our understanding of the role of this modification has been limited by the many challenges of introducing biologically relevant N-terminal truncations with no overhanging starting methionine. Here, we present a method to produce N-terminally truncated variants of α-synuclein that do not carry extra terminal residues. We show that our method can generate highly pure protein to facilitate the study of this modification and its role in physiology and disease. Thanks to this method, we have determined that the first six residues of α-synuclein play an important role in the formation of the amyloids.

A Chemical Mutagenesis Approach to Insert Post-translational Modifications in Aggregation-Prone Proteins.

Neurodegenerative diseases are a class of disorders linked to the formation in the nervous system of fibrillar protein aggregates called amyloids. This aggregation process is affected by a variety of post-translational modifications, whose specific mechanisms are not fully understood yet. Emerging chemical mutagenesis technology is currently striving to address the challenge of introducing protein post-translational modifications, while maintaining the stability and solubility of the proteins during the modification reaction. Several amyloidogenic proteins are highly aggregation-prone, and current modification procedures can lead to unexpected precipitation of these proteins, affecting their yield and downstream characterization. Here, we present a method for maintaining amyloidogenic protein solubility during chemical mutagenesis. As proof-of-principle, we applied our method to mimic the phosphorylation of serine-26 and the acetylation of lysine-28 of the 40-residue long variant of amyloid-ß peptide, whose aggregation is linked to Alzheimer's disease.

The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation.

While aggregation-prone proteins are known to accelerate aging and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that MOAG-4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation-prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age-related protein toxicity.

Man does not live by intrinsically unstructured proteins alone: The role of structured regions in aggregation.

Protein misfolding is a topic that is of primary interest both in biology and medicine because of its impact on fundamental processes and disease. In this review, we revisit the concept of protein misfolding and discuss how the field has evolved from the study of globular folded proteins to focusing mainly on intrinsically unstructured and often disordered regions. We argue that this shift of paradigm reflects the more recent realisation that misfolding may not only be an adverse event, as originally considered, but also may fulfil a basic biological need to compartmentalise the cell with transient reversible granules. We nevertheless provide examples in which structure is an important component of a much more complex aggregation behaviour that involves both structured and unstructured regions of a protein. We thus suggest that a more comprehensive evaluation of the mechanisms that lead to aggregation might be necessary.

The binding of the small heat-shock protein αB-crystallin to fibrils of α-synuclein is driven by entropic forces.

Molecular chaperones are key components of the cellular proteostasis network whose role includes the suppression of the formation and proliferation of pathogenic aggregates associated with neurodegenerative diseases. The molecular principles that allow chaperones to recognize misfolded and aggregated proteins remain, however, incompletely understood. To address this challenge, here we probe the thermodynamics and kinetics of the interactions between chaperones and protein aggregates under native solution conditions using a microfluidic platform. We focus on the binding between amyloid fibrils of α-synuclein, associated with Parkinson's disease, to the small heat-shock protein αB-crystallin, a chaperone widely involved in the cellular stress response. We find that αB-crystallin binds to α-synuclein fibrils with high nanomolar affinity and that the binding is driven by entropy rather than enthalpy. Measurements of the change in heat capacity indicate significant entropic gain originates from the disassembly of the oligomeric chaperones that function as an entropic buffer system. These results shed light on the functional roles of chaperone oligomerization and show that chaperones are stored as inactive complexes which are capable of releasing active subunits to target aberrant misfolded species.

Modulation of amyloid-ß aggregation by metal complexes with a dual binding mode and their delivery across the blood-brain barrier using focused ultrasound.

One of the key hallmarks of Alzheimer's disease is the aggregation of the amyloid-ß peptide to form fibrils. Consequently, there has been great interest in studying molecules that can disrupt amyloid-ß aggregation. While a handful of molecules have been shown to inhibit amyloid-ß aggregation in vitro, there remains a lack of in vivo data reported due to their inability to cross the blood-brain barrier. Here, we investigate a series of new metal complexes for their ability to inhibit amyloid-ß aggregation in vitro. We demonstrate that octahedral cobalt complexes with polyaromatic ligands have high inhibitory activity thanks to their dual binding mode involving π-π stacking and metal coordination to amyloid-ß (confirmed via a range of spectroscopic and biophysical techniques). In addition to their high activity, these complexes are not cytotoxic to human neuroblastoma cells. Finally, we report for the first time that these metal complexes can be safely delivered across the blood-brain barrier to specific locations in the brains of mice using focused ultrasound.

Comparative Studies in the A30P and A53T α-Synuclein C. elegans Strains to Investigate the Molecular Origins of Parkinson's Disease.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.

Rationally Designed Bicyclic Peptides Prevent the Conversion of Aß42 Assemblies Into Fibrillar Structures.

There is great interest in drug discovery programs targeted at the aggregation of the 42-residue form of the amyloid ß peptide (Aß42), since this molecular process is closely associated with Alzheimer's disease. The use of bicyclic peptides may offer novel opportunities for the effective modification of Aß42 aggregation and the inhibition of its cytotoxicity, as these compounds combine the molecular recognition ability of antibodies with a relatively small size of about 2 kD. Here, to pursue this approach, we rationally designed a panel of six bicyclic peptides targeting various epitopes along the sequence of Aß42 to scan its most amyloidogenic region (residues 13-42). Our kinetic analysis and structural studies revealed that at sub-stoichiometric concentrations the designed bicyclic peptides induce a delay in the condensation of Aß42 and the subsequent transition to a fibrillar state, while at higher concentrations they inhibit such transition. We thus suggest that designed bicyclic peptides can be employed to inhibit amyloid formation by redirecting the aggregation process toward amorphous assemblies.

Systematic Activity Maturation of a Single-Domain Antibody with Non-canonical Amino Acids through Chemical Mutagenesis.

Great advances have been made over the last four decades in therapeutic and diagnostic applications of antibodies. The activity maturation of antibody candidates, however, remains a significant challenge. To address this problem, we present a method that enables the systematic enhancement of the activity of a single-domain antibody through the post-translational installation of non-canonical side chains by chemical mutagenesis. We illustrate this approach by performing a structure-activity relationship study beyond the 20 naturally occurring amino acids on a single-domain antibody designed in silico to inhibit the aggregation of the amyloid-ß peptide, a process closely linked to Alzheimer's disease. We found that this approach can improve, by five orders of magnitude, the anti-aggregation activity of the starting single-domain antibody, without affecting its stability. These results show that the expansion of the chemical space available to antibodies through chemical mutagenesis can be exploited for the systematic enhancement of the activity of these molecules.

Small-molecule sequestration of amyloid-ß as a drug discovery strategy for Alzheimer's disease.

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that modifies the properties of their monomeric states. Here, we identify a small molecule (10074-G5) capable of binding and sequestering the intrinsically disordered amyloid-ß (Aß) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aß and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterize this interaction using biophysical experiments and integrative structural ensemble determination methods. We observe that this molecule increases the conformational entropy of monomeric Aß while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aß-associated toxicity, consistent with the mechanism of action identified from the in silico and in vitro studies. These results illustrate the strategy of stabilizing the monomeric states of disordered proteins with small molecules to alter their behavior for therapeutic purposes.

A rationally designed bicyclic peptide remodels Aß42 aggregation in vitro and reduces its toxicity in a worm model of Alzheimer's disease.

Bicyclic peptides have great therapeutic potential since they can bridge the gap between small molecules and antibodies by combining a low molecular weight of about 2 kDa with an antibody-like binding specificity. Here we apply a recently developed in silico rational design strategy to produce a bicyclic peptide to target the C-terminal region (residues 31-42) of the 42-residue form of the amyloid ß peptide (Aß42), a protein fragment whose aggregation into amyloid plaques is linked with Alzheimer's disease. We show that this bicyclic peptide is able to remodel the aggregation process of Aß42 in vitro and to reduce its associated toxicity in vivo in a C. elegans worm model expressing Aß42. These results provide an initial example of a computational approach to design bicyclic peptides to target specific epitopes on disordered proteins.