ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes.

Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes.

Small soluble α-synuclein aggregates are the toxic species in Parkinson's disease.

Soluble α-synuclein aggregates varying in size, structure, and morphology have been closely linked to neuronal death in Parkinson's disease. However, the heterogeneity of different co-existing aggregate species makes it hard to isolate and study their individual toxic properties. Here, we show a reliable non-perturbative method to separate a heterogeneous mixture of protein aggregates by size. We find that aggregates of wild-type α-synuclein smaller than 200 nm in length, formed during an in vitro aggregation reaction, cause inflammation and permeabilization of single-liposome membranes and that larger aggregates are less toxic. Studying soluble aggregates extracted from post-mortem human brains also reveals that these aggregates are similar in size and structure to the smaller aggregates formed in aggregation reactions in the test tube. Furthermore, we find that the soluble aggregates present in Parkinson's disease brains are smaller, largely less than 100 nm, and more inflammatory compared to the larger aggregates present in control brains. This study suggests that the small non-fibrillar α-synuclein aggregates are the critical species driving neuroinflammation and disease progression.

Imaging protein aggregates in the serum and cerebrospinal fluid in Parkinson's disease.

Aggregation of α-synuclein plays a key role in the development of Parkinson's disease. Soluble aggregates are present not only within human brain but also the CSF and blood. Characterizing the aggregates present in these biofluids may provide insights into disease mechanisms and also have potential for aiding diagnosis. We used two optical single-molecule imaging methods called aptamer DNA-PAINT and single-aggregate confocal fluorescence, together with high-resolution atomic force microscopy for specific detection and characterization of individual aggregates with intermolecular ß-sheet structure, present in the CSF and serum of 15 early stage Parkinson's disease patients compared to 10 healthy age-matched controls. We found aggregates ranging in size from 20 nm to 200 nm, in both CSF and serum. There was a difference in aggregate size distribution between Parkinson's disease and control groups with a significantly increased number of larger aggregates (longer than 150 nm) in the serum of patients with Parkinson's disease. To determine the chemical composition of the aggregates, we performed aptamer DNA-PAINT on serum following α-synuclein and amyloid-ß immunodepletion in an independent cohort of 11 patients with early stage Parkinson's disease and 10 control subjects. ß-Sheet aggregates in the serum of Parkinson's disease patients were found to consist of, on average, 50% α-synuclein and 50% amyloid-ß in contrast to 30% α-synuclein and 70% amyloid-ß in control serum [the differences in the proportion of these aggregates were statistically significant between diseased and control groups (P = 1.7 × 10-5 for each species)]. The ratio of the number of ß-sheet α-synuclein aggregates to ß-sheet amyloid-ß aggregates in serum extracted using our super-resolution method discriminated Parkinson's disease cases from controls with an accuracy of 98.2% (AUC = 98.2%, P = 4.3 × 10-5). Our data suggest that studying the protein aggregates present in serum can provide information about the disruption of protein homeostasis occurring in Parkinson's disease and warrants further investigation as a potential biomarker of disease.

The Nt17 Domain and its Helical Conformation Regulate the Aggregation, Cellular Properties and Neurotoxicity of Mutant Huntingtin Exon 1.

Converging evidence points to the N-terminal domain comprising the first 17 amino acids of the Huntingtin protein (Nt17) as a key regulator of its aggregation, cellular properties and toxicity. In this study, we further investigated the interplay between Nt17 and the polyQ domain repeat length in regulating the aggregation and inclusion formation of exon 1 of the Huntingtin protein (Httex1). In addition, we investigated the effect of removing Nt17 or modulating its local structure on the membrane interactions, neuronal uptake, and toxicity of monomeric or fibrillar Httex1. Our results show that the polyQ and Nt17 domains synergistically modulate the aggregation propensity of Httex1 and that the Nt17 domain plays important roles in shaping the surface properties of mutant Httex1 fibrils and regulating their poly-Q-dependent growth, lateral association and neuronal uptake. Removal of Nt17 or disruption of its transient helical conformations slowed the aggregation of monomeric Httex1 in vitro, reduced inclusion formation in cells, enhanced the neuronal uptake and nuclear accumulation of monomeric Httex1 proteins, and was sufficient to prevent cell death induced by Httex1 72Q overexpression. Finally, we demonstrate that the uptake of Httex1 fibrils into primary neurons and the resulting toxicity are strongly influenced by mutations and phosphorylation events that influence the local helical propensity of Nt17. Altogether, our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation, internalization, and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, and neuronal toxicity.

Unraveling the Physicochemical Determinants of Protein Liquid-liquid Phase Separation by Nanoscale Infrared Vibrational Spectroscopy.

The phenomenon of reversible liquid-liquid phase separation of proteins underlies the formation of membraneless organelles, which are crucial for cellular processes such as signalling and transport. In addition, it is also of great interest to uncover the mechanisms of further irreversible maturation of the functional dense liquid phase into aberrant insoluble assemblies due to its implication in human disease. Recent advances in methods based on atomic force microscopy (AFM) have made it possible to study protein condensates at the nanometer level, providing unprecedented information on the nature of the intermolecular interactions governing phase separation. Here, we provide an in-depth description of a protocol for the characterisation of the morphology, stiffness, and chemical properties of protein condensates using infrared nanospectroscopy (AFM-IR).

Soluble amyloid beta-containing aggregates are present throughout the brain at early stages of Alzheimer's disease.

Protein aggregation likely plays a key role in the initiation and spreading of Alzheimer's disease pathology through the brain. Soluble aggregates of amyloid beta are believed to play a key role in this process. However, the aggregates present in humans are still poorly characterized due to a lack of suitable methods required for characterizing the low concentration of heterogeneous aggregates present. We have used a variety of biophysical methods to characterize the aggregates present in human Alzheimer's disease brains at Braak stage III. We find soluble amyloid beta-containing aggregates in all regions of the brain up to 200 nm in length, capable of causing an inflammatory response. Rather than aggregates spreading through the brain as disease progresses, it appears that aggregation occurs all over the brain and that different brain regions are at earlier or later stages of the same process, with the later stages causing increased inflammation.

Squalamine and Its Derivatives Modulate the Aggregation of Amyloid-ß and α-Synuclein and Suppress the Toxicity of Their Oligomers.

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-ß peptide (Aß) and of α-synuclein (αS), which are associated with Alzheimer's and Parkinson's diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aß and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aß and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer's and Parkinson's diseases.

A dopamine metabolite stabilizes neurotoxic amyloid-ß oligomers.

Aberrant soluble oligomers formed by the amyloid-ß peptide (Aß) are major pathogenic agents in the onset and progression of Alzheimer's disease. A variety of biomolecules can influence the formation of these oligomers in the brain, although their mechanisms of action are still largely unknown. Here, we studied the effects on Aß aggregation of DOPAL, a reactive catecholaldehyde intermediate of dopamine metabolism. We found that DOPAL is able to stabilize Aß oligomeric species, including dimers and trimers, that exert toxic effects on human neuroblastoma cells, in particular increasing cytosolic calcium levels and promoting the generation of reactive oxygen species. These results reveal an interplay between Aß aggregation and key biochemical processes regulating cellular homeostasis in the brain.

Environmental Control of Amyloid Polymorphism by Modulation of Hydrodynamic Stress.

The phenomenon of amyloid polymorphism is a key feature of protein aggregation. Unravelling this phenomenon is of great significance for understanding the underlying molecular mechanisms associated with neurodegenerative diseases and for the development of amyloid-based functional biomaterials. However, the understanding of the molecular origins and the physicochemical factors modulating amyloid polymorphs remains challenging. Herein, we demonstrate an association between amyloid polymorphism and environmental stress in solution, induced by an air/water interface in motion. Our results reveal that low-stress environments produce heterogeneous amyloid polymorphs, including twisted, helical, and rod-like fibrils, whereas high-stress conditions generate only homogeneous rod-like fibrils. Moreover, high environmental stress converts twisted fibrils into rod-like fibrils both in-pathway and after the completion of mature amyloid formation. These results enrich our understanding of the environmental origin of polymorphism of pathological amyloids and shed light on the potential of environmentally controlled fabrication of homogeneous amyloid biomaterials for biotechnological applications.

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.

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism.

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-ß (Aß) in Alzheimer's disease and α-synuclein (αS) in Parkinson's disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aß and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.

Rationally Designed Antibodies as Research Tools to Study the Structure-Toxicity Relationship of Amyloid-ß Oligomers.

Alzheimer's disease is associated with the aggregation of the amyloid-ß peptide (Aß), resulting in the deposition of amyloid plaques in brain tissue. Recent scrutiny of the mechanisms by which Aß aggregates induce neuronal dysfunction has highlighted the importance of the Aß oligomers of this protein fragment. Because of the transient and heterogeneous nature of these oligomers, however, it has been challenging to investigate the detailed mechanisms by which these species exert cytotoxicity. To address this problem, we demonstrate here the use of rationally designed single-domain antibodies (DesAbs) to characterize the structure-toxicity relationship of Aß oligomers. For this purpose, we use Zn2+-stabilized oligomers of the 40-residue form of Aß (Aß40) as models of brain Aß oligomers and two single-domain antibodies (DesAb18-24 and DesAb34-40), designed to bind to epitopes at residues 18-24 and 34-40 of Aß40, respectively. We found that the DesAbs induce a change in structure of the Zn2+-stabilized Aß40 oligomers, generating a simultaneous increase in their size and solvent-exposed hydrophobicity. We then observed that these increments in both the size and hydrophobicity of the oligomers neutralize each other in terms of their effects on cytotoxicity, as predicted by a recently proposed general structure-toxicity relationship, and observed experimentally. These results illustrate the use of the DesAbs as research tools to investigate the biophysical and cytotoxicity properties of Aß oligomers.

Rational design of a conformation-specific antibody for the quantification of Aß oligomers.

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.

Effects of sedimentation, microgravity, hydrodynamic mixing and air-water interface on α-synuclein amyloid formation.

The formation of amyloid fibrils is a characterizing feature of a range of protein misfolding diseases, including Parkinson's disease. The propensity of native proteins to form such amyloid fibril, both in vitro and in vivo, is highly sensitive to the surrounding environment, which can alter the aggregation kinetics and fibrillization mechanisms. Here, we investigate systematically the influence of several representative environmental stimuli on α-synuclein aggregation, including hydrodynamic mixing, the presence of an air-water interface and sedimentation. Our results show that hydrodynamic mixing and interfacial effects are critical in promoting several microscopic steps of α-synuclein aggregation and amyloid fibril formation. The presence of an air-water interface under agitation significantly promoted primary nucleation. Secondary processes were facilitated by hydrodynamic mixing, produced by 3D rotation and shaking either in the presence or in the absence of an air-water interface. Effects of sedimentation, as investigated in a microgravity incubator, of α-synuclein lead only to minor changes on the aggregation kinetics rates in comparison to static conditions. These results forward the understanding of α-synuclein fibrillization, paving the way for the development of high-throughput assays for the screening of pharmacological approaches targeting Parkinson's disease.

Infrared nanospectroscopic mapping of a single metaphase chromosome.

The integrity of the chromatin structure is essential to every process occurring within eukaryotic nuclei. However, there are no reliable tools to decipher the molecular composition of metaphase chromosomes. Here, we have applied infrared nanospectroscopy (AFM-IR) to demonstrate molecular difference between eu- and heterochromatin and generate infrared maps of single metaphase chromosomes revealing detailed information on their molecular composition, with nanometric lateral spatial resolution. AFM-IR coupled with principal component analysis has confirmed that chromosome areas containing euchromatin and heterochromatin are distinguishable based on differences in the degree of methylation. AFM-IR distribution of eu- and heterochromatin was compared to standard fluorescent staining. We demonstrate the ability of our methodology to locate spatially the presence of anticancer drug sites in metaphase chromosomes and cellular nuclei. We show that the anticancer 'rule breaker' platinum compound [Pt[N(p-HC6F4)CH2]2py2] preferentially binds to heterochromatin, forming localized discrete foci due to condensation of DNA interacting with the drug. Given the importance of DNA methylation in the development of nearly all types of cancer, there is potential for infrared nanospectroscopy to be used to detect gene expression/suppression sites in the whole genome and to become an early screening tool for malignancy.

Soluble aggregates present in cerebrospinal fluid change in size and mechanism of toxicity during Alzheimer's disease progression.

Soluble aggregates of amyloid-ß (Aß) have been associated with neuronal and synaptic loss in Alzheimer's disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we characterised soluble aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls using a range of sensitive biophysical methods. We used super-resolution imaging and atomic force microscopy to characterise the size and structure of the aggregates present in CSF and correlate this with their ability to permeabilise lipid membranes and induce an inflammatory response. We found that these aggregates are extremely heterogeneous and exist in a range of sizes, varying both structurally and in their mechanisms of toxicity during the disease progression. A higher proportion of small aggregates of Aß that can cause membrane permeabilization are found in MCI CSF; in established AD, a higher proportion of the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells, while there was no detectable change in aggregate concentration. These results show that large aggregates, some longer than 100 nm, are present in the CSF of AD patients and suggest that different neurotoxic mechanisms are prevalent at different stages of AD.

Different soluble aggregates of Aß42 can give rise to cellular toxicity through different mechanisms.

Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer's disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aß42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aß42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aß42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aß42 may contribute to the onset and progression of Alzheimer's disease.

Trodusquemine enhances Aß42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes.

Transient oligomeric species formed during the aggregation process of the 42-residue form of the amyloid-ß peptide (Aß42) are key pathogenic agents in Alzheimer's disease (AD). To investigate the relationship between Aß42 aggregation and its cytotoxicity and the influence of a potential drug on both phenomena, we have studied the effects of trodusquemine. This aminosterol enhances the rate of aggregation by promoting monomer-dependent secondary nucleation, but significantly reduces the toxicity of the resulting oligomers to neuroblastoma cells by inhibiting their binding to the cellular membranes. When administered to a C. elegans model of AD, we again observe an increase in aggregate formation alongside the suppression of Aß42-induced toxicity. In addition to oligomer displacement, the reduced toxicity could also point towards an increased rate of conversion of oligomers to less toxic fibrils. The ability of a small molecule to reduce the toxicity of oligomeric species represents a potential therapeutic strategy against AD.

Identification of Oxidative Stress in Red Blood Cells with Nanoscale Chemical Resolution by Infrared Nanospectroscopy.

During their lifespan, Red blood cells (RBC), due to their inability to self-replicate, undergo an ageing degradation phenomenon. This pathway, both in vitro and in vivo, consists of a series of chemical and morphological modifications, which include deviation from the biconcave cellular shape, oxidative stress, membrane peroxidation, lipid content decrease and uncoupling of the membrane-skeleton from the lipid bilayer. Here, we use the capabilities of atomic force microscopy based infrared nanospectroscopy (AFM-IR) to study and correlate, with nanoscale resolution, the morphological and chemical modifications that occur during the natural degradation of RBCs at the subcellular level. By using the tip of an AFM to detect the photothermal expansion of RBCs, it is possible to obtain nearly two orders of magnitude higher spatial resolution IR spectra, and absorbance images than can be obtained on diffraction-limited commercial Fourier-transform Infrared (FT-IR) microscopes. Using this approach, we demonstrate that we can identify localized sites of oxidative stress and membrane peroxidation on individual RBC, before the occurrence of neat morphological changes in the cellular shape.