Characterizing Prion-Like Protein Aggregation: Emerging Nanopore-Based Approaches.

Prion-like protein aggregation is characteristic of numerous neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. This process involves the formation of aggregates ranging from small and potentially neurotoxic oligomers to highly structured self-propagating amyloid fibrils. Various approaches are used to study protein aggregation, but they do not always provide continuous information on the polymorphic, transient, and heterogeneous species formed. This review provides an updated state-of-the-art approach to the detection and characterization of a wide range of protein aggregates using nanopore technology. For each type of nanopore, biological, solid-state polymer, and nanopipette, discuss the main achievements for the detection of protein aggregates as well as the significant contributions to the understanding of protein aggregation and diagnostics.

Ultrasensitive Detection of Aß42 Seeds in Cerebrospinal Fluid with a Nanopipette-Based Real-Time Fast Amyloid Seeding and Translocation Assay.

In this work, early-stage Aß42 aggregates were detected using a real-time fast amyloid seeding and translocation (RT-FAST) assay. Specifically, Aß42 monomers were incubated in buffer solution with and without preformed Aß42 seeds in a quartz nanopipette coated with L-DOPA. Then, formed Aß42 aggregates were analyzed on flyby resistive pulse sensing at various incubation time points. Aß42 aggregates were detected only in the sample with Aß42 seeds after 180 min of incubation, giving an on/off readout of the presence of preformed seeds. Moreover, this RT-FAST assay could detect preformed seeds spiked in 4% cerebrospinal fluid/buffer solution. However, in this condition, the time to detect the first aggregates was increased. Analysis of Cy3-labeled Aß42 monomer adsorption on a quartz substrate after L-DOPA coating by confocal fluorescence spectroscopy and molecular dynamics simulation showed the huge influence of Aß42 adsorption on the aggregation process.

Aß42 fibril and non-fibril oligomers characterization using a nanopipette.

The Aß42 aggregates with different structures and morphology was investigated through a single molecule label-free technique. To this end, the quartz nanopipettes were functionalized with polyethylene glycol. The set of Aß42- epigallocatechin-3-gallate fibrils with length (from 85 nm to 250 nm) obtained by sonication was detected. The comparison of experimental and computed value of the amplitude of relative current blockade using a geometrical model show that for fibrils longer than 80 nm, the discriminating parameter is their diameter. Then, non-fibril oligomers obtain from Aß42(Osaka) aggregation at different time seed was investigated. The analysis of the amplitude of relative current blockade shows that detected oligomers are smaller than 30 nm regardless the aggregation time. In addition, the wide distributions of the dwell time suggests the polymorph character of the sample.

Combining iontronic, chromatography and nanopipette for Aß42 aggregates detection and separation.

In this work, we aim to capture, detect and analysis at single molecule level Aß42 aggregates. To this end, two strategies of track-etched nanopore membranes functionalization were investigated. The first one uses an aptamer and requires only three steps, whereas the second strategy uses Lecanemab antibodies and requires six steps. Out of the two presented strategies, the second one was found to be the most suitable to detect Aß42 aggregates using a quick current-voltage readout. The resulting single nanopore was then upscale to multipore membranes to capture the Aß42 aggregates before analysis through them through a single-molecule approach. By comparing the species present in the retentate and filtrate, we confirmed the membrane's affinity for the larger Aß42 aggregates present in the sample. We found that chromatographic membranes combined with an ionic diode for binary on/off readout are powerful tools for detecting rare biomarkers before single molecule analysis.

Investigation of α-Synuclein and Amyloid-ß(42)-E22Δ Oligomers Using SiN Nanopore Functionalized with L-Dopa.

Solid-state nanopores are an emerging technology used as a high-throughput, label-free analytical method for the characterization of protein aggregation in an aqueous solution. In this work, we used Levodopamine to coat a silicon nitride nanopore surface that was fabricated through a dielectric breakdown in order to reduce the unspecific adsorption. The coating of inner nanopore wall by investigation of the translocation of heparin. The functionalized nanopore was used to investigate the aggregation of amyloid-ß and α-synuclein, two biomarkers of degenerative diseases. In the first application, we demonstrate that the α-synuclein WT is more prone to form dimers than the variant A53T. In the second one, we show for the Aß(42)-E22Δ (Osaka mutant) that the addition of Aß(42)-WT monomers increases the polymorphism of oligomers, while the incubation with Aß(42)-WT fibrils generates larger aggregates.

Reg-1α, a New Substrate of Calpain-2 Depending on Its Glycosylation Status.

Reg-1α/lithostathine, a protein mainly associated with the digestive system, was previously shown to be overexpressed in the pre-clinical stages of Alzheimer's disease. In vitro, the glycosylated protein was reported to form fibrils at physiological pH following the proteolytic action of trypsin. However, the nature of the protease able to act in the central nervous system is unknown. In the present study, we showed that Reg-1α can be cleaved in vitro by calpain-2, the calcium activated neutral protease, overexpressed in neurodegenerative diseases. Using chemical crosslinking experiments, we found that the two proteins can interact with each other. Identification of the cleavage site using mass spectrometry, between Gln4 and Thr5, was found in agreement with the in silico prediction of the calpain cleavage site, in a position different from the one reported for trypsin, i.e., Arg11-Ile12 peptide bond. We showed that the cleavage was impeded by the presence of the neighboring glycosylation of Thr5. Moreover, in vitro studies using electron microscopy showed that calpain-cleaved protein does not form fibrils as observed after trypsin cleavage. Collectively, our results show that calpain-2 cleaves Reg-1α in vitro, and that this action is not associated with fibril formation.

Loss of glucocorticoid receptor phosphorylation contributes to cognitive and neurocentric damages of the amyloid-ß pathway.

Aberrant cortisol and activation of the glucocorticoid receptor (GR) play an essential role in age-related progression of Alzheimer's disease (AD). However, the GR pathways required for influencing the pathobiology of AD dementia remain unknown. To address this, we studied an early phase of AD-like progression in the well-established APP/PS1 mouse model combined with targeted mutations in the BDNF-dependent GR phosphorylation sites (serines 134/267) using molecular, behavioral and neuroimaging approaches. We found that disrupting GR phosphorylation (S134A/S267A) in mice exacerbated the deleterious effects of the APP/PS1 genotype on mortality, neuroplasticity and cognition, without affecting either amyloid-ß deposition or vascular pathology. The dynamics, maturation and retention of task-induced new dendritic spines of cortical excitatory neurons required GR phosphorylation at the BDNF-dependent sites that amyloid-ß compromised. Parallel studies in postmortem human prefrontal cortex revealed AD subjects had downregulated BDNF signaling and concomitant upregulated cortisol pathway activation, which correlated with cognitive decline. These results provide key evidence that the loss of neurotrophin-mediated GR phosphorylation pathway promotes the detrimental effects of the brain cortisol response that contributes to the onset and/or progression of AD dementia. These findings have important translational implications as they provide a novel approach to treating AD dementia by identifying drugs that increase GR phosphorylation selectively at the neurotrophic sites to improve memory and cognition.

Real-Time Fast Amyloid Seeding and Translocation of α-Synuclein with a Nanopipette.

The detection to α-synuclein (αS) assemblies as a biomarker of synucleinopathies is an important challenge for further development of an early diagnosis tool. Here, we present proof of concept real-time fast amyloid seeding and translocation (RT-FAST) based on a nanopipette that combines in one unique system a reaction vessel to accelerate the seed amplification and nanopore sensor for single-molecule αS assembly detection. RT-FAST allows the detection of the presence αS seeds WT and A53T variant in a given sample in only 90 min by adding a low quantity (35 µL at 100 nM) of recombinant αS for amplification. It also shows cross-seeding aggregation by adding mixing seeds A53T with WT monomers. Finally, we establish the dependence between the capture rate of aggregates by the nanopore sensor and the initial seed concentration from 200 pM to 2 pM, which promises further development toward a quantitative analysis of the initial seed concentration.

Conical nanopores highlight the pro-aggregating effects of pyrimethanil fungicide on Aß(1-42) peptides and dimeric splitting phenomena.

The Aß(1-42) aggregation is a key event in the physiopathology of Alzheimer's disease (AD). Exogenous factors such as environmental pollutants, and more particularly pesticides, can corrupt Aß(1-42) assembly and could influence the occurrence and pathophysiology of AD. However, pesticide involvement in the early stages of Aß(1-42) aggregation is still unknown. Here, we employed conical track-etched nanopore in order to analyse the Aß(1-42) fibril formation in the presence of pyrimethanil, a widely used fungicide belonging to the anilinopyrimidine class. Our results evidenced a pro-aggregating effect of pyrimethanil on Aß(1-42). Aß(1-42) assemblies were successfully detected using conical nanopore coated with PEG. Using an analytical model, the large current blockades observed (>0.7) were assigned to species with size close to the sensing pore. The long dwell times (hundreds ms scale) were interpreted by the possible interactions amyloid/PEG using molecular dynamic simulation. Such interaction could leave until splitting phenomena of the dimer structure. Our work also evidences that the pyrimethanil induce an aggregation of Aß(1-42) mechanism in two steps including the reorganization prior the elongation phase.

Solid-state and polymer nanopores for protein sensing: A review.

In two decades, the solid state and polymer nanopores became attractive method for the protein sensing with high specificity and sensitivity. They also allow the characterization of conformational changes, unfolding, assembly and aggregation as well the following of enzymatic reaction. This review aims to provide an overview of the protein sensing regarding the technique of detection: the resistive pulse and ionic diodes. For each strategy, we report the most significant achievement regarding the detection of peptides and protein as well as the conformational change, protein-protein assembly and aggregation process. We discuss the limitations and the recent strategies to improve the nanopore resolution and accuracy. A focus is done about concomitant problematic such as protein adsorption and nanopore lifetime.

Detection of Amyloid-ß Fibrils Using Track-Etched Nanopores: Effect of Geometry and Crowding.

Several neurodegenerative diseases have been linked to proteins or peptides that are prone to aggregate in different brain regions. Aggregation of amyloid-ß (Aß) peptides is recognized as the main cause of Alzheimer's disease (AD) progression, leading to the formation of toxic Aß oligomers and amyloid fibrils. The molecular mechanism of Aß aggregation is complex and still not fully understood. Nanopore technology provides a new way to obtain kinetic and morphological aspects of Aß aggregation at a single-molecule scale without labeling by detecting the electrochemical signal of the peptides when they pass through the hole. Here, we investigate the influence of nanoscale geometry (conical and bullet-like shape) of a track-etched nanopore pore and the effect of molecular crowding (polyethylene glycol-functionalized pores) on Aß fibril sensing and analysis. Various Aß fibril samples that differed by their length were produced by sonication of fibrils obtained in the presence of epigallocatechin gallate. The conical nanopore functionalized with polyethylene glycol (PEG) 5 kDa is suitable for discrimination of the fibril size from relative current blockade. The bullet-like-shaped nanopore enhances the amplitude of the current and increases the dwell time, allowing us to well discern the fibrils. Finally, the nanopore crowded with PEG 20 kDa enhances the relative current blockade and increases the dwell time; however, the discrimination is not improved compared to the "bullet-shaped" nanopore.

Deconstructing Alzheimer's Disease: How to Bridge the Gap between Experimental Models and the Human Pathology?

Discovered more than a century ago, Alzheimer's disease (AD) is not only still present in our societies but has also become the most common dementia, with 50 million people worldwide affected by the disease. This number is expected to double in the next generation, and no cure is currently available to slow down or stop the disease progression. Recently, some advances were made due to the approval of the aducanumab treatment by the American Food and Drug Administration. The etiology of this human-specific disease remains poorly understood, and the mechanisms of its development have not been completely clarified. Several hypotheses concerning the molecular mechanisms of AD have been proposed, but the existing studies focus primarily on the two main markers of the disease: the amyloid ß peptides, whose aggregation in the brain generates amyloid plaques, and the abnormally phosphorylated tau proteins, which are responsible for neurofibrillary tangles. These protein aggregates induce neuroinflammation and neurodegeneration, which, in turn, lead to cognitive and behavioral deficits. The challenge is, therefore, to create models that best reproduce this pathology. This review aims at gathering the different existing AD models developed in vitro, in cellulo, and in vivo. Many models have already been set up, but it is necessary to identify the most relevant ones for our investigations. The purpose of the review is to help researchers to identify the most pertinent disease models, from the most often used to the most recently generated and from simple to complex, explaining their specificities and giving concrete examples.

Prenatal exposure to low doses of fungicides corrupts neurogenesis in neonates.

Neurogenesis plays a crucial role during neurodevelopment and its dysfunction can lead to neurodevelopmental disorders. A recent hypothesis stipulates that exogenous factors could corrupt this process and predispose to neurodegenerative disorders later in life. The presence of pesticide residues in the diet represents a threat of which we have recently become aware of. Indeed, they could corrupt neurogenesis, especially during gestation, potentially leading to impaired neuronal and synaptic functions. Since the effects of this low-noise contamination have not yet been evaluated on the neurodevelopment, we investigated the impact of fungicide residues on WT mice exposed throughout gestation. Thus, mice were exposed to fungicides, cyprodinil, mepanipyrim and pyrimethanil, alone at 0.1 µg/L during gestation until P3. Besides, another group was exposed to a cocktail of these three fungicides (0.1 µg/L each) for the same time. Exposure was performed through drinking water at the regulatory limit dose of the European countries (0.1 µg/L). No general toxicity was observed in neonates on body and brain weight upon fungicide exposure. However, results showed that gestational exposure to fungicide residues substantially promoted an increase of neural precursor cells at P3. This corrupted neurogenesis was linked to increased levels of ß-catenin, likely through the crosstalk of the PI3K/Akt and Wnt/ß-catenin pathways, both involved in cell proliferation. Fungicide exposure also altered protein expression of PSD95 and NMDA receptors in P3 neonates, two targets of the ß-catenin signaling pathway. Adult neural stem cell extractions from mice treated with the fungicide cocktail, showed an increase proliferation and differentiation combined with a reduction of their migration properties. In addition, in vitro studies on hippocampal primary cell cultures treated with various concentrations of fungicides showed neurotoxic effects. To conclude, corruption of neurogenesis by this chemical assault could be a fertile ground for the development of neurological diseases later in life.

Machine Learning to Improve the Sensing of Biomolecules by Conical Track-Etched Nanopore.

Single nanopore is a powerful platform to detect, discriminate and identify biomacromolecules. Among the different devices, the conical nanopores obtained by the track-etched technique on a polymer film are stable and easy to functionalize. However, these advantages are hampered by their high aspect ratio that avoids the discrimination of similar samples. Using machine learning, we demonstrate an improved resolution so that it can identify short single- and double-stranded DNA (10- and 40-mers). We have characterized each current blockade event by the relative intensity, dwell time, surface area and both the right and left slope. We show an overlap of the relative current blockade amplitudes and dwell time distributions that prevents their identification. We define the different parameters that characterize the events as features and the type of DNA sample as the target. By applying support-vector machines to discriminate each sample, we show accuracy between 50% and 72% by using two features that distinctly classify the data points. Finally, we achieved an increased accuracy (up to 82%) when five features were implemented.

Glucocorticoid receptors signaling impairment potentiates amyloid-ß oligomers-induced pathology in an acute model of Alzheimer's disease.

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis occurs early in Alzheimer's disease (AD), associated with elevated circulating glucocorticoids (GC) and glucocorticoid receptors (GR) signaling impairment. However, the precise role of GR in the pathophysiology of AD remains unclear. Using an acute model of AD induced by the intracerebroventricular injection of amyloid-ß oligomers (oAß), we analyzed cellular and behavioral hallmarks of AD, GR signaling pathways, processing of amyloid precursor protein, and enzymes involved in Tau phosphorylation. We focused on the prefrontal cortex (PFC), particularly rich in GR, early altered in AD and involved in HPA axis control and cognitive functions. We found that oAß impaired cognitive and emotional behaviors, increased plasma GC levels, synaptic deficits, apoptosis and neuroinflammatory processes. Moreover, oAß potentiated the amyloidogenic pathway and enzymes involved both in Tau hyperphosphorylation and GR activation. Treatment with a selective GR modulator (sGRm) normalized plasma GC levels and all behavioral and biochemical parameters analyzed. GR seems to occupy a central position in the pathophysiology of AD. Deregulation of the HPA axis and a feed-forward effect on PFC GR sensitivity could participate in the etiology of AD, in perturbing Aß and Tau homeostasis. These results also reinforce the therapeutic potential of sGRm in AD.

Fungicide Residues Exposure and ß-amyloid Aggregation in a Mouse Model of Alzheimer's Disease.

BACKGROUND: Pesticide residues have contaminated our environment and nutrition over the last century. Although these compounds are present at very low concentrations, their long-term effects on human health is of concern. The link between pesticide residues and Alzheimer's disease is not clear and difficult to establish. To date, no in vivo experiments have yet modeled the impact of this chronic contamination on neurodegenerative disorders. OBJECTIVES: We investigated the impact of fungicide residues on the pathological markers of Alzheimer's disease in a transgenic mouse model. METHODS: Transgenic (J20, hAPPSw/Ind) mice were chronically exposed to a cocktail of residues of cyprodinil, mepanipyrim, and pyrimethanil at 0.1µg/L in their drinking water for 9 months. We assessed the effects of fungicide residues on the pathological markers of the disease including Aß aggregates, neuroinflammation, and neuronal loss. Then, we studied the dynamics of Aß aggregation in vivo via a longitudinal study using two-photon microscopy. Finally, we investigated the molecular mechanisms involved in the production and clearance of Aß peptides. RESULTS: We found that a chronic exposure to three fungicide residues exacerbated aggregation, microgliosis, and neuronal loss. These fungicides also increased vascular amyloid aggregates reminiscent of cerebral amyloid angiopathy between 6 and 9 months of treatment. The mechanism of action revealed that fungicides promoted Aß peptide fibril formation in vitro and involved an in vivo overexpression of the levels of the ß-secretase-cleaving enzyme (BACE1) combined with impairment of Aß clearance through neprylisin (NEP). CONCLUSIONS: Chronic exposure of the J20 mouse model of Alzheimer's disease to a cocktail of fungicides, at the regulatory concentration allowed in tap water (0.1µg/L), strengthened the preexisting pathological markers: neuroinflammation, Aß aggregation, and APP ß-processing. We hypothesize prevention strategies toward pesticide long-term exposure may be an alternative to counterbalance the lack of treatment and to slow down the worldwide Alzheimer's epidemic. https://doi.org/10.1289/EHP5550.

High-Pressure Response of Amyloid Folds.

The abnormal protein aggregates in progressive neurodegenerative disorders, such as Alzheimer's, Parkinson's and prion diseases, adopt a generic structural form called amyloid fibrils. The precise amyloid fold can differ between patients and these differences are related to distinct neuropathological phenotypes of the diseases. A key focus in current research is the molecular mechanism governing such structural diversity, known as amyloid polymorphism. In this review, we focus on our recent work on recombinant prion protein (recPrP) and the use of pressure as a variable for perturbing protein structure. We suggest that the amyloid polymorphism is based on volumetric features. Accordingly, pressure is the thermodynamic parameter that fits best to exploit volume differences within the states of a chemical reaction, since it shifts the equilibrium constant to the state that has the smaller volume. In this context, there are analogies with the process of correct protein folding, the high pressure-induced effects of which have been studied for more than a century and which provides a valuable source of inspiration. We present a short overview of this background and review our recent results regarding the folding, misfolding, and aggregation-disaggregation of recPrP under pressure. We present preliminary experiments aimed at identifying how prion protein fibril diversity is related to the quaternary structure by using pressure and varying protein sequences. Finally, we consider outstanding questions and testable mechanistic hypotheses regarding the multiplicity of states in the amyloid fold.

Pressure Reveals Unique Conformational Features in Prion Protein Fibril Diversity.

The prion protein (PrP) misfolds and assembles into a wide spectrum of self-propagating quaternary structures, designated PrPSc. These various PrP superstructures can be functionally different, conferring clinically distinctive symptomatology, neuropathology and infectious character to the associated prion diseases. However, a satisfying molecular basis of PrP structural diversity is lacking in the literature. To provide mechanistic insights into the etiology of PrP polymorphism, we have engineered a set of 6 variants of the human protein and obtained PrP amyloid fibrils. We show that pressure induces dissociation of the fibrils, albeit with different kinetics. In addition, by focusing on the generic properties of amyloid fibrils, such as the thioflavin T binding capacities and the PK-resistance, we reveal an unprecedented structure-barostability phenomenological relationship. We propose that the structural diversity of PrP fibrils encompass a multiplicity of packing defects (water-excluded cavities) in their hydrophobic cores, and that the resultant sensitivity to pressure should be considered as a general molecular criterion to accurately define fibril morphotypes. We anticipate that our insights into sequence-dependent fibrillation and conformational stability will shed light on the highly-nuanced prion strain phenomenon and open the opportunity to explain different PrP conformations in terms of volumetric physics.

Low doses of bioherbicide favour prion aggregation and propagation in vivo.

Public concerns over the use of synthetic pesticides are growing since many studies have shown their impact on human health. A new environmental movement in occidental countries promoting an organic agriculture favours the rebirth of botanical pesticides. These products confer an effective alternative to chemical pesticides such as glyphosate. Among the biopesticides, the α-terthienyls found in the roots of Tagetes species, are powerful broad-spectrum pesticides. We found that an α-terthienyl analogue with herbicidal properties, called A6, triggers resistant SDS oligomers of the pathogenic prion protein PrPSc (rSDS-PrPSc) in cells. Our main question is to determine if we can induce those rSDS-PrPSc oligomers in vitro and in vivo, and their impact on prion aggregation and propagation. Using wild-type mice challenged with prions, we showed that A6 accelerates or slows down prion disease depending on the concentration used. At 5 mg/kg, A6 is worsening the pathology with a faster accumulation of PrPSc, reminiscent to soluble toxic rSDS-PrPSc oligomers. In contrast, at 10 and 20 mg/kg of A6, prion disease occurred later, with less PrPSc deposits and with rSDS-PrPSc oligomers in the brain reminiscent to non-toxic aggregates. Our results are bringing new openings regarding the impact of biopesticides in prion and prion-like diseases.

A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases.

BACKGROUND: Prion diseases are characterized by the accumulation in the central nervous system of an abnormally folded isoform of the prion protein, named PrP(Sc). Aggregation of PrP(Sc) into oligomers and fibrils is critically involved in the pathogenesis of prion diseases. Oligomers are supposed to be the key neurotoxic agents in prion disease, so modulation of prion aggregation pathways with small molecules can be a valuable strategy for studying prion pathogenicity and for developing new diagnostic and therapeutic approaches. We previously identified thienyl pyrimidine compounds that induce SDS-resistant PrP(Sc) (rSDS-PrP(Sc)) oligomers in prion-infected samples. RESULTS: Due to the low effective doses of the thienyl pyrimidine hits, we synthesized a quaterthiophene-bis-triazine compound, called MR100 to better evaluate their diagnostic and therapeutic potentials. This molecule exhibits a powerful activity inducing rSDS-PrP(Sc) oligomers at nanomolar concentrations in prion-infected cells. Fluorescence interaction studies of MR100 with mouse PrP fibrils showed substantial modification of the spectrum, and the interaction was confirmed in vitro by production of rSDS-oligomer species upon incubation of MR100 with fibrils in SDS-PAGE gel. We further explored whether MR100 compound has a potential to be used in the diagnosis of prion diseases. Our results showed that: (i) MR100 can detect rSDS-oligomers in prion-infected brain homogenates of various species, including human samples from CJD patients; (ii) A protocol, called "Rapid Centrifugation Assay" (RCA), was developed based on MR100 property of inducing rSDS-PrP(Sc) oligomers only in prion-infected samples, and avoiding the protease digestion step. RCA allows the detection of both PK-sensitive and PK-resistant PrP(Sc) species in rodents samples but also from patients with different CJD forms (sporadic and new variant); (iii) A correlation could be established between the amount of rSDS-PrP(Sc) oligomers revealed by MR100 and the duration of the symptomatic phase of the disease in CJD patients; and (iv) Bioassay experiments showed that MR100 can trap prion infectivity more efficiently than P30 drug. CONCLUSIONS: MR100 is a powerful tool not only for studying the prion aggregation pathways regarding oligomeric and sPrP(Sc) species, but also for developing alternative methods for the detection of prion-infected samples. Considering our bioassay results, MR100 is a promising molecule for the development of prion decontamination approaches.