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.

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.

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.

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.

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.

The Volumetric Diversity of Misfolded Prion Protein Oligomers Revealed by Pressure Dissociation.

Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct ß-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation of one specific PrP oligomer, we reveal a large negative activation volume and an increase in both apparent activation enthalpy and entropy. This suggests a transition state ensemble that is less structured and significantly more hydrated than the oligomeric state. Finally, we found that site-specific fluorescent labeling allows monitoring of the transient population of a kinetic intermediate in the dissociation reaction. Our results indicate that defects in atomic packing may deserve consideration as a new factor that influences differences between PrP assemblies and that could be relevant also for explaining the origin of prion strains.

Binding of methylene blue to a surface cleft inhibits the oligomerization and fibrillization of prion protein.

Neurodegenerative protein misfolding diseases, including prionopathies, share the common feature of accumulating specific misfolded proteins, with a molecular mechanism closely related. Misfolded prion protein (PrP) generates soluble oligomers that, in turn, aggregate into amyloid fibers. Preventing the formation of these entities, crucially associated with the neurotoxic and/or infectious properties of the resulting abnormal PrP, represents an attractive therapeutic strategy to ameliorate prionopathies. We focused our attention into methylene blue (MB), a well-characterized drug, which is under study against Alzheimer's disease and other neurodegenerative disorders. Here, we have undertaken an in vitro study on the effects of MB on oligomerization and fibrillization of human, ovine and murine PrP. We demonstrated that MB affects the kinetics of PrP oligomerization and reduces the amount of oligomer of about 30%, in a pH-dependent manner, by using SLS and DSC methodologies. Moreover, TEM images showed that MB completely suppresses fiber formation at a PrP:MB molar ratio of 1:2. Finally, NMR revealed a direct interaction between PrP and MB, which was mapped on a surface cleft including a fibrillogenic region of the protein. Our results allowed to surmise a mechanism of action in which the MB binding to PrP surface markedly interferes with the pathway towards oligomers and fibres. Therefore MB could be considered as a general anti-aggregation compound, acting against proteinopathies.

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.

Secondary Nucleation of Aß Revealed by Single-Molecule and Computational Approaches.

Understanding the mechanisms underlying amyloid-ß (Aß) aggregation is pivotal in the context of Alzheimer's disease. This study aims to elucidate the secondary nucleation process of Aß42 peptides by combining experimental and computational methods. Using a newly developed nanopipette-based amyloid seeding and translocation assay, confocal fluorescence spectroscopy, and molecular dynamics simulations, the influence of the seed properties on Aß aggregation is investigated. Both fragmented and unfragmented seeds played distinct roles in the formation of oligomers, with fragmented seeds facilitating the formation of larger aggregates early in the incubation phase. The results show that secondary nucleation leads to the formation of oligomers of various sizes and structures as well as larger fibrils structured in ß-sheets. From these findings a mechanism of secondary nucleation involving two types of aggregate populations, one released and one growing on the mother fiber is proposed.

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.

Oligomeric-induced activity by thienyl pyrimidine compounds traps prion infectivity.

Accumulation of PrP(Sc), an abnormal form of cellular prion protein (PrP), in the brain of animals and humans leads to fatal neurodegenerative disorders known as prion diseases. Limited protease digestion of PrP(Sc) produces a truncated form called PrP(27-30) that retains prion infectivity and is the main marker of disease targeted in most diagnostic tests. In the search for new anti-prion molecules, drug-screening assays on prion-infected murine cells have been oriented toward decreasing levels of PrP(27-30). In contrast, we screened for drugs promoting multimers of PrP(27-30), illustrating a possible stabilization of mouse PrP(Sc) species, because recent studies aiming to characterize the conformational stability of various prion strains showed that stable recombinant amyloids produced more stable prion strain, leading to longest incubation time. We identified a family of thienyl pyrimidine derivatives that induce SDS-resistant dimers and trimers of PrP(27-30). Bioassays performed on mice brain homogenates treated with these compounds showed that these thienyl pyrimidine derivatives diminished prion infectivity in vivo. Oligomeric-induced activity by thienyl pyrimidine compounds is a promising approach not only to understanding the pathogenesis of prions but also for prion diagnostics. This approach could be extended to other neurodegenerative "prionopathies," such as Alzheimer's, Huntington, or Parkinson's diseases.

Amyloid features and neuronal toxicity of mature prion fibrils are highly sensitive to high pressure.

Prion proteins (PrP) can aggregate into toxic and possibly infectious amyloid fibrils. This particular macrostructure confers on them an extreme and still unexplained stability. To provide mechanistic insights into this self-assembly process, we used high pressure as a thermodynamic tool for perturbing the structure of mature amyloid fibrils that were prepared from recombinant full-length mouse PrP. Application of high pressure led to irreversible loss of several specific amyloid features, such as thioflavin T and 8-anilino-1-naphthalene sulfonate binding, alteration of the characteristic proteinase K digestion pattern, and a significant decrease in the ß-sheet structure and cytotoxicity of amyloid fibrils. Partial disaggregation of the mature fibrils into monomeric soluble PrP was observed. The remaining amyloid fibrils underwent a change in secondary structure that led to morphologically different fibrils composed of a reduced number of proto-filaments. The kinetics of these reactions was studied by recording the pressure-induced dissociation of thioflavin T from the amyloid fibrils. Analysis of the pressure and temperature dependence of the relaxation rates revealed partly unstructured and hydrated kinetic transition states and highlighted the importance of collapsing and hydrating inter- and intramolecular cavities to overcome the high free energy barrier that stabilizes amyloid fibrils.

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.

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.

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.

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.

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.

Microcebus murinus retina: a new model to assess prion-related neurotoxicity in primates.

No effective treatment currently exists for prion diseases and therefore the development of experimental non-human primate models of prion neurotoxicity, to better understand the underlying mechanism and to test new treatments relevant to humans, represents an urgent medical need. However, the establishment of such models is challenging due to animal welfare and cost considerations. We describe here the use of Microcebus murinus retina, in primary cultures and in vivo, as a new experimental primate model to rapidly examine the effects in the central nervous system of PrP(106-126), a neurotoxic fragment of the human prion protein. We demonstrate that PrP(106-126) triggered rod photoreceptor cell loss by apoptosis and a change in morphology of microglial cells in mixed neuronal-glial cultures of retinal cells. In addition, 2days after intravitreal injection of PrP(106-126), retinas showed a significant increase in the number of apoptotic nuclei, mainly in the ganglion cell layer.