Abstract Background In neurodegenerative diseases, the diagnostic value of α-synuclein seed amplification assay (αSAA) in routine clinical practice and its relationship with cognitive performance and fluid biomarkers remains incompletely characterized. In addition, although Alzheimer disease (AD) CSF and blood biomarkers accurately detect amyloid and tau pathology, their ability to identify concomitant α-synuclein (αSyn) pathology remains unknown. Methods We included 398 patients from the multicenter memory clinic ALZAN cohort. All participants underwent CSF and blood sampling with measurement of CSF biomarkers (Aβ42/40, tau, p-tau181) and plasma biomarkers (Aβ42/40, p-tau181, p-tau217, GFAP, NfL). Cognitive assessment was performed using the Mini-Mental State Examination (MMSE). Clinical diagnoses were independently confirmed by two senior neurologists. αSyn status was determined by αSAA (RT-QuIC). Results Of 398 patients, 19/20 patients with Lewy body dementia (LBD) (95.0%) and 32/203 patients with AD (15.8%) were αSAA+. αSAA-positivity presented a sensitivity of 95.0% (95%CI, 75.1-99.9), a specificity of 93.1% (95%CI, 88.3-96.4) for distinguishing LBD from patients without LBD or AD. In the entire cohort, αSAA+ patients showed lower MMSE scores (p<0.01), lower CSF Aβ42/40 ratio (p<0.01), and elevated plasma GFAP (p<0.05). However, within the AD subgroup, only CSF Aβ42/40 differed marginally between αSAA+ and αSAA- patients, whereas CSF total tau, CSF p-tau181, plasma p-tau217, p-tau181, GFAP, and NfL showed no significant differences. Conclusions αSAA accurately detects αSyn pathology in routine memory clinic practice that is not captured by current AD CSF and blood biomarkers. These findings support αSAA as a complementary biomarker for routine diagnosis and patient stratification. What is already known on this topic α-synuclein seed amplification assay (αSAA) has shown high sensitivity and specificity for detecting Lewy body dementia (LBD) in research cohorts, and α-synuclein co-pathology is frequently observed in Alzheimer’s disease (AD). However, it remains unknown whether currently available AD CSF and blood biomarkers can identify patients with α-synuclein pathology in routine clinical practice. What this study adds In a prospective multicenter memory clinic cohort, αSAA showed high sensitivity (95.0%) and specificity (93.5%) for identifying clinically diagnosed LBD and detected αSyn co-pathology in 15.8% of AD patients. Current AD CSF and blood biomarkers did not discriminate between αSAA-positive and αSAA-negative patients, providing little information on concomitant αSyn pathology. How this study might affect research, practice or policy αSAA provides unique biological information by improving the detection of LBD and identifying AD patients with concomitant αSyn co-pathology that is not captured by current CSF and blood biomarkers, thereby improving patient stratification and informing future precision medicine approaches and therapeutic trials.

The accumulation of plastic waste in the environment, breaking down into micro- and nanoplastics, poses significant threats to ecosystem and human health. Plastic particles have been detected in human blood, urine, and placental tissue, indicating widespread exposure. While their long-term health impacts remain unclear, developing brains, especially in fetuses and children, may be vulnerable, potentially resulting in behavioral changes or neurodevelopmental disorders. This study explores the effects of chronic exposure to 23-nm polystyrene nanoplastics at 10 µg/day/kg in wild-type mice across life stages, using exposure levels reflective of human reality. Maternal exposure disrupted critical developmental milestones in pups. In adulthood, exposed mice exhibited Attention-Deficit Hyperactivity Disorder (ADHD)-like traits, including hyperactivity, increased risk-taking behaviors, and impaired motor learning and executive functions. In aging mice, exposure was associated with a lower epileptic threshold, with developing seizures. These behavioral changes were linked to altered gene and synaptic protein expression associated with ADHD and epilepsy. At the cellular level, lifelong nanoplastic exposure caused lysosomal dysfunctions and increased lipofuscin accumulation, indicative of accelerated brain aging. These findings align with the growing prevalence of ADHD and epilepsy in humans, particularly children and the elderly, emphasizing the urgent need to address plastic pollution and its health implications.

This study investigated the impact of pyrimethanil, a fungicide, on the aggregation of amyloid-β 42 (aβ42) peptides in vitro. The findings demonstrated that pyrimethanil accelerated aβ42 aggregation kinetics, as evidenced by thioflavin T (ThT) fluorescence assays in both tube and microplate experiments. A combination of single molecule techniques and molecular dynamics simulations is used to elucidate the complex effects of pyrimethanil on aβ42 aggregation mechanism. Nanopore experiments indicated that pyrimethanil promoted the formation of small oligomers (6-13.5 nm) during the lag phase, which were not detected under control conditions. Confocal fluorescence spectroscopy revealed that pyrimethanil induced the formation of larger β-sheet structured aggregates. In the presence of preformed seeds, pyrimethanil exhibited a dual role by fragmenting existing fibrils into smaller species and enhancing aggregation, likely through combined effects with the newly formed smaller seeds. Molecular dynamics simulations confirmed that pyrimethanil has a higher affinity for fibrils than monomers and weakens monomer-fibril interactions. Overall, this study elucidates the complex effects of pyrimethanil on aβ42 aggregation, involving promotion of primary nucleation, fibril fragmentation, and modulation of monomer-fibril interactions. These findings provide important mechanistic insights into how environmental factors like pesticides may influence amyloid aggregation processes relevant to Alzheimer's disease.

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.

Understanding the mechanisms underlying amyloid-β (Aβ) aggregation is pivotal in the context of Alzheimer's disease. This study aims to elucidate the secondary nucleation process of Aβ42 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 Aβ 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 β-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.

Mammalian prions are neurotropic pathogens formed from PrPSc assemblies, a misfolded variant of the host-encoded prion protein PrPC. Multiple PrPSc conformations or strains self-propagate in host populations or mouse models of prion diseases, exhibiting distinct biological and biochemical phenotypes. Constrained interactions between PrPSc and PrPC conformations confer species specificity and regulate cross-species transmission. The pathogenicity of fibrillar assemblies derived from bacterially expressed recombinant PrP (rPrP) has been instrumental in demonstrating the protein-only nature of prions. Yet, their ability to encode different strains and transmit between species remains poorly studied, hampering their use in exploring structure-to-strain relationships. Fibrillar assemblies from rPrP with hamster, mouse, human, and bovine primary structures were generated and tested for transmission and adaptation in tg7 transgenic mice expressing hamster PrPC. All assemblies, except the bovine ones, were fully pathogenic on the primary passage, causing clinical disease, PrPSc brain deposition, and spongiform degeneration. They exhibited divergent adaptation processes and strain properties upon subsequent passage. Assemblies of hamster origin propagated without apparent need for adaptation, those of mouse origin adapted abruptly, and those of human origin required serial passages for optimal fitness. Molecular analyses revealed the presence of endogenously truncated PrPSc species in the resulting synthetic strains that lack the 90-140 amino acid region considered crucial for infectivity. In conclusion, rPrP assemblies provide a facile means of generating novel prion strains with adaptative/evolutive properties mimicking genuine prions. The PrP amino acid backbone is sufficient to encode different strains with specific adaptative properties, offering insights into prion transmission and strain diversity.

In this work, early-stage Aβ42 aggregates were detected using a real-time fast amyloid seeding and translocation (RT-FAST) assay. Specifically, Aβ42 monomers were incubated in buffer solution with and without preformed Aβ42 seeds in a quartz nanopipette coated with L-DOPA. Then, formed Aβ42 aggregates were analyzed on flyby resistive pulse sensing at various incubation time points. Aβ42 aggregates were detected only in the sample with Aβ42 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 Aβ42 monomer adsorption on a quartz substrate after L-DOPA coating by confocal fluorescence spectroscopy and molecular dynamics simulation showed the huge influence of Aβ42 adsorption on the aggregation process.

The use of single track-etched nanopores to distinguish molecules is limited by the difficulty in comparing independent experiments. This is mainly because the properties (size, surface state) among various equivalent nanopores are not strictly identical. In this work, we propose a way to discriminate different sized gold nanoparticles coated with PEG from 38 to 90 nm using bullet-like shape nanopores with tip diameters of 100 and 130 +/- 30 nm, functionalized with polyethylene glycol (PEG) chains of different lengths (5 and 20 kDa). We show that the two classical parameters of current perturbations (the relative blockade amplitude and the dwell time) caused by the interaction of the nanoparticles with the nanopore allow the discrimination of the nanoparticles using each nanopore independently. Indeed, both the nanopore diameter and the PEG length influence the current blockade parameter, showing that it is possible to distinguish nanoparticles from independent experiments despite the inherent variability of the track-etched nanopores.

The pathogenicity of fibrillar assemblies derived from bacterially expressed recombinant prion protein (rPrP) has been key to the demonstration that prions are infectious proteins responsible for human and animal transmissible spongiform encephalopathies. Yet, their use in identifying which structural PrP features are important for prion biology, including strain properties and capacity to transmit between species, has been hampered by their limited transmissibility de novo. We report the generation of prions with distinct biological characteristics from rPrP assemblies differing only in their primary structure (hamster, mouse and human amino acid sequence). These rPrP assemblies were transmissible to transgenic mice expressing hamster PrP, causing a clinical disease at full attack rate, brain deposition of pathological prion protein PrP Sc and spongiform degeneration. Their adaptation process on serial sub-passaging seemed to depend, as for genuine prions, on the presence of a species/transmission barrier, due notably to PrP sequence mismatch. Remarkably, one of the strains obtained is an unprecedented shortened prion, lacking the 90-140 amino-acid region which is believed to be key to infectivity and structural stability of disease-associated PrP assemblies. Finally, we provide evidence that rPrP prionogenicity lies in the structural organization and/or heterogeneity of the rPrP assemblies. These preparations of rPrP offer unprecedented opportunities for meaningful studies correlating the dynamicity and structures of PrP Sc assemblies to prion pathobiology. Author summary Prions are infectious proteins, causing rapidly progressive neurodegenerative diseases in animals and humans. They are formed from the assisted-refolding and aggregation of the host-encoded prion protein (PrP). During the propagation of the disease, pathological PrP forces normal PrP to adopt its own conformation by a self-templating process. In infected host, different pathological structures of PrP or strains are found, causing diseases with specific biological phenotypes. Prions can also transmit between species. This capacity is limited by a species barrier, which critically depends on the infecting strain and PrP primary structure. How strain biological information is encoded in PrP structural fold remains unknown. We describe here the generation of different bona fide prion strains with markedly distinct adaptation capacities by transmission of refolded assemblies derived from bacterially-derived recombinant PrP (rPrP) of different species. We provide evidence that pathogenicity lies in the structural organization and/or heterogeneity of rPrP assemblies. Pathological PrP from one of the generated strains exhibited unique molecular features, including absence of domains that are thought to be key to prion infectivity, according to most recent ultrastructural studies. Our findings provide new insights for generating prion infectious material and resolving mechanisms of infectivity acquisition during PrP conversion process.

In this work, we aim to capture, detect and analysis at single molecule level Aβ42 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 Aβ42 aggregates using a quick current-voltage readout. The resulting single nanopore was then upscale to multipore membranes to capture the Aβ42 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 Aβ42 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.

The Aβ42 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 Aβ42- 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 Aβ42(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.

Self-assembly of the misfolded Aβ 1-42 peptide is involved in Alzheimer's disease (AD). The oligomeric species formed at an early stage of aggregation are considered as one of the main biomarkers. Nowadays, no tools are available to detect them due to their transient nature and their low concentration (fM) in biofluids such as the cerebro-spinal fluid (CSF). Therefore, our purpose is to propose an approach to detect these species directly in the CSF. Recently, we developed an innovative method to detect preformed α-synuclein oligomers called Real-Time Fast Amyloid Seeding and Translocation (RT-FAST)[1]. The principle is based on the properties of preformed oligomers to promote the aggregation of monomers providing large aggregates faster than the control condition (without oligomers). These newly formed aggregates are detected using a single nanopore by resistive pulse sensing. The latter consists to measure transient ionic current blockages caused by the passage of an aggregate. In this approach, we use a nanopipette as vial to accelerate aggregation (reservoir about 70 µL) and nanopore sensors. Experimentally, current blockades are observed after 120 minutes of aggregation only for the sample containing preformed Aβ 1-42 oligomers in buffer. Further analysis of the current blockade amplitudes allows estimating an aggregate volume in the order of 105 nm3 using a geometrical model. Interestingly, in presence of CSF same trend is observed. However, blockades are detected after 240 minutes of aggregation. One possible explanation could be that the molecules present in CSF slow down aggregation. In addition, CSF proteins decrease the adsorption constant of the Aβ known to accelerate aggregation.

The need for antibody proteins is growing for both diagnosis and therapy. In this work, we combine two strategies based on single nanopore sensing and multipore membrane to separate, detect and identify antibodies. This proof of concept was done for the anti-CD44. In a first step, a single nanopore with a bullet shape were designed using track-etching of polymer film and functionalized, with a PEG spacer and CD44 antigen. The detection of anti-CD44 was evidenced by an inversion of the current rectification. After demonstrating the ability to specifically detect the antiCD44 in a single nanopore, a multipore membrane with cylindrical nanopore was designed following the same protocol and used to elute a solution of anti-CD44.In a second step, the elution product was analysed using a single SiN nanopore showing that the anti-CD44 and CD44 antigen are extracted from the membrane. The analysis of the amplitude of the current blockade shows that the complex is dissociated.

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-β 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 Aβ(42)-E22Δ (Osaka mutant) that the addition of Aβ(42)-WT monomers increases the polymorphism of oligomers, while the incubation with Aβ(42)-WT fibrils generates larger aggregates.

The Aβ(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 Aβ(1-42) assembly and could influence the occurrence and pathophysiology of AD. However, pesticide involvement in the early stages of Aβ(1-42) aggregation is still unknown. Here, we employed conical track-etched nanopore in order to analyse the Aβ(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 Aβ(1-42). Aβ(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 Aβ(1-42) mechanism in two steps including the reorganization prior the elongation phase.

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.

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.

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-β 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-β 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.

Several neurodegenerative diseases have been linked to proteins or peptides that are prone to aggregate in different brain regions. Aggregation of amyloid-β (Aβ) peptides is recognized as the main cause of Alzheimer's disease (AD) progression, leading to the formation of toxic Aβ oligomers and amyloid fibrils. The molecular mechanism of Aβ aggregation is complex and still not fully understood. Nanopore technology provides a new way to obtain kinetic and morphological aspects of Aβ 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 Aβ fibril sensing and analysis. Various Aβ 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.

Single nanopore technology is a versatile tool allowing the sensing, study and discrimination of (bio)macromolecules. The principle of the detection consists to measure transient changes in the ionic current inside the pore caused by the passage of an analyte[1]. This technology is an emerging way to study single protein as well large protein aggregates (such amyloids)[2]. The study and the detection of amyloids is challenging due to their shape (rod-shape) and their transient nature in solution.