Aß oligomers peak in early stages of Alzheimer's disease preceding tau pathology.

INTRODUCTION: Soluble amyloid beta (Aß) oligomers have been suggested as initiating Aß related neuropathologic change in Alzheimer's disease (AD) but their quantitative distribution and chronological sequence within the AD continuum remain unclear. METHODS: A total of 526 participants in early clinical stages of AD and controls from a longitudinal cohort were neurobiologically classified for amyloid and tau pathology applying the AT(N) system. Aß and tau oligomers in the quantified cerebrospinal fluid (CSF) were measured using surface-based fluorescence intensity distribution analysis (sFIDA) technology. RESULTS: Across groups, highest Aß oligomer levels were found in A+ with subjective cognitive decline and mild cognitive impairment. Aß oligomers were significantly higher in A+T- compared to A-T- and A+T+. APOE Îµ4 allele carriers showed significantly higher Aß oligomer levels. No differences in tau oligomers were detected. DISCUSSION: The accumulation of Aß oligomers in the CSF peaks early within the AD continuum, preceding tau pathology. Disease-modifying treatments targeting Aß oligomers might have the highest therapeutic effect in these disease stages. Highlights: Using surface-based fluorescence intensity distribution analysis (sFIDA) technology, we quantified Aß oligomers in cerebrospinal fluid (CSF) samples of the DZNE-Longitudinal Cognitive Impairment and Dementia (DELCODE) cohortAß oligomers were significantly elevated in mild cognitive impairment (MCI)Amyloid-positive subjects in the subjective cognitive decline (SCD) group increased compared to the amyloid-negative control groupInterestingly, levels of Aß oligomers decrease at advanced stages of the disease (A+T+), which might be explained by altered clearing mechanisms.

Disrupted-in-schizophrenia 1 protein aggregates in cerebrospinal fluid are elevated in patients with first-episode psychosis.

AIM: The disrupted-in-schizophrenia 1 (DISC1) protein is a key regulator at the intersection of major signaling pathways relevant for adaptive behavior. It is prone to posttranslational changes such as misassembly and aggregation but the significance of such transformations for human mental illness has remained unclear. We aimed to demonstrate the occurrence of DISC1 protein aggregates in patients with first-episode psychosis (FEP). METHOD: Cerebrospinal fluid samples of patients with FEP (n = 50) and matched healthy controls (HCs; n = 47) were measured by the highly sensitive surface-based fluorescence intensity distribution analysis technology that enables single aggregate detection. RESULTS: We demonstrate that DISC1 protein aggregates are increased in cerebrospinal fluid samples of patients with FEP versus HCs. The concentration was in the low femtomolar range. No correlations were found with specific symptom levels, but the difference was particularly significant in the subset of patients with the diagnoses schizophrenia, unspecified (DSM-IV 295.9) or schizoaffective disorder (DSM-IV 295.70) at 18-month follow-up. DISC1 protein aggregate levels did not significantly change within the 18-month observation interval and were on average higher for individuals carrying the major DISC1 rs821577 allele, before correction. CONCLUSION: The occurrence of protein aggregates in vivo in patients with psychotic disorders has not been previously reported. It underscores the significance of posttranslational modifications of proteins both as pathogenetic mechanisms and as potential diagnostic markers in these disorders.

Oral treatment with the all-d-peptide RD2 enhances cognition in aged beagle dogs - A model of sporadic Alzheimer's disease.

Disease-modifying therapies to treat Alzheimer's disease (AD) are of fundamental interest for aging humans, societies, and health care systems. Predictable disease progression in transgenic AD models favors preclinical studies employing a preventive study design with an early pre-symptomatic treatment start, instead of assessing a truly curative approach with treatment starting after diagnosed disease onset. The aim of this study was to investigate the pharmacokinetic profile and efficacy of RD2 to enhance short-term memory and cognition in cognitively impaired aged Beagle dogs - a non-transgenic model of truly sporadic AD. RD2 has previously demonstrated pharmacodynamic efficacy in three different transgenic AD mouse models in three different laboratories. Here, we demonstrate that oral treatment with RD2 significantly reduced cognitive deficits in cognitively impaired aged Beagle dogs even beyond the treatment end, which suggests in combination with the treatment dependent CSF tau oligomer decrease a disease-modifying effect of RD2 treatment.

Tau protein aggregation associated with SARS-CoV-2 main protease.

The primary function of virus proteases is the proteolytic processing of the viral polyprotein. These enzymes can also cleave host cell proteins, which is important for viral pathogenicity, modulation of cellular processes, viral replication, the defeat of antiviral responses and modulation of the immune response. It is known that COVID-19 can influence multiple tissues or organs and that infection can damage the functionality of the brain in multiple ways. After COVID-19 infections, amyloid-ß, neurogranin, tau and phosphorylated tau were detected extracellularly, implicating possible neurodegenerative processes. The present study describes the possible induction of tau aggregation by the SARS-CoV-2 3CL protease (3CLpro) possibly relevant in neuropathology. Further investigations demonstrated that tau was proteolytically cleaved by the viral protease 3CL and, consequently, generated aggregates. However, more evidence is needed to confirm that COVID-19 is able to trigger neurodegenerative diseases.

Development and Implementation of an Internal Quality Control Sample to Standardize Oligomer-Based Diagnostics of Alzheimer's Disease.

Protein misfolding and aggregation are pathological hallmarks of various neurodegenerative diseases. In Alzheimer's disease (AD), soluble and toxic amyloid-ß (Aß) oligomers are biomarker candidates for diagnostics and drug development. However, accurate quantification of Aß oligomers in bodily fluids is challenging because extreme sensitivity and specificity are required. We previously introduced surface-based fluorescence intensity distribution analysis (sFIDA) with single-particle sensitivity. In this report, a preparation protocol for a synthetic Aß oligomer sample was developed. This sample was used for internal quality control (IQC) to improve standardization, quality assurance, and routine application of oligomer-based diagnostic methods. We established an aggregation protocol for Aß1-42, characterized the oligomers by atomic force microscopy (AFM), and assessed their application in sFIDA. Globular-shaped oligomers with a median size of 2.67 nm were detected by AFM, and sFIDA analysis of the Aß1-42 oligomers yielded a femtomolar detection limit with high assay selectivity and dilution linearity over 5 log units. Lastly, we implemented a Shewhart chart for monitoring IQC performance over time, which is another important step toward quality assurance of oligomer-based diagnostic methods.

Patients with isolated REM-sleep behavior disorder have elevated levels of alpha-synuclein aggregates in stool.

Misfolded and aggregated α-synuclein is a neuropathological hallmark of Parkinson's disease (PD). Thus, α-synuclein aggregates are regarded as a biomarker for the development of diagnostic assays. Quantification of α-synuclein aggregates in body fluids is challenging, and requires highly sensitive and specific assays. Recent studies suggest that α-synuclein aggregates may be shed into stool. We used surface-based fluorescence intensity distribution analysis (sFIDA) to detect and quantify single particles of α-synuclein aggregates in stool of 94 PD patients, 72 isolated rapid eye movement sleep behavior disorder (iRBD) patients, and 51 healthy controls. We measured significantly elevated concentrations of α-synuclein aggregates in stool of iRBD patients versus those of controls (p = 0.024) or PD patients (p < 0.001). Our results show that α-synuclein aggregates are excreted in stool and can be measured using the sFIDA assay, which could support the diagnosis of prodromal synucleinopathies.

Quantitative detection of α-Synuclein and Tau oligomers and other aggregates by digital single particle counting.

The pathological hallmark of neurodegenerative diseases is the formation of toxic oligomers by proteins such as alpha-synuclein (aSyn) or microtubule-associated protein tau (Tau). Consequently, such oligomers are promising biomarker candidates for diagnostics as well as drug development. However, measuring oligomers and other aggregates in human biofluids is still challenging as extreme sensitivity and specificity are required. We previously developed surface-based fluorescence intensity distribution analysis (sFIDA) featuring single-particle sensitivity and absolute specificity for aggregates. In this work, we measured aSyn and Tau aggregate concentrations of 237 cerebrospinal fluid (CSF) samples from five cohorts: Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and a neurologically-normal control group. aSyn aggregate concentration discriminates PD and DLB patients from normal controls (sensitivity 73%, specificity 65%, area under the receiver operating curve (AUC) 0.68). Tau aggregates were significantly elevated in PSP patients compared to all other groups (sensitivity 87%, specificity 70%, AUC 0.76). Further, we found a tight correlation between aSyn and Tau aggregate titers among all patient cohorts (Pearson coefficient of correlation r = 0.81). Our results demonstrate that aSyn and Tau aggregate concentrations measured by sFIDA differentiate neurodegenerative disease diagnostic groups. Moreover, sFIDA-based Tau aggregate measurements might be particularly useful in distinguishing PSP from other parkinsonisms. Finally, our findings suggest that sFIDA can improve pre-clinical and clinical studies by identifying those individuals that will most likely respond to compounds designed to eliminate specific oligomers or to prevent their formation.

Aß oligomer concentration in mouse and human brain and its drug-induced reduction ex vivo.

The elimination of amyloid beta (Aß) oligomers is a promising strategy for therapeutic drug development of Alzheimer's disease (AD). AD mouse models that develop Aß pathology have been used to demonstrate in vivo efficacy of compounds that later failed in clinical development. Here, we analyze the concentration and size distribution of Aß oligomers in different transgenic mouse models of AD and in human brain samples by surface-based fluorescence intensity distribution analysis (sFIDA), a highly sensitive method for detecting and quantitating protein aggregates. We demonstrate dose- and time-dependent oligomer elimination by the compound RD2 in mouse and human AD brain homogenates as sources of native Aß oligomers. Such ex vivo target engagement analyses with mouse- and human-brain-derived oligomers have the potential to enhance the translational value from pre-clinical proof-of-concept studies to clinical trials.

Aß Oligomer Elimination Restores Cognition in Transgenic Alzheimer's Mice with Full-blown Pathology.

Oligomers of the amyloid-ß (Aß) protein are suspected to be responsible for the development and progression of Alzheimer's disease. Thus, the development of compounds that are able to eliminate already formed toxic Aß oligomers is very desirable. Here, we describe the in vivo efficacy of the compound RD2, which was developed to directly and specifically eliminate toxic Aß oligomers. In a truly therapeutic, rather than a preventive study, oral treatment with RD2 was able to reverse cognitive deficits and significantly reduce Aß pathology in old-aged transgenic Alzheimer's Disease mice with full-blown pathology and behavioral deficits. For the first time, we demonstrate the in vivo target engagement of RD2 by showing a significant reduction of Aß oligomers in the brains of RD2-treated mice compared to placebo-treated mice. The correlation of Aß elimination in vivo and the reversal of cognitive deficits in old-aged transgenic mice support the hypothesis that Aß oligomers are relevant not only for disease development and progression, but also offer a promising target for the causal treatment of Alzheimer's disease.

Advancements of the sFIDA method for oligomer-based diagnostics of neurodegenerative diseases.

Early diagnosis of Alzheimer's disease (AD) is of great importance for the development of therapeutics and their application in the clinical environment. Amyloid ß (Aß) oligomers are crucial for the onset and progression of AD and represent a popular drug target, being presumably the most direct biomarker. Efforts to measure Aß oligomers in body fluids are hampered by the low analyte concentration and presence of Aß monomers. The surface-based fluorescence intensity distribution analysis (sFIDA) features both highly specific and sensitive oligomer quantitation as well as total insensitivity towards monomers. In this Review, we highlight structural features of oligomeric and fibrillar Aß. Recent advancements in sFIDA assay development have been the successful automation, adaption for additional biomarkers such as α-synuclein oligomers, and significant improvement of essential assay parameters.

Nanoparticle standards for immuno-based quantitation of α-synuclein oligomers in diagnostics of Parkinson's disease and other synucleinopathies.

Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by symptoms such as rigor, tremor and bradykinesia. A reliable and early diagnosis could improve the development of early therapeutic strategies before death of dopaminergic neurons leads to the first clinical symptoms. The sFIDA (surface-based fluorescence intensity distribution analysis) assay is a highly sensitive method to determine the concentration of α-synuclein (α-syn) oligomers which are presumably the major toxic isoform of α-syn and potentially the most direct biomarker for PD. Oligomer-based diagnostic tests require standard molecules that closely mimic the native oligomer. This is particularly important for calibration and assessment of inter-assay variation. In this study, we generated a standard in form of α-syn coated silica nanoparticles (α-syn-SiNaPs) that are in the size range of α-syn oligomers and provide a defined number of α-syn epitopes. The preparation of the sFIDA assay was realized on an automated platform to allow handling of high number of samples and reduce the effects of human error. The assay outcome was analyzed by determination of coefficient of variation and linearity for the applied α-syn-SiNaPs concentrations. Additionally, the limit of detection and lower limit of quantification were determined yielding concentrations in the lower femtomolar range.

Analysis of anticoagulants for blood-based quantitation of amyloid ß oligomers in the sFIDA assay.

Early diagnostics at the preclinical stage of Alzheimer's disease is of utmost importance for drug development in clinical trials and prognostic guidance. Since soluble Aß oligomers are considered to play a crucial role in the disease pathogenesis, several methods aim to quantify Aß oligomers in body fluids such as cerebrospinal fluid (CSF) and blood plasma. The highly specific and sensitive method surface-based fluorescence intensity distribution analysis (sFIDA) has successfully been established for oligomer quantitation in CSF samples. In our study, we explored the sFIDA method for quantitative measurements of synthetic Aß particles in blood plasma. For this purpose, EDTA-, citrate- and heparin-treated blood plasma samples from five individual donors were spiked with Aß coated silica nanoparticles (Aß-SiNaPs) and were applied to the sFIDA assay. Based on the assay parameters linearity, coefficient of variation and limit of detection, we found that EDTA plasma yields the most suitable parameter values for quantitation of Aß oligomers in sFIDA assay with a limit of detection of 16 fM.

sFIDA automation yields sub-femtomolar limit of detection for Aß aggregates in body fluids.

OBJECTIVES: Alzheimer's disease (AD) is a neurodegenerative disorder with yet non-existent therapeutic and limited diagnostic options. Reliable biomarker-based AD diagnostics are of utmost importance for the development and application of therapeutic substances. We have previously introduced a platform technology designated 'sFIDA' for the quantitation of amyloid ß peptide (Aß) aggregates as AD biomarker. In this study we implemented the sFIDA assay on an automated platform to enhance robustness and performance of the assay. DESIGN AND METHODS: In sFIDA (surface-based fluorescence intensity distribution analysis) Aß species are immobilized by a capture antibody to a glass surface. Aß aggregates are then multiply loaded with fluorescent antibodies and quantitated by high resolution fluorescence microscopy. As a model system for Aß aggregates, we used Aß-conjugated silica nanoparticles (Aß-SiNaPs) diluted in PBS buffer and cerebrospinal fluid, respectively. Automation of the assay was realized on a liquid handling system in combination with a microplate washer. RESULTS: The automation of the sFIDA assay results in improved intra-assay precision, linearity and sensitivity in comparison to the manual application, and achieved a limit of detection in the sub-femtomolar range. CONCLUSIONS: Automation improves the precision and sensitivity of the sFIDA assay, which is a prerequisite for high-throughput measurements and future application of the technology in routine AD diagnostics.

Biofunctionalized Silica Nanoparticles: Standards in Amyloid-ß Oligomer-Based Diagnosis of Alzheimer's Disease.

Amyloid-ß (Aß) oligomers represent a promising biomarker for the early diagnosis of Alzheimer's disease (AD). However, state-of-the-art methods for immunodetection of Aß oligomers in body fluids show a large variability and lack a reliable and stable standard that enables the reproducible quantitation of Aß oligomers. At present, the only available standard applied in these assays is based on a random aggregation process of synthetic Aß and has neither a defined size nor a known number of epitopes. In this report, we generated a highly stable standard in the size range of native Aß oligomers that exposes a defined number of epitopes. The standard consists of a silica nanoparticle (SiNaP), which is functionalized with Aß peptides on its surface (Aß-SiNaP). The different steps of Aß-SiNaP synthesis were followed by microscopic, spectroscopic and biochemical analyses. To investigate the performance of Aß-SiNaPs as an appropriate standard in Aß oligomer immunodetection, Aß-SiNaPs were diluted in cerebrospinal fluid and quantified down to a concentration of 10 fM in the sFIDA (surface-based fluorescence intensity distribution analysis) assay. This detection limit corresponds to an Aß concentration of 1.9 ng l-1 and lies in the sensitivity range of currently applied diagnostic tools based on Aß oligomer quantitation. Thus, we developed a highly stable and well-characterized standard for the application in Aß oligomer immunodetection assays that finally allows the reproducible quantitation of Aß oligomers down to single molecule level and provides a fundamental improvement for the worldwide standardization process of diagnostic methods in AD research.

Application of an Amyloid Beta Oligomer Standard in the sFIDA Assay.

Still, there is need for significant improvements in reliable and accurate diagnosis for Alzheimer's disease (AD) at early stages. It is widely accepted that changes in the concentration and conformation of amyloid-ß (Aß) appear several years before the onset of first symptoms of cognitive impairment in AD patients. Because Aß oligomers are possibly the major toxic species in AD, they are a promising biomarker candidate for the early diagnosis of the disease. To date, a variety of oligomer-specific assays have been developed, many of them ELISAs. Here, we demonstrate the sFIDA assay, a technology highly specific for Aß oligomers developed toward single particle sensitivity. By spiking stabilized Aß oligomers to buffer and to body fluids from control donors, we show that the sFIDA readout correlates with the applied concentration of stabilized oligomers diluted in buffer, cerebrospinal fluid (CSF), and blood plasma over several orders of magnitude. The lower limit of detection was calculated to be 22 fM of stabilized oligomers diluted in PBS, 18 fM in CSF, and 14 fM in blood plasma.

Contact between the ß1 and ß2 Segments of α-Synuclein that Inhibits Amyloid Formation.

Conversion of the intrinsically disordered protein α-synuclein (α-syn) into amyloid aggregates is a key process in Parkinson's disease. The sequence region 35-59 contains ß-strand segments ß1 and ß2 of α-syn amyloid fibril models and most disease-related mutations. ß1 and ß2 frequently engage in transient interactions in monomeric α-syn. The consequences of ß1-ß2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double-cysteine mutant α-synCC, with a disulfide linking ß1 and ß2, is aggregation-incompetent and inhibits aggregation and toxicity of wild-type α-syn. We show that α-syn delays the aggregation of amyloid-ß peptide and islet amyloid polypeptide involved in Alzheimer's disease and type 2 diabetes, an effect enhanced in the α-synCC mutant. Tertiary interactions in the ß1-ß2 region of α-syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach.

Single fibril growth kinetics of α-synuclein.

Neurodegenerative disorders associated with protein misfolding are fatal diseases that are caused by fibrillation of endogenous proteins such as α-synuclein (α-syn) in Parkinson's disease (PD) or amyloid-ß in Alzheimer's disease. Fibrils of α-syn are a major pathological hallmark of PD and certain aggregation intermediates are postulated to cause synaptic failure and cell death of dopaminergic neurons in the substantia nigra. For the development of therapeutic approaches, the mechanistic understanding of the fibrillation process is essential. Here we report real-time observation of α-syn fibril elongation on a glass surface, imaged by total internal reflection fluorescence microscopy using thioflavin T fluorescence. Fibrillation on the glass surface occurred in the same time frame and yielded fibrils of similar length as fibrillation in solution. Time-resolved imaging of fibrillation on a single fibril level indicated that α-syn fibril elongation follows a stop-and-go mechanism; that is, fibrils either extend at a homogenous growth rate or stop to grow for variable time intervals. The fibril growth kinetics were compatible with a model featuring two states, a growth state and a stop state, which were approximately isoenergetic and interconverted with rate constants of ~1.5×10(-4) s(-1). In the growth state, α-syn monomers were incorporated into the fibril with a rate constant of 8.6×10(3) M(-1) s(-1). Fibril elongation of α-syn is slow compared to other amyloidogenic proteins.

Seeded fibrillation as molecular basis of the species barrier in human prion diseases.

Prion diseases are transmissible spongiform encephalopathies in humans and animals, including scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer, and Creutzfeldt-Jakob disease (CJD) in humans. The hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP(C)) to its pathological isoform PrP(Sc), which is accompanied by PrP fibrillation. Transmission is not restricted within one species, but can also occur between species. In some cases a species barrier can be observed that results in limited or unsuccessful transmission. The mechanism behind interspecies transmissibility or species barriers is not completely understood. To analyse this process at a molecular level, we previously established an in vitro fibrillation assay, in which recombinant PrP (recPrP) as substrate can be specifically seeded by PrP(Sc) as seed. Seeding with purified components, with no additional cellular components, is a direct consequence of the "prion-protein-only" hypothesis. We therefore hypothesise, that the species barrier is based on the interaction of PrP(C) and PrP(Sc). Whereas in our earlier studies, the interspecies transmission in animal systems was analysed, the focus of this study lies on the transmission from animals to humans. We therefore combined seeds from species cattle, sheep and deer (BSE, scrapie, CWD) with human recPrP. Homologous seeding served as a control. Our results are consistent with epidemiology, other in vitro aggregation studies, and bioassays investigating the transmission between humans, cattle, sheep, and deer. In contrast to CJD and BSE seeds, which show a seeding activity we can demonstrate a species barrier for seeds from scrapie and CWD in vitro. We could show that the seeding activity and therewith the molecular interaction of PrP as substrate and PrP(Sc) as seed is sufficient to explain the phenomenon of species barriers. Therefore our data supports the hypothesis that CWD is not transmissible to humans.

The amyloid-ß oligomer count in cerebrospinal fluid is a biomarker for Alzheimer's disease.

Recent studies indicate that small amyloid-ß peptide (Aß) oligomers are the major toxic species responsible for development and progression of Alzheimer's disease (AD). Therefore, we suggest that the number of Aß oligomers in body fluids is the most direct and relevant biomarker for AD. Determination of the Aß oligomer content of cerebrospinal fluid (CSF) samples from 14 AD patients and 12 age-matched controls revealed a clear distinction between both groups. All samples of the control group showed homogenously low numbers of Aß oligomers, while the samples of the AD group exhibited significantly higher levels of Aß oligomers. The Aß oligomer numbers correlated with the patients' Mini-Mental State Examination scores. This indicates that the quantity of Aß oligomers in CSF reflects the severity of the disease and that Aß oligomers play a crucial role in AD pathology and in turn can be used as a diagnostic biomarker.

Detection of prion protein particles in blood plasma of scrapie infected sheep.

Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.