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Seeding activities of disease-associated α-synuclein aggregates (αSyn D ), a hallmark of Parkinson's disease (PD), are detectable by seed amplification assay (αSyn-SAA) and being developed as a diagnostic biomarker for PD. Sensitive and accurate αSyn-SAA for blood or saliva would greatly facilitate PD diagnosis. This prospective diagnostic study conducted αSyn-SAA analyses on serum and saliva samples collected from patients clinically diagnosed with PD or healthy controls (HC). 124 subjects (82 PD, 42 HC) donated blood and had extensive clinical assessments, of whom 74 subjects (48 PD, 26 HC) also donated saliva at the same visits. An additional 57 subjects (35 PD, 22 HC) donated saliva and had more limited clinical assessments. The mean ages were 69.21, 66.55, 69.58, and 64.71 years for PD serum donors, HC serum donors, PD saliva donors, and HC saliva donors, respectively. αSyn D seeding activities in either sample type alone or both sample types together were evaluated for PD diagnosis. Serum αSyn-SAA data from 124 subjects showed 80.49% sensitivity, 90.48% specificity, and 0.9006 accuracy (AUC of ROC); saliva αSyn-SAA data from 131 subjects attained 74.70% sensitivity, 97.92% specificity, and 0.8966 accuracy. Remarkably, the combined serum and saliva αSyn-SAA from 74 subjects with both sample types achieved better diagnostic performance: 95.83% sensitivity, 96.15% specificity, and 0.98 accuracy. In addition, serum αSyn D seeding activities correlated inversely with Montreal Cognitive Assessment in males and positively with Hamilton Depression Rating Scale in females and in the < 70 age group, whereas saliva αSyn D seeding activities correlated inversely with age at diagnosis in males and in the < 70 age group. Our data indicate that serum and saliva αSyn-SAA together can achieve high diagnostic accuracy for PD comparable to that of CSF αSyn-SAA, suggesting their potential utility for highly sensitive, accurate, and minimally invasive diagnosis of PD in routine clinical practice and clinical studies., (© 2024. The Author(s).)

α-Synuclein (αSyn) aggregates, detected in the biofluids of patients with Parkinson's disease (PD), have the ability to catalyze their own aggregation, leading to an increase in the number and size of aggregates. This self-templated amplification is used by newly developed assays to diagnose Parkinson's disease and turns the presence of αSyn aggregates into a biomarker of the disease. It has become evident that αSyn can form fibrils with slightly different structures, called "strains" or polymorphs, but little is known about their differential reactivity in diagnostic assays. Here, we compared the properties of two well-described αSyn polymorphs. Using single-molecule techniques, we observed that one of the polymorphs had an increased tendency to undergo secondary nucleation and we showed that this could explain the differences in reactivity observed in in vitro seed amplification assay and cellular assays. Simulations and high-resolution microscopy suggest that a 100-fold difference in the apparent rate of growth can be generated by a surprisingly low number of secondary nucleation "points" (1 every 2000 monomers added by elongation). When both strains are present in the same seeded reaction, secondary nucleation displaces proportions dramatically and causes a single strain to dominate the reaction as the major end product.

Background: Several studies have indicated that skin holds promise as a potential sample for detecting pathological α-Syn and serving as a diagnostic biomarker for α-synucleinopathies. Despite reports in Chinese PD patients, comprehensive research on skin α-Syn detection using RT-QuIC is lacking., Objective: This study aimed to evaluate the diagnostic performance of skin samples using RT-QuIC from PD patients in the Chinese population., Methods: Patients with sporadic PD and controls were included according to the British PD Association Brain Bank diagnostic criteria. The seeding activity of misfolded α-Syn in these skin samples was detected using the RT-QuIC assay after protein extraction. Biochemical and morphological analyses of RT-QuIC products were conducted by atomic force microscopy, transmission electron microscopy, Congo red staining, and dot blot analysis., Result: 30 patients clinically diagnosed with PD and 28 controls with non-α-synucleinopathies were included in this study. 28 of 30 PD patients demonstrated positive α-Syn seeding activity by RT-QuIC assay. In contrast, no α-Syn seeding activity was detected in the 28 control samples, with an overall sensitivity and specificity of 93.3% and 100%, respectively (P < 0.001). Biochemical characterization of the RT-QuIC product indicated fibrillary α-Syn species in PD-seeded reactions, while control samples failed in the conversion of recombinant α-Syn substrate., Conclusion: This study applied RT-QuIC technology to identify misfolded α-Syn seeding activity in skin samples from Chinese PD patients, demonstrating high specificity and sensitivity. Skin α-Syn RT-QuIC is expected to be a reliable approach for the diagnosis of PD., (© 2024. The Author(s).)

The quantification of α-synuclein aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinson's disease. However, multiple species, such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein; these species are likely to coexist in biological samples, and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify individual contributions to the amplification process, recombinant oligomers and preformed fibrils were produced and purified to characterize their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent, but our results reveal that their seeding behavior is very different compared to that of preformed fibrils, in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples is likely to dominate the response in seeding assays.

Introduction: α-syn aggregates represent the pathological hallmark of synucleinopathies as well as a frequent copathology (almost 1/3 of cases) in AD. Recent research indicates a potential role of α-syn species, measured in CSF with conventional analytical techniques, in the differential diagnosis between AD and synucleinopathies (such as DLB). Pioneering studies report the detection of α-syn in blood, however, conclusive investigations are controversial. Ultrasensitive seed amplification techniques, enabling the selective quantification of α-syn seeds, may represent an effective solution to identify the α-syn component in AD and facilitate a biomarker-guided stratification., Areas Covered: We performed a PubMed-based review of the latest findings on α-syn-related biomarkers for AD, focusing on bodily fluids. A dissertation on the role of ultrasensitive seed amplification assays, detecting α-syn seeds from different biological samples, was conducted., Expert Opinion: α-syn may contribute to progressive AD neurodegeneration through cross-seeding especially with tau protein. Ultrasensitive seed amplification techniques may support a biomarker-drug co-development pathway and may be a pathophysiological candidate biomarker for the evolving ATX(N) system to classify AD and the spectrum of primary NDDs. This would contribute to a precise approach to AD, aimed at implementing disease-modifying treatments.

Parkinson's disease (PD) and multiple system atrophy (MSA) are caused by two distinct strains of disease-associated α-synuclein (αSyn D ). Recently, we have shown that olfactory mucosa (OM) samples of patients with PD and MSA can seed the aggregation of recombinant α-synuclein by means of Real-Time Quaking-Induced Conversion (αSyn&#95;RT-QuIC). Remarkably, the biochemical and morphological properties of the final α-synuclein aggregates significantly differed between PD and MSA seeded samples. Here, these aggregates were given to neuron-like differentiated SH-SY5Y cells and distinct inflammatory responses were observed. To deepen whether the morphological features of α-synuclein aggregates were responsible for this variable SH-SY5Y inflammatory response, we generated three biochemically and morphologically distinct α-synuclein aggregates starting from recombinant α-synuclein that were used to seed αSyn&#95;RT-QuIC reaction; the final reaction products were used to stimulate SH-SY5Y cells. Our study showed that, in contrast to OM samples of PD and MSA patients, the artificial aggregates did not transfer their distinctive features to the αSyn&#95;RT-QuIC products and the latter induced analogous inflammatory responses in cells. Thus, the natural composition of the αSyn D strains but also other specific factors in OM tissue can substantially modulate the biochemical, morphological and inflammatory features of the αSyn&#95;RT-QuIC products.

Alpha-synuclein seed amplification assays (αSyn-SAAs) are promising diagnostic tools for Parkinson's disease (PD) and related synucleinopathies. They enable detection of seeding-competent alpha-synuclein aggregates in living patients and have shown high diagnostic accuracy in several PD and other synucleinopathy patient cohorts. However, there has been confusion about αSyn-SAAs for their methodology, nomenclature, and relative accuracies when performed by various laboratories. We compared αSyn-SAA results obtained from three independent laboratories to evaluate reproducibility across methodological variations. We utilized the Parkinson's Progression Markers Initiative (PPMI) cohort, with DATSCAN data available for comparison, since clinical diagnosis of early de novo PD is critical for neuroprotective trials, which often use dopamine transporter imaging to enrich their cohorts. Blinded cerebrospinal fluid (CSF) samples for a randomly selected subset of PPMI subjects (30 PD, 30 HC, and 20 SWEDD), from both baseline and year 3 collections for the PD and HC groups (140 total CSF samples) were analyzed in parallel by each lab according to their own established and optimized αSyn-SAA protocols. The αSyn-SAA results were remarkably similar across laboratories, displaying high diagnostic performance (sensitivity ranging from 86 to 96% and specificity from 93 to 100%). The assays were also concordant for samples with results that differed from clinical diagnosis, including 2 PD patients determined to be clinically inconsistent with PD at later time points. All three assays also detected 2 SWEDD subjects as αSyn-SAA positive who later developed PD with abnormal DAT-SPECT. These multi-laboratory results confirm the reproducibility and value of αSyn-SAA as diagnostic tools, illustrate reproducibility of the assay in expert hands, and suggest that αSyn-SAA has potential to provide earlier diagnosis with comparable or superior accuracy to existing methods., (© 2021. The Author(s).)

The clinicopathological heterogeneity in Lewy-body diseases (LBD) highlights the need for pathology-driven biomarkers in-vivo. Misfolded alpha-synuclein (α-Syn) is a lead candidate based on its crucial role in disease pathophysiology. Real-time quaking-induced conversion (RT-QuIC) analysis of CSF has recently shown high sensitivity and specificity for the detection of misfolded α-Syn in patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In this study we performed the CSF RT-QuIC assay in 236 PD and 49 DLB patients enriched for different genetic forms with mutations in GBA, parkin, PINK1, DJ1, and LRRK2. A subgroup of 100 PD patients was also analysed longitudinally. We correlated kinetic seeding parameters of RT-QuIC with genetic status and CSF protein levels of molecular pathways linked to α-Syn proteostasis. Overall, 85% of PD and 86% of DLB patients showed positive RT-QuIC α-Syn seeding activity. Seeding profiles were significantly associated with mutation status across the spectrum of genetic LBD. In PD patients, we detected positive α-Syn seeding in 93% of patients carrying severe GBA mutations, in 78% with LRRK2 mutations, in 59% carrying heterozygous mutations in recessive genes, and in none of those with bi-allelic mutations in recessive genes. Among PD patients, those with severe GBA mutations showed the highest seeding activity based on RT-QuIC kinetic parameters and the highest proportion of samples with 4 out of 4 positive replicates. In DLB patients, 100% with GBA mutations showed positive α-Syn seeding compared to 79% of wildtype DLB. Moreover, we found an association between α-Syn seeding activity and reduced CSF levels of proteins linked to α-Syn proteostasis, specifically lysosome-associated membrane glycoprotein 2 and neurosecretory protein VGF.These findings highlight the value of α-Syn seeding activity as an in-vivo marker of Lewy-body pathology and support its use for patient stratification in clinical trials targeting α-Syn., (© 2021. The Author(s).)

Aggregated α-synuclein (αSyn) protein is a core pathological feature of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Both PD and DLB demonstrate the presence of diverse intracellular α-synuclein (αSyn) species, including C-terminally truncated αSyn (C-αSyn), although it is unknown how C-αSyn species contribute to disease progression. Using recombinant C-αSyn and PD and DLB brain lysates as seeds in the real-time quaking-induced conversion (RT-QuIC) assay, we explored how C-αSyn may be involved in disease stratification. Comparing the seeding activity of aqueous-soluble fractions to detergent-soluble fractions, and using αSyn 1-130 as substrate for the RT-QuIC assay, the temporal cortex seeds differentiated PD and DLB from healthy controls. In contrast to the temporal cortex, where PD and DLB could not be distinguished, αSyn 1-130 seeded by the detergent-soluble fractions from the PD frontal cortex demonstrated greater seeding efficiency compared to the DLB frontal cortex. Moreover, proteinase K-resistant (PK res ) fragments from the RT-QuIC end products using C-αSyn 1-130 or C-αSyn 1-115 were more obvious in the frontal cortex compared to the temporal cortex. Morphological examinations of RT-QuIC end products showed differences in the size of the fibrils between C-αSyn 1-130 and C-αSyn 1-115, in agreement with the RT-QuIC results. These data show that C-αSyn species can distinguish PD from DLB and suggest diversity in αSyn species across these synucleinopathies, which could play a role in disease progression.

In synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy body (DLB), pathological alpha-synuclein (α-syn) aggregates are found in the gastrointestinal (GI) tract as well as in the brain. In this study, using real-time quaking-induced conversion (RT-QuIC), we investigated the presence of α-syn seeding activity in the brain and colon tissue of G2-3 transgenic mice expressing human A53T α-syn. Here we show that pathological α-syn aggregates with seeding activity were present in the colon of G2-3 mice as early as 3 months old, which is in the presymptomatic stage prior to the observation of any neurological abnormalities. In contrast, α-syn seeding activity was not detectable in 3 month-old mouse brains and only identified at 6 months of age in one of three mice. In the symptomatic stage of 12 months of age, RT-QuIC seeding activity was consistently detectable in both the brain and colon of G2-3 mice. Our results indicate that the RT-QuIC assay can presymptomatically detect pathological α-syn aggregates in the colon of G2-3 mice several months prior to their detection in brain tissue.

Definitive diagnosis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB) relies on postmortem finding of disease-associated alpha-synuclein (αSyn D ) as misfolded protein aggregates in the central nervous system (CNS). The recent development of the real-time quaking induced conversion (RT-QuIC) assay for ultrasensitive detection of αSyn D aggregates has revitalized the diagnostic values of clinically accessible biospecimens, including cerebrospinal fluid (CSF) and peripheral tissues. However, the current αSyn RT-QuIC assay platforms vary widely and are thus challenging to implement and standardize the measurements of αSyn D across a wide range of biospecimens and in different laboratories. We have streamlined αSyn RT-QuIC assay based on a second generation assay platform that was assembled entirely with commercial reagents. The streamlined RT-QuIC method consisted of a simplified protocol requiring minimal hands-on time, and allowing for a uniform analysis of αSyn D in different types of biospecimens from PD and DLB. Ultrasensitive and specific RT-QuIC detection of αSyn D aggregates was achieved in million-fold diluted brain homogenates and in nanoliters of CSF from PD and DLB cases but not from controls. Comparative analysis revealed higher seeding activity of αSyn D in DLB than PD in both brain homogenates and CSF. Our assay was further validated with CSF samples of 214 neuropathologically confirmed cases from tissue repositories (88 PD, 58 DLB, and 68 controls), yielding a sensitivity of 98% and a specificity of 100%. Finally, a single RT-QuIC assay protocol was employed uniformly to detect seeding activity of αSyn D in PD samples across different types of tissues including the brain, skin, salivary gland, and colon. We anticipate that our streamlined protocol will enable interested laboratories to easily and rapidly implement the αSyn RT-QuIC assay for various clinical specimens from PD and DLB. The utilization of commercial products for all assay components will improve the robustness and standardization of the RT-QuIC assay for diagnostic applications across different sites. Due to ultralow sample consumption, the ultrasensitive RT-QuIC assay will facilitate efficient use and sharing of scarce resources of biospecimens. Our streamlined RT-QuIC assay is suitable to track the distribution of αSyn D in CNS and peripheral tissues of affected patients. The ongoing evaluation of RT-QuIC assay of αSyn D as a potential biomarker for PD and DLB in clinically accessible biospecimens has broad implications for understanding disease pathogenesis, improving early and differential diagnosis, and monitoring therapeutic efficacies in clinical trials.

Background: Cerebral deposition of abnormally misfolded and aggregated alpha-synuclein (αSyn) is a neuropathological hallmark of Parkinson's disease (PD). Pathologically aggregated αSyn species of PD (αSyn PD ) can act, in a 'prion-like' manner, as proteinaceous nuclei ('seeds') which are capable of self-templated propagation. This has raised concerns that αSyn PD seeds transmitted iatrogenically between humans may stimulate αSyn pathologies or clinically harmful effects in the recipients. Effective decontamination when reprocessing medical devices could significantly counteract such risks. Steam sterilization at 134°C is recommended as an essential pathogen inactivation step in many reprocessing guidelines for medical devices, and also shows effectiveness against prions, the self-propagating biological agents long thought to exhibit the highest resistance to steam sterilization., Methods: This study examined the reduction in αSyn PD seeding activity in brain tissue homogenates from patients with PD after steam sterilization at 134°C using a specifically adapted real-time quaking induced conversion assay., Findings: Titres of approximately 10 10 50% seeding doses per gram were detected in non-steam-sterilized caudate nucleus tissue of patients with PD by endpoint titration. Five minutes of steam sterilization reduced this titre by only 2.25 ± 0.15 decadic-logarithmic units, with an extension of the sterilization time to 90 min not causing additional inactivation. These findings reveal that αSyn PD species are disease-associated biological agents with seeding activity that has higher resistance to steam sterilization than prions., Conclusion: The remarkable heat resistance of αSyn PD seeds calls for thoroughly validated cleaning and disinfection methods that reliably remove or inactivate possible contaminations of seeding-active αSyn aggregates when reprocessing medical devices., (Copyright © 2020 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein (αSyn). They include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In each disease, it has been proposed that aggregates of αSyn represent different conformational strains of αSyn, leading to self-propagation and spreading from cell to cell. It has been considered that αSyn aggregates grow by seeded polymerization mechanisms. Previously, the mechanism of seed conversion in prion protein aggregation has been exploited by real-time quaking-induced conversion (RT-QuIC) assay. It was further refined by incorporating the fluorescent dye thioflavin-T, which enabled the real-time monitoring of kinetic changes with a highly sensitive detection of seed aggregates present at an extremely low level. In an application for diagnostics, it has been reported that αSyn RT-QuIC exhibits specificity between 82% and 100%, while its sensitivity varies between 70% and 100%, on the basis of a study in which this assay was performed at multiple different laboratories. Furthermore, it has been suggested that the αSyn RT-QuIC method can be applied to study the biochemical characteristics of different αSyn strains among synucleinopathies. In this article, we describe the detailed protocols for αSyn RT-QuIC assays.

Background: The accumulation of aggregated α-synuclein (αSyn) is known as one of the critical reasons to exhibit their variable molecular pathologies and phenotypes in synucleinopathies. Recent studies suggested that the real-time quaking-induced conversion (RT-QuIC) assay is one of the potential methods to detect these αSyn aggregates and could detect the aggregated αSyn in the brain tissue and cerebrospinal fluid (CSF) using the propensity of the prion-like oligomerization., Objective: We tried to optimize the αSyn RT-QuIC assay based on the aggregation of αSyn in brain samples of synucleinopathies by comparing the conditions of the recently reported αSyn RTQuIC assays., Methods: This study applied a highly sensitive RT-QuIC assay using recombinant αSyn (rαSyn) to detect aggregated αSyn in the brain tissue from dementia with Lewy bodies (DLB)., Results: This study compared αSyn RT-QuIC assays under conditions such as beads, rαSyn as a substrate, reaction buffers, and fluorescence detectors. We observed that the addition of beads and the use of 6x His-tagged rαSyn as a substrate help to obtain higher positive responses from αSyn RT-QuIC assay seeding with brain homogenate (BH) of DLB and phosphate buffer-based reaction showed higher positive responses than HEPES buffer-based reaction on both fluorescent microplate readers. We also observed that the DLB BHs gave positive responses within 15-25h, which is faster high positive responses than recently reported assays., Conclusion: This established αSyn RT-QuIC assay will be able to apply to the early clinical diagnosis of αSyn aggregates-related diseases in various biofluids such as CSF., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

RT-QuIC is a shaking-based cyclic amplification technique originally developed in the prion field to detect minute amounts of scrapie prion protein (PrP Sc ). In this study, we applied the RT-QuIC assay to investigate a-synuclein (a-syn) seeding activity in brains of Dementia with Lewy Body (DLB) patients and in brains of G2-3 transgenic mice expressing human a-syn with A53T mutation. The results show that a-syn seeding activity varies between patients with detectable dilutions ranging from 10 -3 to 10 -8 dilutions of brain tissue and is stable under exposures to the cycles of freezing, thawing and sonication. A53T a-syn aggregates from G2-3 transgenic mice greatly favoured A53T recombinant human a-syn as substrates in comparison to wild-type a-syn, suggesting that conformations for wild-type a-syn to be able to adopt are not compatible with that of A53T aggregates from G2-3.

Background: α-Synuclein is a small soluble protein, whose physiological function in the healthy brain is poorly understood. Intracellular inclusions of α-synuclein, referred to as Lewy bodies (LBs), are pathological hallmarks of α-synucleinopathies, such as Parkinson's disease (PD) or dementia with Lewy bodies (DLB)., Main Body: Understanding of the molecular basis as well as the factors or conditions promoting α-synuclein misfolding and aggregation is an important step towards the comprehension of pathological mechanism of α-synucleinopathies and for the development of efficient therapeutic strategies. Based on the conversion and aggregation mechanism of α-synuclein, novel diagnostic tests, such as protein misfolding seeded conversion assays, e.g. the real-time quaking-induced conversion (RT-QuIC), had been developed. In diagnostics, α-synuclein RT-QuIC exhibits a specificity between 82 and 100% while the sensitivity varies between 70 and 100% among different laboratories. In addition, the α-synuclein RT-QuIC can be used to study the α-synuclein-seeding-characteristics of different α-synucleinopathies and to differentiate between DLB and PD., Conclusion: The variable diagnostic accuracy of current α-synuclein RT-QuIC occurs due to different protocols, cohorts and material etc.. An impact of micro-environmental factors on the α-synuclein aggregation and conversion process and the occurrence and detection of differential misfolded α-synuclein types or strains might underpin the clinical heterogeneity of α-synucleinopathies ., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2020.)

Recent work with prion diseases and synucleinopathies indicates that accurate diagnostic methods for protein-folding diseases can be based on the ultrasensitive, amplified measurement of pathological aggregates in biospecimens. A better understanding of the physicochemical factors that control the seeded polymerization of such aggregates, and their amplification in vitro, should allow improvements in existing assay platforms, as well as the development of new assays for other proteopathic aggregates. Here, we systematically investigated the effects of the ionic environment on the polymerization of tau, α-synuclein, and the prion protein (PrP) induced by aggregates in biospecimens. We screened salts of the Hofmeister series, a relative ordering of strongly and weakly hydrated salts that tend to precipitate or solubilize proteins. We found that sensitivities of tau-based assays for Alzheimer's seeds and PrP-based assays for prions were best in weakly hydrated anions. In contrast, we saw an inverse trend with different tau-based assays, improving detection sensitivity for progressive supranuclear palsy seeds by ≈10 6 Hofmeister analysis also improved detection of sporadic Creutzfeldt-Jakob disease prions in human nasal brushings and chronic wasting disease prions in deer-ear homogenates. Our results demonstrate strong and divergent influences of ionic environments on the amplification and detection of proteopathic seeds as biomarkers for protein-folding diseases., Competing Interests: Competing interest statement: M.A.M., E.S., A.K., and B.C. are inventors on a pending patent (US Application No. PCT/US2017/069024).

The diagnosis and treatment of synucleinopathies such as Parkinson disease and dementia with Lewy bodies would be aided by the availability of assays for the pathogenic disease-associated forms of α-synuclein (αSyn D ) that are sufficiently sensitive, specific, and practical for analysis of accessible diagnostic specimens. Two recent αSyn D seed amplification tests have provided the first prototypes for ultrasensitive and specific detection of αSyn D in patients' cerebrospinal fluid. These prototypic assays require 5-13 days to perform. Here, we describe an improved α-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay that has similar sensitivity and specificity to the prior assays, but can be performed in 1-2 days with quantitation. Blinded analysis of cerebrospinal fluid from 29 synucleinopathy cases [12 Parkinson's and 17 dementia with Lewy bodies] and 31 non-synucleinopathy controls, including 16 Alzheimer's cases, yielded 93% diagnostic sensitivity and 100% specificity for this test so far. End-point dilution analyses allowed quantitation of relative amounts of αSyn D seeding activity in cerebrospinal fluid samples, and detection in as little as 0.2 μL. These results confirm that αSyn D seeding activity is present in cerebrospinal fluid. We also demonstrate that it can be rapidly detected, and quantitated, even in early symptomatic stages of synucleinopathy.

Abstract Seeding activities of disease-associated α-synuclein aggregates (αSynD), a hallmark of Parkinson’s disease (PD), are detectable by seed amplification assay (αSyn-SAA) and being developed as a diagnostic biomarker for PD. Sensitive and accurate αSyn-SAA for blood or saliva would greatly facilitate PD diagnosis. This prospective diagnostic study conducted αSyn-SAA analyses on serum and saliva samples collected from patients clinically diagnosed with PD or healthy controls (HC). 124 subjects (82 PD, 42 HC) donated blood and had extensive clinical assessments, of whom 74 subjects (48 PD, 26 HC) also donated saliva at the same visits. An additional 57 subjects (35 PD, 22 HC) donated saliva and had more limited clinical assessments. The mean ages were 69.21, 66.55, 69.58, and 64.71 years for PD serum donors, HC serum donors, PD saliva donors, and HC saliva donors, respectively. αSynD seeding activities in either sample type alone or both sample types together were evaluated for PD diagnosis. Serum αSyn-SAA data from 124 subjects showed 80.49% sensitivity, 90.48% specificity, and 0.9006 accuracy (AUC of ROC); saliva αSyn-SAA data from 131 subjects attained 74.70% sensitivity, 97.92% specificity, and 0.8966 accuracy. Remarkably, the combined serum and saliva αSyn-SAA from 74 subjects with both sample types achieved better diagnostic performance: 95.83% sensitivity, 96.15% specificity, and 0.98 accuracy. In addition, serum αSynD seeding activities correlated inversely with Montreal Cognitive Assessment in males and positively with Hamilton Depression Rating Scale in females and in the

Abstract Background The term rapidly progressive dementia (RPD) with Lewy bodies (rpDLB) is used for DLB patients who develop a rapidly progressive neurological syndrome and have reduced survival. Here, we characterise the clinical, neuropathological, and molecular characteristics of a large rpDLB neuropathological series. Methods We included all RPD patients with a disease duration

Background: The term rapidly progressive dementia (RPD) with Lewy bodies (rpDLB) is used for DLB patients who develop a rapidly progressive neurological syndrome and have reduced survival. Here, we characterise the clinical, neuropathological, and molecular characteristics of a large rpDLB neuropathological series. Methods: We included all RPD patients with a disease duration < 4 years submitted to our prion disease referral centre between 2003 and 2022 who showed Lewy body pathology (LBP) in limbic or neocortical stages as primary neuropathological diagnosis, had no systemic condition justifying the rapid deterioration and were previously neurologically unimpaired. Clinical features were retrieved and compared with Creutzfeldt-Jakob disease (CJD) and rapidly progressive Alzheimer's disease (rpAD) cohorts. Neuropathological and genetic (whole exome sequencing, APOE genotyping, and C9orf72 repeat expansion analysis) characteristics of rpDLB patients were systematically investigated. We scored semi-quantitatively the LBP load and performed a α-synuclein (αSyn) RT-QuIC seeding amplification assay (SAA) on cerebrospinal fluid (CSF) and tenfold serially diluted brain homogenates from different brain areas in rpDLB patients and typical long-lasting Lewy body disease (LBD) with dementia patients as control group. Results: RpDLB patients were older (p = 0.047) and presented more cognitive fluctuations (p = 0.005), visual hallucinations (p = 0.020), neuropsychiatric symptoms (p = 0.006) and seizures (p = 0.032), and fewer cerebellar (p < 0.001) and visual (p = 0.004) signs than CJD ones. Delirium onset was more common than in both CJD (p < 0.001) and rpAD (p = 0.008). Atypical LBD signs (pyramidal, myoclonus, akinetic mutism) were common. All tested patients were positive by CSF αSyn SAA. Concomitant pathologies were common, with only four cases showing relatively "pure" LBP. LBP load and αSyn seeding activity measured through αSyn RT-QuIC SAA were not significantly different between rpDLB patients and typical LBD. We found a likely pathogenic variant in GBA in one patient. Conclusions: Our results indicate that: 1) rpDLB exhibits a distinct clinical signature (2) CSF αSyn SAA is a reliable diagnostic test; 3) rpDLB is a heterogeneous neuropathological entity that can be underlain by both widespread pure LBP, or multiple copathologies 4) rpDLB is likely not sustained by distinct αSyn conformational strains; 5) genetic defects may, at least occasionally, contribute to the poor prognosis in these patients. [ABSTRACT FROM AUTHOR]

Tau neurofibrillary tangles are a hallmark of Alzheimer's disease neuropathological change. However, it remains largely unclear how distinctive Alzheimer's disease tau seeds (i.e. 3R/4R) correlate with histological indicators of tau accumulation. Furthermore, AD tau co-pathology is thought to influence features and progression of other neurodegenerative diseases including Lewy body disease; yet measurements of different types of tau seeds in the setting of such diseases is an unmet need. Here, we use tau real-time quaking-induced conversion (RT-QuIC) assays to selectively quantitate 3R/4R tau seeds in the frontal lobe which accumulates histologically identifiable tau pathology at late disease stages of AD neuropathologic change. Seed quantitation across a spectrum of neurodegenerative disease cases and controls indicated tau seeding activity can be detected well before accompanying histopathological indication of tau deposits, and even prior to the earliest evidence of Alzheimer's-related tau accumulation anywhere in the brain. In later stages of AD, 3R/4R tau RT-QuIC measures correlated with immunohistochemical tau burden. In addition, Alzheimer's tau seeds occur in the vast majority of cases evaluated here inclusive of primary synucleinopathies, frontotemporal lobar degeneration and even controls albeit at multi-log lower levels than Alzheimer's cases. α-synuclein seeding activity confirmed synucleinopathy cases and further indicated the co-occurrence of α-synuclein seeds in some Alzheimer's disease and primary tauopathy cases. Our analysis indicates that 3R/4R tau seeds in the mid-frontal lobe correlate with the overall Braak stage and Alzheimer's disease neuropathologic change, supporting the quantitative predictive value of tau RT-QuIC assays. Our data also indicate 3R/4R tau seeds are elevated in females compared to males at high (≥ IV) Braak stages. This study suggests 3R/4R tau seeds are widespread even prior to the earliest stages of Alzheimer's disease changes, including in normal, and even young individuals, with prevalence across multiple neurodegenerative diseases to further define disease subtypes.

Abstract The alpha-synuclein (aSyn) seed amplification assay (SAA) can identify aSyn aggregates as indicator for Lewy body pathology in biomaterials of living patients and help in diagnosing Parkinson´s disease and dementia syndromes. Our objective was to confirm that qualitative results of aSyn SAA are reproducible across laboratories and to determine whether quantitative findings correlate with patient clinical characteristics. Therefore cerebrospinal fluid samples were re-analysed by aSyn SAA in a second laboratory with four technical replicates for each sample. Kinetic parameters derived from each aggregation curve were summarized and correlated with patient characteristics. We found that qualitative findings were identical between the two laboratories for 54 of 55 patient samples. The number of positive replicates for each sample also showed good agreement between laboratories. Moreover, specific kinetic parameters of the SAA showed a strong correlation with clinical parameters, notably with cognitive performance evaluated by the Montreal Cognitive Assessment. We concluded that SAA findings are highly reproducible across laboratories following the same protocol. SAA reports not only the presence of Lewy pathology but is also associated with clinical characteristics. Thus, aSyn SAA can potentially be used for patient stratification and determining the target engagement of aSyn targeting treatments.

The alpha-synuclein (aSyn) seed amplification assay (SAA) can identify aSyn aggregates as indicator for Lewy body pathology in biomaterials of living patients and help in diagnosing Parkinson´s disease and dementia syndromes. Our objective was to confirm that qualitative results of aSyn SAA are reproducible across laboratories and to determine whether quantitative findings correlate with patient clinical characteristics. Therefore cerebrospinal fluid samples were re-analysed by aSyn SAA in a second laboratory with four technical replicates for each sample. Kinetic parameters derived from each aggregation curve were summarized and correlated with patient characteristics. We found that qualitative findings were identical between the two laboratories for 54 of 55 patient samples. The number of positive replicates for each sample also showed good agreement between laboratories. Moreover, specific kinetic parameters of the SAA showed a strong correlation with clinical parameters, notably with cognitive performance evaluated by the Montreal Cognitive Assessment. We concluded that SAA findings are highly reproducible across laboratories following the same protocol. SAA reports not only the presence of Lewy pathology but is also associated with clinical characteristics. Thus, aSyn SAA can potentially be used for patient stratification and determining the target engagement of aSyn targeting treatments. [ABSTRACT FROM AUTHOR]

Background: Aggregation of α-synuclein (α-syn) is a prominent feature of Parkinson's disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification. Methods: In this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn. Results: We found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates. Conclusions: Our results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts. [ABSTRACT FROM AUTHOR]

Synucleinopathies are characterized by the deposition of alpha-synuclein (α-syn) aggregates in brain tissue. Pathological α-syn aggregates propagate in a prion-like manner and display prion-like biochemical properties. Using RT-QuIC, we measured α-syn seeding activity from brains of Dementia with Lewy body (DLB) patients post autoclave. Here, we show that autoclaving at 121 °C removes one to two log10 of α-syn seeding activity but the remaining 50% seeding dose (SD50) is more than 107/mg tissue. DLB brain samples autoclaved at 132 °C still revealed an SD50 of approximately 106/mg tissue. Our data suggest that DLB α-syn seeds are incompletely inactivated by standard autoclave, thus highlighting the need for evaluating laboratory procedures that fully inactivate them.

Introduction: Dementia with Lewy Bodies (DLB) is the second most common cause of neurodegenerative dementia after Alzheimer’s disease (AD), but the field is still lacking a specific biomarker for its core pathology: alpha synuclein (α-syn). Realtime quaking induced conversion (RT-QuIC) has recently emerged as a strong biomarker candidate to detect misfolded α-syn in DLB. However, the variability in the parameters of the technique and the heterogeneity of DLB patients make the reproducibility of the results difficult. Here, we provide an overview of the state-of-the-art research of α-syn RT-QuIC in DLB focused on: (1) the capacity of α-syn RT-QuIC to discriminate DLB from controls, Parkinson’s disease (PD) and AD; (2) the capacity of α-syn RT-QuIC to identify prodromal stages of DLB; and (3) the influence of co-pathologies on α-syn RT-QuIC’s performance. We also assessed the influence of different factors, such as technical conditions (e.g., temperature, pH, shaking-rest cycles), sample type, and clinical diagnosis versus autopsy confirmation.Methods: We conducted a systematic review following the PRISMA guidelines in August 2022, without any limits in publication dates. Search terms were combinations of “RT-QuIC” and “Lewy Bodies,” “DLB” or “LBD”.Results: Our meta-analysis shows that α-syn RT-QuIC reaches very high diagnostic performance in discriminating DLB from both controls (pooled sensitivity and specificity of 0.94 and 0.96, respectively) and AD (pooled sensitivity and specificity of 0.95 and 0.88) and is promising for prodromal phases of DLB. However, the performance of α-syn RT-QuIC to discriminate DLB from PD is currently low due to low specificity (pooled sensitivity and specificity of 0.94 and 0.11). Our analysis showed that α-syn RT-QuIC’s performance is not substantially influenced by sample type or clinical diagnosis versus autopsy confirmation. Co-pathologies did not influence the performance of α-syn RT-QuIC, but the number of such studies is currently limited. We observed technical variability across published articles. However, we could not find a clear effect of technical variability on the reported results.Conclusion: There is currently enough evidence to test misfolded α-syn by RT-QuIC for clinical use. We anticipate that harmonization of protocols across centres and advances in standardization will facilitate the clinical establishment of misfolded α-syn detection by RT-QuIC.

α-Synucleinopathies, such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy, are a class of neurodegenerative diseases exhibiting intracellular inclusions of misfolded α-synuclein (αSyn), referred to as Lewy bodies or oligodendroglial cytoplasmic inclusions (Papp–Lantos bodies). Even though the specific cellular distribution of aggregated αSyn differs in PD and DLB patients, both groups show a significant pathological overlap, raising the discussion of whether PD and DLB are the same or different diseases. Besides clinical investigation, we will focus in addition on methodologies, such as protein seeding assays (real-time quaking-induced conversion), to discriminate between different types of α-synucleinopathies. This approach relies on the seeding conversion properties of misfolded αSyn, supporting the hypothesis that different conformers of misfolded αSyn may occur in different types of α-synucleinopathies. Understanding the pathological processes influencing the disease progression and phenotype, provoked by different αSyn conformers, will be important for a personalized medical treatment in future. [ABSTRACT FROM AUTHOR]

Abstract Background Multiple system atrophy (MSA) is a neurodegenerative condition characterized by variable combinations of parkinsonism, autonomic failure, cerebellar ataxia and pyramidal features. Although the distribution of synucleinopathy correlates with the predominant clinical features, the burden of pathology does not fully explain observed differences in clinical presentation and rate of disease progression. We hypothesized that the clinical heterogeneity in MSA is a consequence of variability in the seeding activity of α-synuclein both between different patients and between different brain regions. Methods The reliable detection of α-synuclein seeding activity derived from MSA using cell-free amplification assays remains challenging. Therefore, we conducted a systematic evaluation of 168 different reaction buffers, using an array of pH and salts, seeded with fully characterized brain homogenates from one MSA and one PD patient. We then validated the two conditions that conferred the optimal ability to discriminate between PD- and MSA-derived samples in a larger cohort of 40 neuropathologically confirmed cases, including 15 MSA. Finally, in a subset of brains, we conducted the first multi-region analysis of seeding behaviour in MSA. Results Using our novel buffer conditions, we show that the physicochemical factors that govern the in vitro amplification of α-synuclein can be tailored to generate strain-specific reaction buffers that can be used to reliably study the seeding capacity from MSA-derived α-synuclein. Using this novel approach, we were able to sub-categorize the 15 MSA brains into 3 groups: high, intermediate and low seeders. To further demonstrate heterogeneity in α-synuclein seeding in MSA, we conducted a comprehensive multi-regional evaluation of α-synuclein seeding in 13 different regions from 2 high seeders, 2 intermediate seeders and 2 low seeders. Conclusions We have identified unexpected differences in seed-competent α-synuclein across a cohort of neuropathologically comparable MSA brains. Furthermore, our work has revealed a substantial heterogeneity in seeding activity, driven by the PBS-soluble α-synuclein, between different brain regions of a given individual that goes beyond immunohistochemical observations. Our observations pave the way for future subclassification of MSA, which exceeds conventional clinical and neuropathological phenotyping and considers the structural and biochemical heterogeneity of α-synuclein present. Finally, our methods provide an experimental framework for the development of vitally needed, rapid and sensitive diagnostic assays for MSA.

The real-time quaking-induced conversion (RT-QuIC) alpha-synuclein (aSyn) protein kinetic seeding assay has been very useful for detecting pathological aggregates in various synucleinopathies including Parkinson’s disease (PD). This biomarker assay relies on fresh frozen tissue to effectively seed and amplify aSyn aggregating protein. With vast repositories of formalin-fixed paraffin-embedded (FFPE) tissues, it is paramount to harness the power of kinetic assays to unlock the diagnostic potential of archived FFPE biospecimens. However, the major challenge posed by significantly reduced amplification of formalin-fixed tissues in the assay suggests that formalin fixation deterred monomer interaction with the sample seed and depressed subsequent protein aggregation. To overcome this challenge, we developed a kinetic assay seeding ability recovery (KASAR) protocol to maintain the integrity of the tissue and seeding protein. For this, we implemented a series of heating steps with the brain tissue suspended in a buffer composed of 500 mM tris-HCl (pH 7.5) and 0.02% SDS after the standard deparaffinization of the tissue sections. Initially, samples from seven human brain samples, including four samples from patients diagnosed with dementia with Lewy bodies (DLB) and three samples from healthy controls without DLB, were compared to fresh frozen samples under three different, but clinically common sample storage conditions: formalin-fixed, FFPE, and FFPE slices cut 5 µm thick. The KASAR protocol was able to recover seeding activity for all positive samples in all storage conditions. Next, 28 FFPE samples from the submandibular gland (SMG) of patients diagnosed with PD, incidental Lewy body disease (ILBD), or healthy controls were tested with 93% of results replicating when blinded. With samples of only a few milligrams, this protocol recovered the same quality of seeding in formalin-fixed tissue as fresh frozen tissue. Moving forward, protein aggregate kinetic assays, in conjunction with the KASAR protocol, can be used to understand and diagnose neurodegenerative diseases more comprehensively. Overall, our KASAR protocol unlocks and restores the seeding ability of formalin-fixed paraffin-embedded tissues for the amplification of biomarker protein aggregates in kinetic assays.

Background: Increasing evidence supports the use of plasma biomarkers of neurodegeneration and neuroinflammation to screen and diagnose patients with dementia. However, confirmatory studies are required to demonstrate their usefulness in the clinical setting. Methods: We evaluated plasma and cerebrospinal fluid (CSF) samples from consecutive patients with frontotemporal dementia (FTD) (n = 59), progressive supranuclear palsy (PSP) (n = 31), corticobasal syndrome (CBS) (n = 29), dementia with Lewy bodies (DLB) (n = 49), Alzheimer disease (AD) (n = 97), and suspected non-AD physiopathology (n = 51), as well as plasma samples from 60 healthy controls (HC). We measured neurofilament light chain (NfL), phospho-tau181 (p-tau181), and glial fibrillary acid protein (GFAP) using Simoa (all plasma biomarkers and CSF GFAP), CLEIA (CSF p-tau181), and ELISA (CSF NfL) assays. Additionally, we stratified patients according to the A/T/N classification scheme and the CSF α-synuclein real-time quaking-induced conversion assay (RT-QuIC) results. Results: We found good correlations between CSF and plasma biomarkers for NfL (rho = 0.668, p < 0.001) and p-tau181 (rho = 0.619, p < 0.001). Plasma NfL was significantly higher in disease groups than in HC and showed a greater increase in FTD than in AD [44.9 (28.1–68.6) vs. 21.9 (17.0–27.9) pg/ml, p < 0.001]. Conversely, plasma p-tau181 and GFAP levels were significantly higher in AD than in FTD [3.2 (2.4–4.3) vs. 1.1 (0.7–1.6) pg/ml, p < 0.001; 404.7 (279.7–503.0) vs. 198.2 (143.9–316.8) pg/ml, p < 0.001]. GFAP also allowed discriminating disease groups from HC. In the distinction between FTD and AD, plasma p-tau181 showed better accuracy (AUC 0.964) than NfL (AUC 0.791) and GFAP (AUC 0.818). In DLB and CBS, CSF amyloid positive (A+) subjects had higher plasma p-tau181 and GFAP levels than A− individuals. CSF RT-QuIC showed positive α-synuclein seeding activity in 96% DLB and 15% AD patients with no differences in plasma biomarker levels in those stratified by RT-QuIC result. Conclusions: In a single-center clinical cohort, we confirm the high diagnostic value of plasma p-tau181 for distinguishing FTD from AD and plasma NfL for discriminating degenerative dementias from HC. Plasma GFAP alone differentiates AD from FTD and neurodegenerative dementias from HC but with lower accuracy than p-tau181 and NfL. In CBS and DLB, plasma p-tau181 and GFAP levels are significantly influenced by beta-amyloid pathology. [ABSTRACT FROM AUTHOR]

Objective: In the present study, real-time quaking-induced conversion (RT-QuIC) assay was used to evaluate pathologic alpha-synuclein (AS) seeding activity in formalin-fixed paraffin-embedded (FFPE) tissue from the gastrointestinal (GI) tract of Parkinson's disease (PD) patients.Methods: This study was conducted in two parts: Part I. a preliminary autopsy study that included four autopsy-confirmed patients with synucleinopathy (2 PD, 1 dementia with Lewy bodies [DLB], and 1 multiple system atrophy [MSA]) and two normal autopsy controls. Frozen and FFPE tissues of the brain were obtained. Part II. a clinical case-control study that included 20 clinically diagnosed PD patients and matched controls. Surgically resected FFPE tissues from the upper and lower GI tracts were used. The RT-QuIC assay was performed to evaluate pathologic seed amplification using frozen or FFPE tissues. The presence or absence of AS aggregation was confirmed by conventional phosphorylated AS (pAS) immunohistochemistry (IHC).Results: In Part I, RT-QuIC assay showed pathologic AS amplification in frozen and FFPE brain tissues of PD and DLB patients, and FFPE stomach tissue of PD patients but not in the MSA patient and controls. In Part II, pathologic seeding activity was found in 10% (2/20) of the stomach tissues of clinical PD patients but in none of the matched controls. IHC showed pAS-positive staining in 55% of patients (11/20) and 15% of controls (3/20).Conclusion: The present study results showed that the RT-QuIC assay using FFPE tissue of the GI tract was inadequate as a biomarker in PD. [ABSTRACT FROM AUTHOR]

Abstract Alpha-synuclein seed amplification assays (αSyn-SAAs) are promising diagnostic tools for Parkinson’s disease (PD) and related synucleinopathies. They enable detection of seeding-competent alpha-synuclein aggregates in living patients and have shown high diagnostic accuracy in several PD and other synucleinopathy patient cohorts. However, there has been confusion about αSyn-SAAs for their methodology, nomenclature, and relative accuracies when performed by various laboratories. We compared αSyn-SAA results obtained from three independent laboratories to evaluate reproducibility across methodological variations. We utilized the Parkinson’s Progression Markers Initiative (PPMI) cohort, with DATSCAN data available for comparison, since clinical diagnosis of early de novo PD is critical for neuroprotective trials, which often use dopamine transporter imaging to enrich their cohorts. Blinded cerebrospinal fluid (CSF) samples for a randomly selected subset of PPMI subjects (30 PD, 30 HC, and 20 SWEDD), from both baseline and year 3 collections for the PD and HC groups (140 total CSF samples) were analyzed in parallel by each lab according to their own established and optimized αSyn-SAA protocols. The αSyn-SAA results were remarkably similar across laboratories, displaying high diagnostic performance (sensitivity ranging from 86 to 96% and specificity from 93 to 100%). The assays were also concordant for samples with results that differed from clinical diagnosis, including 2 PD patients determined to be clinically inconsistent with PD at later time points. All three assays also detected 2 SWEDD subjects as αSyn-SAA positive who later developed PD with abnormal DAT-SPECT. These multi-laboratory results confirm the reproducibility and value of αSyn-SAA as diagnostic tools, illustrate reproducibility of the assay in expert hands, and suggest that αSyn-SAA has potential to provide earlier diagnosis with comparable or superior accuracy to existing methods.

Background: Idiopathic normal pressure hydrocephalus (iNPH) is a clinico-radiological syndrome of elderly individuals likely sustained by different neurodegenerative changes as copathologies. Since iNPH is a potentially reversible condition, assessing neurodegenerative pathologies in vitam through CSF biomarkers and their influence on clinical features and surgical outcome represents crucial steps. Methods: We measured α-synuclein seeding activity related to Lewy body (LB) pathology by the real-time quaking-induced conversion assay (RT-QuIC) and Alzheimer disease core biomarkers (proteins total-tau, phospho-tau, and amyloid-beta) by immunoassays in the cerebrospinal fluid (CSF) of 293 iNPH patients from two independent cohorts. To compare the prevalence of LB copathology between iNPH participants and a control group representative of the general population, we searched for α-synuclein seeding activity in 89 age-matched individuals who died of Creutzfeldt-Jakob disease (CJD). Finally, in one of the iNPH cohorts, we also measured the CSF levels of neurofilament light chain protein (NfL) and evaluated the association between all CSF biomarkers, baseline clinical features, and surgery outcome at 6 months. Results: Sixty (20.5%) iNPH patients showed α-synuclein seeding activity with no significant difference between cohorts. In contrast, the prevalence observed in CJD was only 6.7% (p = 0.002). Overall, 24.0% of iNPH participants showed an amyloid-positive (A+) status, indicating a brain co-pathology related to Aβ deposition. At baseline, in the Italian cohort, α-synuclein RT-QuIC positivity was associated with higher scores on axial and upper limb rigidity (p = 0.003 and p = 0.011, respectively) and lower MMSEc scores (p = 0.003). A+ patients showed lower scores on the MMSEc (p = 0.037) than A- patients. Higher NfL levels were also associated with lower scores on the MMSEc (rho = -0.213; p = 0.021). There were no significant associations between CSF biomarkers and surgical outcome at 6 months (i.e. responders defined by decrease of 1 point on the mRankin scale). Conclusions: Prevalent LB- and AD-related neurodegenerative pathologies affect a significant proportion of iNPH patients and contribute to cognitive decline (both) and motor impairment (only LB pathology) but do not significantly influence the surgical outcome at 6 months. Their effect on the clinical benefit after surgery over a more extended period remains to be determined. [ABSTRACT FROM AUTHOR]

α-Synuclein (αS) is remarkable for both its extensive conformational plasticity and pathologic prion-like properties. Physiologically, αS may populate disordered monomeric, helically folded tetrameric, or membrane-bound oligomeric states. Pathologically, αS may assemble into toxic oligomers and subsequently fibrils, the prion-like transmission of which is implicated in a class of neurodegenerative disorders collectively termed α-synucleinopathies. Notably, αS does not adopt a single "amyloid fold", but rather exists as structurally distinct amyloid-like conformations referred to as "strains". The inoculation of animal models with different strains induces distinct pathologies, and emerging evidence suggests that the propagation of disease-specific strains underlies the differential pathologies observed in patients with different α-synucleinopathies. The characterization of αS strains has provided insight into the structural basis for the overlapping, yet distinct, symptoms of Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. In this review, we first explore the physiological and pathological differences between conformational states of αS. We then discuss recent studies on the influence of micro-environmental factors on αS species formation, propagation, and the resultant pathological characteristics. Lastly, we review how an understanding of αS conformational properties has been translated to emerging strain amplification technologies, which have provided further insight into the role of specific strains in distinct α-synucleinopathies, and show promise for the early diagnosis of disease. [ABSTRACT FROM AUTHOR]

Objective: Real-time quaking-induced conversion (RT-QuIC) is a novel in vitro acellular seed amplification analysis and has been widely used to detect prion diseases. Due to the similar mechanism of abnormal aggregation of α-synuclein, RT-QuIC has great potential for diagnosing Lewy body diseases. This meta-analysis was performed to evaluate the diagnostic accuracy of RT-QuIC for Lewy body diseases.Methods: This study followed the PRISMA statement. We searched six databases for relevant studies published until February 20, 2022. Meta-analysis was conducted using RevMan 5.3, Stata 17.0, and Meta-Disc 1.4. Subgroup analyses were performed to explore sources of heterogeneity.Results: A total of 16 studies were included in this study. The pooled sensitivity and specificity were 0.91 (95%CI: 0.85-0.94) and 0.95 (95%CI: 0.90-0.97), respectively. The pooled positive and negative likelihood ratios were 17.16 (95% CI: 9.16-32.14) and 0.10 (95% CI: 0.06-0.17), respectively. The pooled diagnostic odds rate and area under the summary receiver operating characteristic curve were 171.16 (95% CI: 66.64-439.62) and 0.97 (95% CI: 0.96-0.99), respectively.Conclusions: This study was the first meta-analysis on RT-QuIC for Lewy body diseases. RT-QuIC is a reliable and accurate method to diagnose Lewy body diseases. [ABSTRACT FROM AUTHOR]

Approximately half of Alzheimer's disease (AD) brains have concomitant Lewy pathology at autopsy, suggesting that α-synuclein (α-SYN) aggregation is a regulated event in the pathogenesis of AD. Genome-wide association studies revealed that the ε4 allele of the apolipoprotein E (APOE4) gene, the strongest genetic risk factor for AD, is also the most replicated genetic risk factor for Lewy body dementia (LBD), signifying an important role of APOE4 in both amyloid-β (Aβ) and α-SYN pathogenesis. How APOE4 modulates α-SYN aggregation in AD is unclear. In this study, we aimed to determine how α-SYN is associated with AD-related pathology and how APOE4 impacts α-SYN seeding and toxicity. We measured α-SYN levels and their association with other established AD-related markers in brain samples from autopsy-confirmed AD patients (N = 469), where 54% had concomitant LB pathology (AD + LB). We found significant correlations between the levels of α-SYN and those of Aβ40, Aβ42, tau and APOE, particularly in insoluble fractions of AD + LB. Using a real-time quaking-induced conversion (RT-QuIC) assay, we measured the seeding activity of soluble α-SYN and found that α-SYN seeding was exacerbated by APOE4 in the AD cohort, as well as a small cohort of autopsy-confirmed LBD brains with minimal Alzheimer type pathology. We further fractionated the soluble AD brain lysates by size exclusion chromatography (SEC) ran on fast protein liquid chromatography (FPLC) and identified the α-SYN species (~ 96 kDa) that showed the strongest seeding activity. Finally, using human induced pluripotent stem cell (iPSC)-derived neurons, we showed that amplified α-SYN aggregates from AD + LB brain of patients with APOE4 were highly toxic to neurons, whereas the same amount of α-SYN monomer was not toxic. Our findings suggest that the presence of LB pathology correlates with AD-related pathologies and that APOE4 exacerbates α-SYN seeding activity and neurotoxicity, providing mechanistic insight into how APOE4 affects α-SYN pathogenesis in AD. [ABSTRACT FROM AUTHOR]

Various disease-associated forms or strains of α-synuclein (αSynD) can spread and accumulate in a prion-like fashion during synucleinopathies such as Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA). This capacity for self-propagation has enabled the development of seed amplification assays (SAAs) that can detect αSynD in clinical samples. Notably, α-synuclein real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) assays have evolved as ultrasensitive, specific, and relatively practical methods for detecting αSynD in a variety of biospecimens including brain tissue, CSF, skin, and olfactory mucosa from synucleinopathy patients. However, αSyn SAAs still lack concordance in detecting MSA and familial forms of PD/DLB, and the assay parameters show poor correlations with various clinical measures. End-point dilution analysis in αSyn RT-QuIC assays allows for the quantitation of relative amounts of αSynD seeding activity that may correlate moderately with clinical measures and levels of other biomarkers. Herein, we review recent advancements in α-synuclein SAAs for detecting αSynD and describe in detail the modified Spearman–Karber quantification algorithm used with end-point dilutions. [ABSTRACT FROM AUTHOR]

Background: Multiple system atrophy (MSA) is a neurodegenerative condition characterized by variable combinations of parkinsonism, autonomic failure, cerebellar ataxia and pyramidal features. Although the distribution of synucleinopathy correlates with the predominant clinical features, the burden of pathology does not fully explain observed differences in clinical presentation and rate of disease progression. We hypothesized that the clinical heterogeneity in MSA is a consequence of variability in the seeding activity of α-synuclein both between different patients and between different brain regions. Methods: The reliable detection of α-synuclein seeding activity derived from MSA using cell-free amplification assays remains challenging. Therefore, we conducted a systematic evaluation of 168 different reaction buffers, using an array of pH and salts, seeded with fully characterized brain homogenates from one MSA and one PD patient. We then validated the two conditions that conferred the optimal ability to discriminate between PD- and MSA-derived samples in a larger cohort of 40 neuropathologically confirmed cases, including 15 MSA. Finally, in a subset of brains, we conducted the first multi-region analysis of seeding behaviour in MSA. Results: Using our novel buffer conditions, we show that the physicochemical factors that govern the in vitro amplification of α-synuclein can be tailored to generate strain-specific reaction buffers that can be used to reliably study the seeding capacity from MSA-derived α-synuclein. Using this novel approach, we were able to sub-categorize the 15 MSA brains into 3 groups: high, intermediate and low seeders. To further demonstrate heterogeneity in α-synuclein seeding in MSA, we conducted a comprehensive multi-regional evaluation of α-synuclein seeding in 13 different regions from 2 high seeders, 2 intermediate seeders and 2 low seeders. Conclusions: We have identified unexpected differences in seed-competent α-synuclein across a cohort of neuropathologically comparable MSA brains. Furthermore, our work has revealed a substantial heterogeneity in seeding activity, driven by the PBS-soluble α-synuclein, between different brain regions of a given individual that goes beyond immunohistochemical observations. Our observations pave the way for future subclassification of MSA, which exceeds conventional clinical and neuropathological phenotyping and considers the structural and biochemical heterogeneity of α-synuclein present. Finally, our methods provide an experimental framework for the development of vitally needed, rapid and sensitive diagnostic assays for MSA. [ABSTRACT FROM AUTHOR]

In synucleinopathies such as Parkinson’s disease (PD) and dementia with Lewy body (DLB), pathological alpha-synuclein (α-syn) aggregates are found in the gastrointestinal (GI) tract as well as in the brain. In this study, using real-time quaking-induced conversion (RT-QuIC), we investigated the presence of α-syn seeding activity in the brain and colon tissue of G2-3 transgenic mice expressing human A53T α-syn. Here we show that pathological α-syn aggregates with seeding activity were present in the colon of G2-3 mice as early as 3 months old, which is in the presymptomatic stage prior to the observation of any neurological abnormalities. In contrast, α-syn seeding activity was not detectable in 3 month-old mouse brains and only identified at 6 months of age in one of three mice. In the symptomatic stage of 12 months of age, RT-QuIC seeding activity was consistently detectable in both the brain and colon of G2-3 mice. Our results indicate that the RT-QuIC assay can presymptomatically detect pathological α-syn aggregates in the colon of G2-3 mice several months prior to their detection in brain tissue.