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Synucleinopathies, including dementia with Lewy bodies (DLB), Parkinson's disease (PD), and multiple system atrophy (MSA), are characterized by the presence of α-synuclein (α-syn) aggregates in the central nervous system. Recent evidence suggests that the heterogeneity of synucleinopathies may be partly explained by the fact that patients may have different α-syn fibrillar polymorphs with structural differences. In this study, we identify nuclease resistant 2'fluoro-pyrimidine RNA aptamers that can differentially bind to structurally distinct α-syn fibrillar polymorphs. Moreover, we introduce a method, AptaFOOT-Seq, designed to rapidly assess the affinity of a mixture of these aptamers for different α-SYN fibrillar polymorphs using next-generation sequencing. Our findings reveal that the binding behavior of aptamers can be very different when they are tested separately or in the presence of other aptamers. In this case, competition and cooperation can occur, providing a higher level of information, which can be exploited to obtain specific 'footprints' for different α-Syn fibrillar polymorphs. Notably, these footprints can distinguish polymorphs obtained from patients with PD, DLB or MSA. This result suggests that aptaFOOT-Seq could be used for the detection of misfolded or abnormal protein conformations to improve the diagnosis of synucleinopathies., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Alpha-synuclein (aSyn) aggregation spreads between cells and underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson's diseases. The mechanisms of cell-to-cell propagation of aggregates, which dictate how aggregation progresses at the network level, remain poorly understood. Notably, while prion and prion-like spreading is often simplistically envisioned as a "domino-like" spreading scenario where connected neurons sequentially propagate protein aggregation to each other, the reality is likely to be more nuanced. Here, we demonstrate that the spreading of preformed aSyn aggregates is a limited process that occurs through molecular sieving of large aSyn seeds. We further show that this process is not facilitated by synaptic connections. This was achieved through the development and characterization of a new microfluidic platform that allows reconstruction of binary fully oriented neuronal networks in vitro with no unwanted backward connections, and through the careful quantification of fluorescent aSyn aggregates spreading between neurons. While this allowed us for the first time to extract quantitative data of protein seeds dissemination along neural pathways, our data suggest that prion-like dissemination of proteinopathic seeding aggregates occurs very progressively and leads to highly compartmentalized pattern of protein seeding in neural networks., (© 2023. The Author(s).)

The role of alpha-synuclein in Parkinson's disease has been heavily investigated since its discovery as a component of Lewy bodies. Recent rodent data demonstrate that alpha-synuclein strain structure is critical for differential propagation and toxicity. Based on these findings, we have compared, for the first time, in this pilot study, the capacity of two alpha-synuclein strains and patient-derived Lewy body extracts to model synucleinopathies after intra-putaminal injection in the non-human primate brain. Functional alterations triggered by these injections were evaluated in vivo using glucose positron emission tomography imaging. Post-mortem immunohistochemical and biochemical analyses were used to detect neuropathological alterations in the dopaminergic system and alpha-synuclein pathology propagation. In vivo results revealed a decrease in glucose metabolism more pronounced in alpha-synuclein strain-injected animals. Histology showed a decreased number of dopaminergic tyrosine hydroxylase-positive cells in the substantia nigra to different extents according to the inoculum used. Biochemistry revealed that alpha-synuclein-induced aggregation, phosphorylation, and propagation in different brain regions are strain-specific. Our findings show that distinct alpha-synuclein strains can induce specific patterns of synucleinopathy in the non-human primate, changes in the nigrostriatal pathway, and functional alterations that resemble early-stage Parkinson's disease., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Aggregated alpha-synuclein (α-Syn) in neurons is a hallmark of Parkinson's disease (PD) and other synucleinopathies. Recent advances (1) in the production and purification of synthetic assemblies of α-Syn, (2) in the design and production of microfluidic devices allowing the construction of oriented and compartmentalized neuronal network on a chip, and (3) in the differentiation of human pluripotent stem cells (hPSCs) into specific neuronal subtypes now allow the study of cellular and molecular determinants of the prion-like properties of α-Syn in vitro. Here, we described the methods we used to reconstruct a cortico-cortical human neuronal network in microfluidic devices and how to take advantage of this cellular model to characterize (1) the prion-like properties of different α-Syn strains and (2) the neuronal dysfunctions and the alterations associated with the exposure to α-Syn strains or the nucleation of endogenous α-Syn protein in vitro., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

An emerging view regarding neurodegenerative diseases is that discreet seeding of misfolded proteins leads to widespread pathology. However, the mechanisms by which misfolded proteins seed distinct brain regions and cause differential whole-brain pathology remain elusive. We used whole-brain tissue clearing and high-resolution imaging to longitudinally map pathology in an α-synuclein pre-formed fibril injection model of Parkinson's disease. Cleared brains at different time points of disease progression were quantitatively segmented and registered to a standardized atlas, revealing distinct phases of spreading and decline. We then fit a computational model with parameters that represent α-synuclein pathology spreading, aggregation, decay, and gene expression pattern to this longitudinal dataset. Remarkably, our model can generalize to predicting α-synuclein spreading patterns from several distinct brain regions and can even estimate their origins. This model empowers mechanistic understanding and accurate prediction of disease progression, paving the way for the development and testing of therapeutic interventions., Competing Interests: Declaration of interests J.H.L. is a founder, consultant, and board member of LVIS., (Copyright © 2022. Published by Elsevier Inc.)

Background: Preclinical rodent models for Parkinson's disease (PD) based on viral human alpha-synuclein (h-αSyn) overexpression recapitulate some of the pathological hallmarks as it presents in humans, such as progressive cell loss and additional synucleinopathy in cortical and subcortical structures. Recent studies have combined viral vector-based overexpression of human wild-type αSyn with the sequential or simultaneous inoculation of preformed fibrils (PFFs) derived from human αSyn., Objective: The goal of the study was to investigate whether sequential or combined delivery of the AAV vector and the PFFs are equipotent in inducing stable neurodegeneration and behavioral deficits., Methods: Here we compare between four experimental paradigms (PFFs only, AAV-h-αSyn only, AAV-h-αSyn with simultaneous PFFs, and AAV-h-αSyn with sequential PFFs) and their respective GFP control groups., Results: We observed reduction of TH expression and loss of neurons in the midbrain in all AAV (h-αSyn or GFP) injected groups, with or without additional PFFs inoculation. The overexpression of either h-αSyn or GFP alone induced motor deficits and dysfunctional dopamine release/reuptake in electrochemical recordings in the ipsilateral striatum. However, we observed a substantial formation of insoluble h-αSyn aggregates and inflammatory response only when h-αSyn and PFFs were combined. Moreover, the presence of h-αSyn induced higher axonal pathology compared to control groups., Conclusion: Simultaneous AAV and PFFs injections are equipotent in the presented experimental setup in inducing histopathological and behavioral changes. This model provides new and interesting possibilities for characterizing PD pathology in preclinical models and means to assess future therapeutic interventions.

While the initial pathology of Parkinson's disease and other α-synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of α-synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analysed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α-synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α-synuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion, and the seeded induction of α-synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α-synucleinopathies is not universally valid., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression., Methods: Alpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed., Results: To study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2-3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture's genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions., Conclusion: The successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments., (© 2021. The Author(s).)

Accumulation and aggregation of α-synuclein (αSyn) trigger neuronal loss in the substantia nigra pars compacta (SNpc), which in turn causes motor symptoms in Parkinson's disease. We previously demonstrated that fatty acid-binding protein 3 (FABP3), an intracellular fatty acid carrier protein, enhances αSyn neurotoxicity in SNpc and motor impairments after intranigral injection of αSyn fibrils. However, the temporal profile of αSyn fibril spread and their toxicity remains unclear. In the present study, we investigated the temporal profile of αSyn fibril spread and its toxicity, which induces intracellular fibril formation. Monomeric and fibrillar aSyn assemblies were labeled with ATTO550 to distinguish the exogenous form from the endogenous species and injected into bilateral striatum in Fabp3 +/+ (wild type) and Fabp3 -/- mice. Accumulation of both monomeric and fibrillar exogenous αSyn in the SNpc was drastically decreased in Fabp3 -/- mice compared to that in the Fabp3 +/+ counterparts. Deletion of Fabp3 also prevented exogenous αSyn fibril-induced seeding of the endogenous αSyn into aggregates containing phosphorylated and filamentous forms in the SNpc. Consistent with these results, loss of dopaminergic neurons and subsequent impaired motor behavior were attenuated in Fabp3 -/- mice. These results highlight the crucial role of FABP3 in pathogenic αSyn accumulation and its seeding ability. Taken together, FABP3 could be a potential therapeutic target against αSyn propagation in synucleinopathies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In synucleinopathies, while motor symptoms are thought to be attributed to the accumulation of misfolded α-synuclein (αSyn) in nigral dopaminergic neurons, it remains to be elucidated how cognitive decline arises. Here, we investigated the effects of distinct αSyn strains on cognition and the related neuropathology in the medial septum/diagonal band (MS/DB), a key region for cognitive processing. Bilateral injection of αSyn fibrils into the dorsal striatum potently impaired cognition in mice. The cognitive decline was accompanied by accumulation of phosphorylated αSyn at Ser129 and reduction of gamma-aminobutyric acid (GABA)-ergic but not cholinergic neurons in the MS/DB. Since we have demonstrated that fatty acid-binding protein 3 (FABP3) is critical for αSyn neurotoxicity in nigral dopaminergic neurons, we investigated whether FABP3 also participates in αSyn pathology in the MS/DB and cognitive decline. FABP3 was highly expressed in GABAergic but rarely in cholinergic neurons in the MS/DB. Notably, Fabp3 deletion antagonized the accumulation of phosphorylated αSyn, decrease in GABAergic neurons, and cognitive impairment caused by αSyn fibrils. Overall, the present study indicates that FABP3 mediates αSyn neurotoxicity in septal GABAergic neurons and the resultant cognitive impairment, and that FABP3 in this subpopulation could be a therapeutic target for dementia in synucleinopathies.

Synucleinopathies are neurodegenerative diseases characterized by the presence of intracellular deposits containing the protein alpha-synuclein (aSYN) within patients' brains. It has been shown that aSYN can form structurally distinct fibrillar assemblies, also termed polymorphs. We previously showed that distinct aSYN polymorphs assembled in vitro, named fibrils, ribbons, and fibrils 91, differentially bind to and seed the aggregation of endogenous aSYN in neuronal cells, which suggests that distinct synucleinopathies may arise from aSYN polymorphs. In order to better understand the differential interactions of aSYN polymorphs with their partner proteins, we mapped aSYN polymorphs surfaces. We used limited proteolysis, hydrogen-deuterium exchange, and differential antibody accessibility to identify amino acids on their surfaces. We showed that the aSYN C-terminal region spanning residues 94 to 140 exhibited similarly high solvent accessibility in these three polymorphs. However, the N-terminal amino acid residues 1 to 38 of fibrils were exposed to the solvent, while only residues 1 to 18 within fibrils 91 were exposed, and no N-terminal residues within ribbons were solvent-exposed. It is likely that these differences in surface accessibility contribute to the differential binding of distinct aSYN polymorphs to partner proteins. We thus posit that the polypeptides exposed on the surface of distinct aSYN fibrillar polymorphs are comparable to fingerprints. Our findings have diagnostic and therapeutic potential, particularly in the prion-like propagation of fibrillar aSYN, as they can facilitate the design of ligands that specifically bind and distinguish between fibrillar polymorphs., Competing Interests: Conflict of interest S. E. is full-time employee of SANOFI R&D., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Alpha-synuclein (α-Syn) aggregation is the primary characteristic of synucleinopathies including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Immunotherapy targeting α-Syn has shown promising results in animal models of the disease. This study investigates the target specificity of three different active vaccines for pathological α-Syn aggregates found in human brain tissue from synucleinopathies., Methods: Guinea pigs were immunised with 3 vaccines developed by United Neuroscience, and IgG fractions purified from the resulting immune sera (IGG-1, IGG-2 or IGG-3) were used to perform immunohistochemical staining of human cases of PD, DLB and MSA. The resulting immunoreactivity was compared to a commercially available α-Syn antibody from Novacastra (NOV) commonly used for diagnostic purposes. Images were captured from the substantia nigra (SN), temporal lobe, internal capsule, insular cortex and putamen and quantified for the percentage area with α-Syn immunoreactivity. Lewy bodies (LB) and Lewy neurites (LN) were further analysed in PD and DLB cases., Results: Vaccine-generated antibodies detected more α-Syn pathology compared to NOV. The levels of α-Syn immunoreactivity varied between brain region and disease type with IGG-3 recognising the highest levels of α-Syn in most cases and in all brain regions that are affected early in disease progression. IGG-3 had a high recognition for glial inclusions found in MSA which are known to have a more compact conformation. Slot blot analysis confirmed the specificity of IGG-3 for native oligomers and fibrillar α-Syn. Higher levels of α-Syn were recognised by IGG-2 in cortical regions, and by IGG-3 in SN of PD and DLB cases. This was due to increased immunolabelling of LNs in these brain regions suggesting that IGG-2 and IGG-3 recognised additional α-Syn pathology compared to IGG-1 and NOV. Whether the unique binding properties of the antibodies produced in guinea pigs will translate in the clinic remains to be addressed, which is the main limitation of this study., Conclusions: These vaccines induce antibodies that bind α-Syn oligomers and aggregates in the human brain and specifically support the choice of the vaccine generating IGG-3 (i.e. UB-312) as a candidate for clinical trials for synucleinopathies.

The pathophysiology of Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and many others converge at alpha-synuclein (α-Syn) aggregation. Although it is still not entirely clear what precise biophysical processes act as triggers, cumulative evidence points towards a crucial role for protein quality control (PQC) systems in modulating α-Syn aggregation and toxicity. These encompass distinct cellular strategies that tightly balance protein production, stability, and degradation, ultimately regulating α-Syn levels. Here, we review the main aspects of α-Syn biology, focusing on the cellular PQC components that are at the heart of recognizing and disposing toxic, aggregate-prone α-Syn assemblies: molecular chaperones and the ubiquitin-proteasome system and autophagy-lysosome pathway, respectively. A deeper understanding of these basic protein homeostasis mechanisms might contribute to the development of new therapeutic strategies envisioning the prevention and/or enhanced degradation of α-Syn aggregates.

Alpha-synuclein inclusions, the hallmarks of synucleinopathies, are suggested to spread along neuronal connections in a stereotypical pattern in the brains of patients. Ample evidence now supports that pathological forms of alpha-synuclein propagate in cell culture models and in vivo in a prion-like manner. However, it is still not known why the same pathological protein targets different cell populations, propagates with different kinetics and leads to a variety of diseases (synucleinopathies) with distinct clinical features. The aggregation of the protein alpha-synuclein yields different conformational polymorphs called strains. These strains exhibit distinct biochemical, physical and structural features they are able to imprint to newly recruited alpha-synuclein. This had led to the view that the clinical heterogeneity observed in synucleinopathies might be due to distinct pathological alpha-synuclein strains.To investigate the pathological effects of alpha-synuclein strains in vivo, we injected five different pure strains we generated de novo (fibrils, ribbons, fibrils-65, fibrils-91, fibrils-110) into the olfactory bulb of wild-type female mice. We demonstrate that they seed and propagate pathology throughout the olfactory network within the brain to different extents. We show strain-dependent inclusions formation in neurites or cell bodies. We detect thioflavin S-positive inclusions indicating the presence of mature amyloid aggregates.In conclusion, alpha-synuclein strains seed the aggregation of their cellular counterparts to different extents and spread differentially within the central nervous system yielding distinct propagation patterns. We provide here the proof-of-concept that the conformation adopted by alpha-synuclein assemblies determines their ability to amplify and propagate in the brain in vivo. Our observations support the view that alpha-synuclein polymorphs may underlie different propagation patterns within human brains.

This review article provides an overview of the different species that α-synuclein aggregates can populate. It also attempts to reconcile conflicting views regarding the cytotoxic roles of oligomers versus fibrils. α-synuclein, while highly dynamic in the monomeric state, can access a large number of different assembly states. Depending on assembly conditions, these states can interconvert over different timescales. The fibrillar state is the most thermodynamically favored due to the many stabilizing interactions formed between each monomeric unit, but different fibrillar types form at different rates. The end distribution is likely to reflect kinetic partitioning as much as thermodynamic equilibra. In addition, metastable oligomeric species, some of which are on-pathway and others off-pathway, can be populated for remarkably long periods of time. Chemical modifications (phosphorylation, oxidation, covalent links to ligands, etc.) perturb these physical interconversions and invariably destabilize the fibrillar state, leading to small prefibrillar assemblies which can coalesce into amorphous states. Both oligomeric and fibrillar species have been shown to be cytotoxic although firm conclusions require very careful evaluation of particle concentrations and is complicated by the great variety and heterogeneity of different experimentally observed states. The mechanistic relationship between oligomers and fibrils remains to be clarified, both in terms of assembly of oligomers into fibrils and potential dissolution of fibrils into oligomers. While oligomers are possibly implicated in the collapse of neuronal homeostasis, the fibrillar state(s) appears to be the most efficient at propagating itself both in vitro and in vivo, pointing to critical roles for multiple different aggregate species in the progression of Parkinson's disease (https://onlinelibrary.wiley.com/page/journal/14714159/homepage/virtual_issues.htm). This article is part of the Special Issue "Synuclein"., (© 2019 International Society for Neurochemistry.)