Chicago sky blue 6B inhibits α-synuclein aggregation and propagation

Abnormal deposition of α-synuclein aggregates in Lewy bodies and Lewy neurites is the hallmark lesion in Parkinsons disease (PD). These aggregates, thought to be the culprit of disease pathogenesis, spread throughout the brain as the disease progresses. Agents that inhibit α-synuclein aggregation and/or spread of aggregates would thus be candidate disease-modifying drugs. Here, we found that Chicago sky blue 6B (CSB) may be such a drug, showing that it inhibits α-synuclein aggregation and cell-to-cell propagation in both in vitro and in vivo models of synucleinopathy. CSB inhibited the fibrillation of α-synuclein in a concentration-dependent manner through direct binding to the N-terminus of α-synuclein. Furthermore, both seeded polymerization and cell-to-cell propagation of α-synuclein were inhibited by CSB treatment. Notably, CSB alleviated behavioral deficits and neuropathological features, such as phospho-α-synuclein and astrogliosis, in A53T α-synuclein transgenic mice. These results indicate that CSB directly binds α-synuclein and inhibits its aggregation, thereby blocking α-synuclein cell-to-cell propagation.This work was supported by National Research Foundation (NRF) Grants funded by the Korean Government (MEST) (NRF-2018R1A5A2025964, NRF2021R1A2C3012681 to S.-J.L.), and the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI19C0256 to S.-J.L.). J.-O.M received a scholarship from the BK21 FOUR education program. MZ was supported by the DFG Collaborative Research Center SFB860 (project B2)

Interaction of Cu(i) with the Met-X3-Met motif of alpha-synuclein: binding ligands, affinity and structural features

The identity of the Cu(i) binding ligands at Met-X3-Met site of AcαS and its role into the affinity and structural properties of the interaction were elucidated by NMR spectroscopy. We provide evidence that the source of ligands for Cu(i) binding to the Met-X3-Met site comes from the N-terminal acetyl group and the Met-1, Asp-2 and Met-5 residues. From the study of site-directed mutants and synthetic peptide models of αS we demonstrated the critical role played by Met-1 and Met-5 residues on the binding affinity of the Cu(i) complex, acting as the main metal anchoring residues. While having a more modest impact in the affinity features of Cu(i) binding, as compared to the Met residues, the N-terminal acetyl group and Asp-2 are important in promoting local helical conformations, contributing to the stabilization of these structures by favoring Cu(i) binding.Fil: Gentile, Iñaki. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; ArgentinaFil: Garro, Hugo Alejandro. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Delgado Ocaña, Susana. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: González, Nazareno. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Strohäker, Timo. Max Planck Institute For Biophysical Chemistry; AlemaniaFil: Schibich, Daniela. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Quintanar, Liliana. Centro de Investigación y de Estudios; MéxicoFil: Sambrotta, Luis Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Zweckstetter, Markus. Max Planck Institute For Biophysical Chemistry; Alemania. Deutsches Zentrum Für Neurodegenerative Erkrankungen; AlemaniaFil: Griesinger, Christian. Max Planck Institute For Biophysical Chemistry; AlemaniaFil: Menacho Márquez, Mauricio Ariel. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnol.conicet - Rosario. Unidad de Direccion; ArgentinaFil: Fernandez, Claudio Oscar. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; Argentina. Max Planck Institute For Biophysical Chemistry; Alemani

Alpha‐synuclein fibrils amplified from multiple system atrophy and Parkinson's disease patient brain spread after intracerebral injection into mouse brain

Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB) are neurodegenerative disorders with alpha-synuclein (α-syn) aggregation pathology. Different strains of α-syn with unique properties are suggested to cause distinct clinical and pathological manifestations resulting in PD, MSA, or DLB. To study individual α-syn spreading patterns, we injected α-syn fibrils amplified from brain homogenates of two MSA patients and two PD patients into the brains of C57BI6/J mice. Antibody staining against pS129-α-syn showed that α-syn fibrils amplified from the brain homogenates of the four different patients caused different levels of α-syn spreading. The strongest α-syn pathology was triggered by α-syn fibrils of one of the two MSA patients, followed by comparable pS129-α-syn induction by the second MSA and one PD patient material. Histological analysis using an antibody against Iba1 further showed that the formation of pS129-α-syn is associated with increased microglia activation. In contrast, no differences in dopaminergic neuron numbers or co-localization of α-syn in oligodendrocytes were observed between the different groups. Our data support the spreading of α-syn pathology in MSA, while at the same time pointing to spreading heterogeneity between different patients potentially driven by individual patient immanent factors

Big data and ICT solutions in the European Union and in China: A comparative analysis of policies in personalized medicine

Introduction: Several countries are either planning or implementing national strategies for the development and integration of Personalized Medicine (PM) into their healthcare systems. Personalized Medicine is an undisputed priority of the European Commission (EC), which has funded the project "Integrating China into the International Consortium for Personalized Medicine" (IC2PerMed), in order to ensure a common basis for Sino-European collaborations. By mapping the current PM landscape in the European Union (EU) and in China, IC2PerMed aims to provide key solutions toward a synergistic and coordinated approach in the field of PM. Methods: An extensive desk research was conducted, aimed at identifying documents on PM-related policies, programs, and action plans in the EU and in China, published up to November 2020. The search was conducted by exploring scientific and gray literature, and official institutional repositories. A descriptive summary condensed the information retrieved for both. Results: Since 2013, the year of publication of the first PM policy by the EC "Use of omics technologies in PM development," several documents have been published. PM is a key element of the policy agenda also in China, which in 2016 integrated PM into the 13th National Five-Year Plan, followed by the publication of several policies on technology infrastructure and big data. Both in the EU and China, especially in recent years, these policies addressed in detail the issues of big data, data interoperability and exchange, while defining the standards of information and communication infrastructures. Conclusions: In order to allow optimal collaboration, it is essential to understand similarities and differences between the respective policy strategies, with particular attention to data management and adopted infrastructures. The results of this project may enable the development of joint Sino-European research and innovation initiatives, promoting developments in the field of PM

Structural characterization of a-synuclein aggregates seeded by patient material

Neurodegenerative diseases share a common underlying pathologic hallmark, the appearance of insoluble protein aggregates in diverse tissues of the nervous system. For many neurodegenerative diseases a common temporal and spatial spreading of the pathology is proposed in analogy to prion disease and discussed under the term “prion-like”. For many diseases the major component of the insoluble protein aggregates is known and aggregation into higher molecular weight amyloid fibrils with intermolecular b-sheet rich cores can be studied in vitro. The aggregation process involves the templated misfolding and aggregation of native monomeric proteins, involving severe conformational changes. An important family of neurodegenerative diseases is caused by the misfolding and aggregation of the protein a-synuclein, the so-called synucleinopathies. asynuclein, which in vivo forms disease-specifically the main component of intracellular inclusions such as Lewy bodies in neurons and cytoplasmatic inclusions in glial cells, undergoes in vitro dramatic conformational changes from a monomeric intrinsically disordered state over transient oligomeric b-sheet rich species into highly ordered asynuclein fibrils. a-synuclein pathology in patients is diverse and there are clinically distinct disease entities with defined pathologic phenotypes among those Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are the best characterized. Similar to prion diseases, key differences within the broad clinical representation of synucleinopathies are thought to be structurally encoded by distinct protein aggregate conformations, referred to as a-synuclein polymorphs. The aim of the study was to amplify a-synuclein aggregates from brain extracts of patients thoroughly diagnosed on the basis of the molecular pathology as well as the clinical symptoms as PD, DLB and MSA, using the established protocol of protein misfolding cyclic amplification (PMCA). A combination of hydrogen-deuterium (HD) exchange coupled to nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) and the specific binding of fluorescent probes to amyloid fibrils was chosen to obtain single-residue resolution of the conformational properties of brain-extract seeded a-synuclein fibrils. The same approach was also applied to two well-characterized in vitro a-synuclein polymorphs, their aggregation was performed in the absence of brain extract seeds following published aggregation procedures and they acted as internal references for benchmarking the methodological approach. On the other hand, the availability of a high-resolution cryo-electron VII microscopy model of the fibrillar core for one of the in vitro a-synuclein polymorphs obtained under high salt conditions, allowed direct correlation of the residue-specific conformational restraints to a structural model, both for in vitro polymorphs of asynuclein as well as brain-extract amplified a-synuclein fibrils of PD, MSA and DLB. Distinct highly ordered conformational features of in vitro a-synuclein fibrils were successfully reproduced, detecting solvent-protected residues with high precision and in agreement with published data. In contrast, a-synuclein fibrils amplified from brain extracts were more flexible and differed structurally from in vitro fibrils. Hydrogendeuterium exchange coupled to NMR spectroscopy identified a common solventprotected core shared among all patient brain derived a-synuclein fibrils for the synucleinopathies PD, MSA and DLB. The solvent-protected fibrillar core was formed by the most hydrophobic residues of a-synuclein. Outside the common core structure, a-synuclein fibrils derived from brain extracts differed disease-specifically in the conformation. Residue-specific conformational differences in core-flanking residues of a-synuclein as well as in defined N-terminal regions were observed. This study establishes a strong correlation between a-synuclein aggregate structure and the disease phenotype for the synucleinopathies Parkinson’s disease, Dementia with Lewy bodies and multiple system atrophy and the data provide further insight in “prion-like” features of neurodegenerative diseases in general and synucleinopathies in particular. The work presented here is a step forward towards the understanding of a-synuclein pathology and hopefully contributes to improved disease diagnosis and treatment of synucleinopathies.Fil: Strohäker, Timo. Centro de Estudios Interdisciplinarios. Universidad Nacional de Rosario; Argentina. Georg-August-Universität Göttingen; Alemani

β-Sheet Augmentation Is a Conserved Mechanism of Priming HECT E3 Ligases for Ubiquitin Ligation

Ubiquitin (Ub) ligases (E3s) catalyze the attachment of Ub chains to target proteins and thereby regulate a wide array of signal transduction pathways in eukaryotes. In HECT-type E3s, Ub first forms a thioester intermediate with a strictly conserved Cys in the C-lobe of the HECT domain and is then ligated via an isopeptide bond to a Lys residue in the substrate or a preceding Ub in a poly-Ub chain. To date, many key aspects of HECT-mediated Ub transfer have remained elusive. Here, we provide structural and functional insights into the catalytic mechanism of the HECT-type ligase Huwe1 and compare it to the unrelated, K63-specific Smurf2 E3, a member of the Nedd4 family. We found that the Huwe1 HECT domain, in contrast to Nedd4-family E3s, prioritizes K6- and K48-poly-Ub chains and does not interact with Ub in a non-covalent manner. Despite these mechanistic differences, we demonstrate that the architecture of the C-lobe Ub intermediate is conserved between Huwe1 and Smurf2 and involves a reorientation of the very C-terminal residues. Moreover, in Nedd4 E3s and Huwe1, the individual sequence composition of the Huwe1 C-terminal tail modulates ubiquitination activity, without affecting thioester formation. In sum, our data suggest that catalysis of HECT ligases hold common features, such as the 13-sheet augmentation that primes the enzymes for ligation, and variable elements, such as the sequence of the HECT C-terminal tail, that fine-tune ubiquitination activity and may aid in determining Ub chain specificity by positioning the substrate or acceptor Ub. (C) 2018 Elsevier Ltd. All rights reserved

Structural heterogeneity of α-synuclein fibrils amplified from patient brain extracts.

Parkinsons disease (PD) and Multiple System Atrophy (MSA) are clinically distinctive diseases that feature a common neuropathological hallmark of aggregated α-synuclein. Little is known about how differences in α-synuclein aggregate structure affect disease phenotype. Here, we amplified α-synuclein aggregates from PD and MSA brain extracts and analyzed the conformational properties using fluorescent probes, NMR spectroscopy and electron paramagnetic resonance. We also generated and analyzed several in vitro α-synuclein polymorphs. We found that brain-derived α-synuclein fibrils were structurally different to all of the in vitro polymorphs analyzed. Importantly, there was a greater structural heterogeneity among α-synuclein fibrils from the PD brain compared to those from the MSA brain, possibly reflecting on the greater variability of disease phenotypes evident in PD. Our findings have significant ramifications for the use of non-brain-derived α-synuclein fibrils in PD and MSA studies, and raise important questions regarding the one disease-one strain hypothesis in the study of α-synucleinopathies

Quaternary structure of patient-homogenate amplified α-synuclein fibrils modulates seeding of endogenous α-synuclein

Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are progressive and unremitting neurological diseases that are neuropathologically characterized by α-synuclein inclusions. Increasing evidence supports the aggregation of α-synuclein in specific brain areas early in the disease course, followed by the spreading of α-synuclein pathology to multiple brain regions. However, little is known about how the structure of α-synuclein fibrils influence its ability to seed endogenous α-synuclein in recipient cells. Here, we aggregated α-synuclein by seeding with homogenates of PD- and MSA-confirmed brain tissue, determined the resulting α-synuclein fibril structures by cryo-electron microscopy, and characterized their seeding potential in mouse primary oligodendroglial cultures. The combined analysis shows that the two patient material-amplified α-synuclein fibrils share a similar protofilament fold but differ in their inter-protofilament interface and their ability to recruit endogenous α-synuclein. Our study indicates that the quaternary structure of α-synuclein fibrils modulates the seeding of α-synuclein pathology inside recipient cells. It thus provides an important advance in the quest to understand the connection between the structure of α-synuclein fibrils, cellular seeding/spreading, and ultimately the clinical manifestations of different synucleinopathies