timer Asked: Apr 20th, 2020

Question Description

I need to present this article in 30 minutes and I thought having a summary would be beneficial in organizing the slides for my presentation. I need a well explained summary that when someone reads it, it would give him a complete idea about whats been mentioned in the article and whats been studied.

I also need to talk about the graphs and diagrams presented in the article, if you guys can give me one sentence or two about each diagram that would be awesome and appreciated it.

Unformatted Attachment Preview

Small molecule inhibits α-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons Jordi Pujolsa,b,1, Samuel Peña-Díaza,b,1, Diana F. Lázaroc,d,e, Francesca Peccatif,g, Francisca Pinheiroa,b, Danilo Gonzálezf, Anita Carijaa,b, Susanna Navarroa,b, María Conde-Giménezh,i, Jesús Garcíaj, Salvador Guardiolaj, Ernest Giraltj,k, Xavier Salvatellaj,l, Javier Sanchoh,i, Mariona Sodupef,l, Tiago Fleming Outeiroc,d,e,m,n, Esther Dalfób,o,p, and Salvador Venturaa,b,l,2 a Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; bDepartament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; cDepartment of Experimental Neurodegeneration, University Medical Center Göttingen, 37073 Göttingen, Germany; dCenter for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, 37073 Göttingen, Germany; e Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, 37073 Göttingen, Germany; fDepartament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; gLaboratoire de Chimie Théorique, Sorbonne Universités, CNRS, F-75005 Paris, France; hDepartment of Biochemistry and Molecular and Cell Biology, University of Zaragoza, 50018 Zaragoza, Spain; iInstitute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, 50018 Zaragoza, Spain; jInstitute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain; kDepartment of Inorganic and Organic Chemistry, University of Barcelona, 08028 Spain; lInstitució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain; mMax Planck Institute for Experimental Medicine, 37075 Göttingen, Germany; nInstitute of Neuroscience, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom; oFaculty of Medicine, University of Vic-Central University of Catalonia (UVic-UCC), 08500 Vic, Spain; and pInstitut de Neurociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain Parkinson’s disease (PD) is characterized by a progressive loss of dopaminergic neurons, a process that current therapeutic approaches cannot prevent. In PD, the typical pathological hallmark is the accumulation of intracellular protein inclusions, known as Lewy bodies and Lewy neurites, which are mainly composed of α-synuclein. Here, we exploited a high-throughput screening methodology to identify a small molecule (SynuClean-D) able to inhibit α-synuclein aggregation. SynuClean-D significantly reduces the in vitro aggregation of wildtype α-synuclein and the familiar A30P and H50Q variants in a substoichiometric molar ratio. This compound prevents fibril propagation in protein-misfolding cyclic amplification assays and decreases the number of α-synuclein inclusions in human neuroglioma cells. Computational analysis suggests that SynuClean-D can bind to cavities in mature α-synuclein fibrils and, indeed, it displays a strong fibril disaggregation activity. The treatment with SynuClean-D of two PD Caenorhabditis elegans models, expressing α-synuclein either in muscle or in dopaminergic neurons, significantly reduces the toxicity exerted by α-synuclein. SynuClean-D–treated worms show decreased α-synuclein aggregation in muscle and a concomitant motility recovery. More importantly, this compound is able to rescue dopaminergic neurons from α-synuclein–induced degeneration. Overall, SynuClean-D appears to be a promising molecule for therapeutic intervention in Parkinson’s disease. | | Parkinson’s disease α-synuclein protein aggregation inhibition dopaminergic degeneration | Interfering with α-Syn aggregation has been envisioned as a promising disease-modifying approach for the treatment of PD (1). However, the disordered nature of α-Syn precludes the use of structure-based drug design for the discovery of novel molecules able to modulate α-Syn aggregation. Therefore, many efforts have focused on the analysis of large collections of chemically diverse molecules to identify lead compounds (10). Recently, we have developed an accurate and robust high-throughput screening methodology to identify α-Syn aggregation inhibitors (11). Here, we describe the properties of SynuClean-D (SC-D), a small molecule identified with this approach (SI Appendix, Fig. S1). We first performed a detailed in vitro biophysical characterization of the inhibitory and disaggregation activities of SC-D and tested its performance in human neural cells. Finally, we validated the effects in vivo in two well-established Caenorhabditis elegans models of PD, which express α-Syn either in muscle cells or in dopaminergic Significance Parkinson’s disease is characterized by the accumulation of amyloid deposits in dopaminergic neurons, mainly composed of the protein α-synuclein. The disordered nature of α-synuclein and its complex aggregation reaction complicate the identification of molecules able to prevent or revert the formation of these inclusions and the subsequent neurodegeneration. By exploiting a recently developed high-throughput screening assay, we identified SynuClean-D, a small compound that inhibits α-synuclein aggregation, disrupts mature amyloid fibrils, prevents fibril propagation, and abolishes the degeneration of dopaminergic neurons in an animal model of Parkinson’s disease. | aggregation Downloaded by guest on April 14, 2020 P arkinson’s disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer’s disease (AD) and is still incurable (1). PD is the most common synucleinopathy, a group of neurodegenerative disorders that includes dementia with Lewy bodies and multiple system atrophy (MSA), among others (2, 3). Although the synucleinopathies are multifactorial disorders, the molecular events triggering the pathogenic breakthrough of the disease converge to the abnormal aggregation of α-synuclein (α-Syn) in dopaminergic neurons (4, 5). α-Syn aggregation also occurs in oligodendrocytes in patients with MSA (6). α-Syn is an intrinsically disordered protein, which is expressed at high levels in the brain. α-Syn function is thought to be related to vesicle trafficking (7). This wild-type protein is the main component of cytoplasmic Lewy bodies (LB) and Lewy neurites (LN) in sporadic PD (8). In addition, dominantly inherited mutations in α-Syn, as well as multiplications of the gene encoding for α-Syn (SNCA), cause familial forms of PD (9). Author contributions: S.V. designed research; J.P., S.P.-D., D.F.L., F. Peccati, F. Pinheiro, D.G., A.C., S.N., M.C.-G., J.G., S.G., and E.D. performed research; J.P., S.P.-D., D.F.L., E.G., X.S., J.S., M.S., T.F.O., E.D., and S.V. analyzed data; and M.S., E.D., and S.V. wrote the paper. Conflict of interest statement: J.P., S.P.-D., M.C.-G., J.S., E.D., and S.V. are inventors on a patent application (PCT/EP2018/054540) related to the compound in this study. This article is a PNAS Direct Submission. Published under the PNAS license. 1 J.P. and S.P.-D. contributed equally to this work. 2 To whom correspondence should be addressed. Email: This article contains supporting information online at 1073/pnas.1804198115/-/DCSupplemental. Published online September 24, 2018. PNAS | October 9, 2018 | vol. 115 | no. 41 | 10481–10486 NEUROSCIENCE Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved August 16, 2018 (received for review April 3, 2018) neurons. The inhibitor reduced α-Syn aggregation, improved motility, and protected against neuronal degeneration. Results SynuClean-D Inhibits α-Syn Aggregation in Vitro. The formation of α-Syn amyloid fibrils can be reproduced in vitro by incubating the recombinant protein. However, fibril growth is very slow and highly variable, complicating drug screening (12). We have implemented a robust high-throughput kinetic assay to screen large chemical libraries in the search for α-Syn aggregation inhibitors (11). The assay uses thioflavin-T (Th-T) as readout of amyloid formation, completing highly reproducible reactions in 30 h. Approximately 14,400 chemically diverse compounds of the HitFinder Collection from Maybridge were screened with this approach. SC-D [2-hydroxy-5nitro-6-(3-nitrophenyl)-4-(trifluoromethyl)nicotinonitrile], a small aromatic compound, was identified as one of the molecules of potential interest (SI Appendix, Fig. S1). Many compounds with promising pharmacological characteristics never become drugs because they are rapidly metabolized in the liver and therefore have low oral bioavailability. SC-D was metabolically stable in the presence of human hepatic microsomes, with an intrinsic clearance of <5 μL·min−1·mg−1 (SI Appendix, Fig. S2). Incubation of 70 μM α-Syn with 100 μM SC-D impacted α-Syn aggregation, as monitored by Th-T fluorescence (Fig. 1A). The analysis of the aggregation curves indicated that the autocatalytic rate constant in the presence of the compound (ka 0.25 h−1) was 25% lower than in its absence (ka 0.33 h−1). SC-D increases t50 by 1.5 h and reduces by 53% the amount of Th-T–positive material at the end of the reaction. By measuring light scattering, we confirmed that the observed changes in Th-T fluorescence reflected an effective decrease in the levels of α-Syn aggregates, with a reduction of 48 and 58% in the scattering signal at the end of the reaction in the presence of SC-D when exciting at 300 and 340 nm, respectively (Fig. 1B). Nanoparticle tracking analysis indicated that the presence of SC-D increased the number of A B E F particles of <100 nm and decreased the formation of large aggregates (150 to 500 nm) (SI Appendix, Fig. S3). Finally, transmission electron microscopy (TEM) images confirmed that samples incubated with SC-D contained smaller and much fewer fibrils per field than untreated samples (Fig. 1 C and D). The inhibitory activity of SC-D was dose-dependent and still statistically significant at 10 μM (1:7 compound:α-Syn ratio), where it reduces the final Th-T signal by 34% (Fig. 1E). We further investigated whether SC-D was active against the aggregation of α-Syn variants associated with PD (1). SC-D was able to reduce by 45 and 73% the amount of Th-T–positive aggregates at the end of the reaction for the H50Q and A30P α-Syn familial variants, respectively (Fig. 1F). The inhibitory activity of SC-D was also assessed using protein-misfolding cyclic amplification (PMCA) (13). Conceptually based on the nucleation-dependent polymerization model for prion replication, PMCA has been recently adapted to amplify α-Syn amyloid fibrils (14). The PMCA technique combines cycles of incubation at 37 °C, to grow fibrils, and sonication, to break fibrils into smaller seeds. In our conditions, a single cycle of amplification was sufficient to generate amyloid-like protease K (PK)-resistant α-Syn assemblies, but the highest levels of protection were attained after four rounds (Fig. 1G). When the same experiment was performed in the presence of SC-D, we observed a substantial decrease in the amount of PK-resistant material (Fig. 1H), indicating that the molecule was interfering with α-Syn template seeding amyloid formation. SynuClean-D Disrupts Preformed α-Syn Fibrils. The progress of α-Syn PMCA reactions can also be monitored by using the Th-T signal as the readout for fibril assembly (15). Consistent with PK resistance analysis, Th-T fluorescence of α-Syn increased significantly after four cycles of PMCA (Fig. 2A). Surprisingly, in the presence of SC-D, the Th-T signal not only did not increase, but C D G H Soluble Downloaded by guest on April 14, 2020 PK- PK+ Step 4 PK- PK+ Step 5 PK- PK+ Soluble PK- PK+ Step 4 PK- PK+ Step 5 PK- PK+ Fig. 1. Effect of SynuClean-D on the aggregation of α-Syn in vitro. (A) α-Syn aggregation kinetics in the absence (black) and presence (blue) of SC-D followed by Th-T–derived fluorescence. (B) Light-scattering signal at 300 and 340 nm, both in the absence (white) and presence (blue) of SC-D. (C and D) Representative TEM images in the absence (C) and presence (D) of SC-D. (E) Inhibition of α-Syn aggregation in the presence of different concentrations of SC-D. (F) H50Q and A30P α-Syn variant aggregation in the absence (white) and presence (blue) of SC-D. (G and H) Bis/Tris gels of PMCA samples in the absence (G) and presence (H) of SC-D, both analyzed after PK digestion. Soluble α-Syn and PMCA steps 4 and 5 are shown. Th-T fluorescence is plotted as normalized means. Final points were obtained at 48 h. Error bars are represented as SE of mean values; **P < 0.01 and ***P < 0.001. 10482 | Pujols et al. We did not detect any perturbations in chemical shifts or peak intensities with respect to the original α-Syn spectrum in the presence of 100 μM concentration of the molecule (SI Appendix, Fig. S4), indicating that SC-D does not bind α-Syn monomers. Induced-fit docking simulations of α-Syn–SC-D revealed four major poses for its interaction with α-Syn fibrils (16): two internal, with SC-D fully inserted in the fibril (poses 1 and 2), and two external, with SC-D partially exposed (poses 3 and 4) (SI Appendix, Fig. S5). In the internal poses, the ligand is sandwiched between two parallel β-sheets of the Greek-key motif and interacts with the side chains of ALA53, VAL55, THR59, GLU61, THR72, and GLY73. The only difference between pose 1 and 2 lies in the orientation of the compound in the binding pocket. PELE (17) interaction energies are stronger for the internal poses, where SC-D binds essentially through dispersion interactions into a solvent-excluded cavity, than for external ones, where SC-D inserts into a surface groove of the fibril. In light of these calculations, we predict that SC-D binds into the core of α-Syn fibrils. MM/GBSA calculations (SI Appendix, Table S1) (18, 19) show that internal binding pose 1 (Fig. 3) exhibits the largest binding energy with the fibril, the computed ΔGbind being −18.4 ± 4.1 kcal· mol−1. The main contribution comes from the van der Waals term, representing roughly 80% of the interaction. This is not surprising given the nature of SC-D, a planar aromatic molecule. Plots of the reduced density gradient versus the density (SI Appendix, Fig. S6A) provide information on the nature of the noncovalent interactions in the system (20). Peaks in the negative and positive regions of the x axis are indicative of attractive and repulsive interactions, respectively. The region around zero corresponds to the weakest noncovalent van der Waals contacts. Though weak, these interactions are present in large number and involve the whole body of the molecule, being the largest contribution to the binding energy. Their spatial extension is shown in SI Appendix, Fig. S6B. For pose 1, the noncovalent interaction plot shows that NEUROSCIENCE Fig. 2. Disaggregational capacity of SynuClean-D. (A) Th-T fluorescence of the different PMCA passes of both treated (blue) and untreated (black) samples with SC-D. (B) Aggregation kinetics of α-Syn after the addition of SCD at different time points. (C and D) Th-T–derived fluorescence (C) and lightscattering (D) assays before and after the addition of SC-D to preformed α-Syn fibrils. (E and F) Representative TEM images in the absence (E) and presence (F) of SC-D. Th-T fluorescence is plotted as normalized means. Error bars are represented as SE of mean values; ***P < 0.001. Downloaded by guest on April 14, 2020 began to decrease after the third cycle. This suggested that SC-D might disrupt newly formed amyloid fibrils. To address the time window in which SC-D is active, we set up aggregation reactions with a constant amount of SC-D at different time intervals. As presented in Fig. 2B, the effect of SC-D on the final amount of amyloid structures was independent of whether it was added at the beginning (4 h), in the middle (12 h), or at the end (18 h) of the exponential phase, or even when the reaction had already attained a plateau (24 h). These results suggested again ability to disrupt/destabilize fibrils. To confirm the fibril-disrupting activity of SC-D, 4-d mature α-Syn fibrils were incubated in the absence or presence of the compound for 24 h. Incubation with SC-D promoted a 43% reduction in Th-T fluorescence emission (Fig. 2C). Moreover, light-scattering measurements indicated a reduction in the amount of detectable aggregates by 29 and 39% at 300 and 340 nm, respectively (Fig. 2D). Consistently, TEM images illustrated how 4-d-incubated α-Syn tended to form big fibrillary clusters (Fig. 2E), which became completely disrupted in the presence of SC-D (Fig. 2F). α-Syn Fibrils Can Accommodate SynuClean-D. To assess if SC-D can bind monomeric and soluble α-Syn, the recombinant protein was isotopically labeled and NMR 1H-15N-HSQC spectra of 70 μM [15N]α-Syn were recorded in the absence and presence of SC-D. Pujols et al. Fig. 3. Characterization of SynuClean-D–fibril interaction. General view (A) and zoom (B) of the most stable binding pose of SC-D on the α-Syn fibril model. PNAS | October 9, 2018 | vol. 115 | no. 41 | 10483 besides van der Waals, an H-bond contact is responsible for the binding of SC-D (SI Appendix, Fig. S6A). SynuClean-D Inhibits the Formation of Intracellular α-Syn Aggregates in Cultured Cells. We tested the potential toxicity of SC-D for human neuroglioma (H4) and human neuroblastoma (SH-SY5Y) cells. For both cell lines, the molecule was innocuous at concentrations as high as 50 μM (Fig. 4A and SI Appendix, Fig. S7). We used a well-established cell model that enabled us to assess α-Syn inclusion formation. H4 cells were transiently transfected with C-terminally modified α-Syn (synT) and synphilin-1, which results in the formation of LB-like inclusions, as we previously described (9). The formation of α-Syn inclusions was assessed 24 h after treatment by immunofluorescence (Fig. 4D). Upon treatment with 1 and 10 μM SC-D, we observed a significant increase in the number of transfected cells devoid of α-Syn inclusions (SC-D, 1 μM: 42.4 ± 1.0%; SC-D, 10 μM: 49.5 ± 4.5%) relative to untreated samples (control: 28.7 ± 2.0%) (Fig. 4B). SC-D treatment also promoted a significant decrease in the number of transfected cells displaying more than five aggregates (SC-D, 1 μM: 35.5 ± 5.0%; SC-D, 10 μM: 32.5 ± 6.6%) relative to control cells (control: 49.6 ± 5.6%) (Fig. 4C). SynuClean-D Inhibits α-Syn Aggregation in a C. elegans Model of PD. Downloaded by guest on April 14, 2020 Next, we tested SC-D in a living system. We used a well-studied nematode model of PD, the strain NL5901, in which human α-Syn fused to the yellow fluorescent protein (YFP) is under control of the muscular unc-54 promoter, transgene pkIs2386 [Punc-54::αSYN::YFP] (21). Muscle expression has been used successfully to model protein-misfolding diseases and to identify modifier genes without considering neuronal effects (21, 22). To determine the effects of SC-D in α-Syn accumulations, animals at the fourth larval stage (L4) (23) were incubated with and without the compound, to analyze the inhibitor efficiency in aged worms at 9 d posthatching Fig. 4. Inhibition of α-Syn aggregate formation in cultured cells. (A) Human neuroglioma cell (H4) survival when incubated with different concentrations of compound (blue) and without (white) the compound. (B and C) Reduction of α-Syn inclusion formation in human cultured cells in the presence of different concentrations of SC-D. (B) Percentage of transfected cells devoid of α-Syn aggregates. (C) Percentage ...
Student has agreed that all tutoring, explanations, and answers provided by the tutor will be used to help in the learning process and in accordance with Studypool's honor code & terms of service.

This question has not been answered.

Create a free account to get help with this and any other question!

Brown University

1271 Tutors

California Institute of Technology

2131 Tutors

Carnegie Mellon University

982 Tutors

Columbia University

1256 Tutors

Dartmouth University

2113 Tutors

Emory University

2279 Tutors

Harvard University

599 Tutors

Massachusetts Institute of Technology

2319 Tutors

New York University

1645 Tutors

Notre Dam University

1911 Tutors

Oklahoma University

2122 Tutors

Pennsylvania State University

932 Tutors

Princeton University

1211 Tutors

Stanford University

983 Tutors

University of California

1282 Tutors

Oxford University

123 Tutors

Yale University

2325 Tutors