Foldamers reveal and validate therapeutic targets associated with toxic α-synuclein self-assembly

Parkinson’s disease (PD) is a progressive neurodegenerative disorder for which there is no successful prevention or intervention. The pathological hallmark for PD involves the self-assembly of functional Alpha-Synuclein (αS) into non-functional amyloid structures. One of the potential therapeutic interventions against PD is the effective inhibition of αS aggregation. However, the bottleneck towards achieving this goal is the identification of αS domains/sequences that are essential for aggregation. Using a protein mimetic approach, we have identified αS sequences-based targets that are essential for aggregation and will have significant therapeutic implications. An extensive array of in vitro, ex vivo, and in vivo assays is utilized to validate αS sequences and their structural characteristics that are essential for aggregation and propagation of PD phenotypes. The study aids in developing significant mechanistic and therapeutic insights into various facets of αS aggregation, which will pave the way for effective treatments for PD.


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All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. All the datasets for experiments in the manuscript and supplementary information will be available in the 'Source Data File'. The data availability statement has been included in the main manuscript as well and it now reads as : 'All the datasets generated and analyzed during the current study are also available from the corresponding author. Source data are provided with this paper. ' The in vitro experiments were carried out with three independent experiments (n = 3) and each independent experiments was repeated four times. For NMR experiments n=1, but at least one NMR experiment was repeated to confirm the reproducibility (data was not shown in the manuscript). Also, the NMR experiments between alpha-synuclein and SK-129 were conducted at different stoichiometric ratios and a consistent trend of the change in the chemical shift volumes was observed as a function of stoichiometric ratios, which supports the reproducibility of the data. Similarly, all CD experiments were conducted one time and the CD experiments between !S and SK-129 (or LUVs, DOPS) were conducted at different stoichiometric ratios and a consistent trend of the change in the CD signals was observed as a function of stoichiometric ratios (of !S and SK-129/DOPS, LUVs), which supports the reproducibility of the data.
The cellular experiments (Proteostat, cell viability etc) were carried out using four independent experiments (n = 4) and each experiment consisted of four technical replicates. For the in vivo experiments (C elegans), the experiments were carried out using at least three independent experiments (n = 3).
No data was excluded from the manuscript.
All attempts for the replication of data were successful. The biophysical, cellular, and C elegans based experiments were replicated at least twice with a different batch of expressed proteins, new cultured cells, or freshly bleached c elegans strains, respectively.
At least one randomization experiment was carried out to monitor the effect of molecules on synuclein aggregation in HEK cells, neuronal cells, and C elegans. The allocation of the samples was carried out randomly without any prior selection. The randomly allocated samples then treated with different experiment conditions to pursue the experiments.
At least one blinding experiment was carried out to monitor the effect of molecules on alpha-synuclein aggregation in HEK cells, neuronal cells, and C elegans. During the blinding experiment, a second member of the lab pursued the blinding part of the experiments and further pursued the experiment. Note that full information on the approval of the study protocol must also be provided in the manuscript. We have specified the dilution used for secondary antibodies in the methods section of the manuscript. The dilution factor for both the primary and secondary antibodies were 1:1000 (v/v) in TBST buffer containing 5% BSA All antibodies (Primary and secondary) are commercially available and are well established. All of the antibodies have been tested, validated, and utilized in multiple study that are published and well documented.
HEK293 cells with transfection and over expression of WT-alpha-synuclein-YFP and A53T mutant alpha-synuclein-YFP were acquired via a generous Gift from Prof. Marc Diamond's lab (University of Texas Southwestern, Dallas, Texas) using a Materials Transfer Agreement. The SHSY 5Y cells were purchased from ATCC (product number: CRL-2266).
HEK293 cells (Overexpressing WT-alpha-synuclein-YFP and A53T mutant alpha-synuclein-YFP) were authenticated by Prof. Marc Diamond's lab (University of Texas Southwestern, Dallas, Texas) and the SHSY 5Y cells were authenticated by ATCC (product number: CRL-2266). Our lab did not authenticate the cell lines.
The testing of the contamination of cell lines were carried out by ATCC (for SHSY 5Y) or by Prof. Marc Diamond's lab (University of Texas Southwestern, Dallas, Texas) (HEK293 modified cell lines). Our lab did not test these cell lines for mycoplasma contamination.
No commonly misidentified cell lines were used in the current study.
In this study, two strains of C elegans were used, including N2 and NL5901. The maintenance of the C elegans strains and their treatment with small molecules were carried out by using well established and published protocols. The C elegans strain were mostly female (male population freequency was less than 0.002). The C elegans strains were purchased from CGC (University of Minnesota) and used in the experiments when they were 3 month old. The primary culture neurons were prepared from Pregnant Sprague Dawley Rats (72-85 days old, mixed male and female), which were purchased from Charles River Laboratories (Strain Code 400) and maintained at the University of Denver Animal facility (AAALAC accredited). The rat embryos (both male and female) extracted from Pregnant Sprague Dawley Rats at the embryonic day 18 were used to prepare primary culture neurons. All animal protocols and experiments were approved by the University of Denver Animal Care and Use Committee (IACUC).
No wild animals were used in the study. Fixed brain tissues were obtained from the Carroll A. Campbell, Jr. Neuropathology Lab (CCNL) brain bank at the Medical University of South Carolina (Dr. Steve Carroll, Director) and from Dr. Greg Gerhardt's laboratory at the University of Kentucky. The alpha-synuclein seeds were extracted from the post mortem brain by following a published protocol.
No ethical approval required for the C elegans strains. All animal protocols and experiments were approved by the University of Denver Institutional Animal Care and Use Committee (IACUC).