Novel Poly-Arginine Peptide R18D Reduces α-Synuclein Aggregation and Uptake of α-Synuclein Seeds in Cortical Neurons
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.1.1. Establishment of Rat Primary Cortical Neurons
2.1.2. Neuronal Cell Plating
2.2. Study Design
2.2.1. Establishment of Neuronal Model of Intracellular α-Syn Aggregation
2.2.2. Assessment of R18D to Inhibit α-Syn Aggregation
2.2.3. Assessment of R18D to Inhibit α-Syn Seed Uptake
2.3. Cell Assays
2.3.1. Cell Death and Cell Viability Assays
LDH Assay
MTS Assay
2.3.2. Thioflavin T (ThT) Assay
2.3.3. Homogenous Time-Resolved Fluorescence (HTRF)
2.4. Microscopy
2.4.1. Scanning Electron Microscopy
2.4.2. Confocal Microscopy
2.5. Statistical Analysis
3. Results
3.1. α-Syn Seeds Induce α-Syn Monomer Aggregation in a Cell-Free Assay
3.2. R18D Reduces α-Syn Monomer Aggregation in a Cell-Free ThT Assay
3.3. R18D Is Non-Toxic to Cortical Neurons at Concentrations Below 0.5 μM
3.4. α-Syn Seeds Induce Intracellular α-Syn Aggregation in Cortical Neurons
3.5. α-Syn Seeds Enter Neurons
3.6. α-Syn Seeds Induce Toxicity in Cortical Neurons
3.7. R18D Reduces Intracellular α-Syn Aggregation in Cortical Neurons
3.8. R18D Reduces α-Syn Seed Uptake in Cortical Neurons
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Cell-Free ThT α-Syn Mononer Aggregation Assay | ||
R18D Concentration Examined | α-Syn Monomer Aggregation—Percentage Inhibition | p Value |
0.05 μM | 28.6% | p < 0.001 |
0.1 μM | 51.0% | p < 0.001 |
0.2 μM | 62.1% | p < 0.001 |
0.25 μM | 61.2% | p < 0.001 |
Intracellular α-Syn Aggregate HTRFAssay | ||
R18D Concentration Examined | α-Syn Aggregation in Neurons—Percentage Inhibition | |
0.0625 μM | 33.5% | p = 0.003 |
0.125 μM | 37.8% | p < 0.0001 |
0.25 μM | 26.9% | p = 0.0015 |
0.5 μM | 17.3% | p = 0.022 |
Confocal Microscopy of Labelled α-Syn Seeds | ||
R18D Concentration Examined | Labelled α-Syn Seed Uptake in Neurons—Percentage Inhibition | |
0.125 μM | 77.74% | p < 0.001 |
0.25 μM | 76.0% | p < 0.001 |
0.5 μM | 67.0% | p < 0.001 |
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Robinson, E.C.; Gorecki, A.M.; Pesce, S.R.; Bagda, V.; Anderton, R.S.; Meloni, B.P. Novel Poly-Arginine Peptide R18D Reduces α-Synuclein Aggregation and Uptake of α-Synuclein Seeds in Cortical Neurons. Biomedicines 2025, 13, 122. https://doi.org/10.3390/biomedicines13010122
Robinson EC, Gorecki AM, Pesce SR, Bagda V, Anderton RS, Meloni BP. Novel Poly-Arginine Peptide R18D Reduces α-Synuclein Aggregation and Uptake of α-Synuclein Seeds in Cortical Neurons. Biomedicines. 2025; 13(1):122. https://doi.org/10.3390/biomedicines13010122
Chicago/Turabian StyleRobinson, Emma C., Anastazja M. Gorecki, Samuel R. Pesce, Vaishali Bagda, Ryan S. Anderton, and Bruno P. Meloni. 2025. "Novel Poly-Arginine Peptide R18D Reduces α-Synuclein Aggregation and Uptake of α-Synuclein Seeds in Cortical Neurons" Biomedicines 13, no. 1: 122. https://doi.org/10.3390/biomedicines13010122
APA StyleRobinson, E. C., Gorecki, A. M., Pesce, S. R., Bagda, V., Anderton, R. S., & Meloni, B. P. (2025). Novel Poly-Arginine Peptide R18D Reduces α-Synuclein Aggregation and Uptake of α-Synuclein Seeds in Cortical Neurons. Biomedicines, 13(1), 122. https://doi.org/10.3390/biomedicines13010122