Novel Poly-Arginine Peptide R18D Reduces α-Synuclein Aggregation and Uptake of α-Synuclein Seeds in Cortical Neurons

Background/Objectives: The role of alpha-synuclein (alpha-syn) pathology in Parkinson's disease (PD) is well established; however, effective therapies remain elusive. Two mechanisms central to PD neurodegeneration are the intracellular aggregation of misfolded alpha-syn and the uptake of alpha-syn aggregates into neurons. Cationic arginine-rich peptides (CARPs) are an emerging class of molecule with multiple neuroprotective mechanisms of action, including protein stabilisation. This study characterised both intracellular alpha-syn aggregation and alpha-syn uptake in cortical neurons in vitro. Thereafter, this study examined the therapeutic potential of the neuroprotective CARP, R18D (18-mer of D-arginine), to prevent the aforementioned PD pathogenic processes through a cell-free thioflavin-T (ThT) assay and in cortical neurons. Methods: To induce intracellular alpha-syn aggregation, rat primary cortical neurons were exposed to alpha-syn seed (0.14 mu M) for 2 h to allow uptake of the protein, followed by R18D treatment (0.0625, 0.125, 0.25, 0.5 mu M), and a subsequent measurement of alpha-syn aggregates 48 h later using a homogenous time-resolved fluorescence (HTRF) assay. To assess neuronal uptake, alpha-syn seeds were covalently labelled with an Alexa-Fluor 488 fluorescent tag, pre-incubated with R18D (0.125, 0.25, 0.5 mu M), and then exposed to cortical neurons for 24 h and assessed via confocal microscopy. Results: It was demonstrated that R18D significantly reduced both intracellular alpha-syn aggregation and alpha-syn seed uptake in neurons by 37.8% and 77.7%, respectively. Also, R18D reduced the aggregation of alpha-syn monomers in the cell-free assay. Conclusions: These findings highlight the therapeutic potential of R18D to inhibit key alpha-syn pathological processes and PD progression.