Fig 7. Effect of a peptide derived from the α3 subunit of the NKA on αSyn fibrils binding and take-up by Neuro-2a cells.

A, Structure of the α3 subunit of the NKA Bos taurus (PDB 4xe5) where the 27 amino acid residues long peptide NKApep corresponding to the extracellular loop previously shown to interact with αSyn fibrils [25] is coloured in red. B, Secondary structure content of NKApep determined by circular dichroism. The CD spectra used for deconvolution is shown in S4C Fig. C, Effect of NKApep on αSyn fibrils binding to the plasma membrane of Neuro-2a cells. αSyn-Alexa488 fibrils (1 μM) were incubated without or with increasing concentrations of NKApep in DMEM for 30 min at 37°C. Neuro-2a cells were next exposed to the mixture for 30 min. Fluorescence was quantified after extensive washing. Representative images are shown in S2B Fig. For each peptide concentration, the mean percentage of Neuro-2a cells with at least 1 αSyn-Alexa488 fibrils foci and its associated standard error value was calculated from 3 independent experiments. The results and the associated significances are expressed relative to fibrils binding in the absence of peptide. D, Effect of NKApep on αSyn fibrils take-up by Neuro-2a cells. αSyn-Alexa488 fibrils (0.5 μM) were incubated with increasing concentrations of NKApep (0–10 μM) in DMEM for 30 min at 37°C. Neuro-2a cells grown in 96-wells plates were exposed to the mixture for 4 hours. After extensive washing trypan blue was added to quench the fluorescence of plasma membrane-bound αSyn fibrils. The amount of internalized αSyn-Alexa488 was measured using a fluorescence plate reader. Means and their associated standard error values were calculated from 4 independent experiments. The results are expressed relative to the internalization in the absence of peptide.