Figure 6.

Aggregation of human α-syn does not spontaneously take place on OLs from PLP-αSyn mice but can be experimentally seeded by challenging with PFFs. (A) Representative immunofluorescence imaging of neurons (NeuN, red) and human α-syn (MJFR1, green) in primary cortical cultures from WT mouse supplemented with OLs from PLP-αSyn mice, untreated (left) or challenged with human α-syn PFF of type 1 (middle, PFF #1) or type 2 (right, PFF #2). Pictures were taken four weeks post treatment and show OLs morphology changes with cellular shrinking upon PFF treatments. (B) Bar graph representing the quantification of MJFR1 area in the different conditions described in A, traducing the OLs surface measurements, with a significant shrinking of these cells upon challenge with PFF (pValues obtained with Holm-Sidak corrected multiple t-tests). (C) Representative immunofluorescence imaging of the synucleinopathy as shown by aggregated α-syn (synF1, red) and hyperphosphorylated pS129-α-syn (EP1536Y, green) in primary cortical cultures from WT mouse (top) and the same cultures supplemented with OLs from PLP-αSyn mice (bottom). These two cultures were untreated (left) or challenged with human α-syn PFF of type 1 (middle, PFF #1) or type 2 (right, PFF #2). (D) Representative image shown in C with a neurite segmentation filter applied (right, pink), allowing the quantification of neuritic synucleinopathy plotted in the bar graph. Total synucleinopathy neurite length is shown for WT primary cortical cultures supplemented or not with WT or PLP-αSyn OLs and challenged or not with PFF of the two types. The extent of the neuronal synucleinopathy is independent of the addition of OLs (p values obtained with Holm-Sidak corrected multiple t-tests), ns: not statistically different. (E) Representative image shown in C with an OLs segmentation filter applied (right, pink), allowing the quantification of synucleinopathy located in the OL cell bodies, plotted in the bar graph. Total OLs synucleinopathy is shown as area measured for WT primary cortical cultures supplemented or not with WT or PLP-αSyn OLs and challenged or not with PFF of the two types (p values obtained with Holm-Sidak corrected multiple t-tests). For each condition 9 fields of 2 independent wells were analyzed corresponding to 5.13 mm² that is, 15% of the total well surface. Results are representative of 2 independent experiments.