Extended Data Fig. 3: GCI-α-Syn is more potent in inducing α-Syn pathology in primary oligodendrocytes.
From: Cellular milieu imparts distinct pathological α-synuclein strains in α-synucleinopathies
a, Oligodendrocytes treated with the same amount of GCI-α-Syn, LB-α-Syn or PFF were sequentially extracted with 1% Triton-X100 lysis buffer followed by 1% sarkosyl lysis buffer, which were combined together as the sarkosyl-soluble fraction. The sarkosyl-insoluble pellets were resuspended in Dulbecco’s PBS by sonication. Both soluble and insoluble fractions were immunoblotted with an antibody against total or pS129 α-Syn. b, Densitometric quantification of insoluble versus soluble α-Syn for experiments described in a (n = 3 biological replicates). c, d, Primary oligodendrocyte cultures were immunostained with antibodies against various cell-type specific markers: CNP (oligodendrocytes), olig2 (oligodendrocytes), Iba1 (microglial cells), NeuN (neurons), GFAP (astrocytes), PLP (oligodendrocytes) at day in vitro 3 (DIV 3) (c) or DIV 9 (d). e, Insoluble phosphorylated α-Syn, induced in primary oligodendrocytes overexpressing α-Syn, was co-stained with antibodies against various cell-type specific markers, demonstrating that the cells with α-Syn pathology are oligodendrocytes. f, Percentage of different type of cells (oligodendrocytes, microglial cells and astrocytes) in oligodendrocyte culture, at DIV 3 (the time point of virus infection), DIV 9 (the time point for misfolded α-Syn treatment) and DIV 23 (the time point for fixation) (n = 3 (DIV3) or 5 (DIV 9, DIV 23) coverslips from three independent experiments). g, Working hypotheses regarding the different cell-type distributions of GCI-α-Syn and LB-α-Syn strains in diseased brains. Hypothesis 1 states that the unique properties of GCI-α-Syn and LB-α-Syn strains determine their different cell-type distributions. The GCI-α-Syn strain (represented by red spheres) is more efficient in inducing α-Syn pathology in oligodendrocytes, whereas the LB-α-Syn strain (green spheres) is more efficient in inducing α-Syn pathology in neurons. Hypothesis 2 states that GCI-α-Syn and LB-α-Syn strains do not have cell-type preferences and that they could both be initiated by the same misfolded α-Syn seeds (grey spheres), but that the different intracellular environments of neurons and oligodendrocytes convert these α-Syn seeds to different strains. Results shown as mean ± s.e.m. **P < 0.01. Scale bars: 100 μm (c, d); 50 μm (e). The experiments shown in a and c–e have been repeated three times with similar results. See Supplementary Table 5 for statistical details. For gel source data, see Supplementary Fig. 1.
