Fig. 2

Elongated axons and collaterals in primary cortical neurons expressing α-Syn associate with higher levels of phosphatidylinositol 4,5-bisphosphate (PI4,5P₂). a Primary cortical cultures from α-Syn^−/− (C57BL/6JOlaHsd) mouse brains, virally transduced either with WT α-Syn, A53T α-Syn, K10,12E α-Syn, or a mock-GFP vector. Cells were fixed at 4 DIV and immunoreacted with antibodies against α-Syn (MJFR1, green), α-tubulin (red) and PI4,5P₂ (white). Direct fluorescence was captured for GFP (green). Bar = 25 μm. b Graph showing the average axon length (in μm); c Total length of collaterals per axon (in μm); and d PI4,5P₂ levels within the axon and its collaterals (per μm² area) quantified by Fiji (Image J) program. Mean ± SE; n > 22 cells; *, P < 0.05. e A primary cortical neuron as in (a), transduced with a mock-GFP (green); immunoreacted with anti GAP-43 (blue) and stained with rhodamine-phalloidin (red). Bar = 10 μm F. A neuron as in (e) expressing WT α-Syn and immunoreacted with anti α-Syn (MJFR1, green) and anti α-tubulin (blue) abs, and stained with rhodamine-phalloidin (red). Shown an entire cell and a zoom on axon collaterals. Bar = 5 μm. No differences in number of growth cones per axon (in μm) were detected. g Graph bar showing quantitation of growth cones per μm axon in WT α-Syn and GFP expressing neurons. Mean ± SD of n = 12–15 cells