Figure 3

Effects of cytosolic lipid depletion on α-syn binding. (A) Using chloroform extraction to fractionate cytosol into three fractions: the top fraction contains the gangliosides or small organic polar molecules, the interface layer contains the proteins and the bottom phase contains lipids solubilised in chloroform. We incubated the synaptic membrane with the two lipid free-fractions, top and interphase (protein) layers, in presence of recombinant α-syn. The lipid-free fractions did not show any significant effects on the Wt and A53T α-syn binding compared to the control condition (α-syn incubated with synaptic membranes in absence of cytosol; Student T-test, p > 0.05) while the A30P α-syn binding was increased (compared to control condition, Student T-test, p < 0.01). (B) Recombinant α-syn (Wt, A30P and A53T) were incubated with synaptosomal membranes in the presence of 1.5 mg/ml cytosol from either KO mice (KO) or from non-transgenic mice (nonTg) for 10 min at 37°C. Binding of normal and mutant human α-syn, measured by the human α-syn specific monoclonal antibody 211, is normalized to that of Wt α-syn in the presence of KO cytosol. (C) Recombinant Wt α-syn was incubated with synaptosomal membranes and C16:0 PAF (0, 10, 100 nM) in the absence (open bars) or presence of delipidated cytosol (closed bars). Inclusion of 100 nM C16:0 PAF significantly increased α-syn binding only in the presence of the delipidated cytosol (compared to corresponding condition without C16:0 PAF, Two-Way ANOVA, p < 0.01, Bonferroni's multiple comparison test p < 0.01, n = 3).