Figure 4

Differential αSyn association to mitochondrial membrane induces fragmentation. (A) Total, cytosolic, and mitochondrial fractions isolated by differential centrifugation of SHSY5Y cells transfected with empty, WT, A30P, or A53T αSyn vectors. Tubulin and VDAC1 were used as controls for cytosolic and mitochondrial fractions, respectively. Upper panel: αSyn enrichment quantified by mitochondrial/cytosolic ratio for control (empty), WT, A30P, or A53T αSyn normalized to the level of αSyn expression. See Figure S8 for complete overexposed gel. Lower panel: immunoblots showing total protein levels from homogenates and quantification of αSyn overexpression levels normalized to WT αSyn. Data is represented as mean ± SEM (n = 3 independent experiments). (B) Dimerization scheme showing fusion protein design to deliver αSyn to mitochondria. WT, A30P, or A53T αSyn were fused in-frame to FRB motif (αSyn-FRB). A vector driving two FKBP domains fused to GFP and an OMM peptide (GFP-FKBP-ActA) that localize to mitochondria was used. FRB and FKBP dimerize in the presence of rapalog, inducing αSyn-FRB delivery to the OMM. (C–F) WT and A53T, but not A30P αSyn, targeting to the OMM induces mitochondrial fragmentation. (C) Human neurons co-transfected with GFP-FKBP-ActA and αSyn-FRB were immunostained for GFP and αSyn to observe the αSyn cytosolic localization in 0 nM rapalog and the mitochondrial delivery in the presence of 500 nM rapalog for 6 h (Scale bar, 10 μm). (D) Neurons co-transfected with WT or A53T αSyn-FRB plus GFP-FKBP-ActA in the presence of 0 or 500 nM rapalog. Inset: axonal magnification. Scale bar, 30 μm; inset, 10 μm. (E) Mean mitochondrial size quantification per axon in 0, 200, or 500 nM rapalog for 6 h normalized to empty vector control. Linear regression, slopes, and deviation from zero were assessed (WT αSyn-FRB: −0.043, p < 0.001; A30P αSyn-FRB: −0.015, p > 0.05; A53T αSyn-FRB: −0.031, p < 0.05). Data is presented as mean ± SEM (n = 15 axons per condition from 3 independent experiments). (F) Probability function distribution of total pooled mitochondrial size measured in WT, A30P, or A53T αSyn-FRB plus GFP-FKBP-ActA neurons in 0 or 500 nM rapalog. (n > 300 mitochondria from 3 independent experiments). See also Figure S6.