FIGURE 9.
Secretion of α-synuclein is mediated by compartments with late endosomal/amphisomal characteristics. A, p25α-expressing PC12 cells were incubated with Alexa 568-conjugated annexin-V on ice before fixation and indirect immunofluorescence with antibodies against cleaved caspase-3. Note widespread annexin-V surface staining in the absence of caspase-3 immunoreactivity. Bar, 10 μm. B, PC12 cell lines labeled with Alexa 488-conjugated annexin-V on ice were analyzed by flow cytometry. The bar graph shows percentage annexin-V positive cells of the whole cell population and represents mean ± S.E. of three independent experiments. C, conditioned medium (CM) from PC12 cells expressing α-synucleinA30P (α-SNCA30P) with or without p25α was centrifuged at 100,000 × g to obtain a pellet (containing exosomes) and a supernatant (sup). Aliquots were then Western blotted with anti-α-SNC (BD Transduction Laboratories) or anti-flotillin-1 (exosome marker) antibodies. The exosome fraction was applied on the gel at 6-fold the relative load of supernatant and medium. The blot is representative of two independent experiments. D–F, PC12-α-SNCA30P cells were differentiated for 2 days and transduced at two different multiplicities of infection with lentivectors constitutively expressing FLAG-tagged Rab27A-Q78L (dominant positive) or Rab27A-T23N (dominant negative). Control wells received a GFP-expressing vector (pLenti). D, after 2 days of transgene induction cell lysates were prepared to show expression of Rab27A using anti-Rab27A mAb 4B12 (arrow points to transgene) or anti-FLAG antibodies. E, conditioned medium was TCA-precipitated and Western blotted with anti-α-SNC antibodies (BD Transduction Laboratories). F, bar graph shows mean ± S.E. of the integrated optical density (IOD) of α-SNC Western blot bands normalized to control-transduced cells (n = 3). * and # denotes a statistic significant increase or decrease, respectively, in α-SNC secretion when compared with control-transduced PC12 cells.