Fig. 7
TrkB-LC3b-Rab7-containing amphisomes are positive for SIPA1L2. a Confocal images of quadruple immunofluorescence performed in rat primary hippocampal neurons stained for SIPA1L2, LC3, TrkB with Rab7. Scale bar is 1 μm. b Confocal images of primary neurons treated with BDNF and stained for pTrkBY515, SIPA1L2, and Bassoon show colocalization of SIPA1L2 and pTrkB Y515 in presynaptic boutons. Scale bar is 5 µm. c Mander’s coefficient calculated for pTrkBY515 and SIPA1L2 in boutons detected by Bassoon staining. For control measurements, the same images were rotated 90º to the right (paired Student’s t test). d, e Representative images in d of rat hippocampal neurons treated with BDNF and stained for pTrkBY515, SIPA1L2 and Synaptophysin1. Arrows indicate boutons where SIPA1L2 is present (black) or absent (red). Note that in the presence of SIPA1L2, pTrkBY515 intensity is higher compared to those from boutons where SIPA1L2 is absent. Quantification is shown in e, circles represent averaged intensities per image (paired Student’s t test for average per image). Averaged intensities are normalized to images acquired from Fc-TrkB-treated neurons. The cumulative frequency distribution is shown in f (n = synaptic boutons). Scale bar is 2 μm. g, h Super-resolution STED imaging performed in rat hippocampal neurons revealed association of TrkB with LC3 at the presynaptic boutons. In h, line profiles from the line in the ROI2 in g. Scale bars 5 µm (overview) and 1 µm (inserts). i, j In i, scheme depicting fractionation protocol performed from rat brain that results in autophagosome (A1), autophagolysosome (A2) and lysosome (L) fraction. Note that Rab7 and SIPA1L2 are only present in the total and A1 fraction (j) according to their presence in amphisomes but not in later stages of the autophagosomal pathway. Lines in dot plot graphs depict data as mean ± SEM. "**" indicates P ≤ 0.01; "***" indicates P ≤ 0.001.
