Fig. 3: Aβ42o treatment induces local mitophagy in dendrites.
a, b Secondary dendritic segments from primary cortical PNs at 21–25 DIV, ex utero electroporated at E15.5 with pCAG-mito-mTagBFP2, pCAG-LAMP1-mEmerald, and pCAG-RFP-LC3 to visualize mitochondria, lysosomes, and autophagosomes, respectively (a). At 21 DIV, neurons were treated with either a vehicle control or Aβ42o and imaged live using time-lapse microscopy every 15 min for 14 h. The yellow boxes label the magnified areas shown in b illustrating that, in control-treated neurons, there is no significant change in LAMP1 + (lysosomes), LC3 + (autophagosomes), or LC3-LAMP1 double-positive (autolysosomes) vesicle dynamics and/or accumulation (see Supplementary Movies S1 and S2). In contrast, in dendrites of Aβ42o-treated cortical PNs, sites of LAMP1-LC3 double-positive autolysosome accumulation often result in loss of mitochondria as indicated by yellow arrows in panel b. c Representative kymographs of mitochondria, lysosomes, and autophagosomes from dendrites in panel a. The yellow arrows point to loss of mitochondrial fluorescent signal, deemed mitophagy events. The red arrow indicates no decrease in mitochondrial signal, despite LAMP1 and LC3 signal, indicating an incomplete mitophagy event. d Detail of an autolysosome engulfing a fragment of mitochondria (135′) following a mitochondrial fission event (105′−120′). e The percentage of dendritic segments showing accumulation of both LC3 + autophagosomes, LAMP1 + lysosomes, or LC3-LAMP1 double-positive autolysosomes. Data are represented by box plots displaying minimum to maximum values, with the box denoting 25th, 50th (median), and 75th percentiles from three independent experiments. ncontrol = 15 neurons; nAβ42o = 24 neurons. f–i Quantification of f LAMP1 intensity, g LC3 intensity, and h Mitochondrial intensity for dendritic segments over the course of the 14 h following treatments. i Mitophagy index defined as the change in mitochondrial fluorescence intensity in dendritic segment with (+) or without (−) LC3-LAMP1 double-positive puncta (autophagosomes) at 14 h. In panels f–i, data are represented as mean + /− SEM based on nControl = 33 dendrites; nAβ42o = 44 dendrites. Statistical significance was performed using a Mann–Whitney test. Exact P values are indicated on the figure when available through Prism software, otherwise, the test significance is provided using the following criteria: **P < 0.01; ****P < 0.0001. Scale bar = 2 μm.
