Fig. 8: Accumulation of axonal autophagosomes and swellings in cortical neurons from BORCS5-KO mice.

a, Cortical neurons isolated from WT and BORCS5-KO E17 mouse embryos¹¹ were transfected with a plasmid expressing soluble GFP. Arrows indicate axonal swellings. Scale bars, 20 μm. The experiment was repeated three times. b, Cortical neurons from WT and BORCS5-KO mice were immunostained for Tau (axon) and MAP2 (soma and dendrites). Arrows indicate the accumulation of Tau-positive aggregates in the swellings. Scale bars, 20 μm. c, Quantification of the number of Tau-positive swellings per unit length of axon from three experiments, such as shown in b. Data are represented as SuperPlots⁷⁷ showing the individual data points, the mean from each experiment and the mean of the means ± s.d. Statistical significance was calculated using an unpaired two-tailed Student’s t-test. BORCS5 KO versus WT ***P < 0.001. d, Axonal swellings (arrows) in BORCS5-KO mouse cortical neurons immunostained with antibodies to Tau and TOMM20 (mitochondria). The four images on the right are magnified views and a 3D rendering of the boxed area. Scale bars, 5 μm. e, Immunofluorescence microscopy and 3D rendering of axonal swellings in cortical neurons from BORCS5-KO mice stained for LC3B (autophagosomes) and CYCS (mitochondria). Scale bars, 5 μm. f, Axons from WT and BORCS5-KO mouse cortical neurons sequentially stained with SPY650-tubulin (tubulin) for 1 h and MitoTracker Green (mitochondria) for 10 min were imaged live on an Airyscan confocal microscope. Arrows point to microtubule swirls in an axonal swelling. Scale bars, 10 μm. g, Zoomed-in view of the boxed area from f shows microtubule swirls in an axonal swelling. Scale bar, 20 μm. All the experiments shown in d–f were repeated three times. h, Phase-contrast microscopy of axons from WT and BORCS5-KO mouse cortical neurons. Notice fragmentation in the BORCS5-KO axons. Scale bars, 5 μm. i, Quantification of axon DI from two independent experiments, such as the one shown in h. Values are the mean ± s.d. from 30 images. Statistical significance was calculated using an unpaired two-tailed Student’s t-test. BORCS5 KO versus WT ***P < 0.001. Neurons shown in this figure were cultured for 7 d (a–g) or 10 d (h and i).