Impairment of the autophagy-lysosome pathway in GBA^-/- medaka.

(A) Ubiquitin, p62, and Cathepsin D immunohistochemistry in the neuronal layer of the optic tectum at 3 months. Almost all of the cells in these figures are neurons. Ubiquitin-positive and p62-positive aggregates were observed in the perikarya of neurons in GBA^-/- medaka at 3 months (arrowheads). Morphologically abnormal organelles with decreased Cathepsin D staining intensity were observed in GBA^-/- medaka (arrows). Scale bars, 10 μm. (B) Transmission electron micrographs of neurons. Left panel: Neuron of a GBA^+/+ medaka. Electron-dense organelles (arrows) are likely lysosomes. N, Nucleus. Scale bar, 2 μm. The inset shows a high-magnification image of electron-dense organelles. Scale bar, 500 nm. Middle panel: Neuron of a GBA^-/- medaka. An aggregate containing filamentous structures (arrowhead) is continuous with an electron-dense organelle. N, Nucleus. Scale bar, 2 μm. Right panel: High-magnification image of an aggregate of filamentous structures (arrowhead) and electron-dense organelles (arrows) in a GBA^-/- medaka. Scale bar, 500 nm. (C) Western blot analysis of ubiquitin, p62, and β-actin (n = 4–7, *p < 0.05, ***p < 0.001). (D) Conforcal microscope images of GBA^-/- medaka. Upper panels: ubiquitin (green) and p62 (red). p62-positive aggregates colocalized with ubiquitin-positive aggregates (white arrowheads). Middle panels: LC3 (green) and p62 (red). p62-positive aggregates (yellow arrows) did not colocalize with LC3 accumulations (yellow arrowheads). Lower panels: Cathepsin D (green) and p62 (red). p62-positive aggregates did not colocalize with Cathepsin D-positive organelles. Nuclei were visualized with DAPI (blue). Scale bars, 10 μm. For all analyses, data are the mean ± SEM.