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. 2017 Dec 4;27(23):3626–3642.e6. doi: 10.1016/j.cub.2017.10.054

Figure 6.

Figure 6

Aggresome-like Formation and Golgi-Mediated Degradation as Signs of Alternative Autophagy Induction in the Dentate Nucleus of DRPLA Mice

(A) (i) TEM image of a dentate nucleus cell in ATN1-FL-65Q mouse containing a membranous aggresome-like (Agg) accumulation in close proximity to the cell nucleus (nuc). Scale bar, 5 μm. (ii) Higher magnification of the inset in (i) containing the aggresome-like (Agg) membranous formation. Scale bar, 500 nm. ly, lysosome; mt, mitochondrion. (iii and iii’) confocal fluorescence image of DN cell in ATN1-FL-65Q;GFP-LC3 mouse brain with p62 positive (red) inclusion inside the nucleus (blue) as well as outside of the nucleus (arrowhead), which also co-localizes with GFP-LC3 (green).

(B) (i) TEM image of a dentate nucleus cell in ATN1-FL-65Q mice; in addition to a large perinuclear aggresome-like (Agg) structure, long membranous formations (arrowhead) with phagophore-like structure are shown at the distal side of the cell soma. Scale bar, 2 μm (note this is a higher-magnification cutout of the image in Figure 2F). (ii) High-magnification TEM image of a similar phagophore-like structure. Scale bar, 300 nm.

(C) Representative TEM images of dentate nucleus cells show double-membrane vesicular structures (arrowheads) in close proximity to the Golgi apparatus (arrow) in ATN1-FL-65Q mice (right), compared to normal Golgi morphology in ATN1-FL-26Q (middle) and wild-type (WT, left) mice. Scale bar, 500 nm

(D and E) Confocal fluorescence microscopy images of dentate nucleus cells showing an enlarged Golgi apparatus (GM130, red) in ATN1-FL-65Q;GFP-LC3 mouse line (left) compared to ATN1-FL-26Q;GFP-LC3 line (middle) and WT;GFP-LC3 (WT, right) (D). Inset in the bottom row shows a higher magnification of a GFP-LC3 (green) positive puncta in close proximity to GM-130 positive structures. Scale bar, 5 μm. Quantification of GM130 signal evidences an increase in the ATN1-FL-65Q;GFP-LC3 mouse line (E). Kruskal-Wallis multiple comparison analysis, mean ± SEM, ∗∗∗p < 0.001

(F and G) Analysis of LaminB1 redistribution into the cytoplasm in DRPLA (17) patient fibroblasts after 48-hr treatment with BafA1 and/or BrefA. BrefA was only added for the last 24 hr. BafA1 and BrefA display an increased localization of LaminB1 into the cytoplasm showing different distribution patterns: diffused after BafA1 treatment only and concentrated in large perinuclear puncta after BrefA treatment. Combined BafA1 and BrefA treatment shows synergistic effect on cytoplasmic LaminB1 localization. Scale bar, 50 μm (F). Automated quantification of area occupied by LaminB1 positive puncta in the cytoplasm was performed using Opera Phenix high-content screening system and Columbus software. Mean ± SEM, two-way ANOVA ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

See also Figure S7.