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

Figure 5.

Figure 5

Lamin-Based Nucleophagy Associates with PolyQ Inclusions in DRPLA Mice and Is Aggravated in Human DRPLA Fibroblasts upon Lysosomal Blockage

(A–C) Representative images of dentate nucleus cells from WT;GFP-LC3 (WT, left), ATN1-FL-26Q;GFP-LC3 (middle), and ATN1-FL-65Q;GFP-LC3 (right) end-stage mice show co-localization (yellow) of GFP-LC3 (green) and LaminB1 (red) in the nucleus (framed arrowheads) and, in particular, in the cytoplasm (full arrowheads) of ATN1-FL-65Q cells (A). The nucleus is visualized in blue. Single-plane images were taken with the confocal laser scanning microscope. Quantification indicates a significant increase in the amount of cytoplasmic LaminB1 (B) and of its co-localization with GFP-LC3 (C) in ATN1-FL-65Q;GFP-LC3 mice compared to WT;GFP-LC3 and ATN1-FL-26Q;GFP-LC3. One-way-ANOVA ∗∗∗p < 0.001, ∗p < 0.05. Scale bar, 5 μm.

(D) Some nuclei in ATN1-FL-65Q;GFP-LC3 show strong degeneration reflected in LaminB1 reorganization forming bright intranuclear puncta. Intranuclear (framed arrowheads) and also cytoplasmic (full arrowheads) LaminB1 puncta colocalize with PolyQ positive puncta. Scale bar, 5 μm.

(E and F) Analysis of LaminB1 redistribution into the cytoplasm in DRPLA patient fibroblasts and age-matched control after 48- and 72-hr treatment with Rap and BafA1. Representative images show cells after 72-hr treatment visualized with α-LaminB1 antibody (E). Upon treatment with BafA1 both control (white) and DRPLA (red) fibroblasts show an increase in LaminB1 positive puncta after 48 and 72 hr, while Rap increased cytoplasmic LaminB1 puncta only after 72 hr in DRPLA fibroblasts (F). In line the effect inhibitory effect of mTOR on proliferation, Rap resulted in significant decrease of relative cell number (p < 0.001 not shown). BafA1 treatment for 48 hr resulted in decreased cell number only in DRPLA samples in contrast to controls, while after 72 hr there was a significant reduction in cell number in both control and DRPLA samples. Automated quantification was performed using Opera Phenix high-content screening system and Columbus software. Mean ± SEM, two-way-ANOVA ∗∗∗p < 0.001. Scale bar, 50 μm.

See also Figure S6.