Figure 4.

Loss of Nuclear Integrity in Mouse and Drosophila Models for DRPLA and in DRPLA Patients’ Fibroblast

(A) TEM images of dentate nucleus cells in wild-type (WT, top) and ATN1-FL-65Q (bottom) mice. The latter is displaying irregular shaped nuclei, where the electron dense structures (heterochromatin) are bulging out at the periphery (arrow) with electron-lucent vacuolar-like structures (arrowheads). nuc, nucleus. Scale bar, 5 μm.

(B and C) Overexpression of Drosophila polyQ Atrophin and LacZ as a control using the UAS-Gal4 system specifically in adult glial and neuronal cells driven by repo and elav promotors. The expression was induced in fully developed adult flies for 14 days by utilizing ubiquitously expressed temperature sensitive Gal4 repressor UbiGal80^TS and inactivated at 29°C. The confocal images of Drosophila LaminB show irregular lamina (arrowheads) in sAtro75QN overexpressing flies compared to LacZ expressing flies (B), which is reflected in significant loss of circularity (ImageJ, particle analysis): mean ± SEM, n = 5, Student’s t test ^∗∗∗p < 0.001 (C).

(D–G) Analysis of nuclear shape dynamics in DRPLA (17) patient fibroblasts and age-matched control after 48-hr treatment with Rap and BafA1. Representative images show folding of nuclear envelope revealed by the aLaminB1 antibody upon treatment with BafA1 in both control and DRPLA (arrows), while Rap induced folding (arrowhead) in DRPLA fibroblasts (D). The structural changes were reflected by a decrease in nuclear size after treatment in both control and DRPLA cells (E). While the nuclear roundness decreased in DRPLA cells, it increased in controls (F). The plot of roundness versus area shows an opposite trend and convergence of controls and DRPLA nuclei upon BafA1 treatment (G). Automated quantification was performed using Opera Phenix high-content screening system and Columbus software. Mean ± SEM, n = 5, two-way-ANOVA ^∗∗∗p < 0.001, ^∗p < 0.05; v1, genotype; v2, treatment. Scale bar, 50 μm.

See also Figure S5.