Nuclear polyQ aggregates induce sustained NE ruptures in primary rat hippocampal neurons. (A) Representative images of primary hippocampal neuron expressing BFP-fill (inverted grayscale), mCherry-cGAS (magenta), and polyQ23-NLS, polyQ74, or polyQ74-NLS (green). Neurons expressing polyQ74 show aggregation in soma, dendrites (black arrows), and nucleus (white arrows). Redistribution of mCherry-cGAS to the nucleus indicates NE rupture (white arrowhead). (B) Quantification of the percentage of neurons shown in A showing intranuclear cGAS accumulation (polyQ23-NLS, polyQ74, polyQ74-NLS; n = 519, 264, 388; N = 3). (C) Representative images of primary hippocampal neurons expressing polyQ23-NLS or polyQ74-NLS (green) and mCherry-NLS (gray) showing loss of nuclear mCherry-NLS enrichment (orange arrow). (D–F) Graphs depict nuclear enrichment of mCherry-NLS signal (D), cumulative frequency distribution of mCherry-NLS ratios (nuclear/cytoplasm; E), and the percentage of neurons with low mCherry-NLS ratio (F; n_polyQ23-NLS = 316 and n_polyQ74-NLS = 259 cells, N = 3). (G) Representative images of hippocampal neurons expressing mCherry-NLS (inverted grayscale and contrast adjusted) and polyQ74 (green) that show aggregation in the cytosol and nucleus (outlined in cyan). Orange arrow indicates loss of nuclear mCherry-NLS enrichment. (H) Proposed model for lamin disruption and sustained NE rupture induced by nuclear aggregates. Failure to restore nuclear integrity could lead to sustained loss of nuclear integrity and prolonged accumulation of NE repair factors at rupture sites. Neurons are outlined in orange. PolyQ74-NLS and polyQ74 signal was gamma adjusted (γ = 0.75; A and G). All scale bars indicate 10 μm. Dots represent averages of independent replicates grouped by shades of gray. *P <0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, assessed by Fischer’s exact test (B and F), unpaired Student’s t test (D), or Mann–Whitney test (E).