Gradual loss of mutant VAPB inclusions in axotomized motor neurons. A-C) Representative images (A) and bar graph (B) showing ATF3 expression in a large number of motor neurons of two mutant VAPB mice that developed progressive motor abnormalities. The mice with a motor phenotype consisted of a mouse from line VM3 (mouse MP1, onset 61 weeks) and line VM1xVM5 (mouse MP2, onset 74 weeks). Non-trangenic (non-tg, n = 4) and transgenic littermates (n = 5) killed at the age of 104 weeks show ATF3 expression in a low proportion of motor neurons. C) Confocal images of motor neurons double-labeled for HA and ATF3 showing the absence of VAPB inclusions in ATF3 expressing motor neurons of mouse MP2. D-G) Gradual loss of mutant VAPB inclusions in axotomized motor neurons. D) Confocal image of lumbar motor neurons of a VM1 mutant VAPB mouse, 7 days after sciatic nerve transection, showing that axotomized motor neurons identified by ATF3 expression (ipsilateral) have no or very small VAPB inclusions as compared to control (contralateral) VM1 motor neurons. E) Representative images of axotomized motor neurons (ipsilateral, ATF3 positive) from VM1 mice killed at different time points after axotomy. Note the gradual reduction of the size of VAPB inclusion following post-axotomy, ultimately leading to diffuse perikaryal HA staining 2–3 weeks post-axotomy. F, G) Bar graphs showing the inclusion size expressed as mean number of pixels per inclusion (F), and the percentage of HA-labeled motor neurons with inclusions (G). Values in F represent means ± SE from more than 20 motor neurons from 2–3 mice. *, **, ***: P < 0.05, P < 0.01, P < 0.001 compared to contralateral (unpaired Student’s t-test). Scale bars: 100 μm, (A), 25 μm (D), 10 μm (C, E).