Fig. 4.

Early abnormal protein aggregation in central nervous system. a Representative fluorescence images show ANXA11 (green) and p62 (red) aggregates in wild-type (+ / +) and mutant (R/R) mice at various ages in spinal cord motor neurons. In mutant mice, ANXA11 aggregates are evident in the cytoplasm, co-localizing with SQSTM1/p62-positive clumps from 2 months of age, with increased prominence as the disease progresses (right). Line-scan plots depict the extent of ANXA11 and p62 co-localization. b Representative fluorescence images of ANXA11 (green) and TDP-43 (red) aggregates in wild-type (+ / +) and mutant (R/R) mice at different ages. TDP-43 mislocalization from the nucleus to the cytoplasm, partially co-localizing with ANXA11 aggregates, is observed in an age-dependent manner in mutant mice. Line-scan plots (right) illustrate a significant increase in ANXA11 and TDP-43 co-localization in mutant mice from 4 months onwards. c DAB (3,3'-diaminobenzidine) staining showed translocation of TDP-43 from nucleus to cytoplasm from 4 months on in the brain of homozygous (R/R) mutant mice. d Western blotting of nuclear and cytosolic fractions from brains demonstrated an increase in cytosolic TDP-43 levels in homozygous (R/R) mutant mice compared to wild-type (+ / +). GAPDH was used as the marker for the cytosolic fraction, and PCNA for the nuclear. Hoechst was used to label nuclei. Scale bars, 10 μm. N = 3 mice, n = 7 sections per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001