Figure 1: TDP-43 mislocalization in the motor neurons of AR2 mice.

(a) Immunohistochemistry for TDP-43 demonstrated that TDP-43 localized to the nucleus in all of the large anterior horn cells (AHCs) in control mice, whereas some AHCs in AR2 mice were devoid of TDP-43 expression (arrows). Sections were counterstained with hematoxylin. Immunofluorescence assays revealed that ADAR2 (red) and TDP-43 (green) both localize to the nucleus in control AHCs, but the AR2 AHCs that lack nuclear TDP-43 immunoreactivity are also devoid of ADAR2 immunoreactivity (see the quantification data in Supplementary Fig. S1). TO-PRO-3 (blue) was used as a cell marker. Scale bars, 20 μm. (b) There was a marked decrease in full-length TDP-43 in the spinal cord lysates of AR2 mice compared with wild-type littermates. Consistent with a marked reduction of α-spectrin (black arrow) in association with the generation of calpain- (black and white arrowhead) and caspase-dependent (white arrow and black arrowheads) α-spectrin breakdown products, levels of active calpain-I and caspase-3 (arrowheads) are increased in the AR2 mouse. Arrow, inactive calpain; LE, long exposure. (c) The ~35-kDa bands in the AR2 mice spinal cord correspond to calpain-I-dependent TDP-43 products (arrowheads) in the lysates of Neuro2a cells treated with calpain-I (Calp-I). The samples of AR2 mice spinal cord were loading of gel with five volumes of wild type. Arrow: full-length TDP-43. (d) TDP-43 immunoreactivity was absent in AHCs that lack ADAR2 (arrows) in AR2 mice at 7 weeks and 26 weeks of age. (e) AHCs with low immunoreactivity for ADAR2 (arrowheads) showed TDP-43 immunoreactivity in the cytoplasm in heterozygous AR2 (AR2H) mice at 17 weeks of age. TDP-43-positive cytoplasmic aggregates were seen as well (inset).