Figure 3: Rescue of abnormal calpain-dependent cleavage of TDP-43 in AR2 mice.

(a) Immunoblot analyses of spinal cord extracts detected neither abnormal TDP-43 fragments nor an increase in activated forms of calpain in the AR2res mice compared with control mice. (b) Levels of the activated forms of calpain-I and II were not different between AR2res and control mice. The results are presented as the ratio to the value from the control mice shown in Supplementary Fig. S3. Means (columns) and s.e.m. (bars) are indicated (n=3; Student’s t-test against the wild-type (WT) value). (c) Immunohistochemical analysis for TDP-43 demonstrated that all AHCs in the AR2res mouse express nuclear TDP-43. Sections were counterstained with hematoxylin. There was normal nuclear TDP-43 immunoreactivity in ADAR2-deficient AHCs (arrow) in the AR2res mouse. (d) Cleavage of TDP-43 by calpain in the brain and spinal cord extracts of Tg (CAST Tg) or knockout (CAST KO) mice for CAST, the endogenous calpain inhibitor protein. Immunoblots show the effects of treatment of the mouse brain or spinal cord extracts with 5 mM Ca²⁺ for the indicated period of time in the presence or absence of 20 mM MDL28170 (calpain inhibitor). The amount of full-length TDP-43 at each time point is expressed as the percentage of that at time 0. Means (symbols) and s.e.m. (bars) are indicated (n=3). (e) Immunohistochemistry for TDP-43 demonstrates that all AHCs express abnormal cytoplasmic TDP-43 immunoreactivity in the CAST KO mouse. Scale bars, 20 μm.