A role for calpain-dependent cleavage of TDP-43 in amyotrophic lateral sclerosis pathology

Abstract

Both mislocalization of TDP-43 and downregulation of RNA-editing enzyme ADAR2 colocalize in the motor neurons of amyotrophic lateral sclerosis patients, but how they are linked is not clear. Here we demonstrate that activation of calpain, a Ca 2 þ -dependent cysteine protease, by upregulation of Ca 2 þ -permeable AMPA receptors generates carboxyterminal-cleaved TDP-43 fragments and causes mislocalization of TDP-43 in the motor neurons expressing glutamine/arginine site-unedited GluA2 of conditional ADAR2 knockout (AR2) mice that mimic the amyotrophic lateral sclerosis pathology. These abnormalities are inhibited in the AR2res mice that express Ca 2 þ -impermeable AMPA receptors in the absence of ADAR2 and in the calpastatin transgenic mice, but are exaggerated in the calpastatin knockout mice. Additional demonstration of calpain-dependent TDP43 fragments in the spinal cord and brain of amyotrophic lateral sclerosis patients, and high vulnerability of amyotrophic lateral sclerosis-linked mutant TDP43 to cleavage by calpain support the crucial role of the calpain-dependent cleavage of TDP43 in the amyotrophic lateral sclerosis pathology.

Dual Vulnerability of TDP-43 to Calpain and Caspase-3 Proteolysis after Neurotoxic Conditions and Traumatic Brain Injury

“…Importantly, our results confirmed the multiple calpain-dependent fragments between 34 to 41 kDa that correspond to the multiple calpain cleavage sites reported by Yamashita et al 12 By comparing major CTFs, we found that both calpain and caspase-3 can in fact generate B25 kDa fragments correlating with the previous reports. 12,28 Furthermore, the calpain-specific CTFs are at 35 kDa, while caspase-3-specific CTFs are at 33 and 12 kDa as identified by our in vitro experiments ( Figure 4A). When comparing major NTFs, we observed that calpain generates 35 kDa N-terminal fragments; in contrast, caspase-3 generated 39 and 12 kDa NTFs (Figures 1 and 2).…”

“…26 The most common underlying biochemical mechanism of TDP-43 cleavage is caspase-dependent producing 35 and 25 kDa fragments have been identified. 7,27 In a recent report, six calpain cleavages of TDP- 43 have also been putatively reported by Yamashita T, et al 12 In the present work, we compared TDP-43 fragments generated by caspase-3 and calpain TDP-43 cleavages ( Figure 3). Our multiple TDP-43 cleavage patterns is generally consistent with previous studies on calpain or caspase-3 proteolysis of TDP-43.…”

“…Similar calpain or caspase-3-mediated major fragments were visualized by Coomassie Brilliant Blue staining or by immunoblotting with internal TDP-43 antibody that can detect both CTFs and NTFs ( Figures 1A and 1D). Both calpain and caspase-3-generated B25 kDa CTFs were reported by Zhang et al 7 and Yamashita T et al 12 However, the 25 kDa band was found only by calpain digestion. We also observed a major 29 kDa fragment after calpain digestion using the internal TDP-43 antibody.…”

“…7,10,11 However, a new study has provided strong evidence of calpain-dependent TDP-43 fragments in the spinal cord and brain of ALS patients and a high vulnerability of ALSlinked mutant TDP-43 to cleavage by calpain. 12 Based on these results, TDP-43 is susceptible to be the substrate of both calpain and caspase-3. Although extensive studies have focused on this protein, it remains unknown whether the proteolysis of TDP-43 by caspase or calpain contributes to generation of pathologic diversity in TDP-43 proteinopathies.…”

Staurosporine Induced Apoptosis Rapidly Downregulates TDP-43 in Glioma Cells

“…Our experiment did not specify exactly which protease is the executer. In addition, some other protease like calpain can cleave TDP-43 in motor neurons of mice (Yamashita et al, 2012), whether they also play a role in the reduction of TDP-43 in glioma cell remains to be investigated.…”