Processing of cdk5 activator p35 to its truncated form (p25) by calpain inacutely injured neuronal cells

Recently, it was shown that conversion of cdk5 activator protein p35 to a C -terminal fragment p25 promotes a deregulation of cdk5 activity, which may contribute to neurodegeneration in Alzheimer's disease. In this study, we p resent evidence that calpain is a protease involved in the conversion of p3 5 to p25. To activate calpain, rat cerebellar granule neurons were treated with maitotoxin (MTX). A C-terminus directed anti-p35 antibody detected tha t p35 conversion to p25 paralleled the formation of calpain-generated alpha -spectrin (alpha-fodrin) breakdown products (SBDP's) in a maitotoxin-dose-d ependent manner. Two calpain inhibitors (MD128170 and SJA6017) reduced p35 processing but were unchanged when exposed to the caspase inhibitor carbobe nzoxy-Asp-CH2OC(=O)-2,6-dichlorobenzene or the proteasome inhibitors (lacta cystin and Z-Ile-Glu(OtBu)Ala-Leu-CHO). p35 protein was also degraded to p2 5 when rat brain lysate was subjected to in vitro digestion with purified m u- and m-calpains. Additionally, in a rat temporary middle cerebral artery occlusion model, p35 processing to p25 again paralleled SBDP formation in t he ischemic core. Lastly, in malonate-injured rat brains, the ipsilateral s ide showed a striking correlation of SBDP formation with p35 to p25 convers ion and tau phosphorylation (at Ser202 and Thr205) increase. These data sug gest that calpain is a major neuronal protease capable of converting p35 to p25 and might play a pathological role of activating cdk5 and its phosphor ylation of tau in Alzheimer's disease. (C) 2000 Academic Press.