Analysis of functional roles of Ca^<2+>-dependent protease calpain in neuronal cell death

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The etiology of Alzheimer's disease (AD) involves Aβ accumulation in the brain. Most therapeutic strategies to date have focused on Aβ metabolism upstream of pathogenesis. However, it is also important to investigate an executor that is located downstream of the pathogenesis, which induces neuronal dysfunction or cell death. We focused on a calpain-calpastatin system in the brain and investigated the relevance of calpain hyperactivation to AD pathogenesis. Calpain is a cytoplasmic cysteine protease, which is activated by intracellular Ca2+ influx, and its protease activity is regulated by the endogenous inhibitory protein calpastatin. However, it is not yet known whether calpain-dependent degradation of endogenous proteins is responsible for neuronal dysfunction or cell death, or which proteins those might be. We therefore attempted to identify substrate proteins of calpain by a proteomics approach using wild-type and calpastain-deficient mouse primary neurons, which were metabolically labeled and then stimulated with glutamate. Candidate protein substrates of calpain were determined by comparing half-lives of the degradation rate of the corresponding proteins between wild-type and calpastatin-deficient neurons based on the radioactivity of spots on 2D-electrophoresis gels. We have thus identified several important proteins as candidate substrates of calpain, which could be responsible for neuronal dysfunction or cell death. We also crossbred calpastatin-deficient mice with AD-model mice (i.e., amyloid precursor protein-transgenic mice) and found that neuronal calpain is not only activated by Aβ accumulation in the brains, but it also mediates Aβ-induced neuritic disorganization and tau phosphorylation in vivo. These results suggest that a calpain inhibitor may be a promising new medication for AD. We are currently exploring the pathological relevance of degradation of the newly identified substrate proteins with calpain activation using AD model mice.