Calpain‐truncated CRMP‐3 and ‐4 contribute to potassium deprivation‐induced apoptosis of cerebellar granule neurons

Increasing evidence shows that calpain‐mediated proteolytic processing of a selective number of proteins plays an important role in neuronal apoptosis. Study of calpain‐mediated cleavage events and related functions may contribute to a better understanding of neuronal apoptosis and neurodegenerative diseases. We, therefore, investigated the role of calpain substrates in potassium deprivation‐induced apoptosis of cerebellar granule neurons (CGNs). Twelve previously known and seven novel candidates of calpain substrates were identified by 2‐D DIGE and MALDI‐TOF/TOF MS analysis. Further, the identified novel calpain substrates were validated by Western blot analysis. Moreover, we focused on the collapsin response mediator proteins (CRMP‐1, ‐2, ‐3 and ‐4 isoforms) and found that CRMPs were proteolytically processed by calpain but not by caspase, both in vivo and in vitro. To clarify the properties of the calpain‐mediated proteolysis of CRMPs, we constructed the deletion mutants of CRMPs for additional biochemical studies. In vitro cleavage assays revealed that CRMP‐1, ‐2 and ‐4 were truncated by calpain at the C‐terminus, whereas CRMP‐3 was cleaved at the N‐terminus. Finally, we assessed the role of CRMPs in the process of potassium deprivation‐triggered neuronal apoptosis by overexpressing the truncated CRMPs in CGNs. Our data clearly showed that the truncated CRMP‐3 and ‐4, but not CRMP‐1 and ‐2, significantly induced neuronal apoptosis. These findings demonstrated that calpain‐truncated CRMP‐3 and ‐4 act as pro‐apoptotic players when CGNs undergo apoptosis.