DIFFERENTIAL REGULATION OF CRMP2 PHOSPHORYLATION AT SER522 AND THR514 IN EXPERIMENTAL MODELS OF CEREBRAL ISCHEMIA

Collapsin response mediator protein 2 (CRMP2) plays a crucial role in neuronal development and signalling. CRMP2-phosphorylation regulation at Ser522 by Cdk5 primes GSK3β-mediated phosphorylation at the critical site Thr514. These CRMP2-phosphorylations have been described to participate in the neuropathogenicity of stroke injury, although, the reported results are controversial. This study aimed to characterize changes in CRMP2 phosphorylation at Ser522 and Thr514 in experimental models of cerebral ischemia to unravel its potential pathogenic role. Transient global and focal cerebral ischemia models in rats were used to perform the experimentation. CRMP2 phosphorylation at Ser522 and Thr514 was evaluated by Western blotting and two-dimensional gel electrophoresis (2-DGE) to resolve their phosphoisoforms. In addition, regional brain distribution was assessed by immunohistochemistry. The results showed a decrease in CRMP2 phosphorylation at Ser522 in cortical and hippocampal CA1 regions and at Thr514 in the cortex, after global ischemia. Whereas both phosphorylations decreased in the infarct core in focal ischemia. 2-DGE identified eight Ser522-phosphorylated CRMP2 isoforms and five overlapping Thr514-phosphorylated isoforms. CRMP2 phosphoisoform pattern differed in focal isquemia, with loss of specific isoforms. Immunohistochemistry revealed no differences in phosphorylated CRMP2 between regions in global ischemia, whereas focal ischemia showed decreased signal in neuronal damage regions. In conclusion, although it has been reported that CRMP2 phosphorylations, such as at Thr514 and Ser522, increase following neuronal damage such as cerebral ischemia, our data do not support this response. Instead, CRMP2 phosphorylation at Thr514 and Ser522 appears to be largely constitutive, with decreased phosphorylation in damaged neuronal regions.