title: Involvement of calpain/p35-p25/Cdk5/NMDAR signaling pathway in glutamate-induced neurotoxicity in cultured rat retinal neurons.
abstract: We investigated possible involvement of a calpain/p35-p25/cyclin-dependent kinase 5 (Cdk5) signaling pathway in modifying NMDA receptors (NMDARs) in glutamate-induced injury of cultured rat retinal neurons. Glutamate treatment decreased cell viability and induced cell apoptosis, which was accompanied by an increase in Cdk5 and p-Cdk5(T15) protein levels. The Cdk5 inhibitor roscovitine rescued the cell viability and inhibited the cell apoptosis. In addition, the protein levels of both calpain 2 and calpain-specific alpha-spectrin breakdown products (SBDPs), which are both Ca(2+)-dependent, were elevated in glutamate-induced cell injury. The protein levels of Cdk5, p-Cdk5(T15), calpain 2 and SBDPs tended to decline with glutamate treatments of more than 9 h. Furthermore, the elevation of SBDPs was attenuated by either D-APV, a NMDAR antagonist, or CNQX, a non-NMDAR antagonist, but was hardly changed by the inhibitors of intracellular calcium stores dantrolene and xestospongin. Moreover, the Cdk5 co-activator p35 was significantly up-regulated, whereas its cleaved product p25 expression showed a transient increase. Glutamate treatment for less than 9 h also considerably enhanced the ratio of the Cdk5-phosphorylated NMDAR subunit NR2A at Ser1232 site (p-NR2A(S1232)) and NR2A (p-NR2A(S1232)/NR2A), and caused a translocation of p-NR2A(S1232) from the cytosol to the plasma membrane. The enhanced p-NR2A(S1232) was inhibited by roscovitine, but augmented by over-expression of Cdk5. Calcium imaging experiments further showed that intracellular Ca(2+) concentrations ([Ca(2+)](i)) of retinal cells were steadily increased following glutamate treatments of 2 h, 6 h and 9 h. All these results suggest that the activation of the calpain/p35-p25/Cdk5 signaling pathway may contribute to glutamate neurotoxicity in the retina by up-regulating p-NR2A(S1232) expression.
authors.affiliation: Institutes of Brain Science, Institute of Neurobiology and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China. yymiao@fudan.edu.cn
authors.forename: Yanying
authors.initials: Y
authors.lastname: Miao
authors.forename: Ling-Dan
authors.initials: LD
authors.lastname: Dong
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Xiao-Chen
authors.initials: XC
authors.lastname: Hu
authors.forename: Xiong-Li
authors.initials: XL
authors.lastname: Yang
authors.forename: Zhongfeng
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology*; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D018698:Glutamic Acid / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D055351:Retinal Neurons / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors
chemical_list: D002154:Calpain; D051360:Cyclin-Dependent Kinase 5; D018698:Glutamic Acid; D009419:Nerve Tissue Proteins; D016194:Receptors, N-Methyl-D-Aspartate; C089495:neuronal Cdk5 activator (p25-p35); C495907:Cdk5 protein, rat