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-Cdk5T15 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 Ca2+-dependent, were elevated in glutamate-induced cell injury. The protein levels of Cdk5, p-Cdk5T15, 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-NR2AS1232) and NR2A (p-NR2AS1232/NR2A), and caused a translocation of p-NR2AS1232 from the cytosol to the plasma membrane. The enhanced p-NR2AS1232 was inhibited by roscovitine, but augmented by over-expression of Cdk5. Calcium imaging experiments further showed that intracellular Ca2+ concentrations ([Ca2+]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-NR2AS1232 expression.

Changes of synapses in experimental autoimmune encephalomyelitis by using Fasudil

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Roles of the miR-137-3p/CAPN-2 gene pair in ischemia-reperfusion-induced neuronal apoptosis through modulation of p35 cleavage and subsequent caspase-8 overactivation

“…Previous in vitro and in vivo studies have shown that CAPN-2 upregulation triggers neuronal apoptosis [20,39]. These studies found that activated CAPN-2 directly and precisely cleaves its substrate, membrane-bound protein p35 into p25, which consequently results in Cdk5 activation in cultured primary neurons and retinal ganglion cells [13,20,39].…”

TFP5 peptide, derived from CDK5-activating cofactor p35, provides neuroprotection in early-stage of adult ischemic stroke

“…However, several large-scale clinical trials have failed to find the expected efficacy of NMDA receptor antagonists in reducing brain injuries303132, and the underlying reason might be the differential roles of NMDA receptor subunits in mediating excitotoxic neuronal death33. Previous studies showed that ischemia induced the phosphorylation of NR2A S1232 which was contributed by CDK5 signaling pathway, and a nonspecific CDK inhibitor roscovitine could inhibit the cell apoptosis3435. In this study, the enhanced p-NR2A S1232 expression after ischemia was inhibited by TFP5, which indicated specific CDK5 inhibitor TFP5 protecting the brain cells after ischemia may be resulted from the inhibition on NR2A-mediated excitotoxicity.…”