Protective effect of cyclin-dependent kinase inhibitors against oxidative stress-induced neuronal death via inhibition of TRPC5-mediated calcium influx in epilepsy

Abstract

Background: Epilepsy is one of the most common neurological disorders caused by recurrent spontaneous seizure which lead neuronal death. Neurons are highly vulnerable to oxidative stress that triggers death signaling in acute brain injuries such as seizures and ischemic and traumatic brain injuries. During oxidative insults, intracellular calcium ion (Ca2+) and zinc ion (Zn2+) play critical roles in neuronal death. However, relationship of oxidative stress and these two ions during neuronal death need to be elucidated. Purpose: The aims of this study are to identify strong neuroprotective molecules against oxidative stress-induced neuronal death and to demonstrate the underlying mechanism for the neuroprotective effect of these drugs. Results: When the primary cultures containing both cortical neurons and astrocytes were exposed to hydrogen peroxide (H2O2), the majority of cells died and increased the release of lactate dehydrogenase (LDH) into the bathing medium. Addition of either NU6027 or indirubin-3’-oxime (I3O) markedly reduced the H2O2-induced cell death. The protective effect of NU6027 or I3O was also observed in cells treated with ZnCl2 or sodium nitroprusside (SNP; a donor of nitric oxide). Interestingly, the drugs selectively protected neurons, while they had no effect on astrocytes. When I analyzed Zn2+ and Ca2+ signals with live cell images of neurons exposed to H2O2, early Zn2+ rises were a prerequisite for late Ca2+ increases. Although both NU6027 and I3O had no effect on Zn2+ rises, they dramatically abrogated Ca2+ rises. On the other hand, N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) blocked both Zn2+ and Ca2+ rises. To figure out the route of Ca2+ responsible for H2O2-induced neuronal death, I treated the cells with several inhibitors for receptors and channels which are permeable to Ca2+. The results showed that H2O2-induced neuronal death was not attenuated by MK801 [N-methyl-D-aspartate (NMDA) receptor antagonist], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX; alpha-amino-3-hydroxy-5-methyl-4-isoxaxolepropionic acid (AMPA)/kainic acid (KA) receptor antagonist], (-)-xestospongin C (inositol trisphosphate receptor antagonist), dantrolene (ryanodine receptor antagonist). Interestingly, 2-aminoethyl diphenylborinate [2-APB; transient receptor potential (TRP) channel blocker] and ML204 (TRPC5 blocker) significantly decreased Ca2+ as well as neuronal death induced by H2O2, suggesting that TRPC may be involved. I found that TRPC5 was exclusively expressed in neurons, and neurons from TRPC5 knock out (KO) mice were resistant to neuronal death by H2O2 compared with wild type (WT) neurons. Additionally, electrophysiological analysis indicated that the NU6027 and I3O directly blocked the basal activity of TRPC5 and Zn2+-mediated TRPC5 activation in human embryonic kidney 293 (HEK293) cells expressing mouse TRPC5 proteins. NU6027 reduced mortality without the effect on seizure severity in KA-induced seizure animal models, in which oxidative stress plays a role in neuronal death. Furthermore, NU6027 significantly attenuated the resultant neuronal death in cerebral cortex and hippocampus of KA-induced seizure rats. Conclusion: This study suggests that NU6027 and I3O directly block TRPC5 channels which mediate Zn2+-dependent Ca2+ influx and oxidative stress-induced neuronal death. Therefore, the inhibition of TRPC5 can be a novel target for developing drugs for epilepsy.