N-Methyl-DD-aspartate Blocks Activation of JNK and Mitochondrial Apoptotic Pathway Induced by Potassium Deprivation in Cerebellar Granule Cells

Xavier Xifró,Anthony Falluel-Morel,Alfredo Miñano,Nicolas Aubert,Rut Fadó,Cristina Malagelada,David Vaudry,Hubert Vaudry,Bruno Gonzalez,José Rodríguez-Alvarez

doi:10.1074/jbc.m504571200

Abstract

During the postnatal development of cerebellum, lack of excitatory innervation from the mossy fibers results in cerebellar granule cell (CGC) apoptosis during the migration of the cells toward the internal granule cell layer. Accordingly, CGCs die by apoptosis when cultured in physiological KCl concentrations (5 mm; K5), and they survive in the presence of depolarizing conditions such as high KCl concentration (25 mm; K25) or N-methyl-D-aspartate (NMDA). We have recently shown that NMDA is able to exert a long lasting neuroprotective effect when added to immature (2 days in vitro) CGC cultures by inhibition of caspase-3 activity. Here we show that NMDA- and K25-mediated neuroprotection is associated with an increase in the levels of Bcl-2, an inhibition of K5-mediated increase in Bax, and the inhibition of the release of apoptogenic factors from mitochondria such as Smac/DIABLO and cytochrome c. Moreover, we have shown that similar effects are observed when c-Jun N-terminal kinases (JNKs) are inhibited and that treatment of CGC cultures with NMDA blocks K5-mediated JNK activation. These results allow us to postulate that the inhibition of JNK-mediated release of apoptogenic factors from mitochondria is involved in the NMDA protection from K5-mediated apoptosis of CGCs.

Highlights

Mitochondria plays a pivotal role in many types of apoptotic responses [5, 6]
Since it has been described that potassium deprivation of mature cerebellar granule cell (CGC) cultures induced a reduction in ⌬⌿m [10, 15], we decided to explore the effect of NMDA or K25 on ⌬⌿m in immature CGC cultures by using the JC-1 red/green ratio
Several evidences have shown that calcium influx is necessary for the antiapoptotic effect of depolarizing agents [3, 28, 29], and we have recently shown that the long lasting neuroprotective effect of NMDA on immature CGC cultures is due to an inhibition of K5-mediated caspase-3 activation [22]

Summary

Introduction

Mitochondria plays a pivotal role in many types of apoptotic responses [5, 6]. The loss of mitochondrial transmembrane potential (⌬⌿m) and the permeabilization of the outer mitochondrial membrane allow the release of apoptogenic factors [7,8,9]. Since JNK inhibition produces similar effects to NMDA on cell viability, caspase-3 activity, release of proapoptotic factors, and ⌬⌿m in CGCs cultured in K5, we explored whether this correlation was observed when the Bax and Bcl-2 protein levels were monitored.

Results

Conclusion