Elsevier

Neurobiology of Disease

Volume 24, Issue 2, November 2006, Pages 296-307
Neurobiology of Disease

Pyruvate protects cerebellar granular cells from 6-hydroxydopamine-induced cytotoxicity by activating the Akt signaling pathway and increasing glutathione peroxidase expression

https://doi.org/10.1016/j.nbd.2006.07.005Get rights and content

Abstract

Parkinson disease (PD) is the second-most common age-related neurodegenerative disease and is characterized by the selective destruction of dopaminergic neurons. Increasing evidence indicates that oxidative stress plays a crucial role in the pathogenesis of idiopathic PD. Anti-oxidant agents including catalase, manganese porphyrin and pyruvate confer cytoprotection to different cell cultures when challenged with 6-hydroxydopamine (6-OHDA). Herein we used rat cerebellar granular cell cultures to ascertain the plausible cellular pathways involved in pyruvate-induced cytoprotection against 0.1 mM 6-OHDA. Pyruvate provided cytoprotection in a concentration-dependent manner (2–10 mM). Consistent with its well-established anti-oxidant capacity, pyruvate (10 mM) prevented 6-OHDA-induced lipid peroxidation by blocking the rise in intracellular peroxides and maintaining the intracellular reduced glutathione (GSH) levels. Further experiments revealed that pyruvate increased Akt, but not extracellular signal-regulated kinase phosphorylation. Moreover, phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated pyruvate-induced cytoprotection indicating that PI3K-mediated Akt activation is necessary for pyruvate to induce cytoprotection. On the other hand, pyruvate also up-regulated glutathione peroxidase mRNA levels, but not those of the anti-oxidant enzymes superoxide dismutase-1 and -2, catalase or the anti-apoptotic oncogenes Bcl-2 or Bcl-xL. In summary, our results strongly suggest that pyruvate, besides the anti-oxidant properties related to its structure, exerts cytoprotective actions by activating different anti-apoptotic routes that include gene regulation and Akt pathway activation.

Section snippets

Cell culture

Primary cultures of cerebellar granular neurons were obtained from dissociated cerebella of 7- to 8-day-old rats (Fernandez-Gomez et al., 2005a). Dissection and dissociation were carried out in Basal Medium Eagle (BME; Life Technology). Tissues were incubated with trypsin for 20 min at 37°C and dissociated by trituration in a medium containing DNase and trypsin. Cells were plated on 96 plastic well dishes or on 60-mm plastic Petri dishes pre-coated with poly-l-lysine (10 g/ml) at a

Pyruvate protects against 6-OHDA-induced cell death

The results shown here confirm and expand the observations that 6-OHDA induces cell death in different models (Galindo et al., 2003, Mazzio et al., 2004, Blum et al., 2001). As shown in Fig. 1, 6-OHDA induced a marked reduction in cell viability of 7 DIV cerebellar granular cultures. To analyze the effects of pyruvate on 6-OHDA-induced cytotoxicity, cells were pre-treated with 0.5–10 mM pyruvate for 1 h before the addition of 0.1 mM 6-OHDA. By 24 h after 6-OHDA addition, pyruvate was observed

Discussion

The results presented in this study are consistent with the hypothesis that pyruvate affords cytoprotection by an anti-oxidant mechanism. Although this is a well-known fact, we have demonstrated for the first time that this alpha-ketoacid confers cytoprotection to cerebellar granular cell cultures challenged with 6-OHDA through a mechanism that involves the up-regulation of glutathione peroxidase expression and the activation of the PI3K/Akt signaling pathway.

Consistent with the cytoprotective

Acknowledgments

We thank Ichiro Ikuta for careful reading and comments on the manuscript. We are grateful to Dr. Maria Ramos and Prof. Norberto Agurirre for discussions and Remedios Sanchis for technical assistance. This study was supported by SAF2002-04721 and SAF2005-07919-C02-01 from CICYT and Sanidad. 04005-00 from JCCM to J.J. PAI-05-017 from JCCM to SC and from Fondo de Investigaciones Sanitarias (FIS) PI051445 to RMS. F.J. F-G, M. G-L and M.D.P. are fellows from JCCM and M.G-F from FIS.

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