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Sodium azide: a useful tool for in vitro studying neurodegenerative diseases.

By SELVATICI R., FALZARANO S., FRANCESCHETTI L., MARINO S., PREVIATI M., LANZONI I. and SINISCALCHI A.

Abstract

The cytochrome c oxidase inhibitor sodium azide (NaN3) can mimic the mitochondrial dysfunction, which is one of the major intracellular lesions of Alzheimer's disease (AD)(Gao, Neurochem Int 2007, 50: 379), as well as the energy depletion which occurs during brain ischemia. In our laboratory the cascade of events taking place following "chemical ischemia" has been examined in brain slices (Cavallini, Neurochem Int 2005, 47: 482; Selvatici, Neurochem Int 2006, 49/8: 729; Siniscalchi, Ann NY Acad Sci 2006, 1090: 445); moreover, the mechanisms of intracellular calcium increase induced by NaN3 in neuronal cultures have been investigated (Marino, Neurotoxicology 2007, 28: 622). In this work, we exposed rat primary cortical neurons to NaN3 in order to study: i) the changes in the expression levels and activity of protein kinases; ii) the features of neuronal death. Western blot analysis of protein kinases C (PKC) revealed a significant decrease in the total levels of alpha and betaI but not in beta2 and epsilon isoforms 30 min after a 10-min treatment with 10 mM NaN3, without any significant change in the levels of betaII and epsilon isozymes. At the same time, alpha and betaII isoforms were activated, as indicated by translocation to the particulate compartment. Twenty-four hours after NaN3, the total labeling of all PKC isoforms, except epsilon, was significantly increased, and all the PKC isoforms were mainly detected in the particulate compartment. The level of p21Ras was increased 30 min after NaN3, and recovered to control values at 24 hours. The phosphorylation degrees of ERK1/2 and of SAPK/JNK, but not of p38 MAPK, were increased both at 30 min and at 24 hours. All the observed changes were prevented by the NMDA receptor antagonist, MK801, 10 µM, suggesting the involvement of glutamate in the action of NaN3. The proportion of viable cells (MTT colorimetric determination) was reduced 24 hours after NaN3 in a concentration-dependent manner. Similarly, JC-1 fluorescence was reduced, indicating mitochondrial damage. Conversely, no nuclear fragmentation was displayed by DAPI labeling, nor by flow cytometry analysis, using Annexin V. In conclusion, the mitochondrial dysfunction induced by NaN3 provides a common platform for investigating the mechanisms of neuronal injury. Exposing neuronal cultures to NaN3 in combination to oxidative stress (e. g., H2O2) may better mimic the AD-like injury, while the combination with glycolysis blockade (e. g., 2-deoxyglucose) may reproduce the ischemic-like injury. Such protocols may also be useful for screening potential protective agents against neuronal death

Publisher: Marc Diederich
Year: 2008
OAI identifier: oai:iris.unife.it:11392/1398746
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