In vitro mitochondrial failure and oxidative stress mimic biochemical features of Alzheimer disease Rita Selvatici a,⇑ , Luca Marani b , Silvia Marino b , Anna Siniscalchi b a Department of Medical Sciences, Sections of Medical Genetics, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy b Department of Pharmacology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy article info Article history: Received 21 February 2013 Received in revised form 13 May 2013 Accepted 16 May 2013 Available online 27 May 2013 Keywords: Sodium azide Hydrogen peroxide Tau GSK3 p35/25 BACE1 abstract Primary cortical neurons exposed to the mitochondrial toxin NaN 3 (0.1–3 mM) were submitted to oxida- tive stress with H 2 O 2 (30–150 lM), to mimic conditions observed in neurodegenerative disorders. The effects of such treatment on a series of parameters useful in characterizing neuronal damage were inves- tigated: (i) the basal release of glutamate, evaluated as 3 H-D-Aspartate efflux, was sharply, concentration- dependently, increased; (ii) the phosphorylation status of intracellular markers known to be involved in the neurodegenerative processes, in particular in Alzheimer disease: tau and GSK3b were increased, as well as the protein level of b-secretase (BACE1) and p35/25 evaluated by Western blotting, while (iii) the cell metabolic activity, measured with the MTT method, was reduced, in a concentration- and time-dependent manner. The latter effect, as well as tau hyperphosphorylation, was prevented both by a mixture of antioxidant drugs (100 lM ascorbic acid, 10 lM trolox, 100 lM glutathione) and by the anti-Alzheimer drug, memantine, 20 lM. Since it is well known that hippocampal cholinergic neurons are particularly affected in Alzheimer disease, the effects of NaN 3 and H 2 O 2 were also studied in electri- cally stimulated rat hippocampal slices, evaluating the 3 H-Choline efflux, as an index of acetylcholine release. The neurotoxic treatment depressed the neurosecretory function and the mixture of antioxidant drugs, as well as memantine, were able to restore it. The neuronal damage induced by the in vitro pro- tocol adopted in the present work displays peculiarities of neurodegenerative disorders, e.g. Alzheimer disease, underlining the role of mitochondrial failure and oxidative stress, which appear to occur upstream the neurodegenerative process; such protocol could be utilized to test the efficacy of neuropro- tective treatments. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Mitochondria play a central role in producing ATP as the source of cellular energy; their functions become less efficient during brain aging, including decreased activities of the electron transport chain (ETC.) enzymes, which in turn leads to enhanced radical oxy- gen species production (Lin and Beal, 2006; Petrozzi et al., 2007). The role of mitochondrial dysfunction and oxidative stress in many age-related neurodegenerative diseases is widely recognized (Lin and Beal, 2006). The most consistent failure in mitochondrial ETC. enzymes reported in Alzheimer disease (AD) has been cytochrome c oxidase (COX, complex IV) (Castellani et al., 2002), whose activity has been found deficient in different brain regions, in particular in the cerebral cortex and hippocampus (Kish et al., 1999; Cottrell et al., 2002). However, it has not been fully clarified whether mitochondrial impairment and oxidative stress are in- volved in the onset and progression of the disorder or are secondary to other phenomena leading to neurodegeneration (Petrozzi et al., 2007; Pickrell et al., 2009). A synergistic link between energy failure and peptide b-amyloid (Ab) accumulation has been shown in sev- eral studies: COX inhibition leads to tau hyperphosphorylation and to Ab deposition; in turn, the activity of COX is inhibited by the increased levels of Ab, thus establishing a vicious circle leading to neuronal death (Müller et al., 2010; Swerdlow, 2012). Sodium azide (NaN 3 ) is a well-known COX inhibitor (Duranteau et al., 1998), which has been utilized over the last 20 years to in- duce metabolic compromise resulting in increase in amyloid pro- duction and changes in tau phosphorylation (Gabuzda et al., 1994; Blass et al., 1990), as well as memory deficit and neurode- generation in rats (Callaway et al., 2002; Berndt et al., 2001). In previous work, carried out in cerebral cortex primary neurons and slices, we used NaN 3 both alone and in combination with the glycolysis blocker, 2-deoxy-glucose, to mimic brain hypoxia and ischemia. Besides its ability to induce mitochondrial failure, evalu- ated as transmembrane mitochondrial potential reduction (mea- sured by JC-1 fluorescence, Selvatici et al., 2009) its effects on various neurochemical parameters were studied in our laboratory: 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.05.005 ⇑ Corresponding author. E-mail address: svr@unife.it (R. Selvatici). Neurochemistry International 63 (2013) 112–120 Contents lists available at SciVerse ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/nci