Strategies for neuroprotection against L‐trans‐2,4‐pyrrolidine dicarboxylate‐induced neuronal damage during energy impairment in vitro

Abstract

Increased levels of extracellular excitatory amino acids and failure of energy metabolism are two conditions associated with brain ischemia. In the present study we have combined the simultaneous inhibition of glutamate uptake and mitochondrial electron transport chain to simulate neuronal damage associated with brain ischemia. Results show that cerebellar granule neurons are not vulnerable to transient glutamate uptake inhibition by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) despite the increase in the extracellular concentration of glutamate, unless they are simultaneously exposed to the mitochondrial toxins 3-nitropropionic acid (3-NP) or sodium azide. Cell damage was assessed by light microscopy observation, by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and by the fluorescent markers for live and dead cells, calcein and ethidium homodimer, respectively. The protective effect of alternative energy substrates, such as pyruvate, acetoacetate, and beta-hydroxybutyrate against PDC-induced neuronal death during 3-NP exposure was studied and compared to the effects of the antioxidant vitamin E, the spin trapper alpha-phenyl-N-tert-butylnitrone (PBN), voltage-dependent calcium channel antagonists, and glutamate receptor antagonists. Results show that neuronal damage can be efficiently prevented in the presence of pyruvate and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, whereas the non-NMDA receptor antagonist NBQX, acetoacetate, vitamin E, and PBN showed partial protection. In contrast, beta-hydroxybutyrate and voltage-dependent calcium channels blockers did not show any protective effect at the concentrations tested.

Pyruvate carboxylation in neurons

“…Several studies have shown a neuroprotective effect of administration of pyruvate or lactate after experimental inhibition of energy metabolism (Desagher et al, 1997;Maus et al, 1999;Schurr et al, 1999;Gramsbergen et al, 2000;Sheline et al, 2000;Garcia and Massieu, 2001). This effect has been attributed to the role of pyruvate or lactate as precursors for acetyl ϳ CoA and to the antioxidant action of pyruvate through which pyruvate is decarboxylated to acetate.…”

Molecular and cellular mechanisms of excitotoxic neuronal death

“…ions and the activation of NMDA receptors by ambient glutamate. Previous studies have shown that neuronal death induced by the accumulation of glutamate and aspartate after inhibition of glutamate transporters is facilitated in the striatum of animals previously treated with the mitochondrial toxin 3-NP, an irreversible inhibitor of complex II of the mitochondrial electron transport chain [219]. Similarly, inhibition of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), by iodoacetate (IOA) facilitates neuronal damage induced by glutamate transport inhibition or by the intrastriatal administration of glutamate [220].…”

Neuroprotective and disease-modifying effects of the ketogenic diet

“…In fact, exogenous administration of β-hydroxybutyrate can reduce brain damage and improve neuronal function in models of brain hypoxia, anoxia, and ischemia (Cherian et al, 1994;Dardzinski et al, 2000;Suzuki et al, 2001Suzuki et al, , 2002Smith et al, 2005). In addition, the other ketone bodies, acetoacetate and acetone, which are β-hydroxybutyrate metabolites and can also serve as alternative energy sources, have similar neuroprotective effects (Garcia and Massieu, 2001;Massieu et al, 2001Massieu et al, , 2003Noh et al, 2006). Interestingly, in rats receiving a ketogenic diet, neuronal uptake of β-hydroxybutyrate is increased after cortical impact injury in comparison with animals receiving a standard diet (Prins et al, 2004).…”