Dimebon does not improve rat brain mitochondrial respiratory coupling

Anthony Markham1, Michael Spedding2, Paul Franklin1. 1University of Sunderland, Sunderland, United Kingdom, 2Servier, Paris, France.

Dimebon is an antihistamine compound that has been proposed to be useful in the treatment of neurodegenerative diseases (Doody et al., 2008). Mitochondria have been proposed to be a target of neuroprotective agents (Bachurin et al., 2003). We wished to investigate the effect of dimebon on brain mitochondria.

Forebrains from female Wistar rats (250-300g) were rapidly removed and placed in isolation buffer containing 220mM mannitol, 60mM sucrose, 5mM Tris-HCl, 0.5mM EGTA and 1mg ml-1 bovine serum albumin (BSA; fatty acid free) pH 7.4. Homogenates were centrifuged at 2,000 rpm for 6 min at 0-4°C and the resulting supernatant decanted off and spun for 8 min at 10,000 rpm. The pellet produced was resuspended in 9 ml of buffer and aliquots layered onto 10 ml ice-cold Percoll solution containing 250mM sucrose, 5mM Tris-HCl, 0.1mM EGTA and 18% (w/v) Percoll, pH 7.4. The resulting density gradient was centrifuged at 10,000 rpm for 45 min. A loose mitochondrial pellet, free from most contamination, was formed at the bottom of the tube with a synaptosomal fraction forming a separate layer at the top of the tube. The two layers were then isolated and centrifuged at 10,000 rpm in isolation buffer minus EGTA (incubation buffer) to remove the Percoll.

Oxygen consumption was measured polarographically (Sweetman & Weetman, 1972) using a Clark-type oxygen electrode (Rank Bros, Bottisham, UK). Respiratory studies were performed after the method of Markham et al., (2004) and recorded on a Kipp and Zonen Flat bed recorder. Calculating the RCI (Respiratory Control Index; a measure of the efficiency of respiratory coupling) assessed mitochondrial integrity. Data were analysed using a one-way ANOVA followed by Dunnett’s post-test.

Dimebon (0.1 - 100μM) had no significant effect on mitochondrial RCI in the presence of either 5 mM glutamate plus 5 mM malate (control 4.1 ± 0.34 vs. 4.02 ± 0.42 for 100μM, n = 4) or 5 mM succinate (control 2.96 ± 0.1 vs. 2.83 ± 0.15 at 100μM, n = 4).

This study provides evidence that dimebon does not influence brain mitochondrial respiration. This differs from other studies in that we have used brain mitochondria whereas others report change in the susceptibility of liver mitochondria to undergo permeability transition (Bachurin et al., 2003).

Bachurin et al., (2003). Ann NY Acad Sci. 993, 334-344.
Doody et al., (2008). Lancet, 372, 207-215.
Markham et al., (2004). Eur J Neurosci. 20, 1189-1196.
Sweetman & Weetman (1972). Exp. Physiol. Biochem. 5, 302-328.