Mitochondrial bioenergetics and cytometric characterization of a synaptosomal preparation from mouse brain cortex.

Mitochondrial function at synapses can be assessed in isolated nerve terminals. Synaptosomes are structures obtained in vitro by detaching the nerve endings from neuronal bodies under controlled homogenization conditions. Several protocols have been described for the preparation of intact synaptosomal fractions. Herein a fast and economical method to obtain synaptosomes with optimal intrasynaptic mitochondria functionality was described. Synaptosomal fractions were obtained from mouse brain cortex by differential centrifugation followed by centrifugation in a Ficoll gradient. The characteristics of the subcellular particles obtained were analyzed by flow cytometry employing specific tools. Integrity and specificity of the obtained organelles were evaluated by calcein and SNAP-25 probes. The proportion of positive events of the synaptosomal preparation was 75 ± 2 % and 48 ± 7% for calcein and Synaptosomal-Associated Protein of 25 kDa (SNAP-25), respectively. Mitochondrial integrity was evaluated by flow cytometric analysis of cardiolipin content, which indicated that 73 ± 1% of the total events were 10 N-nonylacridine orange (NAO)-positive. Oxygen consumption, ATP production and mitochondrial membrane potential determinations showed that mitochondria inside synaptosomes remained functional after the isolation procedure. Mitochondrial and synaptosomal enrichment were determined by measuring synaptosomes/ homogenate ratio of specific markers. Functionality of synaptosomes was verified by nitric oxide detection after glutamate addition. As compared with other methods, the present protocol can be performed briefly, does not imply high economic costs, and provides an useful tool for the isolation of a synaptosomal preparation with high mitochondrial respiratory capacity and an adequate integrity and function of intraterminal mitochondria.

Alcohol hangover effects on brain cortex non-synaptic mitochondria and synaptosomes bioenergetics.

Alcohol hangover (AH) has been associated with oxidative stress and mitochondrial dysfunction. We herein postulate that AH-induced mitochondrial alterations can be due to a different pattern of response in synaptosomes and non-synaptic (NS) mitochondria. Mice received intraperitoneal (i.p.) injections of ethanol (3.8 g/kg) or saline and were sacrificed 6 h afterward. Brain cortex NS mitochondria and synaptosomes were isolated by Ficoll gradient. Oxygen consumption rates were measured in NS mitochondria and synaptosomes by high-resolution respirometry. Results showed that NS-synaptic mitochondria from AH animals presented a 26% decrease in malate-glutamate state 3 respiration, a 64% reduction in ATP content, 28-37% decrements in ATP production rates (malate-glutamate or succinate-dependent, respectively), and 44% inhibition in complex IV activity. No changes were observed in mitochondrial transmembrane potential (ΔΨ) or in UCP-2 expression in NS-mitochondria. Synaptosome respiration driving proton leak (in the presence of oligomycin), and spare respiratory capacity (percentage ratio between maximum and basal respiration) were 30% and 15% increased in hangover condition, respectively. Synaptosomal ATP content was 26% decreased, and ATP production rates were 40-55% decreased (malate-glutamate or succinate-dependent, respectively) in AH mice. In addition, a 24% decrease in ΔΨ and a 21% increase in UCP-2 protein expression were observed in synaptosomes from AH mice. Moreover, mitochondrial respiratory complexes I-III, II-III, and IV activities measured in synaptosomes from AH mice were decreased by 18%, 34%, and 50%, respectively. Results of this study reveal that alterations in bioenergetics status during AH could be mainly due to changes in mitochondrial function at the level of synapses.

Free radical production and antioxidant status in brain cortex non-synaptic mitochondria and synaptosomes at alcohol hangover onset.

Alcohol hangover (AH) is the pathophysiological state after a binge-like drinking. We have previously demonstrated that AH induced bioenergetics impairments in a total fresh mitochondrial fraction in brain cortex and cerebellum. The aim of this work was to determine free radical production and antioxidant systems in non-synaptic mitochondria and synaptosomes in control and hangover animals. Superoxide production was not modified in non-synaptic mitochondria while a 17.5% increase was observed in synaptosomes. A similar response was observed for cardiolipin content as no changes were evidenced in non-synaptic mitochondria while a 55% decrease in cardiolipin content was found in synaptosomes. Hydrogen peroxide production was 3-fold increased in non-synaptic mitochondria and 4-fold increased in synaptosomes. In the presence of deprenyl, synaptosomal H2O2 production was 67% decreased in the AH condition. Hydrogen peroxide generation was not affected by deprenyl addition in non-synaptic mitochondria from AH mice. MAO activity was 57% increased in non-synaptic mitochondria and 3-fold increased in synaptosomes. Catalase activity was 40% and 50% decreased in non-synaptic mitochondria and synaptosomes, respectively. Superoxide dismutase was 60% decreased in non-synaptic mitochondria and 80% increased in synaptosomal fractions. On the other hand, GSH (glutathione) content was 43% and 17% decreased in synaptosomes and cytosol. GSH-related enzymes were mostly affected in synaptosomes fractions by AH condition. Acetylcholinesterase activity in synaptosomes was 11% increased due to AH. The present work reveals that AH provokes an imbalance in the cellular redox homeostasis mainly affecting mitochondria present in synaptic terminals.

Brain cortex mitochondrial bioenergetics in synaptosomes and non-synaptic mitochondria during aging.

Alterations in mitochondrial bioenergetics have been associated with brain aging. In order to evaluate the susceptibility of brain cortex synaptosomes and non-synaptic mitochondria to aging-dependent dysfunction, male Swiss mice of 3 or 17 months old were used. Mitochondrial function was evaluated by oxygen consumption, mitochondrial membrane potential and respiratory complexes activity, together with UCP-2 protein expression. Basal respiration and respiration driving proton leak were decreased by 26 and 33 % in synaptosomes from 17-months old mice, but spare respiratory capacity was not modified by aging. Succinate supported state 3 respiratory rate was decreased by 45 % in brain cortex non-synaptic mitochondria from 17-month-old mice, as compared with young animals, but respiratory control was not affected. Synaptosomal mitochondria would be susceptible to undergo calcium-induced depolarization in 17 months-old mice, while non-synaptic mitochondria would not be affected by calcium overload. UCP-2 was significantly up-regulated in both synaptosomal and submitochondrial membranes from 17-months old mice, compared to young animals. UCP-2 upregulation seems to be a possible mechanism by which mitochondria would be resistant to suffer oxidative damage during aging.