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Kainic acid induces selective mitochondrial oxidative phosphorylation enzyme dysfunction in cerebellar granule neurons: protective effects of melatonin and GSH ethyl ester¹

Department of Pharmacology, University of Padova, 35131 Padova, Italy; and
^* Department of Neuroscience Research, SmithKline Beecham Pharmaceuticals, Harlow, Essex CM19 5AW, U.K.

²Correspondence: Department of Pharmacology, L.go Meneghetti, 2, 35131 Padova, Italy. E-mail: giusti{at}ux1.unipd.it

SPECIFIC AIMS

In this study, we addressed the hypothesis that the potent central nervous system (CNS) excitotoxin kainic acid (KA) causes neuronal death via production of reactive oxygen species (ROS), which leads to mitochondrial dysfunction. The effects of KA on cellular viability, levels of intracellular ROS and mitochondrial oxidative phosphorylation (OXPHOS) enzyme activities have been investigated in vitro on rat cerebellar granule neurons, together with the neuroprotective potential of melatonin and GSH ethyl ester.

PRINCIPAL FINDINGS

1. KA reduces cerebellar granule neuron survival
Treatment of cerebellar granule neurons at 14 days in vitro with KA for 30–60 min resulted in a concentration-dependent reduction in survival after 24 h, as quantified using the viability dye 3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium bromide. Maximal cell death (46.0±4.0% of control) was achieved with a 30 min exposure to 0.5 mM KA; the latter conditions were chosen for all subsequent experiments.

2. KA impairs mitochondrial complex II function
To explore the effects of KA treatment on cellular oxidative function, we extracted mitochondrial OXPHOS enzymes from control and KA-treated granule neurons. Individual OXPHOS enzyme complexes were then resolved by blue native polyacrylamide gel electrophoresis (BN-PAGE) and identified by histochemical staining. Quantitative densitometric analysis of the BN-PAGE-separated complexes revealed a marked (-40%) and significant (P=0.008) decrease in complex II (succinate dehydrogenase; SDH) activity (Fig. 1 ). Smaller, nonsignificant decreases were observed for complex I (NADH ubiquinone-oxidoreductase; NADH) and complex IV (cytochrome oxidase; COX) by histochemical staining and for complexes III and V by Coomassie blue staining (Fig. 1) .

View larger version (16K): [in this window] [in a new window]   Figure 1. Coomassie blue (lanes 1–4) and histochemical enzymatic staining (lanes 5–10) of cerebellar granule neuron OXPHOS enzymes analyzed by BN-PAGE. Complex V (C V, lanes 1, 2) and complex III (C III; lanes 3, 4) protein content expressed as arbitrary units. Lanes 5–10 indicate mitochondrial respiratory chain enzyme activities (NADH, complex I; SDH, complex II; COX, complex IV). Control (white columns); 0.5 mM KA (black columns). Data are means ± SE (n=10). *P < 0.01.

A link between KA receptor engagement and mitochondrial toxicity was suggested by the fact that pretreatment of granule neurons with the KA receptor antagonist DNQX completely prevented the decrease in complex II (SDH) activity. Western blot analysis of mitochondrial complex II revealed that the KA-induced decrease in SDH activity was accompanied by a marked reduction in the catalytic portion of the enzyme, which was also prevented by DNQX (Fig. 2 ). Direct application of KA (up to 500 µM) to isolated brain mitochondria, however, produced no significant alterations in OXPHOS enzyme activities.

View larger version (48K): [in this window] [in a new window]   Figure 2. KA reduced granule neuron content of the catalytic portion of complex II. Cerebellar granule neuron mitochondrial extracts were subjected to Western blot analysis using antibodies against complex II (C II) and complex V (C V). Control (lane 1), 0.5 mM KA (lane 2), 0.5 mM KA + 50 µM DNQX (lane 3).

3. KA stimulates ROS production in cerebellar granule neurons
KA treatment of granule neurons increased ROS production as assessed by 2',7'-dichlorofluorescein, from 4.32 ± 0.15 to 6.85 ± 0.11 pmol/(mg protein·min) (P<0.01). Not unexpectedly, the KA neurotoxicity was completely prevented by DNQX.

4. Antioxidants prevent KA-induced mitochondrial injury and loss of cellular GSH
To provide further support linking KA-induced ROS production and mitochondrial compromise, KA (0.5 mM)-challenged cerebellar granule neurons were either treated concurrently with the established antioxidant melatonin (0.5 mM) or pretreated (30 min before KA) with the GSH delivery agent GSH ethyl ester (5 mM). Melatonin and GSH ethyl ester each counteracted the KA-induced loss in complex II (SDH) activity (103±6% and 99±5% of control, respectively, compared to 60±7% for KA alone), as well as SDH degradation. The pineal hormone significantly (P<0.001) ameliorated KA-induced neuronal death, from 52 ± 4% to 93 ± 2% in KA- and KA+melatonin-treated cells, respectively, and completely prevented the decrease in granule neuron GSH content caused by KA (23±2, 15±1, and 24±2 nmol GSH/mg protein, respectively, in control, KA, and KA+melatonin groups). GSH ethyl ester was equally efficacious in limiting neuronal injury and GSH loss caused by KA.

CONCLUSIONS

A key finding of our study is that exposure of cerebellar granule neurons to KA resulted in a concomitant increase in ROS production and an impairment of mitochondrial OXPHOS enzyme function. In particular, the catalytic portion of mitochondrial complex II was compromised, with consequent loss of SDH activity. The selective KA/AMPA receptor antagonist DNQX prevented both KA-induced ROS overproduction and complex II damage. The data propose mitochondria to be a critical target in KA injury to neurons.

Free radical generation appears to be a central feature in the mechanism of KA-elicited neuronal death in vivo and in vitro. In several CNS neuron populations in culture, KA treatment increased ROS production. ROS overproduction in neurons parallels a sustained increase in intracellular Ca²⁺ with consequent mitochondrial Ca²⁺ accumulation. Although mitochondria are capable of buffering large amounts of Ca²⁺ in response to receptor activation, they do so at the expense of triggering ROS production. This sequence of events has been described in GABAergic cortical and spinal motor neurons. Kainate receptor activation is reported to trigger Ca²⁺entry and generation of ROS in cerebellar granule neurons in vitro, as well. The present findings, however, do not allow one to identify the cellular compartment(s) in which ROS production occurs, and an extramitochondrial site cannot be excluded (Fig. 3 ).

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic diagram of the proposed role of Ca2+, radicals, and mitochondrial dysfunction in KA-mediated neurotoxicity. Prolonged KA receptor activation leads to Ca2+ accumulation in neurons, which can overload mitochondrial buffering capacity with consequent loss of electron transport respiratory chain enzyme activity and ROS generation. Excessive Ca2+ entry could also provoke nitric oxide synthase activation and production of reactive nitrogen species.

KA triggered a series of events that culminated in impaired mitochondrial function of cerebellar granule neurons. Mitochondrial damage was manifested as a specific and marked decrease in complex II activity. Western blot analysis revealed that the catalytic portion of this enzyme was lost in mitochondrial fractions extracted from KA-treated neurons. Such selective enzyme inactivation is not easily explained. Complex II (succinate-ubiquinone-oxidoreductase) is critical for both the Krebs cycle and the mitochondrial respiratory chain, and is the simplest enzyme complex of the electron transport respiratory chain. Two of the four subunits composing complex II constitute the catalytic portion (succinate-dehydrogenase): the 70 kDa flavoprotein and the 27 kDa iron-protein (three nonheme iron-sulfur centers); these project into the matrix. Under the present experimental conditions, detergent extraction for BN-PAGE could lead to loss of the catalytic portion of complex II, made labile by ROS interaction or by Ca²⁺-activated proteases. In this respect, KA effects resemble those of 3-nitropropionic acid and malonate. 3-Nitropropionic acid, an irreversible inhibitor of complex II, induces neuronal apoptosis in vivo and in vitro by a mechanism involving oxidative stress, Ca²⁺ elevation, nitric oxide synthase activation, and production of reactive nitrogen species (RNS). Malonate, although not an irreversible inhibitor of SDH, produces analogous effects including intracellular Ca²⁺ elevation, ROS and RNS production, and apoptosis. Enhanced RNS generation by KA could conceivably also occur in granule neurons due to raised Ca²⁺ levels and activation of nitric oxide synthase (Fig. 3) .

The tight relationship between KA toxicity and ROS production emphasizes the importance of cellular antioxidant defenses in modulating KA neurotoxicity. The cellular level of GSH, a key antioxidant defense molecule, is a key player in determining the sensitivity of cerebellar granule neurons to KA toxicity. Our study clearly demonstrates that increasing cellular GSH levels can protect neurons from mitochondrial damage caused by KA exposure and sustained ROS overproduction. The membrane-permeant GSH delivery agent GSH ethyl ester and melatonin, a free radical scavenger capable of preserving GSH homeostasis via a sparing action on GSH reductase, each increased the GSH concentration and preserved SDH activity.

Our results further strengthen the notion that molecules that maintain glutathione balance in the brain represent putative neuroprotective agents for the treatment of CNS pathologies involving excitotoxicity or where oxidative damage may contribute to mitochondrial dysfunction. Examples include Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0427fje ; to cite this article, use FASEB J. (June 18, 2001) 10.1096/fj.00-0427fje

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