Abstract
Mitochondrial oxidative stress significantly contributes to the underlying pathology of several devastating neurodegenerative disorders. Mitochondria are highly sensitive to the damaging effects of reactive oxygen and nitrogen species; therefore, these organelles are equipped with a number of free radical scavenging systems. In particular, the mitochondrial glutathione (GSH) pool is a critical antioxidant reserve that is derived entirely from the larger cytosolic pool via facilitated transport. The mechanism of mitochondrial GSH transport has not been extensively studied in the brain. However, the dicarboxylate (DIC) and 2-oxoglutarate (OGC) carriers localized to the inner mitochondrial membrane have been established as GSH transporters in liver and kidney. Here, we investigated the role of these carriers in protecting neurons from oxidative and nitrosative stress. Immunoblot analysis of DIC and OGC in primary cultures of rat cerebellar granule neurons (CGNs) and cerebellar astrocytes showed differential expression of these carriers, with CGNs expressing only DIC and astrocytes expressing both DIC and OGC. Consistent with these findings, butylmalonate specifically reduced mitochondrial GSH in CGNs, whereas both butylmalonate and phenylsuccinate diminished mitochondrial GSH in astrocytes. Moreover, preincubation with butylmalonate but not phenylsuccinate significantly enhanced susceptibility of CGNs to oxidative and nitrosative stressors. This increased vulnerability was largely prevented by incubation with cell-permeable GSH monoethylester but not malate. Finally, knockdown of DIC with adenoviral siRNA also rendered CGNs more susceptible to oxidative stress. These findings demonstrate that maintenance of the mitochondrial GSH pool via sustained mitochondrial GSH transport is essential to protect neurons from oxidative and nitrosative stress.
Highlights
Mitochondrial glutathione transport has not been extensively studied within the CNS
cerebellar granule neurons (CGNs) showed a significant reduction of the mitochondrial GSH pool when treated with butylmalonate, but they displayed no decrement in the cytosolic pool (Fig. 2)
A mere ϳ32% reduction in mitochondrial GSH through specific inhibition of DIC leads to a dramatic increase in the susceptibility of CGNs to both oxidative and nitrosative stress-induced apoptosis
Summary
Mitochondrial glutathione transport has not been extensively studied within the CNS. Results: Cerebellar neurons and astrocytes use distinct mechanisms of mitochondrial glutathione transport. ROSinduced alterations in mitochondrial membrane potential and permeability can trigger a positive feedback mechanism known as ROS-induced ROS release, which has the potential to propagate oxidative stress signals from mitochondria to mitochondria, exacerbating cellular injury [5] These findings indicate that mitochondrial free radical scavenging systems, such as the essential antioxidant glutathione (GSH), are critical to protect neuronal cells from mitochondrial oxidative stress. Specific depletion of mitochondrial GSH in astrocytes rendered these glial cells more vulnerable to both oxidative and nitrosative stress [7] Based on these previous studies, mitochondrial GSH appears to be a critical antioxidant that supports both glial and neuronal cell viability. We demonstrate a key role for DIC-dependent mitochondrial GSH transport in the maintenance of the mitochondrial GSH pool and protection of CGNs from oxidative and nitrosative stress
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