Li-Ping Liang

The ketogenic diet increases mitochondrial glutathione levels.

The ketogenic diet (KD) is a high‐fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague–Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of β‐hydroxybutyrate (BHB). KD‐fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet‐fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate‐limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD‐fed rats, as well as up‐regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD‐fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H2O2 production and H2O2‐induced mtDNA damage. Isolated hippocampal mitochondria from KD‐fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H2O2 production and mtDNA damage. Together, the results demonstrate that the KD up‐regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant‐induced damage.

An orally active catalytic metalloporphyrin protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity in vivo.

Parkinsons disease (PD) is an age-related neurodegenerative disease in which the role of reactive oxygen species (ROS) is strongly implicated. The presence of oxidative stress has been detected in human and experimental PD using both direct and indirect indices. Scavenging ROS is, therefore, an important therapeutic avenue for the treatment of PD. Manganic porphyrins are catalytic antioxidants that scavenge a wide range of ROS. In this study, we tested the therapeutic effects of a compound [5,15-bis(methoxycarbonyl)-10,20-bis-trifluoromethyl-porphyrinato manganese (III) chloride (AEOL11207)] belonging to a new generation of lipophilic manganic porphyrins for neuroprotection and oral bioavailability in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of parkinsonism. Groups of adult C57BL/6 mice were administered MPTP with varying subcutaneous or oral dosing regimens of AEOL11207. Neurotoxicity was assessed by measurement of striatal dopamine levels and quantification of tyrosine hydroxylase-positive neurons in the substantial nigra pars compacta one week after the first dose of MPTP. Glutathione depletion, lipid peroxidation, and 3-nitrotyrosine (3-NT) formation were measured as indicators of oxidative stress in the ventral midbrain in vivo. AEOL11207 administered either by subcutaneous or oral routes protected against MPTP-induced dopamine depletion in the striatum as well as dopaminergic neuronal loss, glutathione depletion, lipid peroxidation, and 3-NT formation in the ventral midbrain. Neuroprotection correlated with brain metalloporphyrin concentrations. This is the first demonstration of neuroprotection by an orally active catalytic antioxidant in the MPTP mouse model and suggests its potential clinical utility for the treatment of chronic neurodegenerative diseases such as PD.

Iron-sulfur enzyme mediated mitochondrial superoxide toxicity in experimental Parkinson's disease

Mitochondrial oxidative stress is thought to be an important pathological mediator of neuronal death in Parkinsons disease. However, the precise mechanism by which mitochondrial oxidative stress mediates the death of dopaminergic neurons of the substantia nigra remains unclear. We tested the idea that neuronal damage in the MPTP (1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine) model of Parkinsons disease results, in part, from superoxide radical toxicity via inactivation of an iron‐sulfur (Fe‐S) protein, mitochondrial aconitase. Administration of MPTP in mice resulted in inactivation of mitochondrial aconitase, but not fumarase in the substantia nigra. MPTP treatment mobilized an early mitochondrial pool of iron detectable by bleomycin chelation that coincided with mitochondrial aconitase inactivation. MPTP‐induced mitochondrial aconitase inactivation, iron accumulation and dopamine depletion were significantly attenuated in transgenic mice overexpressing mitochondrial Sod2 and exacerbated in partial deficient Sod2 mice. These results suggest that mitochondrial aconitase may be an important early source of mitochondrial iron accumulation in experimental Parkinsons disease, and that superoxide radical toxicity manifested by oxidative inactivation of mitochondrial aconitase may play a pathogenic role in Parkinsons disease.

Mitochondrial DNA damage and impaired base excision repair during epileptogenesis

Oxidative stress and mitochondrial dysfunction are acute consequences of status epilepticus (SE). However, the role of mitochondrial oxidative stress and genomic instability during epileptogenesis remains unknown. Using the kainate animal model of temporal lobe epilepsy, we investigated oxidative mitochondrial DNA (mtDNA) damage and changes in the mitochondrial base excision repair pathway (mtBER) in the rat hippocampus for a period of 3 months after SE. Acute seizure activity caused a time-dependent increase in mitochondrial, but not nuclear 8-hydroxy-2-deoxyguanosine (8-OHdG/2dG) levels and a greater frequency of mtDNA lesions. This was accompanied by increased mitochondrial H2O2 production and a transient decrease in mtDNA repair capacity. The mtBER proteins 8-oxoguanine glycosylase (Ogg1) and DNA polymerase gamma (Pol gamma) demonstrated elevated expression at mRNA and protein levels shortly after SE and this was followed by a gradual improvement in mtDNA repair capacity. Recurrent seizures associated with the chronic phase of epilepsy coincided with the accumulation of mtDNA damage, increased mitochondrial H2O2 levels, decreased expression of Ogg1 and Pol gamma and impaired mtDNA repair capacity. Together, increased oxidative mtDNA damage, mitochondrial H2O2 production and alterations in the mtBER pathway provide evidence for mitochondrial oxidative stress in epilepsy and suggest that mitochondrial injury may contribute to epileptogenesis.

Acute oxidative stress and systemic Nrf2 activation by the ketogenic diet

The mechanisms underlying the efficacy of the ketogenic diet (KD) remain unknown. Recently, we showed that the KD increased glutathione (GSH) biosynthesis. Since the NF E2-related factor 2 (Nrf2) transcription factor is a primary responder to cellular stress and can upregulate GSH biosynthesis, we asked whether the KD activates the Nrf2 pathway. Here we report that rats consuming a KD show acute production of H(2)O(2) from hippocampal mitochondria, which decreases below control levels by 3 weeks, suggestive of an adaptive response. 4-Hydroxy-2-nonenal (4-HNE), an electrophilic lipid peroxidation end product known to activate the Nrf2 detoxification pathway, was also acutely increased by the KD. Nrf2 nuclear accumulation was evident in both the hippocampus and liver, and the Nrf2 target, NAD(P)H:quinone oxidoreductase (NQO1), exhibited increased activity in both the hippocampus and liver after 3 weeks. We also found chronic depletion of liver tissue GSH, while liver mitochondrial antioxidant capacity was preserved. These data suggest that the KD initially produces mild oxidative and electrophilic stress, which may systemically activate the Nrf2 pathway via redox signaling, leading to chronic cellular adaptation, induction of protective proteins, and improvement of the mitochondrial redox state.

Enhanced hippocampal F2-isoprostane formation following kainate-induced seizures

We attempted to obtain evidence for the occurrence of oxidant injury following seizure activity by measuring hippocampal F2‐isoprostanes (F2‐IsoPs), a reliable marker of free radical‐induced lipid peroxidation. Formation of F2‐IsoPs esterified in hippocampal phospholipids was correlated with hippocampal neuronal loss and mitochondrial aconitase inactivation, a marker of superoxide production in the kainate model. F2‐IsoPs were measured in microdissected hippocampal CA1, CA3 and dentate gyrus (DG) regions at various times following kainate administration. Kainate produced a large increase in F2‐IsoP levels in the highly vulnerable CA3 region 16 h post injection. The CA1 region showed small, but statistically insignificant increases in F2‐IsoP levels. Interestingly, the DG, a region resistant to kainate‐induced neuronal death also showed marked (2.5–5‐fold) increases in F2‐IsoP levels 8, 16, and 24 h post injection. The increases in F2‐Isop levels in CA3 and DG were accompanied by inactivation of mitochondrial aconitase in these regions. This marked subregion‐specific increase in F2‐Isop following kainate administration suggests that oxidative lipid damage results from seizure activity and may play an important role in seizure‐induced death of vulnerable neurons.

Activation of NADPH oxidase and extracellular superoxide production in seizure‐induced hippocampal damage

We sought to determine whether the extracellular compartment contributed to seizure‐induced superoxide (O2.−) production and to determine the role of the NADPH oxidase complex as a source of this O2.− production. The translocation of NADPH oxidase subunits (p47phox, p67phox and rac1) was assessed by immunoblot analysis and NADPH‐driven O2.− production was measured using 2‐(4‐hydroxybenzyl)‐6‐(4‐hydroxyphenyl)‐8‐benzyl‐3,7‐dihydroimidazo [1,2‐α] pyrazin‐3‐one‐enhanced chemiluminescence. Kainate‐induced status epilepticus resulted in a time‐dependent translocation of NADPH oxidase subunits (p47phox, p67phox and rac‐1) from hippocampal cytosol to membrane fractions. Hippocampal membrane fractions from kainate‐injected rats showed increased NADPH‐driven and diphenylene iodonium‐sensitive O2.− production in comparison to vehicle‐treated rats. The time‐course of kainate‐induced NADPH oxidase activation coincided with microglial activation in the rat hippocampus. Finally, kainate‐induced neuronal damage and membrane oxygen consumption were inhibited in mice overexpressing extracellular superoxide dismutase. These results suggest that seizure activity activates the membrane NADPH oxidase complex resulting in increased formation of O2.−.

Persistent impairment of mitochondrial and tissue redox status during lithium‐pilocarpine‐induced epileptogenesis

J. Neurochem. (2010) 115, 1172–1182.

Mitochondrial oxidative stress and epilepsy in SOD2 deficient mice: attenuation by a lipophilic metalloporphyrin

Epileptic seizures are a common feature associated with inherited mitochondrial diseases. This study investigated the role of mitochondrial oxidative stress in epilepsy resulting from mitochondrial dysfunction using cross-bred mutant mice lacking mitochondrial manganese superoxide dismutase (MnSOD or SOD2) and a lipophilic metalloporphyrin catalytic antioxidant. Video-EEG monitoring revealed that in the second to third week of postnatal life (P14-P21) B6D2F2 Sod2(-/-) mice exhibited frequent spontaneous motor seizures providing evidence that oxidative stress-induced mitochondrial dysfunction may contribute to epileptic seizures. To confirm the role of mitochondrial oxidative stress in epilepsy a newly developed lipophilic metalloporphyrin, AEOL 11207, with high potency for catalytic removal of endogenously generated reactive oxygen species was utilized. AEOL 11207-treated Sod2(-/-) mice showed a significant decrease in both the frequency and duration of spontaneous seizures but no effect on seizure severity. A significant increase in the average lifespan of AEOL 11207-treated Sod2(-/-) mice compared to vehicle-treated Sod2(-/-) mice was also observed. Indices of mitochondrial oxidative stress and damage (aconitase inactivation, 3-nitrotyrosine formation, and depletion of reduced coenzyme A) and ATP levels affecting neuronal excitability were significantly attenuated in the brains of AEOL 11207-treated Sod2(-/-) mice compared to vehicle-treated Sod2(-/-) mice. The occurrence of epileptic seizures in Sod2(-/-) mice and the ability of catalytic antioxidant therapy to attenuate seizure activity, mitochondrial dysfunction, and ATP levels suggest that ongoing mitochondrial oxidative stress can contribute to epilepsy associated with mitochondrial dysfunction and disease.

Seizure‐induced formation of isofurans: novel products of lipid peroxidation whose formation is positively modulated by oxygen tension

We have previously shown that seizures induce the formation of F2‐isoprostanes (F2‐IsoPs), one of the most reliable indices of oxidative stress in vivo. Isofurans (IsoFs) are novel products of lipid peroxidation whose formation is favored by high oxygen tensions. In contrast, high oxygen tensions suppress the formation of F2‐IsoPs. The present study determined seizure‐induced formation of IsoFs and its relationship with cellular oxygen levels (pO2). Status epilepticus (SE) resulted in F2‐IsoP and IsoF formation, with overlapping but distinct time courses in hippocampal subregions. IsoF, but not F2‐IsoP formation coincided with mitochondrial oxidative stress. SE resulted in a transient decrease in hippocampal pO2 measured by in vivo electron paramagnetic resonance oximetry suggesting an early phase of seizure‐induced hypoxia. Seizure‐induced F2‐IsoP formation coincided with the peak hypoxia phase, whereas IsoF formation coincided with the ‘reoxygenation’ phase. These results demonstrate seizure‐induced increase in IsoF formation and its correlation with changes in hippocampal pO2 and mitochondrial dysfunction.