Patrick G. Sullivan

Mitochondrial permeability transition in CNS trauma: Cause or effect of neuronal cell death?

Experimental traumatic brain injury (TBI) and spinal cord injury (SCI) result in a rapid and significant necrosis of neuronal tissue at the site of injury. In the ensuing hours and days, secondary injury exacerbates the primary damage, resulting in significant neurologic dysfunction. It is believed that alterations in excitatory amino acids (EAA), increased reactive oxygen species (ROS), and the disruption of Ca2+ homeostasis are major factors contributing to the ensuing neuropathology. Mitochondria serve as the powerhouse of the cell by maintaining ratios of ATP:ADP that thermodynamically favor the hydrolysis of ATP to ADP + Pi, yet a byproduct of this process is the generation of ROS. Proton‐pumping by components of the electron transport system (ETS) generates a membrane potential (ΔΨ) that can then be used to phosphorylate ADP or sequester Ca2+ out of the cytosol into the mitochondrial matrix. This allows mitochondria to act as cellular Ca2+ sinks and to be in phase with changes in cytosolic Ca2+ levels. Under extreme loads of Ca2+, however, opening of the mitochondrial permeability transition pore (mPTP) results in the extrusion of mitochondrial Ca2+ and other high‐ and low‐molecular weight components. This catastrophic event discharges ΔΨ and uncouples the ETS from ATP production. Cyclosporin A (CsA), a potent immunosuppressive drug, inhibits mitochondrial permeability transition (mPT) by binding to matrix cyclophilin D and blocking its binding to the adenine nucleotide translocator. Peripherally administered CsA attenuates mitochondrial dysfunction and neuronal damage in an experimental rodent model of TBI, in a dose‐dependent manner. The underlying mechanism of neuroprotection afforded by CsA is most likely via interaction with the mPTP because the immunosuppressant FK506, which has no effect on the mPT, was not neuroprotective. When CsA was administrated after experimental SCI at the same dosage and regimen used TBI paradigms, however, it had no beneficial neuroprotective effects. This review takes a comprehensive and critical look at the evidence supporting the role for mPT in central nervous system (CNS) trauma and highlights the differential responses of CNS mitochondria to mPT induction and the implications this has for therapeutically targeting the mPT in TBI and SCI.

Modulation of mitochondrial function by endogenous Zn2+ pools

Recent evidence suggests that intracellular Zn2+ accumulation contributes to the neuronal injury that occurs in epilepsy or ischemia in certain brain regions, including hippocampus, amygdala, and cortex. Although most attention has been given to the vesicular Zn2+ that is released into the synaptic space and may gain entry to postsynaptic neurons, recent studies have highlighted pools of intracellular Zn2+ that are mobilized in response to stimulation. One such Zn2+ pool is likely bound to cytosolic proteins, like metallothioneins. Applying imaging techniques to cultured cortical neurons, this study provides novel evidence for the presence of a mitochondrial pool distinct from the cytosolic protein or ligand-bound pool. These pools can be pharmacologically mobilized largely independently of each other, with Zn2+ release from one resulting in apparent net Zn2+ transfer to the other. Further studies found evidence for complex and potent effects of Zn2+ on isolated brain mitochondria. Submicromolar levels, comparable to those that might occur on strong mobilization of intracellular compartments, induced membrane depolarization (loss of Δψm), increases in currents across the mitochondrial inner membrane as detected by direct patch clamp recording of mitoplasts, increased O2 consumption and decreased reactive oxygen species (ROS) generation, whereas higher levels decreased O2 consumption and increased ROS generation. Finally, strong mobilization of protein-bound Zn2+ appeared to induce partial loss of Δψm, suggesting that movement of Zn2+ between cytosolic and mitochondrial pools might be of functional significance in intact neurons.

Cyclosporin A Attenuates Acute Mitochondrial Dysfunction Following Traumatic Brain Injury

Experimental traumatic brain injury (TBI) results in a rapid and significant necrosis of cortical tissue at the site of injury. In the ensuring hours and days, secondary injury exacerbates the primary damage, resulting in significant neurological dysfunction. Recent reports from our lab and others have demonstrated that the immunosuppressant cyclosporin A (CsA) is neuroprotective following TBI. The opening of the mitochondrial permeability transition pore (MPTP) is inhibited by CsA, thereby maintaining the mitochondrial membrane potential and calcium homeostasis in isolated mitochondrial. In the present study we utilized a unilateral controlled cortical impact model of TBI to assess mitochondrial dysfunction in both isolated mitochondria and synaptosomes to elucidate the neuroprotective role of CsA. The results demonstrate that administration of CsA 15 min postinjury significantly attenuates mitochondrial dysfunction as measured using several biochemical assays of mitochondria integrity and energetics. Following TBI, mitochondria isolated from the injured cortex of animals treated with CsA demonstrate a significant increase in mitochondria membrane potential and are resistant to the induction of mitochondrial permeability transition compared to vehicle-treated animals. Similarly, synaptosomes isolated from CsA-treated animals demonstrate a significant increase in mitochondria membrane potential, accompanied by lower levels of intramitochondrial Ca2+ and reactive oxygen species production than seen in vehicle-treated animals. These results suggest that the neuroprotective properties of CsA are mediated through modulation of the MPTP and maintenance of mitochondria homeostasis. Amelioration of cortical damage with CsA indicates that pharmacological therapies can be devised which will significantly alter neurological outcome after injury.

The ketogenic diet increases mitochondrial uncoupling protein levels and activity

Fatty acids are known to enhance mitochondrial uncoupling protein (UCP) activity. We asked whether a high‐fat ketogenic diet (KD) increases UCP levels and activity in hippocampi of juvenile mice. Maximum mitochondrial respiration rates were significantly (p < 0.001) higher in KD‐ versus standard diet (SD)–treated animals, indicating increased UCP‐mediated proton conductance that can reduce reactive oxygen species (ROS) production. Western blots showed significant (p < 0.05) or borderline significant increases in UCP2, UCP4, and UCP5 protein levels, and increased immunoreactivity to these three UCP isoforms was most prominently seen in the dentate gyrus of KD‐fed mice. Finally, we found that oligomycin‐induced ROS production was significantly (p < 0.05) lower in KD‐fed mice than in SD controls. Collectively, our data suggest that a KD may exert neuroprotective effects by diminishing ROS production through activation of mitochondrial UCPs.

Dietary supplement creatine protects against traumatic brain injury.

Creatine, one of the most common food supplements used by individuals at almost every level of athleticism, promote gains in performance, strength, and fat‐free mass. Recent experimental findings have demonstrated that creatine affords significant neuroprotection against ischemic and oxidative insults. The present experiments investigated the possible effect of creatine dietary supplementation on brain tissue damage after experimental traumatic brain injury. Results demonstrate that chronic administration of creatine ameliorated the extent of cortical damage by as much as 36% in mice and 50% in rats. Protection seems to be related to creatine‐induced maintenance of mitochondrial bioenergetics. Mitochondrial membrane potential was significantly increased, intramitochondrial levels of reactive oxygen species and calcium were significantly decreased, and adenosine triphosphate levels were maintained. Induction of mitochondrial permeability transition was significantly inhibited in animals fed creatine. This food supplement may provide clues to the mechanisms responsible for neuronal loss after traumatic brain injury and may find use as a neuroprotective agent against acute and delayed neurodegenerative processes. Ann Neurol 2000;48:723–729

Time Course of Post-Traumatic Mitochondrial Oxidative Damage and Dysfunction in a Mouse Model of Focal Traumatic Brain Injury: Implications for Neuroprotective Therapy

In the present study, we investigate the hypothesis that mitochondrial oxidative damage and dysfunction precede the onset of neuronal loss after controlled cortical impact traumatic brain injury (TBI) in mice. Accordingly, we evaluated the time course of post-traumatic mitochondrial dysfunction in the injured cortex and hippocampus at 30 mins, 1, 3, 6, 12, 24, 48, and 72 h after severe TBI. A significant decrease in the coupling of the electron transport system with oxidative phosphorylation was observed as early as 30 mins after injury, followed by a recovery to baseline at 1 h after injury. A statistically significant (P < 0.0001) decline in the respiratory control ratio was noted at 3 h, which persisted at all subsequent time-points up to 72 h after injury in both cortical and hippocampal mitochondria. Structural damage seen in purified cortical mitochondria included severely swollen mitochondria, a disruption of the cristae and rupture of outer membranes, indicative of mitochondrial permeability transition. Consistent with this finding, cortical mitochondrial calcium-buffering capacity was severely compromised by 3h after injury, and accompanied by significant increases in mitochondrial protein oxidation and lipid peroxidation. A possible causative role for reactive nitrogen species was suggested by the rapid increase in cortical mitochondrial 3-nitrotyrosine levels shown as early as 30 mins after injury. These findings indicate that post-traumatic oxidative lipid and protein damage, mediated in part by peroxynitrite, occurs in mitochondria with concomitant ultrastructural damage and impairment of mitochondrial bioenergetics. The data also indicate that compounds which specifically scavenge peroxynitrite (ONOO) or ONOO−derived radicals (e.g. ONOO− + H+ → ONOOH → †NO2 + †OH) may be particularly effective for the treatment of TBI, although the therapeutic window for this neuroprotective approach might only be 3 h.

Inflammation induces mitochondrial dysfunction and dopaminergic neurodegeneration in the nigrostriatal system

Evidence suggests that chronic inflammation, mitochondrial dysfunction, and oxidative stress play significant and perhaps synergistic roles in Parkinson’s disease (PD), where the primary pathology is significant loss of the dopaminergic neurons in the substantia nigra. The use of anti‐inflammatory drugs for PD treatment has been proposed, and inhibition of cyclo‐oxygenase‐2 (COX‐2) or activation of peroxisome proliferator‐activated receptor gamma (PPAR‐γ) yields neuroprotection in MPTP‐induced PD. Lipopolysaccharide (LPS) induces inflammation‐driven dopaminergic neurodegeneration. We tested the hypothesis that celecoxib (Celebrex, COX‐2 inhibitor) or pioglitazone (Actos, PPAR‐γ agonist) will reduce the LPS‐induced inflammatory response, spare mitochondrial bioenergetics, and improve nigral dopaminergic neuronal survival. Rats were treated with vehicle, celecoxib, or pioglitazone and were intrastriatally injected with LPS. Inflammation, mitochondrial dysfunction, oxidative stress, decreased dopamine, and nigral dopaminergic neuronal loss were observed post‐LPS. Celecoxib and pioglitazone provided neuroprotective properties by decreasing inflammation and restoring mitochondrial function. Pioglitazone also attenuated oxidative stress and partially restored striatal dopamine as well as demonstrated dopaminergic neuroprotection and reduced nigral microglial activation. In summary, intrastriatal LPS served as a model for inflammation‐induced dopaminergic neurodegeneration, anti‐inflammatory drugs provided protective properties, and pioglitazone or celecoxib may have therapeutic potential for the treatment of neuro‐inflammation and PD.

Mitochondrial uncoupling protein-2 protects the immature brain from excitotoxic neuronal death.

Excitotoxic cell death is the fundamental process responsible for many human neurodegenerative disorders, yet the basic mechanisms involved are not fully understood. Here, we exploited the fact that the immature brain is remarkably resistant to seizure‐induced excitotoxic cell death and examined the underlying protective mechanisms. We found that, unlike in the adult, seizures do not increase the formation of reactive oxygen species or result in mitochondrial dysfunction in neonatal brain, because of high levels of the mitochondrial uncoupling protein (UCP2). UCP2 expression and function were basally increased in neonatal brain by the fat‐rich diet of maternal milk, and substituting a low‐fat diet reduced UCP2, restored mitochondrial coupling, and permitted seizure‐induced neuronal injury. Thus, modulation of UCP2 expression and function by dietary fat protects neonatal neurons from excitotoxicity by preventing mitochondrial dysfunction. This mechanism offers novel neuroprotective strategies for individuals, greater than 1% of the worlds population, who are affected by seizures. Ann Neurol 2003

KETONES INHIBIT MITOCHONDRIAL PRODUCTION OF REACTIVE OXYGEN SPECIES PRODUCTION FOLLOWING GLUTAMATE EXCITOTOXICITY BY INCREASING NADH OXIDATION

Dietary protocols that increase serum levels of ketones, such as calorie restriction and the ketogenic diet, offer robust protection against a multitude of acute and chronic neurological diseases. The underlying mechanisms, however, remain unclear. Previous studies have suggested that the ketogenic diet may reduce free radical levels in the brain. Thus, one possibility is that ketones may mediate neuroprotection through antioxidant activity. In the present study, we examined the effects of the ketones beta-hydroxybutyrate and acetoacetate on acutely dissociated rat neocortical neurons subjected to glutamate excitotoxicity using cellular electrophysiological and single-cell fluorescence imaging techniques. Further, we explored the effects of ketones on acutely isolated mitochondria exposed to high levels of calcium. A combination of beta-hydroxybutyrate and acetoacetate (1 mM each) decreased neuronal death and prevented changes in neuronal membrane properties induced by 10 microM glutamate. Ketones also significantly decreased mitochondrial production of reactive oxygen species and the associated excitotoxic changes by increasing NADH oxidation in the mitochondrial respiratory chain, but did not affect levels of the endogenous antioxidant glutathione. In conclusion, we demonstrate that ketones reduce glutamate-induced free radical formation by increasing the NAD+/NADH ratio and enhancing mitochondrial respiration in neocortical neurons. This mechanism may, in part, contribute to the neuroprotective activity of ketones by restoring normal bioenergetic function in the face of oxidative stress.

Synaptic Mitochondria Are More Susceptible to Ca2+Overload than Nonsynaptic Mitochondria

Mitochondria in nerve terminals are subjected to extensive Ca2+fluxes and high energy demands, but the extent to which the synaptic mitochondria buffer Ca2+ is unclear. In this study, we identified a difference in the Ca2+ clearance ability of nonsynaptic versus synaptic mitochondrial populations enriched from rat cerebral cortex. Mitochondria were isolated using Percoll discontinuous gradients in combination with high pressure nitrogen cell disruption. Mitochondria in the nonsynaptic fraction originate from neurons and other cell types including glia, whereas mitochondria enriched from a synaptosomal fraction are predominantly neuronal and presynaptic in origin. There were no differences in respiration or initial Ca2+ loads between nonsynaptic and synaptic mitochondrial populations. Following both bolus and infusion Ca2+ addition, nonsynaptic mitochondria were able to accumulate significantly more exogenously added Ca 2+ than the synaptic mitochondria before undergoing mitochondrial permeability transition, observed as a loss in mitochondrial membrane potential and decreased Ca2+ uptake. The limited ability of synaptic mitochondria to accumulate Ca2+ could result from several factors including a primary function of ATP production to support the high energy demand of presynaptic terminals, their relative isolation in comparison with the threads or clusters of mitochondria found in the soma of neurons and glia, or the older age and increased exposure to oxidative damage of synaptic versus nonsynaptic mitochondria. By more readily undergoing permeability transition, synaptic mitochondria may initiate neuron death in response to insults that elevate synaptic levels of intracellular Ca2+, consistent with the early degeneration of distal axon segments in neurodegenerative disorders.