Bioenergetic restoration and neuroprotection after therapeutic targeting of mitoNEET: New mechanism of pioglitazone following traumatic brain injury.

Mitochondrial dysfunction is a pivotal event in many neurodegenerative disease states including traumatic brain injury (TBI) and spinal cord injury (SCI). One possible mechanism driving mitochondrial dysfunction is glutamate excitotoxicity leading to Ca2+-overload in neuronal or glial mitochondria. Therapies that reduce calcium overload and enhance bioenergetics have been shown to improve neurological outcomes. Pioglitazone, an FDA approved compound, has shown neuroprotective properties following TBI and SCI, but the underlying mechanism(s) are unknown. We hypothesized that the interaction between pioglitazone and a novel mitochondrial protein called mitoNEET was the basis for neuroprotection following CNS injury. We discovered that mitoNEET is an important mediator of Ca2+-mediated mitochondrial dysfunction and show that binding mitoNEET with pioglitazone can prevent Ca2+-induced dysfunction. By utilizing wild-type (WT) and mitoNEET null mice, we show that pioglitazone mitigates mitochondrial dysfunction and provides neuroprotection in WT mice, though produces no restorative effects in mitoNEET null mice. We also show that NL-1, a novel mitoNEET ligand, is neuroprotective following TBI in both mice and rats. These results support the crucial role of mitoNEET for mitochondrial bioenergetics, its importance in the neuropathological sequelae of TBI and the necessity of mitoNEET for pioglitazone-mediated neuroprotection. Since mitochondrial dysfunction is a pathobiological complication seen in other diseases such as diabetes, motor neuron disease and cancer, targeting mitoNEET may provide a novel mitoceutical target and therapeutic intervention for diseases that expand beyond TBI.

MitoNEET (CISD1) Knockout Mice Show Signs of Striatal Mitochondrial Dysfunction and a Parkinson's Disease Phenotype.

Mitochondrial dysfunction is thought to play a significant role in neurodegeneration observed in Parkinson's disease (PD), yet the mechanisms underlying this pathology remain unclear. Here, we demonstrate that loss of mitoNEET (CISD1), an iron-sulfur containing protein that regulates mitochondrial bioenergetics, results in mitochondrial dysfunction and loss of striatal dopamine and tyrosine hydroxylase. Mitochondria isolated from mice lacking mitoNEET were dysfunctional as revealed by elevated reactive oxygen species (ROS) and reduced capacity to produce ATP. Gait analysis revealed a shortened stride length and decreased rotarod performance in knockout mice, consistent with the loss of striatal dopamine. Together, these data suggest that mitoNEET KO mice exhibit many of the characteristics of early neurodegeneration in PD and may provide a novel drug discovery platform to evaluate compounds for enhancing mitochondrial function in neurodegenerative disorders.

Identification of small molecules that bind to the mitochondrial protein mitoNEET.

MitoNEET (CISD1) is a 2Fe-2S iron-sulfur cluster protein belonging to the zinc-finger protein family. Recently mitoNEET has been shown to be a major role player in the mitochondrial function associated with metabolic type diseases such as obesity and cancers. The anti-diabetic drug pioglitazone and rosiglitazone were the first identified ligands to mitoNEET. Since little is known about structural requirements for ligand binding to mitoNEET, we screened a small set of compounds to gain insight into these requirements. We found that the thiazolidinedione (TZD) warhead as seen in rosiglitazone was not an absolutely necessity for binding to mitoNEET. These results will aid in the development of novel compounds that can be used to treat mitochondrial dysfunction seen in several diseases.

Decreased mitochondrial bioenergetics and calcium buffering capacity in the basal ganglia correlates with motor deficits in a nonhuman primate model of aging.

Altered mitochondrial function in the basal ganglia has been hypothesized to underlie cellular senescence and promote age-related motor decline. We tested this hypothesis in a nonhuman primate model of human aging. Six young (6-8 years old) and 6 aged (20-25 years old) female Rhesus monkeys (Macaca mulatta) were behaviorally characterized from standardized video records. Additionally, we measured mitochondrial bioenergetics along with calcium buffering capacity in the substantia nigra and putamen (PUT) from both age groups. Our results demonstrate that the aged animals had significantly reduced locomotor activity and movement speed compared with younger animals. Moreover, aged monkeys had significantly reduced ATP synthesis capacity (in substantia nigra and PUT), reduced pyruvate dehydrogenase activity (in PUT), and reduced calcium buffering capacity (in PUT) compared with younger animals. Furthermore, this age-related decline in mitochondrial function in the basal ganglia correlated with decline in motor function. Overall, our results suggest that drug therapies designed to enhance altered mitochondrial function may help improve motor deficits in the elderly.

Changes in mitochondrial bioenergetics in the brain versus spinal cord become more apparent with age.

The cell is known to be the most basic unit of life. However, this basic unit of life is dependent on the proper function of many intracellular organelles to thrive. One specific organelle that has vast implications on the overall health of the cell and cellular viability is the mitochondrion. These cellular power plants generate the energy currency necessary for cells to maintain homeostasis and function properly. Additionally, when mitochondria become dysfunctional, they can orchestrate the cell to undergo cell-death. Therefore, it is important to understand what insults can lead to mitochondrial dysfunction in order to promote cell health and increase cellular viability. After years of research, is has become increasingly accepted that age has a negative effect on mitochondrial bioenergetics. In support of this, we have found decreased mitochondrial bioenergetics with increased age in Sprague-Dawley rats. Within this same study we found a 200 to 600% increase in ROS production in old rats compared to young rats. Additionally, the extent of mitochondrial dysfunction and ROS production seems to be spatially defined affecting the spinal cord to a greater extent than certain regions of the brain. These tissue specific differences in mitochondrial function may be the reason why certain regions of the Central Nervous System, CNS, are disproportionately affected by aging and may play a pivotal role in specific age-related neurodegenerative diseases like Amyotrophic Lateral Sclerosis, ALS.

N-acetylcysteine amide preserves mitochondrial bioenergetics and improves functional recovery following spinal trauma.

Mitochondrial dysfunction is becoming a pivotal target for neuroprotective strategies following contusion spinal cord injury (SCI) and the pharmacological compounds that maintain mitochondrial function confer neuroprotection and improve long-term hindlimb function after injury. In the current study we evaluated the efficacy of cell-permeating thiol, N-acetylcysteine amide (NACA), a precursor of endogenous antioxidant glutathione (GSH), on mitochondrial function acutely, and long-term tissue sparing and hindlimb locomotor recovery following upper lumbar contusion SCI. Some designated injured adult female Sprague-Dawley rats (n=120) received either vehicle or NACA (75, 150, 300 or 600mg/kg) at 15min and 6h post-injury. After 24h the total, synaptic, and non-synaptic mitochondrial populations were isolated from a single 1.5cm spinal cord segment (centered at injury site) and assessed for mitochondrial bioenergetics. Results showed compromised total mitochondrial bioenergetics following acute SCI that was significantly improved with NACA treatment in a dose-dependent manner, with maximum effects at 300mg/kg (n=4/group). For synaptic and non-synaptic mitochondria, only 300mg/kg NACA dosage showed efficacy. Similar dosage (300mg/kg) also maintained mitochondrial GSH near normal levels. Other designated injured rats (n=21) received continuous NACA (150 or 300mg/kg/day) treatment starting at 15min post-injury for one week to assess long-term functional recovery over 6weeks post-injury. Locomotor testing and novel gait analyses showed significantly improved hindlimb function with NACA that were associated with increased tissue sparing at the injury site. Overall, NACA treatment significantly maintained acute mitochondrial bioenergetics and normalized GSH levels following SCI, and prolonged delivery resulted in significant tissue sparing and improved recovery of hindlimb function.

N-acetylcysteine amide confers neuroprotection, improves bioenergetics and behavioral outcome following TBI.

Traumatic brain injury (TBI) has become a growing epidemic but no approved pharmacological treatment has been identified. Our previous work indicates that mitochondrial oxidative stress/damage and loss of bioenergetics play a pivotal role in neuronal cell death and behavioral outcome following experimental TBI. One tactic that has had some experimental success is to target glutathione using its precursor N-acetylcysteine (NAC). However, this approach has been hindered by the low CNS bioavailability of NAC. The current study evaluated a novel, cell permeant amide form of N-acetylcysteine (NACA), which has high permeability through cellular and mitochondrial membranes resulting in increased CNS bioavailability. Cortical tissue sparing, cognitive function and oxidative stress markers were assessed in rats treated with NACA, NAC, or vehicle following a TBI. At 15days post-injury, animals treated with NACA demonstrated significant improvements in cognitive function and cortical tissue sparing compared to NAC or vehicle treated animals. NACA treatment also was shown to reduce oxidative damage (HNE levels) at 7days post-injury. Mechanistically, post-injury NACA administration was demonstrated to maintain levels of mitochondrial glutathione and mitochondrial bioenergetics comparable to sham animals. Collectively these data provide a basic platform to consider NACA as a novel therapeutic agent for treatment of TBI.

Targeting PPAR isoforms following CNS injury.

A major focus has developed for the discovery of proregenerative and neuroprotective therapeutic agents to help the millions of Americans who receive a CNS injury annually. Tribulations have been encountered along the way due to the complicated set of pathways that are initiated post-injury. To target this complicated multifaceted signaling cascade, the most promising therapeutics target multiple pathways involved in the secondary injury cascade, such as neuroinflammation, the generation of ROS and mitochondrial dysfunction. Compelling experimental data demonstrates that mitochondrial dysfunction is a pivotal link in the neuropathological sequelae of brain injury. A group of PPAR agonists, specifically rosiglitazone and pioglitazone, have shown an extreme amount of promise in the realm of drug discovery for CNS injury due to their ability to increase functional recovery and decrease lesion volumes following injury. The therapeutic effects of these PPAR agonists are thought to be a direct result of PPAR activity however new data is arising that shows some of the effects may be independent of PPAR activity, targeting a novel mitochondrial protein called mitoNEET. In this review, a thorough evaluation of the role of PPAR and mitoNEET in rosiglitazone and pioglitazone mediated neuroprotection will be completed in order to shed light on the mechanism of a new possible therapeutic intervention for CNS injury.