NOVEL TARGETS FOR MITOCHONDRIAL DYSFUNCTION FOLLOWING TRAUMATIC BRAIN INJURY

Mitochondrial dysfunction is a phenomenon observed in models of Traumatic Brain Injury (TBI). Loss of mitochondrial bioenergetics can result in diminished cellular homeostasis leading to cellular dysfunction and possible cellular death. Consequently, the resultant tissue damage can manifest as functional deficits and/or disease states. Therapeutic strategies to target this mitochondrial dysfunction have been investigated for models TBI and have shown promising effects. For this project, we tested the hypothesis that mitoNEET, a novel mitochondrial membrane protein, is a target for pioglitazone mediated neuroprotection. To test this, we used a severe Controlled Cortical Impact (CCI) injury model in mitoNEET null and wild-type mice. We then dosed these animals with pioglitazone or NL-1, which is a compound that has a similar structure to pioglitazone allowing us to hone in one the importance of mitoNEET binding. Wild-type animals treated with the mitoNEET ligands, both pioglitazone and NL-1, had improved mitochondrial function, tissue sparing and functional recovery, compared to mitoNEET null animals. In addition to this specific hypothesis tested, our experiments provided insight casting doubt on the central dogma that mitochondrial dysfunction following TBI is the result of vast oxidative damage and consequential irreversible mitochondrial loss. The data from these studies show that when mitoNEET is targeted with pioglitazone at 12 hours’ post-injury, mitochondrial dysfunction can be reversed. Additionally, when bypassing proteins upstream of Complex I with an alternative biofuel, such as beta-hydroxybuterate (BHB), TBI related mitochondrial dysfunction is once again reversed. This leads to novel hypothesis for future work which posits mitoNEET as a redox sensitive switch; when mitoNEET senses changes in redox, as seen in TBI, it inhibits mitochondrial respiration. When targeted with an agonist/ligand or bypassed with a biofuel TBI mitochondrial dysfunction can be reversed. These studies support the role of mitoNEET in the neuropathological sequelae of brain injury, supporting mitoNEET as a crucial target for pioglitazone mediated neuroprotection following TBI. Lastly, these studies propose a mechanism of TBI related mitochondrial dysfunction which can reversed with pharmacological agents

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

MitoNEET (CISD1) Knockout Mice Show Signs of Striatal Mitochondrial Dysfunction and a Parkinson\u27s 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

Redox Control of Human Mitochondrial Outer Membrane Protein MitoNEET [2Fe-2S] Clusters by Biological Thiols and Hydrogen Peroxide

The human mitochondrial outer membrane protein mitoNEET is a novel target of the type II diabetes drug pioglitazone. The C-terminal cytosolic domain of mitoNEET hosts a redox-active [2Fe-2S] cluster via an unusual ligand arrangement of three cysteine residues and one histidine residue. Here we report that human mitoNEET [2Fe-2S] clusters are fully reduced when expressed in Escherichia coli cells. In vitro studies show that purified mitoNEET [2Fe-2S] clusters can be partially reduced by monothiols such as reduced glutathione, l-cysteine or N-acetyl-l-cysteine and fully reduced by dithiothreitol or the E. coli thioredoxin/thioredoxin reductase system under anaerobic conditions. Importantly, thiol-reduced mitoNEET [2Fe-2S] clusters can be reversibly oxidized by hydrogen peroxide without disruption of the clusters in vitro and in E. coli cells, indicating that mitoNEET may act as a sensor of oxidative signals to regulate mitochondrial functions via its [2Fe-2S] clusters. Furthermore, the binding of the type II diabetes drug pioglitazone in mitoNEET effectively inhibits the thiol-mediated reduction of [2Fe-2S] clusters, suggesting that pioglitazone may modulate the function of mitoNEET by blocking the thiol-mediated reduction of [2Fe-2S] clusters in the protein

Flavin nucleotides act as electron shuttles mediating reduction of the [2Fe-2S] clusters in mitochondrial outer membrane protein mitoNEET

MitoNEET, a primary target of type II diabetes drug pioglitazone, has an essential role in regulating energy metabolism, iron homeostasis, and production of reactive oxygen species in mitochondria. Structurally, mitoNEET is anchored to the mitochondrial outer membrane via its N-terminal transmembrane α-helix. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via three cysteine and one histidine residues. Here we report that the reduced flavin nucleotides can rapidly reduce the mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. In the presence of NADH and flavin reductase, about 1 molecule of flavin nucleotide is sufficient to reduce 100 molecules of the mitoNEET [2Fe-2S] clusters in 4 minutes under aerobic conditions. The electron paramagnetic resonance (EPR) measurements show that flavin mononucleotide (FMN), but not flavin adenine dinucleotide (FAD), has a specific interaction with mitoNEET. Molecular docking models further reveal that flavin mononucleotide binds mitoNEET at the region between the N-terminal transmembrane α-helix and the [2Fe-2S] cluster binding domain. The closest distance between the [2Fe-2S] cluster and the bound flavin mononucleotide in mitoNEET is about 10 Å, which may facilitate rapid electron transfer from the reduced flavin nucleotide to the [2Fe-2S] cluster in mitoNEET. The results suggest that flavin nucleotides may act as electron shuttles to reduce the mitoNEET [2Fe-2S] clusters and regulate mitochondrial functions in human cells